Isolation of IHSS Soil Fulvic and Humic Acids

exerpt from -- Swift, R.S. 1996 Organic matter characterization (chap 35). pp. 10181020. In D.L. Sparks et al. (eds) Methods of soil analysis. Part 3. Chemical methods. Soil
Sci. Soc. Am. Book Series: 5. Soil Sci. Soc. Am. Madison, WI
Soil Science Society of America, used with permission.
Reprints can be obtained from: Roger.S. Swift, CSIRO, PMB 2, Glen Osmond 5064,
Australia.
International Humic Substance Society Method.
A number of methods for the extraction of humic substances from soil using sodium
hydroxide solution have been published. These methods are generally successful and
yield comparable results. The following is a method which has been developed by the
International Humic Substance Society (IHSS) as an acceptable method for the extraction
of humic substances from soils.
It
has
been
clearly
stated
by
IHSS
that
this
is not meant
to
be
a recommended or approved method, but a method that has been found to be
satisfactory for most soil types and one which can be performed in most laboratories. It
produces relatively high yields and can be used as a standard method for comparisons
between and within laboratories. An important component of this method is the use of an
adsorbent resin in the purification process. This can be replaced by dialysis if the resin is
unavailable (See note about dialysis at end of text).
Materials
1. Hydrochloric acid (HCl), 1 M, 6 M
2. Sodium hydroxide, 1 M, 0.1 M
3. Potassium hydroxide (KOH), 0.1 M
4. Potassium chloride (KCl)
5. Hydrofluoric acid (HF) concentrated, 0.3 M
6. XAD-8 resin (Rohm & Haas Co., Philadelphia, PA)
7. Visking dialysis tubing (Visking Co., Chicago, IL) [MWCO (molecular weight cut-off)]
10,000 dalton
Method
Remove roots and sieve the dried soil sample to pass a 2.0-mm sieve. Equilibrate the
sample to a pH value between 1 to 2 with 1 M HCl at room temperature. Adjust the
solution volume with 0.1 MHCl to provide a final concentration that has a ratio of 10 mL
liquid/1 g dry sample. Shake the suspension for 1 h and then separate the supernatant
from the residue by decantation after allowing the solution to settle or by low speed
centrifugation. Save the supernatant (FA Extract 1) for the isolation of fulvic acid using
XAD-8.
Neutralize the soil residue with 1 M NaOH to pH = 7.0 then add 0.1 M NaOH under an
atmosphere of N2 to give a final extractant to soil ratio of 10:1. Extract the suspension
under N2 with intermittent shaking for a minimum of 4 h. Allow the alkaline suspension
to settle overnight and collect the supernatant by means of decantation or
centrifugation. Acidify the supernatant with 6 M HCl with constant stirring to pH = 1.0
and then allow the suspension to stand for 12 to 16 h. Centrifuge to separate the humic
acid (precipitate) and fulvic acid (supernatant - FA Extract 2) fractions.
Redissolve the humic acid fraction by adding a minimum volume of 0.1 M KOH under
N2. Add solid KCl to attain a concentration of 0.3 M [K+] and then centrifuge at high
speed to remove the suspended solids. Reprecipitate the humic acid by adding 6 M HCl
with constant stirring to pH = 1.0 and allow the suspension to stand again for 12 to 16 h.
Centrifuge and discard the supernatant. Suspend the humic acid precipitate in
0.1 M HCl/0.3 M HF solution in a plastic container and shake overnight at room

temperature. Centrifuge and repeat the HCl/HF treatment, if necessary, until the ash
content is below 1%. Transfer the precipitate to a Visking dialysis tube by slurrying with
water and dialyze against distilled water until the dialysis water gives a negative Cl- test
with silver nitrate AgNO3. Freeze dry the humic acid.
Pass the supernatant designated "FA Extract 1" through a column of XAD-8 (0.15 mL of
resin per gram of initial sample dry weight at a flow rate of 15 bed volumes per h).
Discard the effluent, rinse the XAD-8 column containing sorbed fulvic acid with 0.65
column volumes of distilled H2O. Back elute the XAD-8 column with 1 column volume of
0.1 M NaOH, followed by 2 to 3 column volumes of distilled H2O. Immediately acidify the
solution with 6 M HCl to pH = 1.0. Add concentrated HF to a final concentration of
0.3 M HF. The solution volume should be sufficient to maintain the fulvic acid in solution.
Pass the supernatant designated "FA Extract 2" through a column of XAD-8 (1.0 mL of
resin per gram of initial sample dry weight). Repeat the back elution and acidification as
for "FA Extract 1" above. Combine the final eluates from each of the fulvic acid extracts
and pass this solution through XAD-8 resin in a glass column (column volume should be
one-fifth of sample volume). Rinse with 0.65 column volumes of distilled H2O. Back elute
with 1 column volume of 0.1 M NaOH followed by two column volumes of distilled H2O.
Pass the eluate through H+-saturated cation exchange resin [Bio-Rad AG-MP-5 (Bio-Rad,
Richmond, CA) using three times the mole of Na ions in solution]. Freeze dry the eluate to
recover the H+-saturated fulvic acid.
Comments
XAD-8 is a nonionic, macroporous (pore size 25 m), methyl methacrylate ester resin
(see "Fractionation of Humic Substances Adsorption"). Because it is sometimes difficult to
obtain, it may be necessary to use an alternative resin such as Polyclar, which is a crosslinked poly(vinylpyrrolidone) (PVP) (De Nobili et al., 1990; Watanabe & Kuwatsuka, 1991)
or other equivalent resin.
Extensive purification procedures of the resins are required before use. These methods
and methods used to store the resin are detailed by Thurman & Malcolm (1981).
If it is not possible to purify the fulvic acid using resin treatments, exhaustive dialysis
against distilled H2O is an alternative but less satisfactory method of purification. (See
note about dialysis at end of text.). If there is a significant concentration of polyvalent
cations such as A13+ present, these may form insoluble metal-humate complexes as the
solution is neutralized. Therefore, the dialysis should be carried out against dilute HC1
initially until the concentration of any polyvalent cations has been significantly reduced,
before finally dialyzing against distilled H2O. Technically, a fraction obtained in this way
should be referred to as a fulvic fraction, rather than fulvic acid, as it is likely to contain
significant amounts of unbound soil polysaccharide.
REFERENCES
De Nobili, M., G. Bragato, J.M. Alcaniz, A. Puigbo, and L. Comellas. 1990.
Characterization of electrophoretic fractions of humic substances with different
electrofocusing behavior. Soil Sci. 150:763-770.
Thurman, E.M., and R.L. Malcolm. 1981. Preparative isolation of aquatic humic
substances. Environ. Sci. Technol. 15:463-466.
Watanabe, A., and S. Kuwatsuka. 1991. Fractionation of soil fulvic acids using
polyvinyl-pyrrolidone and their ionization difference spectra. Soil Sci. Plant Nutr.
37:611-617.
Note on HF treatment: More recently a filtration step has been added for removal of
clays from the NaOH soil extract. This removes removes ash without HF. The NaOH
extract is filtered through a 0.2 micrometer polyethersulfone membrane filter (Gelman
Supor) under pressure (nitrogen gas). The extract should be filtered twice through the
same membrane. The partial plugging of the filter with the first filtration helps remove

fine clay and eliminates the need for the KOH-KCl and HF-HCl treatments to reduce the
ash content.
Note on dialysis of fulvic acids. Research on dialysis of fulvic acid shows much of
fulvic acid will pass through commercially available dialysis membranes. Dialysis is
probably not a good substitute for the resin treatment.

Cara Kerja
1.
Timbang 10-20 gram tanah ( Top Soil dan Sub Soil ) < 2 mm ( 0-10 cm
dan 10-20 cm ), 10-20 cm gram pupuk kandang ( Sapi dan Ayam ), 10-20
gram tanah Gambut, 10-20 gram pupuk Kompos. Masing sampel
dimasukkan ke dalam Erlenmeyer.
2.
Tambahkan pada masing-masing sampel 50-100 ml NaOH 0.5M
( ekstraksi ).
3.
Kemudian di kocok selama 2 jam atau lebih dengan mesin pengocok
( shaker ).
4.
Setelah selesai pengocokan dibiarkan selama minimal 1 x 24 jam, dan
kemudian suspense yang mengendap dibuang dan supernatant yang
berwarna gelap dipindahkan ke erlenmeyer yang lain.
5.
Supernatan di asamkan dengan HCl 6 N hingga pH 1.0 sambil dilakukan
pengadukan ( fraksionasi ).
6.
Endapan asam humat dipisahkan dari supernatan yang mengandung
asam fulvat dengan sentrifugal.
7.
Endapan asam humat kemudian di suspensi dengan larutan HCl 5% +
HF 5% ( 50 ml ) untuk membersihkan kandungan abu yang terdapat
dalam larutan dan kemudian di sentrifugal ( purifikasi ).
8.
Endapan asam humat di cuci dengan air aquades ( 3x ) ( furifikasi ).
9.
Asam humat yang diperoleh di keringkan dalam oven dengan suhu
0
40 C.
10. Penghitungan persentase ( berat ) asam humat yang terdapat pada
bahan
Pemisahan asam humat dan asam fulvat dari senyawa humat (bahan
humat) dilakukan dengan cara ekstraksi asam basa. Ekstraksi asam basa
didasarkan atas kelarutan asam humat yang larut dalam alkali/basa dan
mengendap dalam asam, sedangkan asam fulvat dapat larut dalam alkali
maupun asam. Contoh tanah kering 60C ditimbang 5 gram lalu dikocok
dengan 2 x 250 ml NaOH 0,1 N, kemudian disentrifuse dan hasil sentrifuse
ditera 500 ml. Untuk memisahkan bahan humat dan nonhumat yang
terapung diatasnya dilakukan penyaringan dengan buncher filter,
sedangkan untuk memisahkan bahan humat dan nonhumat yang
mengendap dilakukan dengan sentrifuse kembali. Bahan humat yang
dihasilkan diturunkan pH-nya dengan HCl 0.1 N sampai pH 2, sehingga
cairan terbagi menjadi dua bagian yaitu yang mengendap sebagai asam
humat dan yang larut sebagai asam fulvat. Sampai tahap ini, kandungan
asam humat dan asam fulvat sudah dapat diperkirakan secara kualitatif.
Asam fulvat dapat diperkirakan berdasarkan tingkat kekeruhan/kejernihan
cairan, dimana semakin jernih berarti kandungan asam fulvatnya semakin
rendah dan sebaliknya, sedangkan asam humat dapat diperkirakan
berdasarkan jumlah endapannya. Asam humat (bagian yang mengendap)

dapat diketahui secara kuantitatif dengan cara disentrifuse 3500 rpm

selama 15 menit dari bahan humat pada pH 2 , sehingga diperoleh
endapan asam humat dan cairan di atas endapan (asam fulvat) yang
kemudian dipisahkan. Asam humat hasil sentrifuse dimasukan secara
kuantitatif ke dalam cawan porselin yang sudah diketahui bobot
kosongnya, kemudian dikeringkan dalam oven 60C sampai bobotnya
konstan lalu ditimbang untuk mengetahui jumlah asam humat yang
dihasilkan.