Journal of the Science of Food and Agriculture J Sci Food Agric 83:783–786 (online: 2003)

DOI: 10.1002/jsfa.1406

Supercritical carbon dioxide extraction for

identification of adulteration of black pepper
with papaya seeds
Paramita Bhattacharjee,1 Rekha S Singhal1∗ and Achyut S Gholap2
1 Food and Fermentation Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai, 400 019,
India
2 Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400 085, India

Abstract: Supercritical fluid extraction (SFE) using carbon dioxide was used to detect the adulteration
of black pepper powder with ground papaya seed. Thin-layer chromatography analysis of the SFE
extracts showed a fluorescent band at 366 nm at Rf 0.172 that proved to be a promising marker for the
presence of papaya seed powder in black pepper powder even at a level of 20 g kg−1 . The straight-chain
aldehydes n-nonanal, n-decanal and n-dodecanal were tentatively identified by gas chromatography–mass
spectrometry analysis as components of this fluorescent marker and were not present in black pepper
extracts.
 2003 Society of Chemical Industry

Keywords: black pepper; papaya seed; adulteration; supercritical carbon dioxide extraction; GC–MS

INTRODUCTION Act (FD&CA)] based on concerns for their toxicity

Spices have been used in processed foods since time
immemorial to enhance consumer appeal. Among
all the spices, black pepper occupies an important
position in the global spice business. In pepper-
growing regions of southwest India, pepper is referred
to as ‘Karuthaponnu’ or ‘black gold’.
Food commodities have always been vulnerable to
fraudulent admixture or adulteration with inexpensive
inferior materials. Standards for pepper are available
from the International Standards Organization (ISO).1
Adulteration has been reported in whole pepper,
ground pepper, essential oil and oleoresins. Most
importantly, whole papaya seeds have been used
as adulterants of black pepper because of their
structural resemblance.2 Undetected adulteration of
black pepper can lead to adulteration of processed
products, such as black pepper powder and oleoresin.
A simple thin-layer chromatography (TLC) method
to detect the adulteration of ground black pepper with
ground papaya seed was developed in our laboratory.
This was based on bluish green fluorescence at 366 nm
at Rf 0.943 of ethylene dichloride extract. This method
could detect papaya at a level of 20 g kg−1 .3
Many solvents have come under increased scrutiny
and regulatory restrictions [such as the Clean Air
Act (CAA), Clean Water Act (CWA), Comprehensive
Environmental Response, Compensation, and Liabil-
ity Act (CERCLA) and Food, Drug, and Cosmetic
and their contributions to air and water pollution. It
is for these reasons that much of the research and
development in green chemistry reaction conditions
is focused on alternative solvents. The use of super-
critical carbon dioxide as a greener solvent has been
actively investigated.4 The present work focuses on
the use of this solvent for the extraction of marker
compounds in adulterated black pepper. The use of
eco-friendly carbon dioxide as an alternative to ethy-
lene dichloride would be proper from the perspective
of green technology, as opposed to the conventional
solvent extraction procedure.
MATERIALS AND METHODS
Materials
Seeds of commercial mature black pepper (Piper
nigrum L) and papaya seeds (Carica papaya L) (var
Washington) obtained from a local market in Mumbai
were used in the present study. All chemicals used
were of analytical grade. Aluminium TLC plates
(10 cm × 10 cm) precoated with silica gel 60 (F254 )
(Merck, Darmstadt, Germany), a CAMAG HPTLC
Linomat (Muttenz, near Basle, Switzerland) and a
Shimadzu QP-5050A GC/MS instrument equipped
with a GC-17A gas chromatograph (Kyoto, Japan) and
DB-1 capillary column were used. For supercritical
fluid extraction (SFE), the SPEED-SFE model from

∗ Correspondence to: Rekha S Singhal, Food and Fermentation Technology Department, Institute of Chemical Technology, University of
Mumbai, Matunga, Mumbai, 400 019, India
E-mail: rekha@foodbio.udct.ernet.in
(Received 28 March 2002; revised version received 5 August 2002; accepted 15 January 2003)

 2003 Society of Chemical Industry. J Sci Food Agric 0022–5142/2003/$30.00 783

P Bhattacharjee, RS Singhal and AS Gholap
Applied Separations (Allentown, PA 18103, USA) was
used.
Methods
The seeds used for the study were cleaned manually
to free them from stones and other undesirable matter
and then ground in an electrically operated grinder
(Crompton Greaves Ltd, Mumbai, India) to ∼0.5 mm
particle size.
SFE of model blends
Model blends of black pepper and papaya seeds in
proportions of 100:0, 98:2, 95:5, 90:10, 80:20 and
0:100 were prepared on a weight basis and ground as
described above. The extraction parameters, such as
temperature and pressure, were optimized to extract
selectively certain marker compounds that are present
in papaya seeds but not in pepper. Samples weighing
approximately 25 g were used in all the extractions.
Pressure was held at two levels, 165–170 bar and
350–355 bar, and temperatures of 45 and 80 ◦ C were
used. Preliminary trials had indicated that a 20 min
static time and a 2 h dynamic time were best for the
extraction, and hence these were maintained in this
work. The extracts were collected in ∼50 ml n-hexane
at −2 to −5 ◦ C at a flow rate of 0.1 l min−1 of carbon
dioxide at the collection end. The optimization of
the parameters was carried out using black pepper
and papaya alone. These optimized conditions were
used in the subsequent trials with the blends. For
TLC analysis, the extract was concentrated on a
Rotavac (Buchi, Flawil, Switzerland) system (28 ◦ C
and 200 mmHg for 7 min) to a volume of ∼0.5 ml. For
gas chromatography–mass spectrometry (GC–MS),
the extracts were further concentrated to a volume of
0.1 ml by slow removal of the collection solvent by a
gentle nitrogen stream.
TLC analysis of the extracts
The concentrated extracts were spotted onto the
aluminium-coated silica gel 60 (F254 ) plates by use
of the Camag Linomat IV. The extracts (20 µl) were
applied to the plates in the form of narrow bands, each
6 mm long; the spacing between consecutive bands
was 8 mm. Nitrogen gas was used at a flow rate of
0.067 µl s−1 for spotting. The plates were run up to
9 cm for 15 min at room temperature (26 ± 2 ◦ C)
in a glass chamber containing ethylene dichloride
as the developing solvent. Of all the solvents tried,
ethylene dichloride gave the best resolution, and hence
necessitated its use in TLC. The plates were then
removed, air-dried and viewed under UV light at
366 nm.
GC–MS analysis of the extracts
The SFE extracts were analysed on a Shimadzu
QP-5050A GC/MS instrument equipped with a GC-
17A gas chromatograph with a flame ionization
detector. A DB-1 (polymethyl siloxane with 1%
phenyl modification) capillary column (length 30 m,
784
internal diameter 0.25 mm, film thickness 0.25 µm)
was used for the analysis. Helium was used as
the carrier gas. 1 µl of sample solution was taken
for analysis and the samples were injected in the
splitless mode. The following temperature program
was used: 60 ◦ C for 5 min; 60 to 200 ◦ C at a rate
of 4◦ C min−1 ; 200 ◦ C for 5 min; 200 to 280 ◦ C at
a rate of 10 ◦ C min−1 ; 280 ◦ C for 25 min. Injector
and interface temperatures were maintained at 210 ◦ C
and 230 ◦ C respectively; ionization voltage was 70 eV;
electron multiplier voltage was 1 kV. Matching of
mass spectral fragmentation patterns with those in the
spectral library (Wiley/NIST Library) provided with
the instrument identified the compounds of interest.
RESULTS AND DISCUSSION
The supercritical carbon dioxide extracts of black
pepper and papaya and their blends were found
to yield characteristic patterns under UV light at
366 nm. Earlier work with ethylene dichloride Soxhlet
extracts had shown the presence of two fluorescent
spots exclusively in papaya seed extract at Rf 0.8 and
0.943, which were absent from black pepper extracts.
However, these bands were also observed in SFE
extracts of black pepper even at the low pressure
of 165 bar (at both 45 ◦ C and 80 ◦ C). To establish
the identity of these bands, their partial purification
from both the pepper and papaya seed extracts
was attempted by silica column chromatography as
reported by Paradkar et al.3 The band with Rf 0.943
was obtained in the initial fraction (first 40 ml) in both
cases, as confirmed by spotting on the silica plate.
This fraction was concentrated for GC–MS analysis
separately for pepper and papaya. The percentage
similarity indices (SI), obtained by matching with
authentic spectra from the library mass spectral
database, are shown in Table 1. A quite similar
composition was obtained for both pepper and papaya
seed extracts for the band with Rf 0.943, with
a number of compounds being common to both
extracts. However, since certain compounds, such as
β-elemene, α-murolene and β-bisabolene, were found
exclusively in pepper, they cannot be used as markers
of adulteration. 3-Eicosene was found in papaya seed
extract alone. But this compound cannot be used as
a marker as it could not be detected as a single band.
The band with Rf 0.8 was obtained in the later column
fractions for both pepper and papaya seed extracts,
and the compounds therein were tentatively identified
by GC–MS analysis and their possible compositions
are listed in Table 2. whereas the spot at Rf 0.8 in
ethylene dichloride extracts of papaya seed could only
be detected at concentrations of 30 g kg−1 and above,
it was possible to detect it at 20 g kg−1 , even under
low-pressure conditions, using SFE.
It was observed that the SFE extracts of papaya seeds
obtained under similar extraction conditions showed
an additional green fluorescent band at Rf 0.172, the
intensity of which was less at 80 ◦ C than at 45 ◦ C,
J Sci Food Agric 83:783–786 (online: 2003)
 Identification of black pepper adulteration

Table 1. MS identification of peaks in the band with Rf 0.943 black pepper, extraction of the latter was carried
out at a pressure of 350–355 bar at 45 and 80 ◦ C.
Tentative
Retention time identification SI No such band could be detected by TLC analysis,
(min) of compound (% probability) whereas for papaya seed extract this band could also
be detected under these high-pressure conditions.
25.26 α-Copaenea 96
This green fluorescent band can thus be considered
25.40 β-Elemeneb 94
26.39 β-Caryophyllenea 96
as a marker for the detection of adulteration of
28.60 β-Selinenea 95 black pepper with papaya seeds. It is known that
28.78 α-Selinenea 95 supercritical carbon dioxide has greater solvating
28.91 α-Muroleneb 94 power and penetrability into solid matrixes than
29.26 β-Bisaboleneb 95 normal liquid solvents. It is reported that certain long-
29.83 δ-Cadinenea 95 chain hydrocarbons produced as a consequence of
30.71 Nerolidola 96 irradiation can be isolated from the lipid matrixes of
37.82 3-Eicosenec 97
irradiated samples by SFE, but not by conventional
a Compound present in SFE extracts of both pepper and papaya. solvent extraction procedures, as in case of irradiated
b Compounds present only in black pepper. cashew nuts (Bhattacharjee and Singhal, unpublished
c Compound present only in papaya seed.
data). SFE has been successfully applied to irradiated
peanuts, instant soup mix powder, roasted pistachio
Table 2. MS identification of peaksa in the band with Rf 0.8 nuts, duck breast, pork meat and German caviar,6
beef,7 freshwater fish (tilapia) and sea-water fish
Retention time Tentative identification SI
(mullet),8 cheese, chocolate and liquid whole egg9
(min) of compound (% probability)
for the extraction of the long-chain hydrocarbons.
18.80 Dodecane 95 Thus, in our present study, we could isolate certain
20.15 trans-2-Undecenal 95 compounds, which would not be obtained by Soxhlet
23.86 2-Undecenal 95 extraction using ethylene dichloride.
27.84 1-Dodecanol 96
The optimized conditions of SFE that allowed the
29.22 Pentadecane 96
32.33 Hexadecane 97 best possible detection of the band with Rf 0.172
38.03 Heptadecane 97 were a sample size of 25 g extruded at 45 ◦ C and
pressure of 165–170 bar for 20 min static time, 2 h
a Present in SFE extracts of both pepper and papaya.
dynamic time with a flow rate of 0.1 l min−1 of
carbon dioxide at the collection end. This band
which is in accordance with the decreased solubility was scraped from the TLC plate and characterized
effect observed in many supercritical systems.5 This by GC–MS (Fig 1). MS identification of the peaks
band could be detected in the SFE extracts of a obtained is given in Table 3 with SI. The compounds
98:2 blend of black pepper:papaya. To check for tentatively identified were n-nonanal, n-decanal and
the possibility of the presence of the same band in n-dodecanal.

(3)

(1)

(2)

10 20 30 40 50 60

Figure 1. GC pattern of supercritical carbon dioxide extract of papaya seed, representing Rf 0.712 on TLC.

J Sci Food Agric 83:783–786 (online: 2003) 785

P Bhattacharjee, RS Singhal and AS Gholap

Table 3. MS identification of peaks in the band with Rf 0.176 on TLC 2 Govindarajan VS, Pepper—chemistry, technology and quality
evaluation. CRC Crit Rev Food Sci Nutr 9:115–225 (1977).
Tentative 3 Paradkar MM, Singhal RS and Kulkarni PR, A new TLC method
Retention time identification SI to detect the presence of ground papaya seed in ground black
Peak noa (min) of compound (% probability) pepper. J Sci Food Agric 81:1322–1325 (2001).
4 Anastus PT and Williamson TC, Green chemistry: an overview,
1 14.192 n-Nonanal 97 in Green Chemistry: Designing Chemistry For the Environment,
2 18.167 n-Decanal 95 ACS Symposium Series 626. American Chemical Society,
3 25.96 n-Dodecanal 95 Washington DC, Chapter 1, p 1–17 (1996).
5 Palmer MV and Ting SS, Applications for supercritical fluid
a With reference to Fig 1. The remaining peaks in the figure have been technology in food processing. Food Chem 52:345–352 (1995).
identified as trisiloxanes and silicate anion tetramers. 6 Lembke P, Börnert J and Engelhardt H, Characterization of
irradiated food by SFE and GC-MSD. J Agric Food Chem
43:38–45 (1995).
CONCLUSION 7 Hampson JW, Jones KC, Foglia TA and Kohout KM, Supercrit-
Adulteration of black pepper powder with papaya ical fluid extraction of meat lipids: an alternative approach to
seed powder at levels of 20 g kg −1 and above can be the identification of irradiated meats. J Assoc Off Anal Chem
detected on the basis of the presence of a fluorescent 73:717–721 (1996).
band at Rf 0.172 at 366 nm in an SFE extract of the 8 Tewfik IH, Ismail HM and Sumar S, A rapid supercritical
fluid extraction method for the qualitative detection of 2-
sample. alkylcyclobutanones in gamma-irradiated fresh and sea water
fish. Int J Food Sci Nutr 50:51–56 (1999).
9 Horvatovich P, Miesch M, Hasselmann C and Marchioni E,
REFERENCES Supercritical fluid extraction of hydrocarbons and 2-
1 Heath HB, The quality control of flavouring materials, in Quality alkylcyclobutanones for the detection of irradiated foodstuffs.
Control in the Food Industry, Vol 4, Ed by Herschdoerfer SM. J Chromatogr A 897:259–268 (2000).
Academic Press, London, p 204 (1987).

786 J Sci Food Agric 83:783–786 (online: 2003)