Talanta 53 (2001) 771 – 782

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Supercritical fluid extraction in herbal and natural product

studies — a practical review
Qingyong Lang, Chien M. Wai *
Department of Chemistry, Uni6ersity of Idaho, Moscow, ID 83844 -2343, USA

Received 26 June 2000; accepted 10 August 2000

Abstract

Due to increasingly stringent environmental regulations, supercritical fluid extraction (SFE) has gained wide
acceptance in recent years as an alternative to conventional solvent extraction for separation of organic compounds
in many analytical and industrial processes. In the past decade, SFE has been applied successfully to the extraction
of a variety of organic compounds from herbs and other plants. This review article presents the practical aspects of
SFE applications in sample preparation, selection of modifiers, collection methods, on-line coupling techniques,
means for avoiding mechanical problems, and approaches to optimization of SFE conditions. SFE can also be used
to clean up pesticides from herb medicines. SFE processes can be modeled to acquire useful information for better
understanding of the extraction, mechanisms and optimization of the extraction procedures. With increasing public
interest in natural products, SFE may become a standard extraction technique for studying herbal, food and
agricultural samples. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: SFE; Review; Herbal; Natural product; Modifier; Pesticide; On-line coupling

1. Introduction one of the most interesting and active research

With the fast development of modern chro-
matographic and spectroscopic techniques, the
chemistry of natural products has made great
progress during the past decades [1]. With better
understanding of natural products, an increasing
number of people has become interested in study-
ing the active natural products as medicines [2], as
food additives [3] or as natural pesticides [4]. The
pharmaceutical studies of natural products are
* Corresponding author. Fax: +1-208-8856173.
E-mail address: cwai@uidaho.edu (C.M. Wai).
areas. Clinical tests have indicated that certain
herbal plants do contain pharmacologically active
ingredients that are effective for treating some
difficult diseases. For example, taxol, a diter-
penoid isolated from the bark of the Pacific yew
tree, showed promising results for the treatment
of ovarian, breast, lung and skin cancers [5–7].
Since pharmacologically active compounds in
herbal plants usually are in low concentrations, a
great deal of research has been done to develop
more effective and selective extraction methods
for recovery of these compounds from the raw
materials. For conventional extraction methods

0039-9140/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
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772 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
such as hydrodistillation (steam distillation) and
solvent extraction, there are few adjustable
parameters to control the selectivity of the extrac-
tion processes. Therefore, developing alternative
extraction techniques with better selectivity and
efficiency are highly desirable. Consequently, su-
percritical fluid extraction (SFE) as an environ-
mentally responsible and efficient extraction
technique for solid materials was introduced and
extensively studied for separation of active com-
pounds from herbs and other plants [8].
The high solvation power of supercritical fluids
(SF) was first reported over a century ago [9].
Demonstration of SFE technology for industrial
applications was reported by Zosel at the Max
Planck Institute for Kohlemforschung in 1969
[10]. In recent years, SFE has received a great deal
of attention as the full potential of this technology
in analytical applications has begun to emerge
[11–13]. Today, SFE has become an acceptable
extraction technique used in many areas. SFE of
active natural products from herbal, or more gen-
erally, from plant materials has become one of the
most important application areas [14,15]. With
the increasing public interest in herbal medicines
and natural products, numerous SFE-related re-
search papers in herbal or natural product studies
have been published in recent years. In this arti-
cle, a practical review of the recent development
in this area will be presented and some of the
interesting research results published within the
last decade will be discussed.
2. Major advantages of SFE technique
Because SFE has several distinct properties, it is
regarded as a promising alternative technique to
conventional solvent extraction methods. Some of
its major advantages are summarized as follows.
(1) SFs have relatively lower viscosity and higher
diffusivity (the diffusivity for SFs is  10 − 4 cm2
s − 1 and for liquid solvents is 10 − 5 cm2 s − 1).
Therefore, it can penetrate into porous solid ma-
terials more effectively than liquid solvents and,
consequently, it may render much faster mass
transfer resulting in faster extractions. For in-
stance, with comparable or better recoveries, the
extraction time could be reduced from hours or
days in a liquid–solid extraction (L–S) to a few
tens of minutes in SFE [16–19]. (2) In SFE, a
fresh fluid is continuously forced to flow through
the samples; therefore, it can provide quantitative
or complete extraction [20]. (3) In SFE, the solva-
tion power of the fluid can be manipulated by
changing pressure (P) and/or temperature (T);
therefore, it may achieve a remarkably high selec-
tivity. This tunable solvation power of SFs is
particularly useful for the extraction of complex
samples such as plant materials [21]. One good
example is the selective extraction of a vindoline
component from among more than 100 alkaloid
compounds from the leaves of Catharanthus
roseus [22]. (4) Solutes dissolved in supercritical
CO2 can be easily separated by depressurization.
Therefore, SFE can eliminate the sample concen-
tration process, which usually is time-consuming
and often results in loss of volatile components
[18]. (5) SFE usually is performed at low tempera-
tures, so it may be an ideal technique to study
thermally labile compounds [23–25] and may lead
to the discovery of new natural compounds [26].
For example, when SFE was used to extract gin-
ger, many undesirable reactions such as hydroly-
sis, oxidation, degradation and rearrangement
could be effectively prevented. Therefore, the
common difficulties for quality assessment in clas-
sical hydrodistillation could be avoided in SFE
[27]. (6) Compared with the 20–100 g of samples
typically required in L–S methods, as little as
0.5–1.5 g of samples are needed in SFE methods
[18,28,29]. It has been reported that from only 1.5
g of fresh plant samples, more than 100 volatile
and semi-volatile compounds could be extracted
and detected by gas chromatography (GC)–mass
spectroscopy (MS), of which more than 80 com-
pounds were in sufficient quantity for accurate
quantifications [18]. (7) SFE uses no or signifi-
cantly less environmentally hostile organic sol-
vents. A SFE method may need no or only a few
milliliters of an organic solvent while a typical
L–S extraction method would require tens to
hundreds of milliliters [16,30]. (8) SFE may allow
direct coupling with a chromatographic method,
which can be a useful means to extract and di-
rectly quantify highly volatile compounds [31–
 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
34]. (9) In large scale SFE processes, the fluid,
usually CO2, can be recycled or reused thus mini-
mizes waste generation. (10) SFE can be applied
to systems of different scales, for instance, from
analytical scale (less than a gram to a few grams
of samples), to preparative scale (several hundred
grams of samples) [26], to pilot plant scale (kilo-
grams of samples) [21] and up to large industrial
scale (tons of raw materials, such as SFE of coffee
beans) [35].
In addition to the advantages mentioned above,
another distinguished advantage of SFE over con-
ventional methods is that SFE can provide more
information pertaining to the extraction processes
and mechanisms. One can use such information to
quantitatively assess or evaluate the extraction
efficiency and then optimize the process accord-
ingly. This will be discussed in more detail later.
3. Fluid materials and modifiers
Of all the gases and liquids studied, CO2 re-
mains the most commonly used fluid for SFE
applications because of its low critical constants
(Tc = 31.1°C; Pc =72.8 atm), its non-toxic and
non-flammable properties, and its availability in
high purity with low cost. Supercritical CO2 has
good solvent properties for extraction of non-po-
lar compounds such as hydrocarbons, while its
large quadrupole moment also enables it to dis-
solve some moderately polar compounds such as
alcohols, esters, aldehydes and ketones.
In order to extract polar compounds, polar SF
materials should be considered. Two such polar
materials that have been successfully used for SFE
of polar compounds from plant samples are
Freon-22 (chlorodifluoromethane) [36,37] and ni-
trous oxide (N2O) [6]. The former was used for
extraction of free carboxylic acids and steroid
compounds, while the latter for extraction of
taxol. However, their application is limited due to
their unfavorable properties with respect to safety
and environmental considerations. Nitrous oxide
can cause explosion and Freon-22 is no longer
available commercially because of its ozone deple-
tion effect in the upper atmosphere.
773
Water as SF material has been reported by
Bröll et al. [38] and mainly used as a reaction
medium. Superheated water (water under pressure
and above 100°C but below its Tc of 374°C) has
been used for extraction of herbal samples [39–
41]. Although superheated water has certain ad-
vantages such as higher extraction ability for
polar compounds, it is not suitable for thermally
labile compounds. If oxygen was not carefully
purged, water at high temperature can be corro-
sive and might cause damage to extraction vessels
[39]. Therefore, in this article we will not include
supercritical water and superheated water
extractions.
The application of modifiers probably is the
simplest yet the most effective way to obtain a
desired polarity of CO2-based fluids. By selecting
a modifier or just simply changing the molar ratio
of a modifier, one can readily manipulate the
properties of the fluids. Usually, addition of a
small amount of a liquid modifier can enhance
significantly the extraction efficiency and, conse-
quently, reduce the extraction time. For example,
to extract essential oils from aromatic plants,
addition of only 0.5 ml of CH2Cl2 to 500 mg
sample in a 2.5-ml SFE cell would reduce the
extraction time from 90 to 30 min, while the
extraction efficiency was consistent with that ob-
tained in 4 h of hydrodistillation [28].
At least 17 modifiers have been studied in SFE
of natural products [8]. Among all the modifiers,
methanol is the most commonly used because it is
an effective polar modifier and is up to 20%
miscible with CO2. It was believed that high per-
centages of methanol could disrupt the bonding
between the solutes and plant matrices [42]. Etha-
nol, though not as polar as methanol, may be a
better choice in SFE of natural products because
of its lower toxicity. Several reports have success-
fully employed ethanol as a modifier in SFE of a
variety of organic compounds from plants
[6,16,43,44]. An interesting finding was that etha-
nol was a more effective modifier for SFE of
linuroon, while methanol was better for SFE of
diuron from plant materials [16].
Depending on the properties of the samples and
the desired compounds, the best modifier usually
can be determined based on preliminary experi-
774 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
mental results. For instance, in SFE of phenolic
acids, methanol was a much more effective
modifier than acetonitrile, acetone, water or
dichloromethane [45]. In SFE of paclitaxel and
baccatin III, among methanol, ethyl acetate,
dichloromethane and diethyl ether,
dichloromethane was the most effective one for
paclitaxel but diethyl ether was the best for bac-
catin III [7]. In another report, 4% of methanol or
chloroform did not result in any improvement on
the recovery of santonin (a sesquiterpene lactone),
but 4% of acetonitrile could increase the recovery
from 38 to 85%, while water-saturated CO2 could
further increase the recovery to 92% [33].
By manipulating the types and ratios of liquid
modifiers, one may obtain different extraction
results. For instance, a stepwise process has been
used in SFE of lemon grass [46]. First the extrac-
tion was performed with 10 and 30% of hexane,
followed by another step with 10 and 20% of
acetone, and finally with 10% of methanol. The
extraction result with 10% of hexane-modified
CO2 was similar to that obtained by steam extrac-
tion, while the result with 30% of hexane was
similar to that obtained with Soxhlet extraction in
hexane. Acetone-modified CO2 could extract addi-
tional compounds, and 10% of methanol modified
CO2 was not selective.
Palma et al. used SFE for investigation of the
active phenolic compounds in grape seeds [47].
With pure CO2 they obtained the first fraction
that mainly contained fatty acids, aliphatic alde-
hydes and sterols, which had a high degree of
antioxidant activity. With 20% methanol-modified
CO2 they extracted the second fraction that
mainly included epicatechin and gallic acid, which
was more agrochemically active. Because SFE
allowed the extraction of the compounds without
exposure to light and air, they found that the
antioxidant properties of the extracts could be
safely conserved.
Although moisture in raw plant materials usu-
ally is undesirable because it may cause clogging
problems, in some cases it could be an advantage
because water may function as a modifier for SFE
of certain compounds. For instance, in SFE of
pesticides from Glycyrrhizae radix or other herbal
medicines, Ling et al. found that 10% of mois-
ture was sufficient to increase the solubility power
of the mixture fluid to its maximum extent [48].
Miyachi et al. [49] also used water as a modifier to
extract a lignan (1-hydroxypinoresinol) from
Fraxinus japonica and other Fraxinus species.
A mixture of methanol and water could also be
employed as modifier. In SFE of flavonoids
(baicalin, baicalein and wogonin) from Scutellaria
radix, Lin et al. [50] observed that, for 1 g of
sample, adding 3 ml of 70% methanol
(methanol:water, 70:30) in 20 ml of CO2 gave
much better extraction than pure methanol. This
might be explained that 30% of water would
further increase the polarity of the modifier, thus
the polar constituents would be extracted more
easily. Janicot et al. [51] also extracted thebaine,
codeine, and morphine by using a mixture of
CO2 –methanol–water (70:24:6 w/w, respectively)
as a modifier.
There are three common ways to introduce a
liquid modifier into the SFE system, using a sec-
ond pump; using pre-mixed fluids from a cylinder;
and direct spiking. Compared with the other two
methods, direct spiking a liquid modifier into the
SFE cell is the simplest and the most economical
method. It also creates less mechanical and repro-
ducibility problems [28,52]. However, if the spik-
ing method is used, one must be very careful to
make sure that the binary fluid is indeed in the
supercritical state [8]. Another common problem
for the spiking method is that most of the
modifier may be flushed out of the sample vessel
in the very beginning of the dynamic extraction
step, which will likely result in inconsistent results
and, therefore, require repeated extractions [17].
Page et al. reported a comprehensive compilation
of phase behavior data for modified CO2 systems
[53].
When a liquid modifier is used, a static extrac-
tion usually is necessary because it can allow the
solvent to modify the sample matrix. Depending
on the composition of the sample and the concen-
tration of the solute, a suitable static extraction
time can vary significantly from one case to an-
other. For example, in order to extract b-carotene
from corn, a 20-min static extraction is required.
Nevertheless, if the same extraction is conducted
with green leaf vegetables, less than 10 min will be
 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
sufficient [17]. However, in SFE of fatty acids and
sterols from plant tissues, 5 – 10 min static extrac-
tions did not result in any enhanced extraction
[37]. Similarly, when using methanol modified
CO2 to extract michellamines A and B, a static
period from 5 to 60 min did not result in any
improved extraction [54], which was believed to
be an indication that such extraction was not
kinetically driven; consequently, the static extrac-
tion period would not be necessary.
In order to understand the modification mecha-
nism, the solute-modifier interactions must be
considered. Such interactions involve dipole–
dipole, dipole-induced dipole, hydrogen bonding
as well as other polarity forces. For predicting the
enhancement effects on matrix-free basis, the at-
tractive and repulsive forces have been modeled in
terms of the specific interactions using the Peng–
Robinson equation-of-state with the covolume-de-
pendent mixing rule [55].
One disadvantage of using a modifier is that it
can cause poor selectivity, i.e. more impurity com-
pounds such as chlorophylls and waxy material
may be extracted with the wanted compounds
[28,56]. If the concentration of a modifier is not
properly selected, a lower recovery might be re-
sulted probably due to the increased liquid
modifier in the collecting solvent that produced a
negative effect on the trapping efficiency [52]. In
fact, not in all SFE processes are modifiers neces-
sary and helpful. For instance, Ashraf-Khorassani
et al. [58] used pure CO2 to extract seven kava
lactones from kava roots, the yield was \90% of
that obtained with a L – S method while addition
of 15% of ethanol had no significant enhancement
effects. It was also reported that even triterpenes
could be extracted under mild SFE conditions
without any modifier [57]. In some other cases, a
modifier would only slightly improve the extrac-
tion yields so that it might not be necessary [22].
4. Sample preparation
In SFE of natural products, fresh plant materi-
als are frequently used if the desired compounds
are volatile or labile. When a fresh sample is
extracted, its high moisture content can cause
775
mechanical difficulties such as restrictor clogging
due to ice formation. One simple yet effective way
to avoid such problems is to mix the sample with
anhydrous Na2SO4. Anhydrous Na2SO4 can im-
prove SFE results because (1) it can provide bet-
ter contact between SFs and samples; (2) it can
reduce the dead volume effects; and (3) it can
retain effectively the moisture [59]. Other re-
searchers, however, believed that silica gel was a
better choice in retaining the moisture for SFE of
fresh samples [23]. Improved SFE results were
also observed as fresh ginger samples were
blended with coarse granulated celite (30–60
mesh) before loading the samples into the SFE
cell for extraction [27].
As reported by Lehotay [60], water is only
 0.3% soluble in supercritical CO2, but could
play an important role in the extraction. If excess
water remained in the extraction vessel, highly
water soluble solutes would prefer to partition
into the aqueous phase and, therefore, the SFE
recovery would be low [61]. Hydromatrix, a pel-
letized diatomaceous earth material, was another
choice of drying agent to absorb water in SFE
[59].
Weathers et al. [61] used acetals such as 2,2-
dimethoxypropane (DMP) as a modifier to re-
move water from paprika samples and to improve
the SFE yield of b-carotene. The reaction of DMP
with water is shown below:
(CH3)2C(OCH3)2 + H2O
“ 2CH3OH+ CH3COCH3
Upon addition of 500-ml of DMP to the sample
in a 7-ml SFE cell, the extraction efficiency of
b-carotene from wet paprika was increased by a
factor of about 2.5 and from dry paprika a factor
of 1.5. This is an indication that the DMP could
act as a ‘drying reagent’ and a modifier as well.
Sometimes, dry plants might be preferred for
SFE. Drying procedures for a plant material
could affect the yield and quality of the extracts.
Ibáñez et al. [62] investigated three drying meth-
ods for rosemary, which included (1) freeze-dry-
ing; (2) oven-drying at 45°C until reaching a
constant weight; (3) vacuum rotary evaporation at
35°C until reaching a constant weight. Freeze-dry-
ing was found to provide the highest quantity of
776 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
rosemary essential oil followed by oven-drying at
45°C and vacuum rotary evaporation. However,
based on the antioxidant activity, drying at ambi-
ent temperature and in a ventilated place was the
method that provided better results.
Sample particle size is a critical factor for a
satisfactory SFE process. Large particles may re-
sult in prolonged extraction because the process
can become diffusion-controlled [63]. A good ex-
ample is the SFE of basil leaves. If the sample
particle size was 0.55 mm, 5 h was not long
enough to achieve a satisfactory extraction. If the
sample was pulverized into 0.17-mm particles, 2 h
was sufficient to complete the extraction [21]. In
fact, pulverizing a sample into fine powder can
speed up the extraction and improve the effi-
ciency, but it may also cause difficulty in main-
taining a proper flow rate. One effective way to
overcome the flow rate problem is to pack the
sample with glass beads or other rigid inert mate-
rials such as sea sand. These rigid particles can
prevent the samples from pressing into solid im-
permeable plugs and thus maintain a desired per-
missibility of the sample bed [7]. A metal filter
installed at the end of the inlet side of the SFE cell
was also helpful to maintain a smooth flow rate
[22] and avoid channeling of the fluid flow [7].
Depending on the distribution of the target
compounds within the sample matrix, a SFE pro-
cess may tail off dramatically after the majority of
the compounds are removed from the matrix sur-
faces at the beginning of the extraction. This is
because the remaining solutes are held inside the
structures of the sample particles and can only be
extracted with a prolonged time. For instance, a
99% removal of a particular compound may re-
quire an extraction time up to ten times that
required to remove the first 50% [64]. Therefore,
using a prolonged extraction time to gain a
slightly higher recovery may not be economically
worthwhile.
5. Extraction conditions
For successful SFE, several factors must be
taken into consideration prior to the experiments.
These factors include the type of sample, method
of sample preparation, type of fluid, choice of
modifiers, method of fluid feeding, and extraction
conditions including pressure, temperature, flow
rate and extraction time. In order to optimize the
SFE conditions, a statistical experimental design
based on a ‘second order central composite de-
sign’ was used and reported by Adasoglu et al.
[65].
In most cases of plant extractions, the diffusion
of the solutes out of the matrix usually is the
limiting step [63]. The diffusion rate of a com-
pound from the sample matrix can be affected by
the following three factors, (1) occupation of the
matrix sites by the SF molecules, which could
reduce the affinity of the matrix for the solutes;
(2) dissolution of the solutes in the SF, which is
directly related to the fluid density; and (3) tem-
perature effects, which can influence the volatility
of the solutes, particularly for those with high
boiling points [16,59].
The solubility of a target compound in a SF is
a major factor determining its extraction effi-
ciency. The solubility is controlled by the sum of
two factors — the volatility of the substance,
which is a function of temperature; and the solva-
tion effect of the SFs, which is a function of fluid
density [8]. The number of polar function groups,
e.g. hydroxyl groups, may affect the volatility of
the solutes thus determine their extractability with
SF CO2. For instance, Stahl et al. [66] were able
to extract steroids with three hydroxyl groups
below 300 bar, but they were unable to extract
those steroids containing four hydroxyl groups, or
three hydroxyl and one acid group, or one pheno-
lic hydroxyl with two other hydroxyl groups.
In SFE of b-carotene from paprika, Weathers
et al. [61] observed that when pressure decreased
from 338 to 250 bar, which corresponded to a
decrease of the fluid density, the extraction effi-
ciency was dramatically reduced. However, in
SFE of lignans, schisandrol A, schisandrol B,
schisandrin A, schisandrin B, and schisandrin C,
from Schisandra chinensis, Choi et al. found that
if the extraction time was sufficiently long (e.g. 30
min 100 mg − 1 plant material), the temperature
(40–80°C) and pressure (135–340 bar) of the SF
CO2 had no distinct effect on the extraction yields
of the targeted lignans. But if the extraction time
 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
was 6 min, increasing the pressure greatly en-
hanced the yields of the lignans [67].
Generally speaking, it is often desirable to ex-
tract the samples right above the point where the
desired compounds just become soluble in the
fluid so that the extraction of other compounds
can be minimized. To achieve a good selectivity
for a SFE process, careful control of the fluid
density is essential. For instance, in SFE of essen-
tial oil from the fruits of archangelica off. Hoffm.,
it was found that at the optimum temperature
(60°C), as the density of CO2 was controlled at 0.3
g ml − 1, practically no other compounds were
extracted except the desired ones [68]. If volatile
compounds are to be extracted, increasing CO2
density to more than 0.80 g ml − 1 would result in
a lower yield for the compounds and lead to the
co-extraction of a significant quantity of triglyce-
ride material, which would deteriorate the quality
of the extract and cause clogging problems
[27,69]. It was also reported that 40°C and 80 atm
were suitable for SFE of geranyl geraniol, a diter-
penoid, without extracting limonoid cedrelone, a
triterpenoid [34]. The optimum conditions for
SFE of essential oil from rosemary, marjoram and
basil leaves are in the ranges of 40 – 50°C and
80–100 bar [21].
By controlling the fluid density, fractionation of
the extracts could be achieved. For instance, a
two-step SFE of antioxidant active compounds,
carnosic acid, carnosol, rosmanol, epirosemanol
and isorosemanol, from rosemary leaves could
separate the oleoresin into two fractions with
different antioxidant activities and essential oil
compositions [62]. The first step was performed at
40°C and 100 bar and second step at 60°C and
400 bar, both with 5-min static followed by 30-
min dynamic extractions.
However, if the targeted compounds are not
that volatile, higher CO2 density may be required.
For instance, in SFE of Pacific yew tree needles,
at 300 atm and 40°C (higher CO2 density) the
target compounds, taxol and baccatin, can be
effectively extracted, but at 100 atm and 40°C
(lower CO2 density), the major portion of the
extract is the waxy compounds [43]. Similar re-
sults were also observed in SFE of vindoline (an
alkaloid) from the leaves of C. roseus, in which a
777
remarkably high yield was obtained at the lowest
temperature (35°C) and the highest pressure (300
bar) tested, i.e. with the highest fluid density [22].
In addition to being used as a drying agent,
silica gels can also be used to couple with SFE
techniques to improve the overall extraction selec-
tivity. As the effluent passes through the silica gel
column, polar compounds will be retained and,
therefore, separated from non-polar constituents
[33]. Cascade separators based on gradient fluid
density can also provide further separations of the
extract. For instance, essential oils with molecular
weight (MW) 140–160 could be nearly completely
separated from cuticular waxes (mainly n-
paraffins ranging from C25 to C35 with mean MW
408–430) by using post-extraction separators. The
waxy compounds are deposited in the first separa-
tor at 0°C and 80 atm [21].
Internal standards were also used in SFE pro-
cesses to assess extraction results and optimize the
extractions [27,33,56], but this method may be
questionable because native and spiked com-
pounds can show different affinities toward ma-
trix sites. Native compounds usually are
distributed throughout the plant matrices, but the
spiked standards are likely only associated with
surface sites. Therefore, high recovery of the
spiked compounds does not necessarily mean high
recovery of the native compounds, and compari-
son of the SFE method with other extraction
methods may be needed [28].
Due to high moisture contents in organic mate-
rials, clogging can be a very common mechanical
problem in SFE of plant samples. As mentioned
above, anhydrous Na2SO4 and silica gel are effec-
tive drying agents to absorb moisture from extrac-
tions. Heating the restrictor to a proper
temperature is another effective means to avoid
the deposition of the extracted compounds so that
clogging problems can be eliminated [28,44]. An
easier yet still effective way is to use a high
concentration of a liquid modifier to increase the
solubility of the organic compounds so that they
will be effectively flushed out into the collecting
vials [5]. For example, in SFE of taxanes from
needles of the English yew tree, Taxus baccata,
when the concentration of methanol was greater
than 5%, restrictors would not be easily clogged
778 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
by a waxy material [5]. Again, the density of CO2
should be carefully determined; otherwise, CO2 at
high density (] 0.80 g ml − 1) may extract signifi-
cant quantities of triglyceride material, which will
cause clogging of the restrictor [27].
One interesting but not commonly used appli-
cation is the in situ trans-esterification and extrac-
tion of certain thermally labile compounds [70].
Without the use of in situ transesterification dur-
ing the static period of the SFE process, those
compounds would undergo thermal isomerization
under analytical GC conditions, thus making the
quantification extremely difficult. Interested read-
ers may refer to the original paper for details [70].
6. Collection methods
In SFE of natural products, a commonly used
collection method is to trap the extract in a liquid
solvent such as CH2Cl2 [28] or ethyl acetate [59].
Keeping the collection vial in water at room
temperature was found helpful in increasing the
recovery of kava lactones [44]. Under cooling
conditions during expansion of a fluid, different
collection solvents may show different collection
efficiencies. For instance, as the collection vial
was cooled to − 8°C, CH2Cl2 and acetone would
still give better recoveries of volatile compounds
than methanol or n-hexane [52]. Compared with
trapping in methanol-ice (− 15°C) or in acetone-
dry ice ( −60°C), trapping in liquid nitrogen (−
170°C) was found to be the most effective way in
collecting volatile compounds [33]. The depth of
the trapping solvent might also affect the collec-
tion results. The same volume of a solvent in a
narrower vial was more effective than in a wider
vial [52].
Solid trapping is another common way for ex-
tract collection. Octadecylsilica (ODS) traps can
be used at 15°C to collect the essential oils and
the bitter principles from hops, and after SFE the
retained compounds can be rinsed out with aceto-
nitrile for recovery [56]. It was also found effective
to retain volatile compounds on an ODS solid
trap at lower temperature (15°C) and then to elute
these compounds with an organic solvent at an
elevated temperature (45°C) [18]. An advantage
for solid trapping is that the selectivity can be
further improved by selective trapping coupled
with selective eluting. For example, polar com-
pounds can be trapped on a silica gel column and
then eluted with proper solvents [33]. Neverthe-
less, there are also some disadvantages for solid
trapping methods. An improperly selected liquid
modifier may result in significant losses of the
wanted compounds because they may be washed
through the trap by the modifier solvent. Unde-
sired reactions were also observed as the elution
was done with improper solvents [17].
Off-line SFE/GC ‘coupling’ is a good alterna-
tive way for highly volatile compounds. A special
sample collection method was proposed by Ibáñez
et al. [71], which consisted of a glass liner of a
Perkin–Elmer programmed temperature vapor-
izer (PTV, 100× 1 mm i.d.) placed inside a stain-
less steel chamber, which was surrounded by a
cooling jacket that could be refrigerated to −
25°C using liquid CO2. The glass liner was filled
with quartz wool at both ends and GasChrom 220
(80/100 mesh) in the middle as the absorbent for
collection of highly volatile components. After
extraction, the liner was introduced into the GC
injector where desorption took place as the tem-
perature increased. Significantly high recoveries of
very volatile compounds were achieved.
7. Clean-up of pesticides from herbal plants and
other samples
In addition to extracting desired compounds
from plants, another very interesting application
of the SFE technique is the extraction or clean up
of pesticides from natural products including
herbal medicines [48,59,72–75]. Because the natu-
ral resource of medicinal plants is limited, manu-
facturers usually get their raw materials through
cultivation of the plants. For instance, millions of
ginkgo trees have been cultivated in Europe,
North America and China [76]. The cultivation of
herbal plants usually takes years and requires the
application of pesticides to control pest damage.
Improper use of pesticides may pollute the soil,
groundwater and cause the accumulation of the
pesticides in the plants. SFE has demonstrated its
 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
ability to serve as a screening or clean-up protocol
for pesticides on a routine basis. Ling et al. devel-
oped a SFE procedure for 13 organochlorine pes-
ticides including heptachlor, aldrin and DDT, and
the mean recoveries for spiked standards were
78–121% [48]. Although the organochlorine pesti-
cide concentrations in marketed herb medicines
were found to be much lower than the FDA
action levels, the SFE method did provide a
screening protocol for the determination of those
harmful compounds. Stefani et al. extracted 92
different pesticides with SF CO2 under milder
conditions (45°C and 189 bar) [74]. A review of
SFE application in removing pesticides (insecti-
cides, fungicides and herbicides etc.) from natural
products was published by Lehotay in 1997 [60].
As Lehotay indicated that based on the solubility
properties of pesticides, SFE using CO2 was well
suited for the extraction of most pesticides with-
out difficulty. SF CO2 undoubtedly provides an
effective means to clean up any pesticide residues
from herb or other plant materials without intro-
ducing any toxic solvent residues.
For collection, the general use of ODS with
acetone elution was found to be the best choice
for SFE trapping of pesticides [75]. The use of the
ODS trap and acetone elution solvent gave the
most consistently high recoveries of the traps and
solvents tested. ODS trap could be achieved with
40-mm particle size contained in 1-ml trap.
8. On-line fractionation and coupling with
chromatographic methods
On-line fractionation is another distinctive ad-
vantage of SFE. By manipulating the SFE condi-
tions, it is feasible to separate the extracted
compounds into two to more groups. For exam-
ple, in SFE of Thymus mastichina L., Blanch et al.
[77] used two on-line separation vessels to obtain
the less and the medium volatile fractions (tem-
perature and pressure were set at 50°C, 150 bar
and 25°C, 50 bar, respectively) and a glass liner of
PTV with a 22.5-mg plug of Tenax TA as adsor-
bent to collect the volatile fraction. The first and
second fractions were recovered by solvent elu-
tion, while the third fraction, which was retained
779
in the glass liner, was directly introduced into a
GC for analysis. The desorption and the transfer
of the retained material to the capillary column
were achieved by raising the injector temperature
to 350°C. This procedure is particularly useful to
analyze complex samples and the loss of the
volatile compounds could be minimized. The au-
thors believed that their procedure could also be
applied in the fractionation of SF extracts of
different samples such as foodstuffs, although the
optimum conditions might be different.
In SFE of azadirachtin A, a natural pesticide,
from neem seed kernels, Ambrosino et al. used a
three-stage separation (fractionation) technique in
a pilot plant scale, and they obtained cuticular
waxes in the first separator and the oil com-
pounds in the second and third separators [47].
On-line coupling of SFE with a chromato-
graphic method is a useful approach for analyzing
volatile compounds. Smith et al. [33] found that
cryogenic focusing (B − 10°C) for retaining
volatile constituents was usually required, that is,
the volatile compounds were deposited and en-
riched at a low temperature and then evaporated
at a high temperature. In addition to the on-line
SFE–GC coupling, other on-line coupled tech-
niques included SFE–SFC, SFE–FTIR and
SFE–MS were also reported [8].
Ramsey et al. [78] reported a SFE–LC–MS
on-line coupling combined with a UV/vis diode-
array detection to obtain a high degree of specific-
ity for analysis of targeted compounds. They used
a high pressure ten port switching valve for the
on-line coupling and successfully determined
drugs at ppb level.
Although on-line coupling technique demon-
strated its merits such as effective for volatile
compounds and easy for automation, neverthe-
less, some disadvantages limited its applications.
These disadvantages include, (1) sample size may
be limited; (2) chromatographic system can be-
come contaminated by highly retained compo-
nents; (3) with inhomogeneous matrices the small
portion analyzed may not be representative of the
bulk sample; (4) it is easier to establish the opti-
mum conditions for SFE in an off-line mode
because the analysis step is independent of the
extraction [33].
780 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
9. SFE modeling
Another distinguished advantage of SFE meth-
ods is that through modeling, one can obtain
more useful information than it can be obtainable
from conventional extraction methods. Modeling
SFE processes can help to better understand the
extraction mechanisms and quickly optimize the
extraction conditions. A large amount of research
has been done in this area [5,21,62,63,79]. Dis-
cussed below is one simple but practically useful
model — the hot-ball model, which was devel-
oped by Bartle et al. in 1990 [80].
Suppose all sample particles are in regular
spherical shapes and the mass of a solute initially
in the sample matrix is M0. After a given extrac-
tion time, t, the remaining mass of the solute in
the matrix becomes M. Then as t ] tc, linear
relation will appear by plotting ln(M/M0) against
t/tc, where tc is the characteristic time that is
related to the diffusion coefficient, D, and the
radius of the spherical sample particle, r, by tc =
r 2/p 2D. This suggests that the extraction rate can
be related to two factors — the diffusivity of the
analyte inside the matrix and the size of the
sample particles.
If SFE is carried out at least as long as the
initial non-exponential period, the mass of the
extracted solute is M1, followed by extraction
over two subsequent time periods to obtain the
solute masses M2 and M3. Then the total mass of
the solute initially in the sample can be calculated
as:
M1 + M22
M0 =
(M2 − M3)
Here M0 is not only useful for mechanism
studies, but it is also the easiest way to estimate
the efficiency of an extraction process [64].
After the M0 is calculated, plotting ln(M/M0)
versus extraction time usually shows two portions
on the extraction curve — an initial falling por-
tion and then a straight-line portion. The initial
fall is believed to include any effects arising from
solubility limitations, particularly the removal of
a solute from the outer surfaces of the particle
matrices. The straight-line portion is indicative of
extraction from deeper within the matrix; there-
fore, it is the most time consuming part of an
extraction. If plotting ln(M/M0) against extraction
time only shows a straight line without steeply
falling at first, then it is believed to be due to
either (1) the low solubility of the solute in the SF
or (2) evaporation of solute from the surface
layers prior to extraction [3]. By using the hot-ball
model, the data acquired from a short extraction
can be used to obtain quantitative analytical in-
formation [64]. For instance, using the kinetic
extraction modeling and with pure CO2, Dean
and Liu [81] predicted that the theoretical yields
(M0) for deoxyschisandrin from the fruit of S.
chinensis was 0.496% (w/w) and for paeonol from
Paeonia suffruticosa was 2.135% (w/w), and their
experimental yields were 0.449 and 1.960%, re-
spectively. The experimental results were well con-
sistent with the predicted values (B10%). When
they used methanol modified CO2 (10% methanol)
as the extraction fluid, they recovered 0.483% of
deoxyschisandrin and 2.346% of paeonol, this
again confirmed the usefulness of the kinetic ex-
traction model. The authors also recommended
an extraction time that was four times of tc be
used for quantitative extraction results.
10. Conclusion
Several years ago, Modey et al. summarized the
applications of SFE in different classes of natural
products such as carotenoids, lipid materials,
flavor and fragrance compounds, triterpenes and
sterols, alkaloids, mycotoxins and others [8]. In
another review paper, Reverchon mainly focused
on the SFE of essential oil and related compounds
[82]. This review article presents the practical as-
pects of SFE such as modifiers, sample prepara-
tion, special considerations for collection,
modeling, and as well as recent developments in
SFE technology for the extraction and separation
of active compounds from herbs and other plants.
Rapid developments in SFE techniques for herbal
and natural product studies are anticipated in the
future. It is hoped that this review may provide a
convenient basis for the general readers who are
interested in understanding and involving in this
new research area.
 Q. Lang, C.M. Wai / Talanta 53 (2001) 771–782
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