Konversi, Volume 6 No.

2, Oktober 2017, 57 – 64 e- ISSN: 2541-3481

EFFECT OF GRAIN SIZE AND ACTIVATION TIME OF ZEOLITE TO

ADSORPTION AND DESORPTION OF NH4OH AND KCL AS MODEL OF
FERTILIZER-ZEOLITE MIX

Muhammad Prasanto Bimantio

Magister Teknik Sistem Fakultas Teknik, Universitas Gadjah Mada
Jl. Teknika Utara No. 3 Barek, Yogyakarta 55281, Indonesia

Email corresponding author: bimantiomp@gmail.com

ARTICLE INFO ABSTRACT

Article history: Zeolites can be used as adsorbent, ion exchange, catalyst, or catalyst
Received: 1-07-2017 carrier. Application of fertilizer use in the zeolite also be one of the
Received in revised form: 3-07- interesting topic. Zeolites in a mixture of fertilizer can use to control
2017 the release of nutrients. The purpose of this research is to study the
Accepted: 8-07-2017 effect of grain size and time of the activation of zeolite to adsorption
Published: 9-10-2017 and desorption of NH4OH and KCl as modeling of ZA and KCl
fertilizer, to obtain the value of adsorption rate constant (ka) and
Keywords: desorption rate constant (kd). This research procedure include: the
Zeolite process of adsorption by adding zeolite with various size and time of
Fertilizer activation into a sealed beaker glass and let the adsorption process
Adsorption occurs for 24 hours. After 24 hours, the solution was filtered, the
Desorption zeolite then put in 100 ml of aquadest into a sealed beaker glass and
Mathematical modelling let the desorption process happened for another 24 hours. Three
samples with the largest difference solution concentrations looked for
the value of the ka and kd. Zeolite configuration with the largest ka is
trialed with fertilizer and compared with the value of ka obtained from
modeling. The result for NH4OH adsorbate, -50+60 mesh 2 hours
configuration zeolite give the largest ka. For KCl adsorbate, -30+40
mesh 4 hours configuration zeolite give the largest ka. The value
between modeling and trials with fertilizers are not much different.

PENGARUH UKURAN BUTIR DAN WAKTU AKTIVASI ZEOLIT

TERHADAP ADSORPSI DAN DESORPSI NH4OH DAN KCL SEBAGAI
MODEL CAMPURAN PUPUK – ZEOLIT

Abstrak - Zeolit dapat digunakan sebagai penjerap, penukar ion, katalis, maupun pengemban katalis.
Aplikasi penggunaan zeolit dalam pupuk juga menjadi salah satu topik yang menarik. Zeolit dalam
campuran pupuk dapat digunakan untuk mengontrol pelepasan unsur hara. Tujuan dari penelitian ini
adalah untuk mempelajari pengaruh ukuran butir dan waktu aktivasi zeolit terhadap adsorpsi dan desorpsi
NH4OH dan KCl sebagai pemodelan dari pupuk ZA dan KCl dengan kalkulasi nilai konstanta laju
adsorpsi (ka) dan nilai konstanta laju desorpsi (kd). Tahap – tahap dalam penelitian ini antara lain:
melakukan proses adsorpsi dengan menambahkan zeolit dengan variasi ukuran dan waktu aktivasi ke
dalam gelas beker tertutup dan biarkan proses adsorpsi terjadi selama 24 jam. Kemudian, larutan
disaring dan zeolit tersebut dimasukkan dalam 100 ml aquadest ke dalam gelas beker tertutup dan
biarkan proses desopsi terjadi selama 24 jam. Tiga sampel dengan selisih konsentrasi terbesar kemudian
dihitung nilai ka dan kd. Konfigurasi zeolit dengan nilai ka terbesar akan di uji dengan pupuk, untuk
dibandingkan dengan nilai ka yang didapat dari pemodelan. Dari hasil penelitian diperoleh untuk
adsorbat NH4OH, konfigurasi zeolit -50+60 mesh dan 2 jam memberikan nilai ka terbesar, yaitu
3,7926x10-4. Sedangkan untuk adsorbat KCl, konfigurasi zeolit -30+40 mesh dan 4 jam memberikan nilai
ka terbesar, yaitu 6,6609x10-4. Hasil antara permodelan dan uji dengan pupuk memberikan nilai ka yang
tidak berbeda jauh.

Kata kunci : zeolit, pupuk, adsorpsi, desorpsi, pemodelan matematis.

Available online at konversi.ulm.ac.id

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Konversi, Volume 6 No. 2, Oktober 2017, 57 – 64
PENDAHULUAN
Mineral zeolit di alam telah banyak
ditemukan dalam beberapa jenis di Indonesia, jenis
mineral zeolit terbanyak adalah klinoptilolit dan
mordenit (Kithome et al., 1998). Zeolit alam
terbentuk karena adanya proses perubahan alam
dari batuan vulkanik. Jumlah dan komposisi kation
dalam zeolit tergantung dari jenis zeolit dan
lingkungan pembentukannya, misalnya mordenit
umumnya banyak mengandung kalsium sedangkan
klinoptilotit mengandung kalium. Zeolit yang
terbentuk pada lingkungan marin, mengandung
banyak natrium sedangkan zeolit yang terbentuk
pada lingkungan vulkanik mempunyai kadar
kalium dan magnesium yang tinggi (Kithome et
al., 1998). Dalam bidang industri, zeolit dapat
digunakan sebagai penjerap, penukar ion, katalis,
maupun pengemban katalis.
Pupuk anorganik seperti urea, KCL, TSP,
NPK, ZA, dan lain lain banyak digunakan dalam
bidang pertanian. Penggunaan pupuk tersebut saat
ini dirasa kurang efektif karena sekitar 40-60%
unsur hara terbuang percuma yang menyebabkan
kerugian ekonomi, rusaknya struktur tanah, dan
mencemari air tanah dan sungai (De Datta, 1987).
Secara tradisional zeolit sudah dikenal oleh
sebagian petani Indonesia. Beberapa petani sudah
memanfaatkan zeolit untuk menambah subur tanah
pertanian dengan jalan menaburkan pada lahan
pertaniannya, penambahan zeolit alam dalam
pupuk dapat mengontrol dan memperlambat
pelepasan unsur hara, karena diinginkan unsur hara
dapat cepat dijerap oleh zeolit namun dapat di-
release kembali dengan lambat (slow release
fertilizer) (Asyari, 2009).
Pada penelitian ini ingin diketahui
konfigurasi zeolit yang dapat memberikan hasil
terbaik dalam mengontrol pelepasan unsur hara
pada pupuk.
Zeolit merupakan mineral kristalin dari
kelompok tektosilikat,yaitu alumino-silikat
terhidrasi dengan kation alkali dan alkali tanah
seperti kalium,natrium,kalsium,dan magnesium
yang mengisi rongga-rongga kerangka alumino-
silikat dan mempunyai struktur tiga dimensi. Zeolit
dicirikan oleh kemampuannya menyerap dan
mengeluarkan air serta menukarkan bagian
kationnya tanpa merubah struktur kristalnya
(Subagyo, 1993).
Penggunaan zeolit pada umumnya
didasarkan pada sifat-sifat kimia zeolit, seperti
adsorpsi, penukar kation, dan katalis. Proses
aktivasi bertujuan untuk meningkatkan sifat-sifat
khusus zeolit dengan membuang unsur pengotor
yang terdapat di dalam zeolit. Ada dua cara yang
digunakan dalam proses aktivasi zeolit, yaitu
pemanasan dan kimia (Rini dan Lingga, 2010).
Available online at konversi.ulm.ac.id
e- ISSN: 2541-3481
Salah satu aspek penggunaan zeolit dalam
bidang pertanian adalah sebagai bahan campuran
pupuk, khususnya pupuk nitrogen. Hal ini
berdasarkan pada selektivitas adsorpsi zeolit yang
tinggi terhadap ion amonium yang mampu
mengefisiensikan penggunaan pupuk kimia
nitrogen sehingga penyerapan pupuk menjadi lebih
efisien. Oleh karena itu zeolit dapat digunakan
sebagai bahan pupuk tersedia lambat (slow release
fertilizer) (Hikmah, 2006).
Penambahan zeolit pada pupuk nitrogen
akan menjerap amonium yang dikeluarkan oleh
pupuk. Jika konsentrasi nitrat dalam tanah
menurun, amonium yang telah dijerap oleh zeolit
akan dilepaskan kembali ke dalam larutan tanah,
dengan cara demikian nitrogen yang diberikan ke
dalam tanah dapat tersedia dalam waktu yang
lama. Pupuk dalam bentuk slow release fertilizers
(SRF), dapat mengoptimalkan penyerapan hara
oleh tanaman dan mempertahankan keberadaan
hara dalam tanah, karena SRF dapat
mengendalikan pelepasan unsur sesuai dengan
waktu dan jumlah yang dibutuhkan tanaman.
Melalui cara ini, pemupukan tanaman, yang
biasanya dilakukan petani tiga kali dalam satu kali
musim tanam, cukup dilakukan sekali sehingga
menghemat penggunaan pupuk dan tenaga kerja
(Suwardi, 1991).
Adsorpsi merupakan salah satu bentuk dari
sorption operation. Ada dua komponen penting
dalam adsorpsi yakni fase bergerak (adsorbate)
dan penjerap (adsorbent). Mekanisme proses
adsorpsi pada dasarnya adalah proses penjerapan
yang terjadi pada permukaan padatan. Proses
transfer massa yang terjadi didasarkan pada
permukaan yang aktif. Mula-mula molekul
mendifusi melalui pori-pori (macropore).
Kemudian melalui mesopore. Kemudian molekul
terjerap dalam pori-pori kecil (micropore) dan
tertahan didalamnya (Mc Cabe, 1985).
Kecepatan transfer bahan dari fase cair ke
padat bergantung pada gradien konsentrasi dan
koefisien transfer massa. Gradien konsentrasi
merupakan ukuran besarnya penyimpangan dari
keadaan setimbang dalam setiap fase.
Kesetimbangan adalah keadaan dimana tidak ada
lagi transfer massa solute diantara kedua fasa
(Foust,1980). Mekaisme transfer massa untuk
system ini dapat dilihat pada Gambar 1.
Gambar 1. Mekanisme Adsorpsi Padat-Cair
58
Konversi, Volume 6 No. 2, Oktober 2017, 57 – 64
Dari Gambar 1 dapat terlihat bahwa selama
proses adsorpsi dan desorpsi, terjadi perubahan
(gradien) pada konsentrasi adsorbate di fase cair.
Dalam hal ini CA* setimbang dengan XA dan XA*
setimbang dengan CA. Namun, laju desorpsi yang
terjadi relatif sangat kecil sehingga untuk
prosesnya akan dilakukan terpisah secara batch
(Rianto, 2012).
Neraca massa adsorbate di dalam zeolit
pada proses adsorpsi adalah (Rianto, 2012):
Rate of mass input – Rate of mass output = Rate of
mass accumulation (1)
Ra – 0 =
( . )
(2)
ka.Ms.(CA-CA*) = M (3)
= ka.(CA-CA )*
(4)
Neraca massa adsorbate di fase cair pada
proses desorpsi adalah (Rianto, 2012:
Rate of mass input – Rate of mass output = Rate of
mass accumulation (1)
( . )
Rd – 0 = (5)
kd.Ms.(CA*-CA) = M (6)
*
= - kd.(CA-CA ) (7)
Ada dua metode yang digunakan untuk
mengetahui persamaan yang menunjukkan
hubungan antara konsentrasi sampel (CA) dengan
kadar sampel dalam zeolit (XA).
Metode pertama yaitu Langmuir,
perhitungan ini didasarkan pada penjerapan satu
lapisan sehingga hanya ada satu lapisan molekul
adsorbate yang dapat dijerap adsorbent. Dengan
demikian kapasitas permukaaan untuk penjerapan
maksimum terbatas dimana semua situs dan
permukaannya homogen.
Persamaan Langmuir:
. .
X = (8)
. konsentrasinya lebih sulit diukur dan tidak efektif
Metode kedua yaitu Freundlich, pendekatan
metode Freundlich ini didasarkan pada anggapan
bahwa tidak hanya satu lapisan molekul adsorbate
saja yang terjerap adsorbent sehingga lapisan
permukaan tidak dibatasi. Setelah permukaan
padatan menjerap satu lapisan molekul adsorbate
maka adsorbate masih bisa terjerap dan
membentuk lapisan seterusnya dimana situs-situs
aktif pada permukaan adsorbent bersifat
heterogen.
Persamaan Freundlich:
X = K .C / (9)
Dalam penelitian ini dicoba penggunaan
zeolit alam karena mudah didapat dan telah banyak
dimanfaatkan untuk berbagai keperluan dan sudah
Available online at konversi.ulm.ac.id
e- ISSN: 2541-3481
banyak penelitian yang dilakukan untuk
mengetahui fungsi lain dari zeolit alam.
Rezaei dan Movahedi (2009) telah
melakukan penelitian mengenai kinetika adsorpsi
dan desorpsi berbagai variasi konsentrasi KCl pada
tanah dengan campuran zeolit dan ion ammonium
pada berbagai konsentrasi di Iran dengan
pendekatan model Elovich. Kinetika adsorpsi dan
desorpsi NH4OH telah dilakukan oleh Kithome, et
al. (1998) dengan variasi pada konsentrasi dan pH
NH4OH pada sampel zeolit berasal dari barat laut
Amerika Serikat menggunakan pendekatan model
Freundlich, parabolic diffusion, Elovich, dan
heterogeneous diffusion.
Huang (2009) telah melakukan penelitian
mengenai adsorpsi NH4OH pada sampel zeolit dari
Tiongkok dengan satu jenis perlakuan aktivasi,
dengan variasi pada waktu kontak, ukuran butir,
jumlah adsorbent, dan keberadaan kation.
Penelitian mengenai peristiwa adsorpsi NH4OH
pada zeolit alam di Indonesia telah dilakukan oleh
Banon dan Suharto (2008) dengan menggunakan
larutan ammonium nitrat sebagai perlakuan
aktivasi sampel zeolit. Pengembangan model
matematis adsorpsi zeolit alam pada peristiwa
adsorpsi boron dalam larutan zirkonium telah
dilakukan oleh Sholikati dan Prayitno (2009).
Penelitian ini dilakukan untuk mengetahui
potensi kegunaan zeolit alam, dengan
memfokuskan pada pembuatan model matematis
peristiwa adsorpsi dan desorpsi NH4OH dan KCl
dengan variasi dua parameter pada aktivasi awal
sampel zeolit dari Yogyakarta.
Dalam penelitian ini digunakan metode
pemodelan untuk mempermudah proses analisis,
karena zat murni mudah diukur konsentrasi awal
dan akhirnya dengan metode titrasi yang sesuai.
Analisis pupuk lebih sulit dilakukan daripada
analisis zat murni, karena pada pupuk banyak
terdapat zat lain atau pengotor sehingga
untuk analisis sampel yang banyak seperti pada
penelitian ini.
Asumsi yang digunakan dalam penelitian
ini adalah: dalam waktu 24 jam, kesetimbangan
adsorpsi padat cair telah tercapai, proses adsorpsi
dilakukan dalam keadaan tertutup untuk
meminimalisir jumlah zat yang terlepas ke udara,
jumlah zat yang terjerap zeolit lebih besar
dibandingkan yang terlepas ke udara, mekanisme
desorpsi adalah murni kebalikan dari mekanisme
adsorpsi.
METODE PENELITIAN
Bahan
Bahan-bahan yang digunakan dalam
penelitian ini antara lain: zeolit alam dari
Kabupaten Gunung Kidul, NH4OH, KCl, pupuk
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Konversi, Volume 6 No. 2, Oktober 2017, 57 – 64 e- ISSN: 2541-3481

ZA dan pupuk KCl yang diperoleh dari UD Usaha HASIL DAN PEMBAHASAN
Tani Jl. Magelang Yogyakarta, dan bahan – bahan Adsorpsi dan Desorpsi NH4OH dan Pupuk ZA
lain untuk proses titrasi: Aquadest, Boraks, HCl, Untuk mendapatkan 3 sampel dengan
Methyl Orange, Metil Merah, K2CrO4, AgNO3, selisih konsentrasi awal dan akhir larutan (%
NaOH, dan NaCl, yang diperoleh dari terjerap) terbesar, maka diambil nilai rerata dari
Laboratorium Teknik Pangan dan Bioproses dan selisih konsentrasi awal dan akhir larutan tiap
Laboratorium Analisis Bahan, Departemen Teknik konfigurasi zeolit yang digunakan pada proses
Kimia, Fakultas Teknik, Universitas Gadjah Mada, adsorpsi.
Yogyakarta. Dari Tabel 1 dapat dilihat konfigurasi zeolit
yang memberikan nilai selisih konsentrasi awal
Cara Penelitian dan akhir larutan, konfigurasi zeolit yang dipilih
Zeolit ditumbuk halus dan kemudian diayak untuk dihitung nilai ka dan kd adalah : -30+40
sampai diperoleh 3 ukuran berbeda (-30+40 mesh, mesh 3 jam, -40+50 mesh 2 jam, dan -50+60 mesh
-40+50 mesh, dan -50+60mesh), kemudian zeolit 2 jam.
tersebut diaktivasi dalam oven bersuhu 250°C
selama 2, 3, dan 4 jam untuk masing masing Tabel 1. Data Nilai Rerata Selisih Konsentrasi Awal dan
ukuran zeolit. Kemudian adsorbate (NH4OH atau Akhir larutan NH4OHpada Berbagai Konfigurasi Zeolit
KCl), sebanyak 100 ml ditambahkan 10 gram
zeolit dengan variasi ukuran dan waktu aktivasi ke Waktu ΔKonsentrasi, %
Ukuran
aktivasi N terjerap
dalam gelas beker tertutup dan dibiarkan proses
2 jam 0,0530 18,88
adsorpsi terjadi selama 24 jam. Setelah 24 jam,
-30+40
larutan disaring, kemudian zeolit tersebut mesh
3 jam 0,0577 20,71
dimasukkan dalam 100 ml aquadest di dalam gelas 4 jam 0,0554 19,71
beker tertutup dan membiarkan proses desopsi
2 jam 0,0548 20,96
terjadi selama 24 jam. Banyaknya sampel yang
-40+50
dijerap oleh zeolit dihitung dari selisih antara mesh
3 jam 0,0471 18,05
konsentrasi larutan sampel sebelum dan sesudah 4 jam 0,0524 18,77
penjerapan, begitu pula untuk banyaknya sampel
2 jam 0,0613 21,09
yang dilepas oleh zeolit. Konsentrasi larutan -50+60
diperoleh dengan metode titrasi yang sesuai untuk 3 jam 0,0596 20,66
mesh
tiap sampel. 4 jam 0,0495 17,56
Tiga sampel dengan selisih konsentrasi
awal dan akhir larutan (% terjerap) terbesar, akan Berdasarkan pada Tabel 1, untuk ukuran
dikalkulasi nilai konstanta laju adsorpsi (ka) dan zeolit yang sama, daya jerap zeolit cenderung
nilai konstanta laju desorpsi (kd) dari konfigurasi semakin menurun seiring dengan lamanya waktu
zeolit yang digunakan pada 3 sampel tersebut pemanasan, hal ini dapat disebabkan karena waktu
dengan cara menambahkan 1,5 gram zeolit ke pemanasan yang terlalu lama akan merusak
dalam 100 ml larutan (NH4OH atau KCl) di dalam permukaan pori pori zeolit sehingga mengurangi
gelas beker tertutup dan dibiarkan proses adsorpsi luas permukaan yang dipakai untuk penjerapan dan
terjadi sampai 1 jam dengan pengambilan sampel ada kemungkinan zeolit tersebut sudah jenuh
tiap 15 menit, waktu adsorpsi dan desorpsi masing sehingga daya jerapnya menurun. Sementara untuk
masing 1 jam dengan pengambilan data sampel waktu pemanasan yang sama, daya jerap zeolit
tiap 15 menit. Setelah 1 jam, larutan disaring, cenderung fluktuatif terhadap ukuran butir zeolit.
kemudian zeolit tersebut dimasukkan dalam 100 Hal ini disebabkan karena pada saat percobaan
ml aquadest di dalam gelas beker tertutup dan ukuran butir yang digunakan tidak 100% seragam
dibiarkan proses desopsi terjadi selama 1 jam diakibatkan pengayakan yang tidak sempurna,
dengan pengambilan sampel tiap 15 menit. sehingga menyebabkan terjadinya hasil yang
Konfigurasi zeolit dengan nilai ka fluktuatif.
terbesarakan di uji dengan pupuk ZA dan KCl, Dari data pada tabel 2 dan 3, dapat dilihat
untuk kemudian dibandingkan dengan nilai ka hasil perhitungan menunjukkan bahwa nilai ka
yang didapat dari permodelan. Komposisi untuk lebih besar daripada nilai kd untuk semua
uji pupuk ZA adalah 1 gram pupuk ZA dicampur konfigurasi zeolit yang ditinjau, semakin besar
dengan 1 gram zeolit di dalam 125 ml NaOH. nilai ka, maka semakin cepat proses adsorpsi
Komposisi untuk uji pupuk KCl adalah 1 gram terjadi. Untuk membandingkan nilai ka yang
pupuk KCl dicampur dengan 1 gram zeolit di diperoleh dari pemodelan ini dengan pupuk
dalam 125 ml Aquadest. Untuk mencari nilai ka sebenarnya, maka dipilih zeolit dengan konfigurasi
dari uji pupuk ini analog dengan cara yang -50+60 mesh 2 jam dan -40+50 mesh 2 jam untuk
dilakukan pada pemodelan. diujicoba dengan pupuk ZA.

Available online at konversi.ulm.ac.id

60
Konversi, Volume 6 No. 2, Oktober 2017, 57 – 64 e- ISSN: 2541-3481

Tabel 2. Data Nilai ka dan kd dengan Pendekatan Langmuir

Zeolit Konstanta Langmuir
ka kd
Ukuran Waktu aktivasi KL X’A
-30+40 mesh 3 jam 3,0445x10-1 3,2889x10-2 2,8013x10-4 4,3834x10-5
-2 -1 -4
-40+50 mesh 2 jam 8,0077x10 1,2513x10 3,5768x10 2,8079x10-5
-50+60 mesh 2 jam 1,4261x10-1 7,0283x10-2 3,8549x10-4 1,1131x10-4

Tabel 3. Data Nilai ka dan kd dengan Pendekatan Freundlich

Zeolit Konstanta Freundlich
ka kd
Ukuran Waktu aktivasi KF n
-30+40 mesh 3 jam 9,9640x10-3 1,0006 2,6943x10-4 4,2162x10-5
-3 -4
-40+50 mesh 2 jam 9,9815x10 1,0004 3,5529x10 2,7791x10-5
-50+60 mesh 2 jam 1,0027x10-2 9,9931x10-1 3,7926x10-4 1,5914x10-4

Tabel 4. Perbandingan Nilai Konstanta Laju adsorpsi (ka)

Zeolit ka Langmuir ka Freundlich
Ukuran Waktu aktivasi Model Pupuk Model Pupuk
-40+50 mesh 2 jam 3,5768x10-4 2,3913x10-4 3,5529x10-4 2,4079x10-4
-50+60 mesh 2 jam 3,8549x10-4 2,6330x10-4 3,7926x10-4 2,6770x10-4

Dari Tabel 4 dapat dilihat perbandingan antara permodelan dan uji sebenaranya memberikan nilai
ka yang tidak berbeda jauh, perbedaan ini dapat disebabkan antara lain karena konsentrasi kandungan
pupuk yang tidak murni dan adanya pengotor pada pupuk tersebut, sehingga nilai ka yang diperoleh lebih
kecil daripada yang diperoleh saat pemodelan.
Dari hasil penelitian ini dapat diketahui bahwa konfigurasi zeolit -50+60 mesh 2 jam memberikan
nilai ka terbesar. Grafik perbandingan perhitungan dengan metode Langmuir dan Freundlich untuk
konfigurasi zeolit -50+60 mesh 2 jam dapat dilihat pada Gambar 3 dan 4.

Gambar 3. Perbandingan Data dan Hasil Perhitungan untuk Konfigurasi Zeolit -50+60 mesh dan 2 jam untuk
adsorbate NH4OH

Gambar 3 menunjukkan bahwa semakin
lama waktu adsorpsi dilakukan, maka semakin
banyak NH4OH yang terjerap dalam zeolit (XA)
sampai suatu saat NH4OH tersebut tidak dapat
terjerap lagi atau sudah dalam keadaan setimbang.
Dari Gambar 4 terlihat hubungan antara
konsentrasi adsorbate di fase cair (CA) sebanding
dengan konsentrasi adsorbate yang terjerap pada
zeolit XA. Dimana semakin banyak sisa adsorbate
di fase cair, maka jumlah adsorbate yang dapat
dijerap oleh zeolit akan semakin banyak pula.

Available online at http://ppjp.ulm.ac.id/journal/index.php/konversi

DOI: 10.20527/k.v6i2.4758 61
Konversi, Volume 6 No. 2, Oktober 2017, 57 – 64 e- ISSN: 2541-3481

Tabel 6.Data Nilai ka dan kd dengan Pendekatan

Langmuir
Konstanta
Zeolit
Langmuir
ka kd,
Waktu
Ukuran KL X’A
aktivasi
-30+40 5,2841 1,8962 6,6609 1,6584
4 jam
mesh x10-2 x10-1 x10-4 x10-4
-40+50 5,8291 1,7192 1.1571 2,1759
2 jam
mesh x10-2 x10-1 x10-4 x10-5
-40+50 5,8017 1,7275 1.2089 4,6696
3 jam
mesh x10-2 x10-1 x10-4 x10-5

Tabel 7. Data Nilai ka dan kd dengan Pendekatan

Freundlich
Konstanta
Gambar 4. Hubungan antara XA vs CA untuk Zeolit
Freundlich
Konfigurasi Zeolit -50+60 mesh 2 jam untuk adsorbate ka kd
Waktu
NH4OH Ukuran KF n
aktivasi
Adsorpsi dan Desorpsi KCl dan Pupuk KCl -30+40 1,0007 9,9972 6,3813 1,6082
4 jam
Untuk mendapatkan 3 sampel dengan mesh x10-2 x10-1 x10-4 x10-4
-40+50 9,9911 1,1110 2,1636
selisih konsentrasi awal dan akhir larutan (% 2 jam 1,0002
mesh x10-3 x10-4 x10-5
terjerap) terbesar, maka diambil nilai rerata dari -40+50 9,9916 1,1806 4,2266
3 jam 1,0002
selisih konsentrasi awal dan akhir larutan tiap mesh x10-3 x10-4 x10-5
konfigurasi zeolit yang digunakan pada proses
adsorpsi. Dari data pada Tabel 6 dan 7, dapat dilihat
Dari Tabel 5 dapat dilihat konfigurasi zeolit hasil perhitungan menunjukkan bahwa nilai ka
yang memberikan nilai selisih konsentrasi awal lebih besar daripada nilai kd untuk semua
dan akhir larutan, konfigurasi zeolit yang dipilih konfigurasi zeolit yang ditinjau, semakin besar
untuk dihitung nilai ka dan kd adalah : -30+40 nilai ka, maka semakin cepat proses adsorpsi
mesh 4 jam, -40+50 mesh 2 jam, dan -40+50 mesh terjadi. Untuk membandingkan nilai ka yang
3 jam. diperoleh dari pemodelan ini dengan pupuk
Berdasar pada Tabel 5, untuk ukuran zeolit sebenarnya, maka dipilih zeolit dengan konfigurasi
yang sama, daya jerap zeolit cenderung fluktuatif -30+40 mesh 4 jam dan -40+50 mesh 3 jam untuk
seiring dengan lamanya waktu pemanasan. diujicoba dengan pupuk KCl.
Sementara untuk waktu pemanasan yang sama,
daya jerap zeolit cenderung fluktuatif terhadap Tabel 8.Perbandingan Nilai Konstanta Laju Adsorpsi
ukuran butir zeolit. Hal ini disebabkan karena (ka)
pada saat percobaan ukuran butir yang digunakan Zeolit ka Langmuir ka Freundlich
tidak 100% seragam diakibatkan pengayakan yang Waktu
Ukuran Model pupuk model Pupuk
tidak sempurna, sehingga menyebabkan terjadinya aktivasi
hasil yang fluktuatif. -30+40 6,6609 2,0728 6,3813 2,0798
4 jam
mesh x10-4 x10-4 x10-4 x10-4
-40+50 1.2089 1,7245 1,1806 1,7294
Tabel 5. Data Nilai Rerata Selisih Konsentrasi Awal dan 3 jam
mesh x10-4 x10-4 x10-4 x10-4
Akhir larutan KCl pada Berbagai Konfigurasi Zeolit Dari Tabel 8 dapat dilihat perbandingan
Waktu ΔKonsentrasi, %
Ukuran
aktivasi N terjerap
antara pemodelan dan uji sebenaranya memberikan
nilai ka yang sedikit berbeda, perbedaan ini dapat
2 jam 0,0196 5,49
-30+40
disebabkan antara lain karena konsentrasi
3 jam 0,0235 6,59 kandungan pupuk yang tidak murni dan adanya
mesh
4 jam 0,0284 7,86 pengotor pada pupuk tersebut.
2 jam 0,0270 7,86
Dari hasil penelitian ini dapat diketahui
-40+50 bahwa konfigurasi zeolite -30+40 mesh 4 jam
3 jam 0,0275 7,65 memberikan nilai ka terbesar. Grafik perbandingan
mesh
4 jam 0,0235 6,29 perhitungan dengan metode Langmuir dan
2 jam 0,0245 6,86 Freundlich untuk konfigurasi zeolite -30+40 mesh
-50+60 4 jam dapat dilihat pada Gambar 5 dan 6.
3 jam 0,0265 7,09
mesh Gambar 5 menunjukkan bahwa semakin
4 jam 0,0275 7,41 lama waktu adsorpsi dilakukan, maka semakin
banyak KCl yang terjerap dalam zeolit (XA)
sampai suatu saat KCl tersebut tidak dapat terjerap
lagi atau sudah dalam keadaan setimbang. Dari

Available online at konversi.ulm.ac.id

62
Konversi, Volume 6 No. 2, Oktober 2017, 57 – 64
Gambar 6 terlihat hubungan antara konsentrasi
adsorbate di fase cair (CA) sebanding dengan
konsentrasi adsorbate yang terjerap pada zeolit XA.
Gambar 5. Perbandingan Data dan Hasil Perhitungan untuk Konfigurasi Zeolit -30+40 mesh 4 jam untuk adsorbate
KCl
Gambar 6. Grafik Hubungan antara XA vs CAuntuk Konfigurasi Zeolit -30+40 mesh 4 jam dengan adsorbate KCl
KESIMPULAN
Kesimpulan yang dapat diambil dari
penelitian ini antara lain:
1. Zeolit dapat digunakan untuk menjerap unsur
hara pada pupuk, sehingga dapat menekan laju
penguapan pupuk ke lingkungan.
2. Peristiwa adsorpsi dan desorpsi pada pupuk
dapat dimodelkan dengan adsorpsi dan
desorpsi dengan larutan murni.
3. Pengaruh ukuran dan waktu aktivasi zeolit
terhadap daya jerapnya sangat bervariasi.
4. Pada permodelan dengan adsorbate NH4OH,
konfigurasi zeolite -50+60 mesh dan 2 jam
memberikan nilai konstanta laju adsorpsi (ka)
terbesar. Sedangkan pada permodelan dengan
adsorbate KCl, konfigurasi zeolite -30+40
mesh dan 4 jam memberikan nilai konstanta
laju adsorpsi (ka) terbesar.
Available online at konversi.ulm.ac.id
e- ISSN: 2541-3481
Dimana semakin banyak sisa adsorbate di fase
cair, maka jumlah adsorbate yang dapat dijerap
oleh zeolit akan semakin banyak pula.
5. Pada penelitian ini nilai konstanta laju adsorpsi
(ka) lebih besar daripada nilai konstanta laju
desorpsi (kd)
DAFTAR SIMBOL
Ms : Massa zeolit (gram)
XA : konsentrasi adsorbate yang terjerap pada
zeolit (gram adsorbate/gram zeolite)
XA* : konsentrasi adsorbate di zeolit yang
setimbang dengan konsentrasi adsorbate
sisa di fasa cair (gram adsorbate/gram
zeolit)
CA : konsentrasi adsorbate di fase cair (gram
adsorbate/liter)
CA* : konsentrasi adsorbate di fase cair yang
setimbang dengan konsentrasi adsorbate di
zeolit (gram adsorbate/liter)
ka : konstanta kecepatan adsorpsi (L/gram
adsorbent.menit)
63
Konversi, Volume 6 No. 2, Oktober 2017, 57 – 64
kd : konstanta kecepatan desorpsi (L/gram
adsorbent.menit
KL : parameter persamaan isotherm Langmuir
X’A : parameter persamaan isotherm Langmuir
KF : parameter persamaan isotherm Freundlich
n : parameter persamaan isotherm Freundlich
DAFTAR PUSTAKA
ASYARI, M.A., 2009, “Penelitian Awal Granulasi
Pupuk Kandang, Pupuk Anorganik, dan
Zeolit sebagai Slow Release Fertilizer”,
Laporan Penelitian, Laboratorium
Teknologi Keramik, Jurusan Teknik Kimia,
Fakultas Teknik, Universitas Gadjah Mada,
Yogyakarta.
BANON, C. DAN SUHARTO, T.E., 2008,
“Adsorpsi Amoniak oleh Adsorben Zeolit
Alam yang Diaktivasi dengan Larutan
Amonium Nitrat”, Jurnal Gradien, 4(2),
354-360, Jurusan Kimia, Fakultas
Matematika dan Ilmu Pengetahuan Alam,
Universitas Bengkulu, Indonesia.
DE DATTA, S.K., 1987, “Advances in Soil
Fertility Research and Nitrogen Fertilizer
Management for Lowland Rice”, Efficiency
of Nitrogen Fertilizer for Rice, pp.27-41,
International Rice Research Institute,
Manila.
FOUST, A.A., 1980, “Principles of Unit
Operation”, 2nd edition, John Wiley And
Sons Inc., New York.
HIKMAH, N., 2006, “Peranan Zeolit Dalam
Pelepasan Nitrogen dari Pupuk Tersedia
Lambat (Slow Release Fertilizers)”,
Departemen Ilmu Tanah dan Sumberdaya
Lahan, Fakultas Pertanian, Institut
Pertanian Bogor.
HUANG, H., XIAO, X., YAN, B., YANG, L.,
2009, “Ammonium removal from aqueous
solutions by using natural Chinese
(Chende) zeolite as adsorbent”, Journal of
Hazardous Materials, 175, 247-252,
Elsevier B.V., Guangzhou.
JEFFERY, G.H., BASSETT, J., MENDHAM, J.,
DENNEY, R.C., 1989, “Vogel’s Textbook
of Quantitative Chemical Analysis”, 5th
ed., pp. 274-275, 286, 300, 348-349,
Longman Scientific & Technical, New
York.
KITHOME, M., PAUL, J.W., LAVKULICH,
L.M., 1998, “Kinetics of Ammonium
Adsorption and Desorption by the Natural
Zeolite Clinoptilolite”, Soil Sci. Soc. Am. J.,
62, 622–629, University of British
Columbia, Vancouver.
MC CABE, W.C., SMITH, J.C., AND HARRIOT,
P., 1985, “Operation of Chemical
Available online at konversi.ulm.ac.id
e- ISSN: 2541-3481
Engineering”, 4 ed., pp. 686-704, Mc Graw
Hill International, New York.
REZAEI, M. & MOVAHEDI NAEINI, S.A.R,
2009, “Effects of Ammonium and Iranian
Natural Zeolite on Potassium Adsorption
and Desorption Kinetics in the Loess Soil”,
International Journal of Soil Science, 4 (2),
27-45, Academic Journal Inc., Golestan
Province.
RIANTO, ADAM KUSUMA, 2012, “Studi
Pengurangan Kadar Air dan Asam Lemak
Bebas pada Minyak Kelapa Sawit dengan
Adsorpsi Menggunakan Zeolit Alam”,
Laporan Penelitian, Laboratorium Proses
Pemisahan, Jurusan Teknik Kimia, Fakultas
Teknik, Universitas Gadjah Mada,
Yogyakarta.
RINI, DIAN KUSUMA & LINGGA, FENDY
ANTHONIUS, 2010, “Optimasi Aktivasi
Zeolit Alam Untuk Dehumidifikasi”,
Skripsi, Jurusan Teknik Kimia, Fakultas
Teknik, Universitas Diponegoro, Semarang.
SHOLIKHATI, S.U. DAN PRAYITNO, 2009,
“Penentuan Kecepatan Adsorpsi Boron
dalam Larutan Zirkonium dengan Zeolit”,
Seminar Nasional SDM Teknologi Nuklir,
5 November 2009, Sekolah Tinggi
Teknologi Nuklir-Batan, Yogyakarta.
SUBAGYO, 1993, “Zeolit: Struktur dan Sifat-
Sifatnya”, Warta Insinyur Kimia, vol. 7,
No. 3, hal.17-23, BKK-PII.
SUWARDI, 1991, “The Mineralogical and
Chemical Properties of Natural Zeolite and
Their Application Effect for Soil
Amandement”, A Thesis for the Degree of
Master Laboratory of Soil Science
Departement of Agriculture Chemistry,
Tokyo University of Agriculture.
SUWARDI, 2002, “Pemanfaatan Zeolit untuk
Meningkatkan Produksi Tanaman Pangan,
Peternakan, dan Perikanan”, Seminar
Teknologi Aplikasi Pertanian Bogor IPB.
TREYBAL, R.E., 1981, “Mass Transfer
Operation”, pp. 565-567, 641-644, Mc
Graw Hill International, New York.
64
 Journal of Environmental Management 317 (2022) 115454

Contents lists available at ScienceDirect

Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman

Research article

Exploring alternate methods for predicting sorption-desorption parameters

for environmental phosphorus loss assessment in poultry litter
impacted soils
Debolina Chakraborty a, Rishi Prasad a, b, *, Dexter B. Watts c, H. Allen Torbert c
a
202 Funchess Hall, Department of Crop, Soil, and Environmental Sciences, Auburn University, Auburn, AL, 36849, USA
b
Department of Animal Science, Auburn University, Auburn, AL, 36849, USA
c
USDA-ARS National Soil Dynamics Lab., 411 S. Donahue Dr., Auburn, AL, 36832, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Repeated applications of poultry litter (PL) in weathered soils cause soil phosphorus (P) accumulation and is a
Poultry litter major environmental concern. Much remains unknown about the distribution of inorganic P (Pi) and organic P
P fractionation (Po) forms along with their retention and release characteristics in soils that have had a history of PL application.
Langmuir isotherm
The objectives of this study were to 1) determine P loss risk by investigating the distribution of Pi and Po forms, P
P sorption
P desorption
sorption-desorption parameters, and soil P saturation ratio (PSR; molar ratio of P/[Al + Fe]); and 2) predict the P
P saturation ratio sorption-desorption parameters using selected soil variables or soil test indicator, PSR. Forty soil samples from 10
random locations within seven pasture fields located in Piedmont soil region were selected and separated into
four depths: 0–5, 5–15, 15–30, and 30–45 cm. These fields received annual PL applications ranging from 26
to 92 kg P ha-1 for the past 10 years. The P forms were determined using sequential P fractionation. Soil P
retention characteristics were determined using Langmuir isotherm. Soil PSR was determined using oxalate
extract (PSROx). Surface 0–5 cm soils had significantly higher concentrations of labile Pi, intermediately
available Pi, organic P pools, higher equilibrium P concentration (EPC), and PSROx compared to the lower
depths, which indicated saturation of the P sorption sites from repeated PL application. Regression analyses
used to predict P sorption-desorption parameters from soil test variables suggest inclusion of multiple soil var­
iables, which may be time-consuming and not always feasible. The strong relationship between P sorption-
desorption parameters and PSROx indicates that PSROx can be effectively used to predict P sorption-desorption
parameters for the Piedmont soils without performing time-consuming batch isotherm experiments. Further
studies would be needed to validate the relationship for other soil types under different environmental
conditions.

1. Introduction
The problem of soil phosphorus (P) accumulation is more severe in
watersheds where greater amounts of manure (such as poultry litter, PL)
above the agronomic recommended rates are generated and cannot be
transported long distances economically (Spiegal et al., 2020; Bryant
et al., 2021). The P accumulation caused due to an imbalance between
manure applications and P removal by crops is environmentally unsus­
tainable and could lead to water quality issues (Harmel et al., 2009;
Sharpley et al., 1999; Higgins et al., 2021; van Dijk et al., 2016). During
the last two decades, several best management practices aimed at
reducing P losses have been adopted to mitigate freshwater
eutrophication (Jarvie et al., 2013; Sharpley et al., 1994). Still, several
watersheds have reported little or no improvement in P loading even
after extensive implementation of best management practices (Bowes
et al., 2011; Meals et al., 2010). Decreasing P loading from heavily
manured soils to prevent further water quality degradation requires a
better understanding of the P forms and retention-release characteristics
of soil P.
Soil P exists in several organic (Po) and inorganic (Pi) forms which
determine their bioavailability and risk of transfer to surface water and
how the management practices may transform P dynamics in the soil
(Nunes et al., 2020; Weihrauch and Opp, 2018; Zhu et al., 2018).
Sequential fractionation of P using acid or alkaline reagents is the most

* Corresponding author. 202 Funchess Hall, Department of Crop, Soil, and Environmental Sciences, Auburn University, Auburn, AL, 36849, USA.
E-mail address: rzp0050@auburn.edu (R. Prasad).

https://doi.org/10.1016/j.jenvman.2022.115454
Received 14 February 2022; Received in revised form 27 May 2022; Accepted 28 May 2022
Available online 11 June 2022
0301-4797/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
D. Chakraborty et al.
widely used technique to quantify the P forms (Hedley et al., 1982). This
chemical extraction technique is then used to separate P into a) labile Pi
pool: highly reactive loosely bound Pi form; b) intermediately avail­
able Pi pool: Pi associated with iron or aluminum or calcium compound;
c) organic P pool: loosely bound Po or humic-fulvic Po; and d) recal­
citrant P pool: residual or non-bioavailable P form (Fu et al., 2021).
The potential for P transport during runoff events depends exten­
sively on the P retention capacity of the soil, soil P status, and the degree
of soil P saturation (Sharpley, 1995; Wang et al., 2015a). Several studies
have shown that the high P sorption capacity of acidic soils has been
related to the crystalline or amorphous Fe and Al oxide surfaces
(Chakraborty et al., 2012; Villapando and Graetz, 2001). Soil P
sorption-desorption characteristics derived using traditional batch P
isotherm are used to determine the P retention capacity of the soil
(Zhang et al., 2012; Zhou and Li, 2001) and are often used for modeling
P transport from agricultural watersheds (Francesconi et al., 2016;
Groenendijk and Kroes, 1999; McCray et al., 2005; Neitsch et al., 2011;
Schoumans et al., 2013). A knowledge gap still exists about the re­
lationships of soil P sorption-desorption parameters with the Pi and Po
forms and soil test variables (such as clay content, total carbon, pH,
extractable- P, Fe, and Al concentrations) (Dou et al., 2009; Dunne et al.,
2020; Hooda et al., 2000Wang et al., 2015b). This knowledge is criti­
cally lacking for the weathered soils of the southeastern U.S, which is a
major poultry-producing region of the nation (Watts et al., 2019).
Another gap that exists for P loss risk assessment is identifying a
cost-effective tool that can be used to predict the P sorption-desorption
parameters, typically determined using batch isotherm method which is
time-consuming, expensive, and not routinely used.
Soil test indicator such as the P saturation ratio (PSR, molar ratio of P
to Fe and Al) is not often used for assessing a site-specific P loss risk but
has the potential to be introduced as an essential cost-effective tool for P
loss assessment and can be derived using routine soil test procedures
(Chakraborty et al., 2011; Chakraborty and Prasad, 2021; Nair et al.,
2010). The PSR is an estimate of soil P availability, as well as the ca­
pacity of soil to bind additional P (Breeuwsma et al., 1995; Sharpley
et al., 2020). Previous field and laboratory studies have demonstrated
that P concentration in leachate or runoff water increases rapidly once
the PSR is above a “threshold level” for that soil type (Dari et al., 2018;
Maguire and Sims, 2002; Nair et al., 2004).
Although P sorption-desorption parameters and PSR are often used
separately to evaluate the risk of P loss from soils, there is limited in­
formation about the relationship between these parameters. This study
will help improve our understanding of P retention and release from PL
impacted soils using the P sorption-desorption parameters and how
these parameters can be alternatively predicted using cost-effective
methods. Thus, the objectives of this study were to: (1) determine the
relationship between the P forms and P sorption-desorption parameters
of PL impacted soils at different depths from 0 to 45 cm; (2) evaluate the
relationship between P sorption-desorption parameters with soil test
variables (such as clay content, total carbon, pH, extractable- P, Fe,
and Al concentrations) and soil test indicator (such as PSR).
2. Materials and methods
2.1. Soil sampling
Forty soil samples were collected from 10 random locations within
seven pasture fields that have received PL applications annually for the
past 10 years. The sampling locations belonged to the southeastern
Piedmont soil region. The Piedmont soil region typically expands from
East Central Alabama into Maryland and Pennsylvania. Management of
the fields consisted of an annual broadcast application of PL at rates
ranging between 26 and 92 kg P ha− 1. No commercial P fertilizer was
applied to the fields for the past 10 years. Multiple soil cores were
collected at each sampling location using a gas-powered core sampling
kit to form a composite sample corresponding to individual depths: 0–5,
2
Journal of Environmental Management 317 (2022) 115454
5–15, 15–30, and 30–45 cm. The predominant soil series at each sam­
pling location was Madison (Fine, kaolinitic, thermic Typic Kanhaplu­
dults) according to the NRCS Web Soil Survey database (Soil Survey
Staff, 2020).
2.2. Chemical analyses
The samples were air-dried, homogenized, and passed through a 2-
mm sieve prior to analyses. The samples were analyzed for pH (1:1
soil/solution ratio), total carbon (TC), and texture (Provin, 2014; Gee
and Or, 2002). Water extractable P (WEP, 1:10 soil/water ratio) was
determined using a flow injection analyzer (FIAlyzer-1000, FIAlab In­
struments Inc.) (USEPA, 1983; Method 365–1). Mehlich 1 (M1) (soil/­
solution ratio of 1:4) extractable P (M1-P), Fe (M1-Fe), Al (M1-Al), Ca
(M1-Ca), and Mg (M1-Mg) were determined using an inductively
coupled argon plasma spectroscopy (ICAP) (Mehlich, 1953). Oxalate
(Ox) extractable P (Ox-P), Fe (Ox-Fe), and Al (Ox-Al) were also obtained
using a soil/solution ratio of 1:50 (McKeague and Day, 1966).
Citrate-bicarbonate-dithionite (CBD) extractable Fe (CBD-Fe) and Al
(CBD-Al) were extracted using a combined solution of Na2S2O4 + 0.3
mol L− 1 Na3C6H5O7 + 1 mol L− 1 NaHCO3 (Mehra and Jackson, 1960).
The Ox-P, Ox-Fe, Ox-Al, CBD-Fe, and CBD-Al were analyzed using ICAP.
Phosphorus fractionations in soils were determined using a modified
Hedley method (Hedley et al., 1982; Ranatunga et al., 2013). The steps
included sequential soil extraction with deionized water, 0.5 M
NaHCO3, 0.1 M NaOH, and 1 M HCl using a soil/solution ratio of 1:60
followed by filtration with 0.45-μm filter paper. The filtrate obtained
from each extract is hereafter referred to as water-Pi, NaHCO3-Pi,
NaOH-Pi, and HCl-Pi. The filtrate was analyzed for inorganic P (ortho­
phosphate) using the Murphy and Riley (1962) procedure on a flow
injection analyzer. Total P (TP) in NaHCO3 and NaOH extracts were
analyzed using ICAP. The differences between TP and Pi in NaHCO3 and
NaOH extracts accounted for the Po in NaHCO3 (NaHCO3-Po), and NaOH
(NaOH-Po) extracts, respectively (Ajiboye et al., 2004, 2007; Dou et al.,
2000). Residual P was determined following Tiessen and Moir (2008).
The sequentially extracted P fractions were classified into four opera­
tionally defined pools: 1) labile Pi represents loosely bound Pi extracted
by water and 0.5 M NaHCO3; 2) intermediately available Pi represents
Pi extracted by 0.1 M NaOH and 1 M HCl; 3) organic P represents Po
extracted by 0.5 M NaHCO3 and 0.1 M NaOH; and 4) recalcitrant P
represents residual P left after removal of labile Pi, intermediately
available Pi, and organic P forms. The sum of labile Pi, intermediately
available Pi, organic P, and recalcitrant P represented the soil’s TP.
Phosphorus sorption isotherms were determined according to
Chakraborty et al. (2012) using 2 g soil in 20 ml of 0.01 M KCl solutions
containing eight different concentrations of P as KH2PO4: 0, 0.1, 1, 5, 10,
25, 50, and 100 mg P L− 1. Two drops of chloroform were added to each
solution in order to prevent microbial activity during the shaking period.
The equilibration was carried out in a reciprocating shaker for 24 h
followed by centrifugation, filtration using a 0.45-μm membrane filter
paper, and analysis for orthophosphate as described above. A
single-point P sorption isotherm was also determined using 2 g of soil
equilibrated with 20 mL of 0.01 M KCl solution containing two drops of
chloroform to prevent microbial activity at saturated P conditions (75
mg P L− 1 as KH2PO4) for 24 h (Bache and Williams, 1971). The soil
suspension was centrifuged, filtered, and analyzed for orthophosphate.
The amount of P sorbed by the soils (P sorption capacity, PSC) was
calculated from the difference between the P concentration in the
equilibrium solution and the initial P added to the soil.
For desorption experiments, the soils that received 100 mg P L− 1 in
the sorption study served as the starting point after removing the filtrate.
The residual soil was then equilibrated with 20 ml 0.01 M KCl in a
reciprocating shaker for 24 h, followed by centrifugation, filtration
using a 0.45-μm membrane filter, and analysis for orthophosphate. The
amount of P in the equilibrium solution was referred to as the net
amount of P desorbed by the soil. Sequentially desorbed P was
D. Chakraborty et al. Journal of Environmental Management 317 (2022) 115454

Table 1
Chemical properties of the soils with four depths (0–5, 5–15, 15–30, and 30–45 cm) used in this study. Data presented in the form of mean ± standard error (n = 40).
Soil properties Soil depth (cm)

0–5 5–15 15–30 30–45

pH 6.1 ± 0.2a 6.2 ± 0.2a 6.3 ± 0.2a 6.3 ± 0.2a

TC (%) 3.8 ± 0.4a 1.5 ± 0.2 b 0.7 ± 0.1c 0.5 ± 0.0c
Sand (%) 52.1 ± 4.8a 50.2 ± 4.9a 41.2 ± 3.7 ab 33.0 ± 3.1 b
Silt (%) 26.1 ± 3.3a 20.0 ± 2.2a 20 ± 2.3a 20.5 ± 2.8a
Clay (%) 21.8 ± 5.0c 29.8 ± 4.8bc 38.8 ± 4.1 ab 46.5 ± 4.2a
WEP (mg kg¡1) 17.1 ± 7.2a 6.6 ± 1.6 ab 2.7 ± 0.9 b 0.3 ± 0.2 b
M1-P (mg kg¡1) 185.0 ± 40.0a 75.2 ± 21.1 b 15.5 ± 4.5bc 4.5 ± 0.7c
Ox-P (mg kg¡1) 672.6 ± 102.2a 255.4 ± 50.0 b 85.5 ± 21.3c 43.6 ± 6.2c
M1-Ca (mg kg¡1) 1691.7 ± 276.0a 701.4 ± 160.6 b 453.2 ± 74.6 b 410.2 ± 61.8 b
M1-Mg (mg kg¡1) 217.9 ± 37.7a 85.5 ± 13.4 b 63.7 ± 7.4 b 69.5 ± 9.5 b
M1-Fe (mg kg¡1) 33.3 ± 6.1a 24.3 ± 4.2a 9.4 ± 0.7 b 9.5 ± 0.9 b
Ox-Fe (mg kg¡1) 1993.6 ± 201.0a 1228.9 ± 163.0 b 790.3 ± 103.2c 859.6 ± 70.9bc
CBD-Fe (mg kg¡1) 24164.4 ± 5701.3 b 27761.9 ± 5922.0 b 37358.1 ± 6042.6 ab 47250.0 ± 5030.9a
M1-Al (mg kg¡1) 223.8 ± 26.3a 211.5 ± 26.8a 147.0 ± 16.2 b 120.4 ± 14.2 b
Ox-Al (mg kg¡1) 779.5 ± 106.8a 682.0 ± 76.1a 628.7 ± 52.2a 684.4 ± 45.8a
CBD-Al (mg kg¡1) 3624.8 ± 636.3 b 4105.7 ± 617.7 b 5218.6 ± 558.1 ab 6058.8 ± 458.6a
PSROx 0.34 ± 0.05a 0.19 ± 0.04 b 0.08 ± 0.02c 0.04 ± 0.01c
#
TC, total carbon; WEP, water extractable P, M1-P, Ca, Mg, Fe, and Al, Mehlich-1 extractable P, Ca, Mg, Fe, and Al; Ox-P, Fe, and Al, oxalate extractable P, Fe, and Al;
CBD-Fe and Al, citrate bicarbonate dithionite extractable Fe and Al; PSROx, P saturation ratio determined using oxalate extract.

determined at each step for seven successive extractions. and proportionately represents the P retention capacity of the soil
(Kleinman et al., 2003).
2.3. Calculations
PSROx = Oxalate-P/[(Oxalate-Fe) + (Oxalate-Al)] (2)

Sorption parameters were calculated using the linear form of the The P, Fe, and Al in Eq. (2) are expressed in moles.
Langmuir adsorption equation following Nair et al. (1998).
2.4. Statistical analyses
C/S = 1/kSmax + C/Smax (1)

Where C is the concentration of P after 24 h equilibration, mg L− 1; S is
the total amount of P sorbed (mg kg− 1) and is the sum of S’ (P sorbed by
the solid phase, mg kg− 1) and So (originally sorbed P on the solid phase,
mg kg− 1); Smax is P sorption maximum, mg kg− 1; and k is a constant
related to the bonding energy, L mg− 1 P. Equilibrium P concentration
(EPC) represents the concentration of P in solution where adsorption
equals desorption and was the value of C when S’ = 0 (Nair and Reddy,
2013; Nair et al., 1998). The maximum buffering capacity (MBC) of soils
is the index of soil resistance to the change of P concentration in soil
solution with the addition or removal of P (Sui and Thompson, 2000).
The MBC of the soil was determined as the product of Smax and k
(Holford, 1979). The P saturation (Psat) of the soils was calculated as the
percentage of Smax that was originally sorbed (So) on the solid phase of
the soil.
The PSR was calculated from the acid ammonium oxalate (PSROx)
because oxalate extracts most of the reactive Al and Fe present in the soil
Generalized linear mixed model procedure (PROC GLIMMIX) in SAS
9.4 (SAS Institute Inc., Cary, NC) was used to determine the fixed effects
of soil depth while treating sampling location as a random effect. Dif­
ferences between means were compared using least square means and
were considered significant at the 0.05 probability level. Empirical re­
lationships between soil parameters were established using simple linear
regression. Principal component analysis (PCA) was used to study the
relationship between the soil P forms, sorption-desorption parameters,
and soil test variables. The PCA was performed using XLSTAT (Addin­
soft, New York, USA). The number of significant components in PCA
analysis was selected based on three criteria a) eigenvalues>1, b) each
component should explain at least 5% of the variance, and c) the cu­
mulative variance is at least 70% (Jolliffe, 2002).
PROC REG in SAS 9.4 was used to predict P sorption-desorption
parameters from commonly used soil test variables. Forward regression
was used with variables considered only for model entry at P = 0.50. The

Table 2
Distribution of phosphorus (P) fractions within the four soil depths (0–5, 5–15, 15–30, and 30–45 cm) for soils used in this study. Data presented in the form of mean ±
standard error (n = 40).
P Fractions (mg kg− 1) Soil depth (cm)

0–5 5–15 15–30 30–45

1. Labile Pi
Water-Pi 40.3 ± 11.9a 13.5 ± 1.9 b 8.1 ± 0.5 b 8.2 ± 0.5 b
NaHCO3-Pi 156.3 ± 25.6a 83.7 ± 22.0 b 28.7 ± 7.9c 12.1 ± 1.1c
2. Intermediately available Pi
HCl-Pi 166.6 ± 40.5a 33.4 ± 9.8 b 11.5 ± 1.0 b 9.0 ± 1.1 b
NaOH-Pi 243.1 ± 35.7a 122.2 ± 19.9 b 61.2 ± 10.6c 41.4 ± 4.4c
3. Organic P
NaHCO3-Po 38.4 ± 6.5a 18.3 ± 2.9 b 12.7 ± 2.0bc 7.4 ± 1.5c
NaOH-Po 150.4 ± 17.4a 85.3 ± 13.1 b 38.1 ± 4.6c 28.2 ± 4.1c
4. Recalcitrant P
Residual-P 339.7 ± 35.0a 261.6 ± 31.7a 251.5 ± 35.7a 277.7 ± 37.3a
#
Water-Pi: water extractable inorganic phosphorus fraction; NaHCO3-Pi and Po, inorganic and organic P extracted using 0.5 M NaHCO3; NaOH-Pi and Po, inorganic and
organic P extracted using 0.1 M NaOH; HCl-Pi, inorganic P extracted using 1 M HCl. Means with same letter between each depth are not significantly different at P <
0.05.

3
D. Chakraborty et al.
Table 3
Phosphorus (P) sorption and desorption parameters for the soils used in this study with four depths (0–5,5-15,15–30, and 30–45 cm). Data presented in the form of
mean ± standard error (n = 40).
P sorption-desorption parameters Soil depth (cm)
0–5
So (mg kg− 1) 30.1 ± 9.8a
EPC (mg L− 1) 4.4 ± 2.4a
S (mg kg− 1) 316.6 ± 78.3 b
Smax (mg kg− 1) 376.6 ± 76.9 b
PSC (mg kg− 1) 238.0 ± 65.3c
Psat (%) 11.7 ± 4.7 a
k (L mg− 1) 0.1 ± 0.1 b
MBC (L kg− 1) 74.0 ± 61.2 b
PDES (%) 62.8 ± 7.3a
#
EPC, equilibrium P concentration; So, originally sorbed P on the solid phase; Psat, P saturation; Smax, P sorption maximum; k, constant related to the bonding energy;
MBC, maximum buffering capacity; S, total amount of P sorbed from Langmuir isotherm; PSC, P sorption capacity using single-point isotherm; PDES, P desorbed
expressed as a percentage of newly sorbed P after the first successive 24-h extraction. Means with same letter between each depth are not significantly different at P <
0.05.
final regression model was selected in which all variables were signifi­
cant at P = 0.05 in the last step (McCray et al., 2012).
3. Results and discussion
3.1. Soil characterization
Soil pH ranged between 5.9 and 6.5, with the texture changing from
sandy clay loam at the surface to clay at a depth of 30–45 cm (Table 1).
Mean M1-P was 185 mg kg− 1 for the surface soils and dropped to 4.4 mg
kg− 1 for 30–45 cm depth. Extractable P (WEP, M1-P, Ox-P) concentra­
tions were significantly higher for the surface 0–5 cm soils than for lower
depths (Table 1). A high concentration of CBD-Fe and Al in the soils
indicates a predominance of crystalline iron oxides (Agbenin, 2003;
Wisawapipat et al., 2009). Mean PSROx for the 0–5 and 5–15 cm depths
were 0.34 and 0.19 and was higher than the threshold PSR of 0.11 as
proposed by Chakraborty et al. (2021) for Alabama Piedmont soils.
Higher PSROx (> threshold PSR of 0.11) for 0–5 and 5–15 cm soils
indicate a higher P loss risk from the soils, and further P loading can be
of environmental concern (Nair, 2014).
3.2. Soil phosphorus fractions and sorption-desorption parameters
Soil P fractions for the four soil depths are presented in Table 2. Both
labile Pi and organic P pool individually constituted 17% of TP, whereas
intermediately available Pi pool constituted the highest proportion
(36%) of TP for the 0–5 cm depth. The proportion of intermediately
available Pi pool decreased to 25%, 18%, and 13% for 5–15, 15–30, and
30–45 cm, respectively. A high concentration of labile Pi on the surface
0–5 cm reflects the impact of manure application history (Cherubin
et al., 2016; Dormaar and Chang, 1995; Neufeldt and Ayarza, 2000).
However, higher concentrations of labile Pi (water-Pi and NaHCO3-Pi)
on the soil surface also raise environmental concerns since P from these
forms can easily release inorganic P into the runoff water during storm
events (Waldrip et al., 2015). Phosphorus is lost from agricultural fields
through three mechanisms: a) attached to the sediment that erodes from
the field; b) dissolved in the surface water during runoff; or c) dissolved
in leachate and carried through the soil profile (Reid et al., 2018).
Phosphorus in the soil is mostly associated in the particulate form with
iron, aluminum, or calcium phosphates or bound to minerals such as
clay or aluminum hydroxides. However, the particulate fraction does
not immediately release chemically bioavailable P forms as it is subject
to deposition to bottom sediments (Baker et al., 2014, 2017). Hence,
reducing P export from agricultural fields associated with dissolved
reactive P forms can help to minimize P loss during runoff events.
The NaOH-Po is often associated with the humic-fulvic fraction and is
Journal of Environmental Management 317 (2022) 115454
5–15 15–30 30–45
6.8 ± 2.4 b 1.6 ± 0.9 b 1.3 ± 0.7 b
1.1 ± 0.3 ab 0.2 ± 0.1 b 0.1 ± 0.0 b
364.9 ± 100.8 b 564.4 ± 95.9 b 872.1 ± 80.0a
413.7 ± 100.1 b 599.1 ± 102.4 b 888.6 ± 78.6a
291.3 ± 81.3bc 462.4 ± 77.8 b 737.2 ± 62.5a
2.9 ± 1.3 b 0.5 ± 0.3 b 0.2 ± 0.1 b
0.2 ± 0.1 b 0.4 ± 0.1a 0.6 ± 0.1a
127.2 ± 97.3 b 312.9 ± 130.6 ab 568.2 ± 127.1a
45.0 ± 5.7 b 27.1 ± 3.1c 17.1 ± 2.0c
generally a more stable form of organic P in soil except under certain
environmental conditions (microbial degradation of the P forms) that
can potentially release inorganic P into the environment (Richardson
and Simpson, 2011). On the other hand, NaHCO3-Po can easily miner­
alize organic P forms in soil (Bowman and Cole, 1978). Hence the high
concentration of NaHCO3-Po in the surface soils can also be of envi­
ronmental concern. The proportion of recalcitrant P fraction increased
from 30% in the 0–5 cm depth to 72% in the 30–45 cm depth (Table 2).
Among all the P fractions, the residual-P or recalcitrant fraction
constituted the highest proportion of TP, followed by the NaOH-Pi
fraction for all the soil depths. The NaOH-Pi fraction represents P forms
attached to Fe and Al hydrous oxides and insoluble Al/Fe phosphate (He
et al., 2006a; Tiessen and Moir, 2008). Phosphorus associated with the
NaOH-Pi fraction can be considered stable P and may not be of envi­
ronmental concern other than when soil pH conditions change (Wright,
2009). The HCl-Pi or Ca bound P fraction concentration was high in 0–5
cm depth and dropped significantly in the subsurface soils (Table 2).
High HCl-Pi fraction in surface soils can be related to surface applica­
tions of PL, considering the large HCl-Pi fraction of PL (He et al., 2006b;
Waldrip-Dail et al., 2009). This further indicates low background levels
of calcium compounds in the soil. Most of the phosphate in the soil is
mostly bound to iron and aluminum oxides, since HCl hydrolyses the
calcium phosphate (Lilienfein et al., 2000).
In a previous study, Richards et al. (1995) observed a significant
increase in water-Pi, NaHCO3-Pi, and NaOH-Pi forms after ten years of P
fertilizer applications in continuous corn production in Canada. How­
ever, Motavalli and Miles (2002) did not find any significant increase in
NaHCO3-Pi form with long-term fertilizer application. In contrast,
manure applications in the continuous corn and wheat cropping systems
resulted in a significant increase in NaHCO3-Pi form. Another long-term
62 years study in a Humic Cambisol showed that the addition of PL
increased Pi contents of all the fractions except the recalcitrant P fraction
(Schmitt et al., 2019). Various studies have shown that the application of
manure or P fertilizer, especially at higher rates, alters the amount and
distribution of P in the various pools of soil P (Qian et al., 2004; Zheng
et al., 2001). Sen Tran et al. (1996) showed that long-term manure
application increased labile P fraction in surface and subsurface soils.
This study showed significantly higher Pi and Po forms in all the fractions
for the surface 0–5 cm soils compared to the lower depths. A higher
concentration of intermediately available Pi in the 0–5 cm depth may be
of environmental concern since P in this form is not readily available for
the plant. However, it could be transferred to the surface or groundwater
(Waldrip et al., 2015).
The P sorption of the PL impacted soils can be best described using
the linearized Langmuir equation (Eq. (1)) as revealed by the linear
fitting of the sorption data (data not shown). The So and EPC were higher
4
D. Chakraborty et al.
Fig. 1. Percentage of sorbed phosphorus (P) remaining after seven successive
sequential desorption in soils with 0–5, 5–15, 15–30, and 30–45 cm depths and
with long-term history of poultry litter applications. Data presented in the form
of mean ± standard error (n = 40).
Fig. 2. Multivariate analysis of phosphorus (P) sorption-desorption parameters
and P fractions. EPC, equilibrium P concentration; So, originally sorbed P on the
solid phase; Psat, P saturation; Smax, P sorption maximum; k, constant related to
the bonding energy; MBC, maximum buffering capacity; S, total amount of P
sorbed from Langmuir isotherm; PSC, P sorption capacity using single-point
isotherm; PDES, P desorbed expressed as a percentage of newly sorbed P
after the first successive 24-h extraction, Water-Pi, inorganic P extracted using
deionized water; NaHCO3-Pi and Po, inorganic and organic P extracted using
0.5 M NaHCO3; NaOH-Pi and Po, inorganic and organic P extracted using 0.1 M
NaOH; HCl-Pi, inorganic P extracted using 1 M HCl.
for the surface 0–5 cm than the lower soil depths (Table 3) and ranged
from 113.2 to 0 mg kg− 1 and 26 to 0.01 mg L− 1, respectively. Signifi­
cantly low EPC for the lower depths indicates that these soils were un­
affected or minimally impacted from manure or fertilizer applications.
Low EPC and So values for the lower depths (15–30 and 30–45 cm) also
indicates that these soils are acting as a P sink. Phosphorus sorption
capacity obtained using a single-point isotherm was comparable to Smax
(Smax = 1.2 PSC + 22.5, R2 = 0.95) and S (S = 1.1 PSC + 79.6, R2 =
0.98). The relationship indicates that the single point isotherm yields
5
Journal of Environmental Management 317 (2022) 115454
similar result and is more convenient than the laborious batch isotherm
method for characterizing the P sorption capacity of Piedmont soils. This
finding aligns with the previous studies on manure impacted Spodosols
(Nair et al., 1998) and Inceptisols (Brock et al., 2007). The P saturation,
Psat was significantly higher for surface 0–5 cm soils than the lower
depths (Table 3). The sorption parameters S, Smax, and PSC were
significantly higher for 30–45 cm compared to 0–5, 5–15, and 15–30 cm
soil depths (Table 3). The maximum buffering capacity, MBC, was
higher for 30–45 cm depth than for 0–5 and 5–15 cm depths, which had
lower MBC values and, hence, were more susceptible to P mobility
(Villapando and Graetz, 2001). The bonding constant k was significantly
less for 0–5 and 5–15 cm depths and increased for the lower depths
(15–30 and 30–45 cm). High S, Smax, PSC, K, MBC, and low So, EPC for
lower soil depths indicates the capacity of the subsoil to retain P. On the
contrary, the surface 0–5 cm soils with high Psat, So, EPC, and lower P
sorption capacity can serve as potential P source during runoff events.
Phosphorus desorbed expressed as a percentage of newly sorbed P after
the first successive 24-h extraction, also referred to as PDES, was
significantly higher for the surface 0–5 cm and decreased down the soil
profile (Table 3). An average 63% of the newly sorbed P was desorbed
from 0–5 cm soils after the first 24-h extraction which decreased to 45%,
27%, and 17% for 5–15, 15–30, and 30–45 cm depths, respectively
(Table 3). High PDES associated with 0–5 cm soils indicate higher
desorption potential of the soils as also indicated by the
above-mentioned sorption parameter (So, EPC, Psat, and MBC).
Percent sorbed P retained by the soil for the seven desorption cycles
for 0–5, 5–15, 15–30, and 30–45 cm depths are shown in Fig. 1.
Sequential desorption on 0–5 cm soils indicated that most of the newly
added P was desorbed after five successive 24-h extractions (Fig. 1).
Whereas, for 5–15, 15–30, and 30–45 cm depths, an average of 22%,
53%, and 68% of the newly sorbed P remained after seven successive 24-
h extractions (Fig. 1). Irreversibility of sorption-desorption processes as
observed for lower depth soils can be related to the shift in P from
Fig. 3. Multivariate analysis of phosphorus (P) sorption-desorption parameters
and commonly used soil test variables. EPC, equilibrium P concentration; So,
originally sorbed P on the solid phase; Psat, P saturation; Smax, P sorption
maximum; k, constant related to the bonding energy; MBC, maximum buffering
capacity; S, total amount of P sorbed from Langmuir isotherm; PSC, P sorption
capacity using single-point isotherm; PDES, P desorbed expressed as a per­
centage of newly sorbed P after the first successive 24-h extraction, WEP, water
extractable P; M1-P, Ca, and Mg, Mehlich-1 extractable P, Ca, and Mg; Ox-P, Fe,
and Al, oxalate extractable P, Fe and Al; CBD-Fe and Al, citrate bicarbonate
dithionite extractable Fe and Al; TC, total carbon.
D. Chakraborty et al. Journal of Environmental Management 317 (2022) 115454

loosely to tightly bound, precipitation of discrete phosphate minerals, a 3.3. Relationship among phosphorus sorption-desorption parameters and
shift from monodendate to bidentate forms of sorbed P, or diffusive easy to measure soil test variables
penetration of surface-sorbed P into the soil components (Munns and
Fox, 1976; Ryden et al., 1977; Riemsdijk et al., 1984; Villapando and The PCA results between sorption-desorption parameters and soil
Graetz, 2001). test variables showed that the first two axes (PC1 and PC2) explained
Principle component analysis between sorption-desorption parame­ 77% of the total variance (Fig. 3). The extractable P (WEP, M1-P, Ox-P)
ters and P fractions showed that the first two axes (PC1 and PC2) was strongly correlated with So, EPC, and PDES and negatively related to
explained 81% of the total variance (Fig. 2). The EPC was strongly Smax, S, PSC, k, and MBC. Similarly, M1-Ca and M1-Mg showed the same
positively correlated with So, Psat, and negatively with Smax. The water- trend with sorption-desorption parameters. The positive correlation of
Pi was strongly correlated with EPC (r = 0.963). Similarly, So and Psat PDES with M1-Ca and So suggests a higher desorption potential of newly
were strongly related to water-Pi along with NaHCO3-Pi. There was a sorbed P in the presence of Ca in the soil. Oxalate extractable Fe and Al
negative relationship between So and k, which is consistent with the used as an index of non-crystalline Fe and Al oxides (Wisawapipat et al.,
previous theory that more saturated the sorption complex is, lower is the 2009) were not related to Smax, S, PSC, or MBC (Fig. 3). There was no
energy of subsequent sorption (Villapando and Graetz, 2001). The Smax relation of Ox-Al with any of the sorption-desorption parameters.
often used as an environmental indicator for P loss (Fu et al., 2021) was However, Ox-Fe was positively related with So and PDES, and negatively
negatively related to the labile Pi, intermediately available Pi, organic P, with k. In contrast, CBD-Fe, CBD-Al, and percent clay content were
PDES, and was positively related with k and MBC (Fig. 2). The PDES was strongly and positively correlated with PSC, Smax, S, k, MBC, and
positively related with So, NaHCO3-Pi, NaOH-Po, and was negatively negatively with PDES. Acid ammonium oxalate extracts both
related with k, MBC, PSC, and Smax. The relationship between the non-crystalline inorganic and organically complexed Fe and Al whereas
sorption-desorption parameters and the P fractions helps us better un­ CBD primarily extracts free Fe oxides, that is, crystalline, poorly crys­
derstand the Pi and Po forms that are related to P retention and release in talline, and non-crystalline Fe oxides (Jackson et al., 1986; McKeague
the soil. and Day, 1966). Previous studies have reported that crystalline Fe and Al
hydrous oxides sorb 10 to 100 times less P than their amorphous com­
ponents (Ryden and Pratt, 1980). Findings of this study indicate the
dominance of crystalline Fe oxides in P retention in Ultisols as observed
Table 4 by previous studies in U.S. Savanna Alfisols (Agbenin, 2003), Danish
Stepwise regression results for the relationships between phosphorus (P) Spodosols (Borggaard, 1986), and in Alfisols of southern Spain (Peña
sorption-desorption parameters and commonly used soil test variables. Only the and Torrent, 1984). Additionally, a negative relation of PDES with
significant variable (P < 0.05) are presented. CBD-Fe and CBD-Al indicates lower P desorption for soils high in crys­
Dependent Independent Steps R2 P>F Parameter talline Fe oxides.
variable variable Estimates We performed a step-wise regression analysis to predict sorption-
PSC CBD-Fe 1 0.84 <0.0001 0.01 desorption parameters using soil test variables (Table 4). The amount
TC 2 0.88 0.002 − 33.93 of P sorbed as indicated by PSC, Smax, and S was best predicted using
Intercept 57.05 CBD-Fe, which explained >80% of the variability (Table 4). The bonding
S CBD-Fe 1 0.87 0.01
constant k was also best predicted by CBD-Fe and TC, explaining 62% of
<0.0001
clay 2 0.89 0.0134 5.56
Intercept − 94.61 the variability in k. The step-wise regression further confirmed that CBD-
EPC M1-Mg 1 0.48 <0.0001 0.07 Fe was the single most important soil variable that plays a dominant role
TC 2 0.60 0.015 − 2.85 in P retention of Piedmont soils. However, for EPC, So, and Psat, M1-Mg
M1-Fe 3 0.70 0.0015 0.09 accounted for most of the variability (Table 4). Multiple regression
Silt 4 0.76 0.068 0.16
pH 5 0.78 0.0448 − 1.73
analysis indicated PDES is associated with M1-Ca, which accounted for
Ox-Al 6 0.83 0.0075 0.00 64% of the total 81% variance.
Intercept 7.66 Since traditional batch isotherms are laborious, time-consuming, and
So M1-Mg 1 0.66 <0.0001 0.23 are not routinely used in commercial soil testing laboratories, soil-
Ox-Fe 2 0.72 0.0108 − 0.01
specific regression models can be developed to predict the sorption-
Intercept − 2.86
Smax CBD-Fe 1 0.85 <0.0001 0.01 desorption parameters. However, multiple soil test variables are
Clay 2 0.87 0.04 4.85 needed to predict the isotherm parameters. Some variables, such as CBD-
Intercept − 30.33 Fe or Ox-Fe are not routinely determined in soil testing laboratories.
PDES M1-Ca 1 0.64 <0.0001 0.02 Hence, it will be beneficial to explore the relationship of sorption-
CBD-Al 2 0.81 − 0.01
desorption parameters with PSR that can be efficiently and cost-
<0.0001
Intercept 50.17
MBC CBD-Fe 1 0.70 <0.0001 0.02 effectively determined from routine soil test and used to predict the
Intercept − 291.14 isotherm parameters.
K CBD-Fe 1 0.54 <0.0001 0.00
TC 2 0.62 0.0073 − 0.07
3.4. Relationship between phosphorus sorption-desorption parameters and
Intercept 0.11
Psat M1-Mg 1 0.54 <0.0001 0.10 phosphorus saturation ratio
Ox-Fe 2 0.63 0.0048 − 0.01
Intercept − 0.26 Soil PSR and Smax can be used for environmental P loss risk assess­
#
EPC, equilibrium P concentration; So, originally sorbed P on the solid phase; ment since soils ability to release sorbed P to the environment is
Psat, P saturation; Smax, P sorption maximum; k, constant related to the bonding dependent on these parameters (Chakraborty et al., 2011; Nair, 2014).
energy; MBC, maximum buffering capacity; S, total amount of P sorbed from For example, soils with high Smax and low PSR may not be of environ­
Langmuir isotherm; PSC, P sorption capacity using single-point isotherm; PDES, mental concern than soils with low Smax and high PSR (greater than the
P desorbed expressed as a percentage of newly sorbed P after the first successive threshold value). The relationship between sorption-desorption param­
24-h extraction; CBD-Fe, citrate bicarbonate dithionite extractable Fe; TC, total eters (EPC, So, S, Smax, PSC, Psat, k, MBC, and PDES) with PSROx is shown
carbon; M1-Ca, Mg, and Fe, Mehlich-1 extractable calcium, magnesium, and in Fig. 4. Fig. 4a and b show that when PSROx is below the threshold
iron; Ox-Fe and Al, oxalate extractable iron and aluminum.
value of 0.11 (based on the findings of Chakraborty et al., 2021 for
Piedmont soils), EPC and So are minimal. However, when the PSROx is
above the threshold value, EPC and So increase linearly with an increase

6
D. Chakraborty et al.
Fig. 4. Relationship between P saturation ratio determined using oxalate extract (PSROx) with a) EPC, equilibrium P concentration; b) So, originally sorbed P on the
solid phase; c) Psat, P saturation; d) PSC, P sorption capacity using single-point isotherm; e) S, total amount of P sorbed from Langmuir isotherm; f) Smax, P sorption
maximum; g) k is a constant related to the bonding energy; h) MBC, maximum buffering capacity; and i) PDES, P desorbed expressed as a percentage of newly sorbed
P after the first successive 24-h extraction.
in PSROx. A similar relation is also observed between Psat and PSROx
(Fig. 4c). The PSROx for most soils in 0–5 and 5–15 cm depths were
greater than the threshold limit resulting in higher EPC, So, and Psat
(Fig. 4a, b and 4c). High PSROx in the surface soils is due to saturation of
the sorption sites and low P retention capacity resulting from the excess
application of P through PL and potentially increasing the risk of P loss
to the surface and subsurface water bodies (Sharpley et al., 1993;
Beauchemin et al., 1996; Dou et al., 2009). The relationship of PSROx
with PSC, S, Smax, k, and MBC (Fig. 4d, 4e, 4f, 4g, and 4h) followed a
logarithmic trend. The soils at the lower depths (30–45 cm) have low
PSROx, higher P retention capacity, higher bonding constant, and greater
buffering capacity. The trend is reversed for the surface 0–5 cm soils
with the least retention capacity, lower bonding constant, and lowest
buffering capacity. The relationship with the desorption parameter
PDES and PSROx (Fig. 4i) shows that the surface soils have higher
desorption potential than lower depths. The difference in desorption
patterns for the different soil depths may be due to the high retention
capacity of the subsurface soils along with the irreversibility of
sorption-desorption reactions (Villapando and Graetz, 2001; Zhou et al.,
1997).
Based on the relationship between PSROx and sorption-desorption
parameters for a soil type under site-specific conditions, Smax or other
isotherm parameters that are often used for P loss assessment can be
estimated cost-effectively and much faster. This information can also be
incorporated into process-based models such as Agricultural Policy
Extender model (APEX) or Soil and Water Assessment Tool (SWAT) for
environmental P loss assessment. However, further studies with
different soil types and from other geographic regions would be needed
to verify the relationship between PSR and the sorption-desorption
parameters.
7
Journal of Environmental Management 317 (2022) 115454
4. Conclusions
The findings of this study helped to understand the distribution of Pi
and Po forms within the soil profile of PL impacted soils and their P
retention and release characteristics. Higher concentrations of labile Pi,
intermediately available Pi, and organic P forms were predominant in
surface soils and decreased down the depth. Phosphorus sorption
described by the Langmuir equation identified So and EPC were higher
for the surface 0–5 cm compared to the lower depths, whereas S, Smax,
PSC, K, and MBC were significantly higher for the subsurface 30–45 cm
compared to the other depths. Stepwise regression between P sorption-
desorption parameters and soil test variables identified CBD-Fe as an
important parameter for P retention. However, predicting other
isotherm parameters such as PDES, EPC, So based on the outcome of the
regression model will need multiple soil test variables which may not be
always feasible.
Soil PSR used for environmental P loss risk assessment indicated that
when PSROx was above the threshold value, EPC and So increased line­
arly with an increase in PSROx. High PSROx in the surface soil indicated
saturation of the sorption sites (high Psat) and low P retention capacity
(PSC, S, and Smax) resulting from repeated application of PL. The soils at
the lower depths (30–45 cm) had low PSROx and significantly higher P
retention capacity (PSC, S, and Smax), higher k, and MBC. Higher pro­
portion of labile Pi, intermediately available Pi, and organic P forms in
the surface soils along with significantly higher EPC, PDES, and PSROx
compared to the subsurface soils indicates environmental P loss risk
during runoff events and further soil P addition can exacerbate P loss
risk. The relationship between PSROx and sorption-desorption parame­
ters can help in better assessment of P loss risk from PL impacted soils.
Additionally, soil test indicators such as PSROx can be used as a practical
D. Chakraborty et al.
means to predict the P sorption-desorption parameters without per­
forming time-consuming and laborious batch isotherm experiments.
Further studies would be warranted to validate the relationship between
PSR and P sorption-desorption parameters for a wide range of soil types
in different parts of the world.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
We thank the farmers of Alabama and extension agents who sup­
ported us in collecting soil samples. We also thank Anjan Bhatta for soil
core collection, Vasyl Fesun for helping with soil processing, and
Gagandeep Kaur for helping with batch isotherm experiments. This
project was financially supported by the Agricultural Research Service,
U.S. Department of Agriculture, under Agreement No. 58-6010-9-011,
the Hatch Program of the National Institute of Food and Agriculture, and
the Alabama Agricultural Experiment Station.
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Empirical modeling of moisture sorption characteristics and mechanical

and barrier properties of cassava flour film and their relation to
plasticizingeantiplasticizing effects
Panuwat Suppakul a, b, *, Buppa Chalernsook a, Bhatama Ratisuthawat a, Sakpipat Prapasitthi a,
Natsaran Munchukangwan a
a
Department of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
b
Center of Advanced Studies for Agriculture and Food (CASAF), Kasetsart University, Bangkok 10900, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Moisture sorption kinetics of cassava flour film was investigated at various relative humidities (RH). Their
Received 1 March 2012 curves were fitted to a Peleg model. This film was also studied for its sorption isotherm. Empirical models
Received in revised form were tested to fit the experimental data. Moisture sorption was more rapid in the initial stages; less
5 May 2012
moisture was adsorbed as adsorption time increased. GAB and Lewicki models were found to be
Accepted 20 May 2012
better-fitted models. Effects of sorbitol concentration and RH on mechanical properties of films were
examined. The tensile strength (TS) of films vastly decreased with increasing sorbitol concentration. The
Keywords:
percentage of elongation increased significantly with increasing sorbitol content; however, it declined
Biopolymer
Edible film
when sorbitol content was above 40 g/100 g. Modulus of elasticity (E) of films displayed a similar trend to
Cassava flour TS. The E of this film was inversely linear to RH, whereas its elongation was exponentially proportional to
Moisture sorption RH. Effects of sorbitol concentration on barrier properties of films were also examined. The water vapor
Mechanical property transmission rate of film was gradually reduced with increasing sorbitol content; however, it dropped
Barrier property dramatically with sorbitol content above 40 g/100 g. The oxygen transmission rate of film significantly
decreased with rising sorbitol content. This is helpful in understanding the performance of edible films
under varying RH conditions and plasticizer contents.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Over the last four decades, extensive research has been under-
taken on the use of edible films and coatings. They have been used as
moisture, gas or solute barriers in food packaging to prolong shelf-
life and improve overall food quality. Potential sources and applica-
tions of edible films and coatings have been reviewed by Debeaufort,
Quezada-Gallo, and Voilley (1998) and Tharanathan (2003). Owing
to the hydrophilic properties of starch and flour, they provide
a minimal barrier to moisture (Kester & Fennema, 1986). Neverthe-
less, these films possess good barrier properties to oxygen, carbon
dioxide and lipids, and protect against lipid oxidation (Banker, 1966).
Cassava (Manihot esculenta Crantz) or tapioca is one of the most
economically important crops in Thailand, Brazil and other tropical
countries. It is an abundant and cheap agricultural source of starch
and flour production. In Thailand, cassava flour consists mostly of
starch (94e97.5 g/100 g dry basis), with a small amount of protein
* Corresponding author. Tel.: þ66 2 562 5058; fax: þ66 2 562 5046.
E-mail address: fagipas@ku.ac.th (P. Suppakul).
(0.3 g/100 g); moisture content typically ranges 13e14 g/100 g. An
amylose content of approximately 17 g/100 g is responsible for the
strong film-formation characteristics of cassava flour film
(Bangyekan, Aht-Ong, & Srikulkit, 2006).
Sorption characteristics of cassava flour film are crucial for the
design, modeling and optimization of its drying, storage and
transport. Sorption isotherms provide information on the
moisture-binding capacity of products at a determined relative
humidity, and are a useful means of analyzing the moisture plas-
ticizing effect and the effect on mechanical properties (Al-
Muhtaseb, McMinn, & Magee, 2002; Bell & Labuza, 2000). Chirife
& Iglesias (1978) reviewed 23 isotherm models and their use for
fitting sorption isotherms of foods and food products.
The phenomena of plasticization and antiplasticization have
begun to receive increasing attention in the last decade from food
scientists and technologists (Chang, Karim, & Seow, 2006). Low-
molecular-mass compounds or diluents, acting as external plasti-
cizers, are an integral part of polymeric systems in which they serve
to enhance the flexibility and workability of the otherwise rigid
neat polymers. Nevertheless, they may serve as mechanical

0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2012.05.013
 P. Suppakul et al. / LWT - Food Science and Technology 50 (2013) 290e297
antiplasticizers when present at low concentrations, leading
instead to the polymer-diluent blends becoming stiffer than the
neat polymer (Sears & Darby, 1982).
Plasticizers are normally added to a polymeric matrix in order to
overcome the film brittleness (Lieberman & Gilbert, 1973). The
properties of cassava films have been extensively studied (Alves,
Mali, Beléia, & Grossmann, 2007; Chang et al., 2006; Famá,
Goyanes, & Gerschenson, 2007; Mali, Sakanaka, Yamashita, &
Grossmann, 2005; Müller, Yamashita, & Laurindo, 2008; Paes,
Yakimets, & Mitchell, 2008; Parra, Tadini, Ponce, & Lugão, 2004;
Souza et al., 2012; The, Debeaufort, Voilley, & Luu, 2009).
However, there is still a lack of comprehensive analysis of sorption
kinetics and information on the plasticizingeantiplasticizing
effects on the properties of cassava films. As exposed to the envi-
ronmental atmosphere, the moisture content of the film will not
equilibrate, resulting in a ceaseless alteration of tensile properties
of the films via moisture sorption (Cho & Rhee, 2002). In addition,
plasticizer concentrations significantly affect both water trans-
mission and oxygen transmission rates of edible films. Conse-
quently, the changes in mechanical and barrier properties of a film
are affected by the rate of moisture adsorption of films and the
concentration of plasticizers, respectively.
The objective of this study is to determine the moisture sorption
characteristics of cassava flour film and the plasticizingeantiplasticizing
effects on mechanical and barrier properties of cassava flour films.
2. Materials and methods
2.1. Materials
Cassava flour was purchased from E.T.C. International Trading
Co., Ltd. (Bangkok, Thailand). Glycerol, sorbitol and polyethylene
glycol with Mw ¼ 400 (PEG-400) were used as plasticizers, and
were obtained from Sigma Chemical Co. (St. Louis, MO). Lithium
chloride (LiCl), magnesium chloride (MgCl2), magnesium nitrate
(Mg(NO3)2), potassium iodide (KI), sodium chloride (NaCl), potas-
sium chloride (KCl) and potassium nitrate (KNO3) were also
purchased from Sigma Chemical Co.
2.2. Preparation of cassava flour film
Cassava flour film solution was prepared by dissolving 5.0 g of
cassava flour in 100 g of aqueous solution, and then heating at its
gelatinization temperature of 70  C while stirring until a viscous
and transparent solution was observed. Weights equivalent to 20,
30, 40 and 50 g/100 g glycerol, sorbitol or PEG-400 were then
added as film plasticizers, to prevent brittleness of the film. The
resulting solutions were degassed in an ultrasonic water bath
(Model 275D; Crest Ultrasonics Corp., Trenton, NJ, USA) for 10 min.
Cassava flour film was cast by pouring film-forming solution
(50 mL) onto a flat 21  29 cm glass plate, which previously
wrapped with a linear low-density polyethylene film. The plates
were dried at 60  C in a ventilated oven for 3 h.
2.3. Moisture sorption kinetics and isotherm
A standard gravimetric methodology (weighing samples equil-
ibrated in thermally stabilized desiccators) was used for determi-
nation of the adsorption kinetics. Film specimens (25  25 mm)
were completely dried in a vacuum oven at 70  C and 76 mmHg for
48 h, and then in a desiccator over P2O5 for 2 weeks. The dried
samples (in triplicate) were placed into desiccators with saturated
salt solutions at 30  C. The salt solutions included LiCl, MgCl2,
Mg(NO3)2, NaCl and KNO3 of known RH: 11.3, 32.4, 51.4, 75.7 and
92.5%, respectively (Greenspan, 1977). At the conditions of relative
291
humidity higher than 60%, a trace of ethanol was used as a fungi-
static agent. Weights of samples as a function of time were
measured; moisture content was then measured by drying in an
oven at 105  C for 3 h (Lane, 1998). A set of experiments was per-
formed in two replications. The results were expressed on a dry-
weight basis as g/100 g dry sample. Water activity (aw) was deter-
mined using a Testo 650 water activity instrument (Testo, Lenz-
kirch, Germany). Moisture adsorption curves of the samples were
fitted to a mathematical model suggested by Peleg (1988):
Mt ¼ M0 þ ½t=ðk1 þ k2 tÞ (1)
where Mt is the moisture after time t; M0 is the initial moisture; and
k1 and k2 are constants. If Mt and M0 are given in g/100 g on a dry
basis, and the time units are in hours (h), then the units of k1 and k2
will be h per g/100 g (dry basis), and the reciprocal of g/100 g (dry
basis), respectively. According to this model, the equilibrium
moisture (ME) is given by:
ME ¼ M0 þ 1=k2 (2)
Similarly, the momentary sorption rate dMt/dt is given by:
dMt =dt ¼ k1 =ðk1 þ k2 tÞ2 (3)
and the initial rate is given by 1/k1. One of the characteristics of Eq.
(1) is that it can be transformed into a linear relationship in the form:
t=½Mt  M0  ¼ k1 þ k2 t (4)
The latter offers a simple way to test the applicability of the
model to sorption data, and to calculate its parameters by linear
regression.
With respect to the test duration, this can be formulated as
follows. Let R be the ratio:
R ¼ ½Mt  M0 =½ME  M0  (5)
It follows from the mathematical structure of Eq. (1) that if, for
example, t1/2 is the time needed to reach [ME  M0]/2, i.e. half the
added moisture to reach equilibrium, then the time to reach any
given level of R, namely tR, is given by:
tR =t1=2 ¼ R=ð1  RÞ (6)
A standard gravimetric methodology was used for determina-
tion of the adsorption isotherms. The film specimens were
prepared and conditioned as described in Section 2.3. The dried
samples in triplicate were equilibrated over saturated salt solutions
inside desiccators at 30  C for 4 weeks. The salt solutions included
LiCl, MgCl2, Mg(NO3)2, KI, NaCl, KCl and KNO3 of known RH: 11.3,
32.4, 51.4, 67.9, 75.7, 83.6 and 92.5%, respectively (Greenspan, 1977).
Moisture content was then measured after drying in an oven at
105  C for 3 h (Lane, 1998). A set of experiments was performed in
four replications. The results were expressed on a dry-weight basis
as g/100 g dry sample. Water activity was determined using a water
activity instrument.
2.4. Moisture sorption isotherm modeling
Isotherm models from the literature (Berg & Bruin, 1981; Toledo,
1991) were selected for modeling the experimental data of
adsorption isotherms of cassava-flour-based baked samples. Those
models are expressed and rearranged as given below.
GAB (GuggenheimeAndersonede Boer) model (Berg & Bruin,
1981):
me ¼ mo Ckaw =ð1  aw Þ ½1 þ ðC  1Þkaw  (7)
292 P. Suppakul et al. / LWT - Food Science and Technology 50 (2013) 290e297
where me ¼ equilibrium moisture content on a dry basis; mo ¼ GAB
monolayer moisture content; C ¼ Guggenheim constant, k ¼ factor
correcting properties of the multiplayer molecules corresponding
to the bulk liquid and aw ¼ water activity.
Lewicki model (Lewicki, 1998):
h i h  i
me ¼ F=ð1  aw ÞG  F= 1  aH
w (8)
where F, G, and H are constants.
Oswin model (Oswin, 1946):
me ¼ k½aw =ð1  aw Þc (9)
where k and c are constants.
Peleg model (Peleg, 1993):
me ¼ a$abw þ c$adw (10)
where a, b, c, and d are constants.
The parameters of the equations were estimated using KyPlot
2.0 for Windows (Keyence Corp., Osaka, Japan). The value of the
root mean square percentage error (%RMS) represents the fitting
ability of a model in association with the number of data points.
2.5. Film thickness measurement
A hand-held digital micrometer (Mitutoyo, Tokyo, Japan) was
used for measuring film thickness. Five readings were taken for each
sample: one at the sample center, and four around the perimeter.
2.6. Effect of sorbitol concentration on mechanical properties of
cassava flour films
Cassava flour film specimens (25  150 mm) with 20, 30, 40 and
50 g/100 g sorbitol were conditioned at 30  1  C and 50  2% RH.
Tensile properties were determined according to ASTM standard
test method D882-02 (ASTM, 2006) using a Testometric Micro 350
(Rochdale, UK). Five samples of each film were tested. The initial
grip separation and crosshead speed were set to 100 mm and
20 mm min1, respectively. The parameters determined were
tensile stress (MPa), elongation (%), and modulus of elasticity (MPa).
2.7. Effect of sorbitol concentration on barrier properties of cassava
flour films
Cassava flour film specimens (25  150 mm) with 20, 30, 40 and
50 g/100 g sorbitol were conditioned at 30  1  C and 50  2% RH.
Barrier properties were determined according to ASTM standard
methods.
2.7.1. Water vapor transmission rate of cassava flour films
Water vapor transmission rate (WVTR) of the films was deter-
mined gravimetrically at 25  1  C using a modified ASTM E96/E96M-
05 method (ASTM, 2008a). A container with silica gel was closed with
a sample of edible film firmly fixed on top. Then the container was
placed in a desiccator over a saturated salt solution of potassium
iodide (KI) having a known relative humidity of 65  2% RH. The films
were weighed daily for 5 d on an analytical balance. Water vapor
transmission rate (WVTR) was calculated according to Eq. (11):
WVTR ¼ x=½t  A (11)
2 1 1
where WVTR is in g m d atm . The term x/t was calculated by
linear regression from the points of weight gain and time, over
a constant rate period. A is the area of the exposed film. Tests were
carried out in triplicate.
2.7.2. Oxygen transmission rate of cassava flour films
Oxygen transmission rate (OTR) of the films was determined
using a model 8500 oxygen permeation analyzer (Systech Instru-
ment, Oxfordshire, UK), according to ASTM method D-3985-05
(ASTM, 2008b) for oxygen gas transmission rate through plastic
films and sheeting using a coulometric sensor. Testing was per-
formed at 25  C using RH of 50  1%. Film samples were cut into
octagonal shapes using a cutting template; each sample was then
lubricated at the rim (CMD Extreme Pressure Lube #3; Chicago
Manufacturing & Distribution Co. Inc., Gagetown, MI, USA) and
attached to the diffusion chamber at the interface between the
upper and lower chambers. Pure oxygen (laboratory grade, Thon-
buri Wattana Ltd., Bangkok, Thailand) was then introduced into the
upper half of the chamber while an oxygen-free carrier gas
(nitrogen) (high-purity grade, Thonburi Wattana Ltd.) flowed
through the lower half of the chamber and was then conveyed to an
oxygen detection sensor by the carrier gas. The response was re-
ported as a transmission rate in cm3 m2 d1 atm1. Tests were
carried out in triplicate.
2.8. Effect of relative humidity on mechanical properties of cassava
flour films
Cassava flour film specimens (25  150 mm) with 30 g/100 g
sorbitol were conditioned in a desiccator containing silica gel beads
for 3 d at 30  1  C to reduce the initial moisture content. Film
specimens were then placed at 30  1  C over saturated salt solu-
tions having known RH (LiCl, MgCl2, Mg(NO3)2, NaCl and KNO3 with
11.3, 32.4, 51.4, 75.7 and 92.5%RH, respectively). At RH conditions
higher than 60%, a trace of ethanol was used as a fungistatic agent.
Tensile properties were determined as described in Section 2.6.
2.9. Statistical analysis
The experiments in solid media were performed in pentaplicate.
Data points were represented by the mean. The data sets were
subjected to analysis of variance (ANOVA) and Tukey’s test at a 0.05
level of significance, using KyPlot 2.0 for Windows.
3. Results and discussion
3.1. Effect of plasticizers on appearance of cassava flour films
Qualitatively, the appearance of all plasticized films was clearer
and more uniform than the control, except for those films con-
taining PEG-400. Unplasticized film yielded a rough surface
appearance, whereas glycerol- and sorbitol-plasticized films were
comparatively smooth (Table 1). This is in agreement with the
study by Laohakunjit and Noomhorm (2004), who investigated rice
starch films. Glycerol- and PEG-400-plasticized films were
unpeelable from the casting surface, whereas sorbitol-plasticized
films were softer with increasing sorbitol concentration. These
macromolecular properties are affected by the plasticizers. Sorbitol
binds with starch molecules very well, so that the cohesive tension
of the molecules becomes weakened; this, in turn, results in a more
flexible structure (Mellan, 1961; Sears & Darby, 1982). Glycerol and
sorbitol are homogenously impregnated within a network of
hydrogen bonds between starch molecules; hence, the film
becomes soft and transparent. PEG-400-plasticized films displayed
a white opaque surface, referred to as “blooming” and “blushing”.
This occurs when the plasticizer concentration exceeds its
compatibility limit in the polymer, causing phase segregation and
physical exclusion of the plasticizer (Aulton, Abdul-Rassak, &
Hogan, 1981; Sakellariou, Rowe, & White, 1986). This might be due
 P. Suppakul et al. / LWT - Food Science and Technology 50 (2013) 290e297 293

Table 1
Appearance of cassava flour film as affected by plasticizer concentration.

Plasticizer type Plasticizer Appearance

concentration (g/100 g)
Control (Cassava Clear, tough, brittle. Different texture on each of the two sides: air side with rougher and tougher texture.
flour film)
Glycerol 20 Clear, transparent. Different texture on each of the two sides: air side stickier, easy to tear. Unable to peel.
30 Clear, transparent. Different texture on each of the two sides: air side stickier, easy to tear. Unable to peel.
40 Clear, transparent, very soft. Different texture on each of the two sides: air side stickier, easy to tear.
Unable to peel.
50 Clear, transparent, very soft. Different texture on each of the two sides: air side stickier, easy to tear.
Unable to peel.
Sorbitol 20 Clear, hard. Different texture on each of the two sides.
30 Clear, soft. Different texture on each of the two sides.
40 Clear, very soft. Different texture on each of the two sides.
50 Clear, very soft, sticky. Texture on each of the two sides was not different.
PEG-400 20 Opaque; some white spots spread on the film; brittle; unable to peel.
30 Opaque; some white spots spread on the film; brittle; unable to peel.
40 Opaque; some white spots spread on the film; brittle; unable to peel.
50 Opaque; some white spots spread on the film; brittle; unable to peel.

to the high molecular weight and relatively low content of hydroxyl
groups within PEG-400 (Laohakunjit & Noomhorm, 2004).
3.2. Moisture adsorption kinetics
The film from cassava flour produced from blending with sorbitol
was homogeneous, clear, smooth, and contained fewer insoluble
particles compared to unplasticized cassava flour film. As a plasti-
cizer, sorbitol effectively decreases internal hydrogen bonding while
increasing intermolecular spacing (Lieberman & Gilbert, 1973) so
that the cohesive tension of the molecules in the attached polymer
chains becomes weakened and the film, in turn, more flexible, soft
and transparent (Laohakunjit & Noomhorm, 2004). The average
thickness of the films was 0.098  0.002 mm. According to
Laohakunjit and Noomhorm (2004), sorbitol can penetrate the
network of starch more quickly and easily to form a stronger and
thicker film with an increasing concentration of sorbitol.
Moisture sorption kinetic curves of sorbitol-plasticized cassava
flour film are depicted in Fig. 1. Moisture adsorption was more rapid
in the initial stages and declined with increasing time. Then the
moisture content of the film reached a plateau, indicating that it
had become equilibrated with the RH in each condition. The higher
the RH, the more time required to reach equilibrium. Measured
sorption kinetic curve data were fitted to either Eq. (1) or Eq. (4).
The fit of Eq. (4) to sorption data of cassava flour film is shown in
Fig. 2. A determination coefficient (R2) of each linearization of
moisture sorption curve of cassava flour film (Table 2) indicates
a good fit. It should be noted that the data shown in the figure were
intentionally taken from the clearly curved part of the sorption
curves. If it is far enough from the curved region where the weight
gains are extremely small, the fit of Eq. (1) or Eq. (4) will be ob-
tained the same values of either k1 or k2 because of
Mt  M0 z constant (Peleg, 1988).
The constants k1 and k2, which were derived from the linear fit,
are shown in Table 2. The coefficients of determination are found to
be very high in all cases (R2 > 0.99); this is an indication of a good fit
to the experimental data. Generally, films containing a higher
concentration of plasticizer, and/or those that were stored at
a higher RH, tend to have lower k1 and k2 values, and vice versa. As
constants associated with mass transfer and maximum moisture
adsorption capacity, the lower the k1, the higher the initial moisture
adsorption rate, and the lower the k2, the higher the moisture
adsorption capacity (Turhan, Sayar, & Gunasekaran, 2002).
In regard to the water affinity of the molecular structure of the
plasticizer, the sorbitol molecule (MW ¼ 182) has six hydroxyl
groups, which strongly interact with moisture by forming hydrogen
bonds. However, the chances of sorbitol interacting with polymeric
starch chains are greater because sorbitol is more similar to the
molecular structure of glucose units. Consequently, sorbitol-
plasticized starch film presented higher intermolecular forces and
showed a lower capacity to interact with water (García, Martino, &
Zarizky, 2000a).
The predictive capability of the model is demonstrated in Table 3
where the constants k1 and k2, which were obtained from the linear
fit, were employed to calculate the moisture content at times well
beyond the range of the regression data. It should be noted, though,

Fig. 1. Moisture sorption curves of cassava flour films with 30 g/100 g sorbitol at various relative humidity as a function of time, A 11.3%RH, - 32.4%RH, : 51.4%RH,  75.7%RH and
* 92.5%RH.
294 P. Suppakul et al. / LWT - Food Science and Technology 50 (2013) 290e297
Fig. 2. Linearization of moisture sorption curves of cassava flour films with 30 g/100 g
sorbitol at various relative humidity as a function of time, A 11.3%RH, - 32.4%RH,
: 51.4%RH,  75.7%RH and * 92.5%RH.
that the moisture content after 24 h remained practically
unchanged, and consequently could be treated as representing
equilibrium conditions. According to the discussed model,
however, the value of the calculated equilibrium moisture was
slightly higher. As can be seen from Table 3, the sorption rates fall
dramatically when these moisture contents are reached and,
therefore, the net weight gain can fall below the accuracy of the
moisture determination method. The times to reach both R ¼ 0.95
and R ¼ 0.99 are longer than what was considered practical. This
experiment was of 48 h duration (Fig. 1), which at least superficially
was very close to the equilibrium level. Peleg (1988) also explained
that the model, as expressed in Eq. (1) or Eq. (4), is only an empirical
model that was not derived from any set of physical laws or
diffusion theories. As to the equilibrium moisture contents, the
model predictions, as previously stated, are significantly higher
than those based on the assumption that true equilibrium is
reached after 24e48 h (Peleg, 1988). Because cassava flour films
become biologically and/or physically unstable after long exposure
to a moist environment, there is no data on their sorption behavior
in a long-term experiment, i.e. on the order of days in this case.
Hence, the question of whether true equilibrium can be reached at
all under such circumstances, and if so, at what moisture levels,
cannot be settled on the basis of the available data (Peleg, 1988).
3.3. Moisture adsorption isotherm
The moisture sorption isotherm curve of sorbitol-plasticized
cassava flour film (Fig. 3) can be classified as a type II sigmoidal
isotherm, which is obtained for soluble materials and shows an
Table 2
Sorption kinetic model constants for cassava flour film with 30 g/100 g sorbitol at
selected relative humidity.
Relative humidity (%) Sorbitol-plasticized cassava flour film
k1a k2b
11.3 0.602 0.279
32.4 0.277 0.166
51.4 0.139 0.102
75.7 0.135 0.059
92.5 0.072 0.023
a
Unit of k1 is hour per g/100 g on dry basis.
b
Unit of k2 is the reciprocal of g/100 g on dry basis.
asymptotic trend as water activity tends toward 1 (Mathlouthi &
Rogé, 2003). Moisture sorption was more rapid in the initial stages,
and a lesser amount of moisture was adsorbed as adsorption time
increased. The higher the RH used, the more pronounced the effect.
The moisture content of sorbitol-plasticized cassava flour film
increased considerably, from 5.85 to 15.43 g/100 g, when the
surrounding RH rose from 32.4 to 75.7%. The equilibrium moisture
content of film dramatically soared above aw ¼ 0.6. This is in accor-
dance with studies by Stading, Rindlav-Westling, and Gatenholm
(2001), Cho and Rhee (2002), and Mali et al. (2005), who found
a similar trend for glycerol-plasticized amylose and amylopectin
films, sorbitol-plasticized soy protein film, and sorbitol-plasticized
cassava starch film, respectively. Sorbitol is a hydrophilic plasticizer
that loosens the structure of film; the addition of this plasticizer
raises the hydrophilicity of film by exposing the hydroxyl groups.
Calculated model constants, coefficient of determination (R2),
and %RMS for each model for sorbitol-plasticized cassava flour film
are represented in Table 4. The GAB model is a semi-theoretical
multilayer sorption model with a physical meaning for each
constant (Timmerman, Chirife, & Iglesias, 2001). In general, it is the
most accepted model for foods or edible materials. Sorbitol-
plasticized cassava flour film presented a monolayer water
content of 0.0455 g/100 g (dry basis). This value is in agreement
with the study by Mali et al. (2005) which reported that monolayer
values of 20 g/100 g- and 30 g/100 g-sorbitol-plasticized cassava
starch films were 0.043 and 0.042 g/100 g (dry basis), respectively,
and the study by Müller et al. (2008) which reported that mono-
layer values of 25e35 g/100 g-sorbitol-plasticized cassava starch
films were 0.046e0.047 g/100 g (dry basis). This value indicates the
maximum amount of water that can be adsorbed in a single layer of
dry film, and is a measure of the number of sorbing sites. The
Lewicki model was developed to be applicable to a high range of aw.
It fitted well with the moisture sorption data at high humidity, and
predicted that the water content tended to infinity as aw reached
1.0 (Lewicki, 1998). Thus, the Lewicki model is considered to be
preferable and more applicable for predicting the moisture
adsorption of film at different RH. The Oswin model provides good
descriptions of the moisture isotherms throughout the entire range
of water activity (Oswin, 1946). However, in this case, maximum %
RMS value was obtained for the Oswin model. The Peleg model can
predict both sigmoid and non-sigmoid isotherms. According to
Peleg (1993), this model fitted as well as or better than the GAB
model, but unfortunately its constants had no physical meaning. In
the case of the Peleg model, the value of R2 was highest and was
similar to the GAB model. Nevertheless, the %RMS from the Peleg
model resulted in a higher value than that of GAB model. Thus, the
GAB model was found to be the best estimator for predicting the
equilibrium moisture content of sorbitol-plasticized cassava flour
film, followed by the Lewicki model.
With the experimental versus predicted moisture content of the
film (data not shown), the adjusted correlation coefficients (radj)
between the actual and the predicted moisture content were 0.9967,
0.9969, 0.9970 and 0.9952 using GAB, Lewicki, Oswin and Peleg
model, respectively. The obtained points lie on the diagonal for low
and intermediate aw levels, indicating low interaction between
components in accordance with their separation in independent
phases as observed during the film drying. At a high level of aw, it can
also be observed that points fall on the diagonal, as a result of the
R2
interaction between water molecules and the polar groups of the film.
0.9996
0.9995
0.9991 3.4. Effect of sorbitol concentration on mechanical properties of
0.9991 cassava flour films
0.9997
Sorbitol-plasticized films with different concentrations had
comparable stress-strain patterns. With 20e30 g/100 g sorbitol, the
 P. Suppakul et al. / LWT - Food Science and Technology 50 (2013) 290e297 295

Table 3
Moisture sorption parameters for cassava flour film with 30 g/100 g sorbitol.

Conditions Initial moisture Moisture content (g/100 g db) Calculatedb ME Calculated sorption rate (%/h)c t1/2d (h) tR (h)d
content (g/100 g db) (g/100 g db)
After time (h) Calculateda Observed Initial After 24 h After 48 h R ¼ 0.95 R ¼ 0.99
30  C, 11.3%RH 0.045 24 3.33 3.25 3.62 1.66 0.011 0.003 2.25 42.68 222.38
30  C, 32.4%RH 0.045 24 5.69 5.56 6.08 3.60 0.015 0.004 1.68 31.86 166.02
30  C, 51.4%RH 0.045 24 9.31 9.17 9.84 7.15 0.021 0.006 1.65 31.33 163.24
30  C, 75.7%RH 0.045 24 15.47 15.32 16.94 7.42 0.056 0.015 2.44 46.29 241.20
30  C, 92.5%RH 0.045 24 38.49 37.73 43.52 13.83 0.186 0.052 3.19 60.68 316.16
a
Calculated by Eq. (1) with k1 and k2 determined from data during the first 20 h.
b
Calculated by Eq. (2) with k2 determined as described above.
c
Calculated by Eq. (3) with k1 and k2 determined as described above.
d
See Eqs. (5) and (6).

typical stress-strain curves of plasticized cassava flour films dis-
played a high resistance to fracture and regarded as brittle material.
The typical stress-strain curve of 40 g/100 g sorbitol-plasticized
cassava flour film clearly demonstrated a large deformation
before fracture and regarded as ductile material. The effect of
sorbitol concentration on tensile properties of the films is shown in
Table 5. Tensile property values (e.g. tensile strength, modulus of
elasticity, and elongation) showed inconsistent dependence on
sorbitol content. All films exhibited yield at strain 3 z 2e3%, which
is in accordance with the study by Rindlav-Westling, Stading,
Hermansson, & Gatenholm (1998) on the mechanical properties
of amylose and amylopectin films. As contained 94e97.5 g/100 g
(dry basis) starch, which composed of 17 g/100 g amylase and 83 g/
100 g amylopectin, in cassava flour, the linear amylose and the
branched amylopectin biopolymers exhibit different behavior with
regard to gelation and development of crystallinity. Rindlav-
Westling et al. (1998) revealed that the crystallinity in the
amylose gel is fully developed during the first period of drying
when the water content is still very high, and thereby also the
mobility. The crystallinity of amylose films, therefore, was only
slightly affected by the air humidity during the film formation or
the addition of a plasticizer. In the absence of a plasticizer, the
amylopectin did not have sufficient time to crystallize before the
water content reached very low values during the drying process,
yielding a totally amorphous biopolymer. The addition of a plasti-
cizer facilitated the mobility of the amylopectin biopolymer over an
extended time so that crystallization could gradually begin
(Rindlav-Westling et al., 1998). As sorbitol concentration increased
from 20 g/100 g to 30 g/100 g, the film showed the highest tensile
strength and modulus of elasticity. The higher stresses are perhaps
due to increased crystalline phase (Myllärinen, Partanen, Seppalla,
& Forsell, 2002). As previously mentioned in regard to the mech-
anism whereby a plasticizer enhances the crystallinity in an
amylopectin biopolymer, the increase in crystallinity made cassava
flour film more brittle, with increased tensile strength and modulus
of elasticity and relatively lower strain. This is because the crys-
talline parts are dense and have a high modulus, while they also
make the network less extendible, thus causing only a small strain.
The highest elongation was revealed at 40 g/100 g of sorbitol. The
mechanism of plasticization of wheat starch by sorbitol was
examined using DMTA and NMR (Gaudin, Lourdin, Le Botlan, Ilari, &
Colonna, 1999). It was concluded that at high concentration (above
27 g/100 g) sorbitol acted as a plasticizer. The presence of 50 g/
100 g sorbitol in cassava flour film resulted in a decrease in the
percent elongation at break. This is probably due to the anti-
plasticizing effect caused by the high level of plasticizer, indicating
stronger interactions between the plasticizer and the amylose/
amylopectin biopolymer that induce a loss of macromolecular
mobility (Shimazu, Mali, & Grossmann, 2007; Souza et al., 2012).

3.5. Effect of sorbitol concentration on barrier properties of cassava

flour films

Sorbitol concentration effect on barrier properties of the films was

shown in Table 5. Water vapor transmission rate of sorbitol-plasticized
cassava flour films ranged from 31 to 37 g m2 d1 atm1. Through the
entire sorbitol concentration regime, the WVTR of cassava flour film
was gradually reduced with increasing sorbitol content. However, it

Table 4
Sorption isotherm model constants for cassava flour film with 30 g/100 g sorbitol.

Isotherm model Constant R2 %RMS

GAB m0 ¼ 0.0455 0.9997 1.6752
C ¼ 9.1273
k ¼ 0.9816
Lewicki F ¼ 6.6381 0.9996 1.8147
G ¼ 0.7992
H ¼ 0.2843
Oswin k ¼ 7.6642 0.9971 6.1998
c ¼ 0.7294
Peleg a ¼ 14.7546 0.9997 2.6413
b ¼ 0.8462
c ¼ 64.4794
Fig. 3. Moisture sorption isotherm of cassava flour film with 30 g/100 g sorbitol at d ¼ 9.0286
30  1  C.
296 P. Suppakul et al. / LWT - Food Science and Technology 50 (2013) 290e297

Table 5
Mechanical and barrier properties and thickness of cassava flour films as a function of sorbitol concentration.

Sorbitol Thickness (mm) Tensile stress at Modulus of Elongation at WVTR (g m2 d1 atm) OTR (cm3 m2 d1 atm)
(g/100 g) peak (MPa) elasticity (MPa) break (%)
20 0.096  0.0015A 14.83  0.69C 1924.88  274.00C 4.13  1.62A 36.68  0.68B 55.33  0.58C
30 0.098  0.0022B 28.65  2.52D 2629.72  205.26D 5.20  0.92B 36.21  0.51B 54.33  1.15BC
40 0.099  0.0028B 9.25  1.20B 971.20  143.78B 28.24  8.79C 36.23  1.61B 51.67  0.58B
50 0.098  0.0023B 5.29  0.78A 365.01  98.22A 5.29  1.50B 31.20  0.55A 48.67  1.53A

Values are represented as mean  standard deviation; means bearing identical letters in the same column are significantly different (p < 0.05).

dramatically dropped when sorbitol content rose above 40 g/100 g
(Table 5). According to Guilbert & Biquet (1996), the addition of
a plasticizer modifies the properties of the edible film by reducing the
intermolecular bonds between the polymer chains, thus increasing
the WVTR of the film. Contrary to this, cassava flour film containing
higher sorbitol concentrations showed lower WVTRs. This is probably
due to sorbitol being less effective in reducing intermolecular
hydrogen bonding between amylose and amylopectin molecules.
Previous research has shown how edible films containing sorbitol as
a plasticizer have lower water vapor permeability than those con-
taining glycerol (Olivas & Barbosa-Cánovas, 2008). The oxygen
transmission rate (OTR) of sorbitol-plasticized cassava flour films
ranged from 47e56 cm3 m2 d1 atm1. The OTR of cassava flour film
significantly decreased with rising sorbitol content (Table 5). With
a molecular weight of 182.17, sorbitol is a relatively larger hydrophilic
molecule compared to glycerol (MW ¼ 92.09). Thus, sorbitol is less
effective in increasing molecular mobility and in facilitating migration
of either water vapor or oxygen molecules (Rodríguez, Osés, Ziani, &
Maté, 2006). According to García, Martino, & Zarizky (2000b), it is
likely that a decrease in biopolymer chain mobility and void spaces in
the film matrix were responsible for a decrease in the OTR of sorbitol-
plasticized cassava flour film. Due to the amylopectin portion of 83 g/
100 g in cassava starch composition, with its branched biopolymer
and, in turn, a highly amorphous network structure, cassava flour
films are expected to provide less of an oxygen barrier.
3.6. Effect of relative humidity on mechanical properties of cassava
flour films
Changes in tensile strength of cassava flour films as a function of
RH are shown in Table 6. An increase in tensile strength of sorbitol-
plasticized film was evident when RH increased from 10 to 50%, and
was followed by a dramatic decrease in tensile stress when relative
humidity reached 90%. Increasing humidification from the dry state
strengthened the films until maximum stress was reached at
a critical aw or moisture content, after which further humidification
weakened the films. Therefore, the antiplasticizing effects of water
on tensile strength were followed by the plasticizing effects when
reaching a critical aw. Modulus of elasticity is the fundamental
measure of the film stiffness: the higher the modulus of elasticity,
the higher the stiffness of the material. The effect of RH on the
modulus of elasticity can be observed in Table 6. With increasing
RH, the modulus of elasticity of sorbitol-plasticized film linearly
Table 6
Mechanical properties of cassava flour films as a function of relative humidity.
Relative Tensile stress Modulus of Elongation
humidity (%) at peak (MPa) elasticity (MPa) at break (%)
11.3 16.05  0.42C 4180.80  273.72E 2.25  0.18A
32.4 22.58  1.12D 3691.40  167.75D 2.37  0.16A
51.4 28.92  0.67E 2604.00  72.78C 5.05  0.57B
75.7 6.11  0.60B 950.40  38.66B 16.52  0.64C
92.5 2.42  0.49A 58.20  5.89A 27.59  1.19D
Values are represented as mean  standard deviation; means bearing identical
letters in the same column are significantly different (p < 0.05).
decreased, symptomatic of plasticization (Chang et al., 2006).
Elongation represents the ductility of the material. Elongation of
sorbitol-plasticized film was exponentially increased with
increasing RH (Table 6). At higher RH conditions, moisture exerts
a plasticizing effect, acting as a mobility enhancer; its low molec-
ular weight leads to a large increase in the molecular mobility of
amorphous and partially crystalline polymers due to an increase in
free volume (van der Berg, 1991).
4. Conclusions
The hydrophilicity of sorbitol and its concentration were found
to be relevant factors in determining the moisture affinity of cas-
sava flour film. Sorbitol-plasticized cassava flour film presented
higher intermolecular forces and showed a capacity to interact with
water. With the predictive capability of Peleg model, the constants
k1 and k2 were employed to calculate the moisture content at times
well beyond the range of the regression data. GAB and Lewicki
models were useful to fit moisture sorption isotherm data. The
application of sorbitol as a plasticizer had a beneficial effect on the
appearance of cassava flour film. Sorbitol-plasticized films were
clearer, smoother and more uniform than unplasticized film. The
percentage of elongation increased significantly with increasing
sorbitol content. However, it dropped when sorbitol content was
above 40 g/100 g. As RH increased, the modulus of elasticity of
sorbitol-plasticized film linearly decreased. Elongation of sorbitol-
plasticized film exponentially increased with increasing RH. The
WVTR and OTR of cassava flour film were reduced with increasing
sorbitol content. The results imply that cassava flour film may have
a potential use as an edible coating and film for food applications by
moisture-resistant modification.
Acknowledgments
This work was fully supported by a fund for the promotion of
research at the Center of Advanced Studies for Agriculture and Food
(CASAF), Kasetsart University. The authors express their thanks and
appreciation for this support.
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 Environmental Technology & Innovation 28 (2022) 102673

Contents lists available at ScienceDirect

Environmental Technology & Innovation

journal homepage: www.elsevier.com/locate/eti

Effect of repeated sorption–desorption on irreversible and

reversible absorption of hydrophobic perfluoroalkyl acids to
freshwater sediment
∗
Huiting Chen a , Martin Reinhard b , Luhua You a , Karina Yew-Hoong Gin a,c ,
a
NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive 1,
#02-01, Singapore 117411, Singapore
b
Department of Civil and Environmental Engineering, Yang & Yamasaki Environment & Energy Building, Stanford University, 473
Via Ortega, Stanford, CA, 94305, United States
c
Department of Civil and Environmental Engineering, National University of Singapore, 1 Engineering Drive 2, E1A
07-03, Singapore 117576, Singapore

article info a b s t r a c t

Article history: Understanding the factors governing irreversible and reversible sorption is essential to
Received 1 April 2022 predict the influence of sediment on contaminant fate and transport in surface waters.
Received in revised form 15 May 2022 This study used sediment of an urban water body to demonstrate irreversible accumula-
Accepted 15 May 2022
tion and reversible absorption of perfluoroalkyl acids (perfluorooctane sulfonate (PFOS),
Available online 21 May 2022
perfluorononanoic (PFNA) and perfluorodecanoic acid (PFDA)) during two sorption–
Keywords: desorption (S–D) cycles. Absorption and desorption isotherms were consistent with
PFOS sorption to irreversibly absorbing glassy sediment organic carbon (SOC) and liquid–liquid
PFNA like partitioning to rubbery SOC. The observed absorption isotherms and the desorption
PFDA isotherms were adequately fitted linear models. Irreversible absorption showed signs of
Sediment saturation and the reversible partitioning diminished during the 2nd S–D cycle causing
Sorption the composite sorption capacity to decrease. Results suggest that under the conditions
Desorption
tested, surface water sediment can act as sink for the studied PFAAs. Rubbery SOC can
Hysteresis
potentially release or absorb contaminants, thus acting as a concentration buffer. S–D
cycling can cause irreversible absorption concentration to increase even when exposure
occurs at lower concentrations (i.e. 5 µg/L). log KOC values of sediment that underwent
S–D cycling overlaps with the range of field date, suggesting that the sediment of surface
water may have experienced some degree of S–D cycling.
© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

1.1. Background

Hydrophobic perfluoroalkyl acids (PFAAs) occur in all types of aquatic environments (Xiao, 2017) including seawa-
ter (Benskin et al., 2012; Sakurai et al., 2010), rivers (Labadie et al., 2007), urban runoff (Nguyen et al., 2012), and urban
fresh water bodies (Chen et al., 2019). Because most of the contaminant mass is associated with sediments (Zareitalabad
et al., 2013; Chen et al., 2016), and sediments could be an important PFAA source (Langberg et al., 2020), understanding

∗ Corresponding author at: NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive 1, #02-01, Singapore
117411, Singapore.
E-mail addresses: cht0831@gmail.com (H. Chen), reinhard@stanford.edu (M. Reinhard), ceeginyh@nus.edu.sg (K.Y.-H. Gin).

https://doi.org/10.1016/j.eti.2022.102673
2352-1864/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.
org/licenses/by-nc-nd/4.0/).
H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

the sediment’s capacity to absorb irreversibly or reversibly is a precondition to assess ecotoxicological impacts (Ankley
et al., 2021), environmental risk and to predict their role as contaminant carrier, sink or source. Key factors that govern
the absorption of PFAAs include: sorbate properties (number of perfluorinated alkyl carbons (N), the type of polar head
group); sediment properties (sediment organic carbon (SOC), clay, and iron oxide, the anion and cation exchange capacity);
and solution conditions (pH, salinity, and Ca2+ and Mg2+ concentrations) (Chen et al., 2019; Zareitalabad et al., 2013;
Ahmed et al., 2020; Ahrens, 2011; Ahrens et al., 2010, 2009; Chen et al., 2012; Du et al., 2014; Higgins and Luthy, 2006;
Prevedouros et al., 2006; Xiao et al., 2019; You et al., 2010; Zhao et al., 2016; Fagbayigbo et al., 2022). Most of these findings
were developed by studying isotherms during the absorption step using native sediment. Some used other materials
including hydrotalcites (Alonso-de-Linaje et al., 2021), and activated carbon (Yu et al., 2012). Absorption of hydrophobic
contaminants is typically hysteretic, i.e. the sorption and desorption isotherms are asymmetric, resulting in irreversibly
sorbed contaminants being resistant to physical, chemical or biological treatments (Xiao et al., 2019). Sorption hysteresis
has been extensively studied (see Di Toro, 1985; Sander et al., 2005; Weber et al., 1992; Kan et al., 1998, 1997 for the
most relevant publications for this study). Theoretically, only the fraction that can desorb from sorbents are considered
bioavailable (Alexander, 2000). Therefore, the degree to which PFAAs absorbed to the sediments will affect the availability
of PFAAs in water and the degree of hysteretic sorption will affect their bioavailability to benthic organisms. However, such
information is significantly lacking in the literature. Balgooyen and Remucal (2022) suggested that sediment desorption
can act as sources of PFASs to receiving waters and observed residue of PFASs in sediments after desorption experiments,
which could indicate sorption hysteresis. Hysteretic sorption of hydrophobic PFAAs has received limited attention (You
et al., 2010; Chen et al., 2016; Zhao et al., 2014; Zhi and Liu, 2018) and, to our knowledge, S–D cycling, has not been
reported. The lack of understanding of this fundamental information needed for determining the transport and fate of
PFAS precludes the establishment of reliable models for the prediction of their fate in the aquatic environment (Naidu
et al., 2020).
This research herein investigated the effect of S–D cycling of selected hydrophobic PFAAs (perfluorooctane sulfonate
(PFOS), perfluorononanoic (PFNA) and perfluorodecanoic acid (PFDA)) on the sorption properties of freshwater sediment.
An S–D cycle refers to a step of absorption experiment followed by a series of desorption experiments. The reversible
and irreversible sorption capacity was evaluated in two consecutive S–D. This is to mimick the more realistic situation
in the environment where sediments could be repeatedly washed and exposed to PFASs. The 1st S–D experiment
was conducted with native sediment and was designed to characterize the initial contamination event. The 2nd S–D
experiment was conducted with samples that underwent the 1st S–D cycle. The reversibly and irreversibly absorbed
fractions were evaluated using the reversible–irreversible dual mode model (DMM) (Di Toro and Horzempa, 1982; Huang
and Weber, 1997). The DMM considers SOC as consisting of rubbery and glassy domains that absorb reversibly or
irreversibly, respectively, whereby sorption by the rubbery domain is a quasi-liquid–liquid partitioning process governed
by a concentration-independent (Henry’s) partition coefficient, π . Absorption to glassy SOC occurs at sites of highly
cross-linked organic material such as kerogen, lignite, or black carbon and follows the Langmuir model (LeBoeuf Eugene
and Weber, 1997; Sander et al., 2006). At low concentrations, the slope, p, of Langmuir’s isotherm is constant. As the
equilibrium concentration increases, sites get saturated and p decreases (Di Toro, 1985; Di Toro and Horzempa, 1982;
Ran et al., 2013; Luthy et al., 1997; Weber et al., 2002; Delle Site, 2001; Sander and Pignatello, 2007, 2005; Huang et al.,
2003; Lu and Pignatello, 2004). The uptake of sorbate can cause swelling of the glassy sorbent and increasing the number
of sorption sites, a process referred to as plasticization (Weber et al., 2002; Lu and Pignatello, 2004, 2002; Delle Site, 2001).
Irreversible absorption is often referred to as to ‘‘desorption resistant’’ to account for the fact that ultimate irreversibility
cannot be ascertained (Pignatello, 2012).
The irreversibly absorbed concentration, qirrev , is determined considering the mass balance (Eq. (1)) (Zhao et al., 2001):

qirrev = qobs − qrev (1)

obs rev
where q is the measured/calculated solid phase concentration, and q is the reversibly absorbed concentration. For
clean sediments that exhibit linear isotherms and reversible sorption, qobs [L/kg] is given by Eq. (2) (irrespective of the
direction of the aqueous concentration, c):

qobs = cKDobs (2)

where KDobs is the distribution coefficients defined as the ratio of observed solid phase concentration to aqueous con-
centration. For sediments that exhibit linear absorption isotherms and contain a reversibly and an irreversibly absorbing
domain, we can write for the absorption step in a first approximation:

∆qobs = (π + p)∆c (3)

where ∆q and ∆c are the changes of the solid phase and aqueous concentrations, respectively. Note that p is necessarily
obs

constant if π is constant. This is difficult to verify experimentally, however. During the desorption steps, ∆c is negative
and hence the change ∆qobs is negative (Eq. (4)). Because theoretically desorption from the irreversible compartment does
not occur, p is equal to zero:

−∆qobs = (π + 0) (−∆c) (4)

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H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

KDobs is the sum of the contributions of reversible and irreversibly absorbed fractions (Eq. (5)) whereby the concentrations
in the two compartments respond differently to ∆c (cf. Eqs. (3) and (4)):
qrev qirrev
KDobs = + (5)
c c
During desorption, as c approaches zero qirrev remains constant, KDobs increases to infinity. During a complete S–D cycle,
i.e., from c = 0 (initiation of absorption experiment) to c = ca (absorption equilibrium/initiation of desorption) and back
(i.e. desorption series till c approaches 0), the initial increase ∆qrev is reversed and the net increase is 0. By contrast, qirrev
is predicted to permanently increase, according to Eq. (6):

∆qirrev = (p)∆c (6)

This study aimed to quantify the increases of ∆qirrev during S–D cycling using hydrophobic PFAAs as model sorbates.
Measurements of KDobs of field sediment lump together qrev and qirrev and, therefore, are highly dependent on the exposure
history. Modeling is difficult because the dependence of p on sediment exposure unknown. Such observations were made
by Kan et al. (Yu et al., 2012; Di Toro, 1985) using naphthalene and tetrachlorobiphenyl as probes. These authors observed
that ∆qirrev , i.e., the irreversible absorbed components, increased stepwise at repeated exposures to low concentrations
until reaching a maximum, as predicted by Eq. (6). Similar observations for PFAAs have not been reported.

1.2. Experimental design, hypotheses and data evaluation

The experimental approach involved in quantifying π and p of the three targeted hydrophobic PFAAs compounds
during two consecutive S–D experiments. Samples of native sediment were spiked at four different concentrations (5, 20,
50, and 100 µg/L). qrev and qirrev were evaluated after batch-wise desorption until the c was at the detection limit (dl)
and constructing the ‘‘continuous desorption isotherm’’ (CDIs). To study changes in the sediment sorption properties, the
isotherms of qtotal , qrev , and qirrev were expressed as isotherms, i.e., as functions of the equilibrium concentrations, c, as
schematically depicted in Fig. 1A and B.
Fig. 1A depicts the hypothesized behavior of the 1st S–D where four native (clean) sediment samples are spiked at
four different concentrations. As observed previously, the resulting absorption isotherms and the CDIs (from the origin
to the dl) are linear (Chen et al., 2016). This range of CDI encompasses the practically relevant range. Fig. 1 A depicts
two continuous desorption isotherms (CDIs) originating at absorption points ② and ③. The microcosms that underwent
the first S–D experiment are re-spiked with the same concentrations. Fig. 1B depicts the behavior during the 2nd S–D
for re-spiking one microcosm. Weaker absorption results in a lower slope, a higher equilibrium concentration (ca2 ) and
a one-point isotherm with a lower slope (Fig. 1B) ④–⑤. The actual experiment discussed below entailed re-spiking the
microcosms at the same concentrations (5, 20, 50, and 100 µg/L) and resulted in four one-point absorption isotherms
originating at four different qD1 0 . π and q0 are evaluated by linearly extrapolating the CDIs to c = 0. The effect of the
D1

1st S–D cycle on the sorption properties is evaluated by comparing the parameters of Eq. (3) obtained during the 1st and
2nd absorption isotherms.
v
qtotal and π are determined at each desorption step n by measuring c and evaluating qre n Eq. (5) Di Toro and Horzempa
(1982):
v
n = π cd(n)
qre (7)
v
where qre
n is the remaining reversibly sorbed concentration after desorption step n. At n = 1, the total of the reversibly
absorbed concentration, qrev , is π ca . It should be noted that with the experimental approach used, slow desorption on
a time-scale of months to years cannot be evaluated. A study that investigated reversible and irreversible sorption of
naphthalene and tetrachlorobiphenyl reported slow desorption (Kan et al., 1997).
D1+2
According to the model (Fig. 1A and B), qD1 0 < q0 indicating that the irreversible concentration increases during the
2nd S–D cycle, as noted previously (Kan et al., 1997; Chen et al., 2016). The CDIs of the 2nd S–D,⑤-⑥ originates at the
D1+2
absorption point ca2 . The increase in q0 during the 2nd S–D is denoted as qD2 0 . The two S–D cycles result in q0 , which
is obtained by the intercepts of the CDIs with the y-axis ((6) in Fig. 1B):

qD1
0
+2
= qD1 D2
0 + q0 (8)
where the superscripts D1 and D2 indicate the 1st and the 2nd desorption experiments, respectively.
The carbon-normalized distribution coefficient, KOC defined in Eq. (9) is often used to compare the sorption behavior
of contaminants in different sediments.
Koc = KDobs /foc (9)
where foc is the mass fraction of SOC. Just as the KDobs ,
the KOC is highly dependent on exposure history. Zareitalabad
et al. (2013) reviewed published log KOC data of PFOS developed in laboratory experiments and derived a ‘‘global’’ log
KOC from reported sediment data and water concentrations. The average log KOC for the published laboratory studies
was 3.0 whereas that derived by ‘‘global’’ aggregation of surface water and sediment concentrations ranged 3.8 to 4.2.
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H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

Fig. 1. Hypothesized (idealized) results of 1st and 2nd absorption and desorption experiments assuming constant sorption properties: (A) 1st S–D
experiment with consecutive desorption isotherm initiated at two different initial absorption points concentrations (ca(1) and ca(2) ). The equilibration
steps are numbered from n = 1 to N, the final desorption step at Cd = dl. (B) 1st and 2nd S–D experiments. Ideal DMM behavior: absorption and
consecutive desorption isotherms (CDIs) are linear and remain constant. Data points are suggested only for the second CDI. The equilibration steps
are numbered from n = 1, the initial absorption point to N, the final measurable desorption step above Cd = dl.

To examine the potential effect of S–D cycling, we compared the range of Koc with values derived from laboratory S–D
cycling experiments.

2. Materials and methods

2.1. Sorbent

Sediments were collected from the benthic layer of an urban reservoir at 5 m depth, air-dried, sieved through a 2 mm-
sieve, homogenized using mortar and pestle and stored in high density polypropylene (PP) bottles. Samples were sent to a
commercial laboratory for characterization (Table SI1). Background PFAA concentrations contained in the native sediment
used for this study were determined using the method of Powley et al. (2005) (SI). Results shown in Table SI2 indicate
that concentrations in the native sediment were less than 3 µg/kg.
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H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

Fig. 2. Experimental design of batch absorption–desorption (S–D) experiment. Arrows leaving the aqueous phase indicate removal of a fraction
of the supernatant and LC-MS/MS analysis. Sediments remain in the centrifuge tubes. Arrows toward the aqueous phase indicate spiking of
individual/mixture of PFAAs at concentration C0 .

2.2. Sorbates and reagents

Perfluorononanoic acid (PFNA, 97%), perfluorodecanoic acid (PFDA, 98%), and perfluorooctane sulfonate (PFOS, potas-
sium salt) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The internal standards [13 C2 ] PFNA, [13 C2 ] PFDA and
[13 C2 ] PFOS were purchased from Wellington Laboratories (Guelph, Ontario, Canada). All the standards were prepared in
HPLC grade methanol.

2.3. Batch experiments and chemical analysis

PFNA, PFDA, and PFOS were used to study sorption reversibility in two consecutive experiments (1st and 2nd S–D cycle)
as depicted schematically in Fig. 2. Step 1 of the 1st S–D cycle involved developing isotherms by spiking at four different
concentrations to the microcosms. Following equilibration and analyzing the equilibrium concentration, microcosms were
extracted sequentially in 8–12 desorption steps. All microcosms were constructed in 50 mL polypropylene (PP) centrifuge
tubes at room temperature (25 ◦ C). The microcosms consisted of 40 ml artificial surface water containing 1 mM NaHCO3
and 1 mM NaCl as background electrolyte.
Samples of sediment of an urban freshwater body are referred to as ‘‘native’’ sediment. Sediment was pretreated by
washing to remove Ca and Mg ions. Triplicate PP tubes were loaded with 5.0 g of air-dried sediments, 40 mL of background
electrolyte, and the tubes were placed horizontally on an orbital shaker (KS 4000i Control, IKA, Germany). After shaking for
24 h at 150 rpm, microcosms were centrifuged at 13,000 g for 20 min, 90% of the supernatant was removed and replaced
with an equal volume of background electrolyte. This washing step was repeated three times. Stock solutions of PFOS,
PFNA and PFDA in mixture were made in methanol at concentrations <0.1% (by volume) in background electrolyte. After
the final washing step on Day 4, supernatant was replaced with the exact volume of PFAA mixture required to obtain the
four initial PFAA concentrations 5, 20, 50, 100 µg/L. The samples were placed into the shaking incubator and equilibrated
for five days. Preliminary tests indicated that 5 d was sufficient to reach equilibrium (Chen et al., 2016). Sediment blanks
without PFAAs and PFAA controls without sediments were also prepared in triplicate in the same manner. Samples were
then centrifuged, one subsample of each supernatant was collected and analyzed as described below.
CDIs were developed using at least eight or more successive desorption points. Each extraction was conducted after
five days of equilibration, as described by Pan et al. (2009). Briefly, after absorption and centrifugation, the 35–40 mL
supernatant was removed and replaced with exactly the same volume of clean background electrolyte. Samples were
again shaken for five days, centrifuged and analyzed. This desorption experiment were repeated eight or more times until
the equilibrium concentration as no longer detectable.
The 1st absorption/desorption experiment was repeated a second time by respiking under the same concentrations.
The CDI of the 2nd S–D was developed by eight to twelve successive batch desorption experiments. At each equilibrium
point, the supernatant volume was measured and collected. A sub-sample was diluted with methanol, spiked with 1.2 ng
absolute of an internal standard (IS) mix (Chen et al., 2016) and analyzed using high performance liquid chromatography
(Agilent 1290 series, CA, USA) coupled with Agilent Jet Stream electrospray ionization tandem mass spectrometry (Agilent
6490), as described previously (Chen et al., 2019). Additional details regarding sample preparation are provided in the
SI. A TARGA C18 column (Sprite, particle size of 5 µm, 2.1 × 40 mm, Higgins, USA) together with a Luna C18 guard
column (particle size of 3 µm, 2 × 100 mm, Phenomenex, USA) were used for the separation of analytes. The solid phase
concentration was determined by mass balance from aqueous phase equilibrium concentrations. Additional microcosms
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H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

Fig. 3. Plot of the solution phase c (A) and (B) solid phase qobs equilibrium concentration profiles of PFOS as a function of desorption steps during
the 1st and 2nd S–D experiment. The solution profiles after spiking the native sediment with 5, 20, 50, and 100 µg/L (indicated by A5, A20, A50,
and A100, respectively, where −1 were in the 1st and −2 were in the 2nd S–D experiment) are depicted in Panel A (a, b, c, d) and the solids
concentrations (qobs ) in Fig. 3 B (e, f, g, h). The solid squares in the upper panels indicate the origin of the CDIs. qirrev as evaluated by extrapolation
of the CDIs to c = 0 (see Fig. 4).

were set up in the same manner, but sacrificed after 1st absorption, 4th desorption and 8th desorption in the first and
second S–D cycles for the determination of solid phase concentrations to rule out mass loss during the experiments. The
blank control showed no significant desorption from the sediments and no significant (less than 5%) reduction of the
PFAAs by absorption to the tube walls. Quality assurance and quality control are the same as previously reported (Chen
et al., 2019), and are summarized in the SI.

3. Results and discussion

3.1. Liquid and solid concentration profiles during the 1st and 2nd S-D cycles

Fig. 3 depicts the equilibrium concentrations, c, and the observed solids concentrations, qobs , of PFOS as a function of
the desorption step during the 1st and 2nd S–D experiment (Fig. 3A). SI-1 and SI-2 depict the same information of PFNA
and PFDA, respectively. The equilibrium concentrations increased after each spiking and decreased exponentially during
the subsequent batch-wise desorption. c decreased to the detection limit while qobs approached qo , (indicated by dashed
line). Fig. 4 depicts the absorption and desorption behavior as isotherms, i.e., as plots qobs vs. c. qirrev . The y-axis intercepts,
D1+2
i.e., qD1
0 and q0 after the 1st and 2nd S–D, respectively, were obtained using OriginPro b9.3.226 using ‘‘instrumental’’
weighting. Error bars are ± standard deviation for both aqueous and solid phase concentrations. The one-point isotherms
originate at the qD10 of the 1st S–D. The desorption resistant solid concentrations increased in all cases, as indicated by the
dashed lines in Fig. 3, Fig. SI-2 and 3.

3.2. Effect of S-D cycling on the reversible and irreversible absorption

Fig. SI-3 depicts the observed four-point isotherms of PFOS, PFNA and PFDA during the 1st S–D cycle fitted to linear
equations. The concentrations of PFOS, PFNA and PFDA in the native sediment were insignificant (1.32, 0.73, 2.45 µg/kg,
respectively) relative to the spiking concentrations (Fig. 3). Table 1 summarizes the slopes of the absorption points
evaluated by linear regression (Slope 1st AI) (Fig. SI-3). The KDobs values were obtained by averaging the q/c ratios of the
individual absorption points and thus represent conditions after the 1st and 2nd absorption steps. Both methods result
in consistent values, as expected for linear isotherms. The slopes increase in the order PFNA < PFOS < PFDA as observed
previously (Chen et al., 2016).
The slopes of four one-point isotherm were evaluated considering qD1 0 as the origin and the absorption points as
the endpoints. The averages of the one-point isotherm slopes are indicated in Table 1. Slopes of the lowest spiking
concentration were 20 to 30% higher than those the three higher concentrations (data not shown). The averaged slopes
of the one-point isotherms obtained during the 2nd S–D were lower than those determined during 1st S–D (Table 1)
indicating weakening sorption.
D1+2
Fig. SI-4 shows the irreversibly absorbed components, qD1 0 and q0 values of PFOS, PFNA and PFDA as a function of
D1+2
absorption equilibrium concentrations (ca ) determined after spiking. qD1 0 and q0 increase with increasing absorption
D1
concentrations and from 1st S–D to 2nd S–D. In the 1st S–D, q0 values were fitted to linear equations. At spiking
concentrations of 100 µg/L, data points of PFOS and PFDA were below the linear fitting curve probably due to saturation
and were therefore excluded. In all cases, the slopes of the irreversible isotherm decreased from the 1st to the 2nd S–D,
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H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

Fig. 4. Absorption equilibrium points after spiking and stepwise desorption resulting in CDIs of PFOS (a, b), PFNA (c, d), and PFDA (e, f) in the
1st (a, c, and e) and 2nd (b, d, and f) absorption–desorption cycles, respectively. The full black squares indicate the absorption-points CDIs after
spiking 5, 20, 50, and 100 µg/L and the origins of the CDIs. Hollow points with different colors indicate desorption-points of the different spiking
concentrations.. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

again suggesting that the irreversibly sorbing domain is showed signs of saturation during the second exposure, especially
at high concentrations.
Fig. 5A, B, and C depict qrev of PFOS, PFNA, and PFDA during the 1st and 2nd S–D experiment isotherm as a function
of the absorption and desorption equilibrium concentration (ca and cd , respectively). The partition coefficients, π , were
v
obtained by correlating qre
n vs. cd(n) (Eq. (4)). The correlations were linear in all cases (R2 = 0.996/0.992, 0.996/0.982,
0.989/0.982 for first/second S–D, respectively) indicate that π remains constant during the CDI experiments. Table 1
summarizes the resulting π values of the 1st and 2nd cycle. The π values decreased by 18, 23, and 25% for PFOS, PFNA
and PFDA, respectively. The p values which were derived from only the origin and the endpoints, decrease in all cases
except for PFNA, where p remained nearly constant. As observed in Chen et al. (2016), PFNA was absorbed to the sediment
relatively weakly and reversibly compared to PFOS and PFDA. Therefore, the hysteretic absorption of PFNA in the 1st S–D
may be less influential in the 2nd S–D. Taken together, the data are consistent with a lower overall sorption capacity
during the 2nd S–D, probably due to strongly sorbing site were occupied by the irreversibly absorbed fraction during the
1st S–D and were no longer available for sorption during the 2nd absorption experiments.
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H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

Table 1
Comparison of the absorption isotherms of the 1st and 2nd S–D cycle (L/kg). π : partition coefficient;
p: distribution coefficient of irreversibly sorbing domain; Slope 1st AI: slope of four-point isotherms
(observed distribution coefficients) of the native sediment; KDobs average of q/c ratio for the absorption
obs
points in the 1st and 2nd S–D experiments; KOC : carbon referenced distribution coefficient; Slope 2nd
AI: averages of the slopes of the four one-point isotherms.
1st S–D experiment (L/kg)
Sorbate π (1) p(2) KDobs (3) Slope 1st AI(4) obs (5)
KOC
PFOS 37.4 27.4 ± 15.6 62.8 ± 18 48.6 910
PFNA 14.1 4.7 ± 1.1 18.3 ± 1.1 18.2 265
PFDA 64.4 72.7 ± 23.1 126.2 ± 14 124 1829

2nd S–D experiment

π p KDobs (3) Slope 2nd AI(6) obs (5)
KOC
PFOS 30.6 18.2 ± 3.6 67.3 ± 16.9 45.5 ± 6.4 975
PFNA 10.9 5.0 ± 2.2 20.7 ± 3.1 16.4 ± 2.2 299
PFDA 48.5 41.3 ± 21.5 121.3 ± 31.2 78.1 ± 13.3 1758
(1)
π : partition coefficients of the soft domain obtained by linear fitting of the reversible absorbed fraction
vs. the liquid concentration in Fig. 5.
p: averaged qD10 /ca ratio q0 /ca ratio for 1st S–D and 2nd S–D experiment, respectively.
(2) D2

(3)
Calculated by averaging the q/c ratio if the absorption points (Fig. 4).
(4)
Slopes of 1st absorption isotherms evaluated by linear (Fig. SI-3).
(5)
KOC = KD /foc where KDobs ; foc = 0.069 (Table SI-1).
(6)
Average slopes of one-point isotherms that resulted from re-spiking at 5, 20, 50, and 100 µg/L in the
second S–D.

Fig. 5. Isotherms of the reversibly absorbed PFOS (A), PFNA (B), and PFDA (C), respectively, constructed from data shown in Fig. 4. Ab-1st and
Ab-2nd: calculated from absorption equilibrium points in the 1st (4 points) and 2nd S–D (one point) experiment, respectively. De-1st and De-2nd
are calculated from desorption equilibrium points in the 1st and 2nd S–D cycle, respectively. qrev = qobs − q0 (based on the geometry shown in Fig. 1).
ca and cd are measured aqueous concentrations at absorption and desorption, respectively. Dashed lines are linear fits to the 1st S–D isotherms.
Solid lines are linearly fitted to the data of the 2nd S–D CDI. Slopes and R2 are indicated in the panels.

3.3. The distribution between the reversibly and irreversibly sorbed fractions

Fig. SI-5 (A, B, C) compares the distribution between qrev and qirrev after the 1st and 2nd absorption experiment. At
the low spiking concentrations (5 and 20 µg/L), the sizes of the bars are approximately equal, indicating that the low
concentrations of the 1st S–D cycle affected the reversible and irreversible sorption behavior relatively little with changes
ranging from 2% to 32%. At spiking concentrations of 100 µg/L, the irreversible uptake increased after the 1st S–D cycle
by 58%, 66% and 13% for PFOS, PFNA and PFDA, respectively. Perhaps, plasticization caused by exposure to the relatively
high concentrations increased sorption in the irreversibly sorbing domain thus to some extent partially compensated for
site saturation. By contrast, the reversible uptake decreased, as indicated in Table 1. Spiking at 50 µg/L resulted in mixed
behavior.

3.4. Comparison of laboratory and field distribution coefficients

Fig. 6 compares laboratory and field log KOC data: it shows that the log K OC values of sediment that underwent
simulated S–D cycling in laboratory experiments overlap with the data ranges observed in the field. A possible explanation
for the relatively high log KOC values observed in the field samples is that contaminated particles entered the surface
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H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

Fig. 6. Organic carbon normalized distribution coefficients of PFOS (A), PFNA (B) and PFDA (C) (log Koc) in relation to the ranges of log Koc observed
in the water body (Chen et al., 2019). Solid and hollow points are Log Koc values calculated from absorption and desorption equilibrium, respectively.
n is the absorption/desorption step. C0 is the initial PFAAs concentrations used in the absorption experiments.

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H. Chen, M. Reinhard, L. You et al. Environmental Technology & Innovation 28 (2022) 102673

water body and released reversibly absorbed contamination into the clean runoff. KOC data obtained by simple one-step
absorption experiments tend to significantly underestimate KD and therefore KOC measurements. One of the possible
reasons for the relatively high log KOC observed in field samples is likely the result of partial removal of labile sorbates,
as suggested previously (Chen et al., 2016). This study demonstrated that S–D cycling could increase this discrepancy.

4. Summary and conclusions

A study was conducted to explore the effect of sorption–desorption cycling on the sorption properties of sediment.
S–D cycling could occur during sediment transport in surface water systems where it is subject to repeated deposition
and remobilization. The investigative approach involved evaluating the hysteretic sorption behavior of three PFAAs, PFNA,
PFOS, and PFDA to native sediment during two consecutive S–D events. As a first approximation, the data was evaluated
using the DMM, which assumes that PFAAs sorb reversibly and irreversibly by soft and glassy SOC domains, respectively.
Observations made within the concentration range explored (5 to approximately 100 µg/L) agreed with this assumption.
Analysis of the irreversible sorption behavior indicated saturation of the glassy domain, especially at the highest spiking
concentrations. Saturation partially compensated by plasticization that appeared to have shifted absorption toward the
irreversible compartment. It is evident that the hysteretic sorption exist in the field since the high log KOC values that
resulted from S–D cycling were within the range of KOC that of those observed in an related water body (Chen et al., 2019)
and aged sediment (e.g. Zareitalabad et al., 2013). This study highlight the effect of hysteretic sorption on the fate of PFAAs
in the aquatic environment and will improve the understanding of bioavailability of absorbed PFAAs in the sediment.

CRediT authorship contribution statement

Huiting Chen: Conceptualization, Methodology, Writing – original draft, Writing – review & editing, Visualization,
Formal analysis, Investigation. Martin Reinhard: Writing – review & editing, Visualization, Supervision. Luhua You:
Methodology, Resources. Karina Yew-Hoong Gin: Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.

Acknowledgments

This research was funded by the Singapore National Research Foundation (NRF) and a grant from the Ministry of
Education through Grant R-302-000-088-750. Publication was supported under the Campus for Research Excellence and
Technological Enterprise (CREATE) program, administered by the NUS Environmental Research Institute (NERI). We are
grateful to PUB, National water agency of Singapore for providing support in this study.

Appendix A. Supplementary data

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.eti.2022.102673.

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 Chemosphere 310 (2023) 136867

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Sorption and desorption of bisphenols on commercial plastics and the effect

of UV aging
Xiaoxin Chen a, Chang-Er Chen b, Xiaoyuan Guo c, Andrew J. Sweetman a, *
a
Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, United Kingdom
b
Environmental Research Institute, School of Environment, Guangdong Provincial Key Laboratory of Chemical Pollution and Environmental Safety and MOE Key
Laboratory of Theoretical Chemistry of Environment, South China Normal University, Guangzhou, 510006, PR China
c
Department of Civil and Environmental Engineering, Faculty of Science and Technology, University of Macau, Macau SAR, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Compared with PA, PVC and EPS, PU

showed a higher sorption capacity for
four types of bisphenols before and after
aging.
• Irradiation has minimal impact for the
sorption behaviour of PU but showed
the highest impact for EPS.
• Desorption efficiency decreased for PU,
PA, PVC and EPS after aging.
• Salinity, DOM and temperature influ­
enced the sorption and desorption
behaviour of plastics in aquatic systems.

A R T I C L E I N F O A B S T R A C T

Handling editor: Keith Maruya Plastics gradually degrade in the natural environment from the effect of irradiation, which can change the
surface properties of plastics and affect the migration behaviour of pollutants. Up to now, studies on the sorp­
Keywords: tion/desorption behaviour of organic pollutants on aged plastics are still limited. In this study, several types of
Plastics commercial plastics (polyurethane (PU), polyamide (PA), polyvinyl chloride (PVC), expanded polystyrene (EPS))
Aging mechanism
were selected to investigate the sorption and release behaviour for four kinds of bisphenols (bisphenol-F, A, B,
Adsorption
AP). The results from Raman spectroscopy and scanning electron microscopy (SEM) analysis showed evidence of
Desorption
Bisphenols oxidization and surface cracks of plastics after irradiation. The sorption behaviour for both fresh and aged
plastics were dominated by hydrophobicity. In addition, the electrostatic force, H-bonding interaction, and π-π
interaction were also the important factors impacting the sorption process. The desorption kinetics behaviour
indicates that desorption becomes faster after aging. Hydrophobicity is also an important factor that affects
desorption behaviour. This study showed that sorption capacity for most fresh and aged plastics was enhanced by
the impact of salinity and dissolved organic matter (DOM). Increased temperature could increase the desorption
of bisphenols on both fresh and aged plastics, which illustrated that warm environments would promote more
pollutants be released from plastics to water bodies.

* Corresponding author.
E-mail address: a.sweetman@lancaster.ac.uk (A.J. Sweetman).

https://doi.org/10.1016/j.chemosphere.2022.136867
Received 5 August 2022; Received in revised form 3 October 2022; Accepted 10 October 2022
Available online 13 October 2022
0045-6535/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
X. Chen et al.
1. Introduction
Commercial plastics are intensively used in daily life owing to their
versatile, flexible, light and durable properties(Hahladakis et al., 2015;
Singla et al., 2020). Globally, over 320 million tons of plastics are pro­
duced each year(Wright and Kelly, 2017). Whilst only a small amount of
plastics are recycled, the majority of plastics are sent to landfills,
incineration plants or even discarded into the natural environment
(Liang et al., 2021). Due to the lack of an effective recovery and man­
agement system, plastic debris is easily transferred to marine, lake, soil
or sediment systems. It has been reported that approximately 5.25 tril­
lion pieces of plastic are present in marine environments, which ac­
counts for 60–80% of marine litter, even reaching 90–95% in some
regions (Singla et al., 2020). These plastic materials can not only cause
physical effects for biota (e.g. entanglement of marine species) (Li et al.,
2018a), but also result in biotoxicity(Wang et al., 2020; Hamlin et al.,
2015). Various chemical additives (e.g. plasticizers, antioxidants, fillers)
are added to polymers to enhance their properties, and as those addi­
tives are not tightly bound to plastics they are easily released to the
environment. Many additives (e.g. phthalates, brominated flame re­
tardants, UV-stabilizers) are endocrine-disrupting chemicals (EDCs)
which have a negative effect on normal hormone function, potentially
affecting growth, causing obesity and even breast cancer (Suhrhoff and
Scholz-Böttcher, 2015). Furthermore, owing to the hydrophobic nature
of many plastics, additives and other organic chemicals released into the
environment could be adsorbed and desorbed by plastics. As a result,
plastics need to be considered a potentially important carrier of con­
taminants to and within the environment.
When plastics enter the environment, they can gradually break into
micro-size (1 μm–5 mm) or even nano-size (<1 μm) particles as a result
of weathering, with photoaging being a vital factor for the degradation
of plastics(Luo et al., 2022). With irradiation, the hydrophobicity of
plastics might be changed, forming oxidation functional groups. In
addition, the surface of plastics might crack, which could affect their
interaction with chemicals in the natural environment. For example, a
study found that the adsorption behaviour of 9-Nitroanthrene (9-Nant)
on polystyrene (PS) and polyethene (PE) microplastics decreased after
artificial aging by UV lamps(Zhang et al., 2020a). This may be related to
the supply of oxygen-containing functional groups, which increased the
surface polarity of plastics and reduced the affinity of hydrophobic
organic pollutants on the surface of plastic particles. However, up to
now, most research concerning aged plastics has focussed on pure
microplastics rather than commercial plastics commonly used in daily
life. It is possible that commercial plastics might show different envi­
ronmental behaviour compared with the pure laboratory microplastics.
Bisphenol A (BPA) is one of the typical EDCs that has commonly been
used as a plasticizer, stabilizer, and antioxidant in plastics (Hahladakis
et al., 2015). It is also the monomer used for polycarbonate (PC) skel­
etons (Sun et al., 2021). As a result, BPA is a widely used chemical and
frequently detected in water bodies(Wu and Seebacher, 2020). Owing to
its toxicity to biota(Wu and Seebacher, 2020; Naveira et al., 2021), BPA
use has been limited as an additive in some regions. As a result, some
bisphenol analogues (e.g. BPF, BPB, BPAP) have been used in plastics as
a substitute for BPA. These bisphenol analogues have been detected in
plastics fibres (Sait et al., 2021), rivers(Wang et al., 2022) and fish
(Barboza et al., 2020). Unfortunately, those analogues can also cause
negative effects in biota and generate the similar toxicity to BPA(Chen
et al., 2016). Nevertheless, studies on the interaction behaviour between
plastics and bisphenol analogues is limited. As a result, the fate,
adsorption/desorption behaviour of bisphenols in the presence of aged
plastics is still unclear.
Therefore, in this study, selected commercial plastic particles
—polyurethane (PU), polyamide (PA), expanded polystyrene (EPS) and
polyvinyl chloride (PVC), have been artificial aged in a Suntest CPS +
chamber for 48 h and 168 h to investigate the sorption/desorption
behaviour of bisphenols (BPF, BPA, BPB, BPAP) on fresh and aged
2
Chemosphere 310 (2023) 136867
plastics. The objectives of this study were to (i) explore the interaction
mechanism of bisphenols on fresh and aged commercial plastics to
clarify the potential relationship between the ageing of plastics and the
uptake/release behaviour of bisphenols; (ii) probe the sorption/
desorption behaviour for different types of plastics to assess the poten­
tial risk for exposure from plastic fragments to the environment; (iii)
examine the influence of environmental conditions(temperature, DOM,
artificial seawater) on the sorption and desorption behaviours of
bisphenols by plastics.
2. Material and methods
2.1. Materials and chemicals
Standards of BPF, BPA, BPB and BPAP (purity >98.0%) were pur­
chased from Sigma-Aldrich Co. LLC (UK), details of the four bisphenols
are shown in Table S1. Acetonitrile (HPLC grade) and Methanol (HPLC
grade) were purchased from Fisher Scientific Ltd (UK). Stock bisphenol
solutions (100 mg/L) were prepared in methanol and kept at 4 ◦ C. The
Humic acid (HA) was purchased from Sigma-Aldrich Co. LLC (UK). The
artificial seawater was obtained from Brightwell Aquatics (USA), the
average concentration of Mg, Ca, K, and Sr are 1290 mg/L, 413 mg/L,
399 mg/L, and 8 mg/L, respectively.
Seven different kinds of commercial plastics (Table S2) related to
food and general use (daily necessities) were collected in a local market
(Guangzhou, China) or “Taobao.com”. The preliminary experiment
(Fig. S1) demonstrated that PU, PA, PVC and EPS had good sorption
ability for bisphenols. This allowed further study of the interaction
behaviour between bisphenols and plastics to be observed along with
the aging process for four selected plastics. The structures of the four
plastics are given in Fig. S2. All plastics were cut into small pieces
approximately 5 × 5 mm.
2.2. Artificial aging and characterization of plastics
A Suntest CPS + xenon chamber (Atlas Material Testing Technology
LLC, USA) was configured at 500 W m− 2, 35 ◦ C for the degradation of
plastic pieces. The wavelength of radiation ranged from 300 nm to 800
nm (including the range of UV-A and part UV-B radiation), and was
performed for 48 h and 168 h respectively, which are similar as exposure
146 and 511 days under the European mean irradiance(Gewert et al.,
2018), the equation is shown in the SI. Raman and SEM were used to
characterize the aged plastics.
2.3. Adsorption experiments
Bisphenols stock solutions (100 mg/L) were diluted to 1 mg/L with
ultrapure water with the volume ratio of methanol kept below 0.01% to
reduce solvent effects. Before the kinetic experiments, three control
experiments were performed: (1) 60 mg original plastic pieces were
added to 15 mL ultrapure water to determine if plastic materials con­
tained and leached bisphenol additives. (2) 15 mL bisphenol solutions
were placed into polypropylene (PP) centrifuge tubes to confirm if the
centrifuge tubes would adsorb bisphenols. (3) 15 mL ultrapure water,
artificial seawater and humic acid water were placed into PP centrifuge
tubes to ensure that the tubes didn’t leach bisphenols. Results showed
that no bisphenols leached from both plastic materials and tubes, the
loss of bisphenols in centrifuge tubes was less than 5%.
For the adsorption kinetic experiments, 60 mg of plastics and 15 mL
1 mg/L bisphenols were added into PP centrifuge tubes. All centrifuge
tubes were placed on the shaker at 180 rpm (25 ± 1 ◦ C). The superna­
tants were collected after 4 h, 12 h, 24 h, 48 h, 72 h, 108 h, 156 h, 204 h,
256 h, 308 h and 360 h, and filtered through a 0.2 μm PTFE membrane
(Fig. S3). In addition, 1 mg/L single solution of each bisphenol were
prepared to investigate the competitive absorption behaviour on plas­
tics. Environmental conditions are important factors in the interaction
X. Chen et al.
between contaminants and plastics. As a result, the sorption experiments
of fresh and aged 168 h plastics were carried out in three different
matrices—ultrapure water, artificial seawater and 15 mg/L humic acid
water to simulate the sorption behaviour of plastics under the impact of
freshwater, seawater and the presence of DOM. Moreover, the impacts of
different temperatures including 12 ◦ C, 25 ◦ C and 35 ◦ C were investi­
gated. Three replicates were conducted for each treatment. The initial
pH values of ultrapure water, artificial seawater and HA water were 6.8
± 0.1, 8.2 ± 0.1 and 6.5 ± 0.1.
2.4. Desorption experiments
After sorption equilibrium was achieved, desorption experiments
were further carried out to investigate the release kinetics of bisphenols
from plastics. Plastics from the sorption experiment were transferred
into centrifuge tubes containing 15 mL ultrapure water, artificial
seawater or 15 mg/L HA water, and shaken at 180 rpm. The desorption
experiments were carried out at 12 ◦ C, 25 ◦ C, 35 ◦ C, respectively. Three
replicates were implemented for each treatment. The supernatants were
collected from 0 h to 156 h.
2.5. Chemical analysis
All samples were analysed using a Shimadzu NexeraX2 UHPLC with
fluorescence detector (Shimadzu, Japan) and a Phenomenex C18 col­
umn (4 μm, 150 × 4.6 mm). Chromatographic separation was performed
with a mobile phase of A: ultrapure water and B: acetonitrile (ACN). The
gradient began at 40% ACN, then increased to 60% ACN within 9 min,
followed by reaching 90% ACN at 12 min and decreased to 40% ACN at
15 min, finally, 5 min of post-ran ensured equilibrium of the column
before the next injection. Samples were prepared in ultrapure water and
methanol (4:1, v/v). Other parameters are available in Table S3.
2.6. Statistical analysis
T-test and ANOVA were used to analyse significant differences and
statistical significance was set as P < 0.05. All statistical analysis was
performed using SPSS 22.0 software and Microsoft Office Excel 2020.
Figures were plotted with Origin 2020.
The adsorption efficiency of bisphenols was calculated according to
Eq. (1):
( )
C0 − Ce
%A= × 100 (1)
C0
where A represents the adsorption efficiency of each bisphenol; C0 and
Ce (μg/L) are the initial concentration and equilibrium concentration of
bisphenols in the solution respectively.
The capacities of 4 types of bisphenols adsorbed to plastics was
calculated to Eq. (2) as below.
C0 − Ce
qe = V (2)
m
where qe (μg/g) refers to the equilibrium adsorption capacity of plastics
for each bisphenol; m (mg) is the mass of plastics and V (mL) is the
volume of the solution used in adsorption experiments.
The adsorption kinetics can be fitted using a pseudo-first order model
and a pseudo-second order model. The linear form of equations are as
follows:
2.6.1. Pseudo-first order model
log (qe − qt ) = log qe − k1 t/2.303 (3)
3
Chemosphere 310 (2023) 136867
2.6.2. Pseudo-second order model
t 1 t
= + (4)
qt k2 q2e qe
where qt (μg/g) is the adsorption amount of bisphenols per gram of
plastics at a given time t (h); k1 and k2 represent the rate constant for
pseudo-first order and pseudo-second order kinetics respectively. The
pseudo-first order model represents a physical process, where the
intermolecular force (van der Waals’ force) is the main interaction be­
tween particles, and the pseudo-second-order model relies on chemical
processes, such as the formation of chemical bonds, electron transfer,
etc. (Wu et al., 2019).
The desorption efficiency of each bisphenol was calculated using:
(
Cdesorb
)
%D = × 100 (5)
Cadsorb
where Cdesorb (μg/L) is the desorption concentration of bisphenols from
plastics and Cadsorb (μg/L) is the adsorbed equilibrium concentration of
bisphenols on plastics at the beginning of the desorption experiment.
3. Results and discussion
3.1. Surface changes and characterization of aged plastics
The Raman spectra (Fig. 1a) showed that there were significant
changes for aged PA and PVC plastics compared with the virgin plastics.
The reduced intensities of stretching (2900 cm− 1), twisting (1301
cm− 1), wagging (1374 cm− 1) and bending (1442 cm− 1) of methylene
(-CH2) suggests the oxidation of PA plastics which would convert C–H
bonding to carbonyl bonding (C– – O) (Uematsu et al., 2021; Zou et al.,
2020). The reduced peak for amide I (C– – O) and amide III (N–H, C–N)
represents amide bending (-C– – O–NH-) breaks during the aging process.
For PVC plastics, there is an obvious reduction for the stretching vi­
bration of carbon-chlorine (C–Cl) (690 cm− 1) and –CH2 (2780-2980
cm− 1) (Kappler et al., 2016), which would be due to oxidation. As for
EPS plastics, Raman spectra showed a slight reduction in benzene ring
intensity. For PU plastics the intense band between 1570 cm− 1 and 1660
cm− 1 with a maximum at 1610 cm− 1 can be ascribed to the –C– – C-
stretching vibrations of aromatic rings. A broadening with increased
aged time could be observed, which illustrates chemical degradation of
aromatic rings (Bruckmoser and Resch, 2014). Changes for PU plastics
were also visually observed as they became yellow with the light irra­
diation (fig. S4). This might be related to oxidation reactions and the
creation of quinone-imide structures (chromophore) (Rosu et al., 2009).
In addition, Photo-Fries rearrangement is another mechanism for the
formation of chromophores (reaction scheme showed in SI).
Fig. 1b illustrates the surface changes after irradiation. The surfaces
of fresh plastics are smooth, whilst in contrast, the surfaces became
rough after aging. For aged PU and PA plastics, surfaces showed more
pits compared with fresh plastics, while the surface change of aged PVC
and EPS were mainly due to the appearance of cracks. The surface
changes observed for plastics after UV-aging was similar to the aged
plastics from previous studies (Fan et al., 2021b; Mao et al., 2020). With
surface erosion, there are more voids appearing compared to the fresh
plastics.
3.2. Sorption and desorption behaviour of bisphenols on plastics
3.2.1. Sorption ability of commercial plastics
Data from a preliminary experiment (Fig. S1), where several types of
commercial plastics were assessed, showed that only a few showed
obvious sorption for bisphenols. This result was different from a previ­
ous study on the sorption behaviour of BPA on a range of plastic particles
(Liu et al., 2019c). Commercial plastics are composed of resins and a
wide range of additives. It is possible that the additives might change the
X. Chen et al.
1
Fig. 1. (a) Raman spectra of plastics. The spectral range of 300–3200 cm−
168 h plastics.
structure or properties of polymers which would lead to different sorp­
tion behaviour compared with pure microplastics. Moreover, the parti­
cle size might also be an important factor affecting the sorption
behaviour. The size of plastic pieces used in this study are much larger
than those used in the previous study (nano-level particles), which
owing to the decrease specific surface area and less sorption sites,
Chemosphere 310 (2023) 136867
(785 nm laser, 15 mW, ×50 objective, 10 s collection). (b) SEM images for fresh and aged
leading to lower sorption capacity (Liu et al., 2019c).
It is noticeable that although two types of PS plastics were firstly
selected for the preliminary study, only the expanded polystyrene (EPS)
showed sorption affinity for bisphenols. It was also found that the weight
of EPS increased 3.6 times (increased from 60 mg to 216 mg) after being
soaked in water for 5 min. This effect was not observed for the other
4
X. Chen et al.
plastics which showed no weight changes. The strong water retention
capacity by EPS was also observed in a previous study on the sorption of
a lipophilic toxin (Costa et al., 2020). The high water retention ability of
plastics could increase their external surface contact area with con­
taminants, which would improve the sorption process and result in
potential sorption for bisphenols.
3.2.2. Sorption kinetics of bisphenols on fresh and aged plastics
The sorption kinetics for BPA, BPF, BPB and BPAP in fresh and aged
plastics are given in Fig. 2. Fresh EPS plastics didn’t adsorb BPF and BPA
in the multi-solute system, which was likely to be related to the
competitive adsorption behaviour. This demonstrated that sorption is
improved in a single solution system rather than a multi-system for EPS
plastics (Fig. S5). Previous studies have suggested that the uptake of
Fig. 2. Sorption kinetics of BPF, BPA, BPB and BPAP on fresh and aged plastics. Error bars are the means ± standard deviation (SD) of three repeats. (60 mg plastics,
15 mL 1 mg/L bisphenols, 25 ◦ C, 180 rpm).
5
Chemosphere 310 (2023) 136867
hydrophobic organic compounds on plastics are expected to be
competitive (Bakir et al., 2012). Chemicals with similar structures show
strong competition, which illustrates the adsorption for compounds are
selective (Pignatello, 1998).
The sorption data has been fitted using both the pseudo-first model
and pseudo-second models (Fig. S6 and Fig. S7), the fitting parameters
are shown in Table S4. The kinetics for fresh and aged plastics were
better described by the pseudo-second-order model (R2 range from
0.979 to 0.999) except for fresh PVC plastics, suggesting that chemical
adsorption was likely to be the dominant factor for the adsorption
process of bisphenols onto plastics. Whereas, fresh PVC plastics could be
better described by the pseudo-first-order model, similar to that in a
previous study (Liu et al., 2019a), which indicates the sorption behav­
iour for fresh PVC plastics was a single sorption process and the
X. Chen et al.
adsorption capacity limited by the sorption sites (Zhang et al., 2018).
The k2 (μg/g.h) value represents the adsorption rate constant which
decreased after the aging process. This suggests that the sorption rate for
bisphenols reduced during the degradation of plastics. It might be due to
the erosion of plastics allowing more bisphenols to diffuse into the in­
ternal plastic structure (Sun et al., 2021), which means that a long time
is needed for bisphenols to reach the binding sites compared with sur­
face sorption.
3.2.3. Sorption mechanism
According to Fig. 3, the equilibrium adsorption efficiency of the four
bisphenols on PVC plastics (except aging for 48 h), EPS plastics (except
fresh) and PA plastics are positively correlated (R2 > 0.9, P < 0.05) with
their octanol/water partition coefficients (logKow), which illustrates that
hydrophobicity was an important factor dominating the sorption
behaviour for most fresh and aged plastics.
For fresh PU plastics (Fig. 3), good correlations (R2 = 0.999, P <
0.05) were evident if the data for BPF were excluded. The logKow of BPA
(3.32) is only slightly higher than BPF (2.91), but the adsorption rate of
BPA (96.29%) on fresh PU plastics was much higher than BPF (88.59%),
which suggested that except for hydrophobicity, there were other factors
that affect the uptake behaviour of BPF onto PU plastics. Surface elec­
trostatic interactions might also be an important factor affecting sorp­
tion ability for bisphenols. As illustrated in Fig. 4a, BPF was present in
both non-ionized form and anionic form at the pH of ultrapure water
(6.8). Whereas, BPA, BPB and BPAP were mostly present in the non-
ionized form (Fig. 4b) as their pKa values (range from 9.50 to 10.27)
are much higher than BPF (7.55). Moreover, plastics are negatively
charged in most water bodies, which indicates that an electrostatic
repulsive-force existed between PU plastic particles and BPF, leading to
the reduced sorption efficiency. Therefore, both hydrophobicity and
electrostatic interaction are important factors affect the sorption
behaviour of bisphenols on fresh PU plastics.
3.2.4. Sorption capacity with the aging of plastics
According to Fig. 5a, the sorption capacity for bisphenols on aged
PVC and EPS plastics are significantly higher (P < 0.05) than fresh
Fig. 3. The correlation between logKow and adsorption efficiency values for four bisphenols partitioning on fresh and aged plastics.
6
Chemosphere 310 (2023) 136867
plastics, which might be due to the cracks in the plastics offering more
available sorption sites. Moreover, according to the Raman data, the
reduction of -Cl for PVC plastics could increase the free volume, which
would further increase the sorption sites for bisphenols on PVC plastics
after irradiation. For EPS plastics the abundance of aromatic rings might
have reduced with the degree of aging, which could reduce the π-π
interaction with bisphenols (Liu et al., 2020). As a result, the sorption
amount decreased with the increased aging degree of EPS plastics. For
PA plastics, the sorption for BPF and BPA reduced after irradiation,
which could be due to the effect of hydrogen bonding interaction.
Several studies have demonstrated that the sorption behaviour of
polyamide plastics strongly relied on hydrogen boding interaction be­
tween the amide group (–CO–NH-) and study chemicals (e.g. sulfa­
methoxazole, 17β-oestradiol, amoxicillin, tetracycline, etc.) (Li et al.,
2018b; Guo et al., 2019; Liu et al., 2019b). As shown in Fig. 1a, the
reduction of amid III (–NH–, –CN–) would weaken the H-bond interac­
tion and further effect the sorption ability for bisphenols (Fig. S8).
Whilst for PU plastics, the sorption ability for most bisphenols did not
change with the aging process, which illustrates that the erosion and
oxidation did not dominate their sorption behaviour.
The sorption capacity for PU plastics was the highest among the four
plastic types. The amorphous regions in polymers are the dominant area
for sorption of HOCs (Guo et al., 2012). The glass transition temperature
(Tg) is the temperature above which segmental mobility of the polymeric
chain begins(Pascall et al., 2005). When temperatures are higher than
Tg, the amorphous regions of plastics are in a rubbery state and in
polymers with high free volume, chain segments can move freely and be
present in a flexible state(Liu et al., 2019b). For temperatures below Tg,
plastics are glassy, and polymers with little void space present a highly
dense structure, which reduce segmental mobility with the chains being
“frozen”(Liu et al., 2019b; Wang et al., 2018). Previous studies have
illustrated that the structure of glassy polymers provides lower sorption
levels of chemicals than in rubbery polymers (Wang et al., 2018; Pascall
et al., 2005). Compared with PU (Tg is around − 17 ◦ C) (Zhang et al.,
2020b), the Tg of PA, PVC and EPS are around 102 ◦ C, 85 ◦ C and 105 ◦ C
(Mueller et al., 2018; Zhou et al., 2020, Samper et al., 2010). Due to the
PU plastics being rubbery they are expected to have a higher free volume
X. Chen et al. Chemosphere 310 (2023) 136867

Fig. 4. (a) Speciation of BPF at different pH values. (b) Speciation of BPA, BPB and BPAP at different pH values.

Fig. 5. (a) The sorption capacity of four bisphenols on PU, PA, PVC and EPS plastics before and after UV- aging. (b) The desorption capacity of four bisphenols on PU,
PA, PVC and EPS plastics before and after UV- aging. Error bars are the means ± standard deviation (SD) of three repeats. (60 mg plastics, 15 mL 1 mg/L bisphenols,
25 ◦ C, 180 rpm). Values not sharing common letters (a–c) are significantly different from each other (p < 0.05).

than PA, PVC and EPS. This is responsible for the excellent adsorption of
the bisphenols compared with the uptake by other polymers.
3.2.5. Desorption of bisphenols on plastics with aging
The time needed to reach desorption equilibrium became shorter for
most plastics after irradiation (Fig. S9). For example, BPF, BPA need
approximately 96 h and BPB, BPAP need 120 h to reach equilibrium on
fresh PVC plastics, which reduced to around 72 h (BPF, BPA, BPAP) and
48 h (BPB) after aging 168 h (one-way ANOVA analysis, P > 0.05).
According to the data in Table 1, the desorption efficiency for both fresh
and aged plastics followed the order: BPF > BPA > BPB > BPAP, which
is negatively correlated with the hydrophobicity.
For most plastics, the desorption efficiency decreased after the aging
process (Table 1). Due to cracks in the plastics, more bisphenols could
diffuse to the inside of the structure (Sun et al., 2021) or the narrow
pores (Fan et al., 2021a) of the plastics, which suggests it is more
difficult for aged plastics to release bisphenols compared with those
bisphenols sorbed on the surface of fresh plastics. As a result, the lower
desorption efficiency was expected after aging. Although the desorption
efficiency of BPF and BPA on PVC plastics decreased sharply after
irradiation, the desorption capacity showed an increasing trend owing to
the increased uptake ability for PVC plastics after aging (Fig. 5b). For
example, the desorption efficiency of BPA on PVC plastics decreased
from 50.5% to 21.8% after aging 48 h, whereas the desorption capacity
increased from 7.8 μg/g to 9.2 μg/g. As a result, the aging of plastics
might suggest that greater pollutant loadings could be released to the
environment.
3.3. Effects of environmental factors
3.3.1. Water matrix
The pH value (8.2) of artificial seawater suggests that BPF was
mostly present in the anionic form, and BPA, BPB, BPAP also showed the
anion potential, so the repulsive-force between bisphenols and plastics

Table 1
Desorption efficiency (%) of bisphenols on fresh and aged plastics.
PU PA PVC EPS

BPF BPA BPB BPAP BPF BPA BPB BPAP BPF BPA BPB BPAP BPF BPA BPB BPAP

Fresh 8.1 3.2 2.0 0.7 21.2 18.4 13.1 5.6 77.4 50.5 35.6 19.8 – – 67.8 32.7
Aging 48 h 7.1 5.0 4.2 3.9 20.2 18.5 11.1 3.2 23.0 21.8 32.1 16.5 9.7 8.2 5.7 4.3
Aging 168 h 7.0 2.8 1.6 0.5 17.2 16.5 10.9 3.8 35.2 20.6 19.3 16.7 24.3 20.9 7.7 12.0

7
X. Chen et al.
would be increased compared with ultrapure water (Fig. 4), which
should lead to the decreased sorption capacity of bisphenols on plastics.
However, the sorption capacity of BPF and other bisphenols increased
for most fresh and aged plastics in seawater (Fig. 6a), which might be
due to the salting-out effect. The salting-out effect would reduce the
solubility of bisphenols in seawater (non-polar organic compounds),
which would enhance the hydrophobic interactions between plastics
and bisphenols, leading to the increased sorption capacity of bisphenols
to plastics (Xiong et al., 2020).
HA is the main component of natural dissolved organic matter
(DOM) (Xiong et al., 2020), and is composed of carboxyl-substituted
benzene rings, hydroxy-substituted benzene rings,
carbonyl-substituted benzene rings, etc. Carboxyl-substituted benzene
rings act as electron acceptors which could bind through n-π interaction
with carbonyl groups of amides (Lara et al., 2021). In addition to that,
the aromatic structure of DOM could interact with EPS polymers via π-π
interaction resulting in a highly conjugated structure (Chen et al., 2018).
HA could also bond with bisphenols by π-π interaction and H-bond
interaction (Lara et al., 2021). As a result, HA can act as a bridge be­
tween bisphenols and plastics (Fig. S10), leading to higher sorption
capacity of bisphenols on plastics (Tourinho et al., 2019). Moreover, the
introduction of HA makes the solution weakly acidic (pH = 6.5) and
reduces the anionic form of BPF (Fig. 4a), which could reduce the
repulsive force and also result in the enhanced sorption capacity
compared with in ultrapure water.
The sorption capacity for fresh and aged plastics showed no signifi­
cant difference (T-test, P > 0.05) in seawater. Whilst the sorption ca­
pacity of bisphenols on most aged plastics decreased compared with
fresh plastics in HA water (T-test, P < 0.05). This might be due to the
reduction of functional groups impacting the interaction behaviour be­
tween plastics and bisphenols/HA after irradiation. For example, the
reduction of amide I (C– – O) and amide III (–NH–, –CN–) after aging
(Fig. 1a) would weaken the bonding of plastics with HA (n-π interaction)
and bisphenols (H-bonding interaction), which could further reduce the
sorption of bisphenols on aged plastics. According to the data in Table S5
and Table S6, hydrophobicity still dominated the sorption behaviour of
most fresh and aged plastics in both seawater and HA water.
According to Fig. 6b, the desorption capacities for most bisphenols
were relatively high in seawater, which might exert potential risks to
aquatic ecosystems since the desorbed contaminants could re-enter the
Fig. 6. (a) The effect of water matrix on the sorption of bisphenols on fresh and aged 168 h plastics. (b) The effect of water bodies on the desorption of bisphenols on
fresh and aged 168 h plastics (n = 3, 60 mg plastics, 25 ◦ C, 180 rpm).
8
Chemosphere 310 (2023) 136867
seawater, sediments, or even organisms (Lu et al., 2020). The desorption
capacity in HA water is low, which might be due to the strong affinity of
HA towards pollutants (Lian et al., 2015), resulting in lower release of
bisphenols to water bodies.
3.3.2. Temperature effect
The sorption capacity for most fresh and aged plastics (Fig. 7a) was
enhanced with increased temperature (one-way ANOVA analysis, P <
0.05), while for aged PVC and EPS plastics, the sorption capacity for
most bisphenols increased at first and then decreased when the tem­
perature was higher than 25 ◦ C, which might be due to the simultaneous
desorption behaviour (Dong et al., 2019; Zhang et al., 2018). In addi­
tion, increased temperature could increase the mobility of molecules,
reducing the bonding between chemicals and plastics (Xu et al., 2019).
For both fresh and aged plastics, the desorption capacity increased with
increased temperature (Fig. 7b), which indicates the potential for
considerable transfer of bisphenols adsorbed to plastics in warmer
regions.
4. Conclusions
In this study, the sorption/desorption behaviour of bisphenols on
plastics were investigated, with special emphasis on the influence of
irradiation/aging and a range of environmental factors. After irradia­
tion, sorption capacity increased for PVC and EPS plastics, whilst the
desorption efficiency showed a decreasing trend after irradiation.
Environmental factors had significant effects on the interaction behav­
iour between bisphenols and plastics. The sorption capacity was higher
for most plastics under artificial seawater and humic acid water
compared to ultrapure water. For both fresh and aged plastics, desorp­
tion quantity increased in artificial seawater and enhanced temperature,
which indicates a potential exposure risk of plastics in the natural
environment.
Author contribution
Xiaoxin Chen – Conceptualization, Formal analysis, draft prepara­
tion, Chang-Er Chen – writing, reviewing, Xiaoyuan Guo - Methodology,
Andrew J. Sweetman - Supervision
X. Chen et al.
Fig. 7. (a) The relationship between temperature and the sorption behaviour of bisphenols on plastics. (b) The relationship between temperature and the desorption
of bisphenols (n = 3, 60 mg plastics, 15 mL ultrapure water, 180 rpm).
Declaration of competing interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
Andrew Sweetman reports a relationship with Lancaster University that
includes: employment.
Data availability
Data will be made available on request.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.chemosphere.2022.136867.
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 Petunjuk Penulisan

DESKRIPSI
Jurnal Farmasi Udayana merupakan jurnal elektronik yang dikelola oleh jurusan
Farmasi FMIPA Udayana. Jurnal ini yang merupakan media publikasi penelitian
dan review article pada semua aspek ilmu farmasi yang bersifat inovatif , kreatif,
original dan didasarkan pada scientific. Artikel yang dimuat dalam jurnal ini
meliputi penemuan obat, sistem penghantaran obat serta pengembangan obat.
Jurnal ini memuat bidang khusus di farmasi seperti kimia medisinal, farmakologi,
farmakokinetika, farmakodinamika, analisis farmasi, sistem penghantaran obat,
teknologi farmasi, bioteknolofi farmasi, obat herbal dan komponen aktif tanaman
serta evaluasi klinik obat.

PEMBACA
Ilmuwan di bidang kimia medisinal, farmasetika dan biofarmasetika,
farmakologi, kimia analisis, farmakologi klinik, mikrobiologi, bioteknologi, kimia
dan statistika

EDITOR
Penanggung jawab : Drs. Ida Bagus Made Suaskara, M.Si
Pengarah : Drs. I Made Satriya Wibawa, M.Si
Anak Agung Bawa Putra, S.Si., M.Si
Dr.rer.nat. IMAG. Wirasauta, M.Si., Apt
Editor :
Ketua Dewan Redaksi : Cokorda Istri Sri Arisanti, S.Farm., M.Si., Apt
Wakil Dewan Redaksi : Ni Kadek Warditiani, S.Farm., M.Sc., Apt
Mitra Bestari:
Ketua : Luh Putu Febryana Larasanty, S.Farm.,M.Sc., Apt
Anggota:
a. Ni PutuAriantari, S.Farm., M.Farm., Apt (Biologi Farmasi)
b. I G. N. Agung Dewantara, S.Farm., M.Sc., Apt (Teknologi Farmasi)
c. Ni Made Pitri Susanti, S.Farm., M.Si. Apt (Kimia Farmasi)

EMAIL

jurnalfarmasiudayana@gmail.com

INFORMASI UNTUK PENULIS DESEMBER 2015 i

PETUNJUK PENULISAN

PENDAHULUAN

Naskah yang diajukan ke jurnal harus memenuhi persyaratan sebagai berikut: (1)
topik artikel akan melewati proses review terlebih dahulu oleh editor, dan (2)
artikel belum dipublikasikan atau akan dipublikasikan seluruhnya atau sebagian di
jurnal lain atau media publikasi yang lain.

Tipe artikel

Artikel hasil penelitian

Review article

Naskah review article harus memuat: judul, abstrak dan kata kunci (3-6 kata),
pendahuluan, pembahasan khusus oleh penulis, kesimpulan, ucapan terima kasih,
daftar pustaka, gambar dan tabel. Tiap pokok bahasan dari pendahuluan sampai
kesimpulan harus diberi nomor. Sub pokok bahasan juga harus dinomori dengan
1.1., 1.2., 1.3., dan seterusnya. Setiap halaman harus diberi nomor dan judul harus
diberi halaman 1.

FAKTOR YANG HARUS DIPERHATIKAN

Conflict of interest
Semua penulis wajib menghindari terjadinya Conflict of interest yang meliputi
pembiayaan atau hubungan dengan orang lain atau badan paling lama tiga tahun
sebelum pengajuan artikel ke jurnal yang dapat mempengaruhi secara langsung
maupun tidak langsung penelitian yang bersangkutan

Contoh hal yang potensial menyebabkan Conflict of interest antara lain pekerja,
konsultan, kepemilikan bahan, honor, pengajuan registrasi/paten, hibah atau
sumber dana yang lain.

Verifikasi Artikel
Artikel yang diajukan ke Jurnal Farmasi Udayana belum pernah dipublikasikan
sebelumnya (kecuali dalam bentuk abstrak atau sebagai bagian dari skripsi), tidak
dalam posisi akan diterbitkan pada jurnal lain, artikel telah mendapat persetujuan
semua penulis yang tercantum di dalam artikel yang bersangkutan dan secara
eksplisit telah mendapat persetujuan dari tempat dimana penulis melakukan
penelitian dan jika diterima, artikel tidak dipublikasikan di tempat lain dalam
bentuk yang sama dalam bahasa Indonesia atau bahasa lainnya untuk menghindari
plagiarisme

Konstribusi
Semua penulis harus berpartisipasi di dalam penelitian dan atau penyipan naskah,
sehingga fungsi dari masing-masing penulis harus didefinisikan.

INFORMASI UNTUK PENULIS DESEMBER 2015 ii

Kepemilikan artikel
Semua penulis harus memiliki peran penting pada setiap tahap pengajuan artikel
yang meliputi: (1) konsep dan desain penelitian, pengolahan data atau
menganalisis atau menginterpretasi data, (2) memperbaiki naskah, (3) menyetujui
draf akhir yang akan dipublikasikan

Perubahan penulis
Pada jurnal ini dimungkingkan untuk menambahkan, pengurangi, mengubah
urutan penulis untuk naskah yang diterima. Hal-hal yang perlu dilakukan antara
lain: membuat permintaan untuk dapat menambahkan, mengurangi atau
mengubah urutan penulis kepada pengelola jurnal yang diajukan oleh
corresponding author yang dicantumkan di dalam naskah yang diajukan dan
meliputi: (a) alasan mengapa nama penulis harus ditambahkan, dikurangi atau
diubah susunannya (b) konfirmasi tertulis (e-mail, fax, surat) dari semua penulis
yang menyatakan persetujuan dengan perubahan tersebut di atas

Bahasa
Penulisan menggunakan bahasa Indonesia sesuai ejaan yang disempurnakan.

PERSIAPAN
Penggunaan program miscrosoft word. File dibuat dalam format asli
menggunakan program miscrosoft word. Teks harus dibuat dalam format satu
kolom, huruf font Times new roman 11, 1 spasi, ditulis dalam kertas ukuran A4.

Struktur Artikel
Sub pokok bahasan-penomoran
Artikel dibagi menjadi pokok bahasan dengan penomoran yang jelas. Sub pokok
bahasan harus diberi nomor 1.1 (kemudian 1.1.1, 1.1.2,...), 1.2 dan seterusnya.
Abstrak tidak dimasukkan dalam sistem penomoran.

Pendahuluan
Nyatakan tujuan dan landasan penelitian, hindari tinjauan pustaka yang terperinci
atau kesimpulan dari hasil penelitian

Bahan dan metode

Ungkapkan bahan dan metode secara terperinci untuk kemungkinan keterulangan
penelitian. Metode yang umum digunakan cukup menunjukkan sumber pustaka,
hanya modifikasi yang relevan yang harus dideskripsikan

Hasil
Pengungkapan hasil harus jelas dan ringkas

Pembahasan
Bagian ini harus merupakan kajian mendalam dari hasil penelitian, jangan
mengulang pengungkapan hasil. Hindari kutipan dan pembahasan yang berlebihan
dari penelitian sebelumnya

INFORMASI UNTUK PENULIS DESEMBER 2015 iii

Kesimpulan
Kesimpulan utama dari penelitian sebaiknya ditampilkan dalam kalimat yang
singkat dan jelas, yang dapat menjadi bagian tersendiri di dalam pokok bahasan
kesimpulan atau menjadi bagian dari pembahasan atau hasil

Appendik

Jika apendik lebih dari satu maka harus dibuat sebagai A, B dan seterusnya.
Persamaan matematika harus diberi nomor terpisah: Pers. (A.1), Pers. (A.2) dan
seterusnya. Hal yang sama juga berlaku untuk tabel dan gambar: Tabel A.1;
Gambar. A.1

Informasi penting dalam struktur artikel

Judul
Ringkas, jelas dan informatif. Jika dimungkinkan hindari pencantuman persamaan
matematika dan singkatan

Nama penulis dan institusi

Ungkapkan institusi tempat bekerja (tempat dimana penelitian dilakukan) di
bawah nama penulis. Tunjukkan institusi penulis dengan supercript di belakang
nama penulis dan didepan nama institusi. Tuliskan alamat lengkap termasuk kode
pos dan nama kota, jika perlu disertakan alamat email masing-masing penulis

Alamat korespondensi
Tunjukkan dengan jelas siapa yang bertanggung jawab terhadap korespondensi
semua tahap dari pengajuan, revisi, publikasi maupun sampai pasca publikasi.
Cantumkan nomor telepon disamping alamat email, kode pos. Kontak terperinci
harus tetap diperbaharui oleh korespondensi penulis

Alamat penulis
Jika alamat penulis berbeda dibandingkan dengan tempat penelitian semula, maka
alamat terbaru atau tetap penulis sebagai catatan kaki dari nama penulis. Alamat
dimana penelitian semula dilakukan oleh penulis tetap digunakan sebagai alamat
utama. Penulisan catatan kaki untuk alamat terbaru maupun alamat tetap
menggunakan supercrip dengan penomoran Arabic

Abstrak
Dibutuhkan abstrak yang jelas, ringkas dan sesuai fakta penelitian. Abstrak harus
menunjukan tujuan penelitian secara tegas, hasil yang penting dan kesimpulan
umum. Untuk memenuhi persyaratan abstrak ini, disarankan untuk tidak
menyertakan tinjauan pustaka, tetapi jika sangat diperlukan wajib mengutip nama
penulis dan tahun. Disamping itu dihindari pencantuman singkatan yang tidak
umum tetapi jika sangat diperlukan maka harus dijelaskan pada awal abstrak itu
sendiri

Gambar
Gambar harus dibuat untuk menyimpulkan isi dari artikel secara jelas untuk dapat
menarik perhatian pembaca yang berasal dari berbagai bidang yang berhubungan

INFORMASI UNTUK PENULIS DESEMBER 2015 iv

dengan farmasi. Gambar harus dibuat dalam bagian terpisah dari artikel. Ukuran
gambar: sediakan gambar dengan minimal setara 531x1328 pixel atau lebih, tetapi
dapat tetap terbaca pada layar 200x500 pixel (pada 91 dpi yang sama dengan 5
x13 cm). Program yang digunakan dapat berupa pdfatau MS Word

Kata kunci
Kata kunci maksimal 6 kata diletakkan langsung di bawah abstrak, hindari
penggunaan frase dan penghubung (dan, dari dan sebagainya)

Singkatan
Deskripsikan singkatan yang tidak umum sebagai catatan kaki pada halaman
pertama artikel. Singkatan yang menjadi keharusan untuk diungkapkan pada
abstrak diwajibkan didefinisikan pada bagian sebelum singkatan tersebut ditulis.
Penulisan singkatan harus konsisten pada seluruh artikel.

Ucapan terima kasih

Cantumkan ucapan terima kasih pada bagian terpisah di bagian akhir artikel
sebelum daftar pustaka, hindari penyertaan ucapan terima kasih pada judul,
sebagai catatan kaki judul atau bagian artikel lainnya. Buatlah rincian orang yang
berkontribusi di dalam penelitian (penerjemah, pengetik atau pembaca dan lain
sebagainya)

Unit
Gunakan satuan internasional (SI). Jika satuan diungkapkan dalam unit yang
berbeda, sebaikknya diungkapkan kesetaraan dengan SI

Tabel
Penomoran tabel diurut berdasarkan urutan munculnya di dalam artikel. Tabel
dibuat dengan tiga garis horisontal, hindari penggunaan garis vertikal dan data
yang diungkapkan di dalam tabel tidak diungkapkan berulang pada bagian lain
dari artikel

Daftar pustaka
Pastikan daftar pustaka tercantum di dalam artikel. Hasil yang belum
dipublikasikan dan personal communication tidak direkomendasikan dimasukkan
di dalam daftar pustaka. Pustaka yang ditandai dengan In Press menunjukan
bahwa artikel tersebut telah disetujui untuk dipublikasikan dan dapat digunakan
sebagai sumber pustaka. Penulisan pustaka mengikuti aturan penulisan pustakan
jurnal ini.

Aturan penulisan pustaka

Daftar pustaka harus diurut berdasarkan alfabetis dan kronologi. Jika terdapat
lebih dari satu sumber yang berasal dari penulis yang sama pada tahun yang sama,
maka harus ditambahkan a, b, c dan seterusnya di belakang tahun terbit.

Penulisan buku
Penulis, A.A., Penulis, B.B., & Penulis, C.C. (tahun terbit). judul buku: sub judul.
(Edisi [jika bukan edisi pertama}). tempat terbit: penerbit

INFORMASI UNTUK PENULIS DESEMBER 2015 v

Contoh:
Buku dengan satu penulis
Nama penulis (tanpa singkatan). (tahun terbit). judul buku. Tempat terbit: penerbit
Reynords Hadi. (2000). Black pioners. Ringwood,Vic: Penguin

Buku dengan banyak penulis

Dua-enam penulis
Dua penulis: kedua penulis. (tahun terbit). judul buku. Tempat terbit: penerbit
Gilbert, R., & Gilbert, P. (1998). Maculinity goes to school. St. Leonards, N.S.W.:
Allen & Unwin
Lebih dari 6 penulis
Setelah nama dan singkatan nama penulis ke-enam gunakan dkk

Buku yang memiliki editor

Broinowski, A. (Ed.) (1990). ASEAN into 1990s. London: Macmillan
Nugent, S.L., Shore, C. (Eds.). (1997). Anthropologyband cultural study. London:
Pluto Press

Buku yang memiliki penulis dan editor

Valery, P. (1957). Oeuvres (J. Hytier, Ed). Paris: Gallimard

Bab yang terdapat di dalam buku

Penulis, singkatan nama penulis. (tahun terbit). judul bab:sub judul. editor. judul
buku. (hal. x-y). tempat terbit: penerbit

Artikel jurnal
Penulis, singkatan nama penulis. (tahun terbit). judul artikel. singkatan jurnal,
volume (issue), halaman

Skipsi/Tesis/Disertasi
Nama penulis, singkatan nama penulis. (tahun terbit). judul. skrispi/tesis/disertasi.
Universitas, kota

Sumber penulisan singkatan jurnal

Index Medicus journal abbreviations: http//www.nlm.nih.gov/tsd/serials/lji.html
List of titlle word abbreviations: http//www.issn.org/2-22661-LTWA-online.php
CAS (Chemical Abstract Service): http//www.cas.org/sent.html

Submission checklist
Daftar isian di bawah ini dapat digunakan untuk memudahkan pemeriksaan akhir
sebelum artikel dikaji oleh editor.
Satu orang penulis ditunjuk sebagai corresponding author:
 alamat email
 kode pos
 nomor telepon atau fax
Semua file yang dibutuhkan telah diupload
 Kata kunci
 Gambar

INFORMASI UNTUK PENULIS DESEMBER 2015 vi

  Tabel (termasuk judul, deskrispi, catatan kaki)
Hal selanjutnya yang harus diperhatikan
 Gunakan penomoran baris (tiap 5 baris) untuk memudahkan pengkajian
naskah
 Naskah telah dicek tata bahasa dan pengucapannya
 Pustaka telah ditulis sesuao format di dalam jurnal ini
 Semua pustaka yang ditulis di dalam daftar pustaka disinggung di dalam
teks
 Izin telah didapat dari untuk materi yang memiliki hak cipta yang berasal
dari sumber lain (termasuk web)

SETELAH ARTIKEL DITERIMA

Perbaikan
Naskah yang telah dikoreksi akan dikirimkan kembali dalam bentuk pdf kepada
corresponding author (melalui alamat email) sehingga penulis dapat mengunduh
untuk keperluan pribadi. Gunakan perbaikan ini untuk mengecek urutan
penulisan, mengedit, menyempurnakan dan memperbaiki tulisan, tabel dan
gambar. Pengiriman naskah yang telah diperbaiki menyertakan koreksi pertama
dari editor ini. Perubahan signifikan dari artikel yang disetujui untuk
dipublikasikan dalam jurnal ini harus mendapat persetujuan dari penerbit. Kami
akan berusaha untuk mempublikasikan artikel anda akurat dan cepat sehingga
diharapkan kami menerima hasil koreksi anda paling lambat 5 hari kerja. Sangat
penting koreksi artikel dilakukan dalam satu kali komunikasi sehingga cermati
hal-hal yang harus dikoreksi sebelum dikirimkan kembali ke editor jurnal.

Naskah yang dipublikasikan

Artikel akan diberikan kepada corresponding author dalam bentuk pdf melalui
email. Penulis akan menerima artikel sesuai format yang terbit di dalam jurnal dan
disertai dengan cover jurnal.

INFORMASI UNTUK PENULIS DESEMBER 2015 vii

DAFTAR ISI

Hal
Halaman Judul …………………………………………………………………………........
Informasi Bagi Penulis di Jurnal Farmasi Udayana ................................................................ i
Petunjuk Penulisan .................................................................................................................. ii
Daftar Isi …………………………………………………………………………………..... viii

1 Uji Aktivitas Adaptogenik Ekstrak Etanol Kulit Batang Bidara (Ziziphus mauritiana
Auct. non Lamk.) dengan Metode Swimming Endurance Test pada Mencit Galur
Balb/C ………………………………………………………………………………… 1
2 Pengaruh Pemberian Ekstrak Etanol Kedondong Hutan Terhadap Volume Organ
Hati Mencit Betina .................................................................................................... ..... 8
3 Pengaruh Kombinasi Asam Oleat dan Minyak Atsiri Daun Cengkeh (Syzygium
aromaticum L.) Sebagai Permeation Enhancer Terhadap Karakter Fisik dan
Pelepasan Ketoprofen dari Matriks Patch Transdermal ………………………………. 11
4 Pengaruh Pemberian Ekstrak Etanol 80% Daun Spondias pinnata Terhadap Volume
Organ Ginjal Mencit Betina …………………………………………………………... 17
5 Validasi Metode Analisis Penetapan Kadar α-mangostin pada Gel Ekstrak Kulit
Buah Manggis (Garcinia mangostana L.) dengan KLT-Spektrofotodensitometri ……. 20
6 Uji Eritema dan Edema Secara In Vivo pada Natrium Lauril Sulfat 10% …………… 25
7 Perbandingan Metode Ekstraksi Maserasi dan Refluks terhadap Rendemen
Andrografolid dari Herba Sambiloto …………………………………………………. 29
8 Efek Pemberian Ekstrak Etanol Daun Spondias pinnata Terhadap Berat Organ Ginjal
Mencit Betina …………………………………………………………………………. 33
9 Optimasi Formula Matriks Patch Ketoprofen Transdermal Menggunakan Kombinasi
Asam Oleat dan Minyak Atsiri Bunga Cempaka Putih (Michelia alba) sebagai
Permeation Enhancer …………………………………………………………………. 37
10 Pemisahan Fraksi Terpenoid dari Ekstrak Etanol 90% Daun Katuk (Sauropus
androgynous (L.) Merr) Menggunakan Kromatografi Kolom ………………………... 45
11 Uji Sifat Fisik Cold Cream Kombinasi Ekstrak Kulit Buah Manggis (Garcinia
mangostana L.), Daun Binahong (Anredera cordifolia), dan Herba Pegagan (Centella
asiatica) sebagai Antiluka Bakar ……………………………………………………... 48
12 Pemberian Ekstrak Etanol Spondias pinnata Terhadap Volume Organ Hati Mencit
Jantan ............................................................................................................................. 53
13 Uji Aktivitas Adaptogenik Ekstrak Etanol Daun Bidara (Ziziphus mauritiana Auct.
non Lamk.) dengan Metode Swimming Endurance Test pada Mencit Galur Balb/C … 56
14 Pengaruh Penggunaan Propilenglikol dan Mentol Terhadap Matrik Patch
Transdermal Ekstrak Air Herba Sambiloto (Andrographis paniculata (Burm. f) Nees. 60
15 Pengaruh Pemberian Fraksi Terpenoid Daun Katuk (Sauropus Androgynus (L.)
Merr) Terhadap Profil Lipid Tikus Putih (Rattus Novergicus, L.) Jantan Galur Wistar
yang Diinduksi Pakan Kaya Lemak ............................................................................... 66
16 Rendemen VCO (Virgin Coconut Oil) yang Diperoleh dengan Penambahan
Enzim Papain dan Bromealin ………………………………………………… 72
17 Stabilitas Formalin Terhadap Pengaruh Suhu dan Lama Pemanasan ………... 76
18 Pengembangan Metode Refluks untuk Ekstraksi Andrografolid dari Herba Sambiloto
(Andrographis paniculata (Burm.f.) Nees) …………………………………………... 82
19 Uji Sitotoksisitas Ekstrak Etanol Limbah Kulit Buah Naga Merah (Hylocereus
polyrhizus) pada Sel Kanker Payudara Secara In Vitro dan In Silico ………………... 91
20 Pemberian Ekstrak Etanol Spondias pinnata Terhadap Volume Organ Ginjal Mencit
Jantan ............................................................................................................................. 98

INFORMASI UNTUK PENULIS DESEMBER 2015 viii

Uji Eritema dan Edema Secara In Vivo pada Natrium Lauril Sulfat 10% (Dewantara, I. G. N. A., Prasetia,
I. G. N. Jemmy, A., Putri, N. N. T. A. N., Arsana, D. A. M. I. P. S., Prabayanti, N. P. M.)
 
UJI ERITEMA DAN EDEMA SECARA IN VIVO PADA NATRIUM LAURIL SULFAT 10%

Dewantara, I. G. N. A.1, Prasetia, I. G. N. Jemmy, A.1, Putri, N. N. T. A. N.1, Arsana. D. A. M. I. P. S.1,

Prabayanti, N. P. M.1
1
Jurusan Farmasi Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Udayana

Korespondensi: I Gusti Ngurah Agung Dewantara

Jurusan Farmasi Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Udayana
Jalan Kampus Unud-Jimbaran, Jimbaran-Bali, Indonesia 80364 Telp/Fax: 703837
Email: agungdp09@gmail.com

ABSTRAK

Surfaktan merupakan suatu molekul dengan rantai hidrokarbon panjang dengan gugus ujung
bersifat polar atau ionik. Surfaktan berfungsi untuk mengangkat dan mengikat kotoran dari suatu
permukaan dengan cara menurunkan tegangan antar muka. Bahan surfaktan sintetik yang sering
digunakan sebagai bahan baku sediaan dipasaran adalah natrium lauril sulfat. Penggunaan surfaktan
sebagai bahan baku sediaan harus diperhatikan, penggunaan bahan yang tidak sesuai akan dapat
menyebabkan efek samping seperti iritasi pada kulit. Penelitian ini bertujuan untuk mengetahui pengaruh
surfaktan sintetik yaitu natrium lauril sulfat 10% sebagai bahan baku sediaan terhadap efek iritasi pada
kulit.
Pengujian iritasi dilakukan secara in vivo dengan menggunakan enam kelinci albino galur New
Zeland dewasa berkelamin jantan. Pencukuran bulu kelinci dilakukan 24 jam sebelum diberikan bahan
uji. Bahan uji diberikan dengan cara patch test tertutup. Pengamatan dilakukan pada jam ke 24, 48 dan 72
setelah pemberian bahan uji. Area uji diperiksa dan diamati perubahannya sebagai reaksi kulit terhadap
bahan uji dan dinilai indeks iritasi kulit dengan cara memberi skor 0 sampai 4 tergantung tingkat
keparahan reaksi kulit yang dilihat. Hasil penelitian menunjukkan bahwa natrium lauril sulfat dengan
konsentrasi 10% memberikan efek iritasi kulit pada jam ke 24, 48 dan 72.

Kata Kunci : surfaktan, uji iritasi, natrium lauril sulfat, in vivo.

1. PENDAHULUAN atau lebih cairan yang tidak bercampur satu

Surfaktan merupakan suatu molekul dengan
rantai hidrokarbon panjang dengan gugus ujung
bersifat polar atau ionik. Bagian rantai
hidrokarbon dari molekul ini bersifat hidrofobik
dan larut dalam cairan non polar, sedangkan
gugus ujung polar/ionik bersifat hidrofilik dan
larut dalam air (Tang, 2011). Surfaktan
berfungsi untuk mengangkat atau mengikat
kotoran dari suatu permukaan dengan cara
menurunkan tegangan antar muka sehingga
dapat melepaskan kotoran yang menempel pada
permukaan bahan. Selain itu surfaktan juga
dapat mendispersikan serta menstabilkan dua
sama lain (Showell, 2006).
Bahan surfaktan sintetik yang sering
digunakan sebagai bahan baku sediaan adalah
natrium lauril sulfat (NLS). NLS merupakan
golongan surfaktan anionik yaitu surfaktan yang
mengandung muatan ion negatif. Jenis surfaktan
ini merupakan surfaktan yang paling banyak
dipakai dalam industri (Permono, 2002).
Pemanfaatan natrium lauril sulfat dalam
formulasi sediaan farmasi sudah sangat luas
digunakan pada sediaan nonparenteral dan
preparat kosmetik. NLS memiliki efek toksisitas
sedang berupa efek akut seperti iritasi kulit,
iritasi mata, iritasi membrane mukosa, dan iritasi

25 
 
Uji Eritema dan Edema Secara In Vivo pada Natrium Lauril Sulfat 10% (Dewantara, I. G. N. A., Prasetia,
I. G. N. Jemmy, A., Putri, N. N. T. A. N., Arsana, D. A. M. I. P. S., Prabayanti, N. P. M.)
 
pada lambung jika tidak sengaja tertelan. Hal ini sediaan uji pada area uji. Setelah dioleskan
berkaitan dengan kemampuan dari kebanyakan bahan uji, area uji lalu ditutup dengan perban
surfaktan untuk dapat merusak membrane yang tidak reaktif dan di plester.
mukosa (Behn, 2005). Setelah 24 jam perlakuan, perban dibuka
Uji keamanan merupakan salah satu dan area uji dibersihkan dengan air untuk
persyaratan sebelum suatu bahan baku dapat menghilangkan sisa bahan uji. Pada jam ke 24,
dijual ke masyarakat umum atau kepasaran. Uji 48 dan 72 setelah pemberian bahan uji, area uji
keamanan dilakukan mencakup pengujian dari kemudian diperiksa dan diamati perubahannya
bahan baku maupun produk akhir. Pengujian sebagai reaksi kulit terhadap bahan uji dan
efek iritasi kulit dari bahan baku atau produk dinilai dengan cara memberi skor 0 sampai 4
akhir sediaan topikal merupakan elemen penting tergantung dari tingkat keparahan reaksi kulit
dari prosedur keamanan (Robinson dan Perkins, yang dilihat (Draize, 1959).
2002). Reaksi iritasi kulit tidak hanya bersifat
lokal pada permukaan kulit rusak saja, tetapi Tabel 1. Skor Derajat Iritasi pada Eritema
juga dapat menyebabkan efek toksik yang dapat REAKSI KULIT SKOR
membahayakan dan mengancam keselamatan
Tanpa eritema 0
jiwa dari penderitanya (Dirjen POM, 1985).
Berdasarkan hal tersebut, maka perlu dilakukan Sangat sedikit eritema (hampir 1
uji iritasi sediaan dengan metode in vivo tidak terlihat)
menggunakan kelinci sebagai hewan uji sebelum Eritema jelas terlihat (diameter 2
pemakaian pada manusia sehingga mencegah 25,1-30 mm)
reaksi hipersensitivitas dan dapat diketahui Eritema sedang (diameter 30,1-35 3
derajat keamanan sediaan yang dihasilkan bagi mm)
konsumen. Eritema berat (gelap merah 4
dengan membentuk eskar,
2. BAHAN DAN METODE diameter > 35 mm)

2.1 Bahan Penelitian Tabel Tabel 2. Skor Derajat Iritasi pada Edema
Bahan-bahan yang digunakan antara lain REAKSI KULIT SKOR
natrium lauril sulfat, aquadest, kelinci albino Tanpa edema 0
galur New Zealand berkelamin jantan, perban, Sangat sedikit edema (hampir 1
plester. tidak terlihat)
Edema jelas terlihat 2
2.2 Metode (ketebalan < 1 mm)
2.2.1Uji Iritasi Pada Kulit Kelinci Edema sedang (tepi naik ± 1 3
Uji iritasi lakukan secara in vivo pada enam mm)
kelinci albino galur New Zeland berkelamin Edema berat (tepi naik lebih 4
jantan dengan metode patch tes dari 1 mm dan meluas keluar
tertutup.Sebelum perlakuan, bulu pada bagian daerah pejanan)
punggung di cukur terlebih dahulu. Pencukuran
ini dilakukan 24 jam sebelum diberi perlakuan (Sani dan Lukmayani, 2010)
pada area uji. Sebelum diberikan perlakuan,
setiap kelinci menerima epidermal abrasi
paralel dengan menggunakan jarum yang steril.
Bahan uji diberikan dengan cara mengoleskan

26 
 
Uji Eritema dan Edema Secara In Vivo pada Natrium Lauril Sulfat 10% (Dewantara, I. G. N. A., Prasetia,
I. G. N. Jemmy, A., Putri, N. N. T. A. N., Arsana, D. A. M. I. P. S., Prabayanti, N. P. M.)
 

3. HASIL

2
Skor  Natrium Lauril 
Sulfat 10% 
1

0
0 24 48 72
Waktu (jam)

Gambar 1. Grafik Hasil Uji Iritasi

Tabel 3. Diameter Indeks Iritasi dan Edema (mm)

Hewan 0 jam 24 jam 48 jam 72 jam

uji Eritema Edema Eritema Edema Eritema Edema Eritema Edema
1 0 0 25,7 0,7 31,2 0,9 30,9 1,1
2 0 0 30,7 0,8 31,1 1,2 31,8 1,3
3 0 0 26,3 0,8 29,8 0,9 30,1 1,1
4 0 0 26,5 0,9 30,6 1,3 30,1 1,3
5 0 0 30,1 0,8 30,5 0,9 30,9 1,4
6 0 0 26,9 0,8 28,1 1,1 30,2 1,2

4. PEMBAHASAN pemberian bahan uji, area uji kemudian

Iritasi adalah gejala inflamasi yang terjadi
pada kulit atau membran mukosa setelah
perlakuan berkepanjangan atau berulang dengan
menggunakan bahan kimia atau bahan lain
(Irsan dkk., 2013). Iritasi kulit disebabkan oleh
suatu bahan yang dapat terjadi pada setiap
orang, tidak melibatkan sistem imun tubuh dan
ada beberapa faktor-faktor yang memegang
peranan seperti keadaan permukaan kulit,
lamanya bahan bersentuhan dengan kulit, dan
konsentrasi dari bahan.
Uji iritasi dilakukan dengan menggunakan
metode patch tes tertutup. Setelah 24 jam
perlakuan, perban dibuka dan area uji
dibersihkan dengan air untuk menghilangkan
sisa bahan uji. Pada jam ke 24, 48 dan 72 setelah
diperiksa dan diamati perubahannya sebagai
reaksi kulit terhadap bahan uji dan dinilai skor
eritema dan edema yang dihasilkan dengan cara
memberi skor 0 sampai 4 sesuai dengan tabel 1
dan tabel 2 tergantung dari tingkat keparahan
reaksi kulit yang dilihat (Draize, 1959).
Hasil pengamatan uji iritasi menunjukkan
bahwa formula dengan natrium lauril sulfat 10%
menyebabkan iritasi pada kulit dilihat dari grafik
yang diperoleh dimana natrium lauril sulfat
10%memiliki skor indeks iritasi pada jam ke-24,
pada jam ke-48 dan pada jam ke-72.
Suatu sediaan sebelum dipasarkan perlu
dilakukan pengujian atau pengecekan terhadap
pH dari bahan baku yang akan digunakan,
dimana perbedaan pH merupakan salah satu hal
yang dapat memicu terjadinya efek samping

27 
 
Uji Eritema dan Edema Secara In Vivo pada Natrium Lauril Sulfat 10% (Dewantara, I. G. N. A., Prasetia,
I. G. N. Jemmy, A., Putri, N. N. T. A. N., Arsana, D. A. M. I. P. S., Prabayanti, N. P. M.)
 
pada kulit seperti eritema dan edema Robinson, M.K and M.A. Perkins. 2002. A
(Tranggono dan Latifah, 2007). Strategy for Skin Irritation Testing.
American Journal of Contact Dermatitis,
5. KESIMPULAN Vol 13, No 1.
Sani, E. P. dan Lukmayani Y. 2010. Sabun
Berdasarkan hasil uji iritasi pada enam Transparan Berbahan Dasar Minyak
kelinci putih galur NewZealand menunjukkan Jelantah serta Hasil Uji Iritasinya pada
bahwa Natrium Lauril Sulfat pada konsentasri Kelinci. Jurusan Farmasi, Universitas
10% mengalami iritasi sedang pada kulit. Islam Bandung.
Showell, Michael S. 2006. Introduction to
UCAPAN TERIMA KASIH Detergents. In: Showell, M.S editor.
Handbook of Detergents Series Part D:
Ucapan terima kasih ditujukan kepada I Gede Formulation. United States of America:
Pasek Budiyadnya yang telah membantu penulis taylor and Francis Group, LLC.
selama melakukan penelitian di Laboratorium Tang, M., Veinardi S. 2011. Pengaruh
Teknologi Farmasi Udayana. Penambahan Pelarut Organik Terhadap
Tegangan Permukaan Larutan Sabun.
DAFTAR PUSTAKA Prosiding Simposium Nasional Inovasi
Pembelajaran dan Sains. Bandung.
Behn, S., AK Taylor, SC Owen, PJ Weller dan
KK Singh. 2005. Sodium Lauryl Sulfate.
In: -Arthur H,K., editor. Handbook of
Pharmaceuticals Excipients 3rd Ed.
London-United Kingdom: Pharmaceutical
Press. 487-480.
Dirjen POM. 1985. Formularium Kosmetika
Indonesia. Jakarta: Penerbit Departemen
Kesehatan RI. HalamanL’
: 22, 84, 356.
Draize, J.H. 1959. Dermal Toxicity. Pages 46-59
in Appraisal of the Safety of Chemicals in
Food, Drugs and Cosmetics. The
Association of Food and Drug Officials of
the United States, Bureau of Food and
Drugs, Austin, TX.
Permono, Ajar. 2002. Membuat Sampo.
Yogyakarta: Puspa Swara.
Tranggono, Retno Iswari dan Fatma Latifah.
2007. Buku Pegangan Ilmu
Pengetahuan Kosmetik. Jakarta: Pt.
Gramedia Pustaka Utama.

28 
 
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 UJI ERITEMA DAN EDEMA
SECARA IN VIVO PADA
NATRIUM LAURIL SULFAT 10%
by I G N Jemmy Anton Prasetia

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 Jurnal Tekonologi Pertanian Volume 5, Nomor 2, Mei 2010

EFEK SUHU TERHADAP MOISTURE SORPTION ISOTHERM

DARI IKAN KERAPU (Epinephelus merra) ASIN KERING DAN
IKAN CAKALANG (Katsuwonus pelamis L) ASAP

The Effect Of Temperature On Moisture Sorption Isotherm

Of Dried Salted Grouper (Epinephelus merra) and
Smoked Skipjack (Katsuwonus Pelamis L)

Albert R. Reo1)
1)
Dosen Fakultas Perikanan dan Ilmu Kelautan

ABSTRACT

Smoked and salted fish are a traditional processing that many

commercially practiced in centers of fishing activity in Indonesia. Free
water content known as water activity (Aw) is used to determine the
ability of water on the destruction process of food. Moisture sorption
isotherm (MSI) of a food product shows the balanced relationship
between the water content and the relative humidity (RH) at a certain
temperature. The purpose of this research is to determine the
adsorption and desorption isotherms of dried salted fish and smoked
skipjack. The MSI curve of dried salted fish and the effect of
temperature on the MSI curves of dried salted fish and smoked
skipjack is compared. Six concentrations of saturated salt solution in
the range of Aw 0.30 to 0.60 at 25˚C ± 2˚C and 10˚C ± 2˚C were
applied in this research. The data were analyzed using the randomized
block design arranged in factorial. Dried salted fish is more
hygroscopic when compared with smoked skipjack. Equilibrium
moisture content of dried salted fish and smoked skipjack were
influenced by temperature at 25˚C. The increase in temperature causes
smaller difference between adsorption and desorption curves.The
temperature did not significantly affect the equilibrium moisture
content but the process.

Keywords: Moisture sorption isotherm, dried salted fish, smoked fish,

grouper, skipjack.

PENDAHULUAN Pengolahan ikan asap

Ikan merupakan komoditi hasil
peikanan yang dikenal sebagai bahan
makanan yang mudah membusuk,
dimana terutama disebabkan oleh
kegiatan bakteri dan enzim. Sifat ini
mengakibatkan ikan tidak dapat
dikonsumsi ditempat yang jauh dari
tempat produksi, maka perlu
dilakukan usaha pengolahan yang
termasuk juga pengawetan (Lakoro,
2009).
merupakan saah satu pengolahan
tradisional yang mempunyai prospek
baik ditinjau dari potensi yang
tersedia, teknk yang sederhana,
kebutuhan modal yang relatif kecil,
tidak terpengaruh oleh keadaan cuaca
juga tidak tergantung pada spesies
dan ukuran ikan. Selain itu
mempunyai rasa yang dapat diterima
oleh rakyat Indonesia pada umumnya,
dan disamping itu pengasinan dan
pengeringan ikan juga salah satu

39
Efek Suhu Terhadap Moisture ......................... A. R. Reo

pengolahan tradisional yang banyak maupun penampakannya selalu

dipraktekkan secara komersial di disukai (Ruus, 2009).
pusat-pusat kegiatan perikanan di Moisture Sorption Isotherm
Indonesia (Adawyah, 2008). suatu produk makanan menunjukkan
Air di dalam bahan pangan hubungan antaran kadar air
terdapat dalam 3 bentuk yaitu: air keseimbangan dari makanan tersebut
bebas yang terdapat dipermukaan dengan tekanan uap air atau
benda dan mudah menguap; air terikat kelembaban relatif keseimbangannya
secara fisik yaitu air yang terikat pada suhu tertentu. Dimana mencakup
menurut sistem kapiler atau air proses adsorpsi dan desorpsi molekul-
adsorpsi, misalnya air yang terdapat di molekul air pada suhu tetap. Proses
jaringan tanama atau air diantara adsorpsi merupakan mobiitas molekul
struktur molekuler, serta air terikat air dari keadaan bebas ke keadaan
secara kimia yaitu air kristal, air polar, terikat dalam bahan, sedangkan proses
dan air terikat dalam sistem dispersi desorpsi merupakan mobilitas
(Muchtadi dkk., 2010). molekul-molekul air dari keadaan
Air yang terdapat dalam terikat ke keadaan bebas (Muchtadi
bentuk bebas dapat menyebabkan dkk., 2010).
terjadinya kerusakan bahan makan Menurut Sing dan Ojha dalam
baik secara mikrobiologis maupun Berhimpon (1990), pengukurn
ensimatis. Tetapi kadar air bukan keseimbangan kadar air penting untuk:
merupakan parameter yang absolut 1. mengetahui perubahan kadar air
untuk dapat dipakai meramalkan produk pada bermacam temperatur
kecepatan jerjadinya kerusakan bahan dan kondisi kelembaban relatif, 2.
makanan. Dalam hal ini dapat menentukan bats minimum kadar air
digunakan pengertian aktivitas air bahan yang dikeringkan.
(Aw) untuk menentukan kemampuan Moisture Sorption Isotherm
air dalam proses –proses kerusakan juga penting untuk mengetahui
bahan makanan (Muchtadi dan perubahan-perubahan selama
Ayustaningwarno, 2010). penyimpanan dan untuk penentuan
Aw mempunyai hubungan erat kebutuhan pengemasan bahan pangan
dengan kelembaban retatif udara yang kering (Muchtadi dkk., 2010).
adalah perbandingan antara tekanan Untuk meningkatkan mutu
uap di udara dengan tekanan uap air ikan asin kering dan ikan asap perlu
jenuh pada suhu yang sama, dimana penelitian-peneitian tentang cara
menunjukkan atmosfir disekeliling pengeringan dan pengemasan produk
material atau larutan. Nilai Aw akhir. Untuk itu data tetang moisture
menunjukkan sifat dari suatu larutan Sorption Isotherm dari produk tersebut
yatu perbandingan antara tekanan uap sangat diperlukan.
air dari larutan dengan tekanan uap air
murni pada suhu yang sama (Muchtadi METODOLOGI PENELITIAN
dan Ayustaningwarno, 2010).
Besar kecilnya Aw dapat Bahan dan Alat
disesuaikan untuk memperolah tingkat Bahan :
pengawetan yang lebih baik. Untuk Bahan yang digunakan dalam
setiap macam bahan pangan, peneltitian ini adalah ikan cakalang
perubahan Aw samapai pada keadaan (Katsuwonus pelamis L) asap dan ikan
yang paling sesuai (optimum) asin kering dari jenis ikan kerapu
bertujuan untuk mendapatkan bahan (Ephinephelus merra), yang dibeli di
pada kondisi yang baik sehingga Pasar Bersehati Manado. Bahan kimia
selama penyimpanan, baik isi, bentuk yang digunakan adalah enam macam
garam untuk mencakup kisaran nilai
 Jurnal Tekonologi Pertanian Volume 5, Nomor 2, Mei 2010
Aw 0,30 – 0,86, pada temperatur 25OC
± 2OC dan 10OC 2OC. Tabel 2
menunjukkan beberapa larutan garam
dan RH yang diberikannya dalam
keadaan jenuh, pada temperatur 25OC
dan 10OC. Bahan kimia lainnya adalah
toluen dan vaselin.
Alat :
Alat yang digunakan dalam
penelitian ini adalah oven, inkubator,
desikator, botol gelas, pisau, sedok
pengaduk, pinggan nilin, cawan petri,
gelas kimia, gelas ukur, tabung raksi,
timbangan o’hause, bender, penjepit,
aluminium foil, lebel selotip, cawan
porselin, karet penghisap, pipet
gondok.
Tata Laksana Penelitian
a. Percobaan Adsorpsi Isotherm
Bagian kepala, ekor, kulit dan
tulang dari ikan yang akan diteliti
dibuan, kemudian ikan dipotong kecil
dan dimasukkan ke dalam botol gelas.
Contoh tersebut kemudian dikeringkan
dengan freeze dryer pada temperatur -
40OC dengan tekanan 0,44 kPa,
selama kurang lebih delapan jam. Ikan
yang telah dikeringkan, digiling
sampai halus, kemudian masukkan ke
dalam botol gelas, disegel dan
selanjutnya disimpan dalam desikator
yang berisi silika gel, sampai
waktunya digunakan.
Desikator (diameter 15 cm)
diisi larutan garam jenuh yang
mempynyai RH tertentu sampai
setengah bagian dasarnya. Untuk
menjamin kejenuhannya, larutan-
larutan tersebut diberikan kristal
garam yang sesuai secara berlebihan
(± 200 gram). Desikator yang telah
berisi larutan garam jenuh ditutup
rapat dan diberi vaselin kemudian di
simpan sampai dengan waktunya akan
digunakan.
kurang lebih 1 gram duplikat
sampel diletakkan dalam Aw disk
yang telah diberikan nomor kode
dengan menggunkan label dan
diletakkan dalam masing-masing
desikator. Untuk mencegah
pertumbuhan mikroorganisme dan
pembusukkan sampel, dietakkan 1 ml
toluen dalam botol kecil pada setiap
desikator yang mempunyai RH 60 %
atau lebih. Karena toluen mudah
menguap maka dapat ditambah jika
telah habis. Percobaan dilakukan pada
temperatur 25OC dan 10OC. Setiap
tujuh hari larutan diperiksa untuk
memperoleh kepastian akan
kejenuhannya. Berat sampel ditimbang
beberapa kali sampai tidak ada
perubahan yang berarti (kurng dari
0,001 gram). Waktu keseimbangan ini
diperkirakan antara tiga minggu
sampai lima minggu. Setelah
keseimbangan beratnya diperoleh,
masing-masing sempel dianalisa kadar
airnya.
b. Percobaan Desorpsi Isotherm
Pada dasarnya percobaan
desorpsi isotherm sama dengan
percobaan adsorpsi isotherm, hanya
sampel yang digunakan dalam
keadaan awal/beku (tanpa
dikeringkan). Bagi sampel yang
kering, untuk kelembaban yang tinggi
(70% atau lebih), dilakukan rehidrasi
dengan menggunakan air suling.
Dalam penelitian ini rehidrasi
dilakukan pada sampel ikan asin
kering.
HASIL DAN PEMBAHASAN
Data Moistore Sorption
Isotherm dari ikan asin kering dan
ikan cakalang asap dapat dilihat pada
Lampiran 1 dan 2 serta pada Gambar 1
sampai 14. Dari keseluruhan isotherm,
terlihat bahwa ikan asin kering bersifat
lebih higroskopis bila dibandingkan
dengan ikan cakalang asap.
Keseimbangan kadar air dari ikan
kering dan cakalang asap dapat
dipengaruhi oleh temperatur, Aw dan
jenis bahan yang dikeringkan. Gambar
9, memperlihatkan bahwa pada
tmperatur yang lebih tinggi
keseimbangan kadar air dari ikan asin
41
Efek Suhu Terhadap Moisture ......................... A. R. Reo

kering dan cakalang asap akan lebih dan desorpsi pada temperatur tertentu.
rendah. Untuk ikan asin peruahan Pada Aw yang sama, kadar air
kadar air lebih kritis dibandingkan seimbang pada proses desorpsi lebih
dengan ikan cakalang asap, dan terjadi besar dari pada proses adsorpsi. Pada
pada Aw sekitra 0,60 ke atas. penelitian ini gejala hesterisis untuk
ikan asin kering terlihat jelas pada Aw
Pengaruh Temperatur Terhadap sekitar 0,60 ke atas dan ika cakalang
Keseimbangan Kadar Air Bahan asp pada Aw 0,30 ke atas. Dengan
Pada temperatur 10OC, kurva menigkatkan temperatur, perbedaan
adsorpsi dan desorpsi ikan asin kering antara kurva adsorpsi dan desorpsi
dan cakalang asap lebih tinggi dari menjadi semakin kecil (Gambar 11
pada temperatur 25OC (Gambar 9 dan sampai 14). Keadaan ini sesuai hasil
10). Dengan kenaikan temperatur penelitian dari Altman dan Benson
pengurangan air akan semakin rendah. (1960) yang meneliti isothermal sorpsi
Ikan asin kering lebih higroskopis air, alkohol dan eter oleh suatu protein
dibandingkan dengan ikan cakalang yang terdenaturasi, Berhimpon (1990)
asap pada temperatur rendah. Hal ini yang meneliti Moisture Sorption
disebabkan pengaruh garam, akan Isotherm dari ikan asin kering dan
lebih mudah menyerap air dari udara Apituley (1991) yang meneliti MSI
sekitarnya pada temperatur yang dari ikan cakalang dan julung-julung
rendah. Caden dalam Apituley (1991) asap.
mengemukakan bahwa kebayakan
bahan pangan akan berkurang sfat
higroskopis jika temperatur dinaikan.
Hal ini juga dikemukakan olah Haynes
(1961) yang meneliti keseimbangan.
Kadar air gabah pada tiga tingkatan
suhu dan Berhimpon (1990) yang
meneliti MSI dari ian asin kering.
Adanya perubahan temperatur
berpengaruh terhadap perubahan
bentuk interaksi molekul air dalam
bahan makanan pangan. Hal tersebut Gambar 1. Grafik Adsorpsi Dari
dijelaskan oleh Rockland dan Nishi Ikan Asin Kering Pada
dalam Taib dkk. (1988), bahwa Temperatur 10OC
perubahan tersebut disebabkan oleh
adanya perubahan energi ikatan
molekul air.
Ikatan hidrogen mempermudah
kemungkinan interaksi berantai di
antara moekul-molekul air, sehingga
membentuk semacam polimer moleku
air. Kecendrungan berpoimerisasi
tersebut, akan menyebabkan molekul
air dapat teradsorpsi olah bahan
pangan sempai pada bagian yang
paling jauh. Gambar 2. Grafik Desorpsi Dari
Histerisis di perlihatkan oleh Ikan Asin Kering Pada
perbedaan harga kadar air seimbang Temperatur 10OC
yang diperolah dari proses adsorpsi
 Jurnal Tekonologi Pertanian Volume 5, Nomor 2, Mei 2010
Gambar 3. Grafik Adsorpsi Dari
Ikan Asin Kering Pada
Temperatur 25OC
Gambar 4. Grafik Desorpsi Dari
Ikan Asin Kering Pada
Temperatur 25OC
Gambar 5. Grafik Adsorpsi Dari
Ikan Cakalang Asap Pada
Temperatur 10OC
Gambar 6. Grafik Desorpsi Dari
Ikan Cakalang Asap Pada
Temperatur 10OC
Gambar 7. Grafik Adsorpsi Dari
Ikan Cakalang Asap Pada
Temperatur 25OC
Gambar 8. Grafik Desorpsi Dari
Ikan Cakalang Asap Pada
Temperatur 25OC
Gambar 13 dan 14,
memperlihatkan bahwa Aw di atas
0,70 kurva adsorpsi ikan cakaang asap
lebih besar dari kurva desorpsi pada
temperatur 10OC. Hal demikian juga
dapat dilihat pada Aw 0,60 sampai
0,70 untuk ikan cakalang asap pada
temperatur 25OC. Penyimpanan dari
keadaan normal tersebut merupakan
penyimpanan hesterisis isotherm. Hal
ini disebabkan pada Aw di atas 0,60
dalam kondisi yang lembab
penyerapan air pada kurva adsorpsi
akan semakin besar dibandingkan
dengan pelepasan air pada kurva
desorpsi. Penyimpanan dari keadaan
norma tersebut akan berbeda pada
setiap kenaikan temperatur.
Turunnya kadar air bahan pada
setiap kenaikan temperatur akan
menyebabkan jumlah air bebas yang
dapat dipergunakan oleh
mikroorganisme berkurang, sehingga
perkembangan mikroorganisme
43
Efek Suhu Terhadap Moisture ......................... A. R. Reo
menjadi terhambat. Sebaliknya bila
temperatur di turunkan, kadar air
bahan akan meningkat sehingga
jumlah air bebas yang diperlukan
mikroorganisme majadi lebih banyak.
Hal ini akan mengakibatkan kerusakan
pada bahan pangan yang disebabkan
oleh pertumbuhan mikroorganisme.
Pengaruh Jenis Ikan Olahan
Terhadap Keseim-bangan Kadar
Air
Dalam penelitian ini digunakan
dua jenis ikan yaitu ikan asin dan ikan
asap, dimana cara pengolahan dari
kedua jenis ikan ini berbeda. Hasil
peneitian menunjukkan bahwa pada
Aw di bawah 0,60 kadar ikan asin
kering pada adsorpsi meupun desorpsi
lebih rendah dari pada ikan cakalang
asap.
Gambar 9. Grafik Adsorpsi Dari
Ikan Asin Kering dan
Ikan Cakalang Asap Pada
Temperatur 10OC dan
25OC
Gambar 10. Grafik Desorpsi Dari
Ikan Asin Kering dan
Ikan Cakalang Asap Pada
Temperatur 10OC dan
25OC
Sedangkan pada keadaan Aw
diatas 0,6 keadaan sebaliknya terjadi.
Dengan demikian diharapkan pada
penyimpanan di antara RH rendah
tingkat keawetan ikan asin kering
lebih baik dari ikan cakalang asap.
Pada Gambar 9 dan 10 dapat dilihat
bahwa peningkatan kadar air yang
sangat tinggi terjadi pada ikan asin
kering, dimana nyata pada Aw di atas
0,6. Hal ini merupakan Aw kritis
untuk ikan asin kering dibandingkan
dengan ikan cakalang asap, dimana
diatas Aw tersebut kerusakan yang
disebabkan oleh pertumbuhan
mikroorganisme akan semakin cepat.
Peningkatan kadar air yang
sanga tinggi pada jenis ikan asin
kering dibandingkan dengan ikan
cakalang asap disebabkan adanya
garam di dalam tubuh ikan akan
menyerap air lebih banyak dan ini
terjadi apabila kelembaban relatif
tinggi (di atas 60%). Menurut Ilyas
(1972), garam dapur mengandung Mg
yang bersifat higroskopis yang dapat
menarik air dari udara menyebabkan
ikan menjadi lembab. Dengan
demikian untuk penyimpanan pada
RH yang tinggi, ikan asin kering perlu
dikemas dengan baik agar kadar
airnya tidak meningkat akibat
penyerapan air dari udara sekitar.
Gambar 11. Grafik Adsorpsi dan
Desorpsi Dari Ikan Asin
Kering Pada Temperatur
10OC
 Jurnal Tekonologi Pertanian Volume 5, Nomor 2, Mei 2010

temperatur 10OC dan 25OC menunjuk

kan bahwa perlakuan jenis ikan dan
temperatur tidak membeikan pengaruh
yang nyata terhadap keseimbangan
kadar air bahan (Tabel 1).

Tabel 1. Hasil Analisis Sidik Ragam

Adsorpsi dari Ikan Asin
Kering dan Cakalang Asap
Pada Temperatur 10OC dan
25OC.
Gambar 12. Grafik Adsorpsi dan
Desorpsi Dari Ikan Asin SK
d
JK Kt Fhit
Ftabel
b 0,5 0,1
Kering Pada Temperatur Kelomp 5 18290,5 3658,11 4,4 2,9 4,5
25OC ok
3
719 43 1* 0 4

Perlaku 5958,11 1986,03 2,6 3,2 5,4

an 1 43 78 9 9 2

Faktor 1 4886,90 4886,90 6,6 4,5 8,6

A 42 42 3* 4 8
1
Faktor 745,823 745,823 1,0 4,5 8,6
B 5 5 2 4 8
1
Interak 5 325,385 325,385 0,4 4,5 8,6
si 7 7 4 4 8
AB

Eror 736,696 736,696

9 9
Total 2 35320,3
3 05

Gambar 13. Grafik Adsorpsi dan Berdasarkan analisis

Desorpsi Dari Ikan keragaman diatas, maka perlakuan
Cakalang Asap Pada jenis ikan dan temperatur
Temperatur 10OC memperlihatkan hasil Fhitung lebih kecil
dari Ftabel. Dengan demikian jenis ikan
dan temperatur tidak memberikan
pengaruh yang nyata terhadap
keseimbangan kadar air. Jenis ikan
memberikan pengaruh yang nyata
terhadap keseimbangan kadar air dan
temperatur tidak memberikan
pengaruh yang nyata, sedangkan
interaksi antara jenis ikan dan
temperatur tidak memberikan
pengaruh yang nyata terhadap
Gambar 14. Grafik Adsorpsi dan keseimbangan kadar air.
Desorpsi Dari Ikan Hasil analisis keragaman untuk
Cakalang Asap Pada desorpsi dari ikan asin kering dan
Temperatur 25OC cakalang asap masing-masing pada
temperatur 10OC dan 25OC
Pengaruh Temperatur dan Jenis
menunjukkan bahwa perlakuan jenis
Ikan Terhadap Keseimbangan
ikan dan temperatur tidak memberikan
Kadar Air
pengaruh yang nyata terhadap
Hasil analisis keragaman untuk
kesembangan kadar air bahan (Tabel
adsorpsi dari ikan asin kering dan
2).
cakalang asap masing-masing pada

45
 Efek Suhu Terhadap Moisture ......................... A. R. Reo

Tabel 1. Hasil Analisis Sidik Ragam 0,3 ke atas dan kurva desorpsi labih
desorpsi dari Ikan Asin tinggi dari kurva adsorpsi.
Kering dan Cakalang Asap - Makin tinggi temperatur, histerisis
Pada Temperatur 10OC dan makin rendah.
25OC. - Penyimpangan histerisis isotherm
Ftabel
SK db JK Kt Fhit
0,5 0,1
terjadi pada daging ikan cakalang
Kelompok 5 18290,5719 3658,114 4,41* 2,90 4,54 asap pada temperatur 10OC dan
Perlakuan 3 5958,1143
3
2,66 3,29 5,42 25OC.
Faktor A 1 4886,9042
1986,037
8 6,54* 4,54 8,68
- Adanya kenaikan temperatur akan
menyebabkan kadar air seimbang
Faktor B 1 745,8235 4886,904 0,94 4,54 8,68
2 semakin berkurang.
Interaksi 1 325,3857 0,51 4,54 8,68
AB 745,8235
- Temperatur tidak memberikan
pengaruh yang nyata sedangkan
Eror 15 736,6969 325,3857
jenis ikan memberikan pengaruh
736,6969 yang nyata terhadap keseimbangan
kadar air.

DAFTAR PUSTAKA
Total 23 35320,305
Adawyah, R. 2008. Pengolahan
Pengawetan Ikan. Bumi
Berdasarkan analisa Aksara.
keragaman di atas, maka perlakuan Apituley, Y.. 1991. Moisture
jenis ikan dan temperatur Sorption Isotherm Dari Ikan
memperlihatkan hasil Fhitung labih kecil Cakalang (Katsuwonus pelamis
dari Ftabel. Dengan demikian jenis ikan L) dan Julung-julung
dan temperatur tidak membekan (Hemirhamphus sp) Asap.
pengaruh yang nyata terhadap Skripsi. Universitas Sam
keseimbangan kadar air. Jenis ikan Ratulangi Fakultas Perikanan.
memberikan pengaruh yang nyata Manado.
terhadap keseimbangan kadar air dan
temperatur tidak memberikan Berhimpon, S. 1990. Studies On
pengaruh yang nyata, sedangkan Salting and Dray-ing of
interaksi antara janis ikan dan Yellowatail (Trachurus
tmperatur tidak memberikan pengaruh mucullochi Nichiols).
yang nyata terhadap keseimbangan Departement of food science and
kadar air. technology, school of applied
bioscience, the niversity of New
South Wales; Tesis. Sydney.
KESIMPULAN Ilyas, S.. 1972. Pengantar
Pengolahan Ikan. Lemba-ga
Berdasarkan hasil penelitian, Teknologi Perikanan. Dirjen
dapatlah disimpukan bahwa: Perikanan. Jakarta.
- Kurva adsorpsi dan desorpsi daging
ikan cakalang asap pada Aw di atas Lakoro, M. 2009. Analisis Mutu Ikan
0.6. Kurva adsorpsi dan desorpsi Roa Asap dengan P..... yang
daging ikan cakalang asap lebih Berbeda di Desa Boras
tinggi dari ikan asin kering pada Kabupaten Banggai Sulawesi
Aw di bawah 0,6. Tengah. Skripsi Fakultas
- Histerisis untuk ikan asin kering Perikanan dan Ilmu Kelautan.
terlihat jelas pada Aw 0,60 ke atas Universitas Sam Ratulangi.
dan ikan cakalang asap pada Aw Manado.
 Jurnal Tekonologi Pertanian Volume 5, Nomor 2, Mei 2010

Muchtadi, T. R. Dan F.
Ayustaningwarno. 2010. Lampiran 2. Data Moisture Sorption
Teknologi Proses Pengolahan Isotherm dari Ikan
Pangan. Alfabeta. Bandung. Cakalang Asap Pada
Suhu 10OC dan 25OC
Muchtadi, T. R., Sugiyono, dan F.
Ayustaningwarno. 2010. Ilmu 1. Data Moisture Sorption Isotherm
Pengetahuan Bahan Pangan. dari Ikan Cakalang Asap Pada
Ikatan Penerbit Indonesia. Suhu 10OC.
Ruus, O. 2009. Pengaruh Kadar Air (BK)
Aw
Konsentrasi Larutan Garam Asorpsi Desorpsi
dan Lama Pengeringan 0.342 8.9117 11.8849
terhadap Mutu Ikan Layang 0.455 11.3841 13.2332
Asin. Skripsi Fakultas Perikanan 0.578 16.4466 16.0791
dan Ilmu Kelautan. Universitas 0.673 19.4689 21.0913
Sam Ratulangi. Manado. 0.752 27.9405 22.7183
0.868 47.5335 42.6067
Thaib, G. G. Said dan Sutedja W..
1988. Operasi Pengeringan
Pada Pengolahan Hasil
Pertanian. Mediyatma Sarana 2. Data Moisture Sorption Isotherm
Perkasa Jakarta. dari Ikan Cakalang Asap Pada
Suhu 25OC.
Kadar Air (BK)
Lampiran 1. Data Moisture Sorption Aw
Asorpsi Desorpsi
Isotherm dari Ikan Asin
0.332 6.9372 9.5743
Kering Pada Suhu 10OC
0.443 9.289 10.8192
dan 25OC 0.543 11.5383 12.6046
0.644 19.6374 16.4181
1. Data Moisture Sorption Isotherm 0.758 19.669 22.5146
dari Ikan Asin Kering Pada Suhu 0.843 32.2135 32.3804
10OC.
Kadar Air (BK)
Aw
Asorpsi Desorpsi
0.342 7.5741 7.9748
0.455 9.9774 11.0285
0.578 15.1432 16.0603
0.673 54.6943 57.9445
0.752 100.2687 103.9824
0.868 173.0715 183.6643

2. Data Moisture Sorption Isotherm

dari Ikan Asin Kering Pada Suhu
25OC.
Kadar Air (BK)
Aw
Asorpsi Desorpsi
0.332 7.0213 7.0116
0.443 8.4655 9.6814
0.543 10.7622 11.1274
0.644 20.8681 25.8898
0.758 83.5085 84.9429
0.843 122.3516 124.9066

47
 e-ISSN 2776-1878 p-ISSN 2086-7816

Formulasi dan Uji Iritasi Sediaan Gel Kombinasi Ekstrak Etanol

Rimpang Kencur (Kaempferia galanga L.) dan Ekstrak Etanol Herba
Pegagan (Centella asiatica (L.) Urban)

Amelia Febriani1*, Ika Maruya1, Fitri Sulistyaningsih1

1)
Program Studi Farmasi, Fakultas Farmasi, Institut Sains Dan Teknologi Nasional (ISTN), Jl. Moh Khafi II Srengseng
Sawah, Jakarta Selatan, 12640, Indonesia
*
Emailkorespondensi: ameliafebriani@istn.ac.id

ABSTRAK

Gel adalah sediaan semipadat yang terdiri dari partikel anorganik yang kecil atau molekul organik yang besar terpenetrasi
oleh suatu cairan. Rimpang kencur (Kaempferia galanga L.) dan herba pegagan (Centella asiatica (L.)Urban) mengandung
senyawa alkaloid, flavonoid, saponin, steroid yang berkhasiat sebagai antiinflamasi. Kombinasi rimpang kencur dan herba
pegagan dilakukan agar dapat meningkatkan efek antiinflamasi. Ekstrak rimpang kencur dan herba pegagan dibuat dengan
metode maserasi menggunakan etanol 70% sebagai pelarut dan diformulasikan dalam bentuk sediaan gel menggunakan
HPMC sebagai gelling agent. Sediaan gel tersebut dibuat menjadi 5 formula, dan memiliki konsentrasi bahan aktif sebesar 2%
dengan perbandingan antara ekstrak kencur dan herba pegagan sebagai berikut yaitu Formula I (0,5:1,5); Formula II (1:1);
Formula III (1,5:0,5); Formula IV (2:0); Formula V (0:2). Gel yang dihasilkan dievaluasi meliputi uji organoleptik,
kemampuan menyebar, homogenitas, pH, viskositas, sifat alir dan uji iritasi dengan metode patch test. Semua sediaan diuji
stabilitasnya dengan metode dipercepat pada suhu kamar dan suhu 40℃. Evaluasi dilakukan setiap 7 hari selama 3 minggu.
Hasil uji stabilitas menunjukkan gel memiliki stabilitas fisik yang baik, berbentuk semipadat, berwarna hijau hingga cokelat
serta homogen, mempunyai nilai daya sebar 1962,5-3419,46 mm2, viskositas antara 15000-60000 cPs, pH 4,90-5,41 dan
daya lekat 24,66 – 59,82 detik. Hasil uji iritasi menunjukkan gel tidak mengiritasi kulit.

Kata Kunci: gel, herba pegagan, rimpang kencur, patch test, stabilitas fisik

Formulation and Irritation Test of Gel Combination of Galangal Rhizome

(Kaempferia galanga L.) and Gotu Kola Herb (Centella asiatica (L.) Urban) Ethanolic Extract

ABSTRACT

Gel is a semi-solid preparation consisting of small inorganic particles or organic molecules which are largely penetrated
by a liquid. Kencur rhizome (Kaempferia galanga L.) and Gotu kola herb (Centella asiatica (L.) Urban) contain a mixture
of alkaloid, flavonoid, saponin, steroid which are efficacious as anti-inflammatory. The combination of kencur rhizome and
Gotukola herb is done to increase the anti-inflammatory effect. Ethanol extract of kencur rhizome and Gotu kola herb were
obtained by maceration method and using 70% ethanol and formulated into gel dosage form using HPMC as a gelling agent.
Each gel preparation made into 5 formulas and had a concentration of 2% active ingredients with a comparison of kencur
extract and Gotu kola herb as follows formula I (0.5:1.5); formula II (1:1); formula III (1.5:0.5); formula IV (2:0); formula
V (0:2). These combinations produced a gel that tested by organoleptic tested, spreading ability, homogeneity, pH, viscosity,
flow properties and safety test with patch test method. All preparations were tested for stability by an accelerated method at
room temperature and the evaluation of a temperature of 40℃ was carried out every week in three weeks times. Stability
test results showed the gel was physically concentrated, has characteristics are semi-solid dosage forms, green to brown
and homogeneous, also can spread until 1962.5-3419.46 mm2, viscosity between 15000-60000 cPs and pH 4.90-5.41 and
stickiness 24.66 – 59.82 seconds. Therefore, the gel is safe to use on the skin.

Keywords: gel, Gotu kola herb, kencur rhizome, patch test, physical stability

PENDAHULUAN Keuntungan sediaan gel apabila digunakan dapat

menimbulkan rasa dingin, sejuk dan mempunyai
Gel adalah sediaan semipadat yang terdiri dari penetrasi yang cepat pada kulit. Gel yang sederhana
partikel anorganik yang kecil atau molekul organik yang terdiri dari satu macam bahan pembentuk gel dalam
besar terpenetrasi oleh suatu cairan (Sayuti, 2015). pelarutnya, bentuk transparan dengan sifat aliran yang

Sainstech Farma Vol 13 No.1, Januari 2020 | 45

Formulasi dan Uji Iritasi Sediaan Gel Kombinasi Ekstrak Etanol Rimpang Kencur (Kaempferia galanga L.) dan Ekstrak Etanol Herb a Pegagan (Centella asiatica (L.) Urban)
| Febriani et al.
umumnya tiksotropik serta penampilan yang cukup
menarik umumnya disukai oleh konsumen (Chiew,
1992).
Pada sediaan topikal, salah satu parameter yang
penting untuk diperhatikan adalah adanya kemungkinan
produk yang diaplikasikan iritasi terhadap kulit.
Iritasi merupakan salah satu reaksi buruk yang
terjadi pada kulit, yang dapat disebabkan oleh beragam
faktor diantaranya lama pemberian, luas area pemberian,
tingkat penetrasi dan ketoksikan dari bahan yang
diaplikasikan. Munculnya iritasi dapat terjadi setelah
beberapa waktu dari pengaplikasian sediaan, ditandai
dengan adanya eritema dan edema dimana eritema
atau kemerahan terjadi karena dilatasi
pembuluh darah pada daerah yang teriritasi, sedangkan
pada edema terjadi perbesaran plasma yang membeku
pada daerah yang terluka (Irsan, 2013). Rimpang kencur
dan herba pegagan yang memiliki senyawa aktif alkaloid,
saponin, flavonoid, tanin dan steroid yang memiliki
aktivitas sebagai antiinflamsi yang dapat digunakan untuk
penyembuhan luka. Rimpang kencur (Kaempferia
galanga L.) merupakan salah satu tanaman obat yang
bernilai ekonomis cukup tinggi sehingga banyak
dibudidayakan dan memiliki potensi untuk
dikembangkan (Nie et al., 2012). Komponen terbesar
yang terkandung dalam rimpang kencur adalah etil p-
metoksinamat, yaitu sebesar 80,05% dan dilaporkan
memiliki aktivitas antiinflamasi dengan menghambat
edema tikus yang diinduksi karagen (Umar et al., 2012).
Menurut penelitian senyawa EPMS pada konsentrasi 1%
dapat menyembuhkan luka selama 2-8 hari, pada
konsentrasi 3% dan 5% dapat menyembuhkan luka 2-9
hari (Fitriani, 2016).
Herba pegagan (Centella asiatica (L.) Urban)
merupakan salah satu tumbuhan obat yang telah
digunakan secara meluas di masyarakat. Pegagan
mengandung bahan aktif seperti triterpenoid glikosida
(terutama asiatikosida, asam asiatat, asam madekasat,
madikassosida) (Hutapea, 1992). Secara empiris herba
pegagan digunakan untuk mengobati radang/inflamasi,
luka, meningkatkan imunitas, menurunkan tekanan darah,
meningkatkan nafsu makan, serta meningkatkan daya
ingat. Menurut penelitian, herba pegagan pada
konsentrasi 1,5% dapat mempercepat penyembuhan luka
pada tikus hiperglikemia dan lebih efektif dibandingkan
dengan salep kombinasi ekstrak herba pegagan dan
ekstrak daun bangun-bangun 5% dan salep komersil
(Kurnawan, 2014).
Pada penelitian ini, ekstrak rimpang kencur dan
herba pegagan dikombinasikan dalam bentuk sediaan gel
yang bertujuan untuk mengetahui efektivitas
antiinflamasi sebagai penyembuhan, dapat bekerja secara
sinergis. Sediaan gel tersebut kemudian dilakukan uji
stabilitas untuk memperoleh informasi mengenai
kemampuan suatu produk obat atau kosmetik untuk
bertahan dalam batas spesifikasi yang diterapkan
sepanjang periode penyimpanan dan penggunaan untuk
menjamin identitas, kualitas, kekuatan dan kemurnian
produk yang diinginkan dalam waktu sesingkat mungkin
dengan cara menyimpan sampel pada suhu tertentu untuk
mempercepat terjadinya perubahan yang bisa terjadi pada
kondisi normal (Djajadisastra, 2004). Selain itu, setiap
produk obat atau kosmetik diharapkan memiliki tingkat
keamanan yang tinggi saat digunakan konsumen sehingga
perlu dilakukan evaluasi keamanan produk yaitu dengan
uji potensi iritasi pada kulit (Mitsui, 1998). Terjadinya
iritasi dikarenakan pH produk obat atau kosmetik tidak
sesuai dengan pH kulit (Rahim & Nofiandi, 2014).
Berdasarkan latar belakang diatas, maka dilakukan
penelitian tentang formulasi dan uji iritasi sediaan gel
dengan kombinasi ekstrak etanol rimpang kencur
(Kaempferia galanga L.) dan ekstrak etanol herba
pegagan (Centella asiatica (L.) Urban) dengan
konsentrasi formula I (0,5:1,5); formula II (1:1); formula
III (1,5:0,5); formula IV (2:0); dan formula V (0:2)
menggunakan HPMC sebagai gelling agent, diharapkan
kombinasi sediaan gel ini memiliki stabilitas fisik gel
yang memenuhi syarat dan tidak mengiritasi kulit.
METODOLOGI PENELITIAN
Penelitian ini dilaksanakan pada bulan Maret sampai
Desember 2018, tempat penelitian antara lain: di Fakultas
Kedokteran Universitas Indonesia untuk pembuatan kaji
etik, di Laboratorium Fitokimia Institut Sains dan
Teknologi Nasional untuk persiapan simplisia,
pembuatan ekstrak dan uji penapisan rimpang kencur dan
herba pegagan, di Laboratorium Farmasetika Institut
Sains dan Teknologi Nasional pembuatan sediaan dan
pengujian stabilitas fisik.
Alat. Alat yang digunakan meliputi lumpang, alu, sudip,
timbangan digital, gelas ukur, beaker glass, cawan
porselen, batang pengaduk, spatel, kaca preparat,
perkamen, kertas saring, evaporator, Viskometer
Brookfield tipe LVT, pH meter (Metrohm), oven
(Memmert)
Bahan. Bahan yang digunakan yaitu rimpang kencur
(Kaempferia galanga L.) dan herba pegagan (Centella
asiatica (L.) Urban) yang diperoleh dari Balai Penelitian
Tanaman Rempah dan Obat (Balitro), etanol 70%, HPMC
(Hydroxy Propyl Methyl Cellulose), propilenglikol, metil
paraben, propil paraben, BHT (Butylated
Hydroxytoluene), aquadest. Bahan uji fitokimia adalah
reagen Mayer, Bouchardat, Dragendorff, FeCl3, gelatin,
AlCl3, amoniak, kloroform, aquadest.
Pembuatan Gel Kombinasi Ekstrak Rimpang Kencur
dan Herba Pegagan. Sediaan gel dibuat sebanyak 5
formula menggunakan kombinasi ekstrak etanol rimpang
kencur dan ekstrak etanol herba pegagan dengan HPMC
sebagai gelling agent. Formulasi gel dapat dilihat pada
Tabel 1.
Sainstech Farma Vol 13 No.1, Januari 2020 | 46
Formulasi dan Uji Iritasi Sediaan Gel Kombinasi Ekstrak Etanol Rimpang Kencur (Kaempferia galanga L.) dan Ekstrak Etanol Herb a Pegagan (Centella asiatica (L.) Urban)
| Febriani et al.
Tabel 1. Formulasi gel
No Bahan
F1
1 Ekstrak rimpang kencur 0,5
2 Ekstrak herba pegagan 1,5
3 HPMC 3 3
4 Propilenglikol 15
5 Metil paraben 0,18
6 Propil paraben 0,05
7 BHT 0,1
8 Aqua dest Ad 100
Uji Stabilitas Fisik Sediaan Gel Kombinasi Ekstrak
Etanol Rimpang Kencur dan Ekstrak Etanol Herba
Pegagan.
Stabilitas Dipercepat. Sediaan gel disimpan pada suhu
diantaranya, suhu rendah 4±2℃, suhu kamar 25±2℃, dan
suhu tinggi 40±2℃ selama 3 minggu, kemudian
dilakukan pengamatan organoleptis, pH, homogenitas,
daya sebar viskositas, sifat alir, sinersis dan daya lekat
(Febriani et al., 2016).
Uji Organoleptik. Pengamatan dilihat langsung secara
visual dari bentuk, warna, dan bau dari sediaan gel yang
dibuat. Dilakukan pada hari ke 0, 7, 14, 21 pada suhu
kamar (Febriani et al., 2016).
Uji Homogenitas. Pengujian homogenitas dilakukan
dengan cara mengoleskan sebanyak 0,1 g gel pada
preparat kaca, sediaan harus menunjukkan susunan yang
homogen (Febriani et al., 2016).
Uji pH. Pengujian pH menggunakan alat pH meter
digital. Sebanyak 5 g gel dilarutkan dengan aquadest
hingga 50 mL. Elektroda pada pH meter dicuci terlebih
dahulu dengan aquadest, selanjutnya dikalibrasi pada
larutan standar pH 4 dan pH 7. Elektroda yang telah
dikalibrasi dicelupkan ke dalam sampel dan diketahui
angka yang ditunjukkan pada pH meter (Remington &
Gennaro, 2000).
Uji Viskositas. Pengujian viskositas dilakukan dengan
menggunakan alat, yaitu Viskometer Brookfield tipe LVT
dipasang spindel nomor 4. Cara pengukurannya yaitu gel
sebanyak 100 mL dimasukkan ke dalam gelas beker,
kemudian spindle dipasang dan dimasukkan sampai batas
yang telah ditentukan. Viskometer dinyalakan dan diputar
dengan rpm 6 sampai jarum viskometer menunjukkan
angka yang konstan.
Uji Sifat Alir. Pengujian sifat alir dilakukan dengan
menggunakan alat yaitu Viskometer Brookfield tipe LVT
dipasang spindel nomor 4. Kecepatan alat dipasang pada
rpm 0,3; 0,6; 1,5; 3; 6; 12 kemudian dibalik menjadi rpm
12; 6; 3; 1,5; 0,6; 0,3. Skala dibaca dengan mengamati
jarum merah saat posisinya telah stabil. Viskositas gel
dihitung menggunakan rumus:
Jumlah (%)
F2 F3 F4 F5
1 1,5 2 0
1 0,5 0 2
3 3 3
15 15 15 15
0,18 0,18 0,18 0,18
0,05 0,05 0,05 0,05
0,1 0,1 0,1 0,1
Ad 100 Ad 100 Ad 100 Ad 100
Viskositas = (skala x faktor koreksi) cPs Rate of Shear
= (rpm faktor spindel x rpm )
Gaya (shearing stress) = (rate of shear x viskositas)
dyne/cm2 ..(3.4)
Faktor perkalian dapat dilihat pada tabel yang sesuai
dengan kecepatan dan spindle yang digunakan.
Perubahan rpm dilakukan agar dapat diukur viskositas
dari berbagai rpm. Sifat alir dari sediaan juga dapat
diketahui dengan cara membuat kurva antara kecepatan
geser (rpm) dengan gaya (dyne/cm2) sesuai dengan data
yang diperoleh kemudian diplotkan pada kertas grafik
antara gaya (x) dengan kecepatan geser (y) kemudian
ditentukan sifat alirnya.
Uji Daya Sebar. Sebanyak 1 g sediaan gel diletakkan di
atas kaca preparat kemudian ditutup menggunakan kaca
preparat lainnya dan diukur diameternya dari lima titik
sudut. Sebanyak 20 g beban diletakkan di atas lapisan gel,
didiamkan selama 1 menit dan dicatat diameter yang
menyebar. Kemudian beban 20 g ditambahkan kembali,
didiamkan selama 1 menit dan dicatat diameter yang
menyebar. Beban 20 g selanjutnya diletakkan di atas gel
hingga beban maksimum di atas gel sebesar ± 100 g, dan
setiap kali beban ditambahkan di atas gel didiamkan
selama 1 menit dan dicatat diameter gel yang menyebar.
Uji daya sebar yang baik berkisar 5 – 7 cm (Voigt, 1994).
Uji Sineresis. Pengujian sineresis dilakukan dengan
menyimpan sampel selama 21 hari yang diamati tiap
minggunya. Pemeriksaan sinersis dilakukan secara visual
dengan mengamati proses terpisahnya cairan dari gel.
Uji Daya Lekat. Pengujian daya lekat dilakukan dengan
cara gel diletakkan di atas dua kaca objek sebanyak 0,25 g,
kemudian ditekan dengan beban 1 kg selama 5 menit.
Setelah itu, kaca objek dipasang pada alat uji lalu
ditambahkan beban 80 g pada alat uji, kemudian dicatat
waktu pelepasan dari kaca objek .
Uji Iritasi Gel Kombinasi Ekstrak Etanol Rimpang
Kencur dan Ekstrak Etanol Herba Pegagan. Uji iritasi
sediaan gel kombinasi ini dilakukan terhadap
sukarelawan dengan menggunakan metode Patch TEST.
Penelitian ini dilakukan pada 13 sukarelawan berumur
rata-rata 18-22 tahun. Pada bagian punggung atas
sukarelawan dibersihkan terlebih dahulu hingga bersih,
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kemudian pada punggung atas sukarelawan dibagi positif yang memberikan warna jingga, hal ini karena
menjadi 5 titik. Setiap titik akan diberikan perlakuan terbentuknya senyawa kompleks antara flavonoid dengan
dengan mengoleskan gel kombinasi dengan konsentrasi AlCl3 (Harborne, 1996). Hasil uji saponin menunjukkan
FI (0,5:1,5); FII (1:1); FIII (1,5:0.5); FIV (2:0); FV (0:2). hasil negatif karena tidak adanya buih yang terbentuk dari
Masing-masing sampel iritan sebanyak ± 80 mg dioleskan pengocokan, baik pada ekstrak maupun pada serbuk
pada bagian punggung atas sukarelawan, lalu ditutup simplisia rimpang kencur, hasil tersebut sesuai dengan
dengan kasa steril kemudian direkatkan dengan plester. penelitian sebelumnya oleh Hasanah et al. (2011). Uji
Selanjutnya diamati pada 30 menit pertama, 24 jam tanin yang menggunakan pereaksi FeCl3 menunjukkan
berikutnya dan 48 jam. Selanjutnya untuk setiap keadaan bahwa ekstrak rimpang kencur memberikan hasil yang
diberi nilai. Data kemudian dinilai berdasarkan kategori negatif karena tidak terbentuk warna hijau atau hitam, hal
terhadap reaksi kulit dan kemudian dihitung untuk ini dikarenakan tidak adanya gugus hidroksil yang ada
memperoleh indeks iritasi primer kulit berdasarkan pada senyawa tanin (Simaremare, 2014). Uji tanin dengan
Primary Irritation Indeks (PII) (Colipa, 1997). menggunakan gelatin menunjukkan hasil positif karena
terbentuk endapan putih, penambahan gelatin untuk
𝐽umlaheritema dan edema jam ke24dan 48 menguji keberadaan tanin, karena tanin memiliki sifat
𝑝𝐼𝐼 = mengendapkan protein. Sedangkan pada uji steroid dan
Jumlah relawan x Jumlah waktu pengamatan triterpenoid memberikan hasil negatif karena tidak
terbentuknya cincin berwarna ungu atau hijau perubahan
HASIL DAN PEMBAHASAN warna yang diinginkan, karena senyawa ini berstruktur
siklik yang kebanyakan berupa alkohol, aldehid, atau
Hasil Maserasi dan Rendemen Ekstrak asam karboksilat yang menyebabkan senyawa ini
Hasil maserasi dari rimpang kencur dan herba cenderung bersifat semipolar sehingga ikatannya dengan
pegagan yang telah dilakukan dengan beberapa tahap dan pelarut etanol yang bersifat polar sangat lemah (Yuliastuti
menghasilkan hasil ekstrak kental dengan rendemen et al., 2017)
rimpang kencur dan herba pegagan masing- masing
sebesar 27,5% dan 21,7% yang berwarna cokelat. Tabel 3. Hasil skrining fitokimia serbuk dan ekstrak herba
pegagan (Centella asiatica (L.) Urban)
Hasil Skrinning Fitokimia.
Hasil skrining fitokimia dari serbuk dan ekstrak Hasil Skrining
Uji Skrining Fitokimia
pada rimpang kencur (Kaempferia galanga L.) dan herba No.
Fitokimia
pegagan (Centella asiatca (L.) Urban) dapat dilihat pada Serbuk Ekstrak
Tabel 2 dan Tabel 3. 1 Alkaloid
Mayer + +
Tabel 2. Hasil skrining fitokimia serbuk dan ekstrak rimpang Bouchardat + +
kencur (Kaempferia galanga L.) Dragendroff + +
2 Flavonoid + +
Hasil Skrining 3 Saponin + +
Uji Skrining 4 Tanin FeCl3
No Fitokimia
Fitokimia Gelatin - +
Serbuk Ekstrak
1 Alkaloid - +
Mayer + + 5 Fenolik - +
Bouchardat + + 6 Uji Steroid + +
Dragendroff + + Keterangan : (+) mengandung senyawa metabolit sekunder
2 Flavonoid + + (-) tidak mengandung senyawa metabolit sekunder
3 Saponin - -
4 Tanin Hasil skrining fitokimia serbuk dan ekstrak etanol
FeCl3 Gelatin - - herba pegagan menunjukkan bahwa herba pegagan
+ + mengandung senyawa aktif yaitu alkaloid, flavonoid,
5 Fenolik - - saponin dan steroid/triterpenoid. Adanya senyawa
6 Uji Steroid / - - alkaloid dengan menggunakan pereaksi Mayer
Tritepenoid menghasilkan endapan hijau lumut, pereaksi Bouchardat
Keterangan : (+) mengandung senyawa metabolit sekunder
menghasilkan endapan cokelat dan pereaksi Dragendroff
(-) tidak mengandung senyawa metabolit sekunder
menghasilkan endapan merah. Adanya senyawa
flavonoid ditunjukkan dengan perubahan warna merah
Hasil uji fitokimia pada ekstrak dan serbuk
atau jingga pada larutan. Adanya senyawa saponin
simplisia menunjukkan bahwa rimpang kencur
ditunjukkan dengan terbentuknya busa stabil selama ± 10
mengandung senyawa aktif yaitu alkaloid, flavonoid dan
menit. Pada uji tanin FeCl3 dan gelatin menunjukkan hasil
tanin gelatin. Adanya senyawa alkaloid dengan
yang berbeda antara serbuk dan ekstrak, hal ini
menggunakan pereaksi Mayer menghasilkan endapan
dikarenakan tanin merupakan senyawa fenolik yang
putih keruh, pereaksi Bouchardat menghasilkan endapan
cenderung larut dalam air dan pelarut polar sehingga
cokelat dan pereaksi Dragendroff menghasilkan endapan
pelarut etanol digunakan dalam ekstraksi yang dapat
merah. Endapan terbentuk karena adanya pembentukan
meningkatkan permeabilitas dinding sel simplisia dan
senyawa kompleks antara ion logam dari reagen dengan
proses ekstraksi menjadi lebih efisien dalam menarik
senyawa alkaloid. Hasil uji flavonoid menunjukkan hasil

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komponen polar hingga semi polar (Harborne, 1996; Uji Organoleptik.

Simaremare, 2014). Hasil uji steroid menunjukkan hasil
positif karena terbentuknya cincin hijau. Hasil skrining
fitokimia pada pegagan sesuai dengan penelitian yang
dilakukan oleh Dwitiyanti et al. (2015).
Hasil pengamatan organoleptik dari stabilitas
sediaan gel kombinasi dari ekstrak rimpang kencur dan
ekstrak herba pegagan menunjukkan kestabilan bentuk,
warna dan bau sediaan setiap minggunya yang disimpan
dan suhu ruang (25℃) dan suhu (40℃). Hasil uji
stabilitas organoleptik sediaan dapat dilihat pada Tabel 4.

Tabel 4. Hasil uji stabilitas organoleptik sediaan

Kamar (25-30℃) Oven (40℃)

Formula/Minggu Ke- 1 2 3 1 2 3
I Bentuk SSP SSP SSP SSP SSP SSP
Warna HK HK HK HK HK HK
Bau BKP BKP BKP BKP BKP BKP
II Bentuk SSP SSP SSP SSP SSP SSP
ORGANOLEPTIK

Warna HK HK HK HK HK HK
Bau BKP BKP BKP BKP BKP BKP
III Bentuk SSP SSP SSP SSP SSP SSP
Warna C C C C C C
Bau BKK BKK BKK BKK BKK BKK
IV Bentuk SSP SSP SSP SSP SSP SSP
Warna C C C C C C
Bau BKK BKK BKK BKK BKK BKK
V Bentuk SSP SSP SSP SSP SSP SSP
Warna H H H H H H
Bau BKP BKP BKP BKP BKP BKP
Keterangan :
SSP = Sediaan Setengah Padat
HK = Hijau Kecokelatan
C = Cokelat
H = Hijau
BKP = Bau Khas Pegagan/Seperti Bau Teh
BKK = Bau Khas Kencur

Uji Homogenitas
Hasil pemeriksaan homogenitas sediaan gel
kombinasi ekstrak rimpang kencur dengan ekstrak herba
pegagan menunjukkan hasil yang baik, tidak terjadi
perubahan homogenitas baik pada penyimpanan di suhu
kamar maupun suhu 40℃ selama 3 minggu. Sediaan gel
dikatakan homogen bila menyebar secara merata, tidak
adanya partikel atau bahan kasar yang dapat diraba dan
dapat diamati oleh mata. Hasil uji stabilitas homegenitas
sediaan dapat dilihat pada Tabel 5.

Tabel 5. Hasil uji stabilitas homegenitas sediaan gel

Suhu (°C) t (minggu) Homogenitas Sediaan

FI F II F III F IV FV
Kamar (25-30) 1 Homogen Homogen Homogen Homogen Homogen
2 Homogen Homogen Homogen Homogen Homogen
3 Homogen Homogen Homogen Homogen Homogen
1 Homogen Homogen Homogen Homogen Homogen
40 2 Homogen Homogen Homogen Homogen Homogen
3 Homogen Homogen Homogen Homogen Homogen

Uji pH 2014), sehingga apabila digunakan aman dan tidak

Hasil pengukuran pH sediaan yang disimpan pada
suhu kamar selama 3 minggu berada di rentang 4,90 –
5,30 (Tabel 6). Hasil pengukuran pH sediaan yang
disimpan pada suhu 40℃ berada direntang 4,97 – 5,37.
Peningkatan pH terjadi pada sediaan namun masih berada
pada rentang pH kulit 4,5 – 6,5 (Rahim & Nofiandi,
mengiritasi kulit. Jika sediaan memliki pH yang terlalu
basa maka dapat menyebabkan kulit menjadi kering,
sedangkan jika pH terlalu asam akan menimbulkan iritasi
kulit. Kondisi penyimpanan yang berbeda tidak begitu
mempengaruhi perubahan pH sediaan. Hasil uji stabilitas
pH sediaan dapat dilihat pada Tabel 6.

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Tabel 6. Hasil uji stabilitas pH sediaan gel

Suhu (°C) t(minggu) pH Sediaan Gel

FI F II F III F IV FV
Kamar (25-30) 1 5,15±0,01 5,23±0,01 5,19±0,02 5,30±0,01 5,30±0,02
2 5,10±0,02 5,22±0,01 5,15±0,01 5,22±0,02 4,97±0,03
3 4,99±0,02 5,12±0,02 4,90±0,02 5,00±0,02 4,90±0,02
40 1 5,19±0,01 5,30±0,02 5,27±0,02 5,35±0,02 5,37±0,01
2 5,07±0,02 5,20±0,02 5,25±0,02 5,28±0,01 5,30±0,01
3 4,97±0,01 5,17±0,01 5,18±0,01 5,23±0,02 5,27±0,01

Uji Daya Sebar menyebabkan semakin besar tahanan yang dialami

Hasil pengamatan uji stabilitas kemampuan
menyebar dari setiap sediaan terdapat pada Tabel 7.
Setiap minggu sediaan pada suhu kamar mengalami
penurunan kemampuan menyebar, sedangkan pada suhu
40°C mengalami peningkatan kemampuan menyebar. Hal
ini disebabkan karena sediaan pada suhu kamar
mengalamai peningkatan viskositas, yang mana akan
sediaan untuk menyebar. Sediaan pada suhu 40℃ bisa
mengalami peningkatan kemampuan menyebar karena
mengalami penurunan viskositas karena pada suhu tinggi
ikatan polimer pada sediaan akan putus mengakibatkan
sediaan semakin encer dan semakin sedikit tahanan pada
sediaan untuk menyebar (Sayuti, 2015) .

Tabel 7. Hasil uji stabilitas kemampuan menyebar sediaan

Formula Suhu (oC) T (minggu) Diameter (mm) r (mm) r2 (mm2) F= πr2 (mm2)
1 57±1,24 28,5 812,25 2550,465
Kamar (25-30) 2 53±0,94 26,5 702,25 2205,065
FI 3 52±1,24 26 676 2122,64
40 1 54±0,94 27 729 2289,06
2 57±0,94 28,5 821,25 2550,465
3 59±0,47 29,5 870,25 2732,585
1 56±1,24 28 784 2461,76
Kamar (25-30) 2 54±0,94 27 729 2289,06
F II 3 50±1,24 25 625 1962,5
40 1 57±1,24 28,5 812,25 2550,465
2 58±0,47 29 841 2640,74
3 60±0,47 30 900 2826
1 55±1,24 27,5 756,25 2374,625
Kamar (25-30) 2 53±1,24 26,5 702,25 2205,065
3 50±1,24 25 625 1962,5
F III 1 56±1,24 28 784 2461,76
40 2 57±1,24 28,5 812,25 2550,465
3 58±1,24 29 841 2640,74
1 52±1,24 26 676 2122,64
Kamar (25-30) 2 52±0,94 26 676 2122,64
F IV 3 50±0,94 25 625 1962,5
1 56±0,94 28 784 2461,76
40 2 59±0,47 29,5 870,25 2732,585
3 61±0,94 30,5 930,25 2920,985
1 54±1,24 27 729 2289,06
Kamar (25-30) 2 54±0,94 27 729 2289,06
FV 3 52±1,24 26 676 2122,64
1 57±0,94 28,5 812,25 2550,465
40 2 57±0,94 28,5 812,25 2550,465
3 60±0,94 30 900 2826

Uji Daya Lekat
Kemampuan daya lekat dilhat dengan menghitung
waktu yang diperlukan untuk memisahkan kedua kaca
objek. Uji daya lekat berhubungan dengan kekentalan
(viskositas). Semakin tinggi viskositas gel, maka waktu
yang diperlukan untuk memisahkan kedua kaca objek
semakin lama. Sebaliknya, semakin rendah viskositas gel,
maka waktu yang diperlukan untuk memisahkan semakin
cepat. Hasil pengamatan uji stabilitas daya lekat dari
setiap sediaan gel yang disimpan pada suhu kamar selama
3 minggu berada direntang 27,15 – 58,02 detik dan pada
suhu 40°C berada direntang 24,66 – 46,56 detik (Tabel 8).
Hasil tersebut sudah memenuhi syarat daya lekat pada
sediaan topikal adalah tidak kurang 4 detik.

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Tabel 8. Hasil uji stabilitas daya lekat sediaan gel

Daya Lekat Sediaan (detik)

Suhu (°C) t (minggu)
FI F II F III F IV FV
Kamar (25-30) 1 58,02±0,80 28,97±0,89 33,42±0,92 35,49±0,92 34,88±0,92
2 39,37±0,92 42,98±0,92 52,23±0,75 51,89±0,64 42,47±0,93
3 30,83±0,75 27,15±0.92 49,05±0,94 46,90±0,84 50,28±0,77
1 33,43±0,93 38,20±0,81 37,25±0,86 46,56±0,76 46,42±0,92
40 2 25,06±0,78 34,00±0,93 36,62±0,89 32,58±0,74 45,02±0,87
3 24,66±0,82 30,60±0,53 31,00±0,92 30,00±0,77 40,78±0,76

Uji Viskositas adanya panas akan memperbesar jarak antar atom

Hasil pengamatan uji stabilitas viskositas sediaan
menunjukkan adanya peningkatan dan penurunan
viskositas pada suhu kamar dan penurunan viskositas
pada suhu 40℃ dengan waktu penyimpanan 3 minggu
(Gambar 1 & 2). Sediaan yang disimpan pada suhu 25℃
(suhu kamar) konsistensinya adanya penurunan dan
peningkatan disebabkan karena menguapnya etanol atau
air, selain itu pemilihan zat pengental, ukuran partikel
yang dapat mempengaruhi konsistensi di dalam sediaan
gel. Sedangkan pada suhu 40℃ mengalami penurunan
viskositas jika suhu dinaikkan. Hal ini dikarenakan
sehingga gaya antar atom berkurang, jarak menjadi
renggang mengakibatkan viskositas menjadi turun. Hal
tersebut juga dapat disebabkan sediaan gel menunjukkan
karakteristik yaitu sinersis yang merupakan proses
keluarnya cairan yang terjerat dalam gel sehingga
memungkinkan cairan untuk bergerak menuju ke
permukaan. (Astuti et al., 2017). Menurut data diatas
dapat disimpulkan suhu dapat mempengaruhi kestabilan
sediaan, dan sediaan tetap berada dalam konsistensi
normal dan dapat dilihat dalam sifat alir sediaan.

Gambar 1. Grafik uji stabilitas viskositas suhu kamar Gambar 2. Grafik uji stabilitas viskositas suhu 40ºC

Uji Sifat Alir
Hasil sifat alir dapat dilihat pada Gambar 3-12.
Berdasarkan hasil pengamatan rheogram sediaan gel
menunjukkan Formula I, II, III, IV dan V memiliki sifat
alir pseudoplastis. Sifat alir pseudoplastis diperlihatkan
oleh sejumlah bahan farmasi yaitu gom alam dan sintesis
seperti disperse cair tragacanth, natrium alginat, metil
selulosa dan beberapa polimer dalam larutan. Sifat alir
pseudoplastis memiliki karateristik yaitu dengan adanya
kenaikan kecepatan geser (rpm) maka viskositasnya akan
turun sehingga sediaan akan mudah mengalir dari wadah.
Sifat alir pseudoplastis mulai pada titik (0,0) atau paling
tidak mendekati pada rate of shear rendah dan tidak ada
yield value. Rheogram lengkung pseudoplastis
disebabkan karena kerja (aksi) shearing terhadap
molekul-molekul bahan yang berantai panjang seperti
polimer-polimer linier. Sifat alir sediaan pada suhu kamar
meningkat setiap minggunya. Peningkatan suhu
mempengaruhi sifat alir sediaan yang menurun setiap
minggunya. Hal tersebut disebabkan karena ikatan
monomer dari molekul–molekul polimer yang ada di
dalam sediaan gel terputus pada suhu tinggi (Martin et al.,
1993).

Gambar 3. Kurva uji stabilitas sifat alir FI suhu kamar Gambar 4. Kurva uji stabilitas sifat alir sediaan FI
suhu 40ºC

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Gambar 5. Kurva uji stabilitas sifat alir FII suhu kamar Gambar 6. Kurva uji stabilitas sifat alir sediaan FII suhu 40ºC

Gambar 7. Kurva uji stabilitas sifat alir FIII suhu kamar Gambar 8. Kurva uji stabilitas sifat alir sediaan FIII suhu 40ºC

Gambar 9. Kurva uji stabilitas sifat alir FIV suhu kamar Gambar 10. Kurva uji stabilitas sifat alir sediaan FIV suhu 40ºC

Gambar 11. Kurva uji stabilitas sifat alir FV suhu kamar Gambar 12. Kurva uji stabilitas sifat alir sediaan FV suhu 40ºC

Uji Sineresis dan V tidak terjadi sineresis (Tabel 9), karena apabila
Berdasarkan hasil pengamatan sinersis gel selama terjadi sineresis dapat mempengaruhi ketidak stabilitas
penyimpanan menunjukkan bahwa formula I, II, III,IV sediaan dan mengakibatkan gel tampak lebih kecil dan

Sainstech Farma Vol 13 No.1, Januari 2020 | 52

Formulasi dan Uji Iritasi Sediaan Gel Kombinasi Ekstrak Etanol Rimpang Kencur (Kaempferia galanga L.) dan Ekstrak Etanol Herb a Pegagan (Centella asiatica (L.) Urban)
| Febriani et al.
padat (Praptiwi, 2014). Sineresis adalah keluarnya air
atau rembesannya cairan dari dalam sediaan dimana air
tidak terikat kuat oleh komponen bahan yang ada. Pada
Tabel 9. Hasil uji sineresis sediaan
T Sineresis Sediaan
Suhu (oC) (minggu) FI
Kamar (25-30) 1 Tidak sineresis
2 Tidak sineresis
3 Tidak sineresis
40 1 Tidak sineresis
2 Tidak sineresis
3 Tidak sineresis
Uji Iritasi Gel
Uji iritasi dilakukan pada 15 orang sukarelawan
untuk mengetahui ada tidaknya reaksi iritasi/alergi. Dari
15 sukarelawan pada awal pengujian, hanya 13
sukarelawan yang memenuhi kriteria inklusi hingga
pengujian berakhir. Dua subjek dikatakan drop out,
karena tidak datang ke tempat penelitian dan menderita
sakit dan sedang minum obat. Berdasarkan hasil
pengamatan terhadap 13 sukarelawan dengan didampingi
dan dikonsultasikan oleh dokter kulit, semua formula
tidak mengiritasi pada semua sukarelawan dan
dinyatakan aman karena dari hasil perhitungan PII
menunjukkan FI, FII, FIII, FIV dan FV dengan nilai 0,05
– 0,08 termasuk kategori tidak berarti (Tabel 10), yang
artinya sediaan gel kombinasi rimpang kencur dan herba
pegagan dapat digunakan secara aman pada kulit.
Tabel 10. Hasil uji keamanan terhadap 13 sukarelawan
Formula Nilai PII Kategori
I 0,08 Tidak berarti
II 0,16 Tidak berarti
III 0,16 Tidak berarti
IV 0,16 Tidak berarti
V 0,08 Tidak berarti
KESIMPULAN
Ekstrak etanol rimpang kencur (Kaempferia
galanga L.) dan Herba Pegagan (Centella asiatica (L.)
Urban) dapat diformulasikan menjadi sediaan gel. Gel
berwarna hijau, hijau kecokelatan, cokelat serta homogen,
memiliki aroma khas kencur dan khas pegagan, memiliki
kemampuan menyebar 1962,5 – 3419,46 mm2 dengan
nilai pH 4,90 – 5,41, daya lekat 24,66 – 59,82 detik,
viskositas antara 15000 – 60000 cPs serta memiliki sifat
alir pseudoplastis. Sediaan gel ekstrak etanol rimpang
kencur (kaempferia galanga L.) dan herba pegagan
(Centella asiatica (L.) Urban) stabil selama 3 minggu
dalam penyimpanan suhu 40℃. Sediaan gel kombinasi
ekstrak etanol rimpang kencur (Kaempferia galanga L.)
dan herba pegagan (Centella asiatica (L.) Urban)
dinyatakan aman digunakan pada kulit dengan nilai pII
0,08 – 0, 16.
fenomena ini, jika sediaan gel didiamkan selama
beberapa saat, maka cairan pembawa dalam matriks akan
keluar atau akan lepas dari matriks (Ningsi et al., 2016).
F II F III F IV FV
Tidak sineresis Tidak sineresis Tidak sineresis Tidak sineresis
Tidak sineresis Tidak sineresis Tidak sineresis Tidak sineresis
Tidak sineresis Tidak sineresis Tidak sineresis Tidak sineresis
Tidak sineresis Tidak sineresis Tidak sineresis Tidak sinersis
Tidak sineresis Tidak sineresis Tidak sineresis Tidak sinersis
Tidak sineresis Tidak sineresis Tidak sineresis Tidak sinersis
UCAPAN TERIMA KASIH
Penulis mengucapkan terima kasih kepada
Kementrian Riset dan Teknologi Dikti yang telah
memberikan dana hibah pada skema PDP 2019.
DAFTAR PUSTAKA
Astuti, D.P, Husni, P., & Hartono, K. (2017). Formulasi
dan uji stabilitas fisik sediaan gel antiseptik tangan
minyak atsiri bunga lavender (Lavandula
angustifolia Miller). Farmaka, 15(1), 1-9.
Chiew, Y.W. (1992). Novel drug delivery system 2nd
edition. New York: Harcel Dekker Inc.
Colipa [The European Cosmetic, Toiletry and Perfumery
Association]. (1997). Product test guidelines for
the assement of human skin compatibility.
European: Colipa.
Djajadisastra, J. (2004). Cosmetic stability. Departemen
Farmasi Fakultas Matematika dan Ilmu
Pengatahuan Alam Universitas Indonesia. Depok:
Seminar Setengah Hari HIKI.
Dwitiyanti, Sediarso, & Kusuma, A.A. (2015). Pengaruh
pemberian fraksi etil asetat ekstrak etanol 70%
Herba Pegagan terhadap penyembuhan luka bakar
pada tikus putih jantan. Media Farmasi, 12(2),
176-185.
Febriani, A, Elya, B, & Jufri, M. (2016). Uji aktivitas dan
keamanan hair tonic ekstrak daun kembang sepatu
(Hibiscus rosa-sinensis) pada pertumbuhan
rambut kelinci. Jurnal Farmasi Indonesia, 8, 259-
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Fitriani, N. (2016). Uji efektivitas gel etil p-
metoksinamat terhadap penyembuhan luka
terbuka pada tikus putih (Rattus norvegicus)
jantan Galur Sprague Dawley. Skripsi. Jakarta:
Universitas UIN Syarif Hidayatullah.
Harborne, J B. (1996). Metode fitokimia penuntun cara
modern menganalisis tumbuhan. Bandung: ITB.
Hasanah, A.N., Nazaruddin, F., Febrina, E., & Zuhrotun
A. (2011). Analisis kandungan minyak atsiri dan
uji aktivitas antiinflamasi ekstrak rimpang kencur
(Kaempferia galanga L.). Jurnal Matematika &
Sains, 16(3).
Hutapea, J.R. (1992). Inventarisasi tanaman obat
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Indonesia edisi I. Jakarta: Departemen Republik
Indonesia.
Irsan, Manggau, M.A., Pakki, E., & Usmar. (2013). Uji
iritasi krim antioksidan ekstrak biji lengkeng
(Euphoria longana Stend) pada kulit kelinci
(Oryctolagus cuniculus). Majalah Farmasi dan
Farmakologi, 17(2).
Kurnawan, E. (2014). Efek salep kombinasi ekstrak daun
bangun-bangun (Coleus amboinicus L.) dan
ekstrak herba pegagan (Centella asiatica
(L.)Urban) terhadap penyembuhan luka eksisi
pada tikus hiperglikemia yang diinduksi aloksan.
Jurnal Mahasiswa Fakultas Kedokteran Untan,
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Fisik Edisi III Jilid 2. Terjemahan Yoshita.
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Nie, Y., Liana, L.K., & Evacuasiany, E. (2012). Pengaruh
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Ningsi, S., Leboe, D.W., & Armaya, S. (2016). Formulasi
dan uji stabilitas fisik gel ekstrak daun binahong
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Praptiwi, Iskandarsyah, & Kuncari, E.S. (2014). Evaluasi,
uji stabilitas fisik dan sineresis sediaan gel yang
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herba seledri (Apium graveolens L.). Buletin
Penelitian Kesehatan, 42(4), 213-222.
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off ekstrak rimpang rumput teki (Cyperus
rotundus L.) sebagai snti jerawat. E-Jurnal
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science and practice of Pharmachy. 20th ed.
Baltimore, Md.: Lippincott Williams & Wilkins.
Sayuti, N.A. (2015). Formulasi dan uji stabilitas fisik
sediaan gel ekstrak daun ketepeng cina (Cassia
alata L.). Jurnal Kefarmasian Indonesia, 5(2), 74-
82.
Simaremare, E.S. (2014). Skrining fitokimia ekstrak
etanol daun gatal (Laportea decumana
(Roxb.)Wedd). Pharmacy, 1(1), 98-107.
Umar, M.I, Asmawi, M Z., Sadikun, A., Atangwho, I.J.,
Yam, M.F., Altaf, R., & Ahmed, A. (2012).
Bioactivity-guided isolation of ethyl-p-
methoxycinnamate, anti-inflammatory
constituent, from Kaempferia galanga L. extracts.
Molecules, 17, 8720-8734.
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Diterjemahkan oleh Soewandhi, S.N. Yogyakarta:
Universitas Gajah Mada.
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Putri, M., & Pradani, K. (2017). Identifikasi
kandungan fitokimia dan Angka Lempeng Total
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Saudi Journal of Biological Sciences (2014) 21, 153–158

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

In vitro evaluation of new functional properties

of poly-c-glutamic acid produced by Bacillus
subtilis D7
Na-Ri Lee a, Tae-Hun Go a, Sang-Mee Lee a, Seong-Yun Jeong b,
Geun-Tae Park c, Chang-Oh Hong a, Hong-Joo Son a,*

a
Department of Life Science and Environmental Biochemistry, Life and Industry Convergence Institute, Pusan National University,
Miryang 627-706, Republic of Korea
b
Department of Medical Life Science, Catholic University of Daegu, Daegu 712-784, Republic of Korea
c
Research and University-Industry Cooperation, Pusan National University, Busan 609-735, Republic of Korea

Received 9 August 2013; revised 10 September 2013; accepted 10 September 2013

Available online 17 September 2013

KEYWORDS
ACE inhibition activity;
Antimicrobial activity;
Moisturizing effect;
Poly-c-glutamic acid
Abstract We investigated the functionality of poly-c-glutamic acid (c-PGA), which is produced by
Bacillus subtilis D7, for its potential applications in medicine and cosmetics. The c-PGA had angio-
tensin-converting enzyme (ACE) inhibition activity. ACE inhibition activity was dependent on the
c-PGA concentration; the highest ACE inhibition activity was observed at 1.25 mg/l of c-PGA.
IC50 (0.108 mg/ml) of the c-PGA was lower than that of standard ACE inhibitory drug, N-[(S)-mer-
capto-2-methylpropionyl]-L-proline (0.247 mg/ml). The c-PGA also had water-holding capacity
and hygroscopicity. Furthermore, the c-PGA inhibited growth of some pathogenic bacteria, includ-
ing Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumonia
and Esherichia coli. The c-PGA exhibited a good metal adsorption capacity; Cr (VI) adsorption
capacity of c-PGA increased with decreasing pH, and the maximal adsorption was observed at
pH 2. Our results suggest that c-PGA may be expected to be widely applied in cosmetics, biomedical
and environmental industries with the feature of being less harmful to humans and the environment.
ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction

* Corresponding author. Tel.: +82 55 350 5544; fax: +82 55 350
5549.
E-mail address: shjoo@pusan.ac.kr (H.-J. Son).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
Poly-c-glutamic acid (c-PGA) is produced by a number of spe-
cies of Bacillus. It is an unusual anionic polyamide that is made
of D- and L-glutamic acid units connected by amide linkages
between a-amino and c-carboxylic groups. The c-PGA is bio-
degradable, edible, water-soluble and non-toxic to humans and
environment. Therefore, it has been suggested for use as biode-
gradable plastics (Tsutomu and Makoto, 2002), flocculants

1319-562X ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
http://dx.doi.org/10.1016/j.sjbs.2013.09.004
154
(Bajaj and Singhal, 2011), biological adhesive (Otani et al.,
1996) and food additives (Lee and Kuo, 2011). For example,
flocculating activity of the c-PGA produced by Bacillus lichen-
iformis CCRC 2826 (Shih et al., 2001) or Bacillus subtilis R23
(Bajaj and Singhal, 2011) has been reported. Some researchers
also reported that the c-PGA could adsorb basic dyes (Inbaraj
et al., 2006).
Hypertension is a common progressive disease leading to
several diseases such as cardiovascular disease, stroke and dia-
betes. Angiotensin-converting enzyme (AC) is a key compo-
nent in rennin angiotensin aldosterone system which
regulates blood pressure (Balasuriya and Rupasighe, 2011).
Inhibition of ACE is a basic approach in the therapy of high
blood pressure in humans. Therefore, screening of potential
ACE inhibitors is expending broadly. ACE inhibitory peptides
have been found in many different food proteins; milk casein,
gelatin, corn gluten, fish proteins and Korean traditional fer-
mented foods (Doenjang, Kanjang) (Ariyoshi, 1993). ACE
inhibitory peptides derived from these food proteins have great
potentials in the development of a novel functional food and
drug for preventing hypertension and therapeutic purposes.
If ACE inhibition activity of the c-PGA was identified, it could
be utilized as a new and harmless drug, since it is edible and
nontoxic toward humans. However, there is no report on the
ACE inhibition activity of the c-PGA. On the other hand,
exposure to external factor, i.e., air humidity, ultraviolet radi-
ation and temperature, as well as endogenous factors, i.e. hor-
mones, may disrupt skin balance between the water content
and lipids on skin. In addition, there is common use of soaps,
detergents and hot water disrupting skin balance (Dal’Belo
et al., 2006). These factors may cause dry skin as well as atopic
dermatitis on skin in severe case. Moisturizers not only in-
crease the skin’s water content but also protect the skin and
encourage an orderly desquamation process that makes the
skin appear smoother. From the cosmetic point of view, there-
fore, the c-PGA could offer a number of cosmetical advanta-
ges if its moisturizing effect would be characterized.
In this study, functionalities of the c-PGA produced by B.
subtilis D7 were screened; especially, ACE inhibition activity
of the c-PGA was investigated in detail to explore its possible
use in the pharmaceutical industry. In addition, various prop-
erties of the c-PGA, such as antimicrobial, moisturizing and
heavy metal adsorbing activities, were also investigated. To
the best of our knowledge, this is the first report highlighting
the ACE inhibition potential of the c-PGA.
2. Materials and methods
2.1. Bacterial strain and culture conditions
B. subtilis D7 used in this study was isolated from Korean tra-
ditional fermented food, Doenjang (Lee 2012). For the produc-
tion of the c-PGA, 100 ml of medium was dispensed into a
300-ml Erlenmeyer flask followed by inoculation with 1 ml of
the strain D7 culture (1.0 · 109 cells/ml) grown in nutrient
broth at 30 C and 200 rpm for 18 h. Cultivation was carried
out at 30 C and 200 rpm in a rotary shaker, and the c-PGA
yield and cell growth were checked at 24 h intervals. The med-
ium used contained the following: 1% L-glutamate, 2% glu-
cose, 0.5% yeast extract, 0.14% K2HPO4 and 0.04%
MgSO4Æ7H2O (Yao et al., 2010). The pH was adjusted to 7.5
prior to sterilization.
N.-R. Lee et al.
2.2. Preparation of c-PGA
The c-PGA was purified by the method of Goto and Kunioka
(1992). Briefly, cells were removed from the culture broth by
centrifugation for 20 min at 17,418g and 4 C. The culture
supernatant was poured into 3 v of ice-cold ethanol and kept
at 4 C for 12 h. The precipitated c-PGA was collected by cen-
trifugation. For further purification, the precipitation step was
repeated three times. The resulting c-PGA was dialysed against
distilled water, and finally freeze-dried for 2 days. Various con-
centrations of the freeze-dried c-PGA were then used to deter-
mine functionality of c-PGA.
2.3. ACE inhibition activity
ACE inhibition activity was measured using the method re-
ported by Cushman and Cheung (1971). 100 ll of substrate
solution (12.5 mM hippuryl-histidyl-leucine acetate) was incu-
bated with 50 ll of sample for 5 min at 37 C, and then 100 ll
of the ACE solution was added and incubated for 30 min at
37 C. The reaction was terminated by adding 250 ll of 1 N
HCl. Hippuric acid was extracted with 1.5 ml of ethyl acetate,
and then the mixture was vortexed for 15 s and centrifuged at
17,418g for 10 min to separate the ethyl acetate layer. A vol-
ume of 500 ll of the supernatant was transferred to another
test tube and evaporated in dry block bath for 1 h at 90 C.
The extracted hippuric acid was redissolved in distilled water
and absorbance was measured at 228 nm. N-[(S)-mercapto-2-
methylpropionyl]-L-proline (Captopril) was used as positive
control. ACE inhibition activity was calculated according to
the following equation:
ACE inhibition activityð%Þ
¼ ½1  absorbance of sample=absorbance of control  100:
The inhibition concentration (IC50) value was calculated
from the plotted graph of inhibition activity versus the concen-
tration of c-PGA. A lower IC50 value indicates the higher inhi-
bition activity.
2.4. Moisturizing effect
Hygroscopy is the ability of a substance to attract and hold
water molecules from the surrounding environment (New-
man et al., 2008). A known concentration of the c-PGA
was put into the desiccator that maintains a high relative
humidity (90%). The desiccator was then incubated at
30 C. During the experiment, the change in the c-PGA
weight was monitored at regular intervals. Glycerol was used
for control. The hydroscopicity of the c-PGA was calculated
as follows:
Hygroscopicity ð%Þ ¼ ½ðW1  W0 Þ=W0   100
where W0 and W1 were initial weight of c-PGA and weight
after moisture adsorption, respectively.
Water-holding capacity is the ability of a substance to re-
tain moisture. A known concentration of the c-PGA was hy-
drated by immersion in droplets of distilled water and
weighed and then incubated at 30 C. During the experiment,
the change in the c-PGA weight was monitored every day. The
water-holding capacity of the c-PGA was calculated as
follows:
In vitro evaluation of new functional properties of poly-c-glutamic acid
Water-holding capacity ð%Þ
¼ ½weight after moisture desorption=initial weight of c
 PGA  100
2.5. Antimicrobial activity
Antimicrobial activity of the c-PGA was evaluated using the
paper disc method (De Beer and Sherwood, 1945). The
microorganisms tested were Gram-negative bacteria (Esche-
richia coli, Salmonella typhimurium, Klebsiella pneumoniae
and Pseudomonas aeruginosa), Gram-positive bacteria (Liste-
ria monocytogenes and Staphylococcus aureus) and yeast
(Candida albicans). The test strains were incubated in nutri-
ent broth at 37 C and 200 rpm for 24 h. The culture broth
was then spread on a nutrient agar plate. The sterile paper
disc (8-mm diameter) was placed on the agar plate and ap-
plied with 1% c-PGA solution. The plate was incubated at
37 C for 2 days and the inhibition zone was observed and
calculated.
2.6. Adsorption of Cr (VI)
A volume of 20 ml of a Cr (VI) solution (100 mg/l) was pre-
pared and adjusted to pH 2–7. The solution was agitated with
2 ml of 1% c-PGA solution at 30 C and 150 rpm for 24 h to
achieve the equilibrium condition. Then sample was taken
from each flask and centrifuged at 17,418g for 15 min. The
supernatant was analysed for the Cr (VI) concentration. The
Cr (VI) concentration in the solution was determined using
the colorimetric method described previously (Clesscerl et al.,
1958). Control experiment was carried out in the absence of
c-PGA. Standard curve was prepared using K2Cr2O7.
All experiments were repeated three times unless otherwise
indicated. The data presented in the tables and figures corre-
sponded to mean values and standard deviation.
3. Results
3.1. Production of c-PGA
Fig. 1 shows the c-PGA production and cell growth by B. sub-
tilis D7 in the medium used. The c-PGA production and cell
growth gradually increased and reached the maximum at
3 days, at which the c-PGA production was 4.7 g/l. The med-
ium became highly viscous due to the production of the c-PGA
along with cell growth.
3.2. ACE inhibition activity of c-PGA
The ACE inhibition activity of the c-PGA is shown in Fig. 2.
The ACE inhibition activity increased with increasing concen-
tration of the c-PGA. The highest ACE inhibition activity was
100% at 1.25 mg/l c-PGA and remained constant thereafter.
Inhibitory concentration (IC50) value of the c-PGA calculated
from Fig. 2 was 0.108 mg/ml. The activity of standard ACE
inhibitory drug (Captopril) was also concentration-dependent
but its IC50 value (0.247 mg/ml) was higher than that of the
c-PGA (Fig. 2).
155
5 3.0
2.5
4
Cell growth (A660)
2.0
PGA (g/l)
3
1.5
2
1.0
1
.5
0 0.0
0 1 2 3 4 5 6
Time (day)
Figure 1 Time courses of c-PGA production and cell growth by
Bacillus subtilis D7 in the medium. Error bars (±S.D.) are shown
when larger than the symbol. d Cell growth and s PGA.
3.3. Moisturizing effect of c-PGA
We investigated some characters of the c-PGA as a moisturiz-
ing agent. As shown in Fig. 3, the hygroscopicity of the c-PGA
increased with increasing the reaction time. Compared to con-
trol that contained glycerol instead of the c-PGA, however, the
hygroscopicity of the c-PGA was low and 86.8% after 72 h. As
shown in Fig. 4, the water-holding capacity of the c-PGA was
reduced as the reaction time increased up to 9 days at which
the water-holding capacity was 56.9%. Similar values of the
water-holding capacity after prolonged reaction were obtained
(data not shown).
3.4. Antimicrobial activity of c-PGA
The antimicrobial activity of the c-PGA is shown in Table 1.
The c-PGA was found to inhibit both Gram-positive (L. mon-
ocytogenes and S. aureus) and Gram-negative (L. monocytoge-
nes, S. typhimurium, K. pneumonia and E. coli) bacteria; S.
aureus was highly inhibited by the c-PGA. One percent of c-
PGA had an antibacterial activity against these bacteria, but
0.1% c-PGA had not (data not shown). Otherwise, the c-
PGA did not exhibit antimicrobial activity against pathogenic
yeast, C. albicans.
3.5. Heavy metal absorption of c-PGA
The influence of pH on the adsorption of Cr (VI) by the c-
PGA is shown in Fig. 5. The absorbed amount of Cr (VI) in-
creased from 5.73 mg/g to 9.12 mg/g with decreasing the initial
pH from 7.0 to 2.0. The influence of the c-PGA concentration
(0.5–5 g/l) on Cr (VI) adsorption is also shown in Fig. 5. The
percentage removal of Cr (VI) increased with increasing the
c-PGA concentration, whereas the amount of Cr (VI) ad-
sorbed per gram of c-PGA was reduced.
4. Discussion
The c-PGA has been known for more than 80 years and many
researchers have investigated the production of c-PGA by bac-
156 N.-R. Lee et al.

120
120

ACE inhibition activity (%)

100

ACE inhibition activity (%)

100

80 80

60 60

40 40

20 20

0 0
0.0 .5 1.0 1.5 2.0 2.5 3.0 0 2 4 6 8 10 12
PGA concentration (mg/ml) Captopril concentration (mg/ml)

Figure 2 Angiotensin-converting enzyme (ACE) inhibition activity of c-PGA and Captopril. Error bars (±S.D.) are shown when larger
than the symbol.

300
teria. Attractive properties of c-PGA, which are water-soluble,
250
edible, safe and environmentally friendly, make it of interest
for applications in the fields of medicine, foods and
bioremediation.
Hygroscopicity (%)

200
ACE plays an important role in the regulating renin–angio-
tensin system of blood pressure. In the rennin angiotensin sys-
150 tem, ACE cleaves the dipeptide portion of angiotensin I from
the C-terminal and produces a potent vasopressor angiotensin
100 II. Angiotensin II induces the release of aldosterone, which
causes the retention of sodium ions by kidney and elevates the
50 blood volume and increased blood pressure (Sun et al., 2009).
Therefore, the inhibition of ACE is a basic approach in the ther-
0
apy of high blood pressure in humans. On the other hand, Kor-
0 12 24 36 48 60 72 84 ean soybean paste, Doenjang is a traditional fermented soybean
Time (hour) food and similar to Japanese Miso. Doenjang not only provides
nutritional value such as essential amino acids and fatty acids
Figure 3 Hygroscopicity of c-PGA and glycerol at 30 C and (Namgung et al., 2010) but also has functionalities such as anti-
90% relative humidity. Error bars (±S.D.) are shown when larger oxidant, ACE inhibition and fibrinolytic activities (Choi et al.,
than the symbol. d PGA and s glycerol. 2007; Park et al., 2008). The quality and functionality of Doenj-
ang are affected by dominant bacteria, B. subtilis and B. lichen-
iformis, during fermentation. In this study, the c-PGA produced
by B. subtilis D7 isolated from Doenjang had the ACE inhibition
120 activity and its IC50 value was 0.108 mg/ml (Fig. 2). To date, the
most potent ACE-inhibitory compounds reported were released
100 from proteins of milk fermented with several strains of Lactoba-
Water-holding capacity (%)

cillus. The ACE inhibition activity of the c-PGA from B. subtilis

D7 was higher than those reported by Leclerc et al. (2002) and
80
Quiros et al. (2005) for whey fractions obtained from casein-en-
riched milk fermented by Lactobacillus helveticus (0.6–1.1 mg/
60
ml) and peptide derived from caprine kefir (0.365 mg/ml). This
result suggests that the ACE inhibition activity of Doenjang
40 might be associated with some compounds of Doenjang, includ-
ing the c-PGA produced by Bacillus strains in Doenjang. In
20 addition, as the c-PGA had ACE inhibition activity, it could
be used as a value-added component to reduce high blood pres-
0 sure as therapeutic drugs. Even though there have been many
0 2 4 6 8 10 12 researches on ACE inhibitory compounds, little is known about
Time (days) the ACE inhibition activity of the c-PGA. Thus, this is the first
report on the ACE inhibition activity of the c-PGA.
Figure 4 Water-holding capacity of c-PGA. Error bars (±S.D.) Moisturizing effect is an important characteristic of a mate-
are shown when larger than the symbol. rial intended for cosmetic application. Ben-Zur and Goldman
In vitro evaluation of new functional properties of poly-c-glutamic acid 157

Table 1 Antimicrobial activity of c-PGA.

Pathogenic microorganism Inhibition zone (mm)a
Gram positive bacteria
L. monocytogenes (a) 11
S. aureus (b) 13
Gram negative bacteria
S. typhimurium (c) 12
K. pneumonia (d) 9
E. coli (e) 11
P. aeruginosa 0
Yeast
C. albicans 0
a
Inhibition zone including paper disc (7 mm).

10 25 12

8 10
20

Removal efficiency (%)

Cr absorbed (mg/g)

Cr absorbed (mg/g) 8
6 15
6
4 10
4

2 5
2

0 0 0
1 2 3 4 5 6 7 8 0 1 2 3 4 5 6
pH PGA concentration (%)

Figure 5 Influence of pH and c-PGA concentration on Cr (VI) adsorption. Error bars (±S.D.) are shown when larger than the symbol.
d Cr adsorbed and s removal efficiency.

(2007) reported that the c-PGA had moisturizing effect and
skin elasticity more than collagen and hyaluronic acid. Also,
water retention of 0.2% c-PGA hydrogel was observed to be
similar to that of 5% glycerol. In this study, the hygroscopicity
of the c-PGA from B. subtilis D7 was 86.8% and its
water-holding capacity was 56.9% (Figs. 3 and 4). These
studies suggest that the c-PGA could have potential material
as alternative to collagen, hyaluronic acid and glycerol in
cosmetic constituent (moisturizer).
Antimicrobial activity is an important characteristic of a
material intended for food and biomedical applications. It
was reported that c-PGA-coated magnetite particles and chito-
san-c-PGA polyelectrolyte complex hydrogels showed antibac-
terial activity against E. coli and S. aureus (Tsao et al., 2010;
Inbaraj et al., 2011). However, little is known about the anti-
microbial activity of pure c-PGA. In this study, the c-PGA
from B. subtilis D7 showed antibacterial activity against L.
monocytogenes, S. typhimurium, S. aureus, K. pneumonia and
E. coli; especially, antibacterial activity was higher against
Gram-positive bacteria than against Gram-negative bacteria.
Although we do not explain the exact mechanism of antibacte-
rial activity of c-PGA, a possible antibacterial mechanism was
reported by Inbaraj et al. (2011). It has been well known that
the proliferation of bacteria is associated with its ability to ad-
here onto material surfaces (bacterial adhesion) through non-
specific interactions such as electrostatic, hydrophobic, van
der Waals forces and specific interactions between the bacterial
cell-membrane receptor and material surface. Inbaraj et al.
(2011) reported that the growth of bacterial cells could be sig-
nificantly reduced by materials that are hydrophilic and anio-
nic (as the net surface charge of bacterial cell is also negative)
and therefore the antibacterial activity of the c-PGA may be
due to the hydrophilic and anionic nature of the c-PGA. Nev-
ertheless, a further study is necessary to elucidate the exact
mechanism of the antibacterial effect of the c-PGA. On the
other hand, the paper disc diffusion assay for the measurement
of antimicrobial activity was used in this study. In this case, it
is important to consider the relationship between the molecu-
lar weight of the c-PGA and the rate of diffusion, which is re-
flected on significance of the results. Therefore, further studies
are also needed to investigate how the diffusion rate of the c-
PGA through a paper disc is influenced by its molecular
weight.
Some researchers reported that the c-PGA could adsorb Hg
(II), Cr (III), Ni (II) and basic dyes (Inbaraj et al., 2006; Yao
et al., 2007). Chromium exists in water supplies in both the
hexavalent and the trivalent state although the trivalent form
rarely occurs in potable water. Hexavalent chromium has been
shown to be carcinogenic by inhalation and is corrosive to tis-
sue (Clesscerl et al., 1958). Therefore, we investigated a poten-
tial as an adsorbent for the removal of Cr (VI). As shown in
Fig. 5, Cr (VI) was strongly adsorbed at a low pH and its
158
adsorption was declined at a higher pH range. Better adsorp-
tion at low pH by the c-PGA may be attributed to a large
number of H+ ions present at low pH which in turn neutralize
the negatively charged adsorbent surface, thereby reducing dis-
turbance to the diffusion of chromate ions (Rao et al., 2002).
As shown in Fig. 5, with increasing c-PGA concentration,
the Cr (VI) removal efficiency was enhanced, whereas the
amount of Cr (VI) adsorbed showed a decline. This result
may be attributed to the adsorption sites remaining unsatu-
rated during the sorption reaction. In other words, a less com-
mensurate rise in Cr (VI) adsorption could occur following the
increase of the c-PGA concentration, probably due to a lower
adsorptive capacity utilization of the c-PGA. In addition, the
particle interaction at high c-PGA concentration may result
in desorption of some weakly bound Cr (VI) from the c-
PGA surface (Inbaraj et al., 2006). In this study, the highest
adsorbed quantity of Cr (VI) by the c-PGA was 21.4 mg/g
(Fig. 5); indicating the lower absorbed quantity as compared
with other c-PGA studies as an adsorbent for the removal of
heavy metal ion (Rao et al., 2002; Mark et al., 2006). Neverthe-
less, our result suggests that the c-PGA may play a role in Cr
(VI) adsorption as an adsorbent even though it has low
adsorption.
In conclusion, the c-PGA produced by B. subtilis D7 had
novel functionalities such as ACE inhibition activity, antibac-
terial activity and moisturizing effect. These results suggest
that the c-PGA could be considered a potential candidate
for moisturizer, therapeutic drug and antimicrobial agent in
the fields of pharmaceutical, food and cosmetics industries.
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 Chemosphere 305 (2022) 135031

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Translating wastewater reuse for irrigation from OECD Guidelines:

Tramadol sorption and desorption in soil-water matrices.
Andrea-Lorena Garduño-Jiménez a, Juan Carlos Durán-Álvarez b, Ruth Silvana Cortés-Lagunes b,
David A. Barrett c, Rachel L. Gomes a, *
a
Food Water Waste Research Group. Faculty of Engineering, University of Nottingham, University Park, Nottinghamshire, NG7 2RD, United Kingdom
b
Instituto de Ciencias Aplicadas y Tecnología, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Coyoacan, Ciudad de México,
04510, Mexico
c
Centre for Analytical Bioscience, Advanced Materials and Healthcare Technologies Division, School of Pharmacy University of Nottingham, University Park,
Nottinghamshire, NG7 2RD, United Kingdom

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• First study of tramadol soil/water par­

titioning at environmentally relevant
conditions. Higher tramadol sorption to Tramadol sorption decreases
• Significantly (p < 0.05) higher tramadol soil from wastewater effluent accordingly with initial tramadol
sorption to soils with higher clay than calcium chloride buffer concentration
content.
• Tramadol sorption is mainly governed Tramadol
by cation exchange.
Calcium Wastewater
• Sorption-desorption hysteresis demon­ effluent
Chloride buffer
strates potential tramadol soil
accumulation.
• Higher tramadol sorption to soil from
wastewater effluent than calcium chlo­
ride buffer.

A R T I C L E I N F O A B S T R A C T

Handling Editor: Klaus Kümmerer Treated and untreated wastewater is often used for agricultural irrigation and, despite the many benefits of this
practice, it poses the risk of biologically active chemical pollutants (such as pharmaceuticals, like tramadol)
Keywords: entering the environment. The partitioning of tramadol between soil/water at environmentally relevant con­
Wastewater reuse centrations is important to understand its environmental toxicity. Kinetics and isotherm sorption studies based
Environmental fate
on the Organisation for Economic Cooperation and Development (OECD) 106 Guideline were undertaken,
Sorption isotherms
ensuring comparability to previous studies. Studies were undertaken in three soils of different characteristics
Soil
Tramadol using aqueous concentrations of tramadol from 500 ng L− 1 (environmentally relevant) to 100 μg L− 1 (compa­
Pharmaceuticals rable to previous studies). Two of the soils presented a significantly (p < 0.05) higher sorption at a lower initial
Agricultural irrigation tramadol concentration (5000 ng L− 1), compared to 20,000 ng L− 1. Hysteresis was observed in all studied soils,
indicating the accumulation of tramadol. Higher sorption to soils correlated with higher clay content, with soil/
water partitioning coefficients (Kd) of 5.5 ± 13.3, 2.5 ± 3.8 and 0.9 ± 3.0 L kg1 for soils with clay contents of
41.9%, 24.5% and 7.4%, respectively. Cation exchange was proposed as the main sorption mechanism for tra­
madol to soils when the pH was below tramadol’s pKa values (9.41 and 13.08). A comparative kinetics study
between tramadol in soil/calcium chloride buffer and soil/wastewater effluent demonstrated significantly higher
(p < 0.05) tramadol sorption to soil from wastewater effluent. This has the environmental implication that clay

* Corresponding author.
E-mail address: rachel.gomes@nottingham.ac.uk (R.L. Gomes).

https://doi.org/10.1016/j.chemosphere.2022.135031
Received 15 February 2022; Received in revised form 9 May 2022; Accepted 17 May 2022
Available online 20 May 2022
0045-6535/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A.-L. Garduño-Jiménez et al.
soils will be able to retain tramadol from irrigation water, despite the organic content of the irrigation water.
Therefore, our studies show that tramadol soil sorption is likely to be higher in agricultural environments reusing
wastewater than that predicted from experiments using the OECD 106 Guideline calcium chloride buffer.
1. Introduction
Tramadol is an opiate widely used since 1995, that provides effective
reduction of moderate to severe pain at relatively low risk, in terms of
addiction and respiratory depression, compared with other opiates
(Gong et al., 2014; Subedi et al., 2019). However, the presence of tra­
madol in the environment is of concern given its known persistence and
effects in terms of acute toxicity (Petrie et al., 2015; Thiebault et al.,
2015). About 30% of the tramadol dose is excreted in the urine as the
parent drug and the rest is excreted as metabolites (Gong et al., 2014).
The widespread use of this drug means that large quantities of un­
changed tramadol can be found globally in municipal wastewater
influent at concentrations of hundreds of ng L− 1 (Du et al., 2021; Petrie
et al., 2015; Silva et al., 2017) to tens μg L− 1 (Petrie et al., 2015). Due to
incomplete removal in conventional wastewater treatment processes
(WWTPs), tramadol is present in effluents at similar concentrations as in
the influent (Petrie et al., 2015, 2017; Silva et al., 2017). After waste­
water effluents are released to receiving surface waters and undergo
dilution and attenuation, tramadol remains at concentrations ranging
from <10 ng L− 1 to >100 μg L− 1 (Campos-Mañas et al., 2019; Petrie
et al., 2017).
Using raw untreated or treated wastewater for irrigation is a wide­
spread global practice (Thebo et al., 2017), and a source for bioactive
chemical pollutants (BACs), such as tramadol, to enter the agricultural
environment (Carter et al., 2019). Transfer of tramadol from wastewater
to soil has been observed, with tramadol having been quantified in
Cameroonian soils at 21 ng g− 1 in the wet season and 1470 ng g− 1 in the
dry season (Kusari et al., 2016). Once in contact with agricultural soil,
the sorption of organic pollutants is a determinant factor in the potential
for subsequent leaching to groundwater (Llamas et al., 2020; Paz et al.,
2016), bioavailability (Goulas et al., 2018; Li et al., 2019) and uptake to
plants, which can lead to pollutants entering the food network (Azanu
et al., 2018; de Santiago-Martín et al., 2020; Kodesova et al., 2019;
Singh, 2021). Sorption to soil is pollutant-dependent (Conde-Cid et al.,
2020; Gregg et al., 2015; Kiecak et al., 2019; Li et al., 2020), therefore it
is important to generate pollutant-specific data.
Tramadol sorption to soil has been barely studied (Barron et al.,
2009; Filep et al., 2021; Stein et al., 2008; Wojsławski et al., 2019) and
discrepancies between its behaviour in batch studies and packed column
tests have been observed (Wojsławski et al., 2019). In batch tests, it was
found to be highly mobile in soils, conversely in packed column studies
tramadol was able to bind strongly to non-structured soil (Wojsławski
et al., 2019). Discrepancies between batch and column tests can be due
to the differing hydraulic conditions and the soil packing itself. How­
ever, one way to bridge these discrepancies is to better emulate envi­
ronmental conditions in batch studies. Bench-scale studies done to date
have been at concentration levels higher than those observed in the
environment or only at the higher end of those observed in wastewater
influent (equating to raw untreated) and effluent (concentration of
tramadol in the liquid phase of batch sorption studies of 25–2500 μg L− 1
(Stein et al., 2008), 125–1000 μg L− 1 (Barron et al., 2009), 250–10,000
μg L− 1 (Paz et al., 2016) and 100–5000 μg L− 1 (Filep et al., 2021)). While
the lower range of these concentrations are relevant for countries where
raw (untreated) wastewater is used for irrigation (Thebo et al., 2017),
they are not so relevant where treated wastewater effluents or surface
waters are used for irrigation. Therefore, it is important to perform
laboratory scale batch-sorption studies at environmentally relevant
concentrations, since sorption is concentration dependent (Paz et al.,
2016). This being due to specific interactions between the dissolved
contaminants and the functional groups on the soil organic matter,
2
Chemosphere 305 (2022) 135031
which are dominant at lower sorbate concentrations (Paz et al., 2016).
To address this knowledge gap, this study evaluated several con­
centrations, at 3 orders of magnitude lower than those previously
studied, corresponding to tramadol concentrations reported in surface
water (i.e., the lowest concentration studied was 500 ng L− 1)
(Campos-Mañas et al., 2019; Petrie et al., 2017). Higher initial con­
centrations of tramadol (the highest was 100 μg L− 1) were also studied
in order to have an order of magnitude in the range of tramadol con­
centrations observed in influent and effluent wastewaters (Barron et al.,
2009; Filep et al., 2021) as well as in the range of tramadol batch
sorption studies published to date. It is important to produce data that is
comparable to previous published studies to support meta-analysis,
hence obtaining a better understating of the sorption behaviour of tra­
madol across different studies. Another consideration is the reality that
wastewater and surface waters contain many other constituents from
BACs, including tramadol, to organic matter and mineral cations, each
with the potential to compete for sorption sites in the receiving soil
(Chefetz et al., 2008; Conde-Cid et al., 2019; Paz et al., 2016) or able to
interact with the sorbate to form complexes with different sorption
properties (Krogh et al., 2008). Therefore, the objectives of this paper
were to:
i. Provide data on batch sorption of tramadol from water to soils which
is comparable to published data for other organic pollutants based on
the OECD 106 Guideline, allowing for a more comprehensive un­
derstanding of the fate of BACs found in wastewater irrigation
systems.
ii. Compare the data obtained using calcium chloride buffer as the
aqueous phase, to a more environmentally relevant aqueous phase
(wastewater effluent) and an environmentally relevant concentra­
tion range of tramadol to better understand its behaviour in the
environment.
2. Materials and method
2.1. Chemicals and reagents
Ammonium bicarbonate (≥99%), formic acid (≥99.5%), and HPLC
grade methanol, water and acetonitrile were acquired from Fisher
(Loughborough, UK). Tramadol analytical standard (>98%, purity) was
bought from Sigma-Aldrich (Dorset, England). Anhydrous calcium
chloride (>97%, purity) was bought from Sigma-Aldrich (Dorset, En­
gland). Tramadol stock solution was prepared by dissolving 4 mg in 4
mL of methanol, and stored at − 20 ◦ C. For each experiment an aliquot of
this main stock was used to prepare a working stock in the tested
aqueous phase at the necessary concentration, ensuring methanol con­
tent was below 5%.
2.2. Soils and wastewater effluent characterisation
Three characterised soils (Soil 1, Soil 2 and Soil 3, Table 1) were
obtained from Lufa-Speyer (Speyer, Germany). Soil was autoclaved
prior to the sorption experiments, and the moisture after autoclaving
was determined gravimetrically (Table 1). Wastewater effluent was
sampled from Stoke Bardolph WWTP (Nottinghamshire, UK) in July
2021. This process plant serves a population equivalent of ~650,000
and utilises a mixture of conventional and advanced activated sludge.
Wastewater samples were filtered through a 1.2 μm microglass fibre
filter paper (Fisher, Leicestershire, UK) and characterised (Table 1).
Wastewater quality parameters were quantified as detailed in Section
A.-L. Garduño-Jiménez et al. Chemosphere 305 (2022) 135031

Table 1 effluent with the endogenous tramadol concentration of 724.7 ± 20.9

Characterised soils (Soil 1, Soil 2 and Soil 3) from Lufa-Speyer (Speyer, Ger­ ng L− 1, 0.01 M CaCl2 buffer solution with 20,000 ng L− 1 tramadol, and
many) and wastewater effluent characterisation. wastewater effluent with the endogenous concentration of 724.7 ± 20.9
MATRIX Soil 1 Soil 2 Soil 3 WASTEWATER ng L− 1 plus spiking at 20,000 ng L− 1. This study was also performed
CHARACTERISTIC EFFLUENT (ON DAY using the parallel method (in triplicate) and the time spans tested were
CA2+ WAS MEASURED) 0.25, 0.5, 0.75, 1, 24, 48 and 72 h.
PH 7.3 ± 6.2 ± 7.5 ± 6.75 (6.99)
0.1 0.3 0.1
2.4. Sample preparation and tramadol quantification
CLAY (%) 41.9 7.4 ± 24.5 –
± 2.7 0.9 ± 1.8
SILT (%) 54.9 90.3 73.9 – Preliminary mass balances were undertaken to determine there was
± 1.0 ± 0.9 ± 2.0 no tramadol sorption to the tubes and stability tests were carried out to
SAND (%) 2.2 ± 2.4 ± 1.5 ± – test for tramadol degradation in the aqueous phase within 72 h. A mass
0.4 0.2 0.4
ORGANIC CARBON 1.55 0.66 1.83 –
balance was carried out to ensure accurate quantification of tramadol in
CONTENT (%) ± ± ± the soil phase by difference, when measuring the tramadol only in the
0.14 0.09 0.25 aqueous phase (Section S2). Control samples consisting of the aqueous
NITROGEN (%) 0.18 0.08 0.23 – phase with no soil, in triplicate were analysed for all and used in the
± ± ±
calculations as the initial concentration of tramadol in the aqueous
0.01 0.02 0.02
CEC (MEQ/100G) 18.7 6.0 ± 17.6 – phase. After sorption tests, the samples were centrifuged for 8 min at
± 1.2 0.9 ± 0.9 4000 rpm and 4 ◦ C, then 1 mL aliquot was placed in LC vials and stored
MOISTURE CONTENT 7.0 ± 4.9 ± 9.6 ± – at 4 ◦ C until analysis (carried out within 24 h) by LC-MS/MS (Section
AFTER 1.4 2.7 2.3 S3). Where the liquid phase was wastewater effluent, the 1 mL aliquot
AUTOCLAVING (%)
TEMPERATURE (◦ C) – – – 19.0 (17.3)
was filtered through 0.22 μm microglass fiber filter paper (Fisher, Lei­
TOTAL SUSPENDED – – – 5 (4) cestershire, UK) prior to analysis. Any losses of tramadol sorbed on the
SOLIDS (MG L− 1) filter were accounted for by comparing to control samples. Analysis was
CHEMICAL OXYGEN – – – 34.58 (11.6) carried out in a triple quadrupole 1290 Series HPLC-MS/MS, using an
DEMAND (MG L− 1)
Agilent jet stream electrospray ioniser (Agilent Scientific, Cheshire, UK).
TRAMADOL (N GL− 1) – – – 724.7 ± 20.9
NA (MG L− 1) – – – 67.4 ± 1.3 The LC-MS/MS method used a C18 Gemini column (3 μm particle
MG (MG L− 1) – – – 22.4 ± 0.4 size, 110 Å pore size, 100 × 3 mm, Phenomenex, Cheshire, UK) at 50 ◦ C
S (MG L− 1) – – – 38.9 ± 1.0 and an injection volume of 10 μL. Mobile phase A was made up of 0.1%
K (MG L− 1) – – – 16.3 ± 0.4 formic acid in water and mobile phase B was 100% acetonitrile. The
CA (MG L− 1) 62.9 ± 2.5
starting mobile phase composition was 95% A and it was ramped to 5%
– – –

S1. The concentrations of BACs and minor cations (concentrations < mg
L− 1) in wastewater can be found in Table S1.
2.3. Batch sorption experiments set up: kinetics and sorption isotherms
Preliminary work was undertaken to determine the adequate soil/
liquid ratio for the sorption tests. In all assays, 1 g of soil and 10 mL of
liquid phase (ratio 1/10) were contacted, in compliance with the OECD
106 Guideline recommendations (OECD Guideline for testing chemicals
No. 106, 2000). Experiments were undertaken in 15 mL falcon tubes
using a horizontal shaker and incubated at 20 ◦ C.
2.3.1. Kinetics and isotherm studies for the three soils using a 0.01 M CaCl2
buffer
For the kinetics experiments, two tramadol concentrations in the
liquid phase were tested: 5000 and 20,000 ng L− 1. The contact time was
set as 1, 2, 3, 7, 24, 48 and 72 h for the lower concentration level, and
0.25, 0.5, 1,3, 7, 24, 48 and 72 h for the higher concentration level. The
parallel method was used as recommended by the OECD 106 Guideline
(OECD Guideline for testing chemicals No. 106, 2000), consisting of
triplicates for each time point prepared in separate falcon tubes and
analysed separately.
To obtain the sorption isotherms, the tested concentrations were of
500, 1,000, 5,000, 20,000, 50,000, and 100,000 ng L− 1, with each
concentration level tested in triplicate. Isotherm samples were analysed
after 72 h contact time. Both the kinetics and isotherm studies were
carried out for the three soils.
2.3.2. Kinetics study comparing 0.01 M CaCl2 buffer to wastewater effluent
as aqueous phase
Kinetics studies to compare sorption of tramadol to soil using 0.01 M
CaCl2 buffer solution against wastewater effluent were carried out using
only Soil 1. Three different aqueous phases were used: wastewater
An over 6 min; this composition was kept for 1.6 min, then it was
ramped back to 95% An over 0.8 min and then kept at 95% A for 1.4
min. The gradient lasted for a total of 10 min and the flowrate was 0.3
mL min− 1. The retention time for tramadol was 2.9 min, and a chro­
matogram and mass spectra can be seen in Figure S2. The drying gas
flow was 7 L min− 1, sheath gas flow was 12 L min− 1, while the source
temperature was 200 ◦ C and the nebuliser pressure was 50 psi. Tramadol
was monitored in positive ionisation mode, using multiple reaction
monitoring (MRM) mode, with a precursor ion of m/z = 264.2 and
quantifier and qualifier ions of m/z = 246.0 and 58.2, respectively.
Fragmentor voltages were 90 and 100 V, collision energies were 4 and
28 eV and the cell accelerator voltages were 7 and 4, for the quantifier
and qualifier ions, respectively. The instrumental limit of detection and
linearity were 0.02 ng mL− 1 and 0.9972, respectively. The intra-day and
inter-day method precision were 0.6% and 0.1%, respectively. The intra-
day accuracy was 99.9%.
2.5. Data analysis
The data from the batch-sorption experiments was analysed using
the soil-liquid partitioning coefficient Kd (OECD Guideline for testing
chemicals No. 106, 2000), with Kd calculated using Equation (1).
m v
Kd = (1)
(1 − m) s
where m is the fraction of tramadol sorbed onto soil particles, v is the
volume of the liquid (mL) phase and s the amount of soil (g)
(Durán–Álvarez et al., 2014). Data was fitted to the linearised form of
the Freundlich and Langmuir models (Equations (2) and (3)).
1
log(qe ) = log Kf + log(Ce ) (2)
n
where Kf is the Freundlich constant or maximum adsorption capacity, 1n
is the slope obtained from plotting log (qe ) against log(Ce ) in the linear

3
A.-L. Garduño-Jiménez et al.
range (Singh, 2016), qe is the equilibrium concentration of the adsorbate
in the soil (mg g− 1), and Ce is the equilibrium aqueous phase concen­
tration of the adsorbate (mg L− 1),
1 1 1 1
= × +
qe KL Cmax Ce Cmax
For the Langmuir linearised model, Cmax is the maximum adsorption
capacity and KL is the Langmuir’s isotherm constant showing the sor­
bate’s affinity for the soil.
The apparent hysteresis index (HI) was calculated as detailed in
Equation (4):
qe d
HI =
qe s
where qe d and qe s are the concentration of tramadol in the soil for the
desorption and sorption experiments, respectively.
3. Results and discussion
3.1. Mass balance
Stability tests demonstrated no tramadol sorption to the falcon tubes
and no tramadol degradation in the liquid phase within 72 h. Control
samples (containing no soil) were used to corroborate the initial con­
centration for all sorption experiments. The results from mass balance
experiments (Table S2) demonstrated that it was possible to accurately
quantify tramadol in the soil and liquid phases by difference between the
tramadol concentration measured in the supernatants obtained
throughout the sorption experiment and the initial concentration
measured in the control samples.
Fig. 1. Kinetics of percentage sorption of tramadol at two initial concentrations: 20,000 ng L−
Chemosphere 305 (2022) 135031
3.2. Tramadol sorption kinetics using calcium chloride buffer as liquid
phase
No significant difference (p < 0.05) was observed in the sorption rate
(3) at 1 or 0.25 h compared to the tramadol sorption at 72 h for any of the
three soils at either concentration level (5000 and 20,000 ng L− 1). Thus,
the sorption of tramadol was deemed to be fast in the tested soils.
However, since variability was observed across the time scale for the
sorption at each point (Fig. 1), a 72 h equilibration time was chosen for
further work. For Soil 1, sorption equilibrium was observed from 7 h for
the lowest initial concentration, and from 24 h for the highest initial
concertation (Fig. 1a). In Soil 2, sorption reached equilibrium from 24 h,
(4) at both initial concentrations (Fig. 1b). For Soil 3, at the lowest initial
concentration, equilibrium was achieved from 24 h, while equilibrium
was observed from 48 h for the highest starting concentration (Fig. 1c).
Previous results have reported equilibrium of tramadol on soils is ach­
ieved after 24 h using 0.01 M CaCl2 as liquid phase (Barron et al., 2009;
Stein et al., 2008; Wojsławski et al., 2019).
For Soil 2 and Soil 3 there was a significant difference in the sorption
percentage depending on the initial tramadol concentration, with
significantly (p < 0.05) higher sorption for the lowest initial concen­
tration (5000 ng L− 1), compared to that observed when 20,000 ng L− 1
was the initial concentration (Fig. 1b and c, respectively); this is
consistent with that previously reported (Paz et al., 2016). Conversely,
sorption of tramadol was not significantly different as a function of the
initial concentration in Soil 1 (Fig. 1a). These results indicate high af­
finity of tramadol to Soil 1, which can be attributed to its high clay and
organic carbon contents, resulting in more sorption sites than in Soils 2
and 3. In fact, comparing the sorption kinetics of Soils 1 and 3 (both with
similar content of organic carbon), equilibrium was faster in the former,
suggesting that clay has a more relevant role in the sorption kinetics
than the organic carbon content, while other soil components, such as
1 1
and 5000 ng L− to the three soils studied; a) Soil 1, b) Soil 2, c) Soil 3.
4
A.-L. Garduño-Jiménez et al. Chemosphere 305 (2022) 135031

sand and silt, have little to no effect in the sorption process.
3.2.1. Tramadol soil affinity depending on soil properties
Higher affinity of tramadol to soil was observed in the order, Soil 1
(pH 7.3) > Soil 3 (pH 7.5) > Soil 2 (pH 6.2) (Table 2). This is to be
expected, as tramadol is ionised at the soil pH, given it has a tertiary
amide moiety which can gain a positive charge at pH < pKa (9.41 and
13.08 (Wishart et al., 2017)) (speciation diagram is presented in
Figure S3). The pH of the aqueous phase was below tramadol’s pKa and
did not vary significantly (p < 0.05) during the experiment (Soil 1: 6.9
± 0.3, Soil 2: 6.7 ± 0.3, Soil 3: 6.8 ± 0.2). As a positively ionised
molecule, tramadol can be attracted to the negatively charged organic
matter and clay particles, as has been previously reported (Stein et al.,
2008; Wojsławski et al., 2019). Higher affinity of tramadol was observed
as the clay content increased, as follows: Soil 1 (41.9%) > Soil 3 (24.5%)
> Soil 2 (7.4%). The cation exchange capacity of the soils was in the
order of Soil 1 (18.7 ± 1.2 meq 100− 1) > Soil 3 (17.6 ± 0.9 meq 100− 1)
> Soil 2 (6.0 ± 0.9 meq 100− 1), which also accounted for the order
observed for tramadol’s sorption affinity to the soils, and is in line with
that reported elsewhere (Jolin et al., 2017). Tramadol has been observed
to present higher sorption to soils with high organic matter content
(Filep et al., 2021; Szabó et al., 2020), due to the capacity of organic
matter to retain positively charged molecules via bonding with the –CO.
O− moieties (Filep et al., 2021). Another sorption mechanism for tra­
madol is partitioning to organic matter, given the organic nature of
tramadol (LogP = 1.39 at neutral pH). The benzene ring can readily
anchor with the soil organic matter, as occurs with other organic pol­
lutants (Hiller and Šebesta, 2017). In the case of the studied soils, clay
content and cation exchange capacity likely have a higher influence on
tramadol sorption, whereas the role of organic carbon remains uncertain
given that the range of the organic carbon content between the soils is
rather narrow (Soil 3: 1.83% > Soil 1: 1.55%, and Soil 2: 0.66%).
3.3. Tramadol sorption-desorption isotherms
3.3.1. Soil-liquid partitioning (Kd values)
The Kd value provides a snap-shot of the ratio of the pollutant in the
aqueous phase when equilibrium has been achieved (OECD Guideline
for testing chemicals No. 106, 2000). In line with that observed in ki­
netics studies, the Kd values were in the order Soil 1 > Soil 3 > Soil 2
(Table 2). For Soil 1, Kd were between 5.1 ± 0.4 and 13.3 ± 6.3 L kg− 1,
whereas for Soil 3 it was between 2.5 ± 0.0–6.0 ± 3.9 L kg− 1, and for
Soil 2 between 0.9 ± 0.0–3.0 ± 2.1 L kg− 1. For Soil 1 and Soil 3, the
sorption behaviour was linear, presenting R2 values above 0.99, indi­
cating that the adsorption sites on the surface of the soil particles were
not fully occupied in the range of the initial concentrations tested. On
the other hand, a non-linear isotherm was obtained for Soil 2, indicating
saturation of adsorption sites. For this soil, the linear range of the
sorption isotherm was found when initial concentrations below 20,000
ng L− 1 were used.
High variability (higher standard deviation between the replicates)
in the sorption kinetics and the Kd values were observed for Soil 1, which
may be attributed to its higher clay content, compared to Soil 2 and Soil
3. It is known that sorption-desorption equilibrium of pharmaceuticals
may be readily achieved with the organic components (presumably
occurring through partition), while cation exchange with clay and other
mineral components leads to a reversible equilibrium (Strawn, 2021).
The Kd values obtained herein are in a similar range to a previous report
on a sandy soil, despite the initial tramadol concentration range in this
reference being up to four orders of magnitude higher (Wojsławski et al.,
2019). However, in the referred study the liquid phase was made up of
0.01 M CaCl2 plus 0.1 g L− 1 sodium azide and the soil:liquid ratio used
was not the same for all soils (Wojsławski et al., 2019), which is not
directly comparable to the 0.01 M CaCl2 solution used in the present
study. On the other hand, The Kd values obtained herein were an order
of magnitude lower than those reported by another study using a silt soil
(Barron et al., 2009). The experimental set-up of (Barron et al., 2009)
was the same as the present study, so the difference in Kd values high­
light the importance of the soil texture on the sorption behaviour of
tramadol besides the content of organic matter.
Out of the three soils studied, the desorption of tramadol was the
highest for Soil 2 at Kd values of 3.3–9.9 (Table 2), being consistent with
the low sorption rate. Considering the low organic carbon and clay
contents in Soil 2, tramadol may be sorbed on slit and sand particles
through weak interactions, which were rapidly broken when fresh CaCl2
solution was added in the desorption step. For all three soils, higher (p <
0.05) desorption Kd values were observed as the initial concentration
was increased (Table 2). In the case of Soil 2, the desorption Kd values of
tramadol were significantly (p < 0.05) higher at concentrations of
20,000 and 50,000 ng L− 1 compared to those obtained when initial

Table 2
Linear sorption and desorption partitioning coefficient (Kd) and hysteresis index (HI) at 72 h equilibrium in the three different soils tested (Soil 1, Soil 2 and Soil 3).
Initial tramadol Kd Kd desorption Soil 1 Kd Kd desorption Soil 2 Kd Kd desorption Soil 3 HI Soil HI Soil HI Soil
concentration in aqueous sorption (Initial concentration sorption (Initial concentration sorption (Initial concentration 1 2 3
phase for the sorption Soil 1 in soil phase ng g− 1) Soil 2 in soil phase ng g− 1) Soil 3 in soil phase ng g− 1)
experiments (ng L− 1)

500 6.8 ± 0.3 2.7 ± 0.2 (1.6 ± 0.0) 1.8 ± 0.0 3.3 ± 0.3 (0.6 ± 0.0) 3.8 ± 0.1 2.6 ± 0.2 (1.1 ± 0.0) 1.03 1.30 1.13
± ± ±
0.07 0.07 0.06
1000 9.4 ± 3.7 2.4 ± 0.1 (2.7 ± 0.1) 1.0 ± 0.0 4.1 ± 0.3 (0.7 ± 0.0) 2.6 ± 0.2 2.1 ± 0.3 (1.7 ± 0.0) 1.15 1.74 1.37
± ± ±
0.05 0.09 0.12
5000 13.3 ± 6.3 2.4 ± 0.2 (16.1 ± 3.0 ± 2.1 3.4 ± 0.1 (4.4 ± 0.0) 6.0 ± 3.9 3.0 ± 0.1 (10.6 ± 1.11 1.66 1.18
0.1) 0.1) ± ± ±
0.04 0.03 0.02
10,000 9.8 ± 5.6 - – - – - - - -
20,000 9.0 ± 6.1 3.3 ± 0.3 (85.2 ± 1.1 ± 0.4 11.2 ± 1.0 (25.3 ± 2.8 ± 0.3 3.2 ± 0.1 (49.5 ± 0.77 0.45 0.76
0.2) 0.1) 0.1) ± ± ±
0.05 0.04 0.02
50,000 5.1 ± 0.4 3.6 ± 0.5 (176.7 ± 1.4 ± 0.4 9.9 ± 0.7 (63.2 ± 2.5 ± 0.0 4.1 ± 0.1 (106.3 ± 0.73 0.44 0.67
2.0) 0.7) 1.2) ± ± ±
0.07 0.03 0.01
100,000 5.5 ± 0.17 4.7 ± 0.3 (341.6 ± 0.9 ± 0.0 - 2.7 ± 0.0 6.2 ± 0.3 (207.6 ± 0.62 - 0.50
3.1) 1.9) ± ±
0.03 0.02

*Initial concentrations for the desorption experiments are not the same for all three soils, given that the soil which was used for the desorption experiments was the one
used for the corresponding initial liquid phase tramadol concentration.

5
A.-L. Garduño-Jiménez et al. Chemosphere 305 (2022) 135031

concentrations were in the range of 500 to 5000 ng L− 1. For Soil 3, 3.3.2. Freundlich and Langmuir isotherms
desorption Kd values were significantly (p < 0.05) different at the three Sorption data was fitted to the Freundlich model, and the values
levels, with the highest values observed at initial concentrations of obtained (Table 3) were consistent with that described in Section 3.3.
50,000 and 100,000 ng L− 1, mid-range values for initial concentrations According to this model, the sorption rate was in the following order:
of 5000 and 20,000 ng L− 1, and the lowest Kd values for initial con­ Soil 1 (KF = 5.23 ng g− 1) > Soil 3 (KF = 2.42 ng g− 1) > Soil 2 (KF = 1.00
centrations of 1000 and 500 ng L− 1. A similar pattern was observed for ng g− 1). Both, Soil 1 and Soil 3, presented R2 values above 0.99, as
Soil 1, with higher desorption Kd values at initial concentrations of opposed to Soil 2 displaying R2 < 0.99 (Fig. 2). The higher correlation
20,000–100,000 ng L− 1, compared to lower values obtained for initial indicates that tramadol is adsorbed to soils as a multilayer, achieving the
concentrations of 500–5000 ng L− 1. This behaviour could be explained maximum adsorption capacity when high initial concentration levels
by two phenomena occurring simultaneously: On one hand, the sorption were tested. The high content of clay of these soils (>20%) favours the
active sites on the soil particles are readily occupied by tramadol mol­ Freundlich type adsorption, as has been previously reported for the
ecules, then the remaining molecules form successive layers that are sorption of cationic organic molecules on clay soils (Stein et al., 2008).
desorbed when fresh CaCl2 solution is added in the desorption step. On For Soil 2, the sorption isotherm reached equilibrium (Fig. 2), indicating
the other hand, tramadol molecules can be rapidly sorbed on the organic the saturation of the active sorption sites, a similar trend was observed
matter and clay, while the remaining molecules, when initial concen­ for concentrations of up to 4 orders of magnitude above those studied
tration is high, are weakly adsorbed on sand and silt particles with
further desorption (Rytwo, 2004).
Hysteresis indices (HI) of <1 were obtained for the three highest Table 3
initial concentration levels (20,000–100,000 ng L− 1), indicating more Freundlich and Langmuir isotherm sorption and desorption parameters for
initial concentrations of 500–100,000 ngL− 1 for the three soils studied (Soil 1,
sorption than desorption (Table 2). The sorption-desorption hysteresis
Soil 2 and Soil 3) KF and KL are the Freundlich and Langmuir sorption co­
effect can also be observed in Fig. 2 for all three soils. Soil 2 presented
efficients, respectively. 1/n is the non-linearity parameter for the Freundlich
the highest desorption rate (Table 2), as expected from its lower clay and isotherm and Cmax is the maximum sorption capacity in the Langmuir model.
organic carbon content. Tramadol displayed higher desorption from Soil
Soil type Freundlich isotherm Langmuir isotherm sorption
3 compared to Soil 1 for most concentration levels, but this difference
parameters parameters
was not significant for all the initial concentration levels (Table 2). The
KF 1/n R2 KL Cmax (μg g− 1) R2
hysteresis has an environmental implication regarding the accumulation
of tramadol in the studied soils, which can result in higher bioavail­ Soil 1 sorption 5.23 0.98 0.9974 28.15 5.55 0.9977
ability in Soil 2. Considering the low biodegradability of tramadol Soil 1 desorption 15.26 0.90 0.9956 – – –
Soil 2 sorption 1.00 0.96 0.9892 15.40 6.93 0.9988
(Bergheim et al., 2012), along with its moderate water solubility (0.75 g
Soil 2 desorption 2.14 0.74 0.9639 –
L− 1), entering the food network through soil biota or plant uptake could Soil 3 sorption 2.42 0.96 0.9971 20.99 6.03 0.9982
be a plausible environmental fate. Soil 3 desorption 8.91 0.84 0.9923 – – –

Fig. 2. Sorption and desorption isotherms of tramadol in the three soils studied; a) Soil 1, b) Soil 2, c) Soil 3.

6
A.-L. Garduño-Jiménez et al.
here (Wojsławski et al., 2019).
The Langmuir sorption model presented similar R2 values to the
Freundlich model (Table 3), indicating tramadol’s sorption mechanism,
in the conditions studied, is also well described by the monolayer
adsorption model. Conversely, the Langmuir model did not fit for the
tramadol sorption (R2 < 0.96) in a previous study using initial concen­
trations of at least 3 orders of magnitude above those used herein
(Wojsławski et al., 2019), highlighting the importance of studies at
environmentally relevant concentrations to identify models that will
best describe environmental behaviour.
3.4. Sorption of tramadol using different liquid matrices
Significantly higher (p < 0.05) tramadol sorption was observed using
treated wastewater spiked with 20,000 ng L− 1 (52.7 ± 2.2%), compared
to that observed using 0.01 M CaCl2 (42.8 ± 2.5%) (Fig. 3). This equates
to linear Kd values of 9.2 ± 0.4 L kg− 1 and 7.5 ± 0.4 L kg− 1 for spiked
effluent and 0.01 M CaCl2, respectively. Competition of Ca2+ for sorp­
tion sites on clay particles (Jolin, 2016) might be a determinant factor in
the sorption of ionised tramadol. The use of 0.01 M of CaCl2 as the
aqueous phase, following the OECD 106 Guideline, is equivalent to
400.78 mg L− 1 of dissolved Ca+2, whereas in the wastewater used in
these experiments, the concentration of calcium was 63.86 mg L− 1 (and
in a similar range to previously reported Ca2+ concentration in other
secondary treated effluent of 74.1 mg L− 1) (Chefetz et al., 2008). Even
considering the potential competition for sorption sites from the sum of
all the major cations in the wastewater, such as sodium and magnesium,
these are still lower than the Ca2+ concentration in the buffer solution
(Table 1). Considering the high load of dissolved organic matter (DOM)
in treated wastewater, a decrease in the sorption of tramadol was ex­
pected, either through the formation of highly polar tramadol-DOM
complexes or because both organic species compete for the active
sorption sites. However, the opposite effect was observed, providing
evidence on the cationic exchange as the dominant adsorption mecha­
nism. It is likely that in the wastewater/soil system the DOM is adsorbed
on the organic domain of soil through partition, while tramadol is
adsorbed mainly through cation exchange in the clay and organic matter
surfaces. Therefore, in the wastewater/soil system, tramadol has lower
competition with dissolved cations than in the CaCl2 solution and thus
higher tramadol sorption was observed. This result highlights the value
Fig. 3. Comparison of percentage sorption kinetics using two different aqueous
phases: wastewater effluent with endogenous tramadol concentration plus
spiked concentration equating to 20,724 ng L− 1 and 20,000 ng L− 1 tramadol in
0.01 M CaCl2.
7
Chemosphere 305 (2022) 135031
added by comparing the sorption of organic pollutant’s under environ­
mentally relevant conditions (i.e., wastewater as the aqueous phase) to
standard conditions following the OECD 106 Guideline. Results
following the OECD Guidelines have great value in terms of comparison
across the literature (allowing for meta-analysis) and acting as a
standardised benchmark. Adding environmentally relevant studies for
comparison can more accurately explain environmental fate.
Wastewater effluent with and without additional spiking of tramadol
to the liquid phase was investigated, equating to low (724.7 ± 20.9 ng
L− 1) and high (20,724.7 ng L− 1) initial concentrations. The higher
sorption rate (64.9% ± 1.8, Kd = 11.3 ± 0.3 L kg− 1) of tramadol to soil
when using treated wastewater effluent with an endogenous tramadol
concentration (no additional spiking), compared to the sorption rates
determined in spiked wastewater effluent is consistent with that
observed using CaCl2 buffer; lower initial concentration of tramadol
means more adsorption sites available on the soil surface (Figs. 3 and 4).
For the CaCl2 buffer system, higher coefficients of variation (CVs) were
observed at the initial 0.25 and 0.5 h times (55.7 and 27.9%, respec­
tively), compared to the CVs at the same time points in the wastewater/
soil system (<6.5%). The higher CVs observed in the CaCl2/soil system
at time points <1 h may be due to the competition taking place between
Ca+2 and the positively charged tramadol, further supporting the
stronger role of cation exchange as the main sorption mechanism for
tramadol when it is at a pH lower than the pKa (see Figure S3 for
speciation diagram).
The pH of the 0.01 M CaCl2 buffer did not vary significantly (p <
0.05) over the 72 h sorption tests, with an average of 7.20 ± 0.05.
Conversely, the pH of the wastewater effluent increased from 6.75 at
time 0 h to 7.40 ± 0.10 at 72 h for samples contacted with soil, and to
7.65 ± 0.10 for control samples where no soil was added. The increase
of pH in the soil/wastewater systems could be attributed to degradation
and partitioning of the DOM to the soil (Wang et al., 1998). This increase
of pH in the wastewater leads to the deprotonation of some tramadol
molecules, even when the pKa value was not surpassed, more neutral
tramadol molecules occurred in the soil/wastewater system when
compared with the soil/CaCl2. In this scenario, at pH values close to 7,
partitioning to the soil organic matter would be happening, along with
cation exchange, which is supported by the higher Kd value observed in
the soil/wastewater system.
4. Conclusion
The results from this work demonstrate that the dominant sorption
mechanism for tramadol to loam soils is ionic interactions with nega­
tively charged particles when the pH of the soils and aqueous phase is
below the pKa value of tramadol. Increased affinity of tramadol was
found in soils with higher clay content (which is negatively charged),
and tramadol also presented a fast <1 h sorption equilibrium under the
conditions studied. Furthermore, higher tramadol sorption to soil was
observed from wastewater effluent than from calcium chloride buffer (as
per OCED 106 Guideline), demonstrating higher competition from cal­
cium ions in the buffer than from dissolved organic matter in waste­
water. This observation demonstrates tramadol’s sorption to soil is
predominantly due to cation exchange rather than hydrophobic parti­
tioning. This has the environmental implication that clay soils will be
able to retain tramadol from irrigation water despite the organic content
of the irrigation water. The lower hysteresis observed for clay soils re­
inforces this observation in that high retention of tramadol will be
achieved in clay soils. This work demonstrates the importance of envi­
ronmentally relevant conditions in batch sorption studies for bioactive
chemical pollutants to better understand their sorption mechanism and
their environmental fate.
When error bars cannot be seen it is because the marker is larger than
the bars.
A.-L. Garduño-Jiménez et al.
Fig. 4. Comparison of percentage sorption kinetics with two different aqueous phases: wastewater effluent endogenously containing 724 ng L−
wastewater effluent with endogenous tramadol concentration plus spiked concentration equating to 20,724 ng L− 1 of tramadol.
Funding
This work was supported by the Engineering and Physical Sciences
Research Council (EPSRC) [grant number EP/M022234], BIO­
TRANSFOROMICS: Bioanalysis to engineer understanding in waste­
water treatment.
Credit author statement
Andrea-Lorena Garduño-Jiménez: Conceptualization, Methodol­
ogy, Validation, Formal analysis, Investigation, Data curation, Writing –
original draft, Visualization, Project administration. Juan-Carlos
Durán-Álvarez: Writing – review & editing. Ruth Silvana Cortes-
Lagunes: Visualization. David A. Barrett: Writing – review & editing.
Rachel L. Gomes: Conceptualization, Methodology, Resources, Writing
– review & editing, Supervision (line management), Project adminis­
tration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The author (RLG) would like to acknowledge the support of Engi­
neering and Physical Sciences Research Council (EPSRC) [grant number
EP/M022234], BIOTRANSFOROMICS: Bioanalysis to engineer under­
standing in wastewater treatment. The authors (AGJ and RLG) would
like to acknowledge the support of the Future Food Beacon of Excellence
at the University of Nottingham. All authors would like to thank Severn
Trent Water Ltd. for the permission to sample wastewater effluent from
Stoke Bardolph wastewater treatment plant.
8
Chemosphere 305 (2022) 135031
1
tramadol and
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.chemosphere.2022.135031.
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0022-202Xj82j7805-0425$02.00jO
THE JOURNAL OF INV ESTI GAT IV E DERMATOLOGY, 78:425-428, 1982 Vol. 78, No.5
Copyright © 1982 by The Williams & Wilkins Co. Prinl.ed in. U. S. A.

Water Sorption-desorption Test of the Skin in Vivo for Functional

Assessment of the Stratum Corneum
HACHIRO TAGAMI, M .D ., YUKO KANAMARU, M.D., KUNIO INOUE, M.D., SHOKO SUEHISA, M.D.,
FUMIO INOUE, M.D., KEIJI IWATSUKI, M.D. , KOHDO YOSHIKUNI , M.D., MIZUHO YAMADA, M.D_

Depart.m.enl of Derrnat.ology, Ha.m.am.alsu Universit.y School of M~edicin.e, Ham.amatsu, Japan.

Based on the evidence from our previous studies that state. Our earlier studies demonstrated that we can evaluate it
we can evaluate the hydration state of the skin surface rapidly and quantitatively without changing the surface prop-
quickly and quantitatively in terms of conductance to erties of the skin by employing an instrument that measures
the high frequency electric current of 3.5 MHz, we have the conductance, i.e., the reciprocal of resistance, to the high
established a simple in vivo function test that furnishes freq uency current of 3.5 MHz [3]. By this method it becomes
information on the hygroscopic property and water- possible to reveal that the water content of the stratum corneum
holding ca pacity of the stratum corneum in a few min- progressively increases from the sw·face to deeper portions and
utes. The test procedure consists of electromeasure- t hat the normal skin sw-face absorbs water instantaneously and
ments before and after application of a droplet of water desorbs it quickly. Although with this method we can appreciate
on the skin for 10 seconds to obtain data on the hygro- that the water content of scaly skin is very low as compared to
scopic property of the skin surface and later serial mea- t he adjacent normal skin, such a difference tends to become
surements at an interval of 30 seconds for 2 min to negligible in a dry ambient condition since the water content in
evaluate the water-holding capacity. Under usual am- the superficial portion of the normal stratum corneum also
bient conditions normal skin surface showed a high rise decreases mruokedly under such conditions. These findings
in conductance just after application of water, which prompted our attempt to improve th e methodology to provide
was followed by a rapid fall-off within 30 seconds, there- much more extensive, functional data on the hydration and
after by gradual return to the prehydration levels b y 2 dehydration kinetics of the stratum corneum in vivo.
min. In this paper we describe a water sorption-desorption test of
By this method we have demonstrated that (i) the the skin t hat furnishes information of the hygroscopicity and
superficial horny layer of normal skin is much less hy- water-holding capacity of the surface horny layer in a short
groscopic and less capable of holding water than the time. By this test it has been disclosed that scaly skin lacks
corresponding deeper portions and that (ii) scaly skin both of these capabilities. In this context we use the following
shows functional defects in both hygroscopicity and wa- words under the definitions given below, i.e., hygroscopicity
ter-holding capacity, between which the former normal- denotes the property of stratum corneum to take up(=sorb)
izes much faster than the latter. water and water-holding capacity, the ability of stratum cor-
neum to retain water opposing a dehydration process (=desorp-
tion) .
The primary function of the epidermis is to produce a pro-
tective sheath, the stratum corneum, that protects the body MATERIALS AND METHODS
from dessication and invasion of various kinds of external
attacks [1]. For this purpose we need a tough yet thin and In strll m ent
flexible barrier membrane. The water content of the stratum A detai1ed descript ion of t he measuring principle was made previ-
corneum plays an important role in providing the skin surface ously [3]. A new model of the instrument that was made by IBS Inc'
with suppleness. In vitro studies with excised pieces of stratum was used. It auto mat ically prints every reading of condu ctance in terms
of /.tU and capacitance in pF on an attached paper 3 seconds after
corneum showed that they are flexible as long as they contain a pplication of a probe on the skin. The printing apparatus works only
more than 10% of water [2]. However, such a situation occms when there is a change of more than 5 p.v in conductance; thus, in case
only in excised pieces of stratum corneum since in vivo the with very low conductan ce values reading should be made directly from
lowermost portion of the stratum corneum which di.rectly faces a digital recorder.
t he water-sa tum ted viable epidermis is always well hydrated, Tlu·oughout this study we have used t he same probe as that used in
while the upper portion is much drier because it is exposed to the previous study [3]. We can increase sensitivity of measurement by
ambient air. Since the water content in the superficial portion . choosing a probe wit h a central electrode larger than 2 mm in diameter,
of the stratum corneum fluctuates depending on the ambient whose extreme sensit ivity, however, becomes a drawback for routine
hUIIl.idity, even normal skin surface may cl·ack fin ely in ar eas measurement.
S in ce the hydration state of the stratum corneum increases gradually
prone to flexing and stretching to develop the clinical feature of fr om t he surface to the lowermost port ion within a thickness of about
"dry skin" or chapping when the thickness of the portion that 20 p., even a small manual pressure on t he probe increases the observed
contains water less than 10% reaches a certain depth. In con- valu e. Therefore, to obtain reproducible resul ts, t he fl exible cable that
trast, even under normal environmental conditions pathological connects the probe with t he recordi ng body was held by the fingers of
horny layer which is defective in barrier function may readily an examiner at a bout 10 cm distance fr om the probe, wh ich was gently
lose water to develop a scaly clinical appearance. lowered on a test area to rest only with its own weight of 80 g on the
U ntil recently we have lacked methodologies that can quickly skin without applying any ad ditional manual pressure.
furnish quantitative information on the skin su.rface hydration
Su.bj ects
Hospital personnel , medical stud ents, and out- and in-patients seen
M anuscript received June 9, 1981; accepted for publication Novem- at t he Department of Dermatology, Hamamatsu University School of
ber 11, 1982. Medicine took part in the study.
This work was supported by Grant-in-aid for Scientific Research
5687007 from the Ministry of Education, Japan and a research grant Test Procedure
from Japan Lydia O'Lear y Memorial Founda tion. The edge of t he test ill·ea at least 1 em in diameter was marked on
Reprint requ ests to: Hac hiro Tagami, M .D., Department of Derma-
tology, Hamamatsu University School of Medicine, 3600 Handa-cho,
Hamamatsu 431-31 Japan. • IBS In c., 47 Motohama-cho, Hamamatsu 430, Japan.

425
426 TAGAMI ET AL Vol. 78, No.5
t he skin with a ball-point pen to avoid mlssmg t he site in repeat
measw'ements since it became impossible to find th e site of artificial
hydration after blotting.
At first a condu ctance-valu e of the test area was measured to obtain striPpir.l g wate1
a prehydration level. Then, a drop of distilled water was placed over
the test area for 10 seco nds. Immediately after blotting the site with a
(lOX) 1 TEMP 22°C
500 R.H. 60%
pad of gauze, a measurement was m ade and was repeated at an interval
of 30 seconds for 2 min. For artificial hydration other aqu eous solutions 11 SUBJECTS
such as saline instead of distilled water can be used since th e resul ts
are not greatly affected by t he presence of electrolytes but only by
exogenous supply of water as reported before [3].

RESULTS
Measurem ents on Normal Skin
As reported before [3], normal skin shows a marked increase ,(~mirV
in conductance to the high frequency current just after blotting
"
of applied water. Most of this increase is lost within 30 seconds
under usual ambient conditions, followed by a gradual return
to a prehydration leveL Thus, pilot experiments were carried
out on neighborjng areas of the flexor aspects of the forearms 0~~--~~~------~O--~60~-1~W
in 6 healthy subjects aged between 30 and 41 yr by changing
the duration of application time of distilled water from 10 to 60
seconds at a room temperature of 24°C and relative humidity FIG 2. Water sorption-desorption test on normal skin surface and
of 50%. that repeated 3 min after adhesive ta pe-stripping of t he horny layer 10
As shown in Fig 1, the desorption curves resembled each times.
other. However, when a water-droplet was applied on the skin
longer than 10 seconds, the increased conductance-values took
longer than 2 min to return to the preapplication level. There-
fore, we have chosen 10 seconds as an application time of
distilled water for the practical reason that the whole test ( J1 IJ) n=97
procedure can be finished in just over 2 min. i o:contro!
. : sca\e
Comparison between the Surfa ce and Deeper Portion. of the
Stratum Corneum G) IIYGr..ostUP I C ITY
200
The test was performed on the same area of the flexor surface
of the forearms of 11 normal subjects aged from 25 to 55 yr \
\
before and after partial stripping of the horny layer to assess I

whether there is any functional difference between the super-

I
/ \
o \-JI\TEH J/OWI f\G CAPl\Cl'l'Y
ficial and deeper portion of the horny layer. \
\
After finishing the first test on the normal skin surface, the I
100
stratum corneum ofthe site was stripped away with an adhesive l
\

tape 10 times. Since the inside of the stratum corneum is more I

hydrated than the surface, an interval of 3 min was allowed
before the start of the next measurement to permit the in-
creased conductance-values to settle at a certain level (Fig 2).

TO 30
• 10 water --4 time (sec.)

400
o 20 FIG 3. Water sorption-desorption test performed in 97 patients with
A 30 various kinds of scaly dermatoses. Solid circles represent a mean value
• 60 from scaly lesions and open circles that from adjacent normal skin. All
t he parameters denoting (1) prehydration level, (2) hygroscopic prop-
erty, and (3) water-holding capacity are decreased in scaly lesions.
300

The results of the second test showed that t here was a promi-
nent increase in conductance immediately after blotting of a
200 droplet of water, pointing out much higher hygroscopic prop-
erty of the stratum corneum at deeper portions than at the
surface. This increase in conductance returned to the prehydJ'a-
100 tion level after 60 seconds, which was somewhat higher than
that obtained on the skin surface, but the difference was not
significant at the 5% level.
Measurements on Scaly Skin
o 30 60 90 120 Fig 3 shows the summarized results of the test performed in
. ti me(SE'c) 97 randomly selected patients with various kinds of scaly der-
FIG L Changes in conductance- value after a pplication of distilled matoses. They consisted of 50 patients with psoriasis, 39 with
water for various periods ra nging from 10 to 60 seconds. Bar represe nts eczematous dermatitis, 3 with tinea corporis, and one each with
standard deviation. subcorneal pustular dermatosis, keratosis follicularis, pityriasis
May 1982 WATER SORPTION-DESORPTION TEST OF THE SKIN IN VIVO 427
(J,J(JI Psoriasis Psorlasis I ~ u ll Psoriasis
~

i Thick S<dles Thi n scales

I ..'' bt>tore
trealmerrt
",21,
~ 0
n=26 2w1< 5wk 7 wk
o :contro l
2CO • : scale 200
O:C.Ontro\
. : scale
,, (PUVA) (no SCdtE')
OJ' , Q 9
. ,\
,
\
"
/\ , q
I \ \
lOJ I \ 100 \

\~
I \
"

/) 0
,
0.
o~~~~~~~~~~
o 30 60
~
9:l
time(s ecJ
al .--.... ~ - ~ --
0 . 0- . 0

b FIG 6. Serial measurements on the same psoriatic lesions dw-ing

a treatment with topical PUV A. Although there are some vru'iations in
FIG 4. Water sorption-desorption test performed on psoriatic le- the results obtained from control skin because of the changes of the
sions. Lesions with thick scales (a) show much decreased parameters ambient conditions, it can be appreciated that hygroscopicity at first
than those with thin scales (b), and water-holding capacity finally return to the control levels. Open
circles represent control values.

Lesions of chronic eczematous dermatitis were also grossly

Eczema Eczema divided into 2 groups. Since in this case scaling was not so
dillusoly scaly lesion part"lly scaly lesion prominent as in psoriasis, they were classified as diffusely scaly
n =15 n: 21,
lesions when scales were observed allover the test area and as
., o :control o: conlf'O( partially scaly lesions if there were only scattered fine scales.
. : sCd le , ; scale
1 \
As shown in Fig 5, again there was a good correlation between
,
the clinical severity of scaling and results, particularly those
obtained just after blotting of applied water.
\
, Such a conelation between the clinical evaluation of scaling
,

~. . f. .
,,
'. ,
§ ·f.3
o __ ~ .
.- • _
a II 60 90
- >lime(sec .J
a b
FIG 5. Water sorption-desorption test carried out on eczematous
lesions, As compared to diffusely scaly lesions (a.), thin scaly ones (b)
show more hygroscopicity but water-holding capacity is still low,
rubra pilaris, pityriasis rosea and Papillon-Lefevre syndrome.
All the measurements were done on both scaly lesions located
at various bodily sites, except for those located on the palms,
soles, scalp, and face, and adjacent uninvolved normal skin.
As illustratively shown in Fig 3, all the functional parameters
measured on scaly skin were invariably lower than those on the
adjacent normal skin, In most cases the prehydration values on
scaly skin were almost zero regardless of the background dis-
orders and the difference between scaly lesions and normal skin
became much more apparent at the reading taken immediately
after blotting of water, indicating lower hygroscopic property of
scales. On scaly skin such slightly increased conductance-values
quickly returned to zero, reflecting defective water-holding
capacity.
Clinical Correlation in Scaly Lesions
The above mentioned impaired functional parameters in
scaly lesions were fUlother analyzed in correlation with t h e
clinical severity, selecting 2 representative dermatoses, i.e., pso-
riasis and eczematous dermatitis.
Psoriatic lesions were arbitrarily grouped into 2 categories,
i.e., thick scaly lesions that showed the presence of thick silvery
scales and thin scaly ones that were accompanied by only thin
scales. The readings made on those with thick scales were
always near zero except for that made just after blotting out of
water when a trace increase in conductance was observed,
whereas the thin scaly lesions showed a moderate increase at
this reading although others were still almost zero. (Fig 4, a
and b) .
and measured results was also observed in the same lesions if
they were followed in the course of successful treatment. Fig 6
shows a representative example noticed in a 27-yr-old patient
with psoriasis in whom topical 8-methoxypsoralen application
11.-
followed by irradiation of long ultraviolet light (topical PUVA)
110
showed a beneficial effect within 7 weeks. As the lesion began
to improve after 2 weeks of treatment, the h ygroscopic property
of the lesional horny layer at flrst began to increase. This trend
became more apparent in the following weeks. At 7 weeks after
initiaion of treatment when the lesion completely regressed, all
the parameters reached the same levels as those on the adjacent
normal skin.
DISCUSSION
The results in the present study show that by employing the
water sorption-desorption test we can perform much better
functional analysis of the stratum corneum in vivo than the
simple measurement of the skin surface hydration state, since
we can obtain important parameters such as (i) in vivo hydra-
tion state, (ii) hygroscopic property and (iii) water-holding
capacity of the stratum corneum in a very short period of time.
If the test area is wide enough, we can increase the precision of
the measurements by repeating the test procedw'e several tlJUes
in neighboring areas.
In vitro kinetic studies on water sorption-desorption by hu-
man stratum corneum gave information on the existence of at
least 2 species of water in stratum corneum [4]; rapidly sorbe~­
desorbed water to the extent of 0.5 mg/ mg stratum corneum IS
a "bound water" which is an energetically bound pool of wate~,
whereas slowly sorbed-desorbed "free water" is a nonenergetl-
cally bound pool of a bulk liquid water which is absorbed up to
12 times dry weight of stratum corneum until the tissue begins
to break down mechanically. It is reasonable to presume that
the measurements of water content in stratum corneum in our
water sorption-desorption test are chiefly concerned with the
amount of the rapidly gained and lost "bound water" in stratum
corneum. We have found that the stratum corneum of the
superficial portion is less hygroscopic and somewhat.iess cap~­
ble of binding water than that of the deeper portIOns. ThiS
seems to be due to a deficit in small-molecular-weight water-
soluble hygroscopic substances that are derived from the viable
428 TAGAMI ET AL
epidermis [5], i.e., so-called "natural moisturizing factor" [6],
which may be leached out from the skin surface by washing or
by taking baths or showers.
The data presented here demonstrate general agreement in
that the stratum corneum of scaly skin is always dry because it
is less hygroscopic and markedly less capable of binding water
t han normal skin. Particularly the defective water-holding ca-
pacity seems to play a crucial role; even eczematous lesions
with inconspicuous fine scales showed an almost complete lack
of this capacity and normalization of the decreased water-
holding capacity of scaly lesions took place only at the end of
successful treatment, while hygroscopicity of such stratum cor-
neum gradually returned to normal with improvement of the
lesions. These fmdings are probably explained by the fact that
normal water-holding capacity requires complete restoration of
stJ'atum corneum with intact structure, whereas hygroscopicity
of the stratum corneum depends on the presence of the hygro-
scopic substances of small molecular weight on the superficial
portion of the stratum corneum being not covered by such
substances as dessicated tissue fluids.
Up to date we have not had in vivo methodologies which are
capable of providing quantitative parameters of "dry skin" [7].
We can apply t his methodology for assessment of such skin; in
fact, some cases of eczematous dermatitis with partially scaly
lesions in the present study were those with eczema craquele
Vol. 78, No.5
which developed from pruritic dry skin occurring in winter. We
can also use this method as a rapid in vivo screening test for
topical agents such as moisturizers and emollients; by this
method we have succeeded in disclosing the moisturizing effect
of 10% urea cream on normal skin (unpublished data). There-
fore we think that it is applicable to many fields and that it
opens a new way for functional analysis of the stratum corneum.
REFERENCES
1. Kligman AM: The biology of t he stratum corn eum, The Epidermis.
Edited by W Montagna, WC Lobitz. New York, Academic Press,
1964, pp 387-433
2. Blank IH: Factors which influence the water content of t he stratum
corneum . J Invest Dermatol 18:433-440, 1952
3. Tagami H, Ohi M, Iwatsuki K, Kanamaru Y, Yamada M, Ichijo B:
Evaluation of the skin surface hydration in vivo by electrical
measurement. J Invest Dermatol 75:500-507, 1980
4. Anderson RL, Cassidy JM, Hansen JR, Yellin W: Hydration of
stratum corneum. Biopolymers 12:2789-2802, 1973
5. Tabachnick J, LaBadie JH: Studies on the biochemistry of epider-
mis. IV. The fTee amino acids, ammonia, urea, and pYl'l'olidone
carboxylic acid content of conventional and germ-free a lbino '
guinea pig epidermis. J Invest Dermatol 54:24-31, 1970
6. Jacobi OK: About the mechanism of moisture regulation in the
horny layer of the skin. Proc Sci Sect Toilet Goods Assoc
31:22-23, 1959
7. Mausner JJ: Moisturization: a critical evaluation. Cosm Toiletries
94:24-27, 1979

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enrollment Course in Dermatology and is oriented primarily to dermatologists involved in patient care.
This postgraduate course is sponsored by the Northwestern University, Cleveland Clinic, and University
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disorders. For registration information and preliminary program, write: Department of Dermatology,
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 Construction and Building Materials 261 (2020) 120007

Contents lists available at ScienceDirect

Construction and Building Materials

journal homepage: www.elsevier.com/locate/conbuildmat

Development of pore structure, moisture sorption and transport

properties in fly ash blended cement-based materials
O. Linderoth ⇑, P. Johansson, L. Wadsö
Building Materials, Lund University, Lund, Sweden

h i g h l i g h t s

Long-term fly ash reactions alter pore structure considerably.

The fly ash reaction does not fill pore volume but refines porosity.

Fly ash replacement lower moisture diffusion coefficients a factor 2–5.

The presence of aggregates has little effect on pore structure.

Sorption isotherms for mortar and concrete can be measured on cement paste.

a r t i c l e i n f o a b s t r a c t

Article history: This study documents how the pore structure develops with time in cement-based materials with vary-
Received 29 February 2020 ing fly ash replacement. Heat production was measured during the first days, water vapour desorption
Received in revised form 15 May 2020 isotherms were determined at different times up to 18 months, and moisture transport properties were
Accepted 18 June 2020
measured after one year. Long-term fly ash reactions alter the pore structure considerably. The fly ash
Available online 13 July 2020
does not fill pore volume but refines porosity. After one year, fly ash replacement resulted in a factor
2–5 lower moisture diffusion coefficients. Aggregates have little effect on the measured pore structure;
Keywords:
sorption isotherms for mortar and concrete can be determined on cement paste.
Pore structure
Sorption
Ó 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license
Moisture transport (http://creativecommons.org/licenses/by/4.0/).
Permeability
SCM
Durability

1. Introduction To reduce the CO2 emissions associated with cement-based

Water is an integral part of cement-based materials; it reacts
with the binder to form hydrated phases that bind the material
together. However, moisture is also closely related to many dura-
bility issues of cement-based materials [1,2]. Processes related to
deterioration such as carbonation, chloride ingress and freeze–
thaw scaling are all moisture dependent. Moisture related
processes also cause microbiological growth and degradation of
adhesives and flooring materials applied to for example a concrete
surface. There is a need to collect data on pore structure and mois-
ture transport for novel binders. This data can be used in computa-
tional models to predict, e.g., drying, shrinkage and service life of
cement-based materials.
⇑ Corresponding author at: Division of Building Materials, Lund University, P O
Box 118, 221 00 Lund, Sweden.
E-mail address: oskar.linderoth@byggtek.lth.se (O. Linderoth).
materials the ordinary Portland cement (OPC) is increasingly
replaced as binder by supplementary cementitious materials
(SCMs). The most commonly used SCMs are waste products from
other industries, and then mainly ground granulated blast furnace
slag (slag) from steel production, and fly ash (FA) from burning of
coal in coal-fired power plants. The addition of SCMs results in
similar reactions products as from OPC hydration, but the SCMs
presence in the binder still change its chemical composition and
reactivity [3,4]. These changes affect the reaction process and by
that the development of most material properties [5], for example
the pore structure and related moisture transport properties [6–
10]. Despite an increasing interest in recent years, more research
on cement-based materials with SCMs is still needed.
The most commonly measured moisture properties are sorption
and transport. The former is generally presented as water vapour
sorption isotherms, showing the physically bound water content
– water in the materials pore structure – as a function of the

https://doi.org/10.1016/j.conbuildmat.2020.120007
0950-0618/Ó 2020 The Author(s). Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
2 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007
relative humidity (RH). Sorption isotherms can be used to follow
the development of the pore structure, and related features such
as the pore volume, pore size distribution and specific surface area.
The first sorption isotherms for cement based materials were pre-
sented by Powers and Brownyard [11] more than 70 years ago.
Their measurements were conducted on material with OPC (of
the types used at that time), as were most subsequent publications
(see for example references [12–17]. Transport of moisture
through the pore structure has been investigated in several studies,
using a number of methods [9–10,12–13,18–19]. The most com-
mon method is probably the cup test (for example in references
[9,12–13]. Moisture transport results are generally presented as
diffusion coefficients over different RH-intervals. As for sorption,
most transport studies have been conducted on materials with
OPC as binder.
In recent years, moisture sorption and transport properties have
been presented also for materials with blended binders. Sorption
measurements on cement pastes and mortars with slag or silica
fume replacement can be found in references [20–27]. Saeidpour
and Wadsö [8,9] and Olsson et al. [10] measured both moisture
sorption and transport for slag or silica blended mortars and
showed that partial replacement of OPC resulted in higher porosi-
ties, but a slower moisture transport; Olsson et al. [10] tried to cou-
ple moisture transport with the pore structure but found no direct
relations. However, the SCM reactions are generally slow com-
pared to the OPC [4,5] and more sensitive to curing temperature
[26,28]. This means that the reaction products of, e.g., fly ash pre-
cipitate in an already formed pore structure which could lead to an
increased heterogeneity of the pore structure. Such an effect has
been indicated in previous studies of blended cement-based mate-
rials, e.g., [6,7]; SCMs refine the critical pore entries – i.e. the small-
est continuous channels in the pore structure – but have less effect
on the total pore volume. This refinement could explain the slow
moisture transport reported for slag and silica blended materials
compared to material with only OPC. Previous studies have also
shown that SCMs change the chemical composition and growth
pattern of calcium silicate hydrate (C-S-H), the main reaction pro-
duct of both the OPC and SCM reactions [29,30]. This too could
affect the pore structure. Whether the effects seen in cements with
slag or silica fume on moisture sorption and transport are similar
in fly ash blended materials have not been shown. In fact, there
are few studies on these properties for fly ash blended materials.
One exception is Xu [31] who presents some sorption measure-
ments in his comprehensive study of fly ash blended systems.
His results indicate that 30% fly ash replacement have little effect
on porosity, but that higher replacement, like slag and silica fume
replacement, increases the pore volume compared to material with
only OPC.
The purpose of this study is to investigate the effects of partial
fly ash replacement on the development of pore structure, mois-
ture sorption and transport properties in cement-based materials
cured under sealed conditions. Heat evolution was measured dur-
ing the first days. Water vapour desorption isotherms were deter-
mined at different times, ranging from three days to 18 months of
curing. The effect of fly ash replacement on these results is dis-
cussed. Water vapour desorption isotherms were measured on
samples of both cement paste and mortar. The effect of aggregates
on the pore structure is discussed. Moisture transport properties
were derived from cup tests on mortar samples after one year of
curing. The transport results are discussed in relation to the pore
volume and size distribution, but also to other effects found by pre-
vious studies. Additional sorption balance measurements were
performed on samples with different w/b ratios, this effect on
the pore structure is discussed.
2. Materials and methods
2.1. Materials
Measurements were conducted on samples of cement paste and
mortar. A commercially available OPC (CEM I 52.5 R) and a
siliceous fly ash (FA) (class F, ASTM C618) were mixed in different
proportions in wt% and used as binders: 100-0, 85-15, 65-35 and
30-70, also referred to as OPC, 15FA, 35FA and 70FA. The difference
in density between the OPC and fly ash meant that the starting
pore volume decreased with increasing replacement. The reduc-
tion was about 2% for 15FA, 4% for 35FA and 8% for 70FA at w/b
0.45. For the mortar a siliceous sand (EN-196-1) with particle sizes
0–2 mm was used as aggregate. The mortars were mixed with a
constant 60 to 40 vol% cement paste to aggregate ratio. The chem-
ical and mineralogical compositions of the raw materials are given
in Appendix A; Tables A.1 and A.2. The mix compositions of the
mortars are given in Table 1.
The mortars were mixed in batches of approximately 25 L using
a free-fall mixer. The fresh material was then divided in five litres
cylindrical PVC plastic moulds, as illustrated in Fig. 1. Entrapment
of air bubbles was prevented by vibrating the moulds on a vibra-
tion table. There were no signs of bleeding or segregation in the
mortars. The moulds were sealed and placed in a water bath at
20 °C. A small hole was drilled in one of the lids so that air could
escape when the moulds were sealed, but also to allow them to
be re-opened at different times to extract material. Once the lid
was fitted the hole was sealed with a vapour tight butyl-tape.
Each time the moulds were opened, the top 10 mm of material
was removed to ensure that the material used for analysis had not
been affected by the surrounding environment. Mortar was cut in
form of 5 mm thick discs using a stone saw. The saw was water-
cooled to prevent heating of the material. After cutting, the discs
were treated differently depending on the measurements they
were to be used for.
For cement paste, three grams were cast in 20 ml plastic vials.
Solids were added to the vial standing on an analytical balance
with a 0.01 g resolution, water was then added using a pipette.
All pastes were made with a w/b-ratio of 0.45. The vials were
sealed with plastic screw caps and placed in a holder on a vortex
mixer (Stuart SA8, Bibby Scientific, UK) set to 1600 rpm for 60 s.
Samples that were to be stored before further use were placed in
a climate chamber at 20 °C and approximately 60% RH. The vials
had a mass loss rate of about 40 mg d1, giving that the loss of water
by leakage was about 0.5% of the mixing water in the present sam-
ples after six months of storage.
2.2. Isothermal calorimetry
Heat evolution was measured by isothermal calorimetry on 3 g
samples of cement paste with increasing levels of fly ash replace-
ment (from 0 up to 70 wt%). The instrument used was an eight cell
TAM Air isothermal calorimeter (Thermometric AB; now TA instru-
ments, USA) set to 20 °C. The calorimeters were regularly cali-
brated electrically with heaters in vials placed in the vial
holders; baselines were determined with empty vials [32]. The
vials containing the samples were placed in the calorimeters
directly after mixing.
2.3. Sorption balance measurement
Water vapour desorption isotherms were measured with sorp-
tion balances [33]. Three instruments were used for the measure-
ments: two DVS intrinsic and one DVS 1000 (Surface Measurement
 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007 3

Table 1
Mortar compositions.

Mortar abbreviation Unit OPC 15FA 35FA 70FA* 35FA-0.32* 35FA-0.55*

1
w/b gg 0.45 0.45 0.45 0.45 0.32 0.55
OPC kg m3 777 646 480 211 572 428
FA kg m3 – 114 259 493 308 230
Water kg m3 349 342 333 317 282 362
Siliceous sand kg m3 1060 1060 1060 1060 1060 1060
*
) Some additional sorption balance measurements were performed on these systems.

Hole
Lid
Rubber seal
Mortar
Pipe
500 mm
100 mm
Fig. 1. Cross-section of the cylindrical moulds in which mortars were cast.
Systems Ltd., UK). In these instruments the mass of a small sample
is continuously measured with an analytical balance during expo-
sure to a predefined sequence of RHs. The desired RH inside the
samples chamber is obtained by a mixture of water vapour satu-
rated and dry nitrogen gas. The relative humidity of the sorption
balances was regularly validated by measurements on saturated
salt solutions and found to be within 1.5% of the set values [34].
The three instruments used here are stable and normally give
equal results for the same material (cf. Appendix B, Fig. B.1).
Cement pastes were measured from three days up to six
months. To avoid drying to the ambient, sample preparation was
conducted inside a container conditioned by wet cloths to approx-
imately 95% RH. The material was crushed with a mortar and pes-
tle (maximal particle size 1 mm) and 40–80 mg were placed in the
sorption balance sample holder. The sample mass was determined
using an analytical balance with a resolution of 0.01 mg. A drop of
de-ionized water was added to the material to achieve close to sat-
urated conditions. Samples that were 14 days or older were stored
for approximately 24h after the addition of water before the start
of the measurement; the sample holder containing the sample
was then placed in a 20 ml plastic vial, sealed with a tight plastic
screw cap. Because the rate of reaction is still significant after three
and seven days, measurements at this age were actually started
after two and six days with a first step at 90% RH lasting for 36 h
(cf. Table 2).
Mortars were measured after six and 18 months.
Approximately-one gram of material was extracted from the cores
of freshly cut 5 mm mortar discs. The material was crushed with a
mortar and pestle (maximal particle size 1 mm), then saturated
with deionized water and placed in 20 ml plastic vials sealed with
plastic screw caps. Like the cement paste samples, the material was
stored in the vials for approximately 24 h before the start of the
measurement. After storage, 40–80 mg of mortar were placed in
the sorption balance vial holder and the measurement was started.
Mortar samples were taken from a larger volume of material. The
binder content of each sample was therefore calculated from the
chemical composition of the binder and the Ca-content of each
samples using the method described in [35]. The results were nor-
malized by the binder content so that results for different samples
could be compared. Normalizing by the binder content introduces
an uncertainty in the absolute level of the desorption isotherms as
the binder content analysis has an uncertainty. In Linderoth and
Johansson [35], Monte Carlo simulation was used to make error
propagation calculations. The calculations indicated a maximal
variation in the binder content of about ±4%. This uncertainty
should be kept in mind when interpreting the results for mortar
in Figs. 8 and 9a. The sorption balance measurements were con-
ducted according to the RH-sequences and times steps in Table 2;
note the rather long measurement times.
The length of each step was adjusted to give a similar proximity
to mass equilibrium. At 50 and 30% RH sorption kinetics were sig-
nificantly slower than at other RH:s, as observed by, e.g., Helsing
Atlassi [21]. Similar observations were also made by de Burgh
and Foster [16] (cf. Fig. 11 in their paper). They suggest that this
is the result of non-Fickian sorption behaviour (i.e., that it does
not follow Fick’s law of diffusion [36] caused by ‘‘a combination
of instantaneous and time-dependent spinodal microstructural
rearrangements” where small pores get smaller and large pores
get larger. According to their measurements, the behaviour
becomes more prominent with decreasing RH in desorption, until
reaching a maximum at approximately 35% RH (at 23 °C). Non-
fickian sorption behaviour is not an issue unique to cement based
materials. It has also been observed for other construction materi-
als, e.g., wood [37,38] and polymers [39]. For cement based mate-
rials, the effect is reduced below 35% RH and at 11% it seems to be
negligible. This RH (11%) was used as the last step in all measure-
ments to differentiate between the physically and chemically
bound water contents [40,41]. The dry state was thus defined as
mass equilibrium with 11% RH at approximately 20 °C. The upper
RH-limit of the sorption balance (about 97%) is an experimental
constraint, as at higher RH water could condense inside the instru-
ment. However, a most of the porosity that empty at higher RH are
air voids and other macro pores that are so coarse they are only
water-filled at complete saturation and therefore not refined by
the binder reactions. The maximal RH measured in this study of
95% means that – especially at early age – part of the coarse capil-
lary porosity is not captured by the sorption balance
measurements.
Some previous studies in which sorption isotherms were deter-
mined with sorption balances used curve fitting and extrapolation
with exponential functions to estimate the mass at infinite time,
e.g., [8,10,14,22]. Although this will probably improve the results,
it does not solve the issues of non-fickian behaviour, as it cannot
be assumed that non-Fickian sorption follows an exponential path
towards equilibrium. However, extrapolation can be useful to
4 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007

Table 2
Relative humidity and time steps for the sorption balance measurements depending on the age of the material.

RH / % 95 90 85 80 75 70 50 30 11
Age / d Time at each step / h
3–7 – 36 – 18 – 18 24 24 44
14–28 36 18 – 18 – 24 36 36 48
180 50 22 22 22 25 35 55 55 50

estimate the final mass at high and low RHs where the non-fickian
behaviour is not as prominent and the time steps used were too
short to close in on equilibrium. Fig. 2a shows an example of curve
fitting and extrapolation towards assumed moisture contents at
infinite time using the following two equations (Eqs. (1) and (2)):
mðtÞ ¼ mf þ ðmf  mi ÞeAðtti Þ
C where d is the Kelvin diameter (or pore diameter in m), c is the sur-
mðtÞ ¼ mf þ ðmf  mi ÞeAðtti Þ
where m(t) (g) is the measured mass of the sample, mf (g) is the final
estimated mass, mi (g) is the mass at the start of the curve fit, t (s) is
the time, ti (s) is the start time for each curve fit, and A and C are
fitting parameters. The curve fit is made from an arbitrary point i
on the curve after the initial rapid change of mass. Note that C is
the only difference between the two equations. In Fig. 2b-c, a des-
orption isotherm derived from the final mass at each step is com-
pared with extrapolated results from Eqs. (1) and (2).
The results in Fig. 2 show that the effects of extrapolation were
negligible (about 3% or less) with both equations. Except for at 30%
RH where the moisture content was, e.g., changed by approxi-
mately 15% with Eq. (2). The isotherms presented in this paper
were all derived from the final measured mass at each step, well-
aware that at particularly 30% RH this does not represent mass
equilibrium.
In desorption, smaller and smaller pores are emptied. The des-
orption branch of the sorption isotherm thus reflects both the pore
volume and pore size distribution of the material. The pore size
distribution should rather be seen as the pore entry size distribu-
tion as many pores are only connected to their ends through smal-
ler openings, forming so called ink-bottle pores [42]. The Kelvin
equation (Eq. (3), [43] relates the relative humidity to pore sizes
under the following assumptions: all pores are cylindrical; there
is no adsorbed layer of water on the pore surfaces; there are water
menisci. In reality, pores can assume any shape, there is always a
layer of adsorbed water, and menisci are only maintained down
to about 45% RH. Calculated pore sizes should thus be seen as esti-
mates. The Kelvin equation is given as:
4cM cos h
d¼ ð3Þ
ð1Þ RT lnðuÞ
ð2Þ
face tension of water (0.0728 J m2), M is the molar volume of water
(17.98  106 m3 mol1), h is the contact angle, R is the gas constant
(8.314 J mol1 K1), u is the RH (Pa Pa1), and T is the absolute tem-
perature (293.15 K). With the Kelvin equation and numbers above,
Fig. 3 presents the pore diameter as a function of relative humidity.
Pores in cement-based materials are often divided into different
categories by their size, as in [44,45], and more recently in [46].
Saeidpour and Wadsö [8] interpreted the mentioned studies to
divide the desorption isotherm into four parts corresponding to
different RH-ranges:

90% Capillary pores

90–80% Inter-hydrate spaces

80–30% Gel pores

30–0% Water in CASAH interlayers
Capillary pores are residuals of the originally continuous water-
filled spaces between cement and aggregate particles. Inter-
hydrate spaces are the most refined capillary pores, similar in size
to the coarsest gel pores. Gel pores are pores intrinsic to the
CASAH, and interlayers are the water-filled spaces between sheets
of CASAH. This division and these definitions are used in this
study, with the slight modification that the largest gel pores (cor-
responding to 80–70% RH) have been separated from the smaller
gel pores (70–30% RH), for example in Figs. 7, 9 and 12.

59.82 0.3 0.3

Measured Measured
0.25 Eq. 1
Extrapolated 0.25 Eq. 2
Extrapolated
59.81
g-11

We /B / g g--11

0.2 0.2
Mass / mg

-
gg

59.8 Measured
/B // g

0.15 0.15
Wee/B

59.79
W

0.1 0.1
Eq. 1
59.78
Eq. 2 0.05 0.05
a) b) c)
0 0
4500 5000 5500 6000 6500 0 50 100 0 50 100
Time / min Relative humidity / % Relative humidity / %

Fig. 2. Measured and extrapolated desorption isotherms of a mortar sample made with 35FA after 18 months of hydration are presented. a) shows an example of how curve-
fitting and extrapolation was done at each RH (here at 85%) where the red and blue solid lines represent the final extrapolated masses with Eqs. (1) and (2). In b) and c) the
measured desorption isotherm is compared with isotherms extrapolated with Eqs. (1) and (2); physically bound water (We) per gram of binder (B) as a function of the relative
humidity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007 5

100 mass loss as a function of time and by calculating the change of

the rate of mass loss per day. Transport properties were evaluated
once this change was 0.003 g day1 per day. Samples were stored
in water baths before the start of the measurements and should be
m

10-1
in desorption, but the degree of capillary saturation could differ
Pore diameter /

between the materials. This could potentially result in that the dif-
ferent materials end up on different desorption scanning iso-
10-2 therms. However, as the highest RH interval for which diffusion
coefficients were evaluated was well below capillary saturation,
this should have little impact on the results. Fig. 4 show curves dis-
10-3 playing mass change as a function of time over two different RH-
intervals in cups with different mortars.
The steady state moisture flux in each RH-interval was deter-
10-4 mined from the measured rate of mass change (Eq. (4)):
0 20 40 60 80 100
Relative humidity / % Dm
qm ¼ ð4Þ
ADt
Fig. 3. Pore diameters calculated from a desorption isotherm with the Kelvin
equation (Eq. (3)) as a function of the relative humidity (the curve is dashed below where qm (g m2 s.1) is the moisture flux, A (m2) is the surface area
45% as the assumptions on which the Kelvin equation are based on are not valid in and Dm/Dt (g s1) is the mass change rate. With the moisture flux,
this region). Fick’s law with vapour content as potential (Eq. (5)) could be used
to calculate diffusion coefficients:

2.4. Cup tests Dv

qm ¼ Dv ð5Þ
Dx
Moisture transport properties in desorption were measured Dv (m2s1) is the diffusion coefficient, Dv (g m3) is difference in
with cup tests using the setup described by Saeidpour and Wadsö vapour content over a distance Dx (m), in this case the sample
[9]. After one year of curing, mortar cores 65 mm in diameter were thickness. However, from the cup tests mean diffusion coefficients
core-drilled out of their moulds (Fig. 1). The cores were cut into were evaluated for each RH-interval. Eq. (5) was therefore rewrit-
5 mm thick mortar discs using a stone saw. Both the core-drill  v is the mean diffusion coefficient between
ten to Eq. (6), in which D
and the stone saw were water-cooled to prevent heating of the two RHs, u1 and u2 (Pa Pa1).
material. The discs were immediately placed in water baths at
20 °C for one week, followed by 24 h in a sealed box conditioned  v Dv
qm ðu1 ; u2 Þ ¼ D ð6Þ
to 95% RH at 20 °C with a saturated salt solution (KNO3). The solid Dx
to liquid ratio in the water baths were about 1 to 200. The discs A potential issue with cup tests is that the vapour contents on
were then placed as lids on plastic cups in which the RH was con- the two sides of the sample are different from those generated
trolled by saturated salt solutions to either 33% (MgCl) or 95% RH by the salt solutions because of external mass transfer resistances.
(KNO3). The salt solutions were prepared by mixing salts (pA qual- On the outside of the cups this was decreased by circulating the air
ity, Merck, USA) with deionized water [47]. The gap between the with fans (1 m s1). The RH at the outer surface of the specimens
cups and the perimeter of the discs were sealed with a butyl tape was therefore assumed to be equal to that of the air in the boxes.
(Platon over-tape, Isola AB, Sweden) to prevent transport other
than through the mortar. Saeidpour and Wadsö [9] used the same
sealant and found that its diffusion coefficient was about 100 times 6 OPC
lower than those of the investigated materials. Their test was
repeated for this study with a similar result (the water vapour dif- 33-75%
4
fusion coefficient of the sealant with vapour content as potential
was about 3.5  1010 m2 s1 at 60–95% RH). After sealing, all cups 15FA
were placed in boxes conditioned to 75% RH with NaCl-solutions. 2
The boxes were made of a high density polyethylene (HDPE) and 35FA
Mass change / g

covered on top with a glass plate resting on an EPDM sealing.

The RH inside the boxes was monitored with relative humidity 0
sensors that were regularly calibrated with saturated salt solu- 35FA
tions. Fans were used to circulate the air in the boxes in order to -2 15FA
keep the RH constant, and to decrease the external moisture trans-
fer resistance. Furthermore, a CO2 absorbent was placed in each
box to minimize the effect of carbonation. Weighing of the cups -4
95-75%
was carried out without removing them from the sealed environ-
ment. Above each cup there was a hole in the glass plate. Between
-6 OPC
weighing the hole was covered by a silicon stopper. A steel wire
went through the stopper and was fitted to the cups in one end.
The other end formed a hook above the glass. By loosening the -8
stopper, the cups could be lifted and hung from a balance above 0 20 40 60 80
the box. An analytical balance with a resolution of 0.01 g was used Time / days
for weighing. An illustration of the setup is given in Saeidpour and
Fig. 4. Curves showing the mass change as a function of time for cups with samples
Wadsö [9] (cf. their Fig. 1). of mortar made with OPC (circles), 15FA (triangles) or 35FA (diamonds). Results are
The cups were weighed regularly until steady-state conditions shown for two RH-intervals: the solid lines correspond to 33–75% RH and the
were achieved. This was evaluated from curves displaying the dotted lines to 95–75% RH.
6 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007
Inside the cups there was a gap of still air between the specimen
surface and the salt solution. To estimate the effect of the air gap
on the diffusion coefficient the mass transfer coefficient (kvi) for
the air gap was calculated according to Eq. (7):
Dair
kvi ¼
dair
where Dair is the diffusion coefficient of water vapour in air
(24.2  106 m2s1 [48] and dair is the thickness of the air gap
(m). The thickness of the air gap varied between 15 and 19 mm. This
gave a kvi of 0.0014–0.0018 m s1. If the mass transfer coefficient is
included in the evaluation of the diffusion coefficient, Eq. (6)
changes to Eq. (8):
Dv
qm ðu1 ; u2 Þ ¼ Dx
Dvþ k1
vi
By this equation, the material with the highest diffusion coeffi-
cient will be most affected by the internal mass transfer coefficient.
In the present study, the diffusion coefficients were changed by 1–
6% depending on the binder composition, RH-interval, and thick-
ness of the air gap. This correction was therefore included in the
evaluations.
3. Results and discussion
3.1. Heat evolution
Fig. 5a-d shows the heat production rate (or thermal power)
and the cumulative heat from samples of cement paste with
increasing fly ash replacement during the first four or seven days
after mixing measured by isothermal calorimetry.
The results in Fig. 5a-d show that both the rate and extent of
reaction decreases with increasing fly ash replacement in the first
week, but that the reaction per gram of OPC increases. There is less
material reacting early on, as manifested by a prolonged induction
period and a lower main peak of reaction in Fig. 5a. The results
reflect the pozzolanic nature of the fly ash, that is, its dependence
on the OPC reaction. Firstly, the pH has to be high enough for the
fly ash particles to dissolve, secondly the OPC has to produce cal-
cium hydroxide with which it can react [4,49]. Little fly ash reac-
tion is likely to take place within the first days after mixing,
instead it functions as a filler, providing additional points of nucle-
ation for the OPC hydrates. This so-called filler effect is well docu-
mented and has been shown to accelerate the OPC reaction while
also creating a more homogenous distribution of hydrates
[50,51]. The OPC reaction is also promoted because the effective
w/c ratio is increased, meaning there is more space and water
available for its reaction. The filler effect is seen in Fig. 5d as an
increase of heat per gram of OPC with increasing fly ash replace-
ment. Despite its accelerating effects on the OPC, it seems that
even modest fly ash replacement result in a decrease of the total
reaction at early age. This decrease is likely to affect the develop-
ment of most material properties, not least the pore structure,
and thereby the moisture transport properties.
3.2. Physically bound water
3.2.1. Effects of fly ash replacement
Fig. 6a-d show water vapour desorption isotherms of cement
pastes made with OPC, 15FA and 35FA at three days up to six
months of reaction. Within the first day after mixing, the initially
continuous water phase between the particles is divided into cap-
illary pores by precipitating hydrates. Further reaction refines the
capillary pores and increase the fraction of inter-hydrate spaces.
This refinement is seen as a flattening of the desorption isotherms
in the high-RH-range (approximately 95–80%) up to six months.
Like the heat evolution in Fig. 5, the isotherms in Fig. 6 show that
a major part of the binder reactions take place within the first day
after mixing; compared to the first three days there are no major
changes in the desorption isotherms up to six months. Fly ash
ð7Þ replacement decreases the reactivity of the binder up to six
months indicated by the higher desorption isotherms at six
months compared to the material with only OPC (Fig. 6d). Up to
six months there is also a decrease of the total measured porosity,
i.e., pores that are water-filled between 11 and 95% RH. However,
35FA is likely the most porous material at both early and later
age, only that at early age there is still a significant porosity that
is empty at RHs higher than 90% or even 95%.
From the results it is evident that fly ash replacement decreases
ð8Þ the early reactivity, but that it increases the reactions that take
place beyond the first month. In the first month, the desorption
isotherms of the fly ash containing mixes are lower than those of
the material with only OPC. Furthermore, the isotherms are steeper
in the high RH range, indicating a larger fraction of coarse porosity
with more pores emptying above 95% RH. Between 28 days and six
months, the pore structures of all the tested materials are mark-
edly refined, indicated by a flattening of the isotherms in the high
RH range (95–80%). For 35FA the measured porosity also increases;
this could be explained by a refinement of pores previously empty-
ing above 95% RH. For the other mixes these pores were already
refined in the first month, which means that their porosities
decrease. Between 28 days and six months, the pore structure
seems to develop most in the fly ash blended pastes, as their des-
orption isotherms change more than that of the cement paste with
only OPC (see Fig. 7a-b). This is likely a consequence of the poz-
zolanic reaction, that is, that little fly ash reacts in the first week
or weeks of curing. However, despite the onset and contribution
of the fly ash reaction (as well as lower initial pore volumes), the
pore structures of 15FA and 35FA are still more porous than that
of the OPC at six months (cf. Fig. 6d and 7a). One explanation for
this is that the pozzolanic reaction does not bind as much water
chemically as the OPC-reaction. Instead, the reaction use water
already bound chemically in Ca(OH)2 (formed by the OPC) to pro-
duce CASAH [5]. This affects the pore size distribution as solid Ca
(OH)2 crystals are replaced by finely porous CASAH gel, so that the
gel pore fraction increases. The development can be seen in Fig. 7a-
b where the desorption isotherms after 28 days and six months in
Fig. 6 are presented in two additional ways; in Fig. 7a as bars divid-
ing the moisture content into different RH-intervals, and in Fig. 7b
normalized by the moisture content at 95% RH. The latter makes it
possible to compare only the shape of the isotherms which reflect
their relative pore size distributions. In Fig. 7a-b it can be seen that
although 15FA has a more porous structure than OPC after six
months, the shape of both desorption isotherms are similar.
Another reason that porosity increases with fly ash replacement
is that the degree of reaction of the fly ash is low even after six
months of reaction [4]. De Weerdt et al. [28] used image analysis
to calculate the degree of reaction of fly ash in cement paste with
30 wt% replacement. According to their results about 35% of the fly
ash had reacted in the first six months. At the same time, about 90%
of the OPC had reacted. One reason for this is that fly ash normally
contains significant amounts of inert (non-reactive) crystalline
phases that lower its ultimate degree of reaction. As seen in
Table A.2 the fly ash used in this study contains approximately
30% of crystalline material. Because of its pozzolanic nature, slow
reaction and content of inert material, it is logical that fly ash
replacement results in a higher porosity regardless of the replace-
ment level and curing time.
For cement-based materials, six months is generally considered
a mature age beyond which little further reaction take place. How-
ever, as presented by De Weerdt et al. [28] the chemically bound
 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007 7

5 500
OPC
10FA
4 20FA 400
Thermal power / mW g-1
B

Cumulative heat / J g -1
30FA

B
40FA
3 50FA 300
60FA
70FA
2 200

1 100
a) b)

0
0 24 48 72 96 0 24 48 72 96 120 144 168
Time / hours Time / hours
5 500
OPC
10FA
4 20FA 400
OPC

30FA OPC
Thermal power / mW g-1

Cumulative heat / J g -1
40FA
3 50FA 300
60FA
70FA
2 200

1 100
c) d)

0
0 24 48 72 96 0 24 48 72 96 120 144 168
Time / hours Time / hours

Fig. 5. a) Heat production rate (thermal power) and b) cumulative heat release, per gram of binder (B); c) thermal power, d) cumulative heat, per gram of OPC. Results for
cement pastes (w/b 0.45) with increasing fly ash (FA) replacement hydrated at 20 °C, measured by isothermal calorimetry.

water seems to be increasing also beyond six months, and if this is
the case it should also effect the pore structure. Olsson et al. [10]
measured desorption isotherms of mortars with OPC and OPC
blended with silica fume or slag after eight months and four years.
Their results indicate that the pore structure continues to develop
for several years. However, in contrast to the material in this study,
their mortars were cured under water saturated conditions. This
means that the binder reactions were not halted by a lack of water
[52], which is likely the case for a low w/b material cured under
sealed conditions, e.g., massive concrete structures. Furthermore,
their measurements were conducted by two different methods:
after eight months by the desiccator method, and after four years
by a sorption balance. It cannot be ruled out that part of the differ-
ences they report as further development of the pore structure are
in fact caused by differences in the two methods. The sample size,
preparation procedures and time-scales are all different between
the desiccator and sorption balance methods, and this could influ-
ence the results.
To study how the microstructure develops beyond six months
in our materials, desorption isotherms were measured for mortars
with OPC, 15FA and 35FA after six and 18 months (Fig. 8a-b). As
mentioned in the method section, normalizing by the binder con-
tent introduces an uncertainty in the absolute level of the mortar
desorption isotherms. To illustrate this effect on the results, three
replicate measurements were made for each mortar at 18 months
(indicated by the error bars in Fig. 8b). Furthermore, additional
measurements were made on mortar with 70FA as binder at
18 months.
The results in Fig. 8a-b are compared in Fig. 9 and indicate that
there is a continued development of the pore structure up to
18 months even as the material is cured under sealed conditions.
As between 28 days and six months, the fly ash blended systems
seem to develop most between six and 18 months, indicated by
further refinement of the capillary porosity. There seems to be
no significant development of the pore structure in the OPC (see
Fig. 9b). This corresponds well with the findings of Yu and Ye
[7], showing that the pore structures of fly ash blended systems
develop most at later ages, but that their porosities are still higher
than that of a reference OPC after two years. The results in Fig. 9
show that whereas the isotherms of 15FA and 35FA are higher
than that of the OPC, the differences have decreased compared
to the previous measurement at six months. From Fig. 9b it even
looks as if 15FA has the most refined pore structure after
18 months. The additional measurements for 70FA (Fig. 8b) show
that 70 wt% fly ash replacement result in an entirely different
pore structure. At this replacement level the onset of the fly ash
8 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007

0.3 0.3

3d 3d
0.25 a) 0.25 b)
7d 7d
14 d 14 d
0.2 0.2
g-1

g-1
/B/B/ g/ g/g

/B/B/ g/ g/g
28 d 28 d
6 mos. 6 mos.
0.15 0.15
ee

ee
WW

WW
0.1 0.1

0.05 OPC 0.05 15FA

0 0
0 20 40 60 80 100 0 20 40 60 80 100
Relative humidity / % Relative humidity / %

0.3 0.3

3d OPC
0.25 c) 0.25 d)
7d 15FA
14 d 35FA
g-1

g-1
0.2 0.2
/B//gg/g

/B//gg/g
28 d
6 mos.
0.15 0.15
W e/B

W e/B
e

e
W

0.1 0.1

0.05 35FA 0.05 6 mos.

0 0
0 20 40 60 80 100 0 20 40 60 80 100
Relative humidity / % Relative humidity / %

Fig. 6. Desorption isotherms of cement paste (w/b 0.45) made with a) OPC, b) 15FA and c) 35FA after three days up to six months of curing at 20 °C in under sealed conditions.
In d) the three binders are compared after six months of curing. All results are presented as physically bound water (We) per gram of binder (B) as a function of the RH.

OPC 28 d

15FA 28 d
11-30% C-S-H interlayers
35FA 28 d 30-70% Gel pores
70-80% Large gel pores
OPC 6 m 80-90% Inter-hydrate spaces
90-95% Capillary pores
15FA 6 m

35FA 6 m a) b)

0 0.1 0.2 0.3 0 0.5 1

We /B / g g-1 Normalized moisture content / -

Fig. 7. Desorption isotherms after 28 days and six months from Fig. 6 presented as a) bars dividing the moisture contents into different RH-intervals and b) bars normalized
by the moisture contents at 95% RH.

reaction will be further delayed as there is so little OPC, and
because of this the reaction could be limited at later age as there
might not be enough Ca(OH)2 left for it to react with. Further-
more, it should be noted that at 70 wt% replacement, the starting
pore volume is reduced by almost 20% compared to the mix with
only OPC because of the difference in density between the fly ash
and OPC. Had the systems been designed so that the starting pore
volume was the same, the differences in, e.g., Fig. 9 would have
been even larger.
The replicate variations shown in Fig. 8b are not insignificant
compared to the development of the pore structure between six
and 18 months. By comparing the shape of the replicate desorption
isotherms it was concluded that variations caused by, e.g., sample
preparation, sampling and the sorption balance itself were negligi-
 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007 9

0.3 0.3

OPC OPC
OPC
0.25 0.25 15FA
15FA 15FA
35FA 35FA
35FA
g-1

g-1
0.2 0.2 70FA
70FA
/B // gg/g

/B // gg/g
0.15 0.15
W e/B

Wee/B
e
W

W
0.1 0.1

0.05 a) 6 mos 0.05 b) 18 mos.

Mortar

0 0
0 20 40 60 80 100 0 20 40 60 80 100
Relative humidity / % Relative humidity / %

Fig. 8. Desorption isotherms of mortars (w/b 0.45) made with OPC, 15FA or 35FA after a) 6 and b) 18 months of curing at 20 °C under sealed conditions. Presented as
physically bound water (We) per gram of binder (B) as a function of the RH. Error bars show the standard deviation of three measurements at 18 months.

Fig. 9. Desorption isotherms at six and 18 months from Fig. 8 here presented as a) bars dividing the moisture contents into different RH-intervals and b) bars normalized by
the moisture contents at 95%

ble. The shape of the isotherm was similar for all replicates, only
the absolute moisture contents varied. The uncertainty in the abso-
lute level of the desorption isotherms seem to mainly arise from
the binder content analysis applied to the mortar residues after
the sorption balance measurement [35]. Similar binder content
analyses were used in previous publications after sorption balance
measurements, e.g., [8,10,14]; however, none of the studies men-
tion the uncertainties the analyses give rise to.
3.2.2. The presence of aggregates
As the desorption isotherms for both cement pastes and mor-
tars are expressed per gram of binder they can be directly com-
pared. Previous studies have reported that aggregates have little
or no effect on this pore structure [8,20]. The mentioned studies
conclude that the only major difference between the microstruc-
tures of cement paste and mortar or concrete is the paste-
aggregate interface, or inter-facial transition zone (ITZ), that
reportedly forms in the presence of aggregates (e.g., [53,54].
Baroghel-Bouny [20] states that this zone should have little impact
on the sorption isotherms as the voids in which the moisture equi-
libration processes described by the sorption isotherms take place
are much smaller than those in the ITZ.
A comparison of the desorption isotherms of cement paste
(Fig. 6d) with those of mortar (Fig. 8a) after six months of curing
show that there are differences. However, normalizing the results
by the moisture content at 95% RH show that the shape of the iso-
therms is very similar (Fig. 10a-c). This indicates that the differ-
ences are mainly caused by variations in the binder content
analysis applied to the mortar samples, rather than the presence
of aggregates. The results thus suggest that sorption isotherms
for mortar and concrete can be measured on cement paste.
3.2.3. Different water to binder ratios
The w/b-ratio has a significant effect on the pore structure,
more so than moderate changes to the binder composition [7–
8,10,20,25]. Fig. 11 presents desorption isotherms measured on
mortar with 35FA and three different w/b-ratios. Fig. 12a-b pre-
sents the results from Fig. 11 as bars divided into different RH-
intervals; in Fig. 12b the results are normalized by the moisture
content at 95% RH.
The results in Figs. 11 and 12 are consistent with the findings of
previous studies and show that the w/b-ratio has a significant
impact on the desorption isotherm and thus on the pore structure;
with higher w/b-ratio the pore volume as well as the fraction of
inter-hydrate spaces and capillary pores increase. Changing the
w/b-ratio is likely to affect the impact of different SCMs on the pore
structure – and thereto related properties – as the available space
and water-filled porosity is changed. Furthermore, even as the w/
10 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007

1 1 1

/- /-
/- /-

/- /-
Paste Paste Paste

content
content

content
Mortar Mortar Mortar
0.8 0.8 0.8
fractions

fractions

fractions
moisture
moisture

moisture
0.6 0.6 0.6
Normalized

Normalized

Normalized
0.4 0.4 0.4

Normalized
Normalized

Normalized
0.2 0.2 0.2

0 0 0
0 50 100 0 50 100 0 50 100
Relative humidity / % Relative humidity / % Relative humidity / %

Fig. 10. Desorption isotherms of cement paste after six months from Fig. 6d and mortar after six months from Fig. 8a normalized by the moisture contents at 95% RH: a) OPC,
b) 15FA and c) 35FA.

0.3 therms presented in this paper. This has also been noted in previ-
ous studies [8,20]; even in second desorption, when the material
0.55
0.25 0.45 has been dried and re-saturated before the measurement [55].
0.32 However, looking more closely, this turns out to be mainly a visual
effect caused by the scale at which the isotherms are presented. For
We / B / g g-1

0.2
example, the differences in moisture content at 30% RH in Figs. 1
1 and 12a are actually in the range of 10–15%.
0.15
3.3. Moisture transport
0.1
Water vapour sorption isotherms measured in desorption
0.05 reflects the materials pore structure but give little information
about its moisture transport properties. Transport of moisture
0 depends not only on the pore volume and size distribution, but also
0 20 40 60 80 100 on the connectivity between pores and the tortuosity of the pore
Relative humidity / % structure. To study the effect of fly ash replacement on moisture
transport, cup tests were performed in desorption on mortar with
Fig. 11. Desorption isotherms of mortars made with 35FA and three different w/b-
ratios: 0.32, 0.45 or 0.55 after 18 months of curing at 20 °C under sealed conditions. three different binders: OPC, 15FA and 35FA as presented in Fig. 13.
Presented as physically bound water (We) per gram of binder (B) as a function of the The mean moisture diffusion coefficients show that fly ash
RH. replacement slows down moisture transport and decreases its
moisture dependence. Slower moisture transport is seen as lower
diffusion coefficients in both RH intervals, and decreasing moisture
b-ratio was changed from 0.55 to 0.32, the isotherms in Fig. 11 look dependence as smaller differences between the result in the tested
similar up to 50% RH. In fact, this seems to be the case for most iso- RH intervals. Although not as pronounced, the presented results

35FA 0.55

11-30% C-S-H interlayers

30-70% Gel pores
35FA 0.45 70-80% Large gel pores
80-90% Inter-hydrate spaces
90-95% Capillary pores

35FA 0.32

0 0.1 0.2 0.3 0 0.5 1

Wee/B
/B/ g/g
g g-1 Normalized
Normalized fractions
moisture /- /-
content

Fig. 12. Desorption isotherms from Fig. 11 here presented as a) bars dividing the moisture contents into different RH-intervals and b) bars normalized by the moisture
contents at 95%
 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007 11

10-7 slight decrease of capillary porosity with FA replacement could

5
be a partial explanation for the lower moisture diffusion coeffi-
OPC cients compared to material with only OPC. Thus, that moisture
4 15FA diffusion coefficients evaluated over the 95–75% RH-interval are
35FA predominately governed by the transport through capillary pores
corresponding to 90–95% RH.
Dv / m 2 s -1

3 Aside from pore sizes and volumes, moisture transport proper-

ties should also depend on the connectivity between pores and the
tortuosity of the pore structure. Previous studies have indicated
2
that both these features are changed by SCM reactions, like the
pozzolanic reaction of fly ash (see, e.g., [7,56,57]. For example,
1 most of the fly ash reacts after most of the OPC reaction has already
occurred. This means that the reaction products of fly ash precipi-
tate in an already formed pore structure. This could lead to an
0 increased heterogeneity of the pore structure as large capillary
0 20 40 60 80 100
Relative humidity / % pores become disconnected, forcing transport to go through smal-
ler openings. Such an effect is shown by Berodier and Scrivener [6],
Fig. 13. Cup test results of mortars made with OPC, 15FA or 35FA after 12 months who studied the pore structures of slag or fly ash blended cement
of curing at 20 °C under sealed conditions. Moisture diffusion coefficients (Dv) as a pastes with mercury intrusion porosimetry. Their results show that
function of the RH. Error bars show the standard deviation of six (OPC, 15FA) or
the critical pore entries – i.e., the smallest continuous channels in
three (35FA) samples.
the pore structure – are refined in the blended systems compared
to that with OPC. At the same time, their total porosities are
similar.
are similar to those reported for mortars with slag or silica fume in Previous studies have also shown that CASAH formed by SCMs
[9,10]. Saeidpour and Wadsö [9] used a similar cup test setup, they have a different chemical composition and structural morphology
found that partial slag or silica fume replacement decreased the than that formed by OPC [29,30]. Since CASAH is the main product
moisture diffusion coefficient by approximately a factor 10 com- of both the OPC and SCM reactions this could possibly explain the
pared to an OPC reference. From the results in Fig. 13 it seems that lower moisture transport coefficients. The main reason for these
35FA gives the largest reduction of diffusion compared to OPC, differences seems to be that most common SCMs contains less
approximately a factor five in the high RH-interval. The error bars CaO and more SiO2 and Al2O3 than OPC (cf. Table A.1). This leads
in Fig. 13 indicate that cup tests are a robust method to measure to the formation of CASAH with a lower Ca to Si ratio (C/S) that
moisture transport, and that the mortars were homogenous. To incorporates more Al [5,58,59]. In turn, this changes the growth
compare the transport results with the pore structure in the same mechanisms of the CASAH, from fibrillar needles growing in one
RH-interval, moisture contents between 95 and 70% RH, in mortars dimension to foils with a two-dimensional growth [60]. Whether
after 18 months of curing, were extracted from the desorption iso- these changes are connected to a slower moisture transport or
therms in Fig. 8b. The moisture contents are presented as bars in not remains to be explicitly shown.
Fig. 14.
The results in Fig. 14 indicate no relationship between the mois-
ture transport ability and pore volume in the same RH-interval, 4. Conclusions
rather the opposite as 35FA has the largest pore volume but the
lowest diffusion coefficient. However, looking only at the capillary Heat and sorption measurements showed that fly ash reacted
porosity corresponding to 90–95% RH it is seen that this porosity slower than OPC and had a limited effect on the pore structure in
decreases with increasing fly ash replacement. As the transport the first month. Beyond the first month the fly ash reaction altered
ability of a pore increases significantly with its cross-section, the the pore structure considerably, and was observed to continue to
do so more than a year after mixing. The differences in porosity
between the fly ash blended mixes and the reference with only
OPC decreased with time, but some remained at 18 months. The
fly ash reaction did not fill the pore volume but refined the capil-
lary porosity, increasing the fraction of inter-hydrate spaces. After
70-80%
one year of curing, the fly ash blended materials showed a factor 2–
80-90%
OPC m
OPC 18 mos. 28.5 16.4 8.1 5 lower moisture diffusion coefficients than the OPC reference,
90-95%
indicating that pore size distribution and connectivity have more
influence on permeability than the total pore volume. The binder
15FA 18 mos.
m 24.7 13.4 7.6 content analysis applied for mortar samples after sorption balance
measurements introduced an uncertainty in the moisture content.
By normalizing the mortar results with the moisture content at
35FA 18 mos.
m 37.5 18.5 6.7 95% RH, the shape of the isotherms could be compared while
avoiding this uncertainty. Sorption balance measurements on
cement paste and mortar at the same age showed that aggregates
had a negligible effect on the pore structure (corresponding to 11–
0 20 40 60 80 100
95% RH), and thus that sorption isotherms for mortar and concrete
Wee/B kg g-1
/B // mg -1
can be measured on cement paste. Additional sorption balance
Fig. 14. Upper part of mortar desorption isotherms (95–70%) after 18 months from
measurements showed that the effect of w/b ratio (0.55, 0.45,
Fig. 8b. The moisture contents corresponding to each RH-interval have been written 0.32) on the pore structure was larger than 15 or 35 wt% fly ash
out. replacement.
12 O. Linderoth et al. / Construction and Building Materials 261 (2020) 120007

CRediT authorship contribution statement Appendix B

O. Linderoth: Conceptualization, Methodology, Formal analysis,

Investigation, Data curation, Writing - original draft, Writing - 0.3
review & editing, Visualization, Project administration. P. Johans-
DVS intrinsic 1
son: Conceptualization, Writing - review & editing, Supervision,
0.25 DVS intrinsic 2
Funding acquisition. L. Wadsö: Writing - review & editing,
DVS 1000
Supervision.
0.2

We / B / g g-1
Declaration of Competing Interest

The authors declare that they have no known competing finan- 0.15
cial interests or personal relationships that could have appeared
to influence the work reported in this paper. 0.1

Acknowledgements
0.05
The authors express their gratitude to SBUF (The Development
Fund of the Swedish Construction Industry), Cementa AB and Skan- 0
ska AB for funding this study (grant number 13146). Cementa AB is 0 20 40 60 80 100
also acknowledged for providing the raw material, including XRF, Relative humidity / %
XRD-Rietveld analyses. The Nanocem Consortium is acknowledged
for accepting this work as a partner project, and Stefan Backe for Fig. B1. Desorption isotherms for cement paste with OPC and w/b 0.4 after six
months of curing measured with three different DVS instruments.
his assistance with the cup test.

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 Chemical Engineering Journal 434 (2022) 134704

Contents lists available at ScienceDirect

Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

Modeling of a cyclic sorption–desorption unit for continuous high

temperature CO2 capture from flue gas
Vanessa F. D. Martins a, Carlos V. Miguel a, Jonathan C. Gonçalves b, Alírio E. Rodrigues b,
Luís M. Madeira a, *
a
Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE), Department of Chemical Engineering, Faculty of Engineering – University of
Porto, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal
b
Laboratory of Separation and Reaction Engineering – Laboratory of Catalysis and Materials (LSRE-LCM), Department of Chemical Engineering, Faculty of Engineering –
University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal

A R T I C L E I N F O A B S T R A C T

Keywords: This study focuses on the modeling of a sorptive unit for CO2 capture at high temperatures, considering as case-
CO2 capture and storage study the data obtained from experiments using a conventional post-combustion stream from coal power plants:
Elovich-type equation 15% CO2/85% N2 at 623 K and 134 kPa. A one-dimensional (1D) heterogeneous dynamic fixed-bed model able to
Sorption kinetics
predict the CO2 equilibrium and kinetics of adsorption on a K-promoted hydrotalcite was successfully developed
bi-Langmuir equation
Adsorption
and implemented in gPROMS® software. The bi-Langmuir model was chosen to describe the experimental
equilibrium data, as it considers the two different adsorption sites on the K-promoted hydrotalcite. A loss of
capacity was observed after the first cycle performed in dry conditions. An Elovich-type expression, accounting
for the change in activation energy of adsorption and desorption for each site as a function of the surface
coverage, was also introduced in the model. The two sites model was used and an excellent fit to the break­
through curves of CO2 adsorption/desorption and temperature histories was obtained. In order to make the
model more robust, it was additionally validated at 573 K, for a total pressure range between 134 and 200 kPa, in
wet conditions. The model validation represents an important advance, since it will allow processes optimization,
and consequently the industrial design.

1. Introduction
The use of fossil fuels has been the basis of the entire world economy,
and its massive use as the main source of energy contributes to the in­
crease of CO2 levels on the atmosphere. CO2 is not the most harmful
greenhouse gas, but it is the most abundant, causing irreversible damage
to the environment, with a drastic impact on the climate change. In
2016, a record increase of CO2 in the atmosphere was registered,
exceeding 402.3 ppm on global average [1], while in 2020 this value
increased even further reaching 412.5 ppm [2].
Carbon capture and storage (CCS) technologies can play an impor­
tant role in reducing CO2 emissions to the atmosphere, as they allow the
capture, transport and storage of CO2 emitted by industrial sources in
appropriate geological formations [3,4]. Absorption is the most used
technology, as it presents high efficiency at low cost. However, the
performance and cost of this technology is compromised when applied
to processes, such as methanation, in which a reaction (and desirably the
CO2 capture) occurs at high temperature [5]. For these cases, in which it
is necessary to capture/separate CO2 at high temperatures, adsorption
can be an interesting alternative; however, it is necessary to choose an
appropriate adsorbent with good CO2 sorption stability under cyclic
operation, a reasonable kinetics and easy regeneration at moderate/high
temperatures. Several sorbent materials have been studied for this
purpose: calcium oxide [6–10], lithium oxides [11,12], lithium zirco­
nates [13–16], lithium silicates [17–19], hydrotalcites [20–35], double
salts [36], and basic alumina [37]. Among the several materials studied,
hydrotalcite-type materials have aroused great interest, since they show
a good CO2 sorption capacity, stability and easy regeneration by tem­
perature or pressure swing for temperatures in the range of 473–673 K
[34,35,38–46]. The use of hydrotalcites combined with reaction pro­
cesses that occur in the presence of water, such as water gas-shift,
reforming or methanation, is an advantage, since under these condi­
tions, the overall process is favored as well as the CO2 capture and
hydrotalcite regeneration [34,39–42,47–52]. In addition to the advan­
tages mentioned, hydrotalcites also have the advantage of being cheaper

* Corresponding author.
E-mail address: mmadeira@fe.up.pt (L.M. Madeira).

https://doi.org/10.1016/j.cej.2022.134704
Received 3 November 2021; Received in revised form 6 January 2022; Accepted 11 January 2022
Available online 17 January 2022
1385-8947/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
V. F. D. Martins et al. Chemical Engineering Journal 434 (2022) 134704

Nomenclature P total pressure (Pa)

PCO2 carbon dioxide partial pressure (Pa)
apM “macro particle” specific area (mpart− 1) qbt,exp experimental adsorption capacity of breakthrough curve
Bii Biot mass number of component i (− ) (mol kg− 1)
Cg,T total bulk phase concentration (mol mfluid− 3) qbt,mod model adsorption capacity of breakthrough curve (mol
Cg,i bulk phase concentration of component i (mol mfluid− 3) kg− 1)
Cp gas mixture molar specific heat at constant pressure (J qeq,CO2 carbon dioxide equilibrium sorption capacity (mol kg− 1)
mol− 1 K− 1) qexp the adsorbed amount obtained experimentally (mol kg− 1)
Cp,i average concentration of component i in the macropores qi adsorbed concentration of component i (mol kg− 1)
(mol mfluid− 3) q*i adsorbed concentration of component i in equilibrium with
C
̂ ps particle specific heat at constant pressure (per mass unit) (J Cp ,i (mol kg− 1)
kgsolid− 1 K− 1) qmax,j maximum capacity for each site j (mol kg− 1)
C
̂ pw wall specific heat at constant pressure (per mass unit) (J qmodel amount in the adsorbed phase predicted by the model (mol
kgwall− 1 K− 1) kg− 1)
Cs,i concentration of component i at the particle surface (mol qtotal adsorption capacity of breakthrough curve (mol kg− 1)
mfluid− 3) R ideal gas constant (J mol− 1 K− 1)
Cv gas mixture molar specific heat at constant volume (J Rp particle radius (mpart)
mol− 1 K− 1) t time (s)
Cv,i molar specific heat of component i at constant volume (J t0 initial time (s)
mol− 1 K− 1) To bulk phase temperature of each cycle at t0 (K)
Cv,ads,i molar specific heat of component i in the adsorbed phase at T temperature (K)
constant volume (J mol− 1 K− 1) tbt,exp experimental breakthrough time (s)
Dax axial dispersion coefficient (mbed2 s− 1) T∞ ambient temperature (K)
dp particle diameter (mpart) Tg bulk phase temperature (K)
Dp,i macropore diffusivity of component i (mpart2 s− 1) Tp solid temperature (K)
Dw bed diameter (mbed) ts sorption stage time (s)
e wall thickness (mbed) tsf step time (s)
Eads,i heat of reaction of component i (J mol− 1) Tw wall temperature (K)
Ea,ads,i,j adsorption activation energy for each component i and site U overall heat transfer coefficient (J s− 1 K− 1 mwall− 2)
j (J mol− 1) u0 superficial velocity (mfluid3 mbed− 2 s− 1)
Ea,des,i,j desorption activation energy for each component i and site yin
CO2 CO2 molar fraction at the system inlet stream (− )
j (J mol− 1) yout
CO2 CO2 molar fraction at the system outlet stream (− )
E0a,ads,i,j initial adsorption activation energy (J mol− 1) yi mole fraction of component i in the fluid phase (− )
E0a,des,i,j initial desorption activation energy (J mol− 1) z axial position (mbed)
in
FCO CO2 molar flow rate at the column inlet (J mol− 1)
2
Greek symbols
Fobj objective function (− )
α capacity factor (− )
hf film heat transfer coefficient between the gas and particle
αw ratio of the internal surface area to the volume of the
(J s− 1 K− 1 mpart− 2)
column wall (mwall− 1)
hw film heat transfer coefficient between the gas and wall (J
α wℓ ratio of the log mean surface to the volume of column wall
s− 1 K− 1 mpart− 2)
(mwall− 1)
kads,i,j adsorption rate constant for each component i and site j
βads,i,j change in activation energy from empty surface to fully
(s− 1)
kdes,i,j desorption rate constant for each component i and site j covered surface for component i on site j (J mol− 1)
(s− 1) βdes,i,j change in activation energy from fully covered surface to
Keqj,CO2 CO2 sorption constant in site j (Pa− 1) empty surface for component i on site j (J mol− 1)
K0eqj,CO2 CO2 pre-exponential factor for the sorption constant on site ΔH heat of adsorption (J mol− 1)
j (Pa− 1) ΔHads,i isosteric heat of adsorption of component i (J mol− 1)
kf film mass transfer coefficient of component i (mfluid3 ε bed porosity (mfluid3 mbed− 3)
i
mbed− 2 s− 1) εp particle porosity (mpores3 mpart− 3)
k0ads,i,j pre-exponential or frequency factor for sorption rate of λ heat axial dispersion coefficient (J s− 1 mbed− 1 K− 1)
bulk gas mixture viscosity (kgfluid mfluid− 1 s− 1)
component i and site j (s− 1)
μ
θ fractional coverage (− )
k0des,i,j pre-exponential or frequency factor for desorption rate of
ρ bulk gas mixture density (kgfluid mfluid− 3)
component i and site j (s− 1) ρp particle density (kgsolid mpart− 3)
L bed length (m) ρw wall density (kgwall mwall− 3)
mads hydrotalcite mass (kg) Ω linear driving force (LDF) factor (− )
N total number of measurements performed (− )

when compared to other materials available for this purpose. Due to the
potential of these adsorbents for large scale applications, a K-promoted
MgO-Al2O3 hydrotalcite was successfully tested in the STEPWISE pilot
installation for SEWGS (Sorption Enhanced Water-Gas Shift) [53].
Several authors proved that the adsorption capacity of CO2 on
hydrotalcite-based adsorbents is promoted by the presence of alkaline-
metals like potassium [21,40,54–56], and that is why this study will
have these materials as its main focus. Other authors discussed the ex­
istence of more than one type of sites within the K-promoted hydro­
talcite structure, suggesting different models to account for the sorption

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V. F. D. Martins et al. Chemical Engineering Journal 434 (2022) 134704

mechanism of CO2 in these materials [24–26,32,33,40,57–59]. Oliveira
et al. [40] determined the CO2 adsorption isotherms in wet conditions at
579, 676, and 783 K, up to 50 kPa on hydrotalcites previously impreg­
nated with potassium and different Mg:Al ratios. A bi-Langmuir model
was proposed by the authors to describe the CO2 sorption capacity in all
the samples prepared [40]. Regarding the kinetics, Ebner and co-
workers [24,25] determined CO2 nonequilibrium isotherms on a K-
promoted hydrotalcite for low CO2 partial pressures and proposed a
chemical kinetic approach to explain the CO2 behavior. The data was
obtained in the absence of water and they observed a rapid initial uptake
followed by slow sorption kinetics due to a complex reaction pathway
involving three connected reversible reactions. More recently, Coenen
et al. study the CO2 adsorption mechanism and kinetics on a KGM30
hydrotalcite under different conditions [50,51]. The authors suggested
that at least four different adsorption sites participate in the adsorption/
desorption of CO2 and H2O. In agreement with other authors they also
verified that the regeneration of the adsorbent with H2O leads to an
increase in the CO2 sorption capacity [51]. In order to describe the CO2
adsorption kinetics, Coenen et al. used an Elovich expression that con­
siders the activation energy of adsorption or desorption as a function of
the surface coverage of the sorbent [33].
The aim of the present work is to implement, for the first time, a one-
dimensional (1D) heterogeneous dynamic fixed-bed model in contin­
uous cyclic operation, able to predict the CO2 adsorption and desorption
on a K-promoted hydrotalcite at different conditions (temperature,
pressure, as well as in wet and dry conditions). The 2-step (adsorption/
regeneration) cyclic experiments are detailed in the previous work
performed by Miguel et al. [48]. The model herein implemented takes
into consideration the overall mass and energy balances, and Ergun
relation for pressure drop. A bi-Langmuir model was selected to describe
the CO2 equilibrium adsorption data, and an Elovich-type equation to
accurately predict the sorption kinetics. The model developed here is an
asset for the study of cyclic processes of CO2 capture, namely to be
applied to PSA (pressure swing adsorption) studies and to processes,
such as the one developed by Miguel et al. [48], which contemplate a
cyclic adsorption/methanation hybrid process (CO2 capture followed by
reactive regeneration for its conversion into methane using renewable
hydrogen – power-to-gas concept). In order to increase the scope of the
model, it was validated under different conditions, in dry and wet
conditions, at different temperatures (573–623 K) and total pressure
range between 134 and 200 kPa (CO2 partial pressure between 20 and
30 kPa). The robustness and validation of the model constitutes a
powerful tool for the CO2 capture process optimization and design.
2. Modeling and simulation
This work has as main goal the development of a dynamic model to
cycles with hydrogen during the (reactive) regeneration stage for CO2
methanation at 573 K and a pressure range between 134 and 200 kPa
[48]. In these methanation cycles (cycle 13 and 16 of the Table S1), H2O
is produced and therefore these cycles are considered to be in wet
conditions. These cycles can thus be used, not only to extend the range of
temperatures and pressures for which the model is valid, but also to
validate the model for wet conditions. The cycles were performed until a
cyclic steady CO2 sorption capacity was reached.
The gPROMS® - general PROcess Modelling System software - was
the software selected to solve the partial differential and algebraic
equations of which consists the model conceived for CO2 capture [60].
2.1. Sorption equilibrium and kinetic models
From the wide range of existing models able to describe the iso­
therms equilibrium data, the bi-Langmuir model was chosen, since it
provides a simplified quantitative description of the physicochemical
heterogeneity of the hydrotalcite surface and can be easily applied for
non-isothermal systems modeling [57–59]. The bi-Langmuir model was
also considered by several authors to fit experimental CO2 adsorption
equilibrium data measured in wet conditions [48,57–59].
The overall CO2 sorption is taking place by a combination of physical
sorption and chemical reaction. The bi-Langmuir model is a straight­
forward model that allows the consideration of two different and inde­
pendent types of sites, in which each one follows a Langmuir behavior
[61]. The bi-Langmuir model is as follows:
[ ]
Keq1,CO2 PCO2 Keq2,CO2 PCO2
qeq,CO2 = α qmax,1 + qmax,2 (1)
1 + Keq1,CO2 PCO2 1 + Keq2,CO2 PCO2
where α is the capacity factor (which takes into account the capacity loss
until reaching the steady/working capacity); other variables have
common meanings and are properly described in the nomenclature
section. Considering site 1 the one responsible for the exothermic
physical adsorption, and that site 2 represents the endothermic chemical
reaction, the CO2 sorption constants Keqj,CO2 (j = site 1 and 2) are given
by the following equations [58]:
( )
− ΔHads
Keq1,CO2 = K0eq1,CO2 × exp (2)
RTp
( )
− Eads
Keq2,CO2 = K0eq2,CO2 × exp (3)
RTp
The parameters that fit the experimental adsorption equilibrium data
were obtained by the minimization of the following objective function,
Fobj:
∑
N

simulate a continuous adsorption/desorption cyclic process using the Fobj = (qexp − qmodel )2 (4)
data obtained by Miguel et al. [48] for the capture of CO2 in a single-
i=1

column unit. In a very succinct way, Miguel et al. proposed a series of The mathematical simulation of gas separation processes requires
experiments (Table S1 of the Supporting Information), using the unit models to describe the adsorption kinetics. In the present study, the
described in detail elsewhere – cf. Fig. S1 [48]. In these experiments, the linear driving force (LDF) model for intraparticle mass transfer was
reactor column was packed in alternating layers of K-promoted hydro­ used, and thus the rate of CO2 adsorption/desorption into the hydro­
talcite pellets (23.89 g) and a Ni-based methanation catalyst spheres talcite particle can be given by the following equations [62,63]:
(7.58 g), as illustrated in Fig. S1 of the Supporting Information; the ( )
∂qCO2
catalyst layers for the purpose of the CO2 adsorption/desorption are = kads,CO2 ,j q*CO2 − qCO2 (5)
∂t
assumed inert.
The set of experiments consisted on the dynamic study of the CO2 ∂qCO2 ( )
sorption behavior on the K-promoted hydrotalcite, using as case-study a = kdes,CO2 ,j q*CO2 − qCO2 (6)
∂t
conventional post-combustion (dry) stream obtained from coal power
plants: 15% CO2/85% N2 mixture. The packed bed was subject to six The kads,CO2,j and kdes,CO2,j parameters correspond respectively to the
sorption–desorption cycles, and the regeneration step with N2 started carbon dioxide adsorption and desorption rate constant for each site j,
when the feed was switched using a 4-way valve placed near the column and can be expressed with an Arrhenius-type equation [33]:
( )
inlet. The total feed flow rate in the adsorption and regeneration steps − Ea,ads,CO2 ,j
was kept constant (i.e. 100 cm3PTN⋅min− 1), at 623 K, and the pressure
0
kads,CO2 ,j = kads,CO2 ,j
× exp (7)
RTg
inside the column was 134 kPa. Miguel et al. performed additional

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V. F. D. Martins et al. Chemical Engineering Journal 434 (2022) 134704

(
− Ea,des,CO2 ,j
) • the film model represents the external mass and heat transfer re­
0
kdes,CO2 ,j = kdes,CO2 ,j
× exp (8) sistances and to account for the internal mass transfer resistance, a
RTg
double LDF model was used;
The Elovich equation was used by several authors to model sorption • the adsorption kinetics behavior was described for the different sites
of different sorbates onto solid sorbents with heterogeneous surfaces based on the kinetic model shown in the previous section;
[32,33,64,65]. Based on these previous studies, an Elovich-type • the bed porosity was considered constant throughout the column,
expression was successfully used to obtain the CO2 adsorption/desorp­ and N2 (used as purge gas to regenerate the bed between break­
tion activation energy as a function of the surface coverage of the sor­ through experiments) was considered not to be adsorbed on the K-
bent [33]: promoted hydrotalcite;
• the Ergun equation was used to relate the pressure drop along the
0
Ea,ads,CO2 ,j = Ea,ads,CO + βads,CO2 ,j θ (9)
2 ,j length of the layered bed with the superficial gas velocity. This
equation also includes the pressure drop dependency with the
0
Ea,des,CO2 ,j = Ea,des,CO 2 ,j
− βdes,CO2 ,j θ (10) packing size, gas viscosity, and gas density (momentum balance):
The fractional coverage (θ) is obtained by the following expression:
∂P 150μ(1 − ε)2 1.75(1 − ε)ρ 2
− = u0 + u0 (19)
qCO2 ∂z ε3 dp2 ε3 dp
θ= (11)
q*CO2
Boundary condition:

2.2. Fixed bed for CO2 capture z=L P = Pexit (20)

In the CO2 capture step, the process is classified as a dynamic

• finally, the energy balance to the gas phase and to the hydrotalcite
adsorption, since the gas continuously passes through the column
pellet was included in the model. Equations (21) and (22) allow the
packed with the K-promoted hydrotalcite and inert, in alternating
determination of the respective gas and solid temperatures, as
layers. The mathematical model development was based on mass, mo­
follows:
mentum, and energy balances (and respective boundary conditions), ( )
according to the equations (12) to (25) [66]. Some assumptions were ∂ ∂Tg ∂Tg ∂Cg,T ( )
λ − u0 Cg,T Cp + εRTg − (1 − ε)apM hf Tg − Tp
taken into consideration in the model; particularly, it was considered: ∂z ∂z ∂z ∂t
4hw ( ) ∂Tg
− Tg − Tw − εCg,T Cv =0 (21)
• axially dispersed plug-flow behavior to describe the gas flow; Dw ∂t
• gradients only in the axial direction of the bed; [ ]
• the mass balances for an axial dispersion fixed-bed plug-flow were ∑
n ∑
n
∂Tp
(1 − ε) εp Cp,i Cv,i + ρp ̂
qi Cv,ads,i + ρp C ps
described following equations (12) to (14): ∂t
i=1 i=1
( ) ( )
∂ ∂y ∂( ) ∂Cg,i apM kfi ( ) ∂Cp,T ∑n
∂q ∑ n
∂q
εDax Cg,T i − uC − ε − (1 − ε) Cg,i − Cs,i = 0 = (1 − ε)εp RTp +ρ ( − ΔHads )i i + ( − Eads )i i + (1
∂z ∂z ∂z 0 g,i ∂t Bii + 1 ∂t ∂t ∂t
i=1 i=1
(12) ( )
− ε)apM hf Tg − Tp
∑
n
(22)
Cg,T = Cg,i (13)
i=1 Boundary conditions:

Cg,i = yi Cg,T (14) z=0 u0,inlet Cg,T,inlet Cp Tinlet = u0 Cg,T Cp Tg − λ

∂Tg
(23)
∂z
Boundary conditions:
∂ Tg
∂yi z=L =0 (24)
z=0 u0,inlet Cg,T,inlet yi,inlet = u0 Cg,i − εDax Cg,T (15) ∂z
∂z

z=L
∂yi
=0 (16) • equation (25) provides the energy balance associated with the wall,
∂z to account the heat transfer between the wall and the surroundings:
∂Tw ( )
• CO2 sorption equilibrium was described by the bi-Langmuir model
̂ p,w
ρw C = αw hw Tg − Tw − αwl U(Tw − T∞ ) (25)
∂t
detailed in the previous section;
• CO2 and N2 ideal gas behavior: Dw
αw = (26)
e(Dw + e)
P = Cg,T RTg (17)
2
αwl = ( ) (27)
• the gas phase composition may not be uniform throughout the (Dw + e)ln Dw+2e

hydrotalcite particle, due to the diffusion phenomena, which occur

Dw

in the pores of the solid material. Considering these phenomena, it

was necessary to accurately describe the mass transfer within the • the temperature was assumed uniform along each adsorbent particle,
particle macropores (mass balance for CO2 on the solid phase): and the heat transfer between the solid phase and the wall was
∂Cp,i ΩDp,i Bii ( ) ρp ∂qi neglected;
= 2 Cs,i − Cp,i − (18) • only the energy exchanges between the column wall with the gas
∂t Rp Bii + 1 εp ∂t
phase and the surroundings were taken into account.

The transport parameters used throughout the previous equations

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V. F. D. Martins et al. Chemical Engineering Journal 434 (2022) 134704

can be obtained through correlations widely used and easily found in the
literature [67]. The correlations used are described in the Supporting
Information.
The equations of the initial conditions can be found on Table 1.
Finally, in order to compare the adsorption capacity between cycles,
the CO2 sorption capacity (qCO2) of the packed-bed was determined as
follows:
( )
in
∫ tsf yout
CO2
FCO 2 0
1 − yin
dts
CO2
qCO2 = (28)
mads
The simulations were carried out in gPROMS® and the partial dif­
ferential equations were discretized using the orthogonal interpolation
method, with third order polynomials, and 20 discretization intervals.
The system of ordinary differential equations with respect to time was
solved using the DASOLV solver from gPROMS®.

3. Results and discussion

Fig. 1. Adsorption isotherms measured on K-promoted hydrotalcite for CO2 at

In the present study a dynamic model to predict the capture of CO2 in
579 K [58] and 633 K [57]. Symbols represent the experimental data, and the
a cyclic unit was conceived. The main bed characteristics are presented solid lines represent the bi-Langmuir model fit.
on Table S2, while the layered bed scheme can be found on Fig. S1. The
properties of the hydrotalcite sorbent are displayed on Table S3 of the
Supporting Information. As case study, a typical post combustion (dry)
stream obtained in coal power plants with a composition of 15% CO2
balanced in N2 at 623 K and 134 kPa was considered.
The design of an adsorption process requires the availability of
reliable equilibrium data and a theoretical model for accurate descrip­
tion of the experimental data measured. The CO2 adsorption equilibrium
data in this K-promoted material have been frequently reported
[23,26,29,50,57–59] considering different models and approaches. In
Fig. 1 are plotted the CO2 adsorption isotherms (amount adsorbed as a
function of CO2 partial pressure), measured by Oliveira [58], and by
Chanburanasiri et al. [57] at 579 K and 633 K, respectively, both in the Fig. 2. Comparison between bi-Langmuir total contribution, physical adsorp­
presence of steam. The solid lines in the same figure correspond to the tion contribution (q1), and chemical reaction contribution (q2) at (a) 579 K, and
fitting of the experimental data using the bi-Langmuir model (Eq. (1)). In (b) 633 K.
Fig. 2a and 2b the lines correspond to the CO2 physical sorption and
chemical reaction contributions in the same material at different tem­
Table 2
peratures as predicted by the model. The optimal bi-Langmuir param­
The bi-Langmuir parameters for CO2 adsorption equilibrium isotherms on K-
eters obtained by minimizing the error between experimental and
promoted hydrotalcite at 579, and 633 K (for a capacity factor, α = 1).
predicted data (Eqs. (1)–(4)) are presented in Table 2.
Component qmax,1 K0eq1, -ΔHads, qmax,2 K0eq2, Eads,
The CO2 experimental data at 579 K and 633 K up to 40 kPa was well
mol⋅kg¡1 CO2 CO2 mol⋅kg¡1 CO2 CO2
described by the model (as can be observed in Fig. 1), which accounts for kPa¡1 kJ kPa¡1 kJ
two different sites that follow different adsorption mechanisms. One site mol¡1 mol¡1
accounts for the physical adsorption, an exothermic spontaneous pro­ CO2 0.47 4.0 × 48 0.60 8.0 × − 140
cess (− ΔHads > 0) attributed to Van der Walls [68,69] and electrostatic 10− 5 109
forces [68], and consequently the capacity must decrease with temper­
ature. Chemical reaction is also spontaneous, (usually) endothermic at
high temperatures and commonly irreversible [68,69]. Fig. 2 shows that results of the cyclic fixed-bed model are compared with the experi­
the contribution of physical adsorption (q1) slightly decreases with mental breakthrough curves (operating conditions on Table S1). As
temperature, as opposed to the chemical reaction (q2) that increases illustrated in Fig. 3 (cycle 0 to 5), Fig. 4 (cycle 0), Fig. 5 (cycle 1), and
with temperature. It is also possible to observe in Fig. 2 that the chemical Fig. S2 (cycle 2 to 5) of the Supporting Information, the experimental
reaction at 579 K has a negligible contribution when compared to the breakthrough curves of CO2 are predicted quite well by the model. The
same at 633 K for the pressure range measured. simulations were performed using the parameters present in Table S4.
For checking the validity of the proposed model, the simulation However, only on cycle 0 the CO2 adsorption was simulated considering
the bi-Langmuir equilibrium parameters present in Table 2, whereas for
the other cycles it was necessary to consider a loss of CO2 capacity factor
Table 1 (1- α) of 38 % on the hydrotalcite. This was experimentally observed by
Initial conditions employed in the breakthrough simulations. Miguel et al. [48] and agrees with what has been observed by other
authors [22,33,40,48,50–52]. This loss of capacity is attributed to the
Initial Conditions
CO2 molecules adsorbed in the first adsorption step (cycle 0), which
yi = Cp,i = qi = 0 for i ∕
= inert cannot be removed by feeding only N2 during regeneration. According
yinert = 1
to the values shown in Table 3, in the cycle 0 a CO2 adsorption capacity
Cp,inert = Cg,T
Tg = Tp = Tw = Tinlet
of ca. 0.53 mol⋅kg− 1 was experimentally obtained (calculated using
P = Cg,T Rg Tg equation (28), which agrees quite well with model predictions), while in

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V. F. D. Martins et al. Chemical Engineering Journal 434 (2022) 134704

Fig. 3. The sorption of 0.15/0.85 CO2/N2 mixture on K-promoted hydrotalcite beads at 623 K and 134 kPa (PCO2 = 20 kPa) from cycle 0 to cycle 5. Open symbols
stand for experimental data and continuous line for the model.

Fig. 4. (a) Adsorption on cycle 0 of a mixture of 0.15/0.85 CO2/N2 over a bed initially full of N2 at 623 K and 134 kPa (PCO2 = 20 kPa); (b) desorption of previously
adsorbed CO2 in flowing N2 at 623 K and 134 kPa (PCO2 = 20 kPa). Open symbols represent experimental data and solid lines simulation results.

Fig. 5. (a) Adsorption on cycle 1 of a mixture of 0.15/0.85 CO2/N2 over a bed initially full of N2 at 623 K and 134 kPa (PCO2 = 20 kPa); (b) desorption of previously
adsorbed CO2 in flowing N2 at 623 K and 134 kPa (PCO2 = 20 kPa). Open symbols represent experimental data and solid lines simulation results.

the other cycles the CO2 adsorption capacity varied between 0.31 and
0.35 mol⋅kg− 1. The steady working capacity was mostly reached at the
end of the second cycle, as can be seen through the comparison of Fig. 5
with Fig. S2. Coenen et al. observed that the regeneration of the
adsorbent with steam promoted a significant increase in the CO2 cyclic
working capacity from 0.3 to 0.53 mol⋅kg− 1 when compared to a dry
regeneration with N2 for the same cycle time [51], which corroborates
the data presented in this study. The time at which breakthrough occurs
for the different cycles can also be found on Table 3. Comparing these
values, it is possible to verify that the breakthrough times are similar for
cycles 1 to 5, varying between 444 and 485 s (variations below 10%).
However, it is in the cycle 0 that, as expected, there is a more significant
difference, since CO2 only breaks after almost 800 s.
The simulated results based on the bi-Langmuir model combined
with the Elovich-type equation fit well most of the experimental results
in terms of adsorption kinetics and capacity during the cycles 0–5,
indicating the quality of the proposed model. As can be seen in Fig. 3, the
desorption step was carried out for longer periods of time, to ensure that
all the CO2 would be desorbed; and it is possible to verify that the model
can predict desorption for long time cycles. The small discrepancies
observed in desorption between the model results and the experiments
on Fig. 4b, Fig. 5b, and Fig. S2 only show some underestimation of the
desorption kinetics in the first 1000 sec of the cycle step.
The thermal positive peak and the sharp breakthrough curves ob­
tained in the adsorption of CO2 are due to the adsorption heat of this
component and its favorable equilibrium isotherm, respectively. In

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V. F. D. Martins et al. Chemical Engineering Journal 434 (2022) 134704

Table 3 behavior of the temperature between cycles 1 and 5 corroborates what

Sorption capacity and breakthrough times comparison for the different cycles at has already been said regarding the steady working capacity being
623 K. reached at the end of the second adsorption cycle (cycle 1). According
Step Cycle qbt,exp qbt,mod tbt,exp, qtotal ¼ α (q1 þ q2) with the bi-Langmuir model considered, the thermal peak is a combi­
mol kg¡1 mol kg¡1 s nation of a physical (exothermic) and a chemical (endothermic)
Adsorption 0 0.5295 0.5289 799 100 (q1 + q2) contribution. If only the site responsible for the endothermic (chemical)
1 0.3411 0.3387 470 0.62 (q1 + q2) contribution remained “available” in cycles 1 to 5, in these cycles the
2 0.3283 0.3313 455 0.62 (q1 + q2) CO2 adsorption would show only negative thermal peaks. On the other
3 0.3147 0.3357 444 0.62 (q1 + q2)
hand, if only the site responsible for the exothermic contribution
4 0.3537 0.3500 460 0.62 (q1 + q2)
5 0.3337 0.3356 485 0.62 (q1 + q2) (physical) remained “available” in cycles 1 to 5, a wider positive peak
Desorption 0 0.3679 0.3420 N.A. 0.62 (q1 + q2) would be expected. None of these limiting situations correctly represents
1 0.3553 0.3428 N.A. 0.62 (q1 + q2) what is observed experimentally, so a capacity factor, α, evenly
2 0.3642 0.3428 N.A. 0.62 (q1 + q2) distributed across the two sites, was considered instead (cf. Table 3).
3 0.3873 0.3486 N.A. 0.62 (q1 + q2)
4 0.3795 0.3517 N.A. 0.62 (q1 + q2)
This assumption corroborates the mechanism suggested by Du et al.
5 0.2851 0.3424 N.A. 0.62 (q1 + q2) [70]. The authors suggested that CO2 is reversibly sorbed in two types of
active sites on K-promoted hydrotalcite in dry conditions: those that
promote the formation of bidentate carbonate (more active sites with
Fig. 6, Fig. 7, and Figs. S3-S5 of the Supporting Information it is possible favorable kinetics and thermodynamics, but less abundant) and others
to observe the temperature history during cycles 0 to 5, and particularly favoring the formation of unidentate and bridged carbonate species, also
that after cycle 0 the temperature behavior changed and remains quite with fast sorption kinetics. The CO2 loss of capacity with cycling can be
similar until cycle 5. The difference between cycle 0 and the other cycles explained by the slow conversion of unidentate and bridged carbonates
is a result of the loss of capacity observed upon the first cycle. Plus, the into irreversibly sorbed bulk polydentate carbonate. Miguel et al. in a

Fig. 6. History of gas temperature along the cycles and measured at 11.82 cm from the top of the column (4th thermocouple in Fig. S1). Open symbols represent
experimental data and solid lines simulation results.

Fig. 7. History of gas temperature measured at 11.82 cm from the top of the column (4th thermocouple in Fig. S1) during the (a) adsorption step on cycle 0, (b)
desorption step on cycle 0; (c) adsorption on cycle 1, and (d) desorption on cycle 1. Open symbols represent experimental data and solid lines simulation results.

7
V. F. D. Martins et al.
previous work proposed this mechanism with two active sites for CO2
sorption with parallel contributions on K-promoted hydrotalcite and
proved that the model can describe accurately the CO2 sorption in wet
and dry conditions for a temperature range between 473 and 573 K [39].
From a global point of view, the mathematical model herein pro­
posed was validated, since it predicts well the CO2 mass and temperature
behavior for cycles 0 to 5 and the potential of K-promoted hydrotalcite
as adsorbent for CO2 capture. In fact, the good prediction of the mass
(both adsorption and desorption) and temperature histories indicates
that the adsorption equilibrium and kinetic model captures the right CO2
behavior for each site.
To predict the CO2 kinetic behavior on the K-promoted hydrotalcite,
the adsorption and desorption steps of cycle 0 were firstly simulated
using the conventional LDF model; it was found that the simulation
results using such model are not suitable to represent this system (data
not shown for brevity). Thus, it was decided to explore the Elovich
model, used to describe the interactions of gas molecules with hetero­
geneous surface sites on the sorbent, accounting for the change in the
activation energy of adsorption and desorption as a function of the
surface coverage.
The change in activation energy modelled with the Elovich-type
equation for the adsorption and desorption kinetics of CO2 is shown in
Fig. 8 (for site 1), and Fig. 9 (for site 2). Using a small desorption con­
stant (conventional LDF model) without the modified Elovich equation
would result in an almost linear weight loss, which would not
adequately describe these experiments with long cycle times. The
Elovich-type kinetic model fitted parameters (k0, E0 and β for adsorp­
tion/desorption in each site) are included on Table S4. All parameters
were defined considering that complete desorption occurs for cycles 1 to
5 after a long desorption time at 623 K under N2, as experimentally
observed. The simulations performed show that the change in activation
energy within one sorption step is about 50 kJ/mol for site 1 and about
10 kJ/mol for site 2, for cycles 0–5.
During the desorption step, the CO2 that has less affinity with the
surface of the K-promoted hydrotalcite begins to be desorbed first, i.e.
with a smaller activation energy. For both sites during the desorption
step the activation energy increases when the surface coverage de­
creases. Coenen and co-workers observed the same behavior for the
activation energy during the CO2 desorption step on a MG30K hydro­
talcite [33].
As illustrated in Fig. 10 and Fig. 11 the kinetic coefficient changes
during the adsorption and desorption step when compared with the
traditional LDF model. When the CO2 coverage ratio is low, the
adsorption kinetic coefficient is large, promoting a very fast adsorption
rate, namely in the beginning of the sorption stage. With the CO2 uptake
increase, kCO2,ads decreases considerably, as observed in Fig. 10a (site 1)
and Fig. 11a (site 2). Similar results were observed by Ebner et al. [25],
Fig. 8. Change in activation energy on site 1 for CO2 adsorption/desorption kinetics on the K-promoted hydrotalcite modelled with the Elovich equation for cycle
0 to 5.
8
Chemical Engineering Journal 434 (2022) 134704
who pointed out that the adsorption rate was 10 times faster than the
desorption one. According to the results obtained in the present work,
the adsorption rate is 8.2 times faster than the desorption for site 1, and
20.2 times faster for site 2.
The developed kinetic model can describe quite well the experi­
mental observations, so that it can be concluded that the equations used
in the process modelling to account for site heterogeneity during the CO2
adsorption and desorption on the hydrotalcite provide an adequate
description.
In order to further extend the model validity in a wider range of
process conditions, simulations were performed at 573 K for a total
pressure range between 134 kPa and 200 kPa, in wet condition. In the
previous study performed by Miguel et al. [48], after the first six cycles
of CO2 capture with normal regeneration with N2, reactive regeneration
cycles with H2 were performed to transform the previously captured CO2
into CH4, keeping the same total feed flow rate (i.e. 100 cm3PTN⋅min− 1)
and feed (15% CO2/85% N2) in the adsorption steps at different con­
ditions. In order to increase the range of temperatures and pressures for
which the model is valid, without further optimization/fittings, addi­
tional simulations were performed and compared with the experimental
data obtained in the representative cycle 13 (573 K and 134 kPa) and
cycle 16 (573 K and 200 kPa) of such work. Before CO2 capture in these
experiments (cycle 13 and 16 of the Table S1), the methanation has been
taking place due to the reactive regeneration and thus steam is being
produced (apart from methane). As already discussed, the steam allowed
to almost recover the initial bed capacity. Several authors observed that
the hydrotalcite recovered approximately 90% of its initial capacity
when in presence of steam and therefore the simulation of cycles 13 and
16 were carried out considering an α of 0.9 [22]. It is important to
mention that the equilibrium model was validated for the temperature
range between 579 and 633 K up to a CO2 partial pressure of 50 kPa (cf.
Fig. 1). For the simulation of cycles 13 and 16 the model was extrapo­
lated to 573 K. Fig. 12a and Fig. 13a show that the model is able to
represent quite well the experimental data obtained for the adsorption
step at 573 K for 134 kPa and 200 kPa of total pressure, respectively.
Comparing with cycle 0 at 623 K and 134 kPa (Fig. 4a), the same profile
shape was obtained in cycle 13 (Fig. 12a), although with different af­
finity because both sorption and reaction kinetics are less favored at 573
K. The model predictions concerning the temperature profiles do not
match so well experimental data (Fig. 12b and Fig. 13b), which is
probably due to differences in the physical and chemical contributions
to the adsorption phenomenon and the fact that parameters fitting was
only carried out at 623 K. Even so, the initial temperature rise and
amplitude are reasonably well predicted.
The obtained values for the bed sorption capacity in the sorption
stage of cycles 13 and 16 were compared to those predicted by the bi-
Langmuir sorption equilibrium (equation (1)). Using the bi-Langmuir
V. F. D. Martins et al. Chemical Engineering Journal 434 (2022) 134704

Fig. 9. Change in activation energy on site 2 for CO2 adsorption/desorption kinetics on the K-promoted hydrotalcite modelled with the Elovich equation for cycle
0 to 5.

Fig. 10. Change in the kinetic parameter on site 1 of the K-promoted hydrotalcite during cycle 0, for: a) CO2 adsorption, and b) CO2 desorption.

Fig. 11. Change in the kinetic parameter on site 2 of the K-promoted hydrotalcite during cycle 0, for: a) CO2 adsorption, and b) CO2 desorption.

Fig. 12. (a) Adsorption on cycle 13 of a mixture of 0.15/0.85 CO2/N2 over a bed initially full of H2 at 573 K and 134 kPa (PCO2 = 20 kPa); (b) history of gas
temperature measured at 11.82 cm from the top of the column (4th thermocouple in Fig. S1) during the adsorption step on cycle 13. Open symbols represent
experimental data and solid lines simulation results.

9
V. F. D. Martins et al.
Fig. 13. (a) Adsorption on cycle 16 of a mixture of 0.15/0.85 CO2/N2 over a bed initially full of H2 at 573 K and 200 kPa (PCO2 = 30 kPa); (b) history of gas
temperature measured at 11.82 cm from the top of the column (4th thermocouple in Fig. S1) during the adsorption step on cycle 16. Open symbols represent
experimental data and solid lines simulation results.
model for the conditions employed in these runs, and considering an α of
90%, the expected adsorption capacity at 20 and 30 kPa are 0.42 and
0.43 mol⋅kg− 1. In cycles 13 (Fig. 12a) and 16 (Fig. 13a) it is observed
that both have very close breakthrough times (565 s for cycle 13 and
610 s for cycle 16), and a similar adsorption capacity (0.41 and 0.42
mol⋅kg− 1, respectively), corroborating the information provided by the
bi-Langmuir model. It can be concluded that the equilibrium and kinetic
models are able to accurately predict the adsorption behavior for the
studied temperature and pressure ranges. These cycles can thus be used,
not only to extend the range of temperatures and pressures for which the
model is valid, but also to validate the model for wet conditions.
4. Future work
In the present work, a useful tool that allows the cyclic study of
adsorption/desorption phenomena in a fixed bed, using a K-modified
hydrotalcite, was developed and validated at different temperatures and
pressures. It is important to clarify that this model refers to the first part
of a work that aims to develop a more complex model to describe the
cyclic CO2 capture/methanation experiments carried out by Miguel et al
[48], being the methanation carried out by reactive regeneration with
hydrogen of the mixed bed (that contains both the CO2-selective sorbent
and a methanation catalyst). Considering the overall process, numerous
issues can influence the sorptive reactor performance in terms of CO2
capture capacity, CH4 productivity and purity. So, the influence of other
species present on flue gas should be assessed (e.g. steam, CO and O2)
and, later, the sorptive reactor must be tested employing an actual flue
gas stream. The same applies in continuous processes of CO2 capture, e.
g. by PSA, where the model can be crucial for process optimization,
having in mind maximization of some key performance indicators like
recovery, purity, or energy usage [71,72]. These issues are crucial for
process design, namely for establishing previous flue gas processing
requirements and to conclude about the suitability of the considered
materials.
The model developed can also be applied to predict the cyclic ca­
pacity in other adsorbents and adsorbates, after being properly charac­
terized; particularly, after obtaining kinetic and equilibrium data. In the
next and second part of this work, the model will also be prepared to
simulate the hybrid cyclic process of CO2 capture/methanation,
considering layered and mixed beds, and/or dual-function materials.
5. Conclusions
In this study a model to describe the cyclic CO2 sorption/desorption
in a packed-bed was successfully implemented considering the mass,
momentum, and energy balances, and appropriated assumptions
involving the specific characteristics of the hydrotalcite-based sorbent.
The bi-Langmuir model was applied to describe the equilibrium
behavior, accounting for two different adsorption sites on the K-pro­
moted hydrotalcite. After the first CO2 adsorption cycle in dry
10
Chemical Engineering Journal 434 (2022) 134704
conditions, the hydrotalcite loses 38% of its capacity and reaches its
working capacity. The model was developed taken into consideration
the capacity change after the first cycle, by applying a capacity factor, α,
of 62%, and simulations show that it correctly represents the experi­
mental data. The kinetics of the CO2 gas molecules interactions with
heterogeneous surface sites on the hydrotalcite was described with an
Elovich-type expression, accounting for the change in activation energy
of adsorption and desorption as a function of the surface coverage for the
two sites. The model used correctly describes the mass and temperature
behavior during the experimental CO2 adsorption and desorption cycles.
So, it is possible to conclude that the equations used in the modeling to
predict the bulk carbonate formation rate, and also the modified Elovich
equation to explain the site heterogeneity during the adsorption and
desorption, offer a satisfactory description of the system. Additionally,
to make the model more robust, it was further validated at a different
temperature (573 K), for a total pressure range between 134 and 200
kPa (PCO2 between 20 and 30 kPa), in wet conditions. The data obtained
in the simulation show that the model is able to successfully predict the
experimental results.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was financially supported by: i) Base Funding - UIDB/
00511/2020 of the Laboratory for Process Engineering, Environment,
Biotechnology and Energy – LEPABE - funded by national funds through
the FCT/MCTES (PIDDAC), ii) Project POCI-01-0145-FEDER-030277,
funded by FEDER funds through COMPETE2020 – Programa Oper­
acional Competitividade e Internacionalização (POCI) and by national
funds (PIDDAC) through FCT/MCTES, iii) Project
“HyGreen&LowEmissions - Tackling Climate Change Impacts: the role of
Green Hydrogen production, storage and use, together with low emis­
sions energy systems”, with the reference NORTE-01-0145-FEDER-
000077, supported by Norte Portugal Regional Operational Pro­
gramme (NORTE 2020), under the PORTUGAL 2020 Partnership
Agreement, through the European Regional Development Fund (ERDF),
and iv) Base-UIDB/50020/2020 and Programmatic-UIDP/50020/2020
Funding of LSRE-LCM, funded by national funds through FCT/MCTES
(PIDDAC).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cej.2022.134704.
V. F. D. Martins et al. Chemical Engineering Journal 434 (2022) 134704

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 Lusty Grace Toding, A. Karim Zulkarnain

OPTIMASI FORMULA DAN UJI IRITASI PRIMER

KUALITATIF PADA KELINCI PUTIH BETINA
DENGAN KRIM W/O EKSTRAK ETANOLIK BUAH
MAHKOTA DEWA [Phaleria macrocarpa
(Scheff.) Boerl.]
OPTIMIZING FORMULA AND QUALITATIVE PRIMARY
IRRITATION TEST IN FEMALE WHITE RABBIT OF W/O
CREAM THE ETHANOLIC EXTRACT OF PHALERIA FRUIT
[Phaleria macrocarpa (Scheff.) Boerl.]

Lusty Grace Toding, A. Karim Zulkarnain

Fakultas Farmasi, Universitas Gadjah Mada

ABSTRAK
Krim w/o ekstrak etanolik buah mahkota dewa [Phaleria macrocarpa (Scheff.)
Boerl.] sebesar 10% telah diuji mempunyai aktivitas sebagai tabir surya. Penelitian ini
bertujuan untuk mengetahui formula optimal, stabilitas fisik, dan aktivitas iritasi primer
krim w/o ekstrak etanolik buah mahkota dewa. Optimasi formula menggunakan metode
simplex lattice design dengan software Design Expert® versi 8.0.7.1 program mixture
design melalui modifikasi komposisi cera alba, span 80, dan minyak mineral. Sebanyak 10
formula krim w/o dievaluasi sifat fisiknya yang meliputi uji viskositas, daya lekat, daya
sebar, pH, rasio volume pemisahan, dan tipe emulsi. Area optimal diperoleh pada
komposisi 18,504% cera alba, 5,231% span 80, dan 44,265% minyak mineral. Berdasarkan
uji-t satu sampel, formula optimal krim w/o yang diperoleh mempunyai respon viskositas
dan daya sebar yang berbeda tidak signifikan dengan nilai prediksi respon yang diberikan
oleh software Design Expert® versi 8.0.7.1, sedangkan respon daya lekat berbeda
signifikan dengan nilai prediksi software. Uji-t berpasangan digunakan untuk mengetahui
stabilitas fisik krim w/o selama penyimpanan. Formula optimal krim w/o stabil selama 4
minggu penyimpanan berdasarkan respon rasio volume pemisahan dan pH serta krim tidak
menimbulkan eritema ataupun edema pada hewan uji.

ABSTRACT
W/O cream of 10 % ethanolic extract from Phaleria fruit [Phaleria macrocarpa
(Scheff.) Boerl.] have been tested have activity as a sunscreen. This study aims to determine
the optimal formula, physical stability and activity of primary irritant w/o cream of
ethanolic extract from Phaleria fruit. Optimization of formulation using simplex lattice
design method with Design Expert® software version 8.0.7.1 mixture design program
through of composition modification of cera alba, span 80, and mineral oil as much as 10
formulas. Evaluation of the physical characteristics of w/o cream that are viscosity,
adhesion, power spread, acidity, ratio of separation volume, and type of emulsion. The
optimal area obtained at 18,504% of cera alba, 5,231% of span 80, and 44,265% of
mineral oil. Based on one sample t-test, the w/o cream of optimation formula has viscosity
and power spread responses which is not different significantly from the predicted value of
responses given by Design Expert® except the adhesion response. Paired t-test used to
determine the physical stability of w/o cream during the storage. In addition to optimal
formula of w/o cream stable for 4 weeks of storage based on the ratio of separation volume
and acidity responses, the cream didn’t cause erythema or edema in animal test.

PENDAHULUAN manusia seperti sunburn, pigmentasi kulit, dan

Radiasi sinar UV dari matahari dapat penuaan dini. Oleh karena itu, dibutuhkan tabir
menimbulkan efek yang merugikan pada kulit surya untuk dapat melindungi kulit dari bahaya sinar

Majalah Farmaseutik, Vol. 11 No. 2 Tahun 2015 321

Optimasi Formula Dan Uji Iritasi …
matahari (Wang et al., 2008). Selain tabir surya
sintetik, terdapat pula tabir surya alami di alam.
Adanya kecenderungan pola hidup kembali ke alam
menyebabkan masyarakat lebih memilih
menggunakan obat alami yang diyakini tidak
memiliki efek samping dan harga yang lebih
terjangkau daripada obat sintetik.
Buah mahkota dewa secara tradisional
digunakan dalam pengobatan berbagai penyakit
(Harmanto, 2004). Penelitian melaporkan bahwa
dalam ekstrak metanolik buah mahkota dewa
mengandung 4,5-dihidroksi-4’-metoksibenzofenon-
3-O-β-D-glukosida (phalerin) (Wahyuningsih et al.,
2005). Phalerin yang terkandung dalam mahkota
dewa merupakan senyawa glikosida benzofenon.
Kerangka dasar phalerin yang berupa benzofenon
ini yang memungkinkan phalerin dapat digunakan
sebagai zat aktif tabir surya. Phalerin memiliki
aktivitas fotoproteksi terhadap radiasi sinar UV
berdasarkan uji in vitro (Pawening, 2009).
Untuk mengembangkan dan
mengoptimalkan pemanfaatannya, ekstrak etanolik
buah mahkota dewa diformulasikan sebagai krim
water in oil (w/o). Krim didefinisikan sebagai cairan
kental atau emulsi setengah padat, baik tipe air
dalam minyak atau minyak dalam air (Ansel, 1989).
Pembuatan krim w/o mengggunakan bahan-bahan
tambahan seperti cera alba, span 80, dan minyak
mineral. Sifat fisis dalam sediaan krim akan
menentukan keefektifan sediaan tersebut saat
diaplikasikan di kulit. Sifat fisis dan stabilitas krim
dipengaruhi oleh bahan-bahan tambahan yang
terdapat dalam krim.
Karena pentingnya memperoleh sediaan
krim dengan sifat fisis dan stabilitas yang baik, maka
penelitian tentang optimasi komposisi bahan-bahan
tambahan krim perlu dilakukan. Simplex lattice design
merupakan metode yang dapat digunakan untuk
optimasi formula pada berbagai jumlah komposisi
bahan yang berbeda (Bolton, 1997). Untuk
mengetahui apakah sediaan krim yang dibuat
mempunyai potensi untuk mengiritasi, dapat
dilakukan dengan uji iritasi pirmer kualitatif
menggunakan hewan percobaan. Iritasi primer
merupakan respon lokal kulit yang terjadi karena
adanya suatu reaksi kulit terhadap zat kimia
misalnya alkali kuat, asam kuat, pelarut, dan
deterjen, sehingga menyebabkan inflamasi atau luka
pada daerah pemejanan (Prottey, 1978; Lu, 1995).
Eritema dan edima merupakan jenis iritasi primer.
Eritema adalah reaksi radang yang berupa warna
kemerahan pada kulit akibat dilatasi pada kapiler
yang disebabkan racun kimia atau sunburn. Edema
adalah akumulasi berlebihan dari cairan serosa atau
air dalam sel, jaringan atau rongga serosa
(Darelanko dan Hollinger, 1995).
322
METODE PENELITIAN
Bahan
Bahan utama yang digunakan dalam
penelitian ini adalah buah mahkota dewa [Phaleria
macrocarpa (Scheff.) Boerl.] yang diperoleh dari
daerah Kulon Progo pada bulan Agustus 2011.
Bahan lain adalah etanol 70% (derajat teknis), plat
silika gel F254 (Merck®), metanol (derajat analisis),
etil asetat (derajat analisis), kloroform (derajat
analisis), cera alba (kualitas farmasi), span 80
(kualitas farmasi), minyak mineral (kualitas farmasi),
metilparaben (kualitas farmasi), propilparaben
(kualitas farmasi), dan akuades.
Alat
Alat yang digunakan antara lain, bejana gelas,
Buchner vacum, penangas air, kompor listrik
(Robusta®), bejana kromatografi, lampu UV, oven
(Shimadzu®), eksikator, neraca analitik
(Adventurer™, Ohaus), alat-alat gelas, cawan
porselen, alat pendispersi ULTRA-TURRAX T25,
waterbath, pot krim, alat uji daya sebar (Laboratorium
Teknologi Farmasi, Fakultas Farmasi UGM), alat uji
daya lekat (Laboratorium Teknologi Farmasi,
Fakultas Farmasi UGM), viscotester VT-04 (Rion Co.
Ltd, Jepang), stopwatch (Alba Digital Stopwatch), dan
alat uji sentrifugasi.
Hewan Uji
Hewan uji yang digunakan adalah kelinci
putih betina yang diperoleh dari kandang Fakultas
Farmasi UGM, Yogyakarta.
Jalannya Penelitian
Determinasi tanaman mahkota dewa
[Phaleria macrocarpa (Scheff). Boerl.] dilakukan di
laboratorium Biologi Farmasi Fakultas Farmasi
UGM dengan mengamati ciri-ciri sampel tanaman
dan mencocokkan ciri-ciri tersebut dengan ciri-ciri
tanaman yang terdapat dalam pustaka.
Sebanyak 600 gram serbuk simplisia buah
mahkota dewa dimaserasi dengan cara direndam
dengan 4 liter etanol 70% teknis dalam maserator.
Maserasi dilakukan selama 4 hari. Maserat yang
dihasilkan disaring menggunakan corong Buchner
dengan bantuan sistem pompa hisap. Kemudian
filtrat yang diperoleh diuapkan di atas penangas air
sampai diperoleh ekstrak kental buah mahkota
dewa. Ekstrak kental yang diperoleh ditimbang dan
dihitung rendemennya kemudian disimpan di dalam
eksikator.
Uji sifat fisika-kimia ekstrak
Uji yang dilakukan adalah pengamatan
organoleptik, susut pengeringan, daya lekat, dan
kromatografi lapis tipis (KLT). Ekstrak yang telah
dilarutkan dalam metanol ditotolkan menggunakan
pipa kapiler diatas plat silika gel F254. Plat KLT
kemudian dielusi menggunakan fase gerak
kloroform:etil asetat (1:4 v/v) dengan pembanding
Majalah Farmaseutik, Vol. 11 No. 2 Tahun 2015
 Lusty Grace Toding, A. Karim Zulkarnain

Uji daya lekat dan daya sebar menggunakan alat

Tabel I. Formula Ekstrak Buah Mahkota Dewa
Nama Komponen Jumlah
Ekstrak buah mahkota dewa 10%
Cera alba optimasi
Span 80 dengan
Minyak mineral SLD
Metilparaben 0,1%
Propilparaben 0,2%
Akuades ad 100%
phalerin. Keberadaan glikosida benzofenon di
dalam ekstrak dideteksi menggunakan sinar UV 254
nm dan UV 366 nm, kemudian dihitung nilai hRf
bercak yang terdapat pada plat KLT.
Formulasi krim w/o ekstrak etanolik buah
mahkota dewa
Formula krim ekstrak etanolik buah
mahkota dewa tipe water in oil (w/o) yang dijadikan
acuan dalam pembuatan krim tabir surya adalah
formula modifikasi dari formula standar yang
diambil dari Howard (1974) seperti ditunjukkan
pada tabel I.
Optimasi formula krim water in oil (w/o)
ekstrak etanolik daging buah mahkota dewa (Phaleria
macrocarpa (Scheff). Boerl.) menggunakan metode
Simplex lattice design. Berdasarkan simplex lattice design
untuk tiga variabel bebas (campuran tiga
komponen) maka dibuat tujuh formula dan tiga
formula validasi pada berbagai komposisi campuran
untuk ketiga komponen yang akan dioptimasi yaitu
cera alba, span 80, dan minyak mineral. Formula
krim dengan metode simplex lattice design ditunjukkan
melalui tabel II.
Uji sifat fisik dan stabilitas krim w/o mahkota
dewa
Uji organoleptik yang dilakukan meliputi
pengamatan warna, bau, konsistensi, homogenitas,
dan pengukuran pH krim. Uji viskositas
menggunakan viskosimeter VT-04, rotor nomor 2.
Tabel II. Formula Krim Ekstrak Buah Mahkota Dewa dengan Metode Simplex lattice design
yang terdapat pada Laboratorium Teknologi
Farmasi UGM. Uji rasio volume pemisahan
menggunakan tabung reaksi berskala sampai skala
tertentu. Jika tidak terdapat pemisahan maka
digunakan uji stabilitas emulsi yang dipercepat
dengan metode sentrifugasi. Uji tipe emulsi
menggunakan metode pengenceran. Uji viskositas,
daya lekat, daya sebar, pH, rasio volume pemisahan,
dan tipe emulsi dilakukan selama 4 minggu
penyimpanan.
Uji iritasi primer kualitatif
Hewan uji yang digunakan adalah kelinci
putih betina dengan waktu percobaan selama 72
jam. Kelinci putih betina yang digunakan sebanyak 3
ekor. Setiap kelinci yang telah dicukur bagian
punggungnya diaplikasikan sebanyak 0,5 gram
formula optimum krim w/o ekstrak etanolik buah
mahkota dewa, kontrol positif krim w/o
benzofenon 3%, dan kontrol negatif yaitu basis
krim w/o, kemudian ditutup dengan menggunakan
kassa. Pengamatan eritema dan edema yang terjadi
dilakukan pada jam ke-24 dan 72 setelah pemejanan
(Lu, 1995).
HASIL DAN PEMBAHASAN
Hasil determinasi menunjukkan bahwa
tanaman yang digunakan dalam penelitian ini adalah
benar merupakan tanaman mahkota dewa [Phaleria
macrocarpa (Scheff). Boerl.]. Rendemen ekstrak
etanolik kental buah mahkota dewa diperoleh
sebesar 15,03% terhadap bobot serbuk kering.
Ekstrak kental etanolik buah mahkota dewa
mempunyai rasa yang pahit, warna coklat tua, bau
khas mahkota dewa, dan konsistensi yang kental.
Susut pengeringan yang diperoleh sebesar 19%
dengan daya lekat rata-rata 4,22 detik. Analisis
kualitatif dengan KLT menunjukkan bahwa ekstrak
etanolik buah mahkota dewa dan phalerin memiliki
hrF sebesar 6,25 dengan pemadaman biru di bawah
UV 254 nm dan berfluoresensi biru di bawah UV
366 nm. Hasil analisis KLT ditunjukkan pada
gambar 1.

Bahan (g) A B C AB AC BC ABC VA VB VC

Ekstrak 15 15 15 15 15 15 15 15 15 15
Cera alba 28,5 22,5 22,5 25,5 25,5 22,5 24,5 28,5 22,5 22,5
Span 80 7,5 13,5 7,5 10,5 7,5 10,5 9,5 7,5 13,5 7,5
Minyak mineral 66 66 72 66 69 69 68 66 66 72
Metil paraben 0,15 0,15 0,15 0,15 0,15 0,15 0,15 0,15 0,15 0,15
Propil paraben 0,3 0,3 0,3 0,3 0,3 0,3 0,3 0,3 0,3 0,3
Akuades ad 150 150 150 150 150 150 150 150 150 150

Majalah Farmaseutik, Vol. 11 No. 2 Tahun 2015 323

Optimasi Formula Dan Uji Iritasi …

Stabilitas fisik krim w/o ekstak etanolik

buah mahkota dewa
Hasil pengukuran viskositas, daya lekat, dan
daya sebar sepuluh formula krim w/o ekstrak
etanolik buah mahkota dewa dapat dilihat pada
tabel III, IV, dan V.

Penentuan formula optimum krim w/o

Gambar 1. Hasil Identifikasi Ekstrak Etanolik Buah
Mahkota Dewa dengan Fase Diam Silika Gel F
254 dan Fase Gerak Kloroform : Etil Asetat (1:4
V/V) yang Diamati Menggunakan (A) UV 254
Nm; (B) UV 366 Nm
Tabel III. Viskositas Sepuluh Formula Krim W/O Ekstrak
Etanolik Buah Mahkota Dewa dengan Variasi
Cera Alba, Span 80, dan Mineral Oil sebagai Bahan
Tambahan
dengan metode Simplex lattice design
menggunakan software Design Expert®
versi 8.0.7.1
Optimasi formula dengan software Design
Expert versi 8.0.7.1 menghasilkan suatu luas area
optimal pada simplex space dengan salah satu titik
memiliki perbandingan formula 18,504% cera alba,
5,231% span 80, dan 44,265% minyak mineral.

Formula Viskositas (dPa.s)

A 263,300 ± 15,280
Validasi A 270,000 ± 20,000
B 236,670 ± 15,275
Validasi B 233,330 ± 15,275
C 161,670 ± 12,583
Validasi C 166,670 ± 15,275
AB 216,670 ± 23,094
AC 231,670 ± 28,431
BC 186,670 ± 32,146
ABC 241,670 ± 38,188

Tabel IV. Daya Lekat Sepuluh Formula Krim W/O Ekstrak

Etanolik Buah Mahkota Dewa dengan Variasi
Cera Alba, Span 80, dan Mineral Oil sebagai Bahan
Tambahan

Formula Daya lekat (detik)

A 0,820 ± 0,015
Validasi A 0,860 ± 0,051
B 0,730 ± 0,017
Validasi B 0,750 ± 0,090
C 0,700 ± 0,020
Validasi C 0,670 ± 0,075
AB 0,780 ± 0,032
AC 0,670 ± 0,060
BC 0,600 ± 0,065
ABC 0,580 ± 0,075

Tabel V. Daya Sebar Sepuluh Formula Krim W/O Ekstrak

Etanolik Buah Mahkota Dewa dengan Variasi
Cera Alba, Span 80, dan Mineral Oil sebagai Bahan
Tambahan

Formula Diameter sebaran (cm)

A 3,150 ± 0,025
Validasi A 3,070 ± 0,058
B 3,300 ± 0,200
Validasi B 3,330 ± 0,058
C 3,200 ± 0,361
Validasi C 3,130 ± 0,115
AB 3,170 ± 0,153
AC 2,830 ± 0,153
BC 3,230 ± 0,153
ABC 3,160 ± 0,115
Gambar 2. Superimposed dari Contour Plot Respon Viskositas,
Keterangan: nilai di atas merupakan nilai rata-rata dari tiga Daya Lekat, dan Daya Sebar Ekstrak Etanolik Buah
kali replikasi ± SD Mahkota Dewa

324 Majalah Farmaseutik, Vol. 11 No. 2 Tahun 2015

 Lusty Grace Toding, A. Karim Zulkarnain

Contour plot respon viskositas, daya lekat, daya sebar
formula krim w/o ekstrak etanolik buah mahkota
dewa, dan formula optimal terletak pada daerah
berwarna kuning pada gambar di bawah, hasil dari
superimposed contour plot viskositas, daya lekat, dan
daya sebar, ditunjukkan melalui gambar 2.
Selain memprediksi komposisi campuran,
software Design Expert juga memberi prediksi nilai
parameter sifat fisik yang menjadi parameter untuk
mendapat formula optimal. Prediksi yang didapat
dari software adalah viskositas sebesar 257,722 dPas,
nilai daya lekat sebesar 0,792 detik, dan nilai daya
sebar sebesar 3,059 cm.
Evaluasi sifat fisik formula optimal krim w/o
ekstrak etanolik buah mahkota dewa
Hasil uji-t satu sampel respon prediksi dan
respon percobaan formula optimum disajikan pada
tabel VI. Tabel VI menunjukkan respon viskositas
dan daya sebar yang berbeda tidak signifikan antara
prediksi software Design Expert® versi 8.0.7.1 dengan
hasil percobaan, sedangkan respon daya lekat
menunjukkan hasil yang berbeda signifikan.
Evaluasi stabilitas fisik krim w/o ekstrak
etanolik buah mahkota dewa dengan
formula optimal
Viskositas
Viskositas krim w/o ekstrak etanolik buah
mahkota dewa cenderung menurun selama 4
minggu penyimpanan. Penurunan viskositas krim
dapat disebabkan karena krim mengalami dehidrasi
sehingga krim menjadi rapuh dan tidak tahan
terhadap pengadukan rotor viskosimeter.
Ketahanan krim dapat berkurang untuk mengalir.
Depolimerisasi emulgator yang terjadi
selama penyimpanan juga dapat menyebabkan
penurunan viskositas krim. Suhu yang tinggi saat
proses pencampuran dimungkinkan juga akan
mengakibatkan emulgator tidak berfungsi secara
Tabel VI. Hasil Uji-T Satu Sampel Formula Optimum Krim W/O Hasil Prediksi Software Dibandingkan dengan Hasil Percobaan
optimal karena mengalami kerusakan pada
strukturnya sehingga tidak dapat menjaga stabilitas
krim dengan baik selama penyimpanan. Hasil
pengukuran viskositas krim w/o ekstrak etanolik
buah mahkota dewa tersaji dalam tabel VII.
Daya lekat
Tabel VIII menunjukkan daya lekat krim w/o
yang disimpan selama 4 minggu naik turun secara
fluktuatif namun tidak menunjukkan perbedaan
yang bermakna.
Daya sebar
Formula optimum krim w/o ekstrak etanolik
buah mahkota dewa mempunyai daya sebar yang
cenderung menaik selama penyimpanan. Karena
viskositas krim yang menurun selama penyimpanan,
tahanan cairan untuk mengalir semakin berkurang
sehingga daya sebar krim meningkat. Hasil
pengukuran daya lekat dapat dilihat pada tabel IX.
Derajat keasaman (pH)
Hasil pengukuran pH krim w/o tersaji pada
tabel X. Formula optimum krim w/o ekstrak
etanolik buah mahkota dewa mempunyai nilai pH
yang stabil selama penyimpanan dari minggu ke-0
sampai minggu ke-4.
Rasio volume pemisahan
Hasil pengamatan selama 4 minggu
menunjukkan bahwa semua krim w/o stabil pada
suhu kamar karena tidak terjadi pemisahan fase
pada ketiga formula krim (F=1). Hal ini dapat
disebabkan karena viskositas krim yang besar
sehingga tidak terjadi pemisahan fase. Hasil
pengukuran respon rasio volume pemisahan
formula optimum krim w/o ekstrak etanolik buah
mahkota dewa tersaji dalam tabel XI.
Hasil uji stabilitas emulsi yang dipercepat
dengan metode sentrifugasi menunjukkan bahwa
dengan meningkatnya kecepatan sentrifugasi,

Respon Prediksi Percobaan Signifikansi 2-tailed Kesimpulan

Viskositas 257,722 dPa.s 246,7 dPa.s 0,080 Berbeda tidak signifikan
Daya Lekat 0,792 detik 0,753 detik 0,048 Berbeda signifikan
Daya Sebar 3,059 cm 2,918 cm 0,065 Berbeda tidak signifikan

Tabel VII. Hasil Uji Stabilitas Fisik Formula Optimal Krim W/O Ekstrak Etanolik Buah Mahkota Dewa

Minggu Viskositas (dPa.s) Daya lekat (detik) Diameter sebaran (cm) pH F

0 246,700 ± 5,774 0,753 ± 0,015 2,918 ± 0,065 5,5 ± 0 1,0 ± 0
1 236,667 ± 5,774 0,713 ± 0,015 2,958 ± 0,014 5,5 ± 0 1,0 ± 0
2 240,000 ± 10,000 0,727 ± 0,025 2,942 ± 0,038 5,5 ± 0 1,0 ± 0
3 230,000 ± 10,000 0,710 ± 0,01 3,000 ± 0,050 5,5 ± 0 1,0 ± 0
4 226,667 ± 11,547 0,727 ± 0,021 3,010 ± 0,036 5,5 ± 0 1,0 ± 0

Majalah Farmaseutik, Vol. 11 No. 2 Tahun 2015 325

Optimasi Formula Dan Uji Iritasi …

Tabel VIII. Data F Rata-Rata ± SD Menit Ke-5 dan Ke-30

dewa merupakan tipe w/o.
Uji Sentrifugasi
Iritasi primer kualitatif
F (menit ke-) Hasil pada tabel XIV menunjukkan bahwa
Kecepatan (rpm) kontrol positif benzofenon 3% dapat menyebabkan
5 30
edema pada kelinci, sedangkan krim w/o ekstrak
1500 1,067 ± 0,058 1,167 ± 0,153 etanolik buah mahkota dewa dan kontrol negatif
3000 1,267 ± 0,058 1,567 ± 0,115 basis tidak menimbulkan iritasi baik eritema
4500 1,633 ± 0,058 1,867 ± 0,058 maupun edema setelah pemejanan pada hewan uji.

KESIMPULAN
Tabel IX. Uji Tipe Emulsi Formula Optimal Krim W/O Optimasi formula krim w/o ekstrak etanolik
Ekstrak Etanolik Buah Mahkota Dewa
buah mahkota dewa menggunakan metode simplex
Minggu ke-
lattice design dengan software Design Expert® versi
Replikasi
0 1 2 3 4
8.0.7.1 menghasilkan suatu area optimal pada
komposisi 18,504% cera alba, 5,231% span
1 - - - - -
80, dan 44,265% minyak mineral. Formula optimal
2 - - - - -
yang diperoleh mempunyai respon viskositas dan
3 - - - - -
daya sebar yang berbeda tidak signifikan dengan
volume pemisahan emulsi juga bertambah (tabel nilai prediksi respon yang diberikan oleh software
XII dan gambar 3). Design Expert® versi 8.0.7.1, sedangkan respon daya
lekat berbeda signifikan. Formula optimal krim w/o
Tipe emulsi stabil selama 4 minggu penyimpanan berdasarkan
Uji tipe emulsi menunjukkan hasil yaitu respon rasio volume pemisahan dan derajat
semua krim tidak dapat terdistribusi merata dalam keasaman. Krim w/o ekstrak etanolik buah mahkota
medium air. Berdasarkan hasil pada tabel XIII dewa tidak menimbulkan eritema ataupun edema
terbukti bahwa krim ekstrak etanolik buah mahkota setelah pemejanan pada hewan uji.

Gambar 3. Perbandingan Volume Pemisahan Krim W/O pada Kecepatan Sentrifugasi 1500, 3000, dan 4500 Rpm Selama 30 Menit

Tabel X. Hasil Pengamatan Uji Iritasi Primer Akibat Pemejanan Senyawa Uji secara Kualitatif

Jam ke-24 Jam ke-72

Kelompok Replikasi
Eritema Edema Eritema Edema
1 - + - +
Benzofenon 3% 2 - + - +
3 - + - +
1 - - - -
Kontrol positif 2 - - - -
3 - - - -
1 - - - -
Kontrol negatif 2 - - - -
3 - - - -

Keterangan : (-) : Belum meninbulkan eritema dan edema

(+) : Sudah menimbulkan eritema dan edema

326 Majalah Farmaseutik, Vol. 11 No. 2 Tahun 2015


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Moisture sorption by dairy powders studied by low-field NMR

Todor Vasiljevic a, Antoinette Toebes b, Thom Huppertz a, b, c, *
a
Advanced Food Systems Research Unit, Institute of Sustainable Industries and Liveable Cities, Victoria University, Werribee campus, Melbourne Australia
b
FrieslandCampina, Amersfoort, the Netherlands
c
Wageningen University & Research, Wageningen, the Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: The influence of moisture sorption on spinespin (T2) relaxation times for skim milk powder (SMP), milk
Received 20 February 2021 protein concentrates (MPC50 and MPC80) and amorphous lactose was studied by low-field nuclear
Received in revised form magnetic resonance (NMR). For amorphous lactose and MPC80, only one main peak was observed in the
9 March 2021
T2 distribution, whereas for SMP and MCP50, two main peaks, relating to the water associated with the
Accepted 11 March 2021
Available online 21 March 2021
amorphous lactose and protein, were observed at aw <0.4; at aw >0.4 lactose crystallised and no peak
relating to water associated with lactose was visible in the T2 spectrum (water of crystallisation for
crystalline lactose has shorter relaxation times, which cannot be detected by low-field NMR). T2 peak
position and peak area for the protein fraction increased with increasing aw. Shifts in the T2 peak position
showed the transition from mono- and bilayer water at low aw to more rapidly exchanging forms of
water at higher aw.
© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction which this occurs is dependent on the components of the product.

Removal of water is one of the most commonly applied pro-
cesses to increase the shelf-life of milk and dairy ingredients,
resulting in powdered dairy products such as skim milk powder
(SMP), whole milk powders (WMP), milk protein concentrate
(MPC) and whey protein concentrate (WPC). During the drying of
these powders, typically by spray-drying, water content is reduced
to values <5% (w/w), leading to water activity (aw) values <0.3.
Water activity is a crucial determinant of powder stability, as it
governs the glass transition temperature (Tg), which relates to, e.g.,
development of stickiness and crystallisation of lactose (Huppertz
& Gazi, 2016; Paterson Brooks, Bronlund, & Foster, 2005; Roos,
2002). Furthermore, development of insolubility, lipid oxidation
and browning during storage also depend strongly on the aw of the
powders (Fan et al., 2018). Therefore, water (de)sorption and water
binding by dairy products is of great scientific and industrial
relevance.
The relationship between water content and water activity, and
therewith Tg, of products is typically expressed in the form of
moisture sorption isotherms. Moisture content (often expressed as
g water per g of dry matter) increases with aw but the manner in
* Corresponding author. Tel.: þ31 6 11187512.
E-mail address: thom.huppertz@frieslandcampina.com (T. Huppertz).
In the aw region 0.0e0.3, most water is in the form of so-called
monolayer water, which is tightly bound to ionic and polar
groups of e.g., proteins. In addition, hydration water around
exposed hydrophobic residues is also present in this aw-region
(Rupley & Careri, 1991). These ‘types’ of water are typically strongly
bound in highly ordered structures (Oleinikova, Smolin, &
Brovchenko, 2007). Rotation and translation of water molecules
in this layer is impaired, which on the other hand impacts nuclear
magnetic resonance (NMR) relaxation time (Kuntz & Kauzmann,
1974). With increasing aw >0.3, layers of hydrogen-bonded water
with progressively decreasing ordered structures, i.e., multilayer
water, start surrounding the aforementioned monolayer water; at
even higher aw (e.g., >0.5) capillary water held in structural
openings can also be present and is again less structured and shows
less impairment of rotation and translation of water molecules
(Oleinikova et al., 2007). These differences in water mobility can be
monitored readily by low-field NMR spectroscopy.
Low-field NMR has been frequently used to assess the molecular
mobility of water as well as water-biopolymer interactions in food
systems (Kou, Molitor, & Schmidt, 1999). In high moisture food
systems, 1H NMR spectrum is dominated by the bulk water signal,
thus providing very little information about water dynamics. On
the contrary, food systems containing small amounts of water are
characterised with a bulk water signal that is greatly depressed or
even absent or greatly reduced and a much slower proton exchange

https://doi.org/10.1016/j.idairyj.2021.105062
0958-6946/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
T. Vasiljevic, A. Toebes and T. Huppertz
due to immobilisation of water (Kou, Dickinson, & Chinachoti,
2000). NMR relaxometry has been used previously to establish
rehydration properties of dairy powders (Davenel, Schuck,
Mariette, & Brule, 2002; Schuck et al., 2002) and to link confor-
mational changes of MPC to water mobility in dry state (Haque,
Bhandari, Gidley, Deeth, & Whittaker, 2011). The relaxometry is
based on differences in transverse relaxation times (T2) of water
molecules governed by molecular motion and exchange with pro-
tons on protein molecules. Therefore, it is possible to identify
different populations of water molecules from their rotational
mobilities and assign them to different environments (Hills,
Manning, & Godward, 1999).
NMR studies on dry dairy systems are very limited mainly due to
difficulties in data interpretation associated with structural and
compositional complexity of samples bearing highly heteroge-
neous proton environments. The aim of the current study thus was
to engage the NMR instrumentation frequently encountered in
commercial settings in the assessment of water mobility in several
commercial dry dairy samples and link that to water sorption
properties of these powders. Main advantage of low-field NMR is in
lower acquisition cost to that of a high-resolution NMR. The water
mobility was assessed in samples equilibrated at different water
activities ranging from <0.1 to 0.85 at 25  C.
2. Materials and methods
2.1. Sample preparation
Low-heat skim milk powder, MPC50 and MPC80 were pro-
duced as described by Gazi and Huppertz (2015). Amorphous
lactose was prepared by dissolving a-lactose monohydrate (Sig-
maeAldrich, St. Louis, MO, USA) in deionised water at 50  C at a
concentration of 25 g 100 g1 water. The solution was subse-
quently flash-frozen in liquid nitrogen, freeze-dried and stored
over phosphorus pentoxide. The absence of crystalline material
was checked by polarised light microscopy. For adjusting the
water activity of samples, aliquots were stored over phosphorus
pentoxide and saturated solutions of lithium chloride, potassium
acetate, magnesium chloride, potassium carbonate, magnesium
nitrate, sodium chloride and potassium chloride. Samples were
stored for 3 weeks at room temperature after which water activity
was determined.
2.2. Determination of moisture sorption isotherms
Moisture sorption isotherms for SMP, MPC50 and MPC80 were
determined at 25  C in the aw range 0.10e0.90 at intervals of 0.05
aw units using a Vapor Sorption Analyzer (VTI-SAþ; TA In-
struments, New Castle, DE, USA). The water content of each sample
was plotted as a function of water activity. The Guggenheim-
Anderson-de Boer (GAB) relationship was established by fitting
the data applying the following equation:
m C$k$aw
¼ (1)
mo ð1  k$aw Þ$ð1  k$aw þ C$k$aw Þ
where m presents the water content (kg of water kg1 of dry
matter), m0 is the monolayer value of water molecules; and C, k are
the constants.
2.3. Low-field NMR
The NMR relaxation measurements were conducted using a
23.5 MHz (0.54 T) MQC23-benchtop NMR (Oxford Instruments,
Abingdon, UK). The NMR system was equipped with a
2
International Dairy Journal 119 (2021) 105062
temperature control device (Air Jet van SP industries mod-
el:XR11851) allowing temperature regulation between 10  C and
40  C. The samples upon equilibration were introduced into the
instrument by filling approximately 2 g of powders into LR-
NMR glass tubes. The measurements were conducted at
25  C. The transverse relaxation time constants (T2) of the
samples were assessed by the Carr-Purcell-Meiboom-Gill
(CPMG) pulse sequence (Carr & Purcell, 1954; Meiboom &
Gill, 1958). The CPMG experiments were carried out using a
relaxation delay of 10 s and 32 consecutive scans for noise
reduction along with a sweep width of 0.1 MHz. A total of 4096
echoes was collected, with a 90e180 pulse gap (t) value of 100
ms. The resultant decays were analysed by Inverse Laplace
Transform in the RI Win-DXP software (V. 1.2.3. Oxford
Instruments).
3. Results and discussion
Moisture sorption isotherms were determined for SMP, MPC50
and MPC80, whereas sorption isotherm data for amorphous lactose
were extracted from Bronlund and Paterson (2004). Sorption iso-
therms are shown in Fig. 1 and the GAB parameters derived
therefrom are shown in Table 1. Only for MPC80 could sorption
isotherms be determined up to aw ¼ 0.9. For the other amorphous
lactose, SMP and MPC50, the occurrence of lactose crystallisation
took place at aw >0.35, >0.55 and >0.65, respectively.
The occurrence of lactose crystallisation during sorption
isotherm measurements and the occurrence thereof at higher aw
with increasing protein and decreasing lactose content is in line
with previous studies, which were reviewed by Huppertz and Gazi
(2016). However, it should be noted that crystallisation at lower aw,
Fig. 1. Sorption isotherms of SMP ( ), MPC50 ( ), MPC80 ( ) samples. Amorphous
lactose line ( ) was plotted based on the information derived from Bronlund and
Paterson (2004).
Table 1
GAB isotherm parameters for SMP, MPC50 and MPC80.a
Sample m0 C K SSE
SMP 0.0345 9.99 1.346 0.0002
MPC50 0.0395 9.99 1.144 0.0003
MPC80 0.0563 14.9 0.881 0.0003
Amorphous lactose 0.0488 3.23 1.16
a
Abbreviations are: m0, monolayer value (kg of water kg1 of dry matter); C, k,
constants; SSE, sum of squared estimate of errors. Parameters for amorphous lactose
were extracted from Bronlund and Paterson (2004).
T. Vasiljevic, A. Toebes and T. Huppertz International Dairy Journal 119 (2021) 105062

but above 0.35, can still occur on longer timescales because of

delayed lactose crystallisation due to the presence of proteins and
salts (Jouppila & Roos, 1994; Omar & Roos, 2007). Lactose crystal-
lisation in dairy powders can occur due to increases in aw when
T > Tg with the rate of crystallisation often described as propor-
tional to T e Tg (Paterson et al., 2005). As Tg increases with
decreasing lactose content (Kelly et al., 2015), the aforementioned
delay in lactose crystallisation for samples with lower lactose
content is in line with expectations.
The T2 spectra for the different samples at different aw values are
shown in Figs. 2 and 3. For amorphous lactose (Fig. 2), the main
peak was observed in the T2-range 104e106 ms, which strongly
increased at aw ¼ 0.32, but decreased again at aw ¼ 0.43. The in-
crease in peak intensity at aw ¼ 0.32 (Fig. 2) is due to moisture
sorption by amorphous lactose and in line with the moisture
sorption isotherm (Fig. 1). Although moisture sorption also occurs
at lower aw (Fig. 1), at this point the adsorbed water is in the field of
monolayer water, which has very short relaxation times, which are
Fig. 2. T2 distribution at different aw levels for (A) amorphous lactose: , <0.1; not detected by low-field NMR. The additional water absorbed at
, 0.144; , 0.217; , 0.323; , 0.432.

Fig. 3. T2 distribution at different aw levels for SMP, MPC50, and MPC80.

3
T. Vasiljevic, A. Toebes and T. Huppertz
Fig. 4. (A) T2 peak position, and (B) T2 peak area of the protein peak as a function of aw
for SMP ( ), MPC50 ( ), MPC80 ( ) and lactose ( ).
aw ¼ 0.32 is in more rapidly exchanging forms at hence more
readily detected.
Hargreaves (1995) observed a decrease in spin-lattice (T1)
relation time with increasing aw up to aw ¼ 0.382, but unfortu-
nately did not report peak intensities. At aw ¼ 0.43, where the
Fig. 5. Derived parameters of linear relationship between log T2 relaxation time (log
ms) and water activity (aw) above 0.4 for SMP ( ; fit,
MPC80 ( ; fit, ).
International Dairy Journal 119 (2021) 105062
lactose has crystallised, a considerable drop in intensity in this
peak was observed. Water of crystallisation, which would be
present in the crystalline lactose, has very short relaxation times
and is not detected in low-field NMR measurements as used in
this study. At aw up to 0.323, a similar peak in the T2-range of
104e106 ms was observed for SMP, MPC50 and MPC80 (Fig. 3). In
addition, however, a notable peak was observed in the T2-range of
101e103 ms. The intensity of this peak increased with concomitant
rise in protein content of the powders (SMP < MPC50 < MPC80)
and was directly related to increasing aw (Fig. 3). In this aw range
mono- and bilayer water and some multilayer water dominate
(Chuy & Labuza, 1994), which explains the short relaxation times.
With increasing aw >0.4, progressive further increases in peak
area as well as shifts of the peaks towards longer relaxation times
were observed (Fig. 3).
For both peak position and peak area, log-linear increases
with increasing aw were observed (Fig. 4). Interestingly, however,
the slope of such curves was highest for SMP, followed by MPC50
and MPC80 (Fig. 5), whereas based on the protein content, the
opposite may have been expected, given that the lactose in the
samples has crystallised and would not contribute. However, the
SMP also contains a notable amount of soluble salts, which are
present at smaller amounts in MPC50 and almost absent in
MPC80 (Cari c, 2002). Particularly at aw >0.5, it has been shown
that the soluble salts fraction of milk powder can contribute very
strongly to moisture sorption, binding in excess of 1 g of water
per g of dry matter at aw >0.8 (Berlin, Anderson, & Pallansch,
1968). This is notably higher than water binding by MPC80
(Fig. 1). Hence, the higher peak areas for SMP compared with
MPC50 and MPC80 in the T2 relaxation profiles (Fig. 4) may be
related to the contribution of the soluble milk salts to water
sorption of the powders. Shifts to larger T2 relaxation times with
increasing aw can be related to more loosely bound multilayer
and capillary water arising at higher water activity (Modig et al.,
2004). In all cases, T2 values remained 3 orders of magnitude
lower than for free water, indicating that notable limitations in
mobility remained.
4. Conclusions
Water mobility was studied in lactose, SMP, MPC50 and MPC80
as a function of aw by low-field NMR spectroscopy. Water associ-
ated with the protein phase and the amorphous lactose phase could
be distinguished based on T2 relaxation times. Log-linear increases
in T2 peak area with increasing aw were observed at aw >0.4, with
strongest shifts found for SMP. These are likely related to the
notable contribution of soluble milk salts to moisture sorption
behaviour of SMP and related products. Overall, low-field NMR
proved to be a very useful technique to study moisture sorption of
dairy powders, providing complementary information next to
sorption isotherms. Because the relaxation times are also
measured, it is possibly to readily obtain insights in water relaxa-
tion and hence the propensity of water in the product to contribute
to physical or chemical instability of the product.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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 Food Chemistry: X 3 (2019) 100050

Contents lists available at ScienceDirect

Food Chemistry: X
journal homepage: www.journals.elsevier.com/food-chemistry-x

Effects of emulsifiers on the moisture sorption and crystallization of

amorphous sucrose lyophiles
Adrienne L. Voelkera, Gianna Verbeeka, Lynne S. Taylorb, Lisa J. Mauera,
⁎

a
Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, IN 47907, United States
b
Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, United States

ARTICLE INFO ABSTRACT

Chemical compounds studied in this article: The crystallization of amorphous sucrose can be problematic in food products. This study explored how emul-
Sucrose (PubChem CID: 5988) sifiers (a range of sucrose esters, polysorbates, and soy lecithin) impact the moisture sorption and crystallization
Stearic ester (PubChem CID: 9898327) of amorphous sucrose lyophiles. Solutions containing sucrose with and without emulsifiers were lyophilized,
Palmitic ester (PubChem CID: 5360814) stored in desiccators, and analyzed by X-ray diffraction, infrared spectroscopy, and polarized light microscopy
Soy lecithin/phosphatidylcholine (PubChem
over time. Moisture sorption techniques, Karl Fischer titration, and differential scanning calorimetry were also
CID: 6441487)
Polysorbate 20 (PubChem CID: 443314)
used. Different emulsifiers had varying impacts on sucrose crystallization tendencies. Polysorbates enhanced
Polysorbate 80 (PubChem CID: 5284448) sucrose crystallization, decreasing both the RH and time at which sucrose crystallized. These lyophiles did not
collapse upon crystallization, unlike all other samples, indicating the likelihood of variations in nucleation sites
Keywords: and crystal growth. All other emulsifiers stabilized amorphous sucrose by up to a factor of 7x, even in the
Amorphous sucrose presence of increased water absorbed and independent of glass transition temperatures, indicating emulsifier
Crystallization structure governed sucrose crystallization tendencies.
Hygroscopicity
Sucrose ester
Lecithin
Polysorbate
Stearic ester
Palmitic ester

1. Introduction
In addition to increasing the sweetness of foods, sucrose contributes
to the structure, texture, dissolution, and/or taste perception of pro-
ducts ranging from various confectioneries and low moisture baked
goods, to powder beverage and seasoning mixtures. The physical state
of the sucrose solid affects many characteristics, including stability,
dissolution, moisture sorption, and many sensory properties, such as
texture and flavor perception (Chirife & Karel, 1974; Mathlouthi,
1995). Amorphous sucrose is often the preferred state for many con-
fectionery products due to the desirable dissolution properties and
softer texture. However, amorphous sucrose has a tendency to crystal-
lize to the more thermodynamically stable crystalline form during
storage. Crystallization can lead to undesirable texture and flavor
changes, impaired solubility, and acceleration of chemical changes such
as oxidation and enzymatic activity in other materials in the food ma-
trix (Buera, Schebor, & Elizalde, 2005; Slade, Levine, & Reid, 1991).
Therefore, sucrose crystallization is a major area of interest in the food
industry, with emphasis placed on the effects of formulations and sto-
rage environments on crystallization kinetics (Buera et al., 2005;
Kinugawa et al., 2015; Saleki-Gerhardt & Zografi, 1994; Thorat, Forny,
Meunier, Taylor, & Mauer, 2017, 2018).
Numerous additives have been shown to disrupt and delay sucrose
crystallization by a variety of mechanisms including: decreasing mo-
lecular mobility (Saleki-Gerhardt & Zografi, 1994), increasing the glass
transition temperature (Tg) and/or viscosity of the co-lyophilized
system (Roe & Labuza, 2005; Roos & Karel, 1991), disrupting the crystal
lattice due to molecular interactions between sucrose and the additive
(Gabarra & Hartel, 1998; Shamblin & Zografi, 1999), and generally
inhibiting nucleation and crystal growth (Carstensen & van Scoik,
1990). More recently, a study of the effects of chloride and sulfate salts
on amorphous sucrose crystallization found that increasing the cation
valency (and corresponding ion hydration shell) delayed or prevented
sucrose crystallization even while decreasing Tg, presumably by al-
tering the water dynamics in the matrix (Thorat et al., 2017). A study
on the effects of a series of mono-, di-, and tri-saccharides on

⁎
Corresponding author.
E-mail addresses: avoelke@purdue.edu (A.L. Voelker), gianna.verbeek@wur.nl (G. Verbeek), lstaylor@purdue.edu (L.S. Taylor), mauer@purdue.edu (L.J. Mauer).

https://doi.org/10.1016/j.fochx.2019.100050
Received 31 January 2019; Received in revised form 16 May 2019; Accepted 5 August 2019
Available online 20 August 2019
2590-1575/ © 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
A.L. Voelker, et al.
amorphous sucrose stability found that saccharides containing regions
of structural similarity as well as structural dissimilarity best inhibited
sucrose crystallization, with these structural relationships seemingly
having a greater influence on the delay of sucrose crystallization than
that of a decrease in Tg due to moisture sorption (Thorat et al., 2018).
While many studies have explored the impact of additives on su-
crose crystallization from the solid state, the role of emulsifiers in al-
tering sucrose crystallization has primarily been studied in solutions
and is not well-defined. Emulsifiers have been shown to alter the
crystallization of compounds by different mechanisms. In solutions,
emulsifiers have been shown to both reduce and increase the primary
nucleation rate of different compounds (Canselier, 1993; van Hook,
1961). Emulsifiers have also been shown to have conflicting effects on
crystal growth rates: the reduction of interfacial tension by the emul-
sifier can increase the crystal growth rate, but the slowing of mass
transfer at the crystal-solution interface due to the presence of the
emulsifier can slow the crystal growth rate (Canselier, 1993; van Hook,
1988; Vasanth Kumar & Rocha, 2009). Some emulsifiers have even
been shown to both increase and decrease the rate of crystallization,
depending on the amount added (Michaels & van Kreveld, 1966). For
example, sodium doecyl (tetrapropylene) benzene sulfonate increased
the rate of lactose crystallization at low levels of addition but decreased
the rate when added in larger amounts (Michaels & van Kreveld, 1966).
Regardless, it is agreed upon that the changes emulsifiers cause on the
adsorbed crystal surface layer are likely to affect secondary nucleation,
and changes in surface energy due to emulsifiers are likely to affect
crystal growth (Canselier, 1993; Hartel & Shastry, 1991; Vasanth
Kumar & Rocha, 2009); however, these concepts have not been shown
to correlate to crystallization from the amorphous state. While under-
standing formulation effects on crystallization from solutions is cer-
tainly important, foods and food ingredients tend to be solids. There-
fore, understanding how emulsifiers alter sucrose crystallization from
the amorphous state is relevant.
The objective of this study was to determine the effects of different
types and concentrations of food-relevant emulsifiers on the crystal-
lization of amorphous sucrose. It was hypothesized that the structure of
the emulsifiers would play a significant role in stabilizing amorphous
sucrose. Emulsifiers containing a region that is structurally similar to
sucrose as well as a structurally dissimilar region were anticipated to
provide the greatest inhibition to sucrose crystallization, consistent
with the concept shown for the efficacy of how different saccharide
structures altered sucrose crystallization (Leinen & Labuza, 2006;
Thorat et al., 2018). The structures and properties of the emulsifiers
used in this study are shown in Table 1. To test the hypothesis, these
emulsifiers were selected to encompass a range of hydrophilic lipophilic
balances (HLB, ~2–17), number of monosaccharide units (0–2),
number of hydroxyl groups (~0–7), molecular weights, thermal and
hygroscopic traits, and structural components.
2. Materials and methods
2.1. Materials
The sucrose used in this study was obtained from Mallinckrodt
Chemicals (Philipsburg, NJ), and the emulsifiers were a series of su-
crose esters (stearic ester 30% (SP30), stearic ester 50% (SP50), stearic
ester 70% (SP70), and palmitic ester 75% (PS750)) varying in the type
of fatty acid as well as the percentage of mono-esters (30–75% as
shown) relative to di- and tri-esters from Sisterna (Roosendaal,
Netherlands); soy lecithin from Modernist Pantry (Eliot, ME); and
polysorbate 20 and polysorbate 80 from Florida Laboratories, Inc. (Fort
Lauderdale, FL). The emulsifiers were chosen based on common usage
in the food industry as well as variable structures of the compounds (as
shown in Table 1).
Desiccators were prepared using phosphorus pentoxide (P2O5)
(Fisher Scientific, Fair Lawn, NJ) to maintain a relative humidity (RH)
2
Food Chemistry: X 3 (2019) 100050
of ~0% or by using the following saturated salt solutions to control the
RH at higher levels: lithium chloride (~11% RH) obtained from
Avantor Performance Materials (Center Valley, PA), potassium acetate
(~23% RH) obtained from Fisher Scientific, and magnesium chloride
(~33% RH) obtained from Fisher Scientific. For use in volumetric one-
component Karl Fischer titrations, Karl Fischer reagents including
HYDRANAL-Composite 2 (titrant), HYDRANAL-Methanol Rapid
(working medium), and HYDRANAL-Water Standard 10 were pur-
chased from Sigma-Aldrich (St. Louis, MO). Water used throughout the
study was deionized and purified using a Barnstead E-Pure ultrapure
water purification system (ThermoScientific, Waltham, MA) with a re-
sistivity at 25 °C greater than 17.5 MΩ·cm.
2.2. Preparation of amorphous samples
Samples were prepared by freeze drying 10% w/v sucrose solutions
with and without 1% and 5% (w/w) of the co-formulated emulsifier in
which both the sucrose and the emulsifier were completely dissolved.
There were 7 co-formulated emulsifier additives (Table 1), each added
at two concentrations (1% and 5% w/w emulsifier/sucrose), giving a
total of 14 dispersion preparations in addition to the control sucrose.
The solutions were frozen at −20 °C for at least 12 h prior to lyophi-
lization. Lyophilization was completed in a VirTis Genesis 25ES freeze
dryer (SP Scientific, Warminster, PA). Samples were initially frozen in
the freeze dryer at −40 °C and 300 mTorr (40 Pa) for 6 h. The freeze
dryer was then held at −40 °C and 150 mTorr (20 Pa) for 24 h to allow
for primary drying to occur. This was followed by an increase in tem-
perature from −40 °C to 20 °C in increments of 10 °C, holding for 9 h at
each step to allow for secondary drying. Finally, a heating step was
completed at 25 °C and 300 mTorr (40 Pa) for 6 h, after which samples
were immediately transferred to desiccators containing P2O5 (~0%
RH). These samples were stored in the desiccators containing P2O5 at
ambient temperature (22 ± 2 °C) until further analysis, and all sub-
sequent sample handling was done in a glove box purged with nitrogen
(to drop the ambient RH to ~5%).
2.3. Storage treatments
To initiate the RH storage treatments, the lyophiles were transferred
from the desiccators containing P2O5 into desiccators containing satu-
rated salt solutions of lithium chloride (~11% RH), potassium acetate
(~23% RH), or magnesium chloride (~33% RH), which were then
stored at 25 °C in a temperature-controlled room. Samples were re-
moved from these desiccators and analyzed periodically over 4 weeks.
A single desiccator was used for each timepoint of analysis to avoid
exposing the samples to ambient RH until the day of their analysis.
Samples were discarded after analysis.
2.4. Determination of crystallinity
A combination of powder X-ray diffraction (PXRD), polarized light
microscopy (PLM), and Fourier-transform infrared spectroscopy (FT-IR)
was used to monitor the physical state of samples over time and to
identify the onset of crystallization of the amorphous lyophiles (Fig. 1).
Lyophiles were analyzed on days 0, 7, 14, 21, and 28. Samples that
were found to be crystalline on day 7 were further analyzed on days 2
and 4 by preparing fresh samples to narrow down the time at which the
onset of crystallization occurred.
2.4.1. Powder X-ray diffraction
PXRD diffractograms were collected using a Rigaku Smartlab dif-
fractometer (Rigaku Corporation, Tokyo, Japan) equipped with a Cu-Kα
radiation source set in Bragg-Brentano geometry and operating at 40 kV
and 40 mA. Samples were analyzed using a scan range of 10–35° 2θ at a
scan speed of 15°/min and a step size of 0.02°. Samples with diffraction
patterns consisting of peaks above a signal-to-noise ratio of 3 were
 Table 1
Properties of sucrose and emulsifiers used in lyophiles. Uppercase superscript letters denote statistical significance between experimental Tgs, and lowercase superscript letters denote statistical significance between
experimental Tms (Tm1s and Tm2s).
A.L. Voelker, et al.

Component Average MW HLB HBD/HBA Number of –OH Starting Physical Structure Amorphous Crystalline
(g/mol) units State at RT
Onset Tg Onset Tm1 Onset Tm2

1 A a
Sucrose 342.3 – 8/11 8 Crystalline solid 56.5 ± 0.5 °C (freeze- 187.9 ± 0.7 °C –
dried)

2
SP30 888.55 6 7/12 5.6–6.3 Crystalline solid – 22 ± 2 °Ce 57 ± 1 °Cb
2
SP50 808.61 11 7/12 6–6.5 Crystalline solid – 19 ± 2 °Ce 46 ± 1 °Cd
2
SP70 728.67 15 7/12 6.4–6.7 Crystalline solid – 18.9 ± 0.9 °Cef 52 ± 2 °Cbc

2
PS750 683.63 16 7/12 6.5–6.75 Crystalline solid – 14 ± 1 °Cf 51 ± 1 °Ccd

Soy Lecithin (Phospha- 643.9 2–7 0/8 0 Amorphous solid 41 ± 2 °CB 190.7 ± 0.5 °Ca –

3
tidylcholine) 3

4,5
Polysorbate 20 1228 16.7 3/26 3 Liquid < −50 °CC −23 ± 2 °Cg –

4,5
Polysorbate 80 1310 15 3/26 3 Liquid < −50 °CC −19.6 ± 0.8 °Cg –

1
Slade et al. (1991).
2
Szűts et al. (2007).
3
Bueschelberger, Tirok, Stoffels, and Schoeppe (2015).
4
Cottrell and van Peij (2015).
5
Amim et al. (2012).
Food Chemistry: X 3 (2019) 100050
A.L. Voelker, et al.
Fig. 1. Analysis of select sucrose lyophiles over time indicating increasing de-
gree of crystallinity, from completely amorphous to completely crystalline,
interceded with increasing degrees of crystallinity of A) Powder x-ray dif-
fractograms, where boxed in peaks are the well-defined crystalline sucrose
peaks (Leinen & Labuza, 2006), B) FT-IR spectra, where crystallinity was
evaluated by the characteristic absorption peaks of crystalline sucrose in the
region of 2800–3800 cm−1 wavenumbers (Lescure, 1995; Mathlouthi, 1995),
and C) PLM images, where birefringence indicates crystallinity.
considered PXRD crystalline. Samples with small peaks above the
baseline were labeled partially crystalline, with increasing peak areas/
intensities related to increasing crystallinity (Fig. 1A). Samples with no
peaks and only an amorphous halo were considered to be PXRD
amorphous.
2.4.2. Fourier-Transform Infrared Spectroscopy
FT-IR (TravelIR HCI, SensIR Technologies, LLC, Danbury, CT) with a
fixed attenuated total reflectance (ATR) accessory was used to monitor
crystallinity of sucrose in the lyophiles using a method described in
Lescure (1995) and Mathlouthi (1995). Briefly, crystalline sucrose can
be identified by characteristic absorption peaks due to hydrogen
bonding in the 2800–3800 cm−1 region. The FT-IR was equipped with a
TGA detector, resolution was set to 4 cm−1, and samples were scanned
64 times from 650 to 4000 cm−1. Spectra of control crystalline and
amorphous sucrose samples were collected and used as comparisons to
verify the physical state of the lyophiles. OMNIC Series Software
(ThermoScientific) was used to analyze the spectra.
4
Food Chemistry: X 3 (2019) 100050
2.4.3. Polarized light microscope
Samples were observed with an Omano polarized light microscope
(Omano, China), and crystal identification was done as described by
Carlton (2011). Briefly, the appearance of birefringence in the lyophi-
lized samples indicated crystallinity. Photographs to document sample
appearance were taken using an iPhone 6 s camera attached to the
microscope eyepiece by an iDu LabCam adapter (Detroit, MI). The
microscope was also paired with a RH-controlled stage (GenRH, Al-
lentown, PA), and crystallization of a subset of samples was observed
over time at 40% RH and ambient temperature (22 ± 2 °C). Timelapse
videos of crystallization were taken using the iPhone 6 s camera.
2.5. Dynamic vapor sorption
Three different moisture sorption profiles of all lyophiles were col-
lected at 25 °C using a SPSx-1μ Dynamic Vapor Sorption Analyzer
(Projekt Messtechnik, Ulm, Germany). For the first moisture sorption
profile, samples (100–200 mg) were placed in a 23-ring sample holder
and held at 0% RH for 48 h in the instrument. Samples were then
analyzed from 0 to 80% RH in 5% RH increments, with a maximum
residence time of 12 h per step and an equilibration end-point criterion
of < 0.001% weight change within 30 min. The moisture sorption
profile of each lyophile was plotted using the percent change in mass at
the end of each RH step as the equilibration moisture gain at that RH.
For the second moisture sorption profile, lyophiles (100–200 mg) were
placed in a 23-ring sample holder, again held at 0% RH for 48 h, and
then the RH was increased to 40% RH, at which samples were held for
96 h or until mass loss indicative of sucrose crystallization had occurred
in all samples. The percent change in mass was plotted versus time to
generate a moisture sorption/desorption profile with time, and the
onset of mass loss was used to identify the onset time of crystallization.
For the third moisture sorption profile, lyophiles were prepared and
handled the same as was done for the second profiling, but then the
samples were held at 33% RH (instead of 40% RH) until mass loss
(indicating crystallization) of most samples had occurred. The percent
change in mass was plotted versus time, and onset of crystallization
data were compared with those from the 40% RH moisture sorption
profile as well as crystallization that occurred in the 33% RH desicca-
tors.
2.6. Moisture content
The moisture contents of all initial lyophiles after exposure to 0%
RH for 2–4 days, as well as all lyophiles that remained amorphous for
the entire 4 weeks of exposure to 11%, 23%, or 33% RH, were de-
termined using a one-component volumetric Karl Fischer titration
method (V20S Volumetric KF Titrator, Mettler-Toledo, LLC, Columbus,
OH). Approximately 50 mg of each lyophile was added directly to the
HYDRANAL-Methanol Rapid working medium to extract water from
the sample. The sample was then titrated using the HYDRA-
NAL-Composite 2 titrant, which allowed moisture content to be mea-
sured in % moisture (wb). Calibration of the Karl Fischer titration
system was completed prior to sample analyses using the HYDRA-
NAL-Water Standard 10.0 (10 mg/g = 1% water content).
2.7. Differential scanning calorimetry
All lyophiles and starting materials were analyzed by differential
scanning calorimetry (DSC) using a DSC 4000 (PerkinElmer, Waltham,
MA). The instrument was calibrated with indium and verified with the
melting point of water. Dry nitrogen was used to purge the system at
20 mL/min. Initial lyophiles that had been exposed to 0% RH for
2–4 days (5–10 mg) were weighed into 50 µL aluminum DSC pans
(PerkinElmer), hermetically sealed, and punctured with a pinhole to
allow water vapor to escape when determining ‘dry’ Tgs. The onset Tg
was determined in a heat-cool-heat protocol from the second scan.
A.L. Voelker, et al.
Samples were first scanned by heating the samples in the DSC from
20 °C to 100 °C at a rate of 20 °C/minute. Samples were cooled to 20 °C
at a rate of 50 °C/minute and held at 20 °C for 3 min to allow the
temperature to equilibrate. A second scan then heated the samples from
20 °C to 100 °C at a rate of 20 °C/minute. All starting ingredients were
also analyzed for Tg or melting point (Tm) using the heat-cool-heat
protocol described above, only varying the temperature range of the
scans based on material. Pyris software (PerkinElmer) was used to
calculate the onset Tg or onset Tm, which was defined as the tempera-
ture in which the endothermic event characterized by a baseline shift
began in the second scan or the temperature in which a sharp en-
dothermic peak began in the second scan, respectively.
2.8. Sample photography
Select samples were analyzed for appearance following crystal-
lization in the second moisture sorption experiment, in which samples
were held at 40% RH in the SPS moisture sorption instrument. These
samples were photographed in a Deep Professional LED Photography
light box and with the polarized light microscope using an iPhone 6 s
camera.
2.9. Scanning electron microscopy
Scanning electron microscopy (SEM) was completed using a NOVA
nanoSEM Field Emission SEM (FEI Company, Hillsboro, OR) to identify
differences in crystal morphology. Lyophiles in which sucrose had
crystallized during storage at 33% RH were applied to double-sided
carbon tape and coated using a platinum target coating system before
analysis.
2.10. Statistical analysis
Samples were analyzed in duplicate for moisture sorption (time of
crystallization), moisture content, Tg, and Tm. Single-variable ANOVA
using SAS 9.4 (SAS Institute, Cary, NC) was used to determine sig-
nificant differences in time of crystallization, moisture content, Tg, and
Tm. Differences were determined using Tukey’s post hoc test for mul-
tiple comparisons at a significance level of α = 0.05. The HLB value,
moisture content (initial, after 4 weeks at 11% RH, and after 4 weeks at
23% RH), Tg, molecular weight, and number of –OH groups were also
plotted vs. time to crystallization to determine Pearson’s correlation
coefficients.
3. Results and discussion
3.1. Stability of amorphous sucrose in RH-controlled desiccators
All of the sucrose lyophiles with and without emulsifiers were in-
itially amorphous, as indicated by PXRD, FT-IR, and PLM, except for the
lyophiles containing the higher concentration (5% w/w) of polysorbate
80. The effects of storage RH on the time to sucrose crystallization in all
lyophiles are summarized in Table 2, wherein it can be seen that dif-
ferent emulsifiers had different effects on the stability of amorphous
sucrose. Most of the lyophiles that were initially amorphous remained
so for the 4 week duration of storage in desiccators at 11% and 23% RH;
however, all lyophiles containing polysorbates at both 1% and 5% (w/
w) concentrations crystallized at these RHs. Increasing the concentra-
tion of either polysorbate and increasing the storage RH both resulted
in shorter time to sucrose crystallization.
More varied times to sucrose crystallization (ranging from 2 days to
2 weeks) were found when the RH in the desiccators was increased to
33%. The lyophiles in which sucrose was fastest to crystallize (by day 2)
at 33% RH included the control and those containing polysorbates,
SP50 1%, and PS750 1%. The lyophiles that were slowest to crystallize
at 33% RH (by day 14) were those containing SP70 5% and PS750 5%.
5
Food Chemistry: X 3 (2019) 100050
Unlike the inverse stability trends seen with increasing polysorbate
concentration resulting in decreased amorphous sucrose stability, it
appeared that increasing the concentration of the sucrose esters tended
to increase amorphous sucrose stability (delay time to crystallization).
Sucrose esters containing higher percentages of mono-esters (instead of
di- and tri-esters) generally resulted in longer amorphous sucrose sta-
bilization. Based on the desiccator studies, the stabilizing trend of the
emulsifiers, as documented by time to crystallization, followed the
general trend: polysorbate 80 5% < polysorbate 20 5% < polysorbate
80 1% < polysorbate 20 1% < sucrose control ≈ SP50 1% ≈ PS750
1% < SP30 1% ≈ SP30 5% ≈ SP50 5% ≈ SP70 1% < soy lecithin
1% ≈ soy lecithin 5% < SP70 5% ≈ PS750 5%. No evidence of crys-
tallization of the emulsifiers was found in PXRD diffractograms over
time. Additional analyses were conducted to better understand the
differing effects of the emulsifiers on sucrose crystallization.
3.2. Moisture content of amorphous sucrose lyophiles
The storage RH and sample moisture content are known to affect
sucrose crystallization (Mathlouthi, 1995). When exposed to environ-
ments with RHs higher than the water activity of the sample, amor-
phous sucrose will absorb moisture, which results in a decrease in the
Tg of the matrix and increase in molecular mobility. If conditions are
favorable, molecular rearrangement and crystallization occur, at which
point moisture is expelled (Makower & Dye, 1956). To enable com-
parisons between moisture contents and amorphous sucrose stability,
the initial moisture contents of all lyophiles and the moisture contents
of lyophiles that remained amorphous after 4 weeks of storage in 11%
and 23% RH desiccators were measured (Table 3). All initial moisture
contents of the lyophiles except for sucrose:SP30 1% were significantly
lower (p < 0.05) than the sucrose control. The low initial moisture
content found in sucrose:polysorbate 80 5% was likely due to the par-
tially crystalline sucrose in the lyophile even immediately after lyo-
philization. Increasing the storage RH to 11% or 23% RH significantly
increased all sample moisture contents but also resulted in no sig-
nificant differences in moisture content between any of the lyophiles,
including the control, at each RH. Taken together, these findings in-
dicate that the addition of an emulsifier altered the moisture diffusion
rates during lyophilization, generally resulting in lower initial moisture
contents than the control, and that the addition of 1 and 5% (w/w) of
the sucrose esters and soy lecithin did not alter the hygroscopicity of the
samples at low storage RHs in desiccators compared to the control.
Although these samples did not crystallize in these conditions, these
data may indicate that matrix effects other than differences in hygro-
scopicity may contribute to the variations in sucrose crystallization
onset times between the formulations.
3.3. Moisture sorption profile and sucrose crystallization
To enable direct comparisons between the samples of moisture
sorption leading up to crystallization as well as the RH at which crys-
tallization occurred (indicated by mass loss (Makower & Dye, 1956)),
moisture sorption profiles were collected from 0 to 80% RH in a
gravimetric moisture sorption instrument (Fig. 2A). A 48 h drying step
at 0% RH was done in the instrument prior to this data collection to
remove significant differences in the initial moisture contents. While
most lyophiles (including the control) crystallized at 40% RH, two
lyophiles exhibited delayed sucrose crystallization (sucrose:SP50 1%
crystallized between 40 and 45% RH, and sucrose:soy lecithin 5% did
not crystallize until 45% RH), and the lyophiles containing polysorbates
crystallized at lower RHs (sucrose lyophiles containing polysorbate 20
and 80 at both 1% and 5% crystallized at 30% and 10–15% RH, re-
spectively; however, sucrose:polysorbate 80 5% was partially crystal-
line initially, so crystallization shown by moisture sorption was af-
fected).
The percent moisture gained before crystallization varied between
A.L. Voelker, et al. Food Chemistry: X 3 (2019) 100050

Table 2
Physical stability of sucrose lyophiles in controlled RH desiccators measured by a combination of PXRD, FTIR, and PLM as well as time of crystallization of
amorphous sucrose lyophiles on exposure to 33% and 40% RH in the SPS instrument and the enhancement compared to the control based on the SPS data. Grayscale
shading of desiccator data indicates timeframe of crystallization. Superscript letters denote statistical significance between times of crystallization.

*Samples that remained amorphous for the entire 4 week desiccator study are marked “A”; length of time prior to evidence of crystallization is indicated otherwise.
PC indicates the onset of crystallization before sample was largely crystalline.

samples, with some formulations crystallizing at lower moisture con-
tents than the control, while others did not crystallize until moisture
contents were higher. The sucrose control gained 6% weight before
crystallizing. The formulations that did not crystallize until moisture
contents surpassed 6% were: sucrose:SP70 5% and sucrose:PS750 5%,
which gained 8% weight, sucrose:SP50 5% and sucrose:soy lecithin 5%,
which gained 7.5% weight, and sucrose:SP70 1%, sucrose:PS750 1%,
sucrose:SP30 5%, and sucrose:soy lecithin 1%, which gained 7%
weight. Lyophiles that sorbed less water than the control prior to su-
crose crystallization were the sucrose:polysorbate 20 and 80 1% and
5% lyophiles, which gained less than 5% and 1% weight before ex-
tensively crystallizing, respectively (though sucrose:polysorbate 80 5%
was partially crystalline initially). Aside from the polysorbate-con-
taining lyophiles, moisture sorption trends at approximately 11% and
23% RH shown in Fig. 2A indicated no differences between lyophiles, in
agreement with the moisture content data (Table 3).
A second set of moisture sorption profiles was collected for samples
exposed to a constant 40% RH after drying (Fig. 2B), and the times at
which mass loss indicative of crystallization occurred in this treatment
are recorded in Table 2. Crystallization differences between lyophiles
have been clearly exhibited in a reasonable timeframe between 32%
and 43% RH (Saleki-Gerhardt & Zografi, 1994; Shamblin & Zografi,
1999), as was the case in previous studies investigating additive effects
on amorphous sucrose stability (Thorat et al., 2017). Here again, dif-
ferences in the amount of water sorbed prior to sucrose crystallization
and the onset time for crystallization were found between the different
emulsifier formulations. As in the desiccator studies, the presence of
polysorbates resulted in faster sucrose crystallization onset times

Table 3
Percent moisture content (wb) of amorphous sucrose lyophiles prior to desiccator storage (Day 0) and samples that remained amorphous after 4 weeks of storage at
11% and 23% RH and onset Tgs of initial (dry) amorphous lyophiles. Uppercase superscript letters on moisture content data denote statistical significance between
percent moisture of each lyophile at the specified timepoint, and lowercase superscript letters on moisture content data denote statistical significance between
percent moisture of the specified lyophile at each timepoint. Superscript letters on Tg data denote statistical significance between Tgs only. Statistical analysis was run
separately for each trial.
Co-formulated Additive Percent Additive Week 0 Week 4 11% RH Week 4 23% RH Week 0

Tg1 Tg2*

Aa Ab Ac BCD
Sucrose – – 2.2 ± 0.1% 3.12 ± 0.07% 5.07 ± 0.08% 56.5 ± 0.5 °C
Sucrose SP30 1% 1.9 ± 0.2% ABa 3.08 ± 0.03% Ab 4.97 ± 0.03% Ac 61 ± 2 °C ABCD
Sucrose SP30 5% 1.05 ± 0.04% Ea 2.99 ± 0.00% Ab 4.7 ± 0.3% Ac 57 ± 2 °C BCD 66 ± 1 °C
Sucrose SP50 1% 1.29 ± 0.03% CDEa 3.10 ± 0.06% Ab 5.005 ± 0.007% Ac 58.4 ± 0.4 °C ABCD
Sucrose SP50 5% 1.14 ± 0.02% DEa 3.0 ± 0.1% Ab 4.9 ± 0.1% Ac 57 ± 3 °C BCD
Sucrose SP70 1% 1.29 ± 0.09% CDEa 2.99 ± 0.02% Ab 4.98 ± 0.05% Ac 64 ± 3 °C A
Sucrose SP70 5% 1.4 ± 0.1% CDEa 3.3 ± 0.1% Ab 4.8 ± 0.1% Ac 58 ± 2 °C ABCD
Sucrose PS750 1% 1.6 ± 0.2% BCa 3.2 ± 0.1% Ab 4.9 ± 0.2% Ac 58 ± 2 °C ABCD
Sucrose PS750 5% 1.08 ± 0.06% DEa 3.0 ± 0.3% Ab 5.1 ± 0.1% Ac 55.1 ± 0.5 °C D
Sucrose Soy Lecithin 1% 1.17 ± 0.04% DEa 3.24 ± 0.08% Ab 5.01 ± 0.01% Ac 62.3 ± 0.9 °C AB
Sucrose Soy Lecithin 5% 1.285 ± 0.007% CDEa 3.1 ± 0.3% Ab 4.9 ± 0.1% Ac 62.5 ± 0.6 °C AB
Sucrose Polysorbate 20 1% 1.51 ± 0.09% BCD – – 61.3 ± 0.8 °C ABC
Sucrose Polysorbate 20 5% 1.06 ± 0.08% DE – – –
Sucrose Polysorbate 80 1% 1.39 ± 0.03% CDE – – 56.1 ± 0.8 °C CD
Sucrose Polysorbate 80 5% 0.9 ± 0.2% E – – –

* Tg2 was found for one sample due to heterogenous nature of the sample at 5% additive.
* No Tg was found for sucrose:polysorbate lyophiles at 5% additive

6
A.L. Voelker, et al. Food Chemistry: X 3 (2019) 100050

A) B)
8 8

7 7

6 6

Percent Moisture Content

Percent Weight Change

5 5
4
4
3
3
2
2
1
1
0

-1 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 90
RH (%) Time (Hours)
Sucrose Control Sucrose:SP30 1% Sucrose:SP30 5% Sucrose Control Sucrose:SP30 1% Sucrose:SP30 5%
Sucrose:SP50 1% Sucrose:SP50 5% Sucrose:SP70 1% Sucrose:SP50 1% Sucrose:SP50 5% Sucrose:SP70 1%
Sucrose:SP70 5% Sucrose:PS750 1% Sucrose:PS750 5% Sucrose:SP70 5% Sucrose:PS750 1% Sucrose:PS750 5%
Sucrose:Soy Lecithin 1% Sucrose:Soy Lecithin 5% Sucrose:Polysorbate 20 1% Sucrose:Soy Lecithin 1% Sucrose:Soy Lecithin 5% Sucrose:Polysorbate 20 1%
Sucrose:Polysorbate 20 5% Sucrose:Polysorbate 80 1% Sucrose:Polysorbate 80 5% Sucrose:Polysorbate 20 5% Sucrose:Polysorbate 80 1% Sucrose:Polysorbate 80 5%

C)

5
Percent Weight Change

0
0 100 200 300 400 500
Time (Hours)
Sucrose Control Sucrose:SP30 1% Sucrose:SP30 5%
Sucrose:SP50 1% Sucrose:SP50 5% Sucrose:SP70 1%
Sucrose:SP70 5% Sucrose:PS750 1% Sucrose:PS750 5%
Sucrose:Soy Lecithin 1% Sucrose:Soy Lecithin 5% Sucrose:Polysorbate 20 1%
Sucrose:Polysorbate 20 5% Sucrose:Polysorbate 80 1% Sucrose:Polysorbate 80 5%

Fig. 2. Moisture sorption profiles of sucrose lyophiles A) from 0 to 80% RH, B) held at 40% RH, and C) held at 33% RH.

(1.5–3.95 h) compared to the control, which did not crystallize until 33% RH desiccators. Aside from these differences, the polysorbates
hour 15 (Fig. 2B, Table 2). The emulsifier formulations that most de- followed the same trends in both the desiccator and SPS experiments,
layed sucrose crystallization were: sucrose:SP70 5%, which crystallized resulting in more rapid sucrose crystallization than the control, and
at hour 27, and sucrose:soy lecithin 5%, which crystallized at hour 24. both experiments found sucrose:SP70 5% to be the most stable lyophile.
All other lyophiles crystallized between hours 14 and 19, times which Differences in crystallization trends between the SPS experiment at
were not significantly different than the control sucrose. Unlike trends a constant 40% RH and the desiccator studies could have been caused
in the desiccator studies, increasing the degree of mono-esters (as op- by the drying step done in the SPS experiment, the passive vs. active
posed to di- and tri-esters) in the sucrose esters did not correlate to headspace differences between the treatments, and/or the higher RH of
increased time before sucrose crystallization occurred (R2 = 0.047), the SPS experiment (40% RH) compared to the 33% RH desiccator. To
and increasing the amount of emulsifier (from 1 to 5% w/w) did not better determine whether the differences in the experiments were due
result in significantly delayed crystallization except for SP70 and soy to the method of storage or specifically as a result of the difference in
lecithin. storage RHs (33% vs. 40%), a third set of sorption profiles was collected
Based on the 40% RH SPS experiment, the stabilizing trend of the in the SPS in which the lyophiles were held at a constant 33% RH
emulsifiers for delaying sucrose crystallization was: polysorbate 80 (Fig. 2C, Table 2). The stabilizing trend of the emulsifiers in this ex-
5% < polysorbate 20 5% < polysorbate 80 1% < polysorbate 20 periment was: polysorbate 80 5% ≈ polysorbate 20 5% < polysorbate
1% < soy lecithin 1% < SP30 1% ≈ SP70 1% < sucrose 80 1% ≈ polysorbate 20 1% < PS750 1% < sucrose control < soy
control ≈ PS750 5% < SP50 5% < SP50 1% < PS750 1% < SP30 lecithin 1% < SP50 1% < SP30 1% < SP70 1% < SP30
5% < soy lecithin 5% < SP70 5% (Fig. 2B, Table 2). This trend dif- 5% < SP50 5% < soy lecithin 5% < SP70 5% < PS750 5%. Other
fered in several places from that found in the 33% RH desiccator ex- than the low stability of sucrose:soy lecithin 1% in both the 33% and
periments (Table 2), with some formulations providing better stability 40% RH SPS experiments (compared to high stability in the 33% RH
and others no longer delaying sucrose crystallization compared to the desiccator), the overall trend of this 33% RH SPS experiment was more
control. Most notably, sucrose:PS750 5% was one of the most stable similar to the 33% RH desiccator experiment than the higher 40% RH
lyophiles in the desiccator experiment but did not significantly delay moisture sorption results, indicating that the difference in RH (33 vs.
sucrose crystallization compared to the control in the 40% RH SPS 40% RH) was likely the main reason for the differing stability trends
experiment. Similarly, the sucrose:soy lecithin 1% formulation was the noted previously. The formulation that exhibited the most RH-depen-
most stable of those containing 1% emulsifier in the desiccators but was dent properties was sucrose:PS750 5%, which was the most stable
not significantly different from the control in the 40% RH experiment. amorphous lyophile (along with sucrose:SP70 5%) in both the 33% RH
Conversely, sucrose:soy lecithin 5% had similar effects to other emul- desiccator and SPS experiments, only partially crystallizing in the
sifiers in the desiccator experiments but was one of the most stable 3 week SPS experiment, but was not significantly different than the
lyophiles in the 40% RH SPS experiment, remaining amorphous until control in the 40% RH SPS experiment. While it is known that RH plays
24 h into the experiment. Sucrose:PS750 1% was also much more suc- a key role in sucrose crystallization (Mathlouthi, 1995; Shamblin &
cessful at stabilizing sucrose in the 40% RH SPS experiment than in the Zografi, 1999), PS750 was the only emulsifier to show such a dramatic

7
A.L. Voelker, et al.
difference in stabilization of amorphous sucrose between 33% and 40%
RH.
3.4. Effect of glass transition temperature on amorphous sucrose stability
The ‘dry’ Tgs of lyophiles in this experiment are shown in Table 3
and Fig. S1. Although previous studies report an increase in Tg as the
underlying reason additives delay crystallization in a variety of food
systems, including sucrose matrices (Roos & Karel, 1991; van Hook,
1961), no significant trends were found between Tg and sucrose crys-
tallization time in the current study (when excluding polysorbates,
R2 = 0.006). The small amount of emulsifier added relative to sucrose
(1% and 5% w/w) did not significantly alter the ‘dry’ Tg compared to
the sucrose control (56.5 °C), except for sucrose:SP70 1%, which had a
Tg of 64 °C. The other difference noted was that the sucrose:SP30 5%
sample had 2 Tgs, presumably due to heterogeneity. Previous studies on
sucrose crystallization in the presence of salts and saccharides have also
shown that there is not a direct relationship between crystallization
onset times and Tg (Thorat et al., 2018). Exposing lyophiles to in-
creasing environmental RHs would be expected to drop the Tgs of all
samples in a predictable manner based on moisture content (according
to models such as the Gordon-Taylor equation), and therefore crystal-
lization would be expected to correlate with moisture content since no
significant differences were found between the majority of the ‘dry’ Tgs
of the lyophiles. However, this was not the case, as shown in Fig. 2.
Discrepancies found in Gordon-Taylor modeling of sucrose:saccharide
lyophiles with varying moisture contents suggested that factors beyond
Tg, specifically structural compatibility of the saccharides with sucrose,
contributed to the stabilization of amorphous sucrose (Thorat et al.,
2018), and the lack of correlation between Tg and crystallization onset
times in sucrose:salt lyophiles was due to ion-water interactions and
possible hydration pockets around the ions in the lyophiles affecting Tg
and plasticization of amorphous sucrose (Thorat et al., 2017).
Despite the lack of evidence that lyophile Tg correlated to delay in
sucrose crystallization, the thermal behaviors of the individual emul-
sifiers were investigated. Experimental values for Tgs of sucrose and soy
lecithin and Tms of sucrose, sucrose esters, soy lecithin, and poly-
sorbates are provided in Table 1. The Tgs of polysorbates were too low
to be measured by this DSC (< −50 °C). Two Tms were found for the
sucrose esters (Fig. S2), which agrees with the report by Szűts, Pallagi,
Regdon, Aigner, and Szabó-Révész (2007), although the range of Tms
found differed. It is important to note that the sample storage tem-
perature (25 °C) was above the melting temperature of some of the
emulsifiers (Table 1), and Tg is always lower than Tm, often by a factor
of Tg/Tm = 2/3 (Sakka & Mackenzie, 1971). Polysorbates are known to
be plasticizers, having Tgs around −61 °C and Tms from −15 to 20 °C
(Amim, Blachechen, & Petri, 2012; Amim, Kawano, & Petri, 2009).
Polysorbates have much lower Tgs and Tms than sucrose or the other
emulsifiers in this study (Table 1). These properties may have led to
more localized plasticization of the sucrose matrix when polysorbates
were added than in matrices with the other emulsifiers, which may
have contributed to the more rapid sucrose crystallization onset times
found in these samples. While the Tgs of other emulsifiers were also
slightly lower than that of sucrose, indicating that if stability is related
to Tg, the samples containing the other emulsifiers should theoretically
have been less stable as well, the Tgs of the emulsifiers in this study
(except polysorbates) were at most 15 °C less than that of sucrose. This
magnitude of difference was small enough that the lowering effect on
the Tg of the lyophiles by these emulsifiers was not significant (Table 3).
Additionally, the Tgs of these lyophiles remained above room tem-
perature.
3.5. Effect of emulsifier structural properties on amorphous sucrose stability
3.5.1. Role of emulsifier structural similarity to sucrose
It has previously been shown that when Tg is not significantly
8
Food Chemistry: X 3 (2019) 100050
affected by additives, the structure of the additive plays the major role
in influencing comparative stabilization of amorphous sucrose against
crystallization (Leinen & Labuza, 2006; Saleki-Gerhardt & Zografi,
1994; Thorat et al., 2018). The stabilizing effect for delaying sucrose
crystallization seems to be best when the additive has a region that is
structurally similar to sucrose, usually a glucose or fructose unit, that is
able to interact with sucrose at the crystal interface and also has a
dissimilar structural region that prevents further incorporation of su-
crose into the crystal lattice (Thorat et al., 2018). When considering the
structures of the emulsifiers used, the sucrose esters had a region that
was most structurally similar to sucrose. Theoretically, the glucose and
fructose units in the sucrose esters could have interacted with sucrose,
and the fatty acid region could have disrupted sucrose crystal growth.
This concept is similar to a report on how raffinose disrupts sucrose
crystallization: the glucose and fructose units on raffinose attach to the
sucrose crystal interface, and the galactose unit disrupts further in-
corporation into the sucrose crystal lattice, slowing crystal growth
(Leinen & Labuza, 2006). However, the success of the sucrose esters at
delaying crystallization was minimal in the 40% RH SPS experiment,
with only the sucrose:SP70 5% and sucrose:PS750 5% formulations
significantly delaying sucrose crystallization time compared to the
control (Fig. 2B, Table 2). These emulsifiers (SP70 and PS750) had a
higher fraction of mono-esters, and thus less di- and tri-esters, than the
other sucrose esters studied. The lower molecular weight of these two
sucrose esters led to a greater contribution of molecules since the
samples were prepared on a weight basis. Assuming the species adsorb
with the head group to sucrose, the presence of the fatty acid tails was
what disrupted crystallization. It does not appear that length of the tail,
and in effect hydrodynamic radius, played a role in efficacy of delaying
crystallization, but rather the prevalence of sucrose head groups de-
termined how effective the sucrose ester was at disrupting crystal-
lization as long as any tail was present.
3.5.2. Role of emulsifier HLB values
While the efficacy of sucrose esters increased with increasing HLB
value, when the HLB values of the other emulsifiers studied were
considered, there was no correlation between the HLB value of emul-
sifiers and crystallization time (R2 = 0.197). The ability of the sucrose
esters to inhibit sucrose crystallization was more likely due to degree of
ester substitution than HLB value since soy lecithin, which has a lower
HLB value than the sucrose esters, was more effective at delaying
crystallization than many of the sucrose esters, and polysorbates, which
have higher HLB values, induced crystallization.
3.5.3. Role of emulsifier structural dissimilarity to sucrose
Although phosphatidylcholine (in soy lecithin) lacks a structurally
similar region to sucrose, which may indicate that it would not be
successful at delaying sucrose crystallization, soy lecithin contains
other phospholipids as well, including phosphatidylethanolamine,
phosphatidylinositol (which contains a monosaccharide unit), phos-
phatidylserine, and phosphatidic acid (Poirier, 2011). The hetero-
geneity of phospholipids found in soy lecithin may have contributed to
the delay of sucrose crystallization seen in this study due to a wider
variety of impurities present in the sample despite the absence of many
structurally similar regions to sucrose (Gabarra & Hartel, 1998; Smythe,
1967).
The polysorbates also lack a structurally similar region to sucrose
and have multiple long hydrophobic chains. However, unlike soy le-
cithin, both polysorbates at both concentrations induced a much faster
rate of crystallization than occurred in the sucrose control (0.1x–0.3x in
the 40% RH SPS experiment) (Fig. 2B, Table 2). The lyophile containing
5% polysorbate 80 was never fully amorphous and crystallized faster
than lyophiles containing polysorbate 20 in all experiments conducted.
The structural differences between these polysorbates (polysorbate 80
contains an oleic acid chain (18:1 n-9) and polysorbate 20 contains a
lauric acid chain (12:0)) suggested that the longer fatty acid side chain
A.L. Voelker, et al.
Fig. 3. Images comparing the A) physical and microscopic (PLM) appearance and B) crystal morphology by SEM micrographs after crystallization of the following
lyophiles: i) sucrose control, ii) sucrose:polysorbate 20 1%, iii) sucrose:polysorbate 20 5%, iv) sucrose:polysorbate 80 1%, and v) sucrose:polysorbate 80 5%.
may have played a role in inducing crystallization. Increasing the
amount of either polysorbate from 1% to 5% resulted in faster sucrose
crystallization.
Interestingly, it was observed that while most lyophiles (including
the control) collapsed before crystallizing, the sucrose:polysorbate
lyophiles did not collapse and did not change much in physical ap-
pearance upon crystallization. PLM and light box images of the physical
polysorbate lyophiles compared to the control that better illustrate this
anomaly are shown in Fig. 3A. The crystallization of these lyophiles at
40% RH was documented using PLM and a RH-controlled microscope
stage, with videos of these events provided in the Supplementary ma-
terial (Figs. S3, S4, and S5). The videos show that while the sucrose
control has a changed morphology when exposed to 40% RH as a re-
sponse to moisture sorption, collapses and becomes rubbery (due to
sorbed moisture lowering the Tg), and then crystallizes, the sucrose:-
polysorbate lyophiles did not undergo the same extent of physical
collapse or plasticization before crystallizing. Collapse precedes crys-
tallization and is caused by decreased viscosity as a response to
moisture sorption (Roe & Labuza, 2005). The lack of collapse in the
polysorbate-containing lyophiles was presumably because the rate of
crystallization was faster than collapse. The videos also suggest that
there was a difference in nucleation between the control and the
polysorbate-containing lyophiles. The control sucrose had few nuclea-
tion sites, which grew larger to eventually completely crystallize the
9
Food Chemistry: X 3 (2019) 100050
sucrose. Conversely, the sucrose:polysorbate lyophiles had a large
number of nucleation sites from which not much crystal growth was
seen under the microscope.
The sucrose and sucrose:polysorbate lyophiles were also viewed by
SEM after they had crystallized (Fig. 3B). Visual observation of these
samples supports the supposition that increased nucleation occurred in
the polysorbate-containing lyophiles. While the surface of the control
was smooth, the crystals from the polysorbate samples had a bumpy
and jagged surface. This rough surface indicated that nucleation was
rampant and crystal growth was more limited in the presence of the
polysorbates. A smaller crystal size and higher surface area also de-
monstrated that crystal growth was less extensive (Canselier, 1993).
The sucrose crystals formed in the polysorbate lyophiles were porous,
as seen in SEM and PLM images, which is consistent with the lack of
collapse and the increased nucleation rate causing formation of many
small crystallites. The formation of this porous crystalline structure
made of very small crystallites generated a large surface area that fa-
cilitated rapid release of moisture from the crystallizing amorphous
sucrose. Without such fast moisture release from the matrix, the water
would have plasticized the remaining amorphous fraction, leading to
the collapse that was observed in the other lyophiles in this study. The
high surface area of the porous crystals has many additional implica-
tions, including altered texture and dissolution. The irregular shape of
the crystals may also indicate heterogeneous nucleation and growth of
A.L. Voelker, et al.
the sucrose on the surface of the polysorbates (Verma, Zeglinski,
Hudson, Davern, & Hodnett, 2018).
3.5.4. Role of emulsifier critical micelle concentration
Another emulsifier property of potential interest is the critical mi-
celle concentration (CMC) since the presence of micelles could con-
tribute to the regions of interaction between the emulsifiers and sucrose
in solution prior to lyophilization. While CMCs of the emulsifiers were
not measured in this study, the concentrations of polysorbates used (1%
and 5%) are greater than reported aqueous CMCs for both polysorbates
20 and 80 (Mahmood & Al-Koofee, 2013; Wan & Lee, 1974), and the
concentrations of sucrose esters used in this study were also above the
CMC since the CMCs of sucrose esters are generally lower than for
polysorbates (Becerra, Toro, Zanocco, Lemp, & Günther, 2008). Because
lecithin has such a low HLB value, it does not have a well-reported CMC
in water. Since micelles were likely formed in all formulations (micelles
were present in solution prior to lyophilization and presumably sur-
factant was trapped in this form following water removal), except
perhaps the sucrose:soy lecithin lyophiles, and polysorbates induced
sucrose crystallization while sucrose esters and soy lecithin delayed or
had no effect on sucrose crystallization, it was concluded that CMC was
not a significant factor in how the emulsifiers altered sucrose crystal-
lization.
3.5.5. Role of emulsifier templating and intermolecular hydrogen bond
lifetime with sucrose
Previous studies on the effect of surfactants on crystallization in the
pharmaceutical industry have shown that, due to their inherent prop-
erties, surfactants with unbranched hydrophobic chains, including
polysorbate 80, are more flexible than surfactants with bulky hydro-
phobic groups and are therefore able to act as a template and align
molecules in the optimal configuration to promote nucleation (Berman,
June Ahn, & Lio, 1995; Chen, Ormes, Higgins, & Taylor, 2015;
Weissbuch, Addadi, Leiserowitz, & Lahav, 1988). Although the studies
cited here describe a templating effect by a hydrophobic chain for a
hydrophobic crystal, the steric properties indicate that the same effect
is worth considering in the case of the numerous nucleation sites and
more rapid sucrose crystallization observed in the sucrose:polysorbate
lyophiles (Figs. 2, S4, and S5 and Table 2). Generally, a surfactant that
is less flexible is unable to have this templating effect and instead in-
hibits nucleation by mass transfer effects. Assuming the templating
theory plays a role in this study, the structural differences between
polysorbates and other emulsifiers may have contributed to the absence
of this effect in the sucrose ester and soy lecithin lyophiles even though
they do not contain exceptionally bulky hydrophobic groups. For ex-
ample, although sucrose esters also have an unbranched hydrophobic
chain, the presence of a sucrose head group caused the sucrose esters to
act more like a raffinose additive in which the sucrose group adsorbs to
the crystal interface and the hydrophobic chain prevents mass transfer
of sucrose into the crystal lattice (Leinen & Labuza, 2006). However, it
is interesting to note that when more di- or tri-esters (unbranched hy-
drophobic chains) were present in the sucrose esters (SP30 and SP50),
the sucrose esters were not as successful at stabilizing the amorphous
sucrose (Fig. 2, Table 2). This may indicate that there is a contradictory
effect between the presence of a sucrose group and the presence of
unbranched hydrophobic chains which prevented the sucrose:SP30 and
sucrose:SP50 lyophiles from being significantly more stable than the
sucrose control. Additionally, soy lecithin also has unbranched hydro-
phobic chains; however, the presence of multiple types of phospholipids
introduces some branched chains and some monosaccharide units, and
the higher prevalence of pi bonds decreases the flexibility of the hy-
drophobic groups. The presence of these bulkier groups in soy lecithin
may have prevented the templating effect seen in the sucrose:poly-
sorbate lyophiles.
While the templating effect is a plausible explanation for the in-
creased sucrose nucleation seen in the sucrose:polysorbate lyophiles, an
10
Food Chemistry: X 3 (2019) 100050
alternative, and possibly more likely, theory is the propensity for hy-
drogen bonding between sucrose and polysorbates (Cui, Zhang, Yin, &
Gong, 2012; Galek, Fábián, Motherwell, Allen, & Feeder, 2007; Verma
et al., 2018). It has been reported that when hydrogen bonding is fa-
vorable between a compound of interest and a heterosurface (poly-
sorbates in the current study), nucleation is promoted in solution due to
the lengthened lifetime of the favorable hydrogen bond (Cui et al.,
2012; Verma et al., 2018). Since the polysorbates have only three hy-
drogen bond donors but have 26 hydrogen bond acceptors, there is a
high propensity for hydrogen bonding between the polysorbates and
the hydrogen bond donor-rich sucrose molecules. The lengthened life-
times of these hydrogen bonds promote more sucrose-sucrose interac-
tions and increase the chance that the crystal nucleus survives (Verma
et al., 2018). Essentially, the polysorbates create a surface which allows
for the clustering and therefore crystallization of the sucrose, which
also accounts for the irregular crystal shape shown in Fig. 3B. While
phosphatidylcholine found in soy lecithin contains no hydrogen bond
donor groups, which could cause it to act like the polysorbates, other
phospholipids contained in soy lecithin contain some hydrogen bond
donor groups and also some monosaccharides units (Poirier, 2011). The
hydrogen bond donor groups on phospholipids may hydrogen bond
with other phospholipids rather than with sucrose, lowering the pro-
pensity for hydrogen bonding with sucrose. Monosaccharides found in
the phospholipids may promote interactions with sucrose as was dis-
cussed in crystallization inhibition by raffinose (Leinen & Labuza,
2006), which is why they effectively delay crystallization despite their
high density of hydrogen bond acceptor groups. Sucrose esters also
contain some hydrogen bond acceptors; however, as previously noted,
sucrose esters contain a structurally similar region to sucrose that al-
lowed them to interact with sucrose and prevent further incorporation
into the crystal lattice. The amorphous sucrose stabilization induced by
the presence of soy lecithin and sucrose esters due to the presence of
monosaccharide units despite the presence of hydrogen bond acceptors
suggested that when a structurally similar region to sucrose is present in
the additive, that emulsifier property outweighed all others when
considering the delay of sucrose crystallization.
4. Conclusion
Different emulsifiers had varying effects on the crystallization rates
of amorphous sucrose, ranging from accelerating to delaying the onset
time of crystallization. Most lyophiles remained amorphous in de-
siccators at low RHs (11 and 23%RH), except for lyophiles containing
polysorbates. Increasing storage RH above 23%RH led to variations in
moisture sorption and crystallization tendencies. The structure of the
emulsifier was considered to be the major factor contributing to crys-
tallization trends in the sucrose:emulsifier lyophiles, and no correlation
was found between moisture sorption, critical micelle concentration, or
Tg and crystallization onset time. Sucrose esters contained a structurally
similar region to sucrose which was able to interact at the crystal in-
terface, and the ester side chains prevented further incorporation into
the crystal lattice, thereby delaying the crystallization of sucrose (by up
to 1.8x that of the control at 40%RH and longer at lower RHs). When
such a region of structural similarity was not present, intermolecular
hydrogen bonding and structural heterogeneity seemed to influence the
sucrose crystallization, contributing to the efficacy of soy lecithin at
delaying sucrose crystallization (by up to 1.6x at 40%RH). Polysorbates
destabilized sucrose crystallization, with crystallization times as low as
0.1x that of the sucrose control at 40%RH, attributed to the long fatty
acid and polyoxyethylene side chains that seemed to have a templating
effect that increased sucrose nucleation and inhibited structural col-
lapse during crystallization. These findings provide insight into me-
chanisms by which emulsifiers alter sucrose crystallization and could be
useful for designing formulations to alter or control the crystallization
of amorphous sucrose in low moisture products.
A.L. Voelker, et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors would like to thank Drs. Vincent Meunier and Laurent
Forny in the Particle Systems Group at Nestlé Research Center,
Lausanne, Switzerland, for providing financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fochx.2019.100050.
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JURNAL ILMU KEFARMASIAN INDONESIA, September 2016, hlm. 135-140 Vol. 14, No. 2
ISSN 1693-1831

Uji Iritasi Sediaan Gel Penyembuh Luka Ekstrak Etanol

Daun Binahong Menggunakan Slug Irritation Test

(Irritation Test of Wound Healing Gel of Ethanolic Extract

of Binahong Leaf Using Slug Irritation Test)

SRI HARTATI YULIANI1, YUMI RAHMADANI2, ENADE PERDANA ISTYASTONO1

1
Farmasi Fakultas Farmasi, Universitas Sanata Dharma, Yogyakarta, Indonesia.
2
PT Genaro Pharmaceuticals.

Diterima 9 November 2015, Disetujui 15 Agustus 2016

Abstrak: Penyembuhan luka adalah proses dinamik yang rumit dan belum sepenuhnya dimengerti.
Proses penyembuhan luka terdiri dari 3 fase yaitu fase inflamasi, fase pembentukan jaringan dan
fase remodeling. Lama fase inflamasi memegang peranan penting pada proses penyembuhan luka.
Fase inflamasi yang panjang akan menyebabkan penundaan proses penyembuhan luka. Iritan dapat
memperpanjang waktu pelepasan interleukin-1 and TNF-α. Hal tersebut akan menyebabkan matriks
ekstraseluler yaitu suatu material penting dalam pembentukan jaringan baru terdegradasi. Sediaan
penyembuh luka tidak boleh mengandung iritan, sehingga semua sediaan penyembuh luka harus
diuji potensi iritasinya. Potensi iritasi gel penyembuh luka ekstrak etanol daun binahong telah diuji
menggunakan slug irritation test. Validasi terhadap metode ini telah dilakukan dengan hasil sensitivitas
dan spesifisitas masing-masing 100% pada nilai batas produksi mukus 8,79%. Metode ini valid untuk
menentukan potensi iritasi terhadap suatu produk. Produksi mukus sediaan gel penyembuh luka ekstrak
etanol daun binahong adalah 4,55% sehingga dapat disimpulkan sediaan tersebut tidak menimbulkan
iritasi pada kulit.

Kata kunci: gel penyembuh luka, ekstrak etanol daun binahong, slug irritation test.

Abstract: Wound healing is a complex of various dynamic processes which are not completely
understood. There are 3 phases in the normal wound healing, i.e. inflammation phase, tissue formation
phase and tissue remodeling phase. The duration of the inflammation phase takes an important part of the
healing process. The prolonged inflammation will delay the healing process. Irritant can lead prolonged
elevation of interleukin-1 and TNF-α. It causes the degradation of extracellular matrix that is an
important material for tissue formation. Wound dressing should not contain irritant material. Therefore,
all wound dressing have to be tested for skin irritation potency. Wound healing gel of ethanolic extract
of binahong leaf has been tested for its irritant potency by slug irritation test. The validation of slug
irritation test resulted both of sensitivity and specificity 100% at the cut-off mucus production of 8.79%.
This method was valid to determine irritant potency of the product. Mucus production of the gel was
4.55%, therefore it can be concluded that the gel did not cause skin irritation.

Keywords: wound healing gel, ethanolic extract of binahong leaf, slug irritation test.

* Penulis korespondensi, Hp. 08995047771

e-mail: yuli_far@usd.ac.id
136 YULIANI ET AL.
PENDAHULUAN
PROSES penyembuhan luka adalah proses dinamis
yang kompleks dan belum sepenuhnya diketahui(1).
Proses ini meliputi tiga fase yaitu fase inflamasi,
fase formasi jaringan dan fase remodeling jaringan(2)
yang terjadi secara berurutan. Fase inflamasi dan
formasi jaringan dapat dipercepat dengan pemberian
obat(3) sedangkan fase remodelling jaringan biasanya
berlangsung dalam jangka yang panjang sampai
dengan 2 tahun(1). Proses penyembuhan luka akan
mengembalikan integritas kulit dengan akibat fibrosis
dan jaringan parut (4). Dewasa ini sudah banyak
dikembangkan sediaan penyembuh luka dengan
bahan aktif herbal diantaranya dalam bentuk salep,
krim, losion dan gel, dan telah terbukti mempunyai
aktivitas(5,6,7,8). Sediaan penyembuh luka yang baru-
baru ini dikembangkan adalah sediaan penyembuh
luka ekstrak binahong (9,10).
Sediaan penyembuh luka tidak boleh mengiritasi
kulit. Iritasi merupakan fenomena inflamasi pada
kulit yang disebabkan oleh senyawa asing. Inflamasi
adalah fase pertama proses penyembuhan luka(2).
Lama inflamasi memegang peranan penting dalam
proses penyembuhan luka. Terjadinya inflamasi
yang berlebihan pada jaringan kulit yang luka akan
menghambat proses perbaikan jaringan(11). Adanya
iritan pada luka akan memperlama pengeluaran pro-
inflammatory cytokines (interleukin-1 dan TNF-α).
Sebagai akibat fase inflamasi akan diperpanjang
sehingga meningkatkan level matrix metalloproteases
(MMPs). MMPs merupakan famili protein yang
mampu mendegradasi extracellular matrices (ECM).
ECM merupakan bahan penting pada proses reepiteliasi
jaringan(12).
European Center for the Validation of Alternative
Methods (ECVAM) mengkampanyekan metode
alternatif untuk menguji keamanan sediaan farmasi
terutama sediaan topikal dengan prinsip 3R (reduce,
refinement and replacement) (13). Penelitian ini
menggunakan prinsip ketiga yaitu replacement
dalam pelaksanaannya. Hewan uji dalam penelitian
ini yaitu kelinci diganti oleh siput telanjang yang
merupakan hewan invertebrata(14) dengan terlebih
dahulu melakukan validasi protokol uji.
BAHAN DAN METODE
BAHAN. Pembuatan Ekstrak Etanol Daun
Binahong. Simplisia daun binahong ditimbang
sebanyak 50 g dan ditambah 500 mL etanol 96%,
suhu penyarian dikendalikan 50 oC menggunakan
waterbath dengan pengadukan konstan 200 rpm
Jurnal Ilmu Kefarmasian Indonesia
selama 90 menit. Sari yang didapat dipisahkan dan
dipekatkan sampai menjadi 25% volume semula.
Pembuatan Sediaan. Formula gel penyembuh
luka ekstrak etanol binahong(9):
R/ Carbopol 940 1,25
Na-CMC 0,5
Ca-alginat 0,5
Trietanolamin 3 (sampai pH 7)
Gliserol 12,5
Asam Borat 0,5
Kalium sorbat 0,2
Ekstrak etanol binahong 5 mL
Aquades qs ad 100
Na-CMC ditaburkan pada air dan diaduk menggunakan
mixer dengan kecepatan putar 400 rpm selama 10
menit, kemudian Ca-alginat ditambahkan dan dengan
kecepatan putar yang sama diaduk selama 10 menit.
Terakhir tambahkan Carbopol 940 ke dalam campuran
dan diaduk sampai homogen. Asam borat dan kalium
sorbat yang telah dilarutkan dalam air ditambahkan ke
dalam campuran di atas dan diaduk sampai homogen.
Gliserol kemudian ditambahkan ke dalam campuran
diaduk sampai homogen. Terakhir trietanolamin
ditambahkan dan diaduk sampai homogen sampai
pH 7. Campuran tersebut adalah basis gel. Basis ini
kemudian disterilisasi menggunakan autoklaf pada
115 oC selama 30 menit. Basis gel yang telah steril
dipindahkan ke laminair air flow. Ekstrak etanol
binahong ditambahkan kedalam basis pada kondisi
aseptis. Kemudian gel penyembuh luka ekstrak etanol
binahong dikemas ke dalam tube di bawah LAF.
Uji Sifat Fisik Gel. Uji Viskositas. Hidrogel
dimasukkan ke dalam wadah dan dipasang pada
portable viscotester. Viskositas diketahui dengan
mengamati gerakan jarum penunjuk viskositas. Uji
dilakukan 24 jam setelah pembuatan hidrogel.
Uji Daya Sebar. Hidrogel ditimbang seberat 2 g
kemudian diletakkan di tengah kaca bulat berskala.
Di atas gel diletakkan kaca bulat lain yang telah
ditimbang dan diberi beban seberat 1 kg selama 3
menit. Tarik kaca bulat sebelah atas dengan beban
seberat 80 g. Dicatat jarak dan waktu yang diperlukan
untuk memisahkan kedua kaca tersebut. Daya sebar
dihitung dengan rumus:
S=MxL/T
S = daya sebar (cm g/detik)
L = jarak tempuh (cm)
M = berat kaca bulat bagian atas (gram)
T = waktu yang dibutuhkan untuk memisahkan kaca
bulat bagian atas dan bawah (detik)
Uji Daya Lekat. Usus babi digunakan untuk
merepresentasikan tekstur dari luka baru. Usus
Vol 14, 2016 Jurnal Ilmu Kefarmasian Indonesia 137
Analisis Data. Uji Slug Irritation Test. Data yang
babi yang telah dibilas dicuci dengan larutan
didapat dari uji iritasi menggunakan siput ditentukan
fisiologis. Usus tersebut kemudian ditempatkan pada
nilai cut-off untuk mengklasifikasikan bahan sebagai
platform. Sebanyak 0,5 g gel ditambahkan di atasnya.
iritan dan non-iritan. Data tersebut dianalisis
Diletakkan pan di atasnya kemudian diberi beban 100
menggunakan paket Classification and Regression
g selama 1 menit. Pada pan yang lain air ditambahkan
Trees (CART) pada aplikasi Statistika Komputasi R
sedikit demi sedikit sampai pan lepas dari permukaan
versi 3.0.2 (https://www.r-project.org/). Luaran dari
usus. Beban yang digunakan untuk melepaskan pan
CART adalah nilai cut-off yang memiliki sensitivitas
menggambarkan bioadesi gel. Bioadesi dihitung
dan spesifisitas terbaik. Berdasarkan nilai cut-off ini
berdasarkan rumus:
sampel diklasifikasikan sebagai senyawa iritan dan
B = W/A
non-iritan. Hasil klasifikasi dibandingkan dengan hasil
B = bioadesi (g/cm2)
menurut HET-CAM.
W= berat air yang diperlukan untuk melepas pan
HET-CAM. Data yang didapat pada uji HET-CAM
dari usus (g)
dihitung menggunakan rumus:
A= luas area pan (cm)
Slug Irritation Test (Uji Iritasi Menggunakan 301 - H 301 - L 301 - C
T= x5+ x7+ x9
Siput Telanjang). Siput yang digunakan dalam 300 300 300
penelitian ini adalah siput dengan berat ± 3 g dan Keterangan:
tidak terdapat luka pada dinding badan siput. Siput T = skor iritasi
ditempatkan pada sebuah petri yang telah diberi H= waktu yang dibutuhkan untuk menimbulkan
sampel sebanyak 1 gram dan dibiarkan selama 60 hemoragi (detik)
menit. Mukus yang dihasilkan siput dikumpulkan dan L= waktu yang dibutuhkan untuk menimbulkan lisis
ditimbang. Sampel yang digunakan adalah aquades (detik)
(kontrol negatif), basis gel (kontrol negatif), 1% SLS C= waktu yang dibutuhkan untuk menimbulkan
dalam basis (kontrol positif) dan gel penyembuh luka koagulasi (detik)
ekstrak binahong. Nilai cut-off dari produksi mukus Hasil yang didapat dari perhitungan kemudian
dicocokan dengan nilai pada Tabel 1(15).
ditentukan mengklasifikasikan sampel ke dalam iritan
atau non iritan.
Hen’s Egg Test Chorioallantoic Membrane. Tabel 1. Kriteria iritasi HET-CAM(15).
Telur ayam leghorn yang telah dibuahi dimasukkan
dalam inkubator dengan suhu 37 oC. Rongga udara No. Skor iritasi HET-CAM Kategori iritasi
telur dipastikan berada di sebelah atas. Telur dirotasi 1. < 0,9 Tidak mengiritasi
selama 10 hari. Pada hari kesepuluh telur diteropong,
2. 1,0-4,9 Iritasi lemah
telur yang tidak dibuahi atau tidak mengandung
embrio hidup dibuang. Rongga udara telur ditandai. 3. 5,0-8,9 Iritasi moderat
Rongga telur yang telah ditandai, digunting cangkang 4. 9,0-21 Iritasi kuat
terluarnya dengan menggunakan gunting steril. Untuk
mempermudah proses ini cangkang dilunakkan
dengan larutan NaCl 0,9% steril. Setelah cangkang HASIL DAN PEMBAHASAN
terluar dibuang, membran terluar telur dibasahi dengan
larutan NaCl 0,9% hangat dan dimasukkan kembali ke Tanaman binahong diketahui mengandung triterpenoid,
dalam inkubator selama 5-20 menit sehingga membran saponin, flavonoid, kuinon, monoterpenoid,
terluar dapat diambil dengan mudah. Setelah membran sesquiterpenoid(16,17). Salah satu senyawa aktif golongan
terluar diambil, dipilih telur yang tidak mengalami triterpenoid yaitu asam ursolat mempunyai aktivitas
kerusakan CAM akibat proses tersebut. Sebanyak 300 anti-inflamasi (18), mengembalikan permeabilitas
mg sampel diletakkan pada CAM, diamkan 20 detik. kulit(19) dan antimikroba(20). Berdasarkan hal tersebut
Setelah 20 detik CAM segera dibersihkan dengan Yuliani membuktikan bahwa ekstrak etanol daun
menggunakan NaCl 0,9% steril. Waktu pengamatan binahong mempunyai aktivitas sebagai penyembuh
selama 300 detik dimulai segera setelah CAM bersih luka(21).
dari sampel. Sebagai kontrol iritan digunakan sodium Ekstrak etanol daun binahong kemudiaan dibuat
lauril sulfat, kontrol negatif adalah air. Percobaan dalam bentuk sediaan gel. Sediaan gel dipilih karena
dilakukan terhadap kontrol iritan, kontrol positif gel memberikan efek hidrasi kepada kulit yang luka
dan sediaan penyembuh luka ekstrak etanol daun sehingga akan mengurangi jaringan nekrosis melalui
binahong. mekanisme apoptosis(22). Selain itu sediaan hidrogel
juga membantu menghilangkan jaringan mati, terasa
138 YULIANI ET AL.
dingin di permukaan luka, menurunkan rasa sakit,
dan meningkatkan penerimaan konsumen(23). Basis
gel diautoklaf terlebih dahulu dan ekstrak dicampur
dibawah kondisi aseptis. Sterilisasi diperlukan karena
salah satu persyaratan sediaan penyembuh luka adalah
steril.
Tabel 2. Sifat fisik gel penyembuh luka
ekstrak etanol binahong.
Sediaan gel penyembuh luka ekstrak etanol
binahong sangat nyaman digunakan pada luka, hal
ini dibuktikan dengan sifat fisik seperti terlihat pada
Tabel 2. Viskositas yang tidak terlalu besar dan daya
sebar yang dimiliki gel penyembuh luka tersebut tidak
menimbulkan rasa sakit saat diaplikasikan karena gel
tersebut bersifat lembut. Gel akan melekat dengan baik
pada kulit yang luka dalam waktu yang cukup untuk
melepaskan obatnya.
Sediaan penyembuh luka tidak boleh bersifat
iritan. Fase inflamasi merupakan fase pertama proses
penyembuhan luka. Fase ini bertujuan untuk mencegah
keluarnya darah dan membentuk perlindungan segera
dari faktor luar dengan terbentuknya agregat oleh
darah. Fase inflamasi diperlukan dalam proses
penyembuhan luka tetapi tidak boleh berlebihan.
Apabila fase inflamasi ini terjadi berlebihan maka
akan merugikan karena akan menghambat proses
reepiteliasi kulit. Iritan akan memperparah inflamasi
yang terjadi pada proses penyembuhan luka sehingga
sediaan penyembuh luka tidak boleh bersifat iritan.
Penelitian ini bertujuan mengetahui sifat iritan
sediaan penyembuh luka ekstrak etanol binahong.
Metode yang digunakan untuk menguji adalah slug
irritation test (menggunakan siput telanjang). Siput
telanjang dipilih sebagai hewan uji karena siput
telanjang merupakan invertebrata. Penggunaan hewan
uji (vertebrata) untuk keperluan uji toksisitas akut
sediaan topikal sudah mulai dihindari. Para penyayang
binatang mulai berkampanye tentang hak-hak hewan,
hal tersebut memaksa peneliti untuk membuat metode
alternatif untuk keperluan uji toksisitas akut. Slug
irritation test yang digunakan merupakan metode
alternatif toksisitas akut (iritasi) pada kulit.
Siput telanjang adalah invertebrata yang
dinding tubuhnya sangat rentan terhadap serangan
fisis dan mekanis. Dinding tubuh siput telanjang
akan mengeluarkan mukus yang berfungsi: (i)
Jurnal Ilmu Kefarmasian Indonesia
Lokomotion; (ii) Lubrikasi; (iii) Mencegah dehidrasi;
(iv) Melindungi tubuh dari kerusakan (14). Siput
dengan berat ± 3 g digunakan dalam penelitian.
Berat siput harus dikendalikan untuk memperkecil
variasi parameter yang diukur. Data dari Tabel 2
memperlihatkan produksi mukus yang disebabkan
oleh air, basis gel, 1% SLS dalam air dan 1% SLS
dalam basis gel sebagai sampel uji. Produksi mukus
siput dengan sampel air bernilai negatif. Hal tersebut
disebabkan siput akan mengabsorbsi air yang
digunakan sebagai sampel.
Data Tabel 3 digunakan untuk menentukan nilai
batas antara senyawa iritan dan non-iritan. Nilai
batas dihitung menggunakan metode Classification
and Regression Tree (ACRT). Sensitivitas dan
spesifisitas protokol dihitung dalam penentuan nilai
batas ini. Sensitivitas adalah jumlah total iritan yang
diklasifikasikan secara benar dibagi dengan jumlah
total sampel. Spesifisitas adalah jumlah total non-
iritan yang diklasifikasikan secara benar dibandingkan
jumlah total sampel.
Protokol slug irritation test dapat digunakan untuk
Tabel 3. Produksi mukus siput telanjang (% berat siput).
1% SLS 1% SLS dalam
No. AirB asis gel
dalam air basis gel
1. -9,11 7,94 16,28 22,72
2. -10,39 6,29 19,13 9,74
3. -15,99 0 19,03 16,33
4. -15,07 0,33 16,11 9,64
5. -19,12 1,96 21,34 16,54
memprediksi potensi iritasi senyawa apabila memiliki
sensitifitas dan spesifisitas lebih dari sama dengan
60%. Nilai batas dalam penelitian ini dihitung
menggunakan perangkat lunak Statistika Komputasi
R. Nilai batas produksi mukus dengan sensitifitas
dan spesifisitas terbaik adalah 8,79%. Sensitifitas
dan spesifisitas untuk nilai batas 8,79% masing-
masing adalah 100% dan 100%. Nilai batas ini berarti
suatu senyawa dikatakan sebagai non-iritan apabila
menyebabkan siput memproduksi mukus < 8,79%
dari berat tubuhnya. Ketepatan dari nilai batas ini
adalah 100%.
Nilai batas yang dihasilkan kemudian digunakan
untuk mengklasifikasikan sampel ke dalam golongan
iritan dan non-iritan berdasarkan jumlah mukus yang
dihasilkan oleh siput. Nilai batas produksi mukus
8,79% dari berat siput menghasilkan klasifikasi
yang diperlihatkan pada Tabel 4. Hasil ini kemudian
dibandingkan dengan klasifikasi iritan sampel
menggunakan metode HET-CAM (Gambar 1). Metode
Vol 14, 2016 Jurnal Ilmu Kefarmasian Indonesia 139
Tabel 4. Klasifikasi senyawa berdasarkan slug irritation test and HET-CAM.

No. Senyawa Slug irritation test HET-CAM

% mukus klasifikasi skor klasifikasi
1. air -13,94 Non-iritan 0 Non-iritan
2. Basis gel 3,30 Non-iritan 0 Non-iritan
3. SLS 1% dalam air 18,51 Iritan 7,58 Iritan
4. SLS 1% dalam basis gel 14,99 Iritan 5,37 Iritan

Gambar 1. Hasil inspeksi visual pada uji HET-CAM untuk air (A) dan basis (B) sebagai kontrol dan SLS 1% dalam air
(C) dan SLS 1% dalam basis (D). Tidak muncul tanda-tanda iritasi pada A dan B. Hemoragi yang muncul ditandai dengan
lingkaran putih pada foto C dan D.

HET-CAM ini adalah metode yang mapan digunakan
untuk mengklasifikasikan potensi iritasi senyawa.
Hasil perhitungan skor HET-CAM dicocokkan
dengan menggunakan Tabel 1 untuk menghasilkan
klasifikasi iritasi berdasarkan HET-CAM seperti
tercantum dalam Tabel 4. Klasifikasi potensi iritasi
senyawa dengan menggunakan slug irritation test dan
HET-CAM memberikan hasil yang sama (Tabel 4).
Sehingga slug irriation test dapat digunakan sebagai
metode alternatif untuk mengukur potensi iritasi suatu
senyawa.
Protokol slug irritation test tersebut kemudian
digunakan untuk mengevaluasi potensi iritasi gel
gel penyembuh luka ekstrak etanol daun binahong
adalah memiliki nilai rata-rata 4,55%. Nilai batas
senyawa dikatakan mengiritasi adalah 8,97%. Jadi gel
penyembuh luka ekstrak etanol daun binahong tidak
mengiritasi kulit.
SIMPULAN
Gel penyembuh luka ekstrak etanol binahong
menyebabkan siput mengeluarkan mukus sebanyak
4,55%, sehingga dapat disimpulkan bahwa sediaan
ini tidak mengiritasi kulit.

Tabel 5. Produksi mukus siput telanjang yang disebabkan

UCAPAN TERIMA KASIH
oleh gel penyembuh luka ekstrak etanol binahong.
Produksi mukus (% Nilai batas Penelitian ini didukung secara finansial oleh Direktorat
No. Klasifikasi Riset dan Pengabdian Masyarakat, Kementerian
berat siput) (%)
1. 5,75 Non-iritan Riset, Teknologi, dan Pendikan Tinggi melaui
2. 5,33 Non-iritan
Penelitian Hibah Kompetensi Tahun 2015 (No. DIPA-
023.04.1.673453/2015).
3. 4,10 8,79 Non-iritan
4. 3,48 Non-iritan DAFTAR PUSTAKA
5. 3,98 Non-iritan
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 Asian Pacific Journal of Tropical Medicine 2015; 8(6): 421–430 421

H O S T E D BY
Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Medicine

journal homepage: http://ees.elsevier.com/apjtm

Original research http://dx.doi.org/10.1016/j.apjtm.2015.05.014

Brazilin from Caesalpinia sappan heartwood and its pharmacological activities: A review

Nilesh P. Nirmal1*, Mithun S. Rajput2, Rangabhatla G.S.V. Prasad3, Mehraj Ahmad4

1
Center for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Health and Food
Sciences Precinct, Block 10, 39 Kessels Road, Coopers Plains, Acherfield Brisbane, QLD 4108, Australia
2
Department of Medical Biochemistry, MGM Medical College, A.B. Road, Indore 452001, MP, India
3
Biomedical and Pharmaceutical Technology Research Group, Nano Research for Advanced Materials, Bangalore 5600040, Karnataka, India
4
Institute of Nutrition, Mahidol University, 999 Phutthamonthon 4 Rd., Salaya, Nakhon Pathom 73170, Thailand

A R TI C L E I N F O ABSTRACT

Article history: Caesalpinia sappan L. (CS) is a plant of Leguminosae family, commonly known as
Received 15 Mar 2015 Brazil or Sappan wood. CS is distributed in Southeast Asia and its dried heartwood has
Received in revised form 20 Apr 2015 been used as traditional ingredient of food or beverages and has a wide variety of me-
Accepted 15 May 2015 dicinal properties. Higher extraction yield of CS wood was achieved with 95% ethanol for
Available online 25 June 2015 2 h. Chemical constituent's investigation of sappan wood resulted in the isolation of
various structural types of phenolic components including one xanthone, one coumarin,
three chalcones, two flavones three homoisoflavonoids and brazilin. Brazilin [(6a S-cis)-7,
Keywords:
11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10(6H)- tetrol], a major and active com-
Brazilin
pound found in CS heartwood. Most of the folkloric uses of brazilin were validated by the
Caesalpinia sappan
scientific studies such as antioxidant, antibacterial, anti-inflammatory, anti-photoaging,
Extraction
hypoglycemic, vasorelaxant, hepatoprotective and anti-acne activity. CS heartwood
Heartwood
extract is safe and did not produce any acute or subacute toxicity in both male and female
Pharamcological activities
rats. Brazilin is the safe natural compound having potential to develop as a medicinal
compound with application in food, beverage, cosmetics and pharmaceutical industries to
screen its clinical use in modern medicine. The information gained could provide the
important and potential approach for pharmaceutical researcher to implicate the knowl-
edge of brazilin in the formulation of new drug and to reveal therapeutic and gaps
requiring future research opportunities. More studies are needed to evaluate the potential
application of brazilin as preservative and coloring agent in food processing industries.

1. Introduction isolation of various structural types of phenolic components

Caesalpinia sappan L. is a plant of Leguminosae family,
commonly known as Brazil or Sappan wood (Figure 1). C.
sappan (CS) is distributed in Southeast Asia and its dried
heartwood has been used as traditional ingredient of food or
beverages [1]. The heartwood of the plant is commonly used for
the extraction of red dye. The heartwood of CS has long been
used in Thai folk medicine to treat tuberculosis, diarrhea,
dysentery, skin infections and anemia [2]. Chemical
constituent's investigation of sappan wood resulted in the
*Corresponding author: Nilesh P. Nirmal, Center for Nutrition and Food Sci-
ences, Queensland Alliance for Agriculture and Food Innovation, University of
Queensland, Health and Food Sciences Precinct, Block 10, 39 Kessels Road, Coopers
Plains, Acherfield Brisbane, QLD 4108, Australia.
E-mail: nirmalnp21@yahoo.co.in
Peer review under responsibility of Hainan Medical University.
including xanthone, coumarin, chalcones, flavones,
homoisoflavonoids, and brazilin etc (Figure 2). Brazilin is the
major compound naturally occurring in the CS heartwood and is
used as a red dye for histological staining [3]. In traditional
Chinese medicine, brazilin is used for treatment of increased
blood circulation, promotes menstruation and exhibit analgesic
and anti-inflammatory potentials [4]. Brazilin have been
reported to possess various biological activities including
antibacterial [5], anti-inflammatory [6], anti-photoaging [7],
hypoglycemic [8,9], vasorelaxant [10,11], anti-allergic [12], anti-
acne [13,14], antioxidant [15] and nuclease activity [16].
Researchers are more interested in finding bioactive com-
pounds from natural origin as drugs, owing to either high cost of
synthetic drug or side effects of the synthetic molecules.
Therefore, there is a continuous evaluation of plant resources for

1995-7645/Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier (Singapore) Pte Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
422 Nilesh P. Nirmal et al./Asian Pacific Journal of Tropical Medicine 2015; 8(6): 421–430
Figure 1. Photograph of Caesalpinia sappan plant with insight showing
fruit, flower, wood and heartwood.
the search of bioactive molecule against non-medicated diseases.
Brazilin is one of the most important bioactive natural com-
pounds from CS heartwood having wide variety of industrial
application including textile, food, beverage and pharmaceutical
area. Since proven medicinal properties and used as dying agent,
the wood has received both domestic and international market
and being exported to USA and Europe from Southeast Asia [17].
Recently, CS heartwood has been listed in the 15th edition of the
Japanese Pharmacopoeia [18]. In this review, we focus mainly on
the different pharmacological activities of brazilin. Isolation
techniques and structure of brazilin were also discussed. The
information gained could provide the important and potential
approach for pharmaceutical researcher to implicate the
knowledge of brazilin in the formulation of new drug.
2. Caesalpinia sappan heartwood
The important part of CS is the heartwood which is pale red,
hard, heavy with even and fine structure. The heartwood is
traditionally used in Indian Ayurveda and Chinese folk medi-
cine. In Thailand, it is mostly used as coloring agent in beverage,
food, garment and cosmetics [19]. A decoction of heartwood is
used in Namya-utai solution which has anti-thirst and cardio-
tonic properties. In Northern Thailand, especially in Chiang Mai,
Nan and Lampang province, CS heartwood decoction is used as
anti-inflammatory agent for the treatment of traumatic disease
and arthritis [20]. The Northern Thai community has a long
history of using decoction of CS heartwood for local
consumption including health promotion and disease
treatment. In Ayurveda, the heartwood is used for vitiated
conditions of pitta which includes skin rashes, burning
sensations, peptic ulcers, excessive body heat, heartburn and
indigestion. It also used as blood purifier and in treatment of
wounds, diarrhea, epilepsy, diabetes etc. CS heartwood is also
used to reduce pain and swelling caused by external injuries
and improvement of complexion [21]. In Chinese folk
medicine, it is mainly used as an emmenagogue, hemostatic,
analgesic and anti-inflammatory for the traumatic disease and
blood flow promoting agent [22]. Additionally, decoction of
CS heartwood is used for the treatment of blood pressure,
burning sensations, cancer, cataract, digestion, dysmenorrhea,
ear diseases, gonorrhea, heart diseases, jaundice, nervous
disorders, obesity, ophthalmic diseases, spermatorrhoea,
stomach aches, syphilis, urinary diseases and vascular diseases.
3. Brazilin
The heartwood contains water soluble flavonoids namely,
brazilin, protosappanin and haematoxylin. Brazilin is the main
homoisoflavonoid constituent found in the CS heartwood, which
is well known as the natural red color dye for staining [3,16].
Brazilin also exhibit different industrial applications.
Therefore, extraction and purification of brazilin is one of the
important steps to achieve high extraction yield and purity,
respectively.
4. Extraction and purification of brazilin
Extraction of phenolic compounds in plant materials is
influenced by their chemical nature, the extraction method
employed, sample particle size, storage time and conditions as
well as the presence of interfering substances [23]. Therefore,
extracts of plant materials are always mixture of different
classes of phenolics that are soluble in the solvent system
used. Solubility of phenolic compounds is governed by the
type of solvent (polarity) used, degree of polymerization of
phenolics, as well as interaction of phenolics with other food
constituents and formation of insoluble complexes [24].
Therefore, there is no uniform or completely satisfactory
procedure that is suitable for extraction of all phenolics or a
specific class of phenolic substances in plant materials [25].
Methanol, ethanol, acetone, water, ethyl acetate and to a lesser
extent propanol, dimethyl formamide and their combinations
are frequently used for the extraction of phenolics. In some
cases, mechanical means to enhance molecular interaction
between the phenolic compounds and solvent are employed.
Shaking, stirring, vortex mixing and sonication are four
commonly used extraction techniques.
Traditional method for the extraction of red dye from sappan
wood used boiling of wood pieces in water. During extraction
few paddy grains are thrown into boiling solution to check the
completion of red dye extraction. If the husk scales off,
extraction is considered sufficient. In general, laboratory
extraction from CS was carried out by using mainly ethanol
(EtOH), methanol (MeOH) and water (H2O) as solvents. Dried
heartwood of sappan was extracted with MeOH for three days at
room temperature [26]. The MeOH extract was concentrated and
partitioned with H2O–MeOH (3:1, 200 mL) and EtOAc
(100 × 3). The EtOAc extract was concentrated and applied to
silica gel column, eluted with CHCl3–MeOH (15:1 to 5:1).
The brazilin containing fraction was collected and loaded on
sephadex LH-20 eluted with MeOH–H2O (9:1) Brazilin was
recrystallized from MeOH–H2O [26]. CS heartwood powder
(700 g) was extracted with methanol (2 L × 3, 1 h each) at
75  C and extract was further concentrated to yield 60 g
orange yellowish residue. The methanolic extract was
dissolved in water and partitioned with hexane and ether. The
ether fraction was further separated on silica gel column with
CHCl3–Me2CO gradient system. Brazilin was purified to
homogeneity with the help of preparative TLC [11]. Xu and
Lee [5] extracted sappan heartwood powder with methanol by
using reflux method. The methanol extract was dissolved in
 Nilesh P. Nirmal et al./Asian Pacific Journal of Tropical Medicine 2015; 8(6): 421–430
water and partitioned with hexane, ether and butanol. The ether
fraction showed highest antibacterial activity was further
subjected to silica gel chromatography and active compound
was eluted with chloroform containing 10% methanol. The
active compound was purified to homogeneity by preparative
thin layer chromatography using chloroform: methanol
(8.5:1.5) solvent system and identified as brazilin by 1H NMR
spectra [5].
Sappan wood was extracted with 95% ethanol for 2 h and
extraction was repeated for three times [27]. The ethanol extract
was dissolved in water and partitioned with petroleum ether,
ethyl acetate and n-butanol. Brazilin was eluted with repeated
silica gel (CHCl3–MeOH 10:1–1:1, v/v) and sphadex LH-20
(MeOH) chromatography from ethyl acetate fraction. Batubara
et al. macerated CS heartwood (500 g) powder in 5 L of MeOH
for 12 h [14]. This process was repeated twice and extract was
concentrated by using rotary evaporator. Crude extract (10 g)
was separated on silica gel column and eluted with hexane,
ethyl acetate and methanol. Brazilin was purified from ethyl
acetate fraction by preparative HPLC using gradient solvent
system from 5% to 100% methanol in 0.05% trifluoroacetic
acid at flow rate of 10 mL/min for 45 min. Nirmal and
Panichayupakarant successively extracted CS powder (500 g)
with 95% ethanol (3L × 3), under reflux conditions for 1 h, to
obtain a dark brown extract (58.5 g) [13]. Further, CS extract
was subjected to isolation of the anti Propionibacterium acnes
(P. acnes) compound using antibacterial assay guided
isolation. The CS extract (10 g) was fractionation on a silica
gel column (7 cm × 24 cm) (1 g extract per 50 g silica gel)
and eluted with a mixture of hexane and ethyl acetate (6:4, v/
v) to give 13 pooled fractions (Fraction 1–13). The fractions
were then subjected to antibacterial assay. The antibacterial
fractions (Fractions 5 and 6) were pooled together and further
purified using a Sephadex LH-20 column (3 cm × 80 cm) us-
ing methanol as eluent to give seven pooled fractions (fractions
A-G). Fraction E with antibacterial activity was re-
chromatographed on a silica gel column (3 cm × 25 cm) and
eluted with a mixture of hexane and ethyl acetate (7:3, v/v) to
obtain the red crystals of brazilin (640 mg), which was identified
by NMR analysis. In general, ethanol and methanol solvent was
extensively used for extraction of CS heartwood powder. The
extract was later on partitioned with different solvents to accu-
mulate the desire compound. Brazilin was generally partitioned
with ether or ethyl acetate.
5. Chemical structure of brazilin
Brazilin [(6a S-cis)-7, 11b-dihydrobenz[b]indeno[1,2-d]py-
ran-3,6a,9,10(6H)-tetrol] (Figure 2) was isolated as red crystal
[13] or amber yellow crystal [14]. Brazilin exhibit a melting point
of 145  C–149  C. The EI mass spectrum of brazilin showed a
molecular weight at m/z 286 corresponding to C16H14O5. The
optical specific rotation value of brazilin was [a]20 D + 118.8
(c = 1.9, MeOH). The UV–visible spectrum showed maximum
absorption at 292 nm. The 1H NMR (500 MHz, CD3OD) and
13
C NMR (150 MHz, CD3OD) spectra of brazilin were shown
in Table 1 [13]. A signal at 144.3 ppm in 13C spectrum,
assigned to C9 atom bonded to a hydroxyl group. The proton
spectrum of brazilin with a group of aromatic signals at the
range between 7.15 and 6.50 ppm, and that of carbinolic
protons at 4.12 and 4.39 ppm [28]. Brazilin shows electronic
423
transition in blue shifted region due to the sp3 carbon atom at
C9 which does not have planarity in this part of the molecule.
Vibrational wave numbers of brazilin together with tentative
assignment is shown in Table 2 [28]. Brazilin does not show
vibrational band at 1 365 cm−1 because of the absence of the
C]O bond.
6. Pharmacological activities of brazilin
CS heartwood extracts have been known for various bio-
logical activities and being tested for unscreened disease.
Pharmacological activities of CS extracts reflect the combination
effects of different phenolic contents. Here, we have discussed
the important biological activities of brazilin (Table 3), which is
the major phenolic compound in CS extract.
6.1. Antioxidant activity
The antioxidant activity of compound have been attributed to
various mechanism including prevention of chain initiation,
binding of transition metal ion catalyst, decomposition of per-
oxides, scavenging of free radicals and prevention of hydrogen
abstraction [29]. The structure of phenolic compounds is a key
determinant of their radical scavenging and metal chelating
activity, which is referred as Structure-Activity Relationships
(SAR) [30]. The chemical activity of phenols in terms of their
reducing properties as hydrogen or electron donating agents
predicts their potential for action as antioxidants [31].
Reductones are also reported to react with certain precursor of
peroxide, thus preventing peroxide formation.
Brazilin showed highest DPPH radical scavenging activity
(IC50 = 57.2 mM) and ferric reduction activity as compared to
standard vitamin E and other compound isolate from sappan
lignum (Tokyo, Japan) including brazilein, sappanchalcone,
protosappanin B and C [15]. The strong reducing power of
brazilin was owing to the presence of an ortho-substituted
diphenol group. Brazilin also showed dose dependent inhibi-
tion of peroxide formation in linoleic acid emulsion during in-
cubation at 50  C for 250 h. Brazilin, isolated from CS
heartwood from Indonesia (IC50 = 8.8 mM) showed near about
similar DPPH radical scavenging activity as compared to stan-
dard (+) catechin (IC50 = 10.2 mM) [14]. Brazilin showed IC50
value of 28.8 mg as determined by ABTS radical scavenging
activity [19]. Dibenzoxocin structure of brazilin was effective
for radical scavenging activity [15].
6.2. Antibacterial activity
A bacterial infection to the living organism is the major
concern of research from decades. Bacteria can be found
everywhere including soil, water and air. Most of the bacteria in
nature are pathogenic and can create serious threat to human
being. Antibiotics are available commercially to combat with
pathogenic bacteria. However, the emergence of bacterial path-
ogens those are resistant to major classes of antibiotic has
created a need of novel antibacterial agents. CS extract have
been tested against different microorganism for its potential
antimicrobial activity. The maximum zone of inhibition was
observed in ethanolic extract against Pseudomonas aeruginosa
(34 mm) followed by Staphylococcus aureus (31 mm), Salmo-
nella typhi (24 mm), Enterobacter aerogens (21 mm), Candida
424 Nilesh P. Nirmal et al./Asian Pacific Journal of Tropical Medicine 2015; 8(6): 421–430

Figure 2. Chemical structure of different compounds isolated from CS heartwood.

 Nilesh P. Nirmal et al./Asian Pacific Journal of Tropical Medicine 2015; 8(6): 421–430 425

Table 1 inhibition of P. acnes growth, lipase inhibitory activity, and

1
H (500 MHz) and 13
C NMR (150 MHz) data of brazilin in CD3OD13. antioxidant activity. In order to isolate the active compound from
CS separation of the extract components was performed by col-
Position Brazilin
umn chromatography and preparative high-performance liquid
dC type dH (J in Hz) chromatography. Brazilin, protosappanin A and sappanone B
1 132.2, CH 7.18, d (8.2) have been isolated from methanolic extracts. Brazilin showed
1a 115.5, C better antibacterial activity [minimum inhibitory concentration
2 109.9, CH 6.46, dd (8.2, 2.5)
(MIC) = minimum bactericidal concentration (MBC) = 0.50 mg/
3 155.7, C
4 104.2, CH 6.28, d (2.5) mL] than protosappanin A (MIC = MBC = 1.00 mg/mL) and
4a 157.8, C sappanone B (MIC = MBC > 2.00 mg/mL). The 50% inhibitory
6 70.8, CH2 3.68, d (11.5) concentration (IC50) for lipase inhibition was lowest for brazilin
3.91, d (11.5) (6 mM), which showed strong inhibition compared with proto-
6a 78.0, C
sappanin A (100 mM) and chloramphenicol (677 mM, positive
7 42.9, CH2 2.76, d (15.8)
3.01, d (15.4) control). The antioxidant activity of brazilin (IC50 8.8 mM) was not
7a 131.3, C significantly different from protosappanin A (9.1 mM) and
8 112.8, CH 6.70, s (+)-catechin (10.2 mM). The antioxidant activity of brazilin and
9 145.6, C protosappanin A were higher than sappanone B (IC50 14.5 mM).
10 145.3, C
11 112.4, CH 6.58, s
Brazilin is considered to have sufficiently potent activity for use as
11a 137.4, C an antiacne agent [34].
12 51.1, CH 3.95, s
6.4. Anti-inflammatory activity
albicans (20 mm), Escherichia coli (15 mm) and minimum zone
of inhibition was observed against Aspergillus niger (14 mm) Inflammation is a protective pathophysiological response to
[32]. Methanol extract of CS heartwood extract showed higher tissue injury caused by physical trauma, hazardous chemicals or
inhibitory activity against methicillin sensitive Staphylococcus microbial agents characterized by heat, redness, pain, swelling and
aureus (MSSA) as well as methicillin resistant Staphylococcus disturbed functions. Inflammation is a complex process and many
aureus (MRSA) (14 mm) than n-butanol, chloroform and different mediators are involved including enzymes (cyclo-
aqueous extracts (8 mm) [33]. Flavonoids in the methanolic oxygenase, nitric oxide synthase, and protease) and their inter-
extracts of CS could be related with the antibacterial activity mediate products and end products. Cyclooxygenase enzymes
against MRSA. (COX-1/COX-2) convert arachidonic acid to the unstable prosta-
Batubara et al. reported that methanol and 50% ethanol glandin intermediates PGG2 and PGH2, which involve in inflam-
extract of CS heartwood showed lower MIC/MBC value against mation [35]. Nitric oxide (NO) a toxic gas has been related with
P. acnes in comparison with other 39 medicinal plants from various diseases such as atherosclerosis, inflammation and
Indonesia [13]. In further study of isolation and identification of carcinogenesis. NO can be formed abnormally by an activation
active compound from CS heartwood against P. acnes, Batubara of bacterial lipo poly saccharide (LPS) in some cell types such as
et al. found that brazilin (MIC/MBC = 0.5 mg/mL) showed macrophages [36]. NO is synthesized by an enzyme nitric oxide
better antibacterial activity than protosappanin A and synthases (NOS), which has 3 different isoforms including
sappanone B [14]. Recently, the active antibacterial principle in endothelial (eNOS), neuronal (nNOS), and inducible (iNOS).
CS heartwood was isolated and identified to be brazilin (MIC/ Among them, iNOS is the main reason for the NO production at
MBC = 15.6/31.3 mg/mL) by using anti-P. acnes guided an inflammatory site [3]. Nuclear factor kappa B (NF-kB) is the
purification [13]. The variation in MIC value of brazilin against most important transcription factor in inflammatory diseases,
P. acnes could be related with bacterial strain and source of activated by LPS. When NF-kB is activated, it induces the
brazilin. Brazilin showed potent activity against antibiotic expression of iNOS as well as synthesis of many pro-
resistant bacteria including MRSA, vancomycin-resistant inflammatory cytokines, such as TNF-a, IL-6, IL-1b and IL-8
enterococci (VRE) and multi-drug resistant Burkholderia [37]. Protein denaturation is one of the causes of inflammations
cepacia (Table 4) [5]. Brazilin showed bactericidal activity where production of auto-antigens occurs in certain inflammatory
against MRSA by inhibiting DNA and protein synthesis. diseases like type Ⅲ hypersensitive reaction [38].
Brazilin also showed strong inhibitory activity against bacteria The inflammatory response must be terminated when no
responsible for dental caries (Streptococcus mutans), longer needed to prevent unnecessary damage to tissues and
periodontal disease (Prevotella intermedia) and bacteria chronic inflammation. Most of the researcher studied anti-in-
causing strep throat (Group A strep). However, brazilin found flammatory activity of brazilin by targeting inhibition of NO
to be less effective against gram-negative bacteria such as production and iNOS suppression. Brazilin showed significantly
Pseudomonas aeruginosa, Klebseilla pneumonia, Escherichia 100% inhibition of lipo poly saccharide (LPS)-induced NO
coli and Enterobacter aerogenes [5]. This compound is active production by J774.1 cell line at 30–100 mM and murine peri-
against a number of medically important bacteria and it has toneal macrophages at 10 mM as compared to standard L-
the potential to be developed into an antibiotic. NMMA (a nitric oxide synthase inhibitor, 44.3% inhibition at
100 mM) and other compound isolate from sappan lignum
6.3. Antiacne activity (Tokyo, Japan) including protosappanin A, B and C [15].
Brazilin completely suppress inducible nitric oxide synthase
In screening experiments for antiacne activity, methanolic and (iNOS) gene expression at 100 mM. Hu et al. reported that
50% ethanolic extracts of CS wood showed the most potent ac- brazilin (10–300 mM) induces hemeoxygenase-1 (HO-1)
tivity out of 28 species of plants extracts. These extracts showed expression and increased bilirubin production in RAW264.7
426 Nilesh P. Nirmal et al./Asian Pacific Journal of Tropical Medicine 2015; 8(6): 421–430

Table 2 macrophage in a dose dependent manner [39]. Brazilin (50–

Raman wave number values (in cm−1, excited at 1 064 nm) and band 300 mM) also showed dose dependent inhibition of NO,
assignment of brazilin. prostaglandin E2 (PGE2), tumor necrosis factor-a (TNF- a),
interleukin (IL)-1b production and iNOS expression in LPS-
Brazilin Tentative assignment
stimulated macrophages. The results suggested brazilin exhibit
252 vw Ring deformation anti-inflammatory activity in macrophages through the action of
423 vw d(ring) + d(CO)
HO-1 induction. In another anti-inflammatory study of brazilin
473 vw d(ring)
490 vw d(ring) in J774.1 cell line Washiyama et al. reported that brazilin
501 vw d(ring) (IC50 = 3.7 mM) inhibit NO production and suppressed iNOS
549 vw d(ring) expression, however there was almost no inhibition in PGE2
642 vw g(CH) (IC50 > 100 mM) [6]. Brazilin (10–40 mM) suppress the iNOS
687 vw g(CH) + d(CC]O) + d(CC-O)
732 mw g(CO) + g(CH) gene expression in dose dependent manner by regulating the
767 w g(CO) + g(CH) LPS-induced transcription factors including nuclear factor-kB
792 vw g(CO) + g(CH) (NF- kB) and activator protein-1 (AP-1) in RAW264.7 macro-
945 vw r(CH2) phage [3]. IC50 value of 24.3 mM was observed for inhibition of
990 vw v(C–C) + v(C–O) NO production in RAW264.7 macrophage. Brazilin contribute
1032 vw Ring stretching
1172 vw d(CCH) + v(C–C) to the dose dependent anti-inflammatory activity of ethanolic
1230 sh v(C–O) + v(C–C) CS extract in human macrophages (THP-1) and osteoarthritis
1260 sh v(C–O) + v(C–C) + d(CH2) (SW1353) chondrocytes [40].
1320 m v(C–O) + d(OCC) + d(CH2)
1451 w v(C]C) + d(ring) + d(COH)
1525 w v(C]C)
6.5. Hypoglycemic activity
1614 s v(C]C)
2858 w v(CH) Type Ⅱ diabetes is characterized by an increase in hepatic
2900 sh v(CH2) glucose production and insulin resistance. Increased gluconeo-
2940 m v(CH2)
genesis is the main cause of increased hepatic glucose in diabetic
3060 mw v(CH) aromatic

Table 3
Different pharmacological activities of brazilin.
Pharmacological activities Methods/Model system References
Antioxidant DPPH radical scavenging assay Sasaki et al.; [15] Batubara et al. [14]
Ferric reduction assay Sasaki et al. [15]
Linoleic acid peroxidation assay Sasaki et al. [15]
Antibacterial Zone of inhibition Kim et al.; [33] Srinivasan et al. [32]
MIC/MBC Xu and Lee; [5] Batubara et al.; [14] Nirmal & Panichayupakaranant [13]
Anti-inflammatory LPS-induced NO production Sasaki et al.; [15] Hu et al.; [39] Washiyama et al.; [6] Wu et al. [40]
Inhibition of iNOS gene expression Sasaki et al.; [15] Hu et al.; [39] Washiyama et al.; [6] Bae et al. [3]
Hemeoxygenase-1 expression Hu et al.; [39] Choi et al. [50]
Bilirubin production Hu et al. [39]
Prostaglandin E2 production Hu et al.; [39] Wang et al. [22]
Tumor necrosis factor-a production Hu et al.; [39] Wu et al.; [40] Wang et al. [22]
Interlukin (IL)-1b production Hu et al.; [39] Wu et al.; [40] Wang et al. [22]
Nucler factor (NF- kB) expression Bae et al.; [3] Wang et al.; [22] Toegel et al.; [1] Lee et al. [7]
Activator protein-1 expression Bae et al. [3]
COX-2 inhibition Wu et al.; [40] Wang et al. [22]
Hypoglycemic activity Modulation of insulin receptor Lee et al.; [41] Kim et al. [26]
Blood glucose You et al. [8]
Protein Kinase C Kim et al. [26]
Glucose transport Khil et al. [42]
Phosphotidylinositol 3-kinase Khil et al. [9]
Pyruvate kinase You et al. [8]
6-phosphofructo-2 kinase You et al. [8]
Vasorelaxant NO and cGMP production Xie et al.; [11] Hu et al. [10]
Activation of Ca2+/calmodulin Hu et al. [10]
NO synthase Hu et al. [10]
Hepatoprotective Inhibition of CCl4 intoxication Moon et al.; [47] Srilakshami et al. [21]
Miscellaneous Luciferase activity Choi et al. [50]
Succinic semialdehyde reductase Baek et al. [49]
activity (anticonvulsant)
Tyrosinase activity Chun et al. [48]
Melan-a cells proliferation Chun et al. [48]
Glioma U87 cell proliferation Lee et al. [51]
Lipase inhibition Batubara et al. [14]
Phospholipase A2 inhibition Hwang et al. [52]
(anti-platelet aggregation)
Nuclease activity Yun et al. [16]
Matrix metalloproteinase inhibition Toegel et al.; [1] Lee et al. [7]
 Nilesh P. Nirmal et al./Asian Pacific Journal of Tropical Medicine 2015; 8(6): 421–430 427

Table 4 brazilin treatment. In the investigation of hypoglycemic

Antibacterial activity of brazilin against various bacteria. mechanism of brazilin on gluconeogensis intermediate
production and gluconeogenic enzyme activity in rat
Bacteria MIC References
(mg/ml)
hepatocytes, You et al. found that brazilin (1–100 mM)
increased the production of F-2,6-BP in hepatocytes, dose
Actinomyces naeslundii >256 Xu and Lee [5]
dependently by elevating intracellular level of fructose-6-
ATCC 12104
Burkholderia cepacia 64 phosphate (F-6-P) and hexose-6- phosphate (H-6-P) [8]. Brazilin
92.443 also significantly increased 6-phosphofructo-2-kinase (PEK-2)
Burkholderia cepacia 32 and pyruvate kinase activity in glucagon treated hepatocytes.
91.452 Increased pyruvate kinase activity also may play a role in the anti-
Enterobacter aerogenes >256
Escherichia coli 256
gluconeogenic action of brazilin.
ATCC 25922
Klebsiella pneumonia >256 6.6. Vasorelaxation activity
ATCC 13883
MRSA 9247922 16
NO serves as a major relaxing factor in the circulatory sys-
MRSA 595445 16
Prevotella intermedia 8 tem. NO is produce from the oxidation of guanidine nitrogen of
2+
ATCC 25611 L-arginine via NOS in Ca dependent or independent manner
Pseudomonas aeruginosa >256 [43]. Medicinal plants have been shown to relax blood vessels via
ATCC 27853 endogenous NO production. CS heartwood extract has been
Salmonella typhimurium 64
used traditionally in folk medicine to promote blood
ATCC 13311
Staphylococcus aureus 16 circulation and as an emmenagogue. Methanolic extract of CS
ATCC 6538 heartwood significantly relaxed the a1-receptor agonist
Staphylococcus epidermidis 8 phenylephrine-precontracted aortic rings, without affecting
ATCC 11490 passive tension of these vessels [11]. Isolation and identification
Staphylococcus 64
epidermidis# 20
of compounds from the methanol extract suggested that brazilin
Streptococcus agalactiae 8 and hematoxylin may be responsible for the vascular relaxant
(Gr. B Strep) A909 effects via endogenous NO and subsequent c GMP formation.
S. mutans NG8 16 The vasorelaxant activity of brazilin was investigated in
Streptococcus pyogenes 4 isolated rat aorta and human umbilical vein endothelial cells
(Gr. A Strep) M1
[10]. Brazilin and CS crude extract relaxed phenylephirine
Streptococcus pyogenes M22 4
Vancomycin resistant 32 induced vasoconstriction and increased cGMP in isolated rat
enterococci# 22 aorta, which was endothelium dependent. Brazilin dose
VRE# 228 16 dependently induced an increase in NO formation, NOS
Propionibacterium acnes 500 Batubara et al. [14]
activity and the influx of extracellular Ca2+ in human
ATCC 6919
Propionibacterium acnes 15.6 Nirmal & umbilical vein endothelial cells. The results suggested that
DMST 14916 Panichayupakarnant [13] brazilin induces vasorelaxation by activating Ca2+/calmodulin
dependent NO synthesis, which intern influenced by increasing
individuals. Fructose-2,6-bisphosphate (F-2,6BP) a gluconeo- intracellular Ca2+ concentration in endothelial blood vessels [10].
genic intermediate, plays a critical role in hepatic glucose output
by regulating gluconeogenisis and glycolysis in the liver [8]. There 6.7. Hepatoprotective activity
are specific rate limiting enzymes in the gluconeogeniss and
glycolytic pathways, which regulate the blood glucose levels eg. Liver injuries induced by CCl4 are the best characterized
pyruvate kinase, protein kinase, protein phosphatase and insulin system of xenobiotic induced hepatotoxicity and commonly
receptor serine kinase [26]. The water extract of CS wood was used for hepatoprotective activities of drugs. The changes
traditionally used for the treatment of diabetic complications in associated with CCl4 induced liver damage are similar to that of
South East Asia. Brazilin reported to enhance glucose acute viral hepatitis. Hepatic injuries are mostly free radical
metabolism in adipose tissue from diabetic KK mice [41] and mediated, therefore medicinal plant extract having potent anti-
decreases blood glucose in diabetic animals [8]. Hypoglycemic oxidant activity, may show potent hepatoprotective activity.
activity of brazilin was found to be based on the stimulation of Plant extract including Phyllanthus nirui Linn. [44], Calendula
peripheral glucose utilization through modulation of insulin officinalis L. flower extract [45] and Daucus carota L [46] have
receptor function [26]. Brazilin inhibit protein kinase C and been shown to protect hepatic injuries induced by CCl4 in
insulin receptors serine kinase, which are involved in the rats. The methanolic and aqueous extract of CS heartwood
regulation of insulin signal pathway [41]. Brazilin induced restored the biochemical levels to normal which were altered
glucose transport in isolated rat epididymal adipocytes [42]. In due to CCl4 intoxication in freshly isolated rat hepatocytes and
their further study of determination of mechanism of brazilin in also in animals [21]. Brazilin from CS heartwood protects
glucose transport from isolated rat epididymal adipocytes, Khil cultured rat hepatocytes from BrCCl3 induced toxicity [47].
et al. reported that brazilin (50 mM) caused an increase in
glucose transport by recruitment of glucose transporter 6.8. Miscellaneous
(GLUT4) from intracellular pools to the plasma membrane via
the activation of phosphotidylinositol 3-kinase (PI3-kinase), not Brazilin and CS heartwood extract also been tested for
by affecting protein synthesis, including GLUT4 synthesis [9]. different biological activities. Brazilin and butanol extract of
Moreover, protein kinase C activity was not influenced by sappan heartwood treated with Melan-a cells showed dose
428 Nilesh P. Nirmal et al./Asian Pacific Journal of Tropical Medicine 2015; 8(6): 421–430
dependent inhibition in their proliferation without apoptotic cell
death might be due to the cell arrest [48]. The decrease in melanin
content and tyrosinase activity was observed in brazilin and CS
butanol extract treated cells with respect to the increasing
concentration. Anticonvulsant activity of brazilin was
determined by using inhibitory activity against two GABA
degradative enzymes namely, succinic semialdehyde
dehydrogenase (SSADH) and succinic semialdehyde reductase
(SSAR). Brazilin inactivated the SSAR activity in a dose
dependent manner, whereas no activity was found against
SSADH [49]. Brazilin induced antioxidant response element,
luciferase activity and heme oxygenase-1 (HO-1) expression in
a dose dependent manner in House Ear Institute-organ of corti 1
(HEI-OC1) cells [50]. Brazilin was found to be more potent than
crude extract and the expression of HO-1 reached a peak at 12 h
after brazilin treatment. The enzyme diminishes the tert-
butylhydroperoxide induced cell death in HEI-OC1 cells. Lee
et al. investigated the effect of brazilin (0–40 mM) on cell pro-
liferation and apoptosis in the glioma U87 cell line [51]. Brazilin
isolated from the methanol extract of the heart wood of CS was
able to inhibit growth of glioma U87 cell by inducing apoptosis
and the induction of apoptosis was associated with the caspase-
related apoptotic pathway. Brazilin showed dose dependent in-
hibition of cell proliferation and induction of apoptosis. There-
fore, effects of brazilin on glioblastoma growth could provide
the potential clinical usefulness of this agent in the treatment of
brain tumors.
Brazilin (IC50 = 6 mM) isolated from CS heartwood from
Indonesia showed higher inhibitory activity against lipase
enzyme as compared to standard (+) catechin (IC50 > 1 750 mM)
and sappanone B (IC50 > 1 650 mM) and protosappanin A
(IC50 = 100 mM) [14]. Moreover, brazilin has been observed to
inhibit platelet aggregation, probably by reducing the activity
of phospholipase A2 and the elevation of intracellular free
calcium concentration [52]. Brazilin form a complex with Cu
(Ⅱ) in the presence as well as the absence of DNA. The Cu
(Ⅱ)-brazilin complex exhibited the nuclease activity for the
pBR322 supercoiled DNA, converting supercoiled forms to
nicked form [16]. As the concentration of both brazilin and Cu
(Ⅱ) increased from 50 to 150 mM, the activity increased.
Ethanolic extract of CS heartwood significantly improved
collagen induced arthritis in rats by decreasing the levels of IL-
1b, IL-6, TNF-a and PGE2 in serum and the expression of COX-
2 and transcription factor NF-kB in paw cartilage [22]. In the
study of MMP inhibitory activity of an ethanolic CS extract in
human osteoarthritis (OA) chondrocytes, Toegel et al. reported
that CS significantly suppressed IL 1b-mediated up regulation
MMP-13 mRNA and protein levels via interruption of NF-kB
(p65/p50) driven MMP-13 promoter activation [1]. Brazilin (5–
100 mM) inhibits ultraviolet B induced matrix
metalloproteinase (MMP-1/3) expression and secretion in a
dose dependent manner by suppressing NF-kB activation in
human dermal fibroblasts [7]. Brazilin also block UVB induced
reactive oxygen species generation in fibroblasts.
7. Toxicology
CS heartwood extracts have been known as safe natural plant
extract and long been used as food and medicinal ingredient.
The aqueous extract of CS heartwood (5 000 mg/kg, single oral
dose) showed no acute toxicity in terms of general behavior
change, mortality or change in gross appearance of internal
organs. Similarly, subacute toxicity was carried out by the daily
oral administration of the CS extract at the doses of 250, 500 and
1 000 mg/kg body weight for 30 days in rats [2]. The results
showed no abnormalities in all treatments as compared to
control including body and organs weight, haematological,
biochemical and histopathological parameters. CS heartwood
extract did not produce any acute or subacute toxicity in both
male and female rats. Nirmal and Prasad reported that brazilin
and brazilin rich extract was found to be non-toxic against
fibroblast cell up to the concentration of 500 mg/mL, respectively
during 24 h of incubation [53]. Hu et al. reported that brazilin did
not cause cytotoxicity bellow 300 mM in RAW264.7 murine
macrophages during 18 h of incubation [40]. Brazilin did not
alter cell viability of human dermal fibroblast up to 100 mM
during 24 h incubation [7].
8. Future directions
Brazilin and CS heartwood extract could be used in food
preservation and preparation of functional food. Chromato-
graphic technique should be optimizing for the higher purifica-
tion yield of brazilin. As brazilin is the major compound in CS,
there is another possibility to prepare brazilin rich extract by
using simple chromatographic technique, which could be
convenient and low cost. More study of brazilin is needed for the
application in food preservative or coloring agent.
9. Conclusions
CS heartwood extract has been used in oriental folk medicine
in Southeast Asia. CS heartwood is also known for its coloring
properties. Ethanol (95%) is the better solvent to obtained high
extraction yield of CS heartwood. CS extract did not produce
any acute or subacute toxicity in both male and female rats.
Brazilin is the major phytochemical found in the heartwood and
is responsible for most of the pharmacological activities of CS
heartwood. Brazilin shows various biological activities
including antioxidant, antibacterial, anti-inflammatory, hypo-
glycemic, hepatoprotective, and vasorelaxation etc. Brazilin has
the potential to develop into a drug and also act as a
nutraceutical.
Conflict of interest statement
We declare that we have no conflicts of interest.
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Article
A Thermal Management Strategy for
Electronic Devices Based on Moisture
Sorption-Desorption Processes
Chenxi Wang, Lingji Hua,
Hongzhi Yan, Bangjun Li,
Yaodong Tu, Ruzhu Wang
rzwang@sjtu.edu.cn

HIGHLIGHTS
A strategy of passive thermal
management based on moisture
sorption-desorption

Proof-of-concept demonstration
of this strategy using MIL-101(Cr)
coating

Higher equivalent enthalpy when

compared with traditional PCMs

A simplified model to guide the

material selection and system
design

The application of traditional phase change materials (PCMs) in electronic thermal

management is limited by their enthalpies. In this work, we adopt sorbents and
their desorption processes to achieve efficient thermal management. The sorbents
with high cyclic water loadings, such as MIL-101(Cr), significantly outperforms the
traditional PCMs, by virtue of their high desorption heat during the temperature-
induced desorption process.

Wang et al., Joule 4, 435–447

February 19, 2020 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.joule.2019.12.005
Article
A Thermal Management Strategy
for Electronic Devices Based on Moisture
Sorption-Desorption Processes
Chenxi Wang,1 Lingji Hua,1 Hongzhi Yan,1 Bangjun Li,1 Yaodong Tu,1 and Ruzhu Wang1,2,*

SUMMARY Context & Scale

Currently, the thermal management of electronics has been drawing growing The efficient thermal
attention. The use of latent heat emerges as an attractive means for intermittent management of electronic
heat dissipation. However, traditional phase change materials (PCMs) are devices is essential, considering
limited by their relatively low enthalpies. In this paper, we demonstrate a pas- overheating is harmful to the
sive thermal management strategy through the desorption process of water in- efficiency and reliability of
side sorbents. The spontaneous adsorption enables self-recovery of working ca- electronic components. In this
pacity at rest mode. The basic guideline for selecting proper sorbents is work, we introduce a new strategy
outlined. In the proof-of-concept test, we coated MIL-101(Cr) powders onto of transient thermal management,
metallic substrates. The coating of 0.288 g achieved a temperature drop using solid sorbents, which can
(maximum 8.6
C) for 25 min at a heating power of 1.5 W. The equivalent absorb moisture from the
enthalpy of the coating can be up to 1,950 J/gcoating, indicating a significant atmosphere and subsequently
improvement compared to that of traditional PCMs. Our results suggest that release moisture at a higher
this strategy could find promising applications in a variety of practical scenes. temperature. During the releasing
process, a huge amount of heat is
extracted to prevent the device
INTRODUCTION
from overheating. The cyclic water
The working performance of electronic devices has continued to grow in recent years, loading is the key criterion in
accompanied by increasing integration levels of components. The poor heat dissipation screening candidate materials.
results in ongoing heat accumulation and elevated temperatures, which is deleterious to Experimentally we demonstrate
the efficiency and reliability of electronic devices.1 Hence, an effective thermal manage- the outstanding performance of
ment method is vital to electronics.2–4 Active cooling strategies achieve good perfor- this strategy. The MIL-101(Cr)
mance at the expense of noise, bulky volume, and extra energy consumption, which coating significantly outperforms
limit their applications in the scenes with strict requirements on space and weight.5,6 traditional phase change
Considering most smart electronics have varying workloads with time rather than being materials. The sorbent coating
operated continuously over long periods, the passive thermal regulation of using latent can also be readily integrated with
heat with phase transition is highly attractive. In this field, extensive efforts have been existing heat dissipation
devoted to solid-liquid phase change materials (PCMs), such as paraffin waxes, salt structures. This work paves a new
hydrates, and fatty acids,7 which absorb latent heat during melting. Even though the pathway for transient thermal
effectiveness of PCMs in electronic thermal management where heat generation is inter- regulation. The challenge to
mittent has been proved by numerous studies,8,9 the wide utilization of PCMs is still large-scale deployment is to find
limited by their comparatively low phase change enthalpies (commonly in the range cost-effective sorbents with
of 50–200 J/gPCM). outstanding sorption and
thermophysical properties.
In nature, mammals can adjust their body temperature by sweating. Inspired by this
biological cooling process, the liquid-vapor phase transition of water is considered
to be a promising candidate for thermal management due to its high latent heat.
However, previous studies based on this principle rely on the active replenishment
of water inside the storage matrixes (e.g., hydrogels).10,11 By contrast, the adsorp-
tion and desorption of water in sorbents12 provide a promising solution to address

Joule 4, 435–447, February 19, 2020 ª 2019 Elsevier Inc. 435

this issue. Specifically, the sorbents are first saturated with water vapor from the sur-
rounding atmosphere.13 Then the temperature swing of electronic devices during
peak load is expected to trigger the release of water from saturated sorbents,
accompanied with the extraction of a large amount of heat. As a result, the target
devices are prevented from overheating. More importantly, the spontaneous
adsorption enables passive cyclic working capacity. Thus, a desorption-based cool-
ing strategy holds great potential for the thermal management of electronics whose
heat generation is intermittent and fluctuant.

From this respect, we herein demonstrate an effective thermal management strategy

based on the desorption of sorbents. The working principle of this strategy and the opti-
mized selection of materials are discussed in this paper. As a proof-of-concept, we
chose MIL-101(Cr) as the sorbent and fabricated a coating layer on a metallic sheet.
The working performance of this coating layer was proved in the subsequent experi-
ments. Arising from the rapid desorption of the sorbent coating, an obvious delay of
the temperature rise of the simulative heater can be observed, which significantly out-
performs traditional PCMs with similar mass. The influence of coating mass, heating po-
wer, and convection condition was discussed in detail. A simplified numerical model was
also developed to compare the working capacity of different sorbents under various
climate conditions, which may guide further improvement.

RESULTS
Working Principle of Thermal Management Using Desorption Process
Figure 1A depicts a schematic working cycle of the sorbent coating layer for electronic
thermal management. The cycle is composed of adsorption and desorption steps.
When the sorbent is in the equilibrium state, its water content (Wd, in gwater/gsorent)
can be determined from the sorption curves in Figure 1B. Herein, the abscissa of
Figure 1B is relative humidity (RH) P/P0, where P is the partial vapor pressure of ambient
air, and P0 is the saturated vapor pressure determined by the sorbent temperature.
Once a temperature rise (Ti to Tii) is encountered, the equilibrium point is expected
to migrate from RHi to RHii due to the increase of the saturated vapor pressure P0 at
an elevated temperature. Hence, the present water content (Wid ) is higher than the
balanced value (Wiid ), then a thermally driven desorption occurs.

With regard to the heat flux, the major heat dissipation fluxes for a common elec-
tronic device include surface radiation and convection. The imbalance between
the generated and dissipated heat results in a rapid increase of the device temper-
ature. An extra heat flux can be realized when the desorption is introduced, as shown
in Figure 1C. Partial heat is extracted due to the desorption process and transferred
into the environment through vapor diffusion. Hence, the ascending trend of tem-
perature can be suppressed. The heat extracted from the device is proportional to
the total amount of the desorbed water, given as:


Qde = md hde Wid Wiid = md hde DWd ; (Equation 1)

where md is the sorbent mass (g), hde is the average desorption enthalpy over the
loading range (J/gwater, heat of desorption when unit gram of water is desorpted),
Wid ,Wiid are the initial and terminal water content (gwater/gsorbent) respectively, and
DWd is the consequent cyclic water uptake (gwater/gsorbent). 1Institute
of Refrigeration and Cryogenics,
Shanghai Jiao Tong University, 800 Dong Chuan
Road, Shanghai 200240, China
In general, the average desorption enthalpy of physisorption is close to the evapo- 2Lead Contact
ration enthalpy of bulk water.14 Therefore, for a certain amount of sorbents, DWd is *Correspondence: rzwang@sjtu.edu.cn
the key metric to the effectiveness of thermal management. https://doi.org/10.1016/j.joule.2019.12.005

436 Joule 4, 435–447, February 19, 2020

Figure 1. Working Principle of the Proposed Thermal Management Strategy
(A) The strategy is composed of the adsorption and desorption cycles. During the adsorption cycle,
the adsorbents capture moisture from surrounding atmosphere. With increasing temperature due
to heavier workloads of the device, the desorption cycle begins.
(B) Schematic sorption curves of two sorbent materials. For a given temperature range, the cyclic
water uptake, DW d, can be determined from the sorption curves. And this parameter is the key
metric to the thermal management capacity.
(C) Energy balance and heat transfer diagram for an electronic device with the desorption process.

Selection of Sorbent Materials

The selection of proper sorbents plays a vital role in determining the practical per-
formance of the proposed thermal management strategy. A rational approach for
selecting sorbents is presented in this section. As discussed above, the cyclic water
uptake is a basic criterion in screening sorbents for electronic cooling. Hence, it
seems that the key target is to realize high water uptakes at various conditions. How-
ever, given the limitation of existing materials, a more feasible solution is adopting
the sorbents whose adsorption performance can be stepped within the specified
range of RH. As show in Figure 1B, we compare two sorbents with similar water up-
takes at the initial state. It can be seen that with the same temperature lift, the
S-shaped sorption curve provides higher cyclic uptake than the linear one. Notably,
once the S-shaped isotherm is chosen, the RH range within which the major uptake
occurs should be taken into consideration. That is, the pressure window should coin-
cide with the specified working range of device temperatures and ambient condi-
tions (especially humidity). Besides, a steep uptake is desirable since it renders sor-
bents sensitive to the temperature swing of electronic devices and facilitates the
desorption process. From a realistic point of view, a fast adsorption kinetics should
be taken into consideration, on account of its benefit to the working performance un-
der periodic workloads.

In addition, some other characteristics are also important, including: (1) high hydro-
thermal and cycling stability, (2) non-corrosive and non-toxic, and (3) favorable ther-
mophysical properties, for e.g., a high thermal conductivity. Traditional sorbents
either achieve most water uptake at a low RH range outside the desired operational

Joule 4, 435–447, February 19, 2020 437

Figure 2. Proof-of-Concept Experiment Using MIL-101(Cr)
(A) Water sorption isotherms of MIL-101(Cr) recorded at 25
C (adsorption, circle symbols; desorption, square symbols), showing a steep uptake in the
P/P 0 range of 0.4–0.55. The hysteresis loop between the adsorption and desorption branch originates from the irreversible capillary condensation.
(B) Digital and SEM images of the prepared binder-based MIL-101(Cr) coating on a 40 3 40 mm 2 aluminum sheet.
(C) Schematic illustration of the home-made test rig used to simulate the electronic device working at heavy workloads. For comparison, another
painted black aluminum sheet was prepared as the baseline sample.

interval due to their strong affinity to water (e.g., zeolites), or possess a low water up-
take over a wide RH range (e.g., silica gels, active carbons). In this content, they are
not suitable for the thermal management application.

Over the past years, MOFs, also known as porous coordinate polymers, have been
widely investigated as a new class of microporous or mesoporous compounds in
the area of gas separation,15,16 chemical catalysis,17,18 and hydrogen storage.19,20
Governed by varied combination of mental and organic building units, the pore dis-
tribution of MOFs can be finely tuned and designed.21

With respect to water adsorption, MOFs exhibit attractive uptake capacity due to
their high porosity and large specific surface area.22 Besides, the steep isotherms
are common for MOFs arising from their high degree of consistency in pore size dis-
tribution. The first generation of MOFs often shows low hydrolytic stability in the
presence of water, but this problem has been solved with the development of a
series of MOFs synthesized in a hydrothermal route. As a consequence, MOFs pro-
vide the opportunity to realize the efficient thermal management based on the prin-
ciple proposed here.

In this research, MIL-101(Cr), first proposed by Ferey,23 is chosen as the candidate

material for electronic cooling by virtue of its well-studied water adsorption behavior
on a molecular level, especially for its high water uptake and a typical S-shaped
isotherm.24 As shown in Figure 2A, the water adsorption-desorption isotherms of
MIL-101(Cr) at 25
C reveals a rapid increase of water uptake in the RH range of
0.4–0.55. At a typical ambient condition (25
C, RH 60%), MIL-101(Cr) can adsorb
up to 1.05 gwater/gsorbent water inside its porous structure. Once MIL-101(Cr) is

438 Joule 4, 435–447, February 19, 2020

subjected to a higher temperature, the balanced water content drops to around
0.1 gwater/gsorbent sharply, and a corresponding cyclic water uptake of 0.95 gwater/
gsorbent is achieved. Besides, MIL-101(Cr) has been demonstrated to be superior
to traditional sorbents in terms of adsorption-desorption kinetics.25 The practical
performance of MIL-101(Cr) in the application of electronic cooling will be further
demonstrated in the next section.

Proof-of-Concept Experiments Using MIL-101(Cr)

The pristine MIL-101(Cr) material was synthesized following a hydrothermal reaction
and received in the powder form (Figures S1–S3; Note S1.1–S1.2). The sorbent was
further processed as a coating on the aluminum sheet (40 3 40 mm2) using a binder-
based method (Note S1.3), considering the necessity of a robust structure in prac-
tical applications. The structure of MIL-101(Cr) was well preserved after the coating
procedure, as proven by the SEM images (Figures 2B, S4, and S5). In order to probe
the influence of sorbent mass, three coated samples with masses of 0.124 g, 0.193 g,
and 0.288 g (10 wt % binder) were prepared. Their respective thicknesses were
198, 323, and 516 mm. Furthermore, the average water uptake of the coated samples
was determined to be 0.92gwater/gsorbent (excluding the inert mass of the binder,
more details in Note S1.5 and Figure S6) at 25
C and RH60%, showing little decrease
when compared with that of the pristine sorbent. This discrepancy may be ascribed
to the determination of the dry coating mass in different measurement experiments.

For comparison, another bare aluminum sheet was painted black to represent the
baseline condition without adsorption process. A home-made test rig with a polyi-
mide resistive heater as the simulative electronic device was fabricated to evaluate
the thermal management performance of coated samples (Figures 2C and S7; Note
S1.6). The heating power can be manually adjusted by the DC power supplier. All
experiments were conducted at a controlled environment condition (Figures S7
and S8).

A simplified energy balance model was developed here to guide the discussion
(more details in Note S2). The governing equation with respect to energy conserva-
tion can be expressed as

 X dTi
_ de +
Qheating = AhðTs  Ta Þ + Asε T 4s  T 4a + mh m i Ci ; (Equation 2)
dt
where Qheating is the heat arising from the Joule effect of the heater (W), A the surface
area of the sorbent coating (m2), h the convection heat transfer coefficient (W/m2.K),
Ts the surface temperature (K), Ta the ambient temperature (K), s the Stefan–
Boltzmann constant (W/m2.K4), ε the emittance of the sample surface, m_ the desorp-
tion rate of the sorbents (g/s), hde the desorption enthalpy (J/gwater).

The last term on the right side represents the sensible heat change of the devices. It
is noteworthy that the desorption heat term mh _ de is only effective during the fast
desorption process.

Prior to experiments, the samples were initially exposed to the environment condi-
tion at 25
C and RH 60% for a long time until reaching both mass and temperature
equilibria, then the power supplier was switched on. The temperature evolution of
the heater and the mass change of samples were recorded simultaneously, as shown
in Figure 3A. Typically, the temperature profile can be divided into the transient
period (including the temperature plateau) where the temperature varies, and the
steady period where the temperature is fixed around the maximum value (Figure S9).

Joule 4, 435–447, February 19, 2020 439

Figure 3. Performance of the MIL-101(Cr) Coating
All experiments are conducted at a typical ambient condition, 25
C and RH 60%.
(A) Temperature evolution of the heater and mass evolution of different samples at a heating power of 1.5 W. The decreasing mass indicates the
desorption process, which brings a prominent thermal management performance.
(B) IR images of samples after a fixed time.
(C) The transient cooling power derivated from the desorption rate.
(D) Comparison of the performance in terms of the effective time. The error based on propagation of instrument uncertainty is determined to be 2%,
indicated by the error bar.
(E) Variation of equivalent thermal resistance for samples with different coating thicknesses. The error based on propagation of instrument uncertainty is
determined to be 2%, indicated by the error bar.
(F) The influence of convection upon the transient heating process and the steady-state temperature. A fan is adopted to enable ambient air to flow
through the upper surface of the coating. The velocity of air is fixed at 0.8 m/s, corresponding to Re = 205.

At a heating power of 1.5 W, the temperature rises rapidly in the initial period. How-
ever, different temperature rising tendencies between the temperature curves of the
baseline and coated samples can be detected after 2.5 min. The temperature rising
rate of the coated samples continues to decrease after the temperature excesses
35
C. This change can be attributed to the initiation of the fast desorption process
of MIL-101(Cr), which confirmed by the mass curves. A temperature plateau is
reached around 54
C for three coated samples. During the plateau period, the sam-
ple temperature maintains a comparatively slow ramp-up. By the end of the desorp-
tion process, the mass change of samples slows down and reaches a steady state ul-
timately. Correspondingly, the sample temperature rises gradually to its peak value.
The maximum temperature differences between the baseline and three coated sam-
ples, reached during the temperature plateau, are 7.9
C, 8.4
C, and 8.6
C,
respectively.

With a deeper reflection on the mass change curves, it appears that for all three
samples, their initial desorption rates are nearly the same and the mass decreases
linearly with identical slop, regardless of the variation of coating thicknesses (Fig-
ure S10). This can be ascribed to the fact that the vapor transport on the gas side
at the air-coating interface is the rate-controlling step of the desorption process.26
Namely, as outlined and validated in the previous literature,27 the influence of
solid-side diffusion and local disequilibrium between the free and adsorbed

440 Joule 4, 435–447, February 19, 2020

moisture, deemed as proportional to the coating thickness, are negligible in our ex-
periments (more details in Note S2). Under this circumstance, the desorption rate of
the coating layers can be expressed by a simplified model:

dWd
m_ = md = K y AðYd  Ya Þ (Equation 3)
dt
where md is the mass of sorbents (g), Wd is the instantaneous water content of
sorbents (gwater/gsorbent), Ky* is the modified mass transfer coefficient in the air
side (kg/m2.s, defined in Equation S9), A is the surface area of the coating (m2).

Humidity ratio Yd of the free moisture inside the sorbent pores (g/kg) can be esti-
mated from the isotherm curves at known temperature and water content. It should
be noted that here Ky* is just correlated with the surface structure and convection
condition. For sorbent materials with S-shaped isotherms, RH of air inside the sor-
bent pores anchors around a constant value when the water content falls along
the steep step of the isotherm. In the initial phase of desorption, Yd increases with
the elevated sorbent temperature, accompanied by an increasing desorption rate.
However, a typical stage of nearly stable temperature, stable humidity ratio in sor-
bent pores and the resultant constant desorption rate would eventually occur,
when a delicate balance is nearly achieved between the heating power and the
heat loss by radiation, convection, and desorption. This quasi-stable state is indi-
cated by the plateau in temperature curves. The contribution of the desorption pro-
cess can be derived from the mass change rate and the desorption enthalpy, as the
third term on the right of Equation 2. Here, the average desorption enthalpy of MIL-
101(Cr) is determined to be 2,400 J/gwater, close to the evaporation enthalpy of
water. As shown in Figure 3C, the desorption power of the three samples shows a
similar tendency and increases rapidly in the initial several minutes. A platform
appears when the desorption power climbs up to a value of about 0.5 W. This
value accounts for 33% of the heating power and is sufficient to demonstrate an un-
ambiguous cooling effect. The quasi-stable state is broken when the water content
of adsorbents drops below the inflection point of isotherms. Then the desorption
power declines and the temperature rises up correspondingly.

Moreover, the plateau temperature is affected by the heating power (see Figures
S11–S13). For a higher heating power, the excess heat needs to be partially compen-
sated by the desorption process. On the other hand, a higher temperature is needed
to facilitate the desorption process. As a result, another equilibrium state is reached
at a higher temperature.

It can be clearly seen from Figures 3A–3C that the delay effect on the peak temper-
ature emergence is more obvious for samples with larger coating mass. To quantita-
tively evaluate the effect of coating mass and heating power on the duration of ther-
mal management, a parameter, effective time te, is defined as the moment when the
coated sample temperature exceeds the baseline. As displayed in Figure 3D, the
effective time is positively correlated with the coating mass. For example, the effec-
tive time of Sample3 is 41.8 min at a heating power of 1 W, while the value of Sample
1 is 26.8 min at the same condition. The dramatic decrease up to 36% confirms the
great influence of coating mass upon the effective time.

Obviously, more coating mass is in favor of extending the transient desorption pro-
cess. However, the sorbent coating works as an extra thermal resistance between the
heat source and ambient at steady state. Here, the equivalent thermal resistance
could be defined as (DT/Q), where DT denotes the temperature difference between

Joule 4, 435–447, February 19, 2020 441

the heater and ambient at steady state (K), Q denotes the heating power of the
heater (W). With the increase of coating mass, the thickness of sorbent coating corre-
spondingly rises, and a slight increase of the thermal resistance can be observed
(Figure 3E). The contradiction between the temperature drop during the transient
period and the temperature rise during the steady period is unavoidable. Hence,
the optimization object is achieving an obvious delay of temperature rise at the
acceptable expense of additional thermal resistance. In this context, the influence
of the coating thickness is discussed in Note S3. The optimal thickness should be
determined according to the specific operational conditions and user demands, us-
ing the proposed numerical model (Note S2).

In addition, a combination with extra active cooling methods can ensure improved
performance in the transient period and reliability in the steady state.28 Here, the in-
fluence of forced convection on the performance of sorbent coating is also investi-
gated. A DC fan was used to provide the forced airflow with an average speed of
0.8 m/s. As shown in Figure 3F, the plateau temperature under free convection is
about 54
C. By contrast, the value under forced convection is about 42
C due to
the enhanced convection heat transfer. Interestingly, the effective time of various
samples are almost unaffected even though a favorable vapor diffusion is expected
under the condition of forced convection. This can be attributed to the lower desorp-
tion temperature since the desorption rate is comprehensively affected by the vapor
diffusion rate and the vapor concentration difference (sensitive to temperature), as
elucidated in Equation 3.

Even though the hydrothermal and cycling stability of pure MIL-101(Cr) has been
widely proved in previous studies,22 the stability of the coating is of great impor-
tance for practical applications. After repeated tests and seven-month aging, the up-
take capacity and structural integrity of the coating is well retained without obvious
degradation. The coating shows its mechanical stability even after a vigorous
shaking (Note S1.7).

Distinguished from previous thermal management methods based on water evapo-

ration, the spontaneous adsorption process of sorbents endows the strategy pro-
posed here with the passive recovery capacity (Figure 4A). To demonstrate the work-
ing performance under intermittent heat generation, the coated sample (Sample1)
was subjected to four successive heating-cooling cycles (heating: 1.5 W for
20 min; cooling: 0W for 60 min). In order to shorten the experimental period, herein
forced convection was introduced to accelerate the adsorption process. The heater
temperature decreases rapidly to be slightly higher than the ambient temperature in
several minutes, once the power supplier is switched off. Then it descends at a much
slower rate which is nearly constant. We attribute this to the mild exothermic adsorp-
tion process. Eventually, the heater temperature recovers to ambient temperature,
indicating the finish of adsorption. It can also be seen from Figure 4B that the work-
ing capacity shows no degradation within the repeated heating period. Here, a long
enough recovery duration is adopted to ensure the complete equilibrium when the
adsorption phase finishes. However, entire saturation is not necessary in the realistic
circumstance of frequent heating cycles. Taking advantage of the fastest part of the
initial adsorption process, followed by desorption of partially saturated sorbents,
has been demonstrated to be beneficial to rapid cycling (Note S4).29

Furthermore, we conducted another cycling experiment during which the heating

power was switched between 0.2 W and 1.5 W, corresponding to the low and
high power consumption state of electronic devices, respectively. Initially, the

442 Joule 4, 435–447, February 19, 2020

Figure 4. Working Performance under Intermittent Heat Generation
(A) The dynamic adsorption curves of coated samples at 25
C and RH 60%. The convection
condition is 0.8 m/s, Re = 205.
(B) An analogy to the electronic device working at periodic workloads. The sorbent can recover its
working capacity through the spontaneous adsorption process. The working performance shows
no degradation over repeated heating and cooling, confirming the cycling working capacity of the
proposed strategy. The dashed lines indicate the data of former cycles.
(C) Switch between the high power (1.5 W) and the low power (0.2 W). The influence of the hysteresis
loop emerges here. The red wireframes indicate the position where enlarged in Figure 4D. The
former one corresponds to the upper panel of Figure 4D and the latter corresponds to the bottom
panel.
(D) The partially enlarged drawings of Figure 4C, as marked by the red wireframes. Different water
contents lead to a discrepancy in the steady-state temperatures.

sorbent was saturated at ambient condition then the power supplier (0.2 W) was
turned on. At the heating power of 0.2 W, the stable sample temperature is about
29
C, slightly higher than the ambient temperature. The equilibrium point can be
further determined from isotherms (Point 1 in Figure 4C). However, different from
the foregoing experiment, the starting point of this cycle locates in the range of hys-
teresis loop. The divergence between adsorption and desorption curves results in an
irreversible water loading after the first desorption cycle, as schematically shown in
the inset of Figure 4C (Point 2–4). The weakened cooling performance in the
following three cycles is due to the decreased cyclic water uptakes under the influ-
ence of hysteresis. Moreover, lower water content and the resulting higher thermal
resistance of the coating lead to a higher temperature in the equilibrium state, as
demonstrated in Figure 4D. Apparently, a large hysteresis loop covering the working
range of sorbents is undesirable. The influence of hysteresis on the cyclic perfor-
mance should be taken into consideration during the selection of optimal sorbents
for electronic thermal management.

In order to demonstrate the superiority of the proposed thermal management strat-

egy based on sorbents than traditional PCMs, we compared the MIL-101(Cr) coating
with commercial form-stable PCMs. A state-of-the-art PCM sheet with a mass of
0.385g was employed as the comparison (Figures S14–S15). In Figure 5A, after
the heater being turned on for 5 min, the temperature of the sample with PCM sheet
shows an inflection point then rise rapidly. The effective time is prolonged from 13.5

Joule 4, 435–447, February 19, 2020 443

Figure 5. Comparison between MIL-101(Cr) and PCMs
(A) Temperature evolution of the heater as a function of heating time at the heating power of 1W. A
commercial state-of-the-art PCM sheet is chosen as the representative of traditional solid-liquid
PCMs. The PCM sheet was adhered to the upper surface of an aluminum sheet, as schematically
shown in Figure 2C.
(B) Comparison of the equivalent enthalpy of MIL-101(Cr) coating with that of form-stable PCMs in
literatures.

to 42 min by replacing the PCM sheet with MIL-101(Cr) coating. It means the sorbent
coating possesses a 310% improvement of thermal management capacity when
compared with the PCM sheet, even though its mass was 25% less than that of
PCM sheet. For a quantitative comparison of thermal management capacity be-
tween sorbents and PCMs, the indicator used here is the equivalent enthalpy,
defined as:

Ql
heq = ; (Equation 4)
mcoating
where Ql is the total latent heat within one working cycle (J) and mcoating is the total
mass of the corresponding monolithic coating (g, including the mass of sorbents and
additives).

As displayed in Figure 5B, we calculated the equivalent enthalpy of MIL-101(Cr)

coating and PCM sheet in this work, as well as other relating PCMs used for
electronic thermal management.30–34 MIL-101(Cr) possesses an equivalent
enthalpy up to 1,950 J/gcoating (corresponding to the cyclic water uptake of 0.81
gwater/gcoating), benefiting from its high cyclic water uptake. As a comparison, the
enthalpies of most PCMs are below 250 J/gPCM. Hence, superior performance of
MIL-101(Cr) is expected in thermal management than PCMs.

A real application for thermal management of electronics was also conducted on a

computing device (ORDROID-XU4, more details in Note S1.6). This device was orig-
inally equipped with a passive heat sink to dissipate excess heat generated by the
chip. For comparison, another heat radiator with MIL-101(Cr) coating (0.743 g)
was fabricated, revealing the readily combination of the proposed strategy with ex-
isting heat dissipation structures. A thermocouple was adhered to the chip shell to
monitor the temperature evolution during operation. The device ran the Android
system and the professional benchmark software (AnTuTu) was employed to drive
the chip working at heavy workloads for 15 min (Figures S16–S18). It can be seen
from Figure 6B that the shell temperature in the case using the coated heat sink in-
creases more slowly than that using the original heat sink. A maximum temperature
reduction of 7
C can be achieved after the stress test for 15 min. Moreover, as shown
in the temperature distribution from IR images, not only the chip but also the

444 Joule 4, 435–447, February 19, 2020

Figure 6. Demonstration of the Practical Application
(A) Digital images of the bare heat radiator and another coated by MIL-101(Cr).
(B) Temperature rising curves of the computing device working at heavy workloads for 15 min. The
insets show the comparative IR images of the computing device with the bare and the coated heat
radiator after running the test code for 15 min.

adjacent PCB board has been controlled at a lower temperature. As a result, the
optimal sorbent coating is a very promising candidate for realistic electronic thermal
management.

DISCUSSION
According to the above experiment results, the MIL-101(Cr) coating has an
outstanding performance at the typical ambient condition. However, it is apparent
that low RH conditions or high working temperatures will decrease the initial water
uptake of MIL-101(Cr), resulting in a reduced working capacity. In this context, the
selection of a sorbent with a lower inflection point of the isotherm will be viable.
Hence, we discuss the performance of different sorbents at various RH conditions us-
ing numerical simulation (see more details in Note S2). For MIL-101(Cr), an obvious
decline of cooling performance at the ambient condition of RH 40% is observed (Fig-
ure S19). By contrast, Co2Cl2BTDD,35 which has an inflection point around 0.3, re-
tained its working capacity. Apparently, the swing of ambient conditions should
also be taken into consideration during the selection of sorbents.

A compilation of some promising materials with their water loadings in specific RH

ranges is given (Figure S20).36–40 The optimal selection of sorbents should be
climate-specific. The RH range in which the major water uptake occurs determines
what climate condition the sorbent is adaptive to. In addition, from the perspective
of a wide environmental adaptability, sorbents with linear water uptakes versus RH,
such as composite sorbent Alg-CaCl240 will be desirable. Even though the temper-
ature plateau vanished when compared to those sorbents with S-shaped isotherms
(Figure S20), the cooling performance of Alg-CaCl2 is still attractive benefiting from
its large water uptake. However, the leakage of salt solution in long-term operations
should be considered. In addition to merely focusing on the high uptake capacities,
a fast adsorption kinetics is also essential for the performance in practical applica-
tions. The optimization of adsorption kinetics, from a better understanding of the ki-
netics mechanism to the rational design of geometric and morphological factors, will
be the focus of future research.

Meanwhile, as previously discussed, the sorbent coating brings an additional ther-

mal conduction resistance. Further improvements can be realized by tailoring the
thermal conductivity of sorbents. The thermal conductivity of pristine sorbents is

Joule 4, 435–447, February 19, 2020 445

comparatively low due to their porous structures, resulting in a higher steady-state
temperature. This problem can be solved by adding thermal conductive additives,
which have been widely utilized in the scenario of PCMs.41,42 The recently reported
compound of MOFs and carbon nanotubes is a good example.43 Such composite
materials offer the opportunity for achieving an increase of adsorption capacity
and a decrease of thermal resistance at the same time.

EXPERIMENTAL PROCEDURES
Full experimental procedures are provided in the Note S1 in the Supplemental
Information.

SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.joule.
2019.12.005.

ACKNOWLEDGMENTS
This work was supported by the Foundation for Innovative Research Groups of the
National Natural Science Foundation of China under the contract no. 51521004.
We thank the members of ITEWA (Innovative Team for Energy Water and Air) of
Shanghai Jiao Tong University (SJTU) for their discussions and suggestions. We
thank Mr. Haoran Liu and Ms. Fangfang Deng for their help during the manuscript
writing. We thank Dr. Xuancan Zhu, Dr. Qiuming Ma, and Dr. Bingchen Zhao for their
assistance during the revision work. Chenxi Wang would like to thank Ms. Siqi Gong
for her help.

AUTHOR CONTRIBUTIONS
Supervision, R.W.; Conceptualization, R.W. and Y.T.; Methodology, C.W. and H.Y.;
Investigation, C.W., L.H., H.Y., and B.L.; Formal Analysis, C.W. and L.H.; Writing-
Original Draft, C.W.; Writing – Review & Editing, R.W. and B.L.

DECLARATION OF INTERESTS
The authors declare no competing interests.

Received: August 18, 2019

Revised: November 11, 2019
Accepted: December 10, 2019
Published: January 22, 2020

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Joule 4, 435–447, February 19, 2020 447
Ontogeny of Water Sorption-Desorption in the
Perinatal Rat
Randall R. Wickett, Julie L. Mutschelknaus, and Steven B. Hoath
College of Pharmacy (RR W, 1M), Division of Pharmaceutics and Drug Delivery; Department of Pediatrics, Division of Neonatology;
and the Perinatal Research Institute (SBH), University of Cincinnati, Cincinnati, Ohio, U.S.A.
In mammals, birth marks a transition to a cold and gaseous
environment that requires rapid physiologic adaptations lim-
iting heat and water loss. In this study, the perinatal Sprague-
Dawley rat was utilized as a model to study the be.havi~r of
water binding to the external body surface followmg birth.
Water sorption and desorption were quantified by measure-
ment of skin surface capacitance following water loading
using a dry ring electrode system. The results indicate that
the external body surface of the newborn rat is highly hydro-
phobic. This hydropho.bi~ity is. manifest~d by. the rapid de-
sorption of water (aml1lotlc flUId) followmg birth as well as
he u~e of electrical measurements to assess skin hydra-
T
tion is well established [1,2]. Whereas such measure-
ments are often referred to as impedance techniques,
conductance or capacitance are the parameters most
commonly measured [3,4]. These methods all rely
on marked increases in the dielectric constant that occur as the
stratum corneum is hydrated. Increasing the dielectric constant
leads to decreased impedance and increased conductance and capaci-
tance.
Tagami and co-workers [4,5] have used conductance measure-
ments in a water sorption-desorption test to investigate the ability of
the stratum corneum to hold water in "i"o. In this method, the sk111
is first hydrated with liquid water and then water evaporation is
monitored by measuring conductance as a function of time as the
surface dries. Using this technique, Tagami and co-workers have
shown a reduced water-holding capacity in the skin of newborn
infants [6] and in psoriatic scale [7] and an increase in the water-
holding capacity of normal stratum corneum following treatment
with glycerol [4]. . been shown to c.orrelate to skin water content hI "i"o [3,8] and
In this study, we have modified Tagami's water sorption-desorp-
tion test by measuring skin surface capacitance rather than conduct-
ance following water exposure. This approach has been used to
determine the ontogeny of water-holding capacity in the neonatal
rat. Results are presented relative to the effects of birth, developmen-
tal age, tape stripping, somatic growth, and extraction with orga~ic
solvents. These data are interpreted within the context of a potential
thermoregulatory role of the fetal periderm during adaptation to
postnatal life.
MATERIALS AND METHODS
Materials Timed-gestation Sprague-Dawley rats were purchased
from Zivic-Miller Laboratories, Inc. (Zelienople Park, PA) on the
Manuscript received June 24, 1992; accepted for publication December
4,1992.
Reprint request to: Dr. Steven B. Hoath, Division of Neonatology, De-
partment of Pediatrics, University of Cincinnati College of Medicine, 231
Bethesda Avenue, Cincinnati 45267-0541.
0022-202X/93/$06.00 Copyright © 1993 by The Society for Investigative Dermatology, Inc.
407
marked limitation of water sorption after birth. Post-natally,
this hydrophobic effect is gradually lost over the first 3 d of
life. Somatic growth retardation results in retention of this
surface property, whereas extraction of the skin surface with
acetone abolishes it. Morphologic and functional (water bind-
ing) studies performed after stripping the stratum corneum
with ac.rylic a?hesive strongly suggest a physiologic role for
the penderm m determining this postnatal hydrophobic ef-
fect. These data are interpreted as evidence for a novel ther-
moregulatory mechanism in the rat during adaptation to
post-natal life.] I'lIIest Dermatoi100:407-411, 1993
fifteenth day of pregnancy and housed in our vivarium. Mothers
were given water and routine laboratory chow ad libitum. Delivery
occurred rout111ely on day 21-22 of gestation and litters were un-
disturbed until the time of experimentation. Acetone was purchased
from Fisher Scientific. Adhesive tape (Scotch Transparent Tape
600) was purchased from 3M Company, St. Paul, MN. The DPM
9003 instrument was obtained from the NOVA Technology Corpo-
ration, Gloucester, MA.
Capacitance Measurements Skin surface capacitance measure-
ments were made with a NOVA Technology Corporation Model
DPM 9003 dermal phase meter. Thi~ device emits a 1-Mhz span of
simultaneously ~roduced frequenCIes, producing a differential
current source US111g a controlled rise time. Two concentric brass
electrodes are separated by a non-conducting resin. Capacitance is
calcul.ated at se~e~al frequencies from the signal-phase delay using a
p~opnetary clup 111 .the lIlstrument. The capacitance readings are
dl:ectly r~lated to picoFarads (pF) of capacitance in the volume of
sk111 that IS effectively measured . Conductance and capacitance have
measurements uSlllg thiS device have been shown to correlate to
clinical scaling and cracking grades [9]. The probe head is connected
to th~ mam ~ody of the 111strument through a flexible cable and
contallls a spnng to ensure consistent contact pressure on the skin .
Protocol for the Water Sorption/Desorption Test The abil-
ity o~ the stratum corneum to sorb and desorb water was assessed by
modIficatIOn of the standard test procedure described by Tagami [5] .
All measurements were performed using fetal or neonatal animals
~naintained ~t a ':nest temperature" of 34 °C [10,11] in a C-100
111cubator (Air-Shields, Hatboro, P A). Pups were placed in individ-
ual, ~erforated, polystyrene cups prior to measurement. Baseline
capaCItance measurements were obtained over the low mid-dorsal
area using the NOVA DPM 9003 instrument. The animals were
then gently lifted between thumb and forefinger and inverted, and
the low dorsal area submerged in double-distilled water for 10 sec-
onds. Following submersion, excess water on the dorsum was re-
mov~d with three quick, successive blots using sterile gauze sponges
(Cunty, Kendall Company, Boston, MA). Serial measurements
408 WICKETT ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

600

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I
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g 300
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'"
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a 20 40 70 100
Seconds

A
600

'"
CIII 500
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UJ 400
'is.
III
I
0
c:
OIl
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OIl
OIl
0
A

a 20 40 70 100
Seconds

B
Figure 1. A) Ontogenetic profile of the water SOrption/ desorption test in
the neonatal rat. Consecutive water sorption/desorption tests were per-
formed using littermate rat pups beginning on day 0 and continuing
through day 3 of life. Values represent mean ± standard error, n = 6.
Error bars for the day 3 animals are contained within the symbol. All peak
capacitance measurements differed significantly from each other, p <
0.05, analysis of variance. B) Effect of epidermal tape stripping on the
water sorption/desorption profile. In this representative experiment, six
rat pups (littermates to animals shown in A) received a single tape strip-
ping of the low dorsal epidermis on the day of birth (day 0). Serial water
sorption/desorption tests were performed on subseq\l.ent days. Standard
errors were maximal for the day 0 animals and for clarity are shown for
this day only. Adhesive tape stripping on day 0 resulted in peak capaci-
tance readings that were indistinguishable from measurements per-
formed on subsequent days.

were taken every 20 seconds thereafter and the data plotted using
the onset of water exposure as time O.
Experimental Protocols The ontogenetic profile of the water
sorption/desorption test was evaluated in littermate rats on post-
natal days 0-3. The day of birth was routinely designated day 0 in
all experiments. In this study, litters were divided at birth into two
groups with the first group used to measure tbe normal, unper-
turbed, developmental profile of the water sorption/ desorption test.
This test was performed on the day of birth and at 24-h intervals for
the subsequent three post-natal days. The second group received
B
tape stripping of the epidermis on the day ofbitth only. Tape strip- Figure 2. A) Representative photomicrograph of alkaline-expanded
ping was performed approximately 20 min prior to the first sorp- stratum corneum in the newborn rat. Cryostat sections of dorsal epider-
tion/ desorption test using Scotch clear transparent tape as described mis were obtained from rat pups on the day of birth and stained with
previously [5] except each animal received tape stripping once only. methylene blue. The stratum corneum was expanded under alkaline (pH
The sorption/desorption test was then performed exactly as on the 12.5) conditions and the number of stratum corneum layers were counted
littermate controls on post-natal day 0-3. The effect of adhesive directly from photomicrographs. The post-natal persistence of the granu-
tape stripping on the morphology of the stratum corneum was ex- lar periderm is apparent at the upper border of the stratum corneum. B)
amined using cryostat sections of dorsal epidermis stained with Effect of tape stripping to remove the granular periderm. Rat pups (litter-
mates of animals in A) received adhesive tape stripping once over the low
methylene blue and exposed to alkaline conditions according to the
dorsal e?idermis prior to obtaining frozen sections of the epidermis and
method of MacKenzie [12]. alkaline expansion of the stratum corneum. This representative photOmi-
For studies of postnatal adaptation, fetal animals were delivered crograph shows the effect of tape stripping to remove the overlying gran-
by Caesarean section on gestational day 21 following anesthesia of ular periderm exposing the underlying stratum corneum.
 VOL. 100, NO. 4 APRIL 1993
1000
900 ~
. 33 .0
~ Amnion
800
..,C'" 700
! 600 \ '"
~
UJ
1
500
g
400
I i
300
'"
i
()
200
100
0 31 . 0
0 30 60 90 120
SECONDS
Figure 3. Skin capacitance readings in the fetal rat following pre-term
delivery. Skin surface capacitance was measured followin g blotting of
amniotic fluid in fetal rats delivered by Caesarian section on day 21 of
gestation. Pups exhibited a rapid decrease in skin capacitance reaching a
baseline by 30-60 seconds, whereas measurement of the inner surface of
the amnion at 120 seconds showed persistently high capacitance read-
ings. Simultaneous skin temperatures measured over the dorsal epider-
mal surface using an infrared thermometer are also shown . Values repre-
sent mean ± SEM, n = 10.
th e mothers with methoxyflurane (Pitman-Moore, W as hington
Crossing, NJ). Pups were delivered prematurel y, one at a time, and
placed in a C -100 incubator at 34 C. The a mnio~ was removed,
0
blotted WIth gauze, and placed to one side. Baselme capaCI tance
measurements were obtained over the low dorsal surface of the fetal
pups 5 to 10 seconds after blotting the epidermis with sterile gauze.
Simultaneous skin temperature readings were obtained using an
infrared thermometer. Subsequent readings were taken every 30
seconds thereafter four times. Capacitance measurements were also
performed on the inner surface of the amnion at the 2-min time
point.
The effect of postnatal growth retardation on the ontogeny of
water sorption/ desorption was assessed using the paradigm of con-
trolled litter size described by Widdowson [13J . In routine experi-
ments, one litter of rat pups (n = 12) was pared to four pups at birth
and th e remaining eight pups cross-fostered to another litter of
eight born on the same day. This distribution schema resulted in a
litter of four (growth acceleration) and a litter of 16 (growth retard-
ation) . D aily water sorption/desorption tests were performed on
the original 12 littermate pups along with determination of body
weights.
The effect of ace tone extraction to alter skin surface capacitance
and the water sorption/desorption test was assessed using postnatal
littermate animals. Briefly, the extraction was performed in a C-l 00
incubator with the rat pups maintained individually in styrofoam
cups on sterile gauze pads. Rats were treated over the low dorsal
region with absolute acetone-soaked cotton swabs essentially as de-
scribed by Menon et at [14J. Treated animals received five individual
acetone treatments spaced approximately 1 min apart with each
treatment lasting a total of 10 seconds. Control animals were un-
treated . A water sorption/desorption test was performed 60 min
after the last acetone treatment.
RESULTS
The ontogeny of water sorption-desorption was assessed in the neo-
natal rat by skin surface capacitance readings. Initial water sorption-
desorption tests were performed on littermate rat pups beginning on
the day of birth and continuing throu gh day 3 of life. As shown in
Fig lA, there was a progressive increase in peak capacitance readings
over the first 3 d after birth . Of interest, the peak values obtained on
postnatal day 3 correlated very closely. In a representative experi-
ment on postnatal day 3, for example, peak capacitance values at the
20-second time point were recorded as 572, 572, 572, 574, and 574
pF in six littermate rat pups. The low variability in these data is
appa rentl y not du e to saturatio n of the instrument because the de-
vice is capable of measurin g much higher values (cf. Fig 3).
WATER SORPTIO N-DESORPTION IN THE PERINATAL RAT 409
-<
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13
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12
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11
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i ~ 9 '\
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~ing
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:£ 6
5
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Postnatal Age (Days)
~igu.re 4. Representative growth curves of fast-growing and slow-grow-
mg htters. As described in Materials alld Methods, newborn littermate rat
pups were ?istribut~d at birth to litters of four (fast growing) versus 16
(slow growmg). Senal body weights were obtained at 24-h intervals for
the three subsequent days. Values represent mean ± SEM .
The effect of ad hesive tape stripping on stratum corneum water
sorptl?n-desorptl?n ~s shown in Fig lB. Rat pups were subjected to
ad heSIve tape. stnp'pmg one of the low dorsal epidermis several
hours followm g bIrth and serial water sorption-desorption tests
were performed on subsequent days. Tape stripping abolished the
~lormal ontoge ny of.response and resulted in pea k capacitance read-
Ings on the day of btrth th at were indistinguishable from measure-
ments performed on subsequent days.
As shown in Fig 2A, alkaline exposure results in expansion of the
stratum corneum and the number of stratum corn eum laye rs can be
counted d~rectly from photomIcrographs [12J . The gra nular peri-
derm I?emsts post-natally m the newborn rat and is clearly visible
overl>:lI1g th e upper layer o.f corneocytes in Fig 2A. A single tape
stnppmg, however, results m removal of the peridermal layer and
exposure. of the underlym g stratum corneum. To quantitate this
effect: a smgle lItter ~f 12 pups was di.vided into two groups at birth
and SIX ammals receIved tape stnjJPll1g once over the dorsal epi-
derm~s. All a11lmals were then sacnficed and frozen sections of epi-
dermIS were processed for alkalll1e expansion of the stratum cor-
neum [12J . The number of stratum corneum laye rs were determined
d~rectl y fr?m the resultmg photomicrographs. There was no sig-
11Ificant dIfference between the control and tape stripped pups
(control, 16.5 ± 0.7 layers versus tape stripped, 17.2 ± 1.0 layers;
mean ± SEM, n = 6 per group) .
Th: presence of a hydroph obic boundary laye r in the newborn
o:gal1lsm has potentIal thermoregulatory significance as a mecha-
I1Ism of cu:tatlmg I~eat loss due to evaporation. Figure 3 shows a
representatIve expe nm e l~t 111 whIch fetal rats were delivered prema-
turely by Caesa~lan sectIon on day 21 of gestation and serial skin
capacItance readIngs we re ~aken every 30 seconds following birth.
As shown, fo ll owlIlg blottll1g of the dorsal epidermis, there was a
rapId. decrease 111 skm sur.face capacitance readings, which reached
basel me values at approXImately 60 seconds after delivery. At 120
seconds after bIrth, meas urement of the inner sur face of the blotted
fetal membranes (amnion) showed extrem ely high values (962 ± 9
pF, mean ± SEM~ n = 10) consistent w ith a wet, non-hydrophobic
surface. ConcOImt~nt meas urements of skin surface temperature
USIng a noncontact mfrared th ermometer rose from initial readings
to plateau levels by approximately 30 seconds.
To evaluate the effect of somatic growth on the ontogenetic
profile of th~ water sorption-desorption test, newborn littermate rat
pups were dlstr.lbutedat birth to litters of four (fast-growing) versus
16 (slow-~rowmg) . FIgure 4 shows representative growth curves of
fast-growmg and slow-growing litters . Water sorption-desorption
tests ~ere perform ed on the day of birth and at 24-h intervals until
the thIrd postnatal day . As shown in Fig SA, no significant differ-
ence was observed between gro ups on the day of birth (day 0).
M easuremenr on subsequent days, however, showed the fast-grow-
ing animals moved through the norma l ontogenetic sequence at an
---
410 WICKETT ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

700 ~ 700
Day 0 Day 1
600 j 600
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Figure 5. A-D. Effect of postnatal growth retardation test. A-D show the results of water sorption/desorption tests performed on pups from the
growth accelerated and growth retarded litters shown in Fig 4. On the day of birth (A ), the water sorption/ desorption profiles exhibit a blunted, barely
distinguishable peak with no Significant difference between trea tment groups. Twenty-four hours later (B), peak readings are Significantly elevated in the
growth-accelerated group compared to the growth-retarded litter, p < 0.01. On post-natal days 2 and 3 (C and D), the growth-accelerated group has
reached a maximum, whereas the growth-retarded group continues to exhibit a slow but steady increase in peak capacitance. Values are means with
standard errors shown for the peak capacitance readings only. Error bars for the fast-growing animals on postnatal days 2 and 3 are contained within the
symbols.

accelerated rate compared to th e slow-growing animals (Fig B - D) . water to the stratum corneum [3]. In other experiments, it has been
This was manifested by high er peak capacitance values in the water shown th at increased capacitance parallels increased conductance as
sorption-desorptio n test on eac h day following birth. the less hydrated outer layers of the stratum corneum are removed in
The apparent h ydrophobic character of the superficial skin sur- se rial tape-stripping experiments [4,7]. In this study, we measured
face in rats durin g the early neonatal period supports the potential skin surface capacitance in the late fetal and early neonatal rat under
role of lipids actin g as a water barrier. Figure 6 shows the effect of experimental conditions designed to assess water binding at the
extraction of the skin with ace to ne prior to performance of th e organism-environmental interface.
water sorptio n-desorption test. As shown, ace tone treatment re-
sulted in a marked increase in peak capacitance readings compared 500 -
to untreated littermate co ntrol s.
DISC USSION C'"
.!!l
400
The proper functioning of the mammalian stratum corneum in-
'"
>
'5
C'
w
volves co mpl ex interactions with molecular water [15] . On th e one ~I I
hand, th e stratum co rn eum must be relatively impermeable to water I 300
I
<I>
to prevent excess water loading before birth and dessication after g
birth. On th e oth er hand, w ater must be bound within the stratum ~
u
200
corneum to maintain th e fl ex ibility necessary for free body move- '"
C.
I

ment and to prevent fissuring and scaling [16] . Moreover, nearly '"
()

every biophysical property measureabl e at th e skin surface (elec tri- 100 Control
cal, mechanical, thermal, chemical) is influenced by the water con- 0 20 40 60
tent of the stratum co rneum.
SECONDS
Numerous methods have been devised to evaluate th e hydration
state of the skin surface [2,4,17]. One increasingly popular tech-
Figure 6. Effect of acetone extraction on water sorption/desorption.
nique involves th e measurement of skin impedance using dry sur- Skin surface capacitance profiles were obtained on littermate rat pups at
face electrodes [4]. This method is based upon the dramatic change 24 h of age following acetone treatment of the low dorsal epidermis.
in th e electrical properties of the stratum corneum when moistened. Acetone treatment results in a marked increase in peak capacitance read-
In gene ral , the primary electrical quantities measured are conduct- ings follOwing water exposure compared to untreated littermate controls.
ance or capacitance, both of which increase following addition of Values are mean ± SEM, n = 6 for each group.
VOL. 100, NO.4 APRIL 1993
Our data strongly support the hypothesis that the newborn rat
p ossesses a superficial, hydrophobic barrier th at results in blunted
water sorption-desorption postnatally. In term human infants, stud-
ies of water sorption-desorption using conductance techniques have
revealed a similar hydrophobic skin surface (6). Rather than viewing
this effect as a deficiency of stratum corneum water-holding capac-
ity we propose that a hydrophobic surface would confer a selective
th~rmoregulatory advantage on the newborn organism by decreas-
ing heat loss ass?ciated with evaporation .. At birth, for exa.mple,
mechanisms to lunlt the adsorptIOn or facIlitate the desorption of
amniotic fluid wou ld be highly protective against evaporative heat
loss (Fig 3). Moreover, after birth, the neonatal rat is intermittently
exposed to other hazards in the form of surface contammatlon WIt!l
urine or the potential cooling effects of maternal saliva (18). In tlllS
view, the ex ternal surface of the organism functions not only to
"keep water in" but also to "keep water off." . .
W ater flux is an important component in the regulatIOn of skm
surface temperature, and, consequently, peripheral heat flux. In the
newborn organism, central thermoregulatory mechanisms a:e 1I1ef-
ficient and peripheral mechanisms m ay be relatively more Impor-
tant. In humans, for example, the temperature gradient between the
skin and air is ti ghtly correlated with metabolic rate, whe:eas ab~o­
lute core temperature shows no relation (19). In rats, there IS consid-
erable evidence that factors regulating the temperature of the ma-
ternal-infant interface determine the length of lactation (18).
Physical stroking, for example, results in surface cooling and, by
hypothesis, a prolongation .of lactat1C:>n [~O). Our results are consist-
ent with an additIOnal phYSical contributIOn to the control of surface
temperature early in life, namely, the presence of a superfiCIal hy-
drophobic layer overlying the stratum corneum. . .
The localization of this barrier to the granular periderm IS consist-
e nt with the results of both functional (Fig 1) and structural (Fig 2)
studies following simple tape stripping. In other work (21), we have
demonstrated that the inner layer of periderm, as originally de-
scribed by Stern et at (22), persists postnatally for several days a~ter
birth. This layer invests the rat at birth and, hypothetically, conSlS.ts
of terminally differentiated, non-dividing cells linked to form a thl.n
cellular membrane. This interfacial structure presumably expen-
enceS lateral expansion following birth associated with the growth
of the organism. Breakdown of this layer in fas t-growing a~imals
would provide a simple explanation of the results shown 111 Fig
SA-D. In slow-growing animals with a relative paucity of penph-
eral insulation, the persistence of a hydrophobic surface may be of
thermoregu latory benefit. . 15.
In the postnatal rat, the transition from a fluid to a gaseous ~nvI­
ronment is accompanied by spontaneous proteolysis of fil aggnn to
free amino ac ids such as urocanic ac id and pyrrolidone carboxylic
acid (23). These molecules, particularly the latter, ar.e considered .t o
be important natural moisturizers that impart plastiCIty and flexlbl!-
ity to the stratum corneum (23,24). The presence of a hydrophobiC
peridermal coveru~g overlymg the develo~m g stratum co:neum IS
consistent With thiS process and, hypothetically, would aid 111 hy-
dration of the outermost corneum layers by acting as a temporary
barrier to transepidermal water loss . . 19.
Figure 6 shows the ability of acetone to disrupt the wat.er ~arn~r
properties of the skin and support an important role for lipids m tillS
regard. Previous work by Elias and co-workers have clea~ly Impli-
cated stratum corneum lipids in formation of an effective water
barrier (25) . Whether the periderm possesses specific lipids impor-
tant in maintaining this type of function is undetermined. . the perinatal rat.] Invest Dermatoll00:400-406, 1993 (this issue)
In summary, the measurement of skin capacitance has been uti-
lized to assess the dynamics of water sorption and desorption to the
surface of the perinatal rat. The results indicate that the rat at birth
exhibits a superficial hydrophobicity that is gradually lost as a func-
tion of somatic growth. Attempts to localize this effect strongly
implicate the fetal periderm as a thin, terminally differentiated,
hydrophobic membrane that persists for a limited time postnatally.
This layer envelops the m aturing stratum corneum, separates the
perinatal organism from its environment, and subserves potential
thermoregulatory and/or other se nsory functions.
WATER SORPTION-DESORPTION IN THE PERINATAL RAT 411
We tlrauk Reiko Tatwka for expert techuical assis/atl Ce and Mary Brutmer for
aSSiS(atlCe itl prepara(iotl of the matH/script. This study was supported, itl part, by the
Childretl's Hospital Research FOlltldatiorl and, itl part, by the Natiotlal Institute of
Child Health atld H,mlan Developmetlt Grant HD-20748 (SBH).
REFERENCES
1. T agami H, Ohi M, Iwatsuki K, Kanamaru Y, Yamada M, Ichijo B:
Evaluation of th e skin surface hydration in vivo by electrical mea-
surement.] Invest DermatoI 75:500-507, 1980
2. Leveque ]L, De Rigal J: Impedance methods for studying skin mois-
turization. ] Soc Cosmet Chern 29:419 - 428, 1983
3. Masuda Y, Nishikawa M, Ichijo B: New methods of measuring capaci-
tance and resistance of very high loss materials at high frequencies.
IEEE Trans Instr Meas IM-29:28-36, 1980
4. Tagami H: Impedance measurement for evaluation of the hydration
state of the skin surface. In: Leveque ]-L (ed.). Cutaneous Investiga-
tion in Health and Disease - Noninvasive Methods and Instrumen-
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5. Tagami H, Kanamaru Y, Inoue K, et al: Water sorption-desorption test
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] Invest Dermatol 78:425-428, 1982
6. Saijo S, T agami H: Dry skin of newborn infants: functional analysis of
the stratum corneum. Pediatr Dermatol 8:155 -159, 1991
7. Tagami H, Yoshikuni K: Interrelationship between water-barrier and
reservoir functions of pathologic stratum corneum. Arch Dermatol
121:642-645, 1985
8. Moseley H, English ]SC, Coghill GM, MacKie RM: Assessment and
use of a new skin hygrometer. Bioeng Skin 1: 177 - 192, 1985
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Infec Contr 18:194 -200, 1990
10. Adolph EF: Ontogeny of physiological regul ations in the rat. Q Rev
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11. Donnelly MM, Hoath SB, Pickens WL: Early metabolic consequences
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in neonate rat epidermis.] Invest Dermatol 77:278-282, 1981
13. Widdowson EM, McCance RA: Some effects of accelerating growth.
Proc R Soc London B 152:188-206, 1960
14. Menon GK, Feingold KR, Mao-Qiang M, Schaude M, Elias PM: Struc-
tural basis for the barrier abnormality following inhibition ofHMG
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219,1992
Blank IH: Factors which influence the water content of the stratum
corneum.] Invest Dermatol 18:433-440, 1952
16. Obata M, T agami H: Electrical determination of water content and
concentration profile in a simulation model of in vivo stratum cor-
neum. ] Invest Dermatol 92:854 - 859, 1989
17. Triebskorn A, Gloor M, Greiner F: Comparative investigations on the
water content of the stratum corneum usin g different methods of
measurement. Dermatologica 167:64-69, 1983
18. Leon M, Croskerry PG, Smith G K: Thermal control of mother-young
contact in rats. Physiol Behav 21:793-811, 1978
Adamsons K, Gandy GM, James LS: The influence of thermal factors
upon oxygen consumption of the newborn human infant.] Pediatr
66:495 - 508, 1965
20. Sullivan RM, Shokrai N, Leo n M: Physical stimulation reduces the
body temperature of infant rats. Dev Psychobiol 21 :225 - 235, 1988
21 . Hoath SB, T anaka R, Boyce ST: Rate of stratum corneum formation in
22. Stern 1B, Dayton L, Duecy J: The uptake of tritiated thymidine by the
dorsal epidermis of the feta l and newborn rat. Anat Rec 170:225-
234,1971
23. Scott IR, Harding C R: Filaggrin breakdown to water binding com-
pounds during development of the rat stratum corneum is controlled
by the water activity of the environment. Dev BioI 115:84-92, 1986
24. Laden K, Spitzer R: Identification of a natural moisturizi ng agent in
the skin. ] Soc Cosmer Chern 18:351-360, 1967
25. Elias PM, Menon GK: Structural and lipid biochemical correlates of
the epidermal permeability barrier. Adv Lipid Res 24:1-26,1991
 Grain & Oil Science and Technology 4 (2021) 149–164

Contents lists available at ScienceDirect

Grain & Oil Science and Technology

journal homepage: http://www.keaipublishing.com/en/journals/grain-and-oil-
science-and-technology/

Sorption equilibrium moisture and isosteric heat of Chinese wheat bran

products added to rice to increase its dietary fibre content
⁎
Xingjun Li a, , Xu Han a, Lisi Tao a,b, Ping Jiang a, Wen Qin b
a
Academy of the National Food and Strategic Reserves Administration, Beijing 100037, China
b
College of Food, Sichuan Agricultural University, Ya'an 625014, China

A R T I C L E I N F O A B S T R A C T

Article history: More and more people concern about supplementing dietary food with wheat bran to increase fibre con-
Received 5 May 2021 tent, but there has been little study of the sorption isotherms and isosteric heats of wheat bran fibre
Received in revised form 16 August products, which are important to the quality and storage durability of such commodities. This study col-
2021 lected equilibrium moisture content (EMC) and equilibrium relative humidity (ERH) data on six Chi-
Accepted 1 September 2021 nese wheat bran products via the static gravimetric method and analysed their sorption isosteric
heats. Results showed that all six wheat bran products had sigmoidal isotherms. The data were best
Keywords: fitted by polynomial, modified GAB, modified Oswin, and modified Halsey models. The relative safe
Moisture sorption isotherm moisture contents of the six wheat bran products were 12.20%–13.86% wet basis (w.b.). The heat of va-
Wheat bran porization of wheat bran products approached the latent heat of pure water at around a moisture con-
Isosteric heat of sorption tent (MC) of 22.5% and approximately 2450 kJ/kg. A process of extrusion and ultrafine grinding
Solid surface area reduced the solid surface area and sorption isosteric heats of the monolayer, multilayer and condensed
Multilayer water region water regions in the wheat bran products. In the temperature range of 10–37 °C, the EMC of sorption in
Thermomechanical properties monolayer, multilayer and condensed water regions decreased with increasing temperature. The milled
rice to which 1%–3% 200-mesh wheat bran product was added maintained its texture when made into
cooked rice and its thermomechanical properties when made into rice dough. After processing by extru-
sion and ultrafine grinding, wheat bran products have a lower solid surface area and lower monolayer
water content (Mm) and sorption isosteric heat values. Some 3% 200-mesh wheat bran product can be
added to cooked rice for increasing its fibre content and making it more nutritious.

1. Introduction Similarly, 160 million had hypertension, 160 million had

Some plant cell-wall polysaccharides that are found in
dietary fibre-rich foods have been implicated in the modu-
lation of the human immune system [1], and have antitu-
mor, antiviral, and anti-inflammatory bioactivities [2].
China has a population of 1.4 billion and is developing
high rates of modern civilized diseases that were once
only common in developed countries. According to a re-
port [3] on nutrition and chronic disease among the
Chinese population, in 2014, 225 million and 125 million
individuals were overweight and obese, respectively.
dyslipidaemia, and 94 million had abnormal blood sugar
levels. These nutrition and health survey data can be attrib-
uted to insufficient levels of dietary fibre being ingested by
the general Chinese population over the past 12 years. Ce-
real dietary fibre can prevent and alleviate diabetes, abnor-
mal plasma cholesterol and cardiovascular disease, as well
as rectal cancer and obesity [4, 5]. Soluble dietary fibres
such as synthetic polydextrose and phytoextractive inulin
[6] are beneficial to human health although costly, but a re-
cent report [7] showed that dysregulated microbial fermen-
tation of inulin can induce cholestatic liver cancer.

⁎ Corresponding author.
E-mail address: lixingjun888@aliyun.com (X. Li).

http://dx.doi.org/10.1016/j.gaost.2021.09.001
2590-2598/© 2021 Henan University of Technology. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Li et al.
Arabinoxylan can be extracted from wheat bran using an al-
kaline solution [8], which has environmental implications.
Thus, this paper explores wheat bran products that can be
obtained by extrusion and ultrafine-grinding processes.
Fibrous foods can regulate colon function due to their
physical properties, in part because of their ability to bind
water [9]. The moisture absorptive isotherm, which de-
scribes the relationship between water activity (aw) or the
equilibrium relative humidity (ERH) and the equilibrium
moisture content (EMC) of a food under constant tempera-
ture and pressure, has been widely used to study the
water-binding characteristics of food materials [10]. The
committee of the American Society of Agricultural and Bio-
logical Engineering revises the technical standard data of
“Moisture relationships of plant–based agricultural prod-
ucts” every five years [11]. This is a collection of EMC/
ERH data with isotherm-fitting equations of important
agricultural products and their processed derivatives, in-
cluding dietary fibre-rich products such as rice bran,
wheat bran, corn bran, oat bran and soy bran [11]. Reddy
and Chakraverty [12] measured the EMC of raw bran and
parboiled bran in paddies at 13, 30, and 40 °C in the
range of 20%–80% relative humidity (RH). In the past
decade, the moisture sorption isotherms and thermody-
namic properties of dietary fibre-rich food materials have
been determined, such as grapes [13], apples [14], tea
[15], guar [16], soybean [17], peas [18], cassava [19] and
peppercorns [20]. The EMC/ERH data of such products
are useful dietary fibre resources for the drying, safe storage
and transportation of food. As a food, the main problems in
wheat bran products are poor flavour and taste. Thus, food
processing technology, such as extrusion and ultra-grinding
processes, is being used to overcome these deficiencies.
However, few reports have investigated the EMC/ERH
data of wheat bran products after extrusion and ultra-
grinding processes.
Many empirical and semi-empirical mathematical
models describing the sorption behaviour of foodstuffs
are available in the literature. The criteria used for
selecting the most appropriate sorption model are its fit-
ness to experimental data and its simplicity [21]. Knowing
the sorption isotherms at different temperatures and the
Table 1
The constitutions of wheat bran samples.
Samples Moisture (% w.b.) Protein (%)
a f
Rough bran 11.41 ± 0.17 17.31 ± 0.13
Fine bran 10.78 ± 0.12b 17.60 ± 0.08e
100-Mesh 7.44 ± 0.20e 18.34 ± 0.39d
200-Mesh 7.18 ± 0.16e 20.81 ± 0.03c
300-Mesh 7.87 ± 0.10d 21.54 ± 0.46b
Active bran 8.39 ± 0.14c 25.35 ± 0.06a
Notes: DF, dietary fibre; data represent the mean ± SD of triplicates; the same column values followed by different superscript letters are significantly
different at P ≤ 0.05, according to Duncan's multiple range test.
150
Grain & Oil Science and Technology 4 (2021) 149–164
best fitting EMC equations makes it possible to calculate
thermodynamic functions like sorption isosteric heat. The
thermodynamic functions of water sorption in dried food-
stuffs have drawn great interest because they provide a
more thorough interpretation of the sorption isotherm phe-
nomenon and help to better understand the sorption mech-
anism [22]. The objectives of the present study were: (1) to
determine the moisture sorption isotherms of six wheat
bran products at 10–35 °C and 11%–96% RH, (2) to evalu-
ate the best sorption isotherm model for fitting to experi-
mental data, and (3) to analyse the isotherm types and
safe moisture contents. In addition, the sorption isosteric
heat and solid surface area parameters were determined
and the effects of adding 200-mesh bran products to
cooked rice and rice flour dough were evaluated. Ulti-
mately, the aim of this study was to demonstrate the effi-
cacy of modifying the matrix structure of these wheat
bran products via extrusion and ultrafine-grinding process
to improve their composition, product output and shelf-
life stability.
2. Materials and methods
2.1. Wheat bran dietary fibre products
Coarse bran in large lamellar shape and fine bran in tiny
lamellar shape were supplied by local wheat flour mills.
Three kinds of wheat bran (100-, 200-, and 300-mesh)
were provided by Rongmeier Biological Technology Co.,
Ltd., Qingzhou, China, and obtained by enzymolysis, extru-
sion and ultrafine grinding. Briefly, whole bran was sprayed
with an enzyme solution of xylanase and cellulase and
enzymolysed at 36–38 °C for 4 h. The product was then
extruded at 80 Pa and 120 °C for 60 s. Finally, the extrusion
product was ground in a 75-kW electric horizontal ultrafine
pulverizer at 1440 r/min. Active bran with 37.5% dietary
fibre was provided by Heima Grain and Oil Industry Co.,
Ltd., Hebei Province, China, remoisturized and extruded
from fine wheat bran using an intermeshing co-rotating
twin-screw extruder, then milled using an ultrafine grinder.
The constitutions of the six wheat bran products were
shown in Table 1.
Fat (%) DF (%) Ash (%)
c a
2.84 ± 0.03 43.45 ± 0.33 2.64 ± 0.04b
2.86 ± 0.06c 42.51 ± 0.45b 1.75 ± 0.03e
2.95 ± 0.13c 41.52 ± 0.61bc 2.39 ± 0.04d
3.27 ± 0.08b 41.15 ± 0.40cd 2.47 ± 0.01c
3.61 ± 0.15a 40.56 ± 0.29d 2.49 ± 0.06cd
2.80 ± 0.06c 37.34 ± 0.45e 5.67 ± 0.04a
X. Li et al. Grain & Oil Science and Technology 4 (2021) 149–164

2.2. Determination of moisture absorption isotherm
The static gravimetric method was used to measure the
equilibrium moisture content of wheat bran products ac-
cording to the method of Li [23] with some modification.
Briefly, 27 wide-mouth 250-mL glass bottles, each contain-
ing 65 mL of saturated salt solution, were kept in a
temperature-controlled cabinet to maintain nine groups of
different ERH levels in the specified range. A total of 135
bottles were used in an experiment to determine five sorp-
tion isotherms for each sample. Each sample of wheat
bran product (5.0000 g) was placed in a small bucket
(3 cm in diameter and 4 cm in height) made of 300-mesh
nylon gauze and hung on a copper wire pothook under a
rubber plug into a wide-mouth glass bottle 2–3 cm above
the saturated salt solution. The bottles, with the samples ex-
posed to saturated vapour, were maintained at five temper-
atures (10, 20, 25, 30, 35 °C). After 17 days, we began
weighing the sample-containing buckets every other day.
The measurements continued until the change in mass be-
tween two successive readings was less than 2 mg. The
moisture content (MC) of the sample at this constant-mass
stage was defined as the EMC and was determined using
the oven-drying method [24]. The lower the exposure tem-
perature, the longer the samples were left to equilibrate.
However, wheat bran products exposed to the saturated po-
tassium nitrate and potassium chloride solutions for
3–6 days at higher temperatures were susceptible to fungal
growth and were removed immediately if any mould was
visually observed.
2.3. Analysis of the sorption data
The experimental EMC/ERH data were used to construct
isotherm curves in Kaleidagraph for Mac v4.1.3 software,
with the ERH and EMC data represented on the x- and y-
axes, respectively. Eight equations were used to fit models
to the EMC data of the wheat bran products (Table 2).
Fitting was conducted by non-linear regression analysis
in SPSS v17.0 for Windows. The criteria used to determine
the best fit to the EMC/ERH data were the determination
coefficient (R2), residual sum of squares (RSS), standard
error (SE), and mean relative percentage error (MRE),
which were calculated using Eqs. (1)–(4), respectively.

2
R2 ¼ 1−∑ni¼1 mi −mpi =∑ni¼1 ðmi −mmi Þ2 (1)

2
RSS ¼ ∑ni¼1 mi −mpi (2)
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

2 ffi
SE ¼ ∑ni¼1 mi −mpi =ðn−1Þ (3)
 
100 n mi −mpi 
MRE ð%Þ ¼ ∑ (4)
n i¼1 mi 
In the equations above, mi is the experimental value, mpi
is the predicted value, mmi is the average of the experimen-
tal values, and n is the number of observations. The fit of a
model to the EMC/ERH data was considered satisfactory if
the MRE was <10% [25, 26].
Classification of the moisture isotherm types of the
wheat bran products was conducted based on the general-
ized D'Arcy and Watt (GDW) model provided by Furmaniak
et al. [27]. In the GDW model (Table 2), m0 is the maximum
sorption value on the primary centres and w is a parameter
determining the ratio of molecules bonded to primary cen-
tres to those converted into secondary ones. K and k are ki-
netic constants. For type II (sigmoidal) and type III
isotherms, parameter w > 0 and K is greater or less than
unity, respectively.

Table 2
Moisture sorption equations used in this study.
Models Formula References
BET ðAþB∙T Þ∙C∙RH [28]
M ¼ ð1−RH Þð1−RHþC∙RH Þ
m B∙C∙RH
GAB M ¼ ð1−B∙RHM Þð1−B∙RHþC∙B∙RH Þ
[29]
GDW m0 ∙K∙RH 1−k∙ð1−wÞ∙RH [27]
M ¼ 1þK∙RH ∙ 1−k∙RH
h i
MCPE RH ¼ exp −A∙ exp ð−C∙M Þ
BþT
[30]

3-MGAB  12 [31]

2þðCTÞðM Þ
½2þðCTÞðMA −1Þ −4ð1−C=T Þ
A −1 − 2

RH ¼
h2Bð1−C=T Þ i
MHAE RH ¼ exp − exp MðAþB∙T Þ
C
[23]

MHE RH = 1 − exp [−A ∙ (T + B) ∙ M ] C

[32]
MOE RH ¼ 1þ½ðAþB∙T
1
Þ=M C
[33]
Polynomial M = A ∙ RH3 + B ∙ RH2 + C ∙ RH + D ∙ RH2 ∙ T + E ∙ RH ∙ T + F ∙ T + G [34]
Notes: GAB, Guggenheim-Anderson-de Boer; GDW, generalized D'Arcy and Watt; MCPE, modified Chung-Pfost equation; 3-MGAB, Modified GAB;
MHAE, modified Halsey equation; MHE, modified Henderson equation; MOE, modified Oswin equation; RH, equilibrium relative humidity (decimal);
M, moisture content (% d.b.); T, temperature (°C); Mm, monolayer moisture; A, B, C, D, E, F, G, K, k, m0 and w are the equation parameters.

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The moisture content required for the safe storage of
wheat bran products was analysed using the polynomial
equation in Table 2 [34].
2.4. Determination of the isosteric heat of sorption
where S is the surface area of the particles (m2/g solid), Mm
is the monomolecular layer water content (% dry basis), NA
is the Avogadro number (6.02 × 1023 mol-1), SW is the area
of a water molecule (10.6 × 10−20 m2), and MW is the mo-
lecular weight of water (18 g/mol).

The isosteric heat of sorption (hs) is the amount of energy 2.6. Texture profile analysis of cooked rice containing 200-mesh
required to change a unit mass of product from liquid to va- wheat bran product
pour at a particular temperature and water activity [35].
The hs for wheat bran products was assayed according to Two indica rough rice varieties, Yandao (YD) and
previous reports [36, 37] and calculated using the following Zhunliang You (ZLY), and one japonica variety, Wuyou
equations: (WY), were milled into rice and used for cooking tests
 with additions of 0, 1%, 3%, or 5% of 200-mesh wheat
hs p dT ∂RH 
¼1þ s ∙ ∙ (5) bran product. The YD rough rice was stored at room tem-
hv RH dps ∂T M
perature for two years, while the rough rice of ZLY and
WY was stored at 20 °C for half a year. The rough rice was
hv ¼ 2501:33−2:363T (6) milled into rice using a miller (LJJM 5588, Taizhou Grain
Instrument Company, China) by milling 150 g of rough

6  1025 6800 rice for 30 s.
ps ¼ ∙ exp − (7) The texture of the cooked rice was analysed using a hard-
ð273:15 þ T Þ5 T þ 273:15
ness and adhesiveness analyser (RHS-1A, Japanese Satake
 Manufacturing Co., Ltd., Suzhou, China) according to the
dps ps 6800
¼ ∙ −5 (8) method described by Liu et al. [39]. Five replicate aliquots
dT ðT þ 273:15Þ T þ 273:15 were tested on each sample and the values recorded from
( the resulting compression cycle for each test were used for
  )
∂RH  1 B∙C A þ B∙T C−1 texture profile analysis, which included the attributes of
¼ −n C o2 ∙ M (9)
∂T M
M hardness, adhesiveness, elasticity, and adhesiveness-to-
1 þ AþB∙T
M hardness ratio.

∂RH  A∙RH 2.7. Mixolab measurement of rice flour dough containing 200-
 ¼ ∙ exp ð−C∙M Þ (10) mesh wheat bran product
∂T M ðT þ BÞ2

where hs is the isosteric heat of sorption (kJ/kg), hv is the la-
tent heat of vaporization of free water (kJ/kg), and ps is the
saturated vapour pressure (Pa). Eq. (5) was used to calcu-

late the hs-to-hv ratio from dps/dT and ∂RH 
∂T M , which can be
determined using Eqs. (8) and (9), respectively. The hv of
free water in Eq. (6) is dependent on temperature. The sat-
urated vapour pressure (ps) was calculated using Eq. (7).
The term depends on the sorption isotherm equation used,
with this study adopting the modified Oswin equation
(MOE; Eq. (9)) and the modified Chung-Pfost equation
(MCPE; Eq. (10)).
2.5. Solid surface area
The solid surface area of the wheat bran particles was
analysed using the formula described by Moraes et al. [38]:
N A  Sw
S ¼ Mm  ¼ 3533:3M m (11)
Mw
The YD, WY and ZLY rice varieties were milled into rice
flour and mixed with 0, 1%, 3% or 5% of 200-mesh wheat
bran product for use in pasting behaviour tests. The milling
was performed using a high-speed universal pulverizer
(Kewei Yongxing Instrument Co., Ltd., Beijing, China),
which was rated at 1000 W, a 26,000 r/min rotation rate
and a 100–120-mesh degree of grinding. The rice flour
was kept at −20 °C until use.
The mixing and pasting behaviours of rice flour
dough were measured using a Mixolab (Chopin Tech-
nologies, Villeneuve la Garenne, France), as described
by Liu et al. [39] and Rosell et al. [40]. To perform
assays at a constant hydration, 55–60 g samples of
different rice flour types were placed into the Mixolab
bowl. Dough samples weighing 90 g with a 60% water
level (14% moisture basis) were evaluated. The param-
eters of interest were the protein weakness in the
constant-temperature phase (C1−C2), amylase activity
(C3 /C4 ), starch breakdown (C 3−C4 ), starch setback

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(C 5 −C 4), dough development time (DDT) and dough 3. Results and analysis
stability time (DST).
3.1. Experimental EMC/ERH data for wheat bran fibre prod-
2.8. Statistical analysis ucts

The results were statistically analysed using SPSS (ver-
sion 17.0, SPSS Inc., Chicago, USA). One-way analysis of
variance (ANOVA) and independent-samples t-tests were
used to compare multiple and pairs of means, respectively.
To observe the effects of several factors, a general linear
model–univariate method was used to compare the means
by the least significant difference (LSD) tests. Statistical sig-
nificance was declared at P < 0.05.
The equilibrium moisture contents of sorption were ob-
tained at nine relative humidities (ranging from 11.3%–
96.0%) and five temperatures (10, 20, 25, 30, and 35 °C)
for coarse, fine, 100-mesh, 200-mesh, 300-mesh, and active
wheat bran products, then fitted with MCPE models, as
shown in Fig. 1. The sorption curves for all wheat bran sam-
ples were sigmoidal in shape. At a constant temperature,
the EMC increased with increases in ERH, especially at

30 30

EMC (% d.b.)
EMC (% d.b.)

20 20

10 10

Coarse bran Fine bran

0 0

30 30

EMC (% d.b.)
EMC (% d.b.)

20 20

10 10

100-Mesh 200-Mesh
0 0

30
EMC (% d.b.)

30
EMC (% d.b.)

20
20

10 10

300-Mesh Active bran

0 0
0 20 40 60 80 0 20 40 60 80

ERH (%) ERH (%)

Fig. 1. The moisture sorption isotherms of six wheat bran products fitted with MCPE models.

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ERH = 70%. At a constant ERH, the EMC decreased with
increasing temperature. With increasing bran fineness, the
isotherms appeared to fall.
3.2. Fitting of sorption equations to experimental sorption data
The statistical parameters used to compare the model fits
(R2, RSS, SE, and MRE) were given in Tables 3, 4 and 5. Re-
sidual plots were also used to evaluate the goodness-of-fit of
the models. The commonly used equations, namely MCPE,
3-MGAB, MHAE, and MOE, as well as GAB and our devel-
oped polynomial, all provided the good fits to the experi-
mental sorption isotherm data of wheat bran products in
the range of 11.3%–96.0% ERH. The MHE equation pro-
vided the worst fits to most of the experimental data,
displaying a range of residual errors and high MRE values
of 10.08%–15.71% in the RH = f(M, T) form and
10.35%–15.70% in the M = f(RH, T) form.
Further comparisons of the better sorption equations
were made for seven sets of wheat bran product isotherm
data (Table 6), and average values of the R2 and error pa-
rameters (RSS, SE, and MRE) were calculated. For the
form of M = f (RH,T), the equations were ranked in the fol-
lowing order of accuracy (from highest to lowest): polyno-
mial, 3-MGAB, MOE, GAB, MHAE, MCPE, and MHE. In the
case of the RH = f (M,T) form, the order was 3-MGAB,
MOE, MHAE, MCPE, and MHE. The polynomial model in
the form of M = f (RH,T) and the 3-MGAB and MOE models
in the form of RH = f (M,T) well described the equilibrium
moisture data of the six species of wheat bran products in
the ERH range of 11.3%–96.0%. The coefficients of the
best-fitting equations are summarized in Tables 3 and 5.

Table 3
Fitting of equations of the form RH = f (M,T) to the moisture sorption isotherms of wheat bran products.
Equation parameters Statistical parameters
Models Samples Residue plot
A B C RSS SE R2 MRE (%)
MCPE Coarse bran 434.381 46.283 0.181 0.04153 9.89E-04 0.9873 5.0663 R
Fine bran 475.621 55.304 0.176 0.05338 1.27E-03 0.9836 6.2221 R
100-Mesh 538.419 79.553 0.172 0.09968 2.37E-03 0.9694 12.5791 P
200-Mesh 550.667 93.618 0.176 0.11090 2.64E-03 0.9659 13.4232 P
300-Mesh 680.007 92.916 0.181 0.09080 2.16E-03 0.9722 11.7264 P
Active bran 582.874 85.423 0.193 0.02637 6.28E-04 0.9919 4.0020 R
Average 448.469 67.172 0.176 0.05556 1.32E-03 0.9829 8.3322 R
3-MGAB Coarse bran 7.954 0.763 512.177 0.05616 1.337E-03 0.9828 5.9352 R
Fine bran 7.990 0.767 522.646 0.05603 1.334E-03 0.9828 6.0354 R
100-Mesh 6.752 0.836 1026.341 0.04214 1.003E-03 0.9871 6.7507 R
200-Mesh 6.079 0.866 874.076 0.04416 1.051E-03 0.9865 7.3503 R
300-Mesh 6.812 0.833 1346.039 0.03533 8.412E-04 0.9892 6.5335 R
Active-bran 7.041 0.756 438.341 0.03627 8.634E-04 0.9888 5.7261 R
Average 6.721 0.821 581.485 0.02684 6.389E-04 0.9918 4.5318 R
MHAE Coarse bran 5.347 −1.682E-02 2.166 0.04787 1.139E-03 0.9853 9.9669 R
Fine bran 5.219 −1.497E-02 2.122 0.05837 1.389E-03 0.9821 9.8229 R
100-Mesh 4.796 −1.216E-02 2.022 0.02507 5.969E-04 0.9923 5.6668 R
200-Mesh 4.271 −1.119E-02 1.877 0.03167 7.539E-04 0.9903 7.4625 R
300-Mesh 4.984 −1.037E-02 2.104 0.01954 4.652E-04 0.9941 5.5735 R
Active-bran 4.585 −9.489E-03 2.042 0.07973 1.898E-03 0.9756 13.3538 P
Average 4.508 −1.324E-02 1.934 0.01957 4.659E-04 0.9941 5.3282 R
MHE Coarse bran 5.222E-05 55.887 2.043 0.07838 1.866E-03 0.9759 9.4143 R
Fine bran 5.045E-05 62.186 2.016 0.08747 2.083E-03 0.9732 10.0823 R
100-Mesh 7.108E-05 84.927 1.815 0.13270 3.159E-03 0.9593 15.6865 P
200-Mesh 8.491E-05 105.519 1.729 0.13770 3.279E-03 0.9578 15.7178 P
300-Mesh 4.382E-05 104.581 1.937 0.13300 3.167E-03 0.9592 15.1874 P
Active bran 6.431E-05 82.522 1.949 0.03922 9.337E-04 0.9879 5.1685 R
Average 9.569E-05 69.095 1.798 0.08040 1.914E-03 0.9754 11.2955 R
MOE Coarse bran 14.003 −8.605E-02 3.071 0.02887 6.874E-04 0.9912 5.3351 R
Fine bran 14.023 −8.084E-02 3.030 0.03679 8.759E-04 0.9887 5.6957 R
100-Mesh 13.089 −6.676E-02 2.800 0.05147 1.225E-03 0.9842 9.3427 R
200-Mesh 11.993 −5.779E-02 2.615 0.05763 1.372E-03 0.9823 9.9274 R
300-Mesh 12.923 −5.397E-02 2.937 0.04749 1.131E-03 0.9854 9.0559 R
Active bran 11.677 −5.248E-02 2.909 0.02398 5.709E-04 0.9927 6.2326 R
Average 12.669 −7.321E-02 2.729 0.01942 4.623E-04 0.9941 4.7194 R
Notes: Average is the average EMC data of six wheat bran products; P and R represent patterns and randomness, respectively. The notes below are the
same.

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Table 4
Fitting of GAB equations of the form M = f (RH,T) to the moisture sorption isotherms of wheat bran products.
GAB parameters Statistical parameters
Samples Residue plot
Mm K C RSS SE R2 MRE (%)
Coarse bran 6.613 0.835 97.658 101.0890 2.4069 0.9592 7.7622 R
Fine bran 6.871 0.826 66.434 62.6231 1.4911 0.9743 7.3926 R
100-Mesh 6.559 0.848 66.397 112.9151 2.6885 0.9616 6.2677 R
200-Mesh 5.371 0.907 1.07E+06 117.8520 2.8059 0.9699 6.9025 R
300-Mesh 6.132 0.873 7.89E+07 37.1071 0.8835 0.9883 5.1483 R
Active bran 6.504 0.789 23.997 28.0960 0.6689 0.9829 5.8611 R
Average 6.096 0.855 53.878 48.3834 1.1519 0.9818 6.1959 R

Table 5
Fitting of polynomial equations to the moisture sorption isotherms of wheat bran products.
Polynomial equation parameters Statistical parameters
Samples
A B C D E F G RSS SE R2 MRE (%)
Coarse bran 100.292 −99.369 37.379 −0.9454 0.8282 −0.2199 6.006 39.5684 1.0400 0.9841 4.7571
Fine bran 98.498 −107.069 45.727 −0.5293 0.4624 −0.1543 4.484 22.6365 0.5960 0.9907 4.1345
100-Mesh 71.671 −44.483 11.323 −1.0020 0.7958 −0.1777 7.947 39.9221 1.0500 0.9864 3.6707
200-Mesh 131.391 −114.603 31.121 −1.3590 1.1570 −0.2399 6.074 84.3848 2.2200 0.9784 7.3394
300-Mesh 114.319 −112.317 39.206 −0.6677 0.5884 −0.1482 5.259 25.6047 0.6740 0.9919 4.7232
Active bran 74.106 −89.969 47.185 −0.1649 0.0831 −0.0483 1.039 8.1715 0.2151 0.9951 3.1752
Average 91.967 −88.038 35.030 −0.6603 0.5080 −0.1368 4.385 9.3625 0.2464 0.9965 3.5408

Table 6
Best-fitting equations for wheat bran product data.
Models Equation RSS SE R2 MRE (%) Order
M = f (RH,T) GAB 73.8483 1.7583 0.9738 6.5506 4
MCPE 139.2196 3.3147 0.9550 11.0416 6
MOE 56.5710 1.3469 0.9806 5.6875 3
MHE 124.6577 2.9681 0.9579 11.9113 7
MHAE 76.0619 1.8109 0.9721 7.2926 5
3-MGAB 68.0027 1.6191 0.9762 5.5581 2
Polynomial 33.7676 0.8886 0.9887 4.5639 1
RH = f (M,T) MCPE 0.0700 0.0017 0.9785 8.8975 4
MOE 0.0396 0.0009 0.9878 7.3864 2
MHE 0.1018 0.0024 0.9687 11.0532 5
MHAE 0.0403 0.0010 0.9876 8.1709 3
3-MGAB 0.0435 0.0010 0.9867 6.2858 1

These calculated coefficients can be used to describe the
process of wheat bran product dehydration and improve
the physical control of moisture during storage.
As shown in Table 4, compared to fine bran, extrusion
and ultrafine grinding reduced the Mm value and tended
to increase the value of the K constant in the GAB equation.
3.3. Prediction of moisture sorption isotherms by the best fitting
equation
The predicted sorption isotherms of the wheat bran prod-
ucts at 20 °C and 30 °C based on the MOE, MHAE, and MCPE
models were displayed in Fig. 2. Temperature had a relatively
strong effect on the sorption isotherms predicted by the MOE
and MHAE models at ERH values higher than 30% and 45%,
respectively, whereas the effect was weak at lesser values.
The isotherms predicted by the MCPE model could distin-
guish temperature effects in the range of 11.3%–96.0% ERH.
The sorption isotherms predicted by the three models
showed that, in comparison to the coarse and fine bran,
the wheat bran products subjected to ultrafine grinding,
like the 200-mesh and active bran, had lower EMC values
at a given temperature and 60%–70% ERH. With increasing
temperature at a certain EMC, the 200-mesh and active
bran products showed higher ERH values than the coarse
and fine bran products.
Table 7 showed the classification of the moisture sorp-
tion isotherms of wheat bran products. In the range of

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MOE MHAE MCPE

35 35
30 30
25 25
E MC (% d.b.)

EMC (% d.b.)
20 20
15 15
10 10
5 5
Coarse bran Coarse bran Coarse bran
0 0
35 35

30 30

25 25
EMC (% d.b.)

EMC (% d.b.)
20 20

15 15

10 10

5 5
Fine bran Fine bran Fine bran
0 0
40 40
35 35
30 30

EMC (% d.b.)
EMC (% d.b.)

25 25
20 20
15 15
10 10
5 200-Mesh 5
200-Mesh 200-Mesh
0 0
30 30

25 25

EMC (% d.b.)
EMC (% d.b.)

20 20

15 15

10 10

5 5
Active bran Active bran Active bran
0 0
0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
ERH (%) ERH (%) ERH (%)

Fig. 2. The 20 °C and 30 °C moisture sorption isotherms of wheat bran products predicted by MOE, MHAE, and MCPE models.

10–35 °C, parameter w in the GDW model was >0, and K
was >1.0, indicating that the moisture sorption isotherms
of all six wheat bran products belonged to type II.
Table 8 showed the safe storage EMCs of wheat bran
products at 25 °C. Wheat bran products are often stored in
air at a humidity range of 60%–70%. At 25 °C and 70%
ERH, the relatively safe storage EMCs of coarse and fine
brans were predicted to be 13.27% and 13.47% by the poly-
nomial, 14.07% and 14.21% by the MGAB, 13.69% and
13.86% by the MOE model, respectively. After enzymolysis,
extrusion and ultrafine grinding, these three equations pre-
dicted the relative safe storage EMCs of 200- and 300-
mesh products to be 12.57% and 13.26%, 13.16% and
13.89%, 13.34% and 13.97%, respectively. The correspond-
ing values in active bran were 11.99%, 12.46% and 12.03%,
respectively. At ERH of 65% or 70%, in comparison to
coarse and fine bran products, the safe moisture content of
200-mesh and active bran products significantly decreased.

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Table 7
Classification of moisture sorption isotherms of wheat bran products.
Parameters of GDW model Statistical parameters
Samples Isotherm type
m0 (% d.b.) K k w R2 MRE (%)
Coarse bran 11.837 8.151 0.931 0.288 0.9727 6.7455 II
Fine bran 10.708 10.398 0.899 0.403 0.9796 6.3528 II
100-Mesh 5.945 276.556 0.834 1.222 0.9619 6.3021 II
200-Mesh 6.703 32.019 0.924 0.701 0.9707 6.5652 II
300-Mesh 7.514 40.479 0.899 0.679 0.9895 4.7379 II
Active bran 11.035 5.812 0.882 0.349 0.9876 4.5393 II
Average 7.898 15.963 0.884 0.649 0.9829 5.8478 II
Note: Average, the average EMC data of six wheat bran products.

Table 8
Safe EMCs of wheat bran products at 25 °C.
EMC (% w.b.)
Samples Polynomial MGAB MOE GAB Mean2
ERH 65% ERH 70% ERH 65% ERH 70% ERH 65% ERH 70% ERH 65% ERH 70% ERH 65% ERH 70%
a
Coarse bran 12.15 13.27 13.07 14.07 12.81 13.69 12.54 13.64 12.64 ± 0.39 13.67 ± 0.33a
Fine bran 12.35 13.47 13.20 14.21 12.98 13.86 12.78 13.88 12.83 ± 0.36a 13.86 ± 0.31a
100-Mesh 12.36 13.70 12.66 13.79 12.88 13.82 12.62 13.77 12.63 ± 0.21a 13.77 ± 0.05a
200-Mesh 11.04 12.57 11.98 13.16 12.24 13.34 11.57 12.82 11.71 ± 0.52bc 12.97 ± 0.34b
300-Mesh 11.88 13.26 12.76 13.89 12.96 13.97 12.42 13.62 12.51 ± 0.47ab 13.69 ± 0.32a
Active bran 11.07 11.99 11.56 12.46 11.28 12.03 11.38 12.32 11.32 ± 0.20c 12.20 ± 0.23c
Average1 11.56 12.81 12.20 13.28 12.21 13.13 11.91 13.04 11.97 ± 0.31b 13.07 ± 0.20b
Notes: 1Average, the average EMC data of six wheat bran products. 2Data are presented as the mean ± SD from four values calculated from polynomial,
MGAB, MOE, and GAB models. For different bran species at the same ERH, values followed by different superscript letters are significantly different at
P ≤ 0.05, according to Duncan's multiple range test.

The general linear model–univariate analysis further 3.4. Isosteric heat of sorption in wheat bran products
showed that both the species of wheat bran product and
the ERH significantly influenced the safe moisture content The isosteric heat of sorption (hs) was calculated using
(Table 9). The safe EMCs of fine bran, 200-mesh, and active Eqs. (5)–(10). The influence of moisture content on the hs
bran products at ERH 65%–70% were 13.34%, 12.34%, and values of wheat bran products, as determined by the sorp-
11.76%, respectively. Thus, extrusion and ultrafine grinding tion isotherms, was shown in Fig. 3. When the MOE
processes decreased the safe EMC of wheat bran products. model was employed to calculate the hs of wheat bran prod-
ucts, Eq. (9) was adopted. At MC values <8%, hs at
Table 9 20–30 °C increased rapidly with increases in moisture con-
Safe moisture content of different species of wheat bran products at dif- tent in the coarse, fine and active bran products. Further in-
ferent ERHs. creases in MC resulted in a decrease in hs, which was rapid
Factors Levels Safe moisture content (% w.b.) in the range of 8%–25% MC but became stable above 25%
Bran species Coarse bran 13.16 ± 0.11ab MC. At MC < 15%, temperature only had a small effect
Fine bran 13.34 ± 0.11a on the hs values. Above MC 15%, hs appeared to correlate
100-Mesh 13.20 ± 0.11ab negatively with temperature. After extrusion and ultrafine
200-Mesh 12.34 ± 0.11c
300-Mesh 13.10 ± 0.11b
grinding, the sorption isosteric heat curves of the wheat
Active bran 11.76 ± 0.11d bran products fell.
Average 12.52 ± 0.11c When the MCPE model (Eq. (10)) was adopted, hs de-
ERH (%) 65 12.23 ± 0.06b creased parabolically and rapidly as the MC of the wheat
70 13.32 ± 0.06a
bran products increased up to 22.5%. Above 22.5%, further
Notes: Average, the average EMC data of six wheat bran products. For dif- increases in MC led to a smooth decrease in hs. At MC values
ferent bran species and two equilibrium relative humidity (ERH), values
in the studied range, hs was negatively correlated with tem-
followed by different superscript letters are significantly different
at P ≤ 0.05, according to Duncan's multiple range test and t-test, perature. The hs values of the four wheat bran products
respectively. were close to those of pure water at an MC of 25%. From

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MOE MCPE

3800 3800
3600 3600

3400 3400

3200 3200
3000 3000

Is osteric heat of sorption (kJ/kg)

Isosteric heat of sorption (kJ/kg)

2800 2800

2600 2600
Coarse bran Coarse bran
2400 2400
3600 3600

3400 3400

3200 3200

3000 3000

2800 2800

2600 2600
Fine bran Fine bran
2400 2400
3400 3400

3200 3200

3000 3000

Isosteric heat of sorption (kJ/kg)

Isosteric heat of sorption (kJ/kg)

2800 2800

2600 2600
200-Mesh 200-Mesh
2400 2400
3400 3400

3200 3200

3000 3000

2800 2800

2600 2600
Active bran Active bran
2400 2400
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35

MC (% d.b.) MC (% d.b.)

Fig. 3. Changes in the 20 °C and 30 °C isosteric heat of moisture sorption in wheat bran products, as predicted by MOE (left) and MCPE (right) equations.

5% to 20% MC, the wheat bran products made by extrusion isosteric heat values of monolayer, multilayer, and con-
and ultrafine grinding had lower isosteric heat values than densed water with 0.20, 0.70, and 0.96 water activity (aw)
untreated products at a given temperature. values in the temperature range of 10–37 °C, respectively.
General linear model–univariate analysis (Table 10)
3.5. Sorption isosteric heat in the monolayer, multilayer and showed that the wheat bran product species and tempera-
condensed water ture had significant influences on the sorption equilibirum
moisture content (EMC) and isosteric heat in the mono-
The MOE, MHAE and MCPE models in the form of M = f layer, multilayer and condensed water of wheat bran prod-
(RH, T) were used to calculate the EMC and sorption ucts. When compared with coarse and fine bran, the EMCs

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Table 10
Comparison of EMC and isosteric heat of sorption in the monolayer, multilayer and condensed water in wheat bran product.
EMC of sorption (% w.b.) Sorption isosteric heat (kJ/kg)
Factors Levels
Monolayer Multilayer Condensed water Monolayer Multilayer Condensed water
ab bc d a a
Bran species Coarse bran 7.01 ± 0.16 13.80 ± 0.14 25.48 ± 0.31 3356 ± 23 2672 ± 9 2476 ± 3a
Fine bran 7.16 ± 0.16a 13.97 ± 0.14ab 25.59 ± 0.31d 3250 ± 23b 2653 ± 9a 2470 ± 3ab
100-Mesh 6.74 ± 0.16bc 13.97 ± 0.14a 26.51 ± 0.31c 3078 ± 23c 2601 ± 9c 2463 ± 3c
200-Mesh 5.53 ± 0.16e 13.64 ± 0.14c 28.71 ± 0.31a 3039 ± 23cd 2577 ± 9d 2456 ± 3d
300-Mesh 6.56 ± 0.16c 14.17 ± 0.14a 27.67 ± 0.31b 3008 ± 23de 2572 ± 9d 2456 ± 3d
Active bran 6.27 ± 0.16cd 12.09 ± 0.14e 22.17 ± 0.31e 2992 ± 23e 2597 ± 10c 2466 ± 3bc
Average 6.24 ± 0.16d 13.29 ± 0.14d 25.78 ± 0.31d 3155 ± 23f 2625 ± 9b 2466 ± 3bc
Temp. (°C) 10 7.16 ± 0.16a 14.35 ± 0.14a 27.02 ± 0.31a 3112 ± 23a 2633 ± 9a 2497 ± 3a
15 6.92 ± 0.16ab 14.07 ± 0.14b 26.65 ± 0.31ab 3115 ± 23a 2629 ± 9a 2486 ± 3b
20 6.69 ± 0.16bc 13.79 ± 0.14bc 26.30 ± 0.31bc 3118 ± 23a 2621 ± 9a 2475 ± 3c
25 6.47 ± 0.16cd 13.53 ± 0.14cd 25.95 ± 0.31cd 3123 ± 23a 2613 ± 10a 2463 ± 3d
30 6.25 ± 0.16de 13.27 ± 0.14de 25.60 ± 0.31de 3130 ± 23a 2606 ± 9a 2452 ± 3e
35 6.05 ± 0.16e 13.01 ± 0.14ef 25.26 ± 0.31e 3138 ± 23a 2599 ± 9a 2441 ± 3f
37 5.96 ± 0.16e 12.91 ± 0.14f 25.12 ± 0.31e 3141 ± 23a 2596 ± 9a 2440 ± 3f
Notes: Average, the average EMC data of six wheat bran products. Data are presented as the mean ± SD. For different bran species and temperatures, the
same column values followed by different superscript letters are significantly different at P ≤ 0.05, according to Duncan's multiple range test.

of monolayer water in extruded and ultrafine-ground sam-
ples, such as the 100-, 200-, and 300-mesh bran products,
significantly decreased, while the EMCs of condensed
water in these samples significantly increased. The EMCs
of the monolayer, multilayer and condensed water in the
active bran product were significantly lower than those of
fine bran. The EMCs of the monolayer, multilayer and con-
densed water in all wheat bran products significantly de-
creased with increasing temperature.
The heat of sorption at the monolayer water for the six
wheat bran products were in the range of
2990–3380 kJ/kg water, compared to approximately
2610 kJ/kg in multilayer water and 2465 kJ/kg in
condensed water. When compared with coarse and fine
bran, the sorption isosteric heat of monolayer, multilayer
and condensed water in extruded and ultrafine-ground
samples significantly decreased. In the range of 10–37 °C,
the sorption isosteric heat of condensed water in the
wheat bran products decreased significantly with increas-
ing temperature, but the sorption isosteric heat of mono-
layer and multilayer water did not change significantly.
3.6. Solid surface area in wheat bran products
The GAB, 3-MGAB and BET models in the form of
M = f(RH, T) were used to calculate the Mm and solid
surface area of the wheat bran product particles. In con-
trast to the GAB model, the temperature effect was con-
sidered in the 3-MGAB model, and the Mm values under
the same condition were similar when calculated by
GAB and 3-MGAB. At a given temperature, extrusion
and ultrafine grinding treatments tended to decrease
the Mm values (Table 11). The Mm values of the 200-
and 300-mesh bran products were significantly lower
than those of fine bran. With increases in temperature,
the Mm values of a given bran product tended to de-
crease.
At a given temperature, extrusion and ultrafine grinding
treatments significantly decreased the surface area of the
granular solids in the wheat bran products (Table 11). Com-
pared with coarse and fine bran, the 200-mesh bran product
had the lowest solid surface area at 10–30 °C. With increas-
ing temperature, the solid surface area of wheat bran prod-
ucts tended to decrease.
General linear model–univariate analysis (Table 12)
was used to further show the effects of product species,
temperature, and equation type on the Mm values and sur-
face areas of the granular solids. From coarse bran to 300-
mesh bran, the Mm values significantly decreased with the
strength of ultrafine grinding treatment. The Mm value at
35 °C was significantly lower than those at 10–30 °C.
The Mm values calculated by the GAB and MGAB equa-
tions were similar and 2.8%–21.9% higher than those cal-
culated by BET.
From coarse bran to 300-mesh bran, the surface area of
the granular solid significantly decreased with the strength
of ultrafine grinding treatment. When calculated by GAB
and MGAB equations, the surface area of the granular
solid at 35 °C was significantly lower than those at
10–30 °C, but the surface area calculated by BET signifi-
cantly decreased with increasing temperature. The surface
areas of the granular solid calculated by the GAB and
MGAB equations were similar and were 8.1%–20.7%
higher than those calculated by BET.

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X. Li et al. Grain & Oil Science and Technology 4 (2021) 149–164

Table 11
The Mm values and surface area of wheat bran products.
Parameters Samples 10 °C 20 °C 25 °C 30 °C 35 °C
a ab a ab
Mm (% w.b.) Coarse bran 6.97 ± 0.35 7.09 ± 0.37 7.38 ± 0.49 7.05 ± 0.33 6.80 ± 0.27a
Fine bran 7.07 ± 0.51a 7.45 ± 0.37a 7.35 ± 0.42a 7.48 ± 0.51a 6.42 ± 0.57abc
100-Mesh 7.01 ± 0.85a 7.13 ± 0.78abc 7.09 ± 0.49ab 6.89 ± 0.58abc 6.43 ± 0.35ab
200-Mesh 5.87 ± 0.24b 5.66 ± 0.170d 5.31 ± 0.14c 6.29 ± 0.44bc 6.28 ± 0.56abc
300-Mesh 6.40 ± 0.29b 6.35 ± 0.25c 6.61 ± 0.32ab 6.39 ± 0.24c 6.03 ± 0.25bc
Active bran 6.86 ± 0.27a 6.77 ± 0.32abc 7.19 ± 0.30a 6.55 ± 0.34bc 6.11 ± 0.46abc
Average 6.55 ± 0.22a 6.60 ± 0.32bc 6.44 ± 0.21b 6.26 ± 0.22c 5.73 ± 0.18c
Surface area (m2/g solid) Coarse bran 246.1 ± 12.3a 250.3 ± 13.1ab 260.9 ± 17.2a 249.0 ± 11.5ab 240.2 ± 9.7a
Fine bran 249.7 ± 18.1a 263.4 ± 12.9a 259.7 ± 14.7a 264.4 ± 18.0a 226.9 ± 20.2ab
100-Mesh 247.8 ± 30.1a 251.9 ± 27.5abc 250.5 ± 17.1ab 243.6 ± 20.6abc 227.3 ± 12.3ab
200-Mesh 207.3 ± 8.4b 200.0 ± 6.0d 187.7 ± 4.8c 222.4 ± 15.7bc 222.0 ± 19.8ab
300-Mesh 226.3 ± 10.1a 224.2 ± 8.9c 233.7 ± 11.4ab 225.7 ± 8.4c 212.9 ± 8.7b
Active bran 242.3 ± 9.5a 239.1 ± 11.4bc 254.0 ± 10.7a 231.3 ± 11.9bc 215.7 ± 16.1ab
Average 231.6 ± 7.6a 233.0 ± 11.2bc 227.4 ± 7.3b 221.1 ± 7.7abc 202.6 ± 6.5ab
Notes: 3-MGAB equation was used to calculate the Mm and surface area. Average, the average EMC data of six wheat bran products. Data are presented
as the mean ± SD. For different bran species at the same temperature, the same column values followed by different superscript letters are significantly
different at P ≤ 0.05, according to Duncan's multiple range test.

Table 12
Effects of bran species, temperature and models on the Mm and surface area values of the granular solids of wheat bran products.
Mm (MGAB and GAB) Surface area (MGAB and GAB) Mm (BET) Surface area (BET)
Factors Levels
(% d.b.) (m2/g solid) (% d.b.) (m2/g solid)
Bran species Coarse bran 7.02 ± 0.09ab 247.8 ± 3.1ab 6.14 ± 0.16a 216.8 ± 1.4b
Fine bran 7.13 ± 0.09a 251.7 ± 3.1a 6.10 ± 0.16a 219.8 ± 1.4a
100-Mesh 6.87 ± 0.09b 242.9 ± 3.1b 5.88 ± 0.16ab 201.3 ± 1.4d
200-Mesh 5.84 ± 0.09e 206.0 ± 3.1e 5.68 ± 0.16bc 183.9 ± 1.4f
300-Mesh 6.31 ± 0.09d 223.1 ± 3.1d 5.68 ± 0.16bc 206.1 ± 1.4c
Active bran 6.67 ± 0.09c 235.4 ± 3.1c 5.47 ± 0.16cd 198.9 ± 1.4d
Average 6.35 ± 0.09d 224.5 ± 3.1d 5.29 ± 0.16d 195.1 ± 1.4e
Temp. (°C) 10 6.68 ± 0.07a 235.9 ± 2.6a 5.92 ± 0.13a 218.3 ± 1.2a
20 6.74 ± 0.07a 238.2 ± 2.6a 5.81 ± 0.13ab 207.5 ± 1.2b
25 6.76 ± 0.07a 238.9 ± 2.6a 5.75 ± 0.13ab 202.1 ± 1.2c
30 6.65 ± 0.07a 234.9 ± 2.6a 5.68 ± 0.13ab 196.6 ± 1.2d
35 6.15 ± 0.07b 217.4 ± 2.6b 5.59 ± 0.13b 191.2 ± 1.2e
Model GAB 6.57 ± 0.05a 232.1 ± 1.6a
MGAB 6.62 ± 0.05a 234.1 ± 1.6a
Notes: Average, the average EMC data of six wheat bran products. Data are presented as the mean ± SD. For different bran species and temperatures, the
same column values followed by different superscript letters are significantly different at P ≤ 0.05, according to Duncan's multiple range test. For GAB
and MGAB models, values followed by different superscript letters are significantly different at P ≤ 0.05, according to t-test.

3.7. Effect of 200-mesh wheat bran product addition on the tex-
ture of cooked rice and the thermomechanical properties of rice
flour dough
In the present study, the hardness, adhesiveness, balance
and elasticity of the three Chinese varieties of cooked rice
respectively were 4.06–5.86 kgf, 0.51–0.76 kgf, 0.09–0.19
kgf, and 0.69–0.72 kgf in the control samples, while the tex-
ture attributes of those with added wheat bran were
4.88–7.21 kgf, 0.23–1.29 kgf, 0.06–0.38 kgf, and
0.67–0.73 kgf, respectively. The addition of 1%, 3% or 5%
of 200-mesh wheat bran to rice increased the hardness of
cooked rice and appeared to increase its adhesiveness,
especially for milled WU and ZLY rice stored for half a
year, without a significant influence on its elasticity. Gen-
eral linear model–univariate analysis further showed that
the three varieties of milled rice had different texture pa-
rameters when cooked; in comparison to the control sam-
ple, the addition of 1%–5% 200-mesh bran product to rice
increased its cooked hardness and maintained or even in-
creased the adhesiveness-to-hardness ratio and adhesive-
ness (Table 13).
The addition of 200-mesh bran product affected the
thermomechanical properties of rice flour dough at con-
stant hydration. The addition of 1%–3% of this product to
rice flour almost maintained the dough development time
(DTT), amylase activity (C3/C4), starch breakdown (C3–
C4) and starch retrogradation (C5–C4). These additions

160
X. Li et al.
Table 13
Effects of rice variety and content of 200-mesh bran product on the texture of cooked rice.
Hardness
Factors Levels
(kgf)
Rice variety YD 6.37 ± 0.09a
WY 6.39 ± 0.09a
ZLY 5.03 ± 0.09b
Addition amount (%) CK 5.05 ± 0.10c
1 5.87 ± 0.10b
3 5.99 ± 0.10b
5 6.82 ± 0.10a
Notes: YD, Yandao; WY, Wuyou; ZLY, Zhunliang You; CK, the control sample without wheat bran addition. Data are presented as the mean ± SD. For
rice varieties and the addition amount of 200-mesh wheat bran product, the same column values followed by different superscript letters are signifi-
cantly different at P ≤ 0.05, according to Duncan's multiple range test.
seemed to decrease the dough stability time (DST), starch
gelatinization maximum torque (C3), and gelatinization
speed (β) while increasing the torque (C1–C2) and speed
(α) of protein weakness during a Mixolab test. A 5% addi-
tion decreased the β, γ (enzymatic degradation speed) and
C3 values. The effect of adding 200-mesh bran product is es-
pecially useful for improving the thermomechanical proper-
ties of rice flour dough processed from WU and ZLY rice
stored for half a year. General linear model–univariate anal-
ysis (Table 14) showed the differences between the
thermomechanical properties of three varieties of rice
flour dough. After addition of 1%–3% 200-mesh bran prod-
uct, in comparison to the control sample, the DDT, C3/C4
and C3–C4 of the rice dough were maintained, but the
DST, C3 and β decreased significantly, suggesting that
such addition decreased the starch gelatinization speed
and maximum torque of rice dough. The 5% addition in-
creased C1–C2 and α but decreased γ, implying an increase
in protein weakness speed and, subsequently, a reduction
in enzymatic degradation speed.
4. Discussion
The prophylactic value attributed to certain food fibres
in regulating colonic function has received the attention of
the food industry [41–43]. Thus, we measured the hygro-
scopic properties of wheat bran products processed by ex-
trusion and ultrafine grinding. The moisture absorption
isotherm of wheat bran products modeled by MCPE showed
a linear region at ERH of 20%–80%, while the MOE and
MHAE models showed this at ERH of 20%–70%.
The physical structure and theoretical surface area play
important roles in determining the water-binding proper-
ties of a specific product [28, 38]. We calculated the solid
surface area of wheat bran products to explore this relation-
ship. In the temperature range of 10–35 °C and when using
the GAB and MGAB models, the specific surface areas of the
coarse, fine, 100–300-mesh, and active bran products were
247.8, 251.7, 242.9–223.1, and 235.4 m2/g solid,
Grain & Oil Science and Technology 4 (2021) 149–164
Adhesiveness Ratio of adhesiveness to hardness Elasticity
(0.1 kgf) (0.1) (0.1 kgf)
4.61 ± 0.53b 0.83 ± 0.20b 6.94 ± 0.07b
5.18 ± 0.53b 1.59 ± 0.20a 7.19 ± 0.07a
8.65 ± 0.53a 1.73 ± 0.20a 6.88 ± 0.07b
5.98 ± 0.61b 1.23 ± 0.23b 7.02 ± 0.08ab
6.33 ± 0.61ab 1.10 ± 0.23b 6.93 ± 0.08b
4.79 ± 0.61c 1.00 ± 0.23b 7.06 ± 0.08a
7.49 ± 0.61a 2.19 ± 0.23a 7.01 ± 0.08ab
respectively. These values are smaller than those of grapes
(481.3 m2/g solid) and apples (544.6 m2/g solid) at 30 °C
[14]. The large surface area of many biopolymers can be at-
tributed to the existence of an intrinsic microporous struc-
ture in these food materials. We further found that the
surface areas of wheat bran products calculated by the
GAB and MGAB equations were 8.1%–20.7% higher than
those calculated by BET. The BET estimates decreased sig-
nificantly with increasing temperature. The extruded and
ultrafine-ground wheat bran products had smaller solid sur-
face areas than fine bran, making them taste more smooth
and soft.
When dietary fibre and its associated water progresses
through the gastrointestinal tract, free water is released
from the fibre surface, leaving multiple and single layers
of water. The loosely held water of multilayers readily in-
creases stool weight [44, 45]. In the present study, com-
pared with fine bran, with increases in the strength of
extrusion and ultrafine grinding, the EMC of multilayer
and condensed water in the 100-, 200-, and 300-mesh
wheat bran products increased in the temperature range
of 10–37 °C, while the EMC of monolayer water decreased.
The sorption isosteric heats of monolayer, multilayer and
condensed water significantly decreased with decreases in
bran particles. Thus, the extrusion and ultrafine grinding
treatments changed the physical characteristics of wheat
bran fibre by reducing the solid surface area of particles
and the sorption isosteric heat of the monolayer, multilayer
and condensed water. Hence, the function of the multilayer
water region should be further evaluated.
The energy required by different fibres to absorb water is
of physiological interest [45]. The present study shows that
the isosteric heats of sorption are differential quantities that
change inversely with the amount of water vapour
adsorbed by the solid. All wheat bran samples had signifi-
cantly higher sorption isosteric heats in the monolayer re-
gion than in the condensed water region, indicating that
the water held by the monolayer region is more reliable
than that held by the condensed water region. As the
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 X. Li et al.

Table 14
Effects of rice variety and content of 200-mesh bran product on the thermomechanical properties of rice flour dough.
DDT DST C1–C2 C3 C3/C4 C3–C4 C5–C4 α β γ
Factors Levels
(min) (min) (0.1 Nm) (Nm) (Nm) (Nm) (−0.1 Nm/min) (0.1 Nm/min) (−0.1 Nm/min)
Rice variety YD 3.41 ± 0.63b 6.51 ± 0.25b 7.63 ± 0.08a 2.41 ± 0.02a 1.31 ± 0.02a 0.58 ± 0.02a 1.13 ± 0.02a 0.57 ± 0.04c 3.22 ± 0.07b 0.54 ± 0.04a
WY 6.88 ± 0.63a 8.71 ± 0.25a 3.35 ± 0.08c 2.02 ± 0.02c 1.16 ± 0.02b 0.27 ± 0.02c 0.81 ± 0.02b 0.25 ± 0.04a 3.22 ± 0.07b 0.47 ± 0.04a
ZLY 7.61 ± 0.63a 9.12 ± 0.25a 4.45 ± 0.08b 2.10 ± 0.02b 1.29 ± 0.02a 0.47 ± 0.02b 0.80 ± 0.02b 0.41 ± 0.04b 4.60 ± 0.07a 0.53 ± 0.04a
Addition amount (%) CK 4.94 ± 0.73b 9.18 ± 0.28a 4.62 ± 0.09b 2.31 ± 0.02a 1.23 ± 0.02b 0.43 ± 0.02b 0.93 ± 0.02b 0.71 ± 0.05c 4.33 ± 0.09a 0.37 ± 0.04a

162
1 6.12 ± 0.73ab 7.11 ± 0.28d 4.91 ± 0.09a 2.18 ± 0.02b 1.22 ± 0.02b 0.39 ± 0.02b 0.98 ± 0.02a 0.52 ± 0.05b 3.59 ± 0.09b 0.40 ± 0.04a
3 5.94 ± 0.73ab 7.70 ± 0.28c 4.98 ± 0.09a 2.13 ± 0.02c 1.25 ± 0.02b 0.43 ± 0.02b 0.94 ± 0.02ab 0.19 ± 0.05a 3.63 ± 0.09b 0.67 ± 0.04b
5 6.86 ± 0.73a 8.47 ± 0.28b 5.07 ± 0.09a 2.08 ± 0.02d 1.31 ± 0.02a 0.51 ± 0.02a 0.80 ± 0.02c 0.23 ± 0.05a 3.18 ± 0.09c 0.61 ± 0.04b
Notes: YD, Yandao; WY, Wuyou; ZLY, Zhunliang You; CK, the control sample without wheat bran addition. Mixolab assays were carried out at a constant water level (60% flour basis). Data are presented
as the mean ± SD of triplicate experiments. For rice varieties and the addition of 200-mesh wheat bran product, the same column values followed by different superscript letters are significantly different
at P ≤ 0.05, according to Duncan's multiple range test. DDT, dough development time (min); DST, dough stability time (min); C1–C2, protein weakening (0.1 Nm); C3, starch gelatinization maximum
torque (Nm); C3/C4, amylase activity; C3–C4, starch breakdown (Nm); C5–C4, starch retrogradation (Nm); α, protein weakness speed due to heating (−0.1 Nm/min); β, gelatinization speed
(0.1 Nm/min); γ, enzymatic degradation speed (−0.1 Nm/min).
Grain & Oil Science and Technology 4 (2021) 149–164
X. Li et al.
temperature increased from 10 °C to 37 °C, the sorption
isosteric heats in the monolayer region of the wheat bran
products showed an increasing trend, while those in the
multilayer water region decreased slightly, but in the con-
densed water region they significantly decreased. With in-
creases in the strength of extrusion and ultrafine grinding,
the sorption isosteric heats in the monolayer, multilayer
and condensed water regions of the wheat bran products
were all reduced, indicating that the wheat bran particle
size affects the water adsorption behaviour in these water
regions, thereby affecting their physiological function. The
effect of changes in the porosity of wheat bran fibre on
water adsorption in the multilayer water region deserves
further study by magnetic resonance imaging.
In the evaluation of cooked rice for texture and consumer
acceptability, hardness and adhesiveness are two important
parameters [46]. In the present study, cooked rice contain-
ing 1%–3% of 200-mesh bran product maintained its adhe-
siveness, elasticity and adhesiveness-to-hardness ratio. This
suggests that it is feasible to increase the dietary fibre
content of cooked rice by adding wheat bran products.
The hardness and adhesiveness of cooked rice are consid-
ered to be related to the hydration process of starch
granules [47]. Thus, in the present study, a Mixolab device
was used to evaluate the mixing and pasting behaviour
of rice flour dough after addition of 200-mesh bran product.
Despite decreases in the speed and maximum torque
of starch gelatinization in the rice dough, the addition of
1%–3% 200-mesh bran product to the rice flour processed
from rough rice after being stored for half a year maintained
its DDT, C1–C2, C3/C4, C3–C4 and C5–C4 values. This indi-
cates that such modification maintains the texture of
cooked rice and the thermomechanical properties of rice
flour dough.
5. Conclusions
All six Chinese wheat bran products had sigmoidal iso-
therms, to which the good fitting models were the polyno-
mial, 3-MGAB, MOE and MHAE equations. The relative
safe moisture contents of 200-mesh and active bran prod-
ucts were 12.97% and 12.20% w.b., respectively.
The extrusion and ultrafine grinding process changed the
physical characteristics of wheat bran products by reducing
the solid surface area of the particles and the sorption
isosteric heats in the monolayer, multilayer and condensed
water regions. In the temperature range of 10–37 °C, the
EMC in the monolayer water region of wheat bran products
decreased with decreasing particle size, while the concen-
tration of water in the condensed water region increased.
The sorption isosteric heats of monolayer, multilayer and
condensed water significantly decreased with decreasing
bran particle size. The effect of the porosity of wheat bran
163
Grain & Oil Science and Technology 4 (2021) 149–164
fibre on the function of the multilayer water regions de-
serves further study.
The milled rice processed from the rough rice after being
stored for half a year to which 1%–3% 200-mesh wheat
bran product was added maintained its texture when
cooked, while rice flour dough made from this mixture
retained good thermomechanical properties.
Author Contributions
Xingjun Li: Writing, reviewing & editing, methodology,
investigation, supervision, results interpretation, data
curation; Xu Han: Methodology, data collection; Lisi Tao:
Methodology, data collection; Ping Jiang: Methodology;
Wen Qin: Supervision, reviewing.
Conflits of Interest
The authors declare that there are no conflicts of interest.
Acknowledgement
The authors would like to acknowledge the Operating
Expenses of Basic Scientific Research Project of Central
Public-interest Scientific Institution, China (JY2007) and
the Special Fund for Grain Scientific Research in the Public
Interest of the State Administration of Grains, China
(201313001-03-01). We appreciate Mr. Liang Zhang and
Ruiping Bai for giving sincerely suggestion and helps. We
also appreciate the editor and three anonymous reviewers
giving invaluable suggestion.
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 LWT - Food Science and Technology 167 (2022) 113866

Contents lists available at ScienceDirect

LWT
journal homepage: www.elsevier.com/locate/lwt

Moisture sorption isotherms and thermodynamic properties of tiger nuts:

An oil-rich tuber
Zhen-shan Zhang *, Xiao-dan Li , Hui-jie Jia , Yu-lan Liu
College of Food Science and Technology, Henan University of Technology, NO.100, Lianhua Street, Zhengzhou, 45001, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: The moisture adsorption isotherms of tiger nuts were determined by a gravimetric static method at 15, 25, 35 ◦ C
Tiger nut within the water activity varying from 0.113 to 0.979. Ten mathematical models were selected to fit the
Adsorption isotherm adsorption data using non-linear regression technique. Thermodynamic properties such as isosteric heat, sorption
Thermodynamic properties
entropy and Gibbs free energy were evaluated. The results indicated that equilibrium moisture content of tiger
Isosteric heat
Gibbs free energy
nuts tended to increase with increasing water activity and decreasing temperature. The isotherm curves of tiger
nuts exhibited a Type II sigmoidal shape. Peleg model was the most suitable for describing the experimental
sorption behavior, followed by the Smith and GAB models. The adsorption surface area and monolayer moisture
content of tiger nuts obtained from GAB model were 166.37–149.74 m2/g solid and 4.71–4.24 H2O/100 g solid,
respectively, which decreased with increasing temperature. Moreover, the isosteric heat of adsorption decreased
exponentially with the increase of equilibrium moisture content, while the Gibbs free energy was opposite.
Adsorption entropy was negative in value and it decreased sharply and then rose slowly as the moisture content
increased, having a minimum value (6.43 J/mol K) at a moisture content of 11 g/100 g d.b.

1. Introduction
Cyperus esculentus L. is a perennial herb that belongs to the Cyper­
aceae family, having a spherical tubers at the end of its rhizomes
(Clemente-Villalba et al., 2021). It is widespread in tropical and
temperate zones, such as Africa, Spain, North America and China (Ezeh
et al., 2014). In some countries, this plant is considered as a weed, but it
is widely growing as a domesticated food crop in others. The tubers of
Cyperus esculentus L. are commonly known as tiger nut, earth almonds,
or “chufa” in Spanish (Lopez-Cortes et al., 2013). It can be consumed
raw, roasted or ground to make beverages. In addition, tiger nuts con­
sumption has been reported to help boost blood circulation, improve
male fertility and sexual performance, prevent stroke and heart disease,
and reduce colon cancer risk (Udefa et al., 2020; Yang et al., 2016). More
importantly, tiger nut is quite special because it contains much more oil
(24.49%) than common underground tubers such as potato (0.16%),
yam (0.20%), sweet potato (3.88%) and cassava (0.3%) (Sanchez-Za­
pata et al., 2012). The oil content of tiger nut is comparable to even
higher than that of soybean, which is the most important source of
edible oil in the world. Moreover, the yield of tiger nut is approximately
four times as high as that of soybean, up to 19000 kg ha− 1
(Clemente-Villalba et al., 2021). The literature reveal that tiger nut oil
possesses the similar fatty acid composition with olive and hazelnut oils,
but a higher content of tocopherol and phytosterol (Vega-Morales et al.,
2019). Therefore, tiger nut is considered as a potential alternative to
other oilseeds to satisfy the increasing demand for edible oil, especially
in China.
Since tiger nuts grow in the soil, this makes them highly susceptible
to contamination by pathogenic microorganisms that adhere very easily
to surface and pore of tiger nuts and cannot be completely eliminated in
the following process (Muhammad et al., 2019). When the storage
conditions are not appropriate, tiger nut is prone to deteriorate under
the action of microorganisms and enzymes (Kizzie-Hayford et al., 2021).
As it is known, the growth of mold depends on environmental condi­
tions, such as relative humidity and temperature. Generally, extended
shelf-life of food can be achieved by reducing the moisture content to
levels below those required by microorganisms (Lee & Lee, 2008).
Therefore, it is necessary to assess the relationship between moisture
content of product and relative humidity of the atmosphere at certain
temperature. In fact, the relationship can be described by moisture
sorption isotherms.
The moisture sorption isotherm is an extremely valuable tool to

* Corresponding author. College of Food Science and Technology, Henan University of Technology, NO.100, Lianhua Street, Zhengzhou, 45001, PR China.
E-mail address: zzsan010@haut.edu.cn (Z.-s. Zhang).

https://doi.org/10.1016/j.lwt.2022.113866
Received 25 May 2022; Received in revised form 3 July 2022; Accepted 7 August 2022
Available online 10 August 2022
0023-6438/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Z.-s. Zhang et al. LWT 167 (2022) 113866

understand the mechanism of water sorption, optimize the drying pro­ Table 1
cess, determine critical moisture level, and select packaging material. The water activities (aw) of saturated salt solutions at different temperatures for
There are numerous theoretical, semi theoretical models and empirical 15, 25 and 35.
equations have been proposed to describe moisture sorption character­ Salt aw (15 ◦ C) aw (25 ◦ C) aw (35 ◦ C)
istics of foods. However, no equation could fit data for all types of foods Lithium Chloride 0.113 0.113 0.113
since the sorption depends on food matrix (Collazos-Escobar et al., Potassium Acetate 0.234 0.225 0.216
2022). In addition, the sorption isotherm is also important to calculate Magnesium Chloride 0.333 0.328 0.321
different thermodynamic properties, such as isosteric heat of sorption, Potassium carbonate 0.432 0.432 0.432
Magnesium Nitrate 0.559 0.529 0.499
sorption entropy and Gibbs free energy, which provide information
Sodium Bromide 0.607 0.576 0.546
about food structure, energy requirements and molecular state of water Potassium Iodide 0.710 0.689 0.670
within the products (Sánchez-Torres et al., 2021; Tunç & Duman, 2007). Sodium Chloride 0.756 0.753 0.749
Moisture sorption isotherm have been reported for many agricultural Ammonium Chloride 0.799 0.786 0.779
products, such as coffee beans (Collazos-Escobar et al., 2022), Camellia Potassium Chloride 0.859 0.843 0.830
Potassium sulfate 0.979 0.973 0.967
oleifera seeds (Zhu et al., 2021), wheat malt (Silva et al., 2021), Cucumis
melo L. seeds (Mallek-Ayadi et al., 2020), cocoa beans(Collazos-Escobar
et al., 2020) and whole chia seeds (Arslan-Tontul, 2020b). However, the mathematical equation widely found in the literature. Table 2 summa­
information on the sorption isotherms of tiger nut is quite limited. rizes the expression of the models used in this study to describe the
Djomdi et al. (2007) investigated the water adsorption of tiger nuts relationship between equilibrium moisture content and water activity.
during soaking. while Chukwu and Ajisegiri (2011) only determined the Xeq and aw are equilibrium moisture content and water activity,
moisture sorption isotherms of tiger nuts at 34 ◦ C. Considering the fact respectively, while A, B, C and D are the model coefficients. The curve
that there is a fundamental difference in the moisture absorption and fitting and regression analysis were carried out by a mathematical
water absorption of materials due to the nature and source of water, and analysis software (Origin 2018; Origin Lab, Northampton, USA). The
there is no study on the thermodynamic properties of moisture sorption model coefficient and fit parameter are listed in Table 3. The goodness of
isotherms of tiger nuts. The objective of the present works is to inves­ each model fitting the experimental data was evaluated by the factors,
tigate and model the moisture adsorption isotherms of tiger nut at including coefficient of correlation (R2, Eq. (1)), root of mean square
different temperatures (15, 25 and 35 ◦ C), and determine the thermo­ error (RMSE, Eq. (2)), and percent average relative deviation (P, Eq.
dynamic properties including isosteric heat, entropy, surface area and (3)).
Gibbs free energy using the best fitted sorption isotherm model.
N (
∑ )2
Xe − Xp
2. Materials and methods 2
R =1 − i=1
(1)
∑N
(Xe − X e )2
2.1. Materials i=1

√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
√
Tiger nut was purchased from Henan academy of agricultural sci­ √1 ∑ N
( )2
ences, Zhengzhou, China. For adsorption experiment, tiger nuts with RSME = √ Xe − Xp (2)
N i=1
uniform size were selected by hand. The average dimensions of 30
random tiger nuts were determined as follow: 13.68 ± 0.64 mm length, N ⃒ ⃒
12.95 ± 0.24 mm width, and 11.47 ± 0.37 thickness. The proximate 100 ∑ ⃒Xe − Xp ⃒
P= ⃒ ⃒ (3)
composition was analyzed according to the standard methods of AOCS N i=1 ⃒ Xe ⃒
(1997). The contents of oil, protein, starch, soluble sugar, ash and
moisture were detected to be 23.64 ± 1.25, 5.62 ± 0.32, 26.3 ± 1.47, where Xe and Xp are the experimental and predicted moisture content
19.51 ± 0.47, 2.77 ± 0.14, and 7.13 ± 0.50% respectively. Prior to values, respectively. Xe is the average experimental values. N is the
adsorption experiment, the tiger nuts were washed with sterile water to number of observations.
remove the dirt on the surface, and then dried in an air circulating oven
at 65 ◦ C until its weight remains constant.
Table 2
2.2. Adsorption procedure The model equation selected for fitting the sorption isotherms of tiger nuts.
Model type Equation Reference
The equilibrium moisture content of tiger nut was determined at ABaw
BET
15 ◦ C, 25 ◦ C and 35 ◦ C using the static gravimetric method (Malle­ Xeq = Brunauer et al. (1938)
(1 − aw )[1 + (A − 1)aw ]
k-Ayadi et al., 2020). Twenty grams of tiger nut were placed in a poly­ Smith Xeq = A − B[ln(1 − aw )]
Smith (1947)
thene mesh bag, which was hanged above the saturated salt solution
GAB Xeq =
contained in a glass jar. The jar was tightly closed and placed in a ABCaw
van den Berg and Bruin
temperature controlled cabinet (±0.2 ◦ C). The sample was weighted (1 − Caw )(1 − Caw + BCaw ) (1981)
daily until the mass difference was less than 0.001g. Finally, the sample Caurie Xeq = exp(A + Baw )
Caurie (1970)
was dried at 105 ◦ C for 24 h in order to determine the equilibrium Halsey )1 Halsey (1948)
moisture content. Each experiment was carried out in triplicate. Eleven ( A B
Xeq = −
saturated salt solutions were prepared with a wide range of water ac­ lnaw
Henderson Xeq = A[− ln(1 − aw )]B
tivities from 0.113 to 0.979. The salt solutions used and their corre­ Henderson (1952)
sponding water activities at 15 ◦ C, 25 ◦ C and 35 ◦ C were taken from Oswin ( a )
w
Xeq = A Oswin (1946)
reported data (Greenspan, 1977) and given in Table 1. 1 − aw
Peleg Xeq = A(aw )B + C(aw )D
Peleg (1993)
2.3. Mathematical treatment White Eiring 1
Xeq = Castillo et al. (2003)
A + Baw
Chung and ( B )
The data of moisture adsorption behavior for tiger nuts at three Xeq = Aln Chung and Pfost (1967)
Pfost lnaw
different temperature (5 ◦ C, 25 ◦ C and 35 ◦ C) were modeled using 10

2
Z.-s. Zhang et al. LWT 167 (2022) 113866

Table 3
Fitted parameters of the models applied to the moisture sorption isotherms of tiger nuts.
Model type Temp. (oC) Model fit parameters Model coefficients

R2adj RSME P A B C D

Smith 15 0.994 0.006 2.785 0.030 0.079

25 0.995 0.005 3.882 0.032 0.069
35 0.996 0.003 3.281 0.032 0.062
BET 15 0.973 0.012 12.115 0.018 9.992
25 0.982 0.008 10.028 0.019 8.801
35 0.982 0.007 9.354 0.020 7.599
GAB 15 0.992 0.006 7.175 0.047: Xm − 6.8 × 1044 0.877
25 0.989 0.006 7.890 0.045: Xm 3.07 × 1044 0.862
35 0.990 0.005 6.970 0.042: Xm − 2.4 × 1043 0.853
Caurie 15 0.963 0.015 15.374 − 4.117 3.000
25 0.972 0.010 10.877 − 3.932 2.642
35 0.971 0.009 9.676 − 3.853 2.430
Halsey 15 0.944 0.018 17.984 0.001 2.654
25 0.946 0.015 16.770 0.001 2.652
35 0.955 0.011 13.898 0.001 2.615
Henderson 15 0.944 0.008 9.571 16.021 1.275
25 0.946 0.006 6.976 21.539 1.369
35 0.955 0.005 6.628 27.375 1.435
Oswin 15 0.944 0.013 11.194 0.092 0.340
25 0.946 0.010 10.365 0.085 0.336
35 0.955 0.007 7.792 0.079 0.337
Peleg 15 0.999 0.002 2.442 0.111 0.509 0.250 6.003
25 0.997 0.003 3.180 0.107 0.535 0.195 5.628
35 0.998 0.002 2.929 0.106 0.549 0.164 5.902
White Eiring 15 0.944 0.006 7.159 21.237 − 18.633
25 0.946 0.006 7.887 22.281 − 19.200
35 0.955 0.005 6.964 23.583 − 20.110
Chung and Pfost 15 0.944 0.011 12.370 0.068 − 0.065
25 0.946 0.007 8.852 0.065 − 0.056
35 0.955 0.006 7.206 0.062 − 0.050

2.4. Determination of adsorption surface area (q )( 1 )

The specific surface area of adsorption has an important role in
determining the water binding properties of foods (Tunç & Duman,
2007). The adsorption surface area of the samples was determined ac­
cording to the value of monolayer moisture content using Eq. (4) (Fan
et al., 2015). The monolayer moisture content was obtained from the
GAB model.
Xm NA AH2 O
S= = 3.53 × 103 Xm
M H2 O
where S is the adsorption surface area (m2/g d.b.), Xm is the monolayer
moisture content (g water/g d.b.), NA is Avogadro’s number (6.02 ×
1023 molecules/mol)/g, AH2O is the area of water molecule (1.06 ×
10− 19 m2), and MH2O is the molecular weight of water (18 g/mol).
2.5. Determination of isosteric heat of sorption
According to literature (Polatoğlu et al., 2011), the isosteric heat of
sorption (Qst) is the energy released in the process of sorption of water
molecules on a surface of the adsorbent, which is also an indicator of the
state of water absorbed by the solid material. The isosteric heat of
sorption (Qst) is numerically equal to the net isosteric heat of sorption
(qst) plus the heat of vaporization of water (HL, 43 kJ/mol) at the system
temperature (Moreira et al., 2008), as shown in Eq. (5).
Qst = qst + HL
The net isosteric heat of sorption (qst) is the energy amount needed to
change the state of a product unit mass from liquid to vapor at constant
temperature and water activity (Mallek-Ayadi et al., 2020). It could be
calculated from the experimental data using the Clausius-Clapeyron
equation (Eq. (6)) by plotting the sorption isotherm as the natural log­
arithm of water activity versus 1/T (Fan et al., 2015).
(6)
st
ln(aw ) = − +C
R T
where qst is the net isosteric heat of sorption (J/mol), T is the absolute
temperature (K), R is the gas constant (8.314 J/mol K) and C is the
constant.
2.6. Determination of sorption entropy
(4)
Entropy change plays an important role in energy analysis of food
processing systems since it proportional to the number of available
sorption sites at a specific energy level (Koua et al., 2014). The differ­
ential entropy of sorption (ΔS) was determined by fitting Eq. (7) to
equilibrium moisture content from the best-fitting equation:
qst ΔS
ln(aw ) = − + (7)
RT R
ΔS can be calculated by plotting ln(aw) and 1/T for certain moisture
content and then determined the intercept as ΔS/R. This procedure was
repeated for different moisture content to determine the dependence of
sorption entropy upon the moisture content.
2.7. Determination of Gibbs free energy
The change in Gibbs free energy (ΔG) can be used to assess the af­
finity of sorbents for water and gives information about the spontaneous
(5)
process of adsorption. It was calculated from Eq. (8) by water activity
that generated from the best-fitting model at certain temperatures
(Moreira et al., 2008; Sánchez-Torres et al., 2021).
ΔG = RT ln(aw ) (8)

3
Z.-s. Zhang et al.
3. Results and discussion
3.1. Adsorption isotherm of tiger nuts
The moisture adsorption isotherm of tiger nuts at different temper­
atures (15 ◦ C, 25 ◦ C and 35 ◦ C) is shown in Fig. 1. The equilibrium
moisture content increased slowly at lower water activity, while
increased sharply in the case of water activity greater than 0.7. The
isotherm curves was sigmoid shape and showed type II behavior ac­
cording to Brunauer classification (Brunauer et al., 1940). This isotherm
is typical for biological products including seeds, grains and fruits
(Mallek-Ayadi et al., 2020; Rosa et al., 2021; Silva et al., 2021; Sya­
maladevi et al., 2009). Based on water activity, the sorption isotherm
can be divided into three different parts, that is range A (aw = 0–0.2),
range B (aw = 0.2–0.7) and range C (aw = 0.7–1.0) (Toğrul & Arslan,
2007). In the range A, the adsorption belonged to monolayer adsorption
and water molecular was strongly bound on the polar sites via hydrogen
bonds (Sablani et al., 2009). The moisture content corresponded to the
critical content for excellent storage stability of food. In the range B,
there was a linear relationship between moisture content and water
activity due to the structural changes in matrix and the binding of water
to the components (Arslan-Tontul, 2020a). In this region, a multilayer
adsorption occurred and the water molecules were bound together to
filled the micropores in matrix. As with those in region A, the water
molecules were not in free form, and their participation in biochemical
reaction are largely limited. In the range C, water molecules penetrate
into the pores and voids in the cells, are mechanically trapped in the void
space, and eventually condense into water. The water is expressed as
free water and the water molecules are active enough to participate in
biochemical reaction (Fan et al., 2015). At the water activity greater
Fig. 1. Experimental adsorption isotherms of tiger nuts and fiting with Peleg model (a), Smith model (b) and GAB model (c) at 15, 25 and 35 ◦ C.
4
LWT 167 (2022) 113866
than 0.7, a small rise in water activity caused an adsorption of higher
amount of water. This indicated that the range is highly unstable in term
of spoilage.
In addition, as shown in Fig. 1, the equilibrium moisture content of
tiger nuts decreased with increasing temperature at a constant water
activity. This phenomenon may be explained by the fact that the in­
crease in temperature rises the energy level and activity of water mol­
ecules. It will result in faster molecular motion and greater
intermolecular distances. As a result, the water molecules become less
stable and more easily detached from the water binding sites of the food.
This phenomenon has been reported in previous studies (Arslan-Tontul,
2020b). In addition, according to the reports of Rosa et al. (2021) and
Leonardo Betiol et al. (2020), this behavior can be attributed to the
reduction in the number of active sites as consequence of physico­
chemical changes induced by temperature. Kizzie-Hayford et al. (2021)
confirmed that storage temperature had a great influence on the prox­
imate composition of tiger nuts.
It is well known the sorption capacity of a material is closely related
to its chemical composition and structure. At the highest water activity
and temperature examined (aw = 0.967, T = 35 ◦ C), 100 g of tiger nuts
adsorbed nearly 32 g of H2O. This results suggested that tiger nuts are
more hygroscopic than most of the reported grains, such as wheat (Li
et al., 2011) and quinoa (Arslan-Tontul, 2020a), when water activity is
high. The higher hydrophilicity of tiger nuts may be due to its high
soluble sugar content (19.51 ± 0.47%), which has a hydrophilic struc­
ture. In fact, at high water activities, the water molecules can be avail­
able as a solvent of low molecular weight solutes (Collazos-Escobar
et al., 2022). Meanwhile, it was found that when the water activity was
less than 0.7, the equilibrium moisture content of tiger nuts was equal to
or less than that of the aforementioned grains. This phenomenon can be
Z.-s. Zhang et al.
explained by the high content of oil (approximately 24%) in tiger nuts,
which reduced the number of active sites during monolayer adsorption.
Similar phenomenon was also found in previous studies (Arslan-Tontul,
2020b; Toğrul & Arslan, 2007).
3.2. Fitting of sorption model to experimental data
A large number of equations have been reported in literature that can
be used to describe the sorption isotherm of food. In this paper, 10
common models were selected for simulating the experimental sorption
data of tiger nuts by non-linear regression. The adequacy and goodness
of fit of each model was evaluated by coefficient of correlation (R2), root
of mean square error (RMSE), and percent average relative deviation
(P). The results of the modeling are shown in Table 3. Statistically, the
model is acceptable when the value of R2 is higher than 0.98, while the
values of P and RMSE is less than 10 and 1, respectively (Arslan-Tontul,
2020b). Based on this standard, three models, namely Peleg, Smith and
GAB, gave better fit to the experimental data (as shown in Fig. 1).
However, The Peleg model provided the best explanation for the
adsorption isotherms of tiger nuts at three different temperatures
examined due to having the highest R2 and lowest RMSE and P values,
followed by the Smith and GAB model. As can be seen form Fig. 1(a),
Peleg and Smith models are adequately fitted to the experimental data
over the entire range of measured water activity. Peleg model was found
to be applicable to different materials and was the best one to describe
the sorption isotherms of several oily seed, such as soybean (Gowen
et al., 2007), papaya seed (Rosa et al., 2021) and camellia seed (Zhu
et al., 2021). However, the coefficient of Peleg and Smith models had no
any physical meaning. The selection of a model should consider not only
the goodness of fit, but also the number of parameters and their physical
meaning (Sánchez-Torres et al., 2021). A small number of parameters
can increase the freedom of the identification procedure, while the
physical meaning of parameters can help one to better interpret and
understand the physical phenomenon by described. In this regard, GAB
model seems to be more practical in describing the absorption isotherm
of tiger nuts, although there is a significant lack of fit at low water ac­
tivity (aw<0.4).
3.3. Monolayer moisture content
Monolayer moisture content (Xm) is the amount of water that is
strongly adsorbed to specific sites at the food surface. It is considered an
important value to assure food stability for long-term storage (Kurozawa
et al., 2015). Below the moisture level, the adsorbed water cannot be
used as solvent and the rate of deterioration reactions including lipid
oxidation and enzyme activity, is minimized.
Monolayer moisture content can usually be calculated according to
the theoretical definition of GAB and BET models. However, due to the
lack of fit for the BET model, the GAB model was used in this paper to
calculate the monolayer moisture content. As shown in Table 3, the
value was determined to be 4.71, 4.49 and 4.24 g H2O/100 g solid at
15 ◦ C, 25 ◦ C and 35 ◦ C, respectively. The monolayer moisture content of
tiger nuts was similar to that of several grain and oil-rich seeds reported
in the literature. Lazouk et al. (2015) determined the monolayer mois­
ture content of rapeseed, sunflower and linseed were 3.0, 4.9 and 6.0
g/100 g seed (d.b.), respectively. Arslan-Tontul (2020a, 2020b) found
the monolayer moisture content of whole chia seed and quinoa grains
calculated by the GAB model to be 2.70–2.91 and 4.31–5.25 g H2O/100
g solid, respectively.
Tiger nut was more hydrophilic at low temperatures, because the
monolayer moisture content tended to decrease with increasing tem­
perature. Similar phenomena was also observed by other researchers in
the adsorption isotherms of wheat (Silva et al., 2021) and loquat seeds
(Moreira et al., 2008). It could be explained by the fact that as the
temperature increases, the water molecules became activated due to
increased energy levels, which caused them to become unstable and
5
LWT 167 (2022) 113866
detach from the water binding sites of the tiger nuts, resulting in a lower
monolayer moisture content.
3.4. Isosteric heat of sorption
The isosteric heat of sorption (Qst) is an important thermodynamic
parameter that can be used to understand the interaction between
moisture and food materials, as well as to analyze the drying process and
the stability of foods under given storage conditions (Arslan-Tontul,
2020a). The net isosteric heat of sorption was calculated by the
Clausius-Clapeyron equation. The adsorption isosteric curve for tiger
nuts at different moisture contents is presented in Fig. 2. While Fig. 3
describes the effect of equilibrium moisture content on the value of
isosteric heat of sorption (Qst).
As seen from Fig. 3, the isosteric heat of sorption is a positive value in
the studied moisture content range (Xeq = 0.10–0.35 g/g d.b.), indi­
cating that the moisture adsorption process undergoes an endothermic
reaction. In addition, the isosteric heat of sorption is closely related to
equilibrium moisture content. The isosteric heat of adsorption at high
moisture content is noticeably lower than that at low moisture content.
This phenomenon was widely present in many foods such as pork liver
(Sánchez-Torres et al., 2021),. Cottonseeds (Tunç & Duman, 2007),
Cucumis melo L. seeds (Mallek-Ayadi et al., 2020), and chia seeds
(Arslan-Tontul, 2020b).
At the lowest moisture content (1.0%, d.b.), the isosteric heat of
adsorption was the highest (58.86 kJ/mol). This may be due to the fact
that in the case of low moisture content, there are many active polar sites
on the surface of the food material which bind to the water molecules
and form a single molecular layer. Therefore, the energy required to
remove the strength of water bound was high.
As the moisture content increased to 15% d.b., the isosteric heat of
adsorption was drastically reduced to 45.6 kJ/mol, and thereafter
remained approximately constant. At higher moisture content (>15% d.
b.), the isosteric heat of sorption is slightly higher than the vaporization
energy of pure water. This indicated that the energy of binding between
the water molecules and the sorption sites is higher than the energy
which holds the water molecules together in the liquid phase. In this
state, water molecules exist in the food in free form. Zhu et al. (2021)
confirmed that the component of food has an impact on the moisture
sorption behavior. The binding energy between water molecules and
food material decreased with increasing oil content. Moreover, the high
sorption heat of food means that more energy is required to vaporize the
water in food product during the drying process. This study shows that it
is more economical to control the final moisture to about 10% for the
drying of tiger nuts.
Fig. 2. Adsorption isosteric curve of tiger nuts.
Z.-s. Zhang et al.
Fig. 3. The relationship between isosteric heat of adsorption and equilibrium
moisture content.
The relationship between the isosteric heat of adsorption and equi­
librium moisture content of tiger nuts can be expressed by an empirical
exponential model (Eq. (9)). This model fits the experimental data well,
with a coefficient of determination of 0.994 and an average relative
deviation of 0.35%.
( )
Qst = 45.51 + 17.82 exp − 28.89Xeq (9)
3.5. The differential entropy of sorption
Differential entropy is related to the number of available sorption
sites at a specific energy level and the spatial arrangement between the
water and food matrix (Collazos-Escobar et al., 2022; Silva et al., 2021).
It can be used to characterize the degree of disorder in a given system
(Silva et al., 2021). The effect of equilibrium moisture content on the
entropy of tiger nuts is presented in Fig. 4. As seen in Fig. 4, the dif­
ferential entropy decreases with increasing moisture content until 11
g/100 g d.b., and then increases with increasing moisture content.
Fig. 4. The relationship between the entropy and the equilibrium mois­
ture content.
6
LWT 167 (2022) 113866
Similar trends have previously been reported by others in the studies of
pork liver (Sánchez-Torres et al., 2021), coffee (Collazos-Escobar et al.,
2022), mint leaves (Dalgic et al., 2012) and apple (Moraes et al., 2008).
At lower moisture content (less than 11 g/100 g d.b.), there is a loss of
rotational movement of water molecules as the available sorption sites
become saturated, thus increases in moisture content lead to a decrease
in the entropy, and to disorder in the system (Kurozawa et al., 2015).
However, when moisture contents higher than 11 g/100 g d.b., the
adsorption entropy increased slightly with the increase of moisture
content, which could be ascribed to the increase in moisture content
allowing more water molecules to be adsorbed into the multilayer, and
the water with lower binding energy and higher mobility for the rotation
and translation (Silva et al., 2021). The value of adsorption entropy was
lowest (6.43 J/mol.K) at the moisture content of 11 g/100 g d.b., which
means that water molecules are more trapped and less able to participate
in spoilage reaction. The moisture content is considered to be the suit­
able level of retained moisture for tiger nuts during the drying process.
3.6. Gibbs free energy
The Gibbs free energy represents the maximum amount of energy
released in a process under constant temperature and pressure (Silva
et al., 2021). In the adsorption process, the Gibbs free energy can be used
to estimate the affinity between a solid matrix and water and to deter­
mine whether moisture sorption is spontaneous. Generally, if ΔG is
negative, the process is regarded as spontaneous, conversely, the process
is considered to be non-spontaneous (McMinn et al., 2007). The varia­
tion of Gibbs free energy with moisture content at different temperatures
is given in Fig. 5. It is observed that the ΔG increased with increasing of
equilibrium moisture content and tending to stabilize at higher levels of
moisture content. In general, the ΔG of tiger nuts varied from − 11.3 to
0.15 kJ/kg.
The negative values of ΔG confirmed that the moisture adsorption of
tiger nuts is a spontaneous process, i.e. no energy is needed to be
absorbed from the environment to ensure that the reaction occurs. For
moisture contents below 10 g/100 g d.b., the Gibbs free energy
decreased steeply with increasing moisture content, which may be
attributed to the weakening of the affinity between the water and the
absorbent material. Similar results were obtained for quinoa grains
(Arslan-Tontul, 2020a). It is worth noting the Gibbs energy decreased as
the temperature increased at a given moisture content, suggesting that
there is a temperature-dependent on the Gibbs free energy. However,
Fig. 5. The relationship between the Gibbs free energy and the equilibrium
moisture content.
Z.-s. Zhang et al. LWT 167 (2022) 113866

the temperature dependence is so weak that the curves at different Table 4

temperatures almost overlap. The relationship between the Gibbs free Model parameters of Gibbs free energy at different temperature.
energy and the equilibrium moisture content can be fitted using an Parameters 15 ◦ C 25 ◦ C 35 ◦ C
exponential model (Eq. (10)) and the model parameters are given in
A − 14.269 − 13.430 − 13.115
Table 4. Base on this model, the variation trend of moisture in tiger nuts B − 0.035 − 0.036 − 0.036
can be predicted at specific temperature and pressure. C − 0.236 − 0.151 − 0.073
( ) R2 0.997 0.995 0.996
Xeq RSME 0.141 0.153 0.146
ΔG = Aexp +C (10)
B

3.7. Adsorption surface area

The specific surface area is an important indictor in determining the

water binding properties of food materials. The adsorption surface area
of tiger nuts was calculated by monolayer moisture content obtained
from GAB model. The values of surface area were 166.37, 158.46, and
149.74 m2/g solid for the temperatures of 15, 25 and 35 ◦ C, respectively.
This result was consistent with those previously reported by Labuza
(1968), who indicated that surface area value of food products are
within the range of 100–250 m2/g of sample. As shown in Fig. 6, there is
a remarkably negative linear relationship between the specific surface
area and the temperature (R2 = 0.9992), which indicated that the total
surface area available for hydrophilic binding decreased with increasing
temperature. Similar tendency was also found in the moisture adsorp­
tion of sucuk (Polatoğlu et al., 2011) and texturized soy protein (Cassini
et al., 2006). This behavior has been described as a reduction in the
number of active sites due to physical and chemical changes induced by
temperature (Fan et al., 2015; Polatoğlu et al., 2011). According to
Arslan-Tontul (2020b), the surface interaction, structure and chemical
composition affected the moisture sorption capacity of seeds. It was
reported that temperature has an important influence on microbial
growth and enzymatic activity, which affected the chemical composi­
tion of food materials during storage (Kizzie-Hayford et al., 2021).
Moreover, the lower temperature encouraged more water molecular
adsorbed at the polar sites to condense down during capillary conden­
sation and filled the entire void space, and resulted in the swelling of the
sample. It is well known that the adsorption and desorption of moisture
are accompanied by expansion and shrinkage of the material, respec­
tively (Mallek-Ayadi et al., 2020).
4. Conclusion
The equilibrium moisture content of tiger nuts have been determined
at 15, 25, 35 ◦ C using a serial of saturated salt solutions with different
water activities (0.113–0.979). The equilibrium moisture content
increased with increasing water activity and decreased with increasing
temperature. The adsorption isotherm obtained in this investigation had
a sigmoid shape and the behavior belonged to the type II according to
Brunauer classification. Among the ten equations selected, Peleg, Smith
and GAB models gave better fit to the experimental data, but Peleg
model was found to be the most suitable one for describing the
adsorption isotherm of tiger nuts. The monolayer moisture content and
adsorption surface area tended to decrease with increasing temperature,
indicating tiger nuts are more hydrophilic at low temperatures. The
adsorption entropy first decreased and then increased with the increase
of moisture content. The isosteric heat was positive and the Gibbs free
energy was negative over the entire moisture range studied, suggesting
that the moisture adsorption process of tiger nuts is a spontaneous
exothermic reaction. Moreover, both isosteric heat and Gibbs free en­
ergy were exponentially related to the equilibrium moisture content. It is
recommended that a moisture content of 11 g/100 g d.b. is more suitable
for the storage of tiger nuts.
CRediT authorship contribution statement
Fig. 6. The relationship between the specific surface area and the temperature.
Writing – original draft, Writing – review & editing, Funding acquisition.
Xiao-dan Li: Methodology, Validation, Investigation, Data curation.
Hui-jie Jia: Methodology, Investigation. Yu-lan Liu: Project adminis­
tration, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
The work was supported financially by National Key Research and
Development Plan of China (2019YFD1002605), National Natural Sci­
ence Foundation of China (32172261) and Major Science and Tech­
nology Projects of Henan (211100110100–3).
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 Pharmaceutical Biology

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Antioxidant, antibacterial, and anti-inflammatory

activities of standardized brazilin-rich Caesalpinia
sappan extract

Nilesh Prakash Nirmal & Pharkphoom Panichayupakaranant

To cite this article: Nilesh Prakash Nirmal & Pharkphoom Panichayupakaranant (2015)
Antioxidant, antibacterial, and anti-inflammatory activities of standardized brazilin-rich Caesalpinia
sappan extract, Pharmaceutical Biology, 53:9, 1339-1343, DOI: 10.3109/13880209.2014.982295

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Pharm Biol, 2015; 53(9): 1339–1343
! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.982295

ORIGINAL ARTICLE

Antioxidant, antibacterial, and anti-inflammatory activities of

standardized brazilin-rich Caesalpinia sappan extract
Nilesh Prakash Nirmal1,2 and Pharkphoom Panichayupakaranant2,3
1
Excellent Research Laboratory, Phytomedicine and Pharmaceutical Biotechnology Excellence Center, Faculty of Pharmaceutical Sciences, Prince of
Songkla Univesity, Hat-Yai, Songkhla, Thailand, 2Department of BioPharmacy, Faculty of Pharmaceutical Science, Burapha University, Saensuk,
Muang, Chonburi, Thailand, and 3Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Center for
Excellence, Prince of Songkla University, Hat-Yai, Songkhla, Thailand

Abstract Keywords
Context: Brazilin is a major active principle of Caesalpinia sappan L. (Leguminosae or Fabaceae). Anti-denaturation, natural preservative,
For industry aspects, brazilin-rich extract (BRE) has been prepared and standardized to contain sappan wood
39% w/w brazilin. BRE may have more advantages than brazilin in term of a lower-cost
production process. History
Objectives: To investigate the antioxidant, antibacterial, and anti-inflammatory activities of BRE.
Material and methods: BRE was prepared by a simple one-step purification of the crude ethanol Received 10 June 2014
extract of C. sappan heartwood (CSE) using a DiaionÕ HP-20 column. The antioxidant activities Revised 15 October 2014
were determined using three methods, including DPPH radical scavenging, reducing power, Accepted 27 October 2014
and b-carotene bleaching assays, at concentration ranges of 1–10, 10–100, and 10–100 mg/mL, Published online 13 April 2015
respectively. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration
(MBC) of BRE (15.6–1000 mg/mL) against Gram-positive and Gram-negative bacteria were
determined by the broth microdilution method. Anti-inflammatory activity of BRE (0.1–5 mg/mL)
was evaluated as anti-denaturation activity using bovine serum albumin as a substrate.
Results and discussion: On the basis of b-carotene bleaching assay, BRE showed antioxidant
activity with an EC50 value of 60.5 mg/mL, which was almost equal to that of pure brazilin
(52.1 mg/mL). Gram-positive bacteria were more sensitive to all tested samples than Gram-
negative bacteria. BRE possessed higher antibacterial activities than CSE, but lower than
brazilin. MIC/MBC values of 62.5–125/125 and 250–500/250–500 mg/mL were obtained for
BRE against Gram-positive and Gram-negative bacteria, respectively. A low concentration
(0.1 mg/mL) of brazilin, BRE, and CSE showed anti-inflammatory activity by inhibiting protein
denaturation up to 46.8, 54.1, and 61.9%, respectively.

Introduction xanthone, and brazilin (Chen et al., 2008). A decoction of

the heartwood has been traditionally used as an emmenagogue,
Phenolic compounds from plants are known to possess
anti-inflammatory, and hemostatic agent in Southeast Asia
antioxidant activities in terms of free radical scavenger,
(Zhao et al., 2008). The wood extract has also been used as a
reducing power, and quencher of singlet oxygen formation
natural preservative in chili paste (Saraya et al., 2009).
(Duh et al., 2001). It is well known that antioxidant compounds
Brazilin [(6aS,11bR)-7,11b-dihydro-6H-indeno[2,1-c]
can protect against several degenerative diseases, and have
chromene-3,6a,9,10-tetrol] (Figure 1) is a major active
potential use as a dietary supplement. In addition, many food or
compound of C. sappan that has antibacterial, anti-inflam-
functional food producers are interested in a natural antioxi-
matory, and antioxidant activities (Bae et al., 2005; Batubara
dant to improve the nutritional quality of processes foods or to
et al., 2010; Hu et al., 2009; Sasaki et al., 2007; Xu & Lee,
prepare functional food (Wijeratne et al., 2006).
2004). However, the major limitation of the use of pure
Caesalpinia sappan L. (Leguminosae or Fabaceae) heart-
brazilin is its high cost of production, due to the number of
wood has been traditionally used as a natural coloring agent in
steps and time involved for purification. Brazilin, therefore, is
beverages, food, cosmetics, and garments. The heartwood
used as an indicative marker for preparation and standard-
contains various types of phenolic components, such as
ization of brazilin-rich extract (BRE) from C. sappan
dibenzoxocins, flavones, homoisoflavonoids, chalcones,
heartwood (Nirmal & Panichayupakaranant, 2014).
In order to investigate the full potential of the standardized
Correspondence: Pharkphoom Panichayupakaranant, Department of BRE as natural food preservatives and nutraceuticals, we now
Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical
Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, report on the antioxidant, antibacterial, and anti-inflammatory
Thailand. Tel/Fax: +66 74 428220. E-mail: pharkphoom.p@psu.ac.th activities of BRE containing 39% w/w brazilin.
1340 N. P. Nirmal & P. Panichayupakaranant
Figure 1. Chemical structure of brazilin.
Materials and methods
Chemicals and reagents
Folin–Ciocalteu reagent, 2,2-diphenyl-1-picryl hydrazyl
(DPPH), sodium carbonate, sodium chloride, trichloroacetic
acid, and potassium ferricyanide were from Fluka Chemie AG
(Buchs, Switzerland). Ferric chloride, quercetin, ascorbic
acid, b-carotene, linoleic acid, and BSA were from Sigma-
Aldrich (St. Louis, MO). Tween-40 was from E. Merck
(Darmstadt, Germany). All solvents were HPLC and analyt-
ical grade and from Labscan Asia (Bangkok, Thailand).
Plant material
Caesalpinia sappan heartwoods were collected from
Chonburi province, Thailand, in February 2012. A voucher
specimen (specimen no. SKP 098 03 19 01) was identified by
Associate Professor Pharkphoom Panichayupakaranant and
has been deposited at the herbarium of the Faculty of
Pharmaceutical Sciences, Prince of Songkla University,
Thailand. The plant material was washed and dried at 60  C
for 24 h in a hot air oven, and was reduced to powder using a
grinder, and the powder passed through a sieve no. 45.
Preparation of BRE
BRE was prepared and standardized to contain 39% w/w
brazilin. The dried CSE (25 g) was dissolved in 35% ethanol
(3 L) to obtain a solution after filtering. The solution was
loaded on a DiaionÕ HP-20 column (12  50 cm, 1 g CSE per
60 g resin) and eluted with 35% ethanol. The first 2 L fraction
was discarded and another fraction (4 L) that contained
brazilin was collected. The collected fractions were evapo-
rated to dryness to obtain a reddish extract of BRE.
Determination of the total phenolic content
The total phenolic content was determined with the Folin–
Ciocalteu reagent according to the method of Nirmal
and Benjakul (2011). Appropriately diluted extract (0.1–
5.0 mg/mL; 1 mL) was added with 0.2 mL of two-fold diluted
Folin–Ciocalteu reagent and mixed thoroughly. After 3 min,
3 mL of 2% sodium carbonate solution was added.
After standing for 30 min at room temperature (25 ± 2  C),
the absorbance was measured at 760 nm using a
Pharm Biol, 2015; 53(9): 1339–1343
UV-spectrophotometer (Genesys-6, Thermo-Scientific,
Madison, WI). The concentration of total phenolic compound
in CSE and BRE was calculated from the standard curve of
quercetin with the range of 0.01–0.1 mg/mL and expressed as
g quercetin equivalent (QE)/ 100 g dry extract powder.
DPPH radical scavenging assay
DPPH radical scavenging activity was determined according
to the method of Nirmal and Benjakul (2011) with some
modifications. Samples (1.5 mL) at concentration ranges of
1–10 mg/mL were added to 1.5 mL of 0.15 mM DPPH in 95%
ethanol. The reaction mixtures were allowed to stand for
30 min at room temperature (25 ± 2  C) in the dark and the
absorbance was measured at 517 nm using the UV-spectro-
photometer. Quercetin (1–7 mg/mL) was used as a standard.
The sample blank at each concentration was prepared in the
same manner except that ethanol was used instead of the
DPPH solution. Control was prepared with DPPH solution
and ethanol without any sample. The percentage inhibition of
DPPH radical was calculated from following equation:


Percentage inhibition of DPPH radical ¼ 1  Asample =Acontrol  100
Radical scavenging potential was expressed as IC50 values
which represents the sample concentration at which 50% of
the radicals are scavenged.
Determination of reducing power
The reducing power was determined as described by Duh et
al. (2001). The samples (0.01–0.1 mg) in phosphate buffer
(1.0 mL, 0.2 M, pH 6.6) were mixed with 1% potassium ferric
cyanide (1 mL), and the mixtures were incubated at 50  C for
20 min. After incubation, 1 mL of 10% trichloroacetic acid
was added and the mixtures were centrifuged at 2500 rpm for
10 min. The upper layer of the solution (1 mL) was mixed
with distilled water (1 mL) and 0.1% ferric chloride (0.2 mL)
and the absorbance was read at 700 nm using the UV-
spectrophotometer. Higher absorbance of the reaction mixture
indicated greater reducing power. Ascorbic acid and quercetin
were used as the standard and subjected to the same procedure
mentioned above instead of extract.
b-Carotene bleaching assays
The antioxidant activity of extracts was evaluated by using
the b-carotene–inoleate model system (Panichayupakaranant
et al., 2010). b-Carotene (10 mg) was dissolved in 10 mL of
chloroform. The carotene–chloroform solution (0.2 mL) was
transferred into a flask containing linoleic acid (20 mg) and
Tween-40 (200 mg). Chloroform was removed under vacuum
using a rotary evaporator at 40  C for 5 min. The resulting
mixture was slowly diluted with 50 mL of distilled water and
mixed thoroughly with vigorous shaking to form an emulsion.
Aliquots (5 mL) of the emulsion were transferred into a series
of tubes containing 0.2 mL of samples solution. Immediately
after the addition of the emulsion to each tube, the zero time
absorbance was measured at 470 nm. The tubes were placed
in a water bath at 50  C and monitored spectrophotometrically
by measuring the absorbance at 470 nm over a 60-min period.
DOI: 10.3109/13880209.2014.982295 Standardized brazilin-rich C. sappan extract 1341

Quercetin was used as the reference antioxidant. Blank
samples devoid of b-carotene were prepared for back-
ground subtraction. A control sample contained 0.2 mL of
distilled water instead of sample solution was prepared.
All tests were performed in triplicate. The antioxidant
activity was expressed as inhibition percentage with reference
to the control after 60 min incubation using the following
equation.
AA ¼ 100ðBRC  BRSÞ=BRC
where AA is the antioxidant activity; BRC is the bleaching
rate of the control ¼ (ln(a/b)/60); BRS is the bleaching rate in
the presence of sample ¼ (ln(a/b)/60); a is the absorbance at
time 0; b is the absorbance at 60 min.
Anti-inflammatory assay
Anti-inflammatory activity of BRE was evaluated as anti-
denaturation activity using bovine serum albumin as a
substrate. Anti-denaturation activities of the samples were
determined according to the method of Williams et al. (2008)
with slight modification. A stock solution of 0.2% (w/v) BSA
was prepared in the Tris-acetate buffer, pH 6.8. The test
compounds (0.1–5 mg/mL) were prepared in methanol. The
reaction mixture consists of 150 mL of test samples and
2850 mL of BSA solution was incubated at room temperature
for 15 min. Test tubes were heated at 72  C for 4 min and then
cooled at room temperature for 20 min. The absorbance of
these solutions was determined by the UV-spectrophotometer
at a wavelength of 660 nm. The percentage inhibition of
denaturation or precipitation of the BSA from the sample
solution was calculated from following equation:
% inhibition of denaturation ¼ ð1  Asample =Acontrol Þ  100
The control consists of 2850 mL of BSA solution with 150 mL
respectively. The MIC was defined as the lowest concentra-
tion of the compound to inhibit the growth of microorganisms
and the MBC was defined as the lowest concentration of the
compound required to kill the microorganisms.
Statistical analysis
All analyses were performed in triplicate and a completely
randomized design (CRD) was used. Analysis of variance
(ANOVA) was performed, and mean comparisons were done
by Duncan’s multiple range tests. For pair comparisons, a
t-test was used. P values less than 0.05 were considered to be
statistically significant.
Results and discussion
The antioxidant activities of a compound have been attributed
to various mechanisms, including prevention of chain initi-
ation, binding of transition metal ion catalyst, decomposition
of peroxides, scavenging of free radicals, and prevention of
hydrogen abstraction. In this study, the antioxidant activities
of BRE, CSE, and the pure brazilin were determined using
three different assay systems, including DPPH radical
scavenging, reducing power, and b-carotene bleaching assays.
On the basis of DPPH radical scavenging method, increase
of concentrations of all samples resulted in increase of DPPH
radical scavenging activity (Figure 2). BRE showed higher
DPPH radical scavenging activity than CSE, but lower than
brazilin and quercetin. The IC50 values of 6.2, 7.3, 5.6, and
3.5 mg/mL were calculated for BRE, CSE, brazilin, and
quercetin, respectively. These results agree with the results
CSE BRE Brazilin Quercetin
140
120
% Inhibition of DPPH radical

of methanol. Diclofenac was used as the standard drug.

100
Microorganisms and media
80
Pseudomonas aeruginosa (DMST 15442) and Salmonella
60
typhimurium (DMST 562) were obtained from the
Department of Medical Science Center, Thailand. 40
Escherichia coli (ATCC 25922), Staphylococcus aureus
20
(ATCC 25923), and S. epidermidis (ATCC 14990) were
obtained from the Department of Microbiology, Faculty of 0
Medicine, Prince of Songkhla University, Thailand. Bacillus 0 2 4 6 8 10 12
subtilis was from Faculty of Science, Prince of Songkhla Concentration (µg/mL)
University. Muller–Hinton agar (MHA), Muller–Hinton broth,
Brain–Heart infusion (BHI), and agar were from Becton Figure 2. Percentage inhibitions of DPPH radicals by BRE, CSE, and
brazilin at different concentrations, n ¼ 3 (Nirmal & Benjakul, 2011).
Dickinson (Franklin Lakes, NJ).
Table 1. Radical scavenging activity of BRE, CSE,
Antibacterial assay and brazilin determined by b-carotene bleaching
All bacteria were incubated in BHA at 37  C for 24 h. Inocula assay (Panichayupakaranant et al., 2010).
were prepared by mixing a few bacteria colonies with sterile
Samples EC50 (mg/mL)
ringer solution (0.85% NaCl) and comparing the turbidity
with the standard 0.5 McFarland solution, which were CSE 92.2 ± 0.9
BRE 60.5 ± 0.2
equivalent to 108 CFU/mL. Brazilin 52.1 ± 0.4
The minimum inhibitory concentration (MIC) and min- Quercetin 17.4 ± 0.4
imum bactericidal concentration (MBC) were determined by
the broth microdilution assay (NCCLS, 2008). Ampicillin and The results are expressed as mean ± SD (n ¼ 3).
BRE, brazilin-rich extract; CSE: crude ethanol
DMSO were used as positive and negative controls, extract of C. sappan heartwood
1342 N. P. Nirmal & P. Panichayupakaranant Pharm Biol, 2015; 53(9): 1339–1343

from b-carotene bleaching assay. BRE showed almost equal
radical scavenging activity to brazilin, but higher activity
than that of CSE (Table 1). Although BRE contained only
39% w/w brazilin, it showed comparable radical scavenging
activities to brazilin. This implies that BRE contains other
radical scavenging compounds besides brazilin.
Determination of the total phenolic content of BRE and
CSE found that BRE contained a lower total phenolic content
(50.3 ± 1.0 g QE/100 g of extract powder) than that of CSE
(97.8 ± 3.3 g QE/100 g of extract powder). This implied that
about half of the phenolic compounds in the crude extract had
been eliminated by treatment with the DiaionÕ HP-20 column
without any significant reduction of the antioxidant and
reducing activities. This also implied that brazilin contributed
more radical scavenging activities than any other phenolic
compounds in the BRE. In addition, CSE may contain a
greater content of polar phenolic compounds than BRE that
were removed, so that the radical scavenging activity of CSE
was markedly lower than that of BRE, especially in the
b-carotene–linoleic acid assay system. These results indicated
that BRE and brazilin have more advantages than CSE for
their application as the antioxidant in the products containing
oil–water emulsion systems.
The reducing capacity of a compound may also serve as a
significant indicator of its potential antioxidant activity
(Duh et al., 2001). The reducing properties associated with
the presence of reductones such as ascorbic acid, which is
capable of breaking the free radical chain by donating a
hydrogen atom. Reductones are also reported to react with
certain precursor of peroxide, thus preventing peroxide
formation (Jayaprakasha et al., 2001). Determination of
reducing powers of BRE CSE and brazilin found that the
reducing powers of all samples were in a dose-dependent
manner (Figure 3). In all tested concentrations, BRE
possessed equal reducing power to brazilin, but higher than
CSE. At low concentrations (10 and 50 mg/mL), both BRE
and brazilin exhibited lower reducing power than the ascorbic
acid. However, at high concentration of 100 mg/mL, they
showed comparable activity to ascorbic acid. The reducing
powers of BRE and CSE were also not related to the total
3 100
AA Brazilin BRE
phenolic content of the extracts. All antioxidant activities
suggested that BRE possessed the comparable activities to the
pure brazilin and higher than CSE. BRE can be, therefore,
considered as an alternative natural antioxidant agent.
Protein denaturation is a process in which proteins lose
their tertiary structure and secondary structure by the
application of external stress during food processing and
post-harvest treatments (Adarsh et al., 2011). Most biological
proteins lose their biological function when denatured.
The compounds inhibiting denaturation greater than 20%
over the range of concentration were considered as having
anti-inflammatory properties (Williams et al., 2008). In
this study, BRE, CSE, and brazilin, at a concentration of
0.1 mg/mL, were capable of inhibiting denaturation of albu-
min by 54.1, 61.9, and 46.8%, respectively. Surprisingly, an
increase of sample concentrations resulted in decrease anti-
inflammatory property (Table 2). However, this phenomenon
was coincident with the previous reports (Adarsh et al., 2011;
Duganath et al., 2010; Williams et al., 2008), which
demonstrated that the relationships between concentrations
and anti-denaturation effects of most plant extracts were
controversial. The results suggested BRE, CSE, and brazilin
may have the anti-inflammatory property approximately
by 50% inhibition, if they are used at low concentration
as 0.1 mg/mL.
Evaluation of antibacterial activities of BRE, CSE, and
brazilin found that all of them possessed antibacterial activity
against both Gram-positive and Gram-negative bacteria
Table 2. Anti-denaturation activity of BRE, CSE, and brazilin (Williams
et al., 2008).
Samples Concentrations (mg/mL) % Inhibition
Brazilin 0.1 46.8 ± 2.6
0.5 34.8 ± 3.9
5 21.5 ± 4.5
BRE 0.1 54.1 ± 1.3
0.5 46.8 ± 5.2
5 24.7 ± 1.3
CSE 0.1 61.9 ± 1.9
0.5 48.2 ± 1.9
5 27.5 ± 1.2
Diclofenac sodium 10 30.7 ± 3.2
50 71.5 ± 1.3
88.9 ± 1.3
CSE

2.5 a a a The results are expressed as mean ± SD (n ¼ 3). BRE, brazilin-rich

extract; CSE, crude ethanol extract of C. sappan heartwood.
Absorbance (700 nm)

2
b
a Table 3. Antibacterial activity of BRE, CSE, and brazilin against Gram-
1.5
positive and Gram-negative bacteria (NCCLS, 2008).
b b
1
c Concentrations (mg/mL)
Microorganisms
0.5 a b CSE BRE Brazilin Ampicillin
b c
MIC MBC MIC MBC MIC MBC MIC MBC
0
10 50 100 S. aureus 250 250 125 125 62.5 62.5 0.12 0.12
S. epidermidis 125 250 62.5 125 31.3 62.5 0.12 0.12
Concentration (µg/mL) B. subtilis 125 125 62.5 125 31.3 62.5 0.12 0.12
S. typhimurium 250 500 250 250 125 125 250 250
Figure 3. Reducing power of ascorbic acid, brazilin, BRE, and CSE at P. aeruginosa 250 500 250 250 125 125 0.48 0.48
different concentrations. Small letters within the same concentration of E. coli 1000 1000 500 500 250 250 31.3 31.3
different samples indicate significant difference (p50.05). AA, ascorbic
acid; BRE, brazilin-rich extract; CSE, crude ethanol extract of C. sappan n ¼ 3. BRE, brazilin-rich extract; CSE, crude ethanol extract of
heartwood. C. sappan heartwood.
DOI: 10.3109/13880209.2014.982295
(Table 3). Gram-positive bacteria were more sensitive to all
tested compounds than Gram negative bacteria. The antibac-
terial activities of BRE and CSE were related to the content of
brazilin in the extracts. BRE and CSE contained only 39% and
20% w/w brazilin, respectively. Thus, brazilin showed the
highest antibacterial activity with MICs/MBCs values of
31.3–250/62.5–250 mg/mL, followed by BRE (MICs/MBCs
values of 62.5–500/125–500 mg/mL), and CSE (MICs/MBCs
values of 125–1000/125–1000 mg/mL), respectively (Table 3).
This indicated that brazilin plays an important role in
antibacterial activity of CSE or BRE. The bactericidal effect
of brazilin could be attributed to the ability to inhibit DNA
and protein synthesis in cells (Xu & Lee, 2004).
Conclusion
The results from this study support the potential use of BRE
for antioxidant, anti-inflammatory, and antibacterial proposes
in food and nutraceutical applications. Regarding an industrial
application, BRE has more advantages than the use of pure
brazilin in terms of a simple and low-cost production process.
Acknowledgements
The authors thank Dr. Brian Hodgson for assistance with the
English language.
Declaration of interest
The authors have declared that there are no conflicts of
interest. This work was supported by Prince of Songkla
University, Thailand under postdoctoral fellowship program
and the Higher Education Research Promotion and National
Research University Project of Thailand, Office of the Higher
Education Commission.
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