Vous êtes sur la page 1sur 23

Journal of Chromatography A, 1163 (2007) 2–24

Review

Supercritical fluid extraction in plant essential and volatile oil analysis


Seied Mahdi Pourmortazavi ∗ , Seiedeh Somayyeh Hajimirsadeghi
Faculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, P.O. Box 16765-3454, Tehran, Iran
Received 21 February 2007; received in revised form 1 June 2007; accepted 4 June 2007
Available online 17 June 2007

Abstract
The use of supercritical fluids, especially carbon dioxide, in the extraction of plant volatile components has increased during two last decades
due to the expected advantages of the supercritical extraction process. Supercritical fluid extraction (SFE) is a rapid, selective and convenient
method for sample preparation prior to the analysis of compounds in the volatile product of plant matrices. Also, SFE is a simple, inexpensive, fast,
effective and virtually solvent-free sample pretreatment technique. This review provides a detailed and updated discussion of the developments,
modes and applications of SFE in the isolation of essential oils from plant matrices. SFE is usually performed with pure or modified carbon
dioxide, which facilitates off-line collection of extracts and on-line coupling with other analytical methods such as gas, liquid and supercritical
fluid chromatography. In this review, we showed that a number of factors influence extraction yields, these being solubility of the solute in the fluid,
diffusion through the matrix and collection process. Finally, SFE has been compared with conventional extraction methods in terms of selectivity,
rapidity, cleanliness and possibility of manipulating the composition of the extract.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Supercritical fluid extraction; Essential oils; Antioxidant; Cartenoids; Terpenes; Volatile components

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Introduction to supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Solubility and mass-transfer rate of plant oils in supercritical fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4. Effect of matrix on supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5. Effect of extraction parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.1. Effect of pressure and temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.2. Effect of modifiers on supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.3. Effect of extraction time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.4. Effect of flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.5. Sample particle size and packing density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5.6. Effect of water in supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
5.7. Drying effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Abbreviations: AHF, adhyperforin; AIDS, acquired immunodeficiency syndrome; AZA-A, azadirachtin A; BHT, butylated hydroxytoluene; CER, constant
extraction rate; CWO, cedarwood oil; YCER , concentration of the oil in the supercritical phase; CC-SFE, countercurrent supercritical fluid extraction; DAD, diode-array
detection; DPPH, 1,1-diphenyl-2-picrylhydrazyl; kf , external mass-transfer coefficients; FID, flame ionization detection; GC-O, gas chromatography-olfactometery;
HD, hydrodistillation; HS-SPME, headspace solid-phase microextraction; HF, hyperforin; HM, hydrocarbon monoterpene; HS, hydrocarbon sesquiterpene; LC-CO2 ,
liquid carbon dioxide; MCER , mass of extract at constant extraction rate; MS, mass spectrometry; MEKC, micellar electrokinetic chromatography; MWHD, microwave
assisted hydrodistillation; ODS, octadecylsilica; OM, oxygenated monoterpene; OS, oxygenated sesquiterpene; Q, solvent flow rate; RPLC, reversed-phase liquid
chromatography; Re, Reynolds number; SDE, simultaneous distillation–extraction; SFC, supercritical fluid chromatography; SFE, supercritical fluid extraction;
SJW, St. John’s Wort (Hypericum perforatum L.); S/F, solvent-to-feed ratio; SPE, solid-phase extraction; S-HS, static headspace; tCER , time of the CER period; Y* ,
solubility; YCER , oil concentration at the tCER ; YieldTOTAL , total amount of solute collected for the entire measuring time
∗ Corresponding author. Tel.: +98 2122952285; fax: +98 2122936578.

E-mail address: pourmortazavi@yahoo.com (S.M. Pourmortazavi).

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.06.021
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 3

6. Collection of extracted analyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15


6.1. Solvent collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
6.2. Solid-phase collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
6.3. Collection in empty vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
6.4. Novel collection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
6.5. On-line coupling of supercritical fluid extraction with chromatographic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
7. Extraction of oxygenated compounds from plant materials by supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
8. Extraction of terpenes and sesquiterpenes by supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
9. Comparison of supercritical fluid extraction to conventional methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

1. Introduction 2. Introduction to supercritical fluid extraction

Essential oils represent a small fraction of a plant’s compo- Supercritical fluids have been used as solvents for a wide
sition but confer the characteristic for which aromatic plants variety of applications such as essential oil extraction [3], metal
are used in the pharmaceutical, food and fragrance industries. cation extraction [7,8], polymer synthesis [9] and particle nucle-
Essential oils have a complex composition, containing from a ation [10,11]. In practice, more than 90% of all analytical
few dozen to several hundred constituents, especially hydrocar- supercritical fluid extraction (SFE) is performed with carbon
bons (terpenes and sesquiterpenes) and oxygenated compounds dioxide (CO2 ) for several practical reasons. Apart from having
(alcohols, aldehydes, ketones, acids, phenols, oxides, lactones, relatively low critical pressure (74 bar) and temperature (32 ◦ C),
acetalse, ethers and esters). Both hydrocarbons and oxygenated CO2 is relatively non-toxic, non-flammable, available in high
compounds are responsible for the characteristic odors and purity at relatively low cost, and is easily removed from the
flavors. The proportion of individual compounds in the oil com- extract. In the supercritical state, CO2 has a polarity compara-
position is different from trace levels to over 90% (δ-limonene ble to liquid pentane and is, therefore, best suited for lipophilic
in orange oil). The aroma’s oil is the result of the combination of compounds. The main drawback of CO2 is its lack of polarity
the aromas of all components. Trace components are important, for the extraction of polar analytes [12].
since they give the oil a characteristic and natural odor. Thus, In the 1990s, some reports were published about the choice of
it is important that the natural proportion of the components is N2 O as extraction fluid for analytical SFE [13,14]. This fluid was
maintained during extraction of the essential oils from plants by considered better suited for polar compounds because of its per-
any procedure [1]. manent dipole moment. One of the applications where N2 O has
Steam distillation has traditionally been applied for essential shown significant improvements when compared to CO2 is for
oils recovery from plant materials. One of the disadvantages example the extraction of polychlorinated dibenzodioxins from
of the hydrodistillation methods is that essential oils undergo fly ash [13]. Unfortunately, this fluid has been shown to cause
chemical alteration and the heat-sensitive compounds can easily violent explosions when used for samples having high organic
be destroyed. Therefore, the quality of the essential oil extracts content and should, therefore, be used only when absolutely
is extremely impaired [2]. necessary [13,14].
The extraction of essential oil components using solvents at Other more exotic supercritical fluids which have been used
high pressure, or supercritical fluids, has received much atten- for environmental SFE are SF6 and freons. SF6 is a non-polar
tion in the past several years, especially in food, pharmaceutical molecule (although easy polarizable) and as a supercritical fluid,
and cosmetic industries, because it presents an alternative to it has been shown to selectively extract aliphatic hydrocarbons
conventional processes such as organic solvent extraction and up to around C-24 from a mixture containing both aliphatic and
steam distillation [3]. aromatic hydrocarbons. Freons, especially CHClF2 (Freon-22),
The increasing use of vegetable extracts by the food, cos- has on several occasions been shown to increase the extraction
metic, and pharmaceutical industries can make the extraction efficiency compared to conducting extractions with CO2 [15].
of essential oils using supercritical carbon dioxide an attractive Although supercritical H2 O has often been used for the
technology compared to conventional processes with respect to destruction of hazardous organics, the high temperature and
the product quality [4–6]. The knowledge of the mass-transfer pressure needed (T > 374 ◦ C and P > 221 bar) together with the
mechanism, the kinetics parameters and the thermodynamics corrosive nature of H2 O at these conditions, has limited the pos-
restrictions of the extraction conducted in a bed of vegetable sible practical applications in plant oil analysis [16]. H2 O at
material can be used to economically evaluate the extrac- subcritical conditions has, however, been shown to be an effec-
tion process. This requires information on the thermodynamic tive fluid for the extraction of several classes of essential oil. In
restrictions of the system vegetable material/CO2 . On the other 2000, Gamiz-Garcia et al. [17] tested a static–dynamic subcrit-
hand, the understanding of the various process variables and ical water extraction for the isolation of fennel oil. Their results
how they can be connected to a theoretical model to describe showed that, subcritical water extraction is an efficient method
the extraction kinetics are also desirable. in terms of quantitative composition of the extract.
4 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

Ethane, propane, ethylene, dimethyl ether, etc. have also been Jaubert et al. [27] chose the ternary system CO2 –
recommended as solvents under sub- and supercritical condi- limonene–citral as a model system in order to study the
tions for extraction [18,19]. extraction of terpenes from lemon oil using supercritical carbon
Catchpole et al. [20] extracted ginger, black pepper, and dioxide. Extractions were performed at several pressures and
chilli powder using near-critical carbon dioxide, propane, and temperatures to evaluate the influence of these parameters on
dimethyl ether on a laboratory scale to determine the overall the separation efficiency. They used a theoretical model, based
yield and extraction efficiency for selected pungent components. on a modified Peng–Robinson equation of state to understand
They also determined the volatile contents of ginger and black the thermodynamic and mass-transfer aspects of the extraction
pepper extracts. Extraction of all spice types was carried out column. The critical parameters and the acentric factors of
with acetone to compare overall yields. Their results showed limonene and citral were estimated by group contribution meth-
that subcritical dimethyl ether was as effective at extracting the ods. They applied the method developed by Abdoul et al. [28],
pungent principles from the spices as supercritical carbon diox- which allows the calculation of binary interaction parameters
ide, although a substantial amount of water was also extracted. (kij ) to terpenic compounds. The extraction experiments were
Subcritical propane was the least effective solvent. All sol- simulated using this model, and the extraction profiles were
vents quantitatively extracted the gingerols from ginger. The accurately reproduced. On the other hand, Gaspar et al. [29]
yields of capsaicins obtained by supercritical CO2 and dimethyl measured the solubility of borage, echium, and lunaria oils
ether were similar and approximately double that extracted by in compressed CO2 using the dynamic method. Pressure and
propane. The yield of piperines obtained by propane extraction temperature were varied from 60 to 300 bar and 10 to 55 ◦ C,
of black pepper was low at ∼10% of that achieved with dimethyl respectively. Their measured solubilities of echium, borage,
ether and CO2 , but improved with increasing extraction tempera- and lunaria oils in compressed CO2 are presented in Table 1.
ture. Mohamed et al. [21] explored supercritical extraction using As shown in this table, at a given temperature, the solubility
ethane and CO2 for the recovery of the methylxanthines caf- increases with the increase of pressure, as a direct result of the
feine and theobromine and cocoa butter from cocoa beans using increased solvent density. They showed that the effect of extrac-
a high-pressure apparatus. Their finding showed that the extrac- tion temperature is also similar for all oils. At low pressures
tion yields of cocoa butter obtained with ethane are much higher (60 and 100 bar), an increase of temperature leads to a decrease
than those obtained with CO2 because of the higher solubility of of solubility, and the opposite is observed at the highest tested
this fat in ethane. A pronounced effect of pressure on the extrac- pressure (300 bar). At 200 bar, there is an improvement in solu-
tion of methylxanthines and cocoa butter was observed for both bility when increasing the temperature from 10 to 25 ◦ C. Also,
solvents. The methylxanthines in cocoa beans were slightly more they compared the solubilities to those of other vegetable oils
soluble in ethane than in CO2 probably because of co-solvency and were correlated using the density-based model proposed by
effects of cocoa butter, which was extracted more easily using Chrastil [29]. They predicted by the model, a linear relationship
supercritical ethane. Despite the higher cost of ethane, its critical between the logarithm of solubility and the logarithm of
pressure is lower than that of CO2 , and the higher butter solu- solvent density was obtained. Average deviation between their
bility could render ethane a viable solvent through lower energy measured and calculated solubility did not exceed 14%.
costs.

3. Solubility and mass-transfer rate of plant oils in Table 1


∗ ) in compressed
Results of experimental study of solubility for seed oils (Cexp
supercritical fluid
CO2 [29]

There are many variables to be considered in SFE and P (bar) T (◦ C) ρ (kg/m3 ) ∗ (kg/m3 )
Cexp
method development can seem a daunting task. The choice Echium Borage Lunaria
of extraction conditions has largely been determined empir-
60 10 883.8 3.17 1.90 0.49
ically, which is time consuming. One initial area that must 25 190.5 0.11
be assessed is the solubility of the analyte to be extracted 40 149.2 0.00
in the supercritical extracting fluid. This can be investigated 55 129.6 0.00
by spiking an inert medium, usually celite or sand, with the 100 10 921.9 4.78 2.80 0.64
analyte of interest. In addition to providing an indication of 25 819.5 3.01 1.75 0.52
the solubility of the analyte in the supercritical fluid, addi- 40 629.3 0.81 0.26 0.09
tional information is also obtained relating to the efficiency 55 327.1 0.02 0.13 0.00
of collection of the analyte after depressurization. The time 200 10 980.8 7.72 5.07 1.10
consuming nature of even these simple experiments has led 25 915.2 8.61 5.77 1.70
40 840.8 7.31 4.39 1.21
several groups of workers to propose techniques for modeling
55 755.5 5.27 2.05 0.65
analyte solubility [22]. Fundamentals, theory and equations of
states for estimating the solubility of various compounds can 300 10 1020.2 10.20 6.93 1.40
25 966.8 13.44 9.87 2.88
be found in the literature [23–26]. In this review we will focus 40 910.3 14.56 9.90 2.84
on the investigations involving the solubility of plant volatile 55 850.6 16.35 9.96 3.23
components.
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 5

behavior is similar to that of the system linalool + CO2 . This


behavior shows that the best conditions for supercritical extrac-
tions in both systems will be at pressures >90–100 bar and a
temperature near the critical temperature of carbon dioxide in
order to obtain the maximum amount of product. Their results
were in agreement with those of kinetic studies which fixed
the best pressure for the extraction or deterpenation of citrus
oil peel at about 200 bar [31]. Also, they successfully mod-
eled the solubilities using equations of state (Peng–Robinson,
Soave–Redlich–Kwong, 3P1T, Dohrn and Prausnitz non-polar)
and a semi-empirical equation (Chrastil model). They obtained
generalized parameters for the Peng–Robinson equation of state
for each system. These parameters were independent of temper-
ature, and they reproduce successfully all data available in the
Fig. 1. Comparison of solubility values for the linalool in supercritical CO2
obtained by different studies (values obtained by Berna et al.: (䊉) at 318.15 K, literature. The results showed that the solubility of limonene in
() at 328.15 K. Values obtained from Iwai work: () at 313.15 K, () at supercritical carbon dioxide was higher than the solubility of
323.15 K, () at 333.15 K). (From ref. [30] with permission.) linalool.
On the other hand, Sovova et al. [32] investigated fatty oil
Berna et al. [30] measured the solubilities of essential oil com- influence on the solubility of limonene in CO2 under pressures
ponents of orange in supercritical carbon dioxide. In their study, 8–12 MPa at 313.2 K. They measured solubility in CO2 using
the solubilities of pure limonene and linalool in compressed car- the dynamic method both for limonene and for the mixture of
bon dioxide were measured using a flow apparatus at 318.2 and limonene and blackcurrant seed oil. The concentration of fatty
328.2 K and pressures ranging from 69 to 111 bar. The obtained oil in the vapor phase was found to be negligible in comparison
values of these solubilities are shown in Figs. 1 and 2. From the with the concentration of limonene. Limonene was distributed
values that were obtained, Berna et al. showed that the solubil- between the liquid phase, rich in fatty oil, and the vapor phase,
ity of limonene in supercritical carbon dioxide is higher than the rich in CO2 , and its equilibrium concentration in the latter
solubility of linalool at the same conditions but that at higher decreases with the diminishing limonene-to-oil ratio in the satu-
pressures they approach each other. Also, both systems show rator. Also, there was a steep increase of the limonene partition
a sudden increase in the solubility at pressures up to approxi- coefficient with pressure between 8 and 10 MPa, near the criti-
mately 80 bar. For the linalool–CO2 system, it can be noticed cal pressure of the binary mixture of limonene and CO2 . Their
that when the temperature rises at pressures under 80 bar, the applied thermodynamic model was the Soave–Redlich–Kwong
solubility increases but at pressures over 80 bar, the solubility cubic equation of state with either the one fluid linear van der
decreases. Waals mixing rule or with the MHV2 mixing rule. Extraction
The comparison between their results and previous studies for pressures should be approximately 20% larger than the criti-
the system limonene + CO2 showed that the solubility increases cal pressure of the essential oil + CO2 binary mixture and rather
when the temperature rises at pressures under 80 bar but at pres- tight packing of the ground seed in the bed should be applied.
sures over 80 bar, it decreases. Also, it can be observed that this Catchpole and Proells [33] measured the solubilities of lipids
typically found in marine oils and seed oil refining byproducts in
subcritical R134a to determine whether R134a could be a viable,
low-pressure alternative to supercritical CO2 . The measured sol-
ubilities of squalene, oleic acid, soya oil, and deep sea shark
liver oil in subcritical R134a in a countercurrent packed column
apparatus over the temperature range 303–353 K at 60 bar. Sol-
ubility measurements were also made over the pressure range
40–200 bar at 343 K for shark liver oil and oleic acid. Their
results indicated that, the solubilities of all solutes in R134a
are low, ranging from 0.8 to 10 g solute/kg solvent. The sol-
ubilities increased almost linearly with increasing temperature
at fixed pressure and increased logarithmically with increasing
pressure at fixed temperature. The recorded strong temperature
dependence of the solubility allows for two-stage fractionation
of extracts. They used a linear solvation energy relationship
approach to correlate the enhancement factors of the solutes
Fig. 2. Comparison of solubility values for the limonene in supercritical carbon
as a function of the solvent polarity/dipolarizability factor and
dioxide obtained at different studies (values from Berna et al.: (䊉) at 318.15 K.
Values from Iwai et al.: () at 313.15 K, () at 323.15 K, () at 333.15 K. obtained linear relationships. However, the dependence of the
Values from Matos et al.: () at 323.15 K. Values from Di Giacomo et al.: () enhancement factor on other solute–solvent parameters could
at 323.15 K). (From ref. [30] with permission.) not be determined.
6 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

Table 2
Spline parameters for the assays performed at 66.7 bar and 288.15 K, used to choose the adequate flow rate [34]

Q (×105 kg/s) tCER per 60 s MCER (×108 kg/s) YCER (× 103 kg/kg) YieldCER (%) YieldTOTAL (%)

0.62 159 6.67 10.8 0.71 1.47


1.08 93 2.05 18.9 1.03 1.73
1.50 106 2.92 19.5 1.64 3.11
2.15 75 3.05 18.3 1.45 2.24
3.16 60 3.77 11.9 1.44 1.96

table, the amount of solute collected up to the end of the CER


period (YieldCER ) along with the total amount of solute collected
for the entire measuring time (YieldTOTAL ) are shown.
The effect of temperature on solubility is complex, due to
the combination of two variables, density and vapor pressure.
The vapor pressure of the solute increases with temperature,
causing an elevation in solubility. However, decreasing of sol-
vent density may cause decreases of the solute solubility. The
dominant effect will depend on the magnitude of each effect
on the others for each system. Higher solubility values were
obtained at 66.7 bar in the range of 288.15–293.15 K. There-
fore, the increase in the solubility for this range of temperatures
should mainly be due to the increase in the vapor pressure of
Fig. 3. The effect of solvent flow rate on the mass ratio of solute in the fluid
phase at the bed outlet at T = 288.15 K and P = 66.7 bar. (From ref. [34] with
the solute. For essential oils the vapor pressure is low, however,
permission.) small changes in temperature can cause significant changes in
solubility. For example, a 5◦ increase in the temperature (from
288.015 to 293.015 K) at 66.7 bar caused an increase in solubil-
Sousa et al. [34] measured the solubility of the essential oil
ity of 14%. However, the same increase in temperature, but from
present in L. sidoides Cham. in liquid CO2 by the dynamic
293.15 to 298.15 K, resulted in a reduction in solute solubility of
method, in which the solvent is saturated by the solute as it
42%. Therefore, in the first case, the dominant effect was solute
flows through the bed of solids at a predetermined constant flow
vapor pressure, while in the second it was density. The effects of
rate. Table 2 reports their results and Fig. 3 shows the effect
both temperature and pressure on solute solubility can be shown
of solute in the supercritical phase at the measuring cell out-
in Fig. 4. The overall saturation curves at 66.7 bar and various
let. Their finding showed that for experiments accomplished at
extremely low solvent flow rates, the effects of axial dispersion
were important, resulting in a smaller value of YCER (the con-
centration of the oil in the supercritical phase at the outlet of the
column). At high solvent flow rates, smaller values of YCER were
obtained due to shorter residence times. As shown in Fig. 3, sol-
vent flow rates in the vicinity of 1.5 × 10−5 kg/s is appropriate to
be used for measuring solubility, using the dynamic method for
the L. sidoides + CO2 system. The variation in the thermophys-
ical properties of the solute and the solvent was relatively small
due to the narrow interval of both temperature and pressure in
this study. Based on this, the solubility for the L. sidoides + CO2
system was measured at solvent flow rates in the vicinity of Fig. 4. The influence of temperature on the overall saturation curves at 66.7 bar.
1.5 × 10−5 kg/s. Table 3 shows the measured solubility. In this (From ref. [34] with permission.)

Table 3
Solubility measured by the dynamic method for the pseudo-ternary system [34]

T (K) P (bar) Q (×10−5 kg/s) Y* (×10−3 kg/kg) YieldCER (%) YieldTOTAL (%)

283.15 66.7 1.60 13.4 1.34 2.19


288.15 66.7 1.50 19.5 1.64 3.11
288.15 78.5 1.60 17.8 1.63 3.03
293.15 66.7 1.53 22.7 1.76 3.21
293.15 78.5 1.60 20.1 1.45 2.75
295.65 66.7 1.53 19.0 1.55 3.29
298.15 66.7 1.57 13.2 1.16 2.82
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 7

temperatures (Fig. 4) reflect, as expected, the behavior of solu- predict. The physical structure of the matrix is of critical impor-
bility, since the slope of each overall saturation curve is directly tance, as the extraction efficiency is related to the ability of the
proportional to solubility. Also, investigations on the effect of supercritical fluid to diffuse within the matrix. For that reason,
pressure on solubility for temperatures of 288.15 and 293.15 K the extraction conditions of the same group of oils may differ
showed that, solubility decreases by enhancing pressure from from one matrix to another.
66.7 to 78.5 bar. The behavior is consistent with the literature on As a general rule, decreasing the particle size of solid matrices
solid–fluid equilibria [35]. leads to a higher surface area, making extraction more efficient.
From the industrial point of view, optimization of the pro- Yet, excessive grinding may hinder the extraction due to re-
cesses that employ supercritical fluids not only requires the adsorption of the analytes onto matrix surfaces (this could be
knowledge of thermodynamic parameters (solubility and selec- avoided by increasing the flow rate) and pressure drop inside
tivity) but also requires consideration of mass-transfer rate the extraction chamber. On the other hand, environmental agents
[36,37]. may affect the composition and essential oil contents of matrix.
Ferreira and Meireles [38] modeled the mass-transfer mech- Esmelindro et al. [44] assess the influence of light intensity
anism for extraction of black pepper essential oil using (plants exposed to direct sun and in controlled lighting condi-
supercritical carbon dioxide by the extended Lack’s plug flow tions), and the age of leaves (6–24 months) on the characteristics
model developed by Sovova (Sovova’s model) [39]. Their pro- of the extracts of mate tea leaves obtained from carbon diox-
cedure was used to evaluate the parameters of the Sovova’s ide at high pressures. Samples of mate were collected in an
model from experimental data—it quantitatively described the experiment conducted under agronomic control at Industria e
experimental data for the majority of conditions analyzed. Their Comercio de Erva-Mate Barão, Brazil. Quantitative analysis of
finding indicated that temperature and pressure levels used in caffeine, theobromine, phytol, vitamin E, squalene, and stigmas-
their work did not change mass-transfer coefficient values and terol was performed, and the results showed that field variables
is a function of the bed characteristic and initial amounts of exert a strong influence on the liquid yield and on the chemical
solute. Germain et al. [40] showed that, the external mass- distribution of the extracts.
transfer coefficients (kf ) during supercritical fluid extraction of
high-solubility solutes, under solvent up-flow conditions and 5. Effect of extraction parameters
low superficial velocities, can be small because of the nega-
tive influence of natural convection phenomena. They used a 5.1. Effect of pressure and temperature
shrinking-core model for mass transfer to estimate best-fit val-
ues of kf for data on SFE of lipids from pre-pressed rape seeds. Four parameters are extremely helpful in the understanding
Values of kf at a high Reynolds number (Re = 14.1) were sim- of solute behavior in supercritical media, and thus in executing
ilar when using solvent up-flow or down-flow, but kf at lower successful analytical supercritical fluid extractions [45,46]: (i)
Re (1.57) was 3.6 times smaller when using solvent up-flow the miscibility or threshold pressure [47,48], which corresponds
than that predicted from a literature correlation for down-flow to the pressure at which the solute partitions into the supercritical
conditions. These kf values are consistent with values estimated fluid, (ii) the pressure at which the solute reaches its maximum
by fitting literature data, or gathered from various sources under solubility, (iii) the fractionation pressure range, which is the
similar, non-adequate conditions (solvent up-flow under low Re) pressure region between the miscibility and solubility maximum
for the extraction of both fatty and essential oils. pressures (in this interval it is possible to extract selectivity one
solute by choosing the correct pressure) and (iv) a knowledge
4. Effect of matrix on supercritical fluid extraction of the physical properties of the solute, particularly its melting
point (in fact most solutes dissolve better when they are in their
Different factors such as the particle size, shape, surface area, liquid state, i.e. above their melting point).
porosity, moisture, level of extractable solutes and the nature of To illustrate the difference between the threshold pressure and
the matrix will affect the supercritical fluid extraction results. the solubility maximum pressure, the solubility–pressure curve
Similarly, the interactions between solutes and active sites of of naphthalene is given in Fig. 5 [46]. This solute is slightly solu-
the matrix can necessitate strict extraction conditions. The suc- ble in CO2 at 75 bar (threshold pressure) as the pressure increases
cess of a SFE method not only depends on the extraction step the solubility rises, especially around 90 bar, up to its maximum
itself (i.e. nature of the supercritical fluid and choice of extraction value. The fluid pressure is the main parameter that influences the
parameters) but also on the matrix considered (a pretreatment extraction efficiency. An elevation of this pressure at a given tem-
may be recommended) as well as on the analyte trapping system perature results in an increase in the fluid density (Fig. 6), which
[41–43]. Consequently, SFE must be regarded as a four-stage means an enhanced solubility of the solutes. Consequently, the
process: (1) desorption of the compound from the matrix with higher the extraction pressure, the smaller is the volume of fluid
(2) subsequent diffusion into the matrix, (3) solubilization of necessary for a given extraction. For example, one needs to dou-
the analyte by the supercritical fluid, and (4) sweeping out of ble the volume of CO2 in order to extract 70% of diuron herbicide
the extraction cell by the fluid. Each part of the process has to from a contaminated soil when working at 110 bar instead of
be carefully optimized in order to obtain quantitative and repro- 338 bar (Fig. 7) [49]. However, high pressure is not always rec-
ducible recoveries. Most of the time, the first step remains most ommended for complex matrices owing to the higher solubility
difficult to control, as solute–matrix interactions are difficult to of solutes when the pressure is elevated, resulting in complex
8 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

Fig. 7. Variation of the extraction yield of diuron from a polluted soil vs. the
volume of CO2 percolated at different pressures (( ) 110, () 235, (䊉) 338 bar).
Fig. 5. Variation of the solubility of naphthalene with the pressure of supercrit- Extraction conditions: extractant, CO2 –CH3 CN (90:10, v/v), extraction cell,
ical CO2 at 45 ◦ C. (From ref. [46] with permission.) 25 cm × 4.6 mm I.D.; temperature, 100 ◦ C; flow rate of liquid CO2 , 16.5 ml/min.
(From ref. [49] with permission.)
extracts and difficult analysis. On the other hand, it must be
borne in mind that the presence of co-extracted solutes can dra- and its volatility (which rises with increasing temperature). For
matically change the solubility level of the solute of interest. At example, when the temperature increases from 80 to 120 ◦ C, the
a constant pressure, the density of CO2 decreases when the tem- extraction recovery of diuron from soil with methanol-modified
perature is increased. This effect becomes more pronounced as CO2 is enhanced from 75 to 99% [51].
the fluid compressibility increases, as shown in Fig. 6. Tempera- Baysal and Starmens [52] studies supercritical carbon diox-
ture also affects the volatility of the solute. Hence, the effect of a ide of carvone and limonene from caraway seed. Their results
temperature elevation is difficult to predict because of its depen- showed that pressure and temperature have main effects on
dence on the nature of the sample. For a non-volatile solute, the extraction efficiency. They showed that at moderate tem-
a higher temperature would result in lower extraction recovery peratures just above the critical temperature of CO2 (31.1 ◦ C),
owing to a decrease in solubility thus the distribution coefficient extraction yield for limonene is considerable at pressures just
of phenol between water and supercritical CO2 decreases when above the critical pressure of CO2 (73.8 bar). Below this value,
the fluid temperature rises from 25 to 30 ◦ C [50]. On the other hardly any limonene is obtained from the seed matrix, regardless
hand, for a volatile solute, there is a competition between its sol- of the temperature applied during the extraction procedure. At
ubility in CO2 (which decreases as the temperature increases) elevated temperatures, a pressure of up to 125 bar is required to
extract limonene in only small quantities. Further increasing of
the pressure yield resembles those found for limonene.
Careri et al. [53] used a chemometric approach to inves-
tigate the effects of different parameters on the supercritical
extraction of carotenoids from spirulina Pacifica algae. Their
results showed that the temperature of the supercritical fluid
did not influence extraction efficiency. However, the pressure
of the supercritical fluid plays an important role in the SFE
of carotenoids from Spirulina pacifica algae. Reverchon et al.
[54] extracted volatile oil from rose concrete, using different
pressures and temperatures of supercritical fluids. The results
showed at the highest extraction densities (for example 100 bar
and 40 ◦ C) that large quantities of paraffins and steroptens were
co-extracted with the rose volatile oil. Therefore, lower pres-
sure and temperature were used for SFE of the rose concrete.
Hamburger et al. [55] studied the effect of supercritical pressure
on yield of extracted substances from three medicinal plants
(marigold, hawthorn and chamomile). They reported that, at
pressures above 300 bar, the yields of total extract increased.
Increased yield of non-volatile lipophilic compounds, such as
Fig. 6. Pressure–density diagram for carbon dioxide. The shaded area cor- faradiol esters, are achieved at pressure above 300 bar. Brachet
responds to the experimental domain of supercritical phase extraction and et al. [56] used central composite designs in the study of three
chromatography. (From ref. [46] with permission.) tropane alkaloids: hyoscyamine and scopolamine from Datura
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 9

Fig. 8. All-trans-lycopene and cis-lycopene contents (expressed as percentage


of the total lycopene) in the tomato supercritical fluid extracts performed in the Fig. 9. Overall extraction curve for marigold extraction with near-critical CO2
0.55–0.90 g/ml density range. Error bars are indicated at each density of CO2 at various conditions. (From ref. [59] with permission.)
used. (From ref. [57] with permission.)
and subcritical extractions at ms /m0 = 64 and 119 mg/kg of seeds.
arborea hairy roots and cocaine from coca leaves. They showed The most convenient extraction was gained using an ms /m0 ratio
that for cocaine extraction, pressure had a weak influence on of 119 rather than 64. For supercritical extraction, a separation
cocaine recovery. The influence of pressure and temperature on of cuticular waxes from oil was performed in the pilot plant.
extraction yields is fairly identical for hyoscyamine and scopo- In 2005, Campos et al. [59] investigated extraction of
lamine. At higher pressure, increasing the extraction temperature marigold (Calendula officinalis) oleoresin with supercritical car-
from 40 to 100 ◦ C yielded significant increases in hyoscyamine bon dioxide. They showed that, the temperature effect in the
and scopolamine recoveries, despite the CO2 density decrease. process yield is complex due to the combined effect of solvent
At low pressure, the effect of temperature is less pronounced. density and solute vapor pressure. These opposite effects must be
These results confirm that SFE is governed by the volatility of evaluated to observe crossing of the yield isotherms. In Fig. 9, a
analytes as well as by fluid density. constant yield at 15 MPa with increasing temperature (from 303
Gmez-Prieto et al. [57] proposed a procedure for the SFE of to 313 K) is observed, while at 20 MPa the extract yield increases
all-trans-lycopene from tomato using carbon dioxide at 40 ◦ C with temperature going from 299 to 313 K. This behavior is an
without modifier. Their method minimizes the risk of degra- indication that the solute vapor pressure is the dominant effect at
dation via isomerization and oxidation of health-promoting 20 MPa, while at 15 MPa the difficulty to observe the dominant
ingredients, such as lycopene. The effects of different experi- effect is a suggestion that the crossover region (yield isotherms)
mental variables on the solvating power of the supercritical fluid is close to 15 MPa, for the temperatures studied.
were evaluated in terms of both the selectivity for and the yield Gaspar [60] studied the effect of the extraction pressure
of the extraction of all-trans-lycopene. Fig. 8 gives the relative and temperature on the extraction of essential oils and other
amounts of the cis and all-trans forms of lycopene obtained at the co-extracted components (cuticular waxes) from oregano (Ori-
different densities investigated in the range over which lycopene ganum virens L.) bracts by compressed CO2 from 50 to 300 bar
was extracted (i.e. 0.55–0.90 g/ml). The amount of the trans- and 300 to 320 K, respectively. Moderate conditions, using sol-
lycopene extracted increases (and the corresponding cis form vent densities between 0.3 and 0.5 kg m−3 , were found to be
content decreases) if the extraction pressure increases. From sufficient for efficient extraction of essential oils. The use of
these results, it seems clear that the enhancement of the fraction- higher pressures and temperatures, despite slight advantages
ation of trans-lycopene requires a proper choice of CO2 density. for the rate of extraction and yields of essential oils extraction,
An extract 88% all-trans-lycopene and 12% cis-lycopene could led to significant co-extraction of waxes and, consequently, to
be produced. extracts with lower essential oil content. For CO2 densities below
Ambrosino et al. [58] applied a new supercritical extraction 0.25 kg m−3 , selective extraction of individual essential oils was
methodology to extract azadirachtin A (AZA-A) from neem attained. At these low-density conditions, the lighter and more
seed kernels. They used supercritical and liquid carbon dioxide volatile hydrocarbons were preferentially extracted.
(CO2 ) as extractive agents in a three-separation-stage supercrit- Canela et al. [61] studied the supercritical fluid extraction
ical pilot plant. They carried out comparisons by calculating the of fatty acids and carotenoids from the Microalgae spirulina
efficiency of the pilot plant with respect to the milligrams per Maxima with carbon dioxide, assessing the effect of pressure
kilogram of seeds (ms /m0 ) of extracted AZA-A. Conventional and temperature on the yield and chemical composition of the
pressure extraction on raw seeds (320 MPa) led to a low yield in extracts. Their experiments were conducted at temperatures
oil (8%) together with the lowest concentration of AZA-A per of 20–70 ◦ C and pressures of 15–180 bar. Statistical analysis
kilogram of oil and also to the lowest concentration of AZA-A showed that neither the temperature nor the pressure signifi-
per kilogram of seeds (44 mg/kg of seeds). Compared to con- cantly affected the total yield, but both the temperature and the
ventional pressure extraction, both supercritical and subcritical pressure affected the extraction rate, and temperature was more
pressures gave rise to a greater enrichment of AZA-A. Signif- significant than pressure upon the SFE. The extracts were rich in
icant differences were evident by comparison of supercritical essential fatty acids and carotenes, and at 100 bar and 45 ◦ C the
10 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

extract contained no carotenes. Also, temperatures larger than a cylinder tank of modified CO2 , but this is much more expensive
50 ◦ C degraded the carotenes, as expected. and, as the tank is emptied, the content of modifier tends to
increase.
5.2. Effect of modifiers on supercritical fluid extraction Kohler et al. [67] investigate the effects of modifier concentra-
tion and its nature on the supercritical carbon dioxide extraction
The nature of the modifier depends on the nature of the solute of artemisinin and artemisinic acid from Artemisia annua L.
to be extracted [62]. For example, the extraction of diuron is They selected methanol, ethanol, methanol–water (50/50, v/v)
considerably enhanced with methanol instead of acetonitrile as and toluene as modifiers based on preliminary tests and of their
a modifier [63]. A reasonable starting point consists of selecting solvating power for artemisinin. Methanol, ethanol and toluene
a modifier that is a good solvent in its liquid state for the target gave similar results, however, the use of toluene presents a major
analyte. It should be noted that the addition of large amounts of drawback due to its high boiling point and concomitant longer
modifier will change the critical parameters of the mixture [64], evaporation times. Methanol–water gave unsuccessful results
as shown in Fig. 10 for methanol–carbon dioxide mixtures [64]. based on the slopes of extraction kinetic curves. This result can
As a result, binary mixtures of carbon dioxide and an organic be explained by the low solvating power of water for artemisinin.
solvent are often used in a subcritical state, where the diffusion In order to improving supercritical CO2 extractability of
coefficients are smaller than in a supercritical state. Modifiers hyoscyamine and scopolamine hydrochloride salts, Choi et al.
can be introduced as mixed fluids in the pumping system with a [68] investigate the effect of methanol and water on the extrac-
second pump and mixing chamber [65], or by simply injecting tion yields. It was found that addition of methanol drastically
the modifier as a liquid into sample before extraction [66] (the increased the extraction yield of hyoscyamine and scopolamine.
later way being less successful because it leads to concentration However, water did not show any significant influence on the
gradients within the matrix). Alternatively, one may use directly extractabilities of hyoscyamin, although it slightly increased
in the case of scopolamine. The poorer result for water rela-
tive to methanol may be due to the fact that water could not
sufficiently improve the polarity of CO2 as much as methanol,
since only 0.3% (v/v) of water can be completely miscible with
CO2 . They also studied the effects of basified modifiers (diethy-
lamine) added to methanol and water on analyte extractability.
The addition of 10% diethylamine to methanol or water dramat-
ically enhanced the extraction of hyoscyamine and scopolamine
hydrochloride compared to using pure methanol or water. This
result may be due to the fact that the salts are changed to
free bases by minor addition of methanol or with the diethy-
lamine, allowing supercritical CO2 to more easily dissolve the
free bases. Ethanol, water and an equimolar mixture of these
two solvents were chosen as co-solvents for supercritical car-
bon dioxide extraction of stevia glycosides by Pasquel et al.
[69]. The results indicated that due to the high polarity of water
in comparison with ethanol, an increase in glycosides solubil-
ity resulted. Comparison of the yields for the experiments using
water as co-solvent showed that regardless of whether it was
used with or without ethanol, water increased the solubility of
the glycosides. Monteiro et al. [70] investigated the CO2 extrac-
tion of bacuri fruit. They showed that the co-solvent influenced
the extraction yield of soluble material from shells of the fruit
in two different ways. First, owing to its polarity, the co-solvent
favored dissolution of the polar substances present in the bacuri
shells. Second, the co-solvent diluted the extract, diminishing
its viscosity, thereby enhancing the flow of the extract through
the extractor.
Cocero and Garcia [71] studied supercritical fluid extraction
of sunflower oil with carbon dioxide in a pilot plant at 30.0 MPa
and 40 ◦ C, using different amount of methanol, ethanol, butanol
and hexanol as co-solvent. Comparing the co-solvent extraction
experimental data with SFE using neat CO2 , it was found that
the use of 10% (w/w) of a co-solvent increases oil solubility 10-
Fig. 10. Variations of the critical pressure and temperature of CO2 –CH3 OH fold. They used a mathematical model to investigate the effects
mixtures with the molar fraction of methanol. (From ref. [64] with permission.) of co-solvents on two adjustable parameters, i.e. equilibrium
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 11

coefficient and mass-transfer parameter. Their results showed Phellandrene was the most prominent compound in all extracts
that equilibrium parameter increases with co-solvent concentra- obtained with the mixture of ethanol and water as modifier, but
tion. Butanol gives the higher values which are exactly the same the relative abundance varied from 9.2 to 30.7% depending on
result obtained comparing oil solubility. Solute recovery values extraction conditions. The relative proportion of both sesquiter-
using methanol values are higher than when using hexanol and pene hydrocarbons and oxygenated sesquiterpenes decreased
ethanol. This is explained by comparing the mass-transfer val- when the polarity of the solvent was increased by the mixture of
ues, methanol values being the lowest. Also, they showed that ethanol and water in comparison to liquid CO2 extracts obtained
mass transfer increases with co-solvent concentration depend- without any modifier.
ing on the nature of co-solvent. This variation is probably caused Palma et al. [73] extracted white grape seeds by sequential
by the change of physical properties in the supercritical mix- supercritical fluid extraction. Their results showed that, modifier
ture. Kerrola and Kallio [72] investigated the effect of adding has a main effect on the SFE process and by increasing the
various quantities of water and a mixture of ethanol and water polarity of the supercritical fluid using methanol as a modifier
(1:1, v/v) to the carbon dioxide extraction system on the rel- of CO2 , it was possible to fractionate the extracted compounds.
ative amounts of the volatile components of angelica roots. They obtained two fractions: the first, which was obtained with
Their results showed that the yields of recovered components pure CO2 , contained mainly fatty acids, aliphatic aldehydes, and
increased when an aliquot of water was added to the system. sterols. The second fraction, obtained with methanol-modified
After the extraction had completed and carbon dioxide released, CO2 , had phenolic compounds, mainly catechin, epicatechin,
the water co-solvent formed a separate phase. The increase in and gallic acid.
volume was considered as a positive factor. The co-solvent was Pourmortazavi et al. [74] studied the supercritical fluid extrac-
suggested to act as the primary solvent, thus enhancing the dif- tion of aerial parts of Perovskia atriplicifolia Benth. In this
fusivity within the matrix. The mixture of ethanol and water research, the effect of different modifiers at a constant pressure
appeared to increase the proportion of monoterpene hydrocar- (100 atm) and temperature (35 ◦ C) on the extraction efficiency
bons when compared with water alone used as modifier and was also evaluated. Fig. 11 shows the effect of different volumes
pure liquid CO2 without a modifier. The effect of the co-solvent of modifiers on the main contents of P. atriplicifolia essential
added on extractability of a compound varied considerably. ␤- oil. Fig. 11a shows the effect of methanol (1 and 5%, v/v) on the

Fig. 11. Effects of different volume of modifiers [(a) methanol, (b) ethanol, (c) dichloromethane and (d) n-hexane] on the main contents of Perovskia atriplicifolia
Benth. essential oil these main compounds are: (1) ␣-pinene; (2) ␦-3-carene; (3) 1,8-cineole + limonene; (4) camphor; (5) ␤-caryophyllene; (6) ␣-humulene; (7)
unknown compounds with large retention number (the extraction pressure was 100 atm and extraction temperature was 35 ◦ C). (From ref. [74] with permission.)
12 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

composition of P. atriplicifolia essential oil. Methanol decreased Table 4


the number of extracted compounds in comparison with the Effect of dynamic extraction time on the composition of fennel oils obtained by
SFE (at static time of 25 min and pressure 200 and 350 bar) [78]
extraction by pure supercritical CO2 from 22 compounds to
11 compounds. Also, using 1% methanol as modifier increase Pressure (bar) 200 200 350 350
content of ␣-pinene, ␤-pinene, ␤-caryophyllene, ␦-3-carene, ␣- Temperature (◦ C) 45 45 45 45
Dynamic time (min) 30 45 30 45
humulene and decrease the percent of 1,8-cineole + limonene
and camphor in the extracted essential oil. Methanol increased Compound Run1 Run2 Run3 Run4
the percent of co-extracted compounds with large retention num- ␣-Pinene 1.32 0.89 1.05 1.00
ber (unknown compounds) in the oil composition. Ethanol was Sabinene 0.89 – – –
also tested for extraction of essential oil from P. atriplicifolia. Limonene 9.34 7.94 7.26 7.17
(z)-␤-Ocimene 1.39 0.79 1.01 –
Results showed that addition of ethanol (Fig. 11b) enhanced ␥-Terpinene 0.77 0.83 – –
the concentration of ␣-pinene, camphene, ␤-pinene, myrcene, Fenchone 9.25 8.45 9.93 8.36
␦-3-carene, and decrease the content of 1,8-cineole + limonene, Estragole 2.94 3.07 3.09 2.81
camphor and ␤-caryophyllene in the plant oil. However, these (E)-Anethol 72.30 76.64 77.67 72.70
results showed that addition of %1 ethanol, as modifier was Germacrene D 1.78 1.38 – 1.61
more effective than 5% ethanol. Dichloromethane as modi-
fier (Fig. 11c) decreased the number of extracted compounds
from 22 with pure supercritical CO2 to 8 and 9 compounds in flow rate 1 ml/min (60 ◦ C and at 367 bar), about 83.2% of the
the presence of 1 and 5% (v/v) dichloromethane, respectively. total extractable hyperforin (HF) and 88.3% extractable adhy-
Dichloromethane also increased the concentration of ␣-pinene, perforin (AHF) were extracted in the first hour, and about 95.3%
␤-pinene, ␦-3-carene, 1,8-cineole + limonene, ␣-humulene and extractable HF and 97.2% extractable AHF were extracted in the
reduced the concentration of camphor in the P. atriplicifplia first 1.5 h. No more than 4% HF and AHF were extracted within
essential oil composition. However, dichloromethane increased another hour after the 1.5 h dynamic extraction. Thus, they used
the content of co-extracted compounds with large retention 10 min static extraction followed by 1.5-h dynamic extraction
indices (unknown compounds) in the essential oil composition. for all samples.
Furthermore, it was found that dichloromethane was a selec- Pourmortazavi et al. [77] showed the influence of the dynamic
tive modifier for the extraction of 1,8-cineole + limonene and extraction time on the composition of the essential oil of
␦-3-carene from P. atriplicifolia. By using hexane as modifier Juniperus communis L. leaves that was studied by perform-
(Fig. 11d), the number of identified compounds decreased from ing extraction with supercritical carbon dioxide this consists
22 to 12 and 7 compounds (hexane volumes were 1 and 5%, of a static extraction of 25 min, followed by 20 and 30 min
v/v, respectively). In the presence of hexane as modifier the of dynamic extraction time. Results showed that increasing
percent of ␣-pinene, ␤-pinene, ␦-3-carene, ␣-terpenyl acetate dynamic extraction time at constant pressure 350 atm, enhances
increased and the percent of 1,8-cineole + limonene and cam- the content of heavy compounds with large retention indices in
phor decreased in comparison with extraction by pure carbon the plant oil. They also tested a static–dynamic SFE approach
dioxide. The result of these studies showed that changing modi- for the isolation of fennel oil [78]. A static extraction period (for
fier type and identity could significantly affect the selectivity of enhancing sample–extractant contact thus favoring the attain-
the extraction process. ment of the portion equilibrium) was carried out, followed
by a dynamic extraction period in which extractant passed
5.3. Effect of extraction time continuously through the extraction chamber. The influence
of the dynamic extraction time on the composition of the
It is important to maximize the contact of the supercriti- essential oil was studied by performing extraction with super-
cal fluid solvent with the sample material in order to enhance critical carbon dioxide consisted of a static extraction step of
the efficiency of SFE. Several variables that influence the sol- 25 min, followed by 30 and 45 min of dynamic extraction.
vent contact with sample material include flow rate, SFE time, The results are shown in Table 4. It was found that increased
and SFE mode (static with no follow-through or dynamic with dynamic extraction time enhance the extraction of most of the
follow-through). It was reported [75] that, a 10–20 min static compounds.
extraction prior to dynamic extraction improved the extract
recoveries in SFE extraction of aflatoxins. Cui and Ang [76] 5.4. Effect of flow rate
developed a small-scale supercritical fluid extraction system for
the selective extraction of phloroglucinols from St. John’s Wort The speed of the supercritical fluid flowing through the
(SJW) leaf/flower mixtures using carbon dioxide. The extraction cell has a strong influence on the extraction efficiencies. The
efficiency was investigated as influenced by pressure, temper- slower the fluid velocity, the deeper it penetrates the matrix.
ature, time, and modifier. They optimized condition of SFE at The fluid speed can be expressed by the linear velocity, which
367 bar and 50 ◦ C. Samples were held in static extraction for is strongly dependent on the flow rate and the cell geome-
10 min, followed by a dynamic extraction for 90 min at the flow try. For a given extraction cell, the flow rate can be easily
rate of 1 ml/min. In this study, they showed static extraction changed by using a new restrictor with a different inside diam-
longer than 10 min did not increase extraction efficiency. At the eter. Decreasing the flow rate resulted in a lower linear velocity
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 13

Fig. 13. Effect of solvent flow rate on the extraction yield vs. the specific amount
of solvent (Q) at 150 bar, 45 ◦ C. (From ref. [80] with permission.)

Fig. 12. Effect of solvent flow rate on the extraction yield vs. extraction time at
150 bar, 45 ◦ C. (From ref. [80] with permission.) matrix effects. Sample particle size and vessel packing density
uniformity in day-to-day operations may be a factor in some
and usually in increased extraction. For example, 14 C-labeled SFE applications that achieve variable results [81].
linear alkylbenzenesulphonates were better extracted by super- Louli et al. [82] extracted parsley seed oil with supercritical
critical CO2 modified with methanol (40 mol%) (at 380 bar and carbon dioxide at different conditions. Fig. 14 presents their
125 ◦ C) from a sludge-amended soil with a liquid CO2 flow results about the effect of particle size on the extraction rate. As
rate of 0.45 ml min−1 (mean recovery 90.8 ± 1.3%) instead of it was expected, the extraction rate increases by decreasing the
1.2 ml min−1 (mean recovery 75.6 ± 1.1%), the same volume size of the seeds. This is due to the higher amount of oil released
of fluid being used in each instance [79]. Higher flow rates as the seed cells are destroyed by milling. Moreover, after milling
result in a decrease in the recovery either by using an ele- the diffusion paths in the solid matrix become shorter resulting
vated pressure drop though the extraction cell this phenomenon in a smaller intra-particle resistance to solute diffusion.
probably occurred during the extraction of diuron from a con- Sabio et al. [83] extracted tomato skins and their mixtures
taminated soil with a CO2 –methanol (90:10, v/v) mixture, or with seeds by supercritical CO2 extraction. The results of their
by increasing analyte loss during decompression of the fluid. experimental study at a pressure of 300 bar and temperature of
Papamichail et al. [80] studied the SFE of oil from milled celery 60 ◦ C (and a flow rate of 0.792 kg/h) for two different parti-
seeds using CO2 as a solvent. They investigated the effect of cle sizes are shown in Fig. 15. From this figure, it is evident
flow rate of CO2 on the extraction rate of celery seeds. They that the smaller particle size reduces the yield obtained in the
showed that the increase of the solvent flow rate leads to the extractions. Because of their small specific surface area, large
increase of the amount of oil extracted versus extraction time
(Fig. 12). The amount of the extracted oil per kilogram of CO2
used is higher for the lower flow rate due to the intra-particle
diffusion resistance. This, actually, has as a result the smaller
slope of the extraction curve in Fig. 13 for the higher flow
rate.

5.5. Sample particle size and packing density

In general, decreasing particle size in SFE creates more sur-


face area and benefits extraction, but it also may hinder extraction
if the analytes re-adsorb on matrix surfaces. Hawthorne and co-
worker [81] discussed elution of the analytes from the matrix in
the vessel, to determine the rate-limiting step in SFE. A higher
flow rate can help reduce partitioning back onto matrix sites
Fig. 14. Effect of particle size of Parsley seed samples on the extraction rate
if this is the limiting factor (otherwise, solubility factors are (data are presented as the extraction yield vs. the specific amount of solvent (Q)
limiting). Larger particles, decreased packing density, smaller at 10 MPa, 318 K, and a solvent flow rate of 1.1 kg CO2 /h). (From ref. [82] with
sample size, and a wider extraction vessel reduce potential permission.)
14 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

favors the dispersion of the analytes in the matrix and the sample
homogenization [85].
Leeke et al. [86] extracted Origanum vulgare L. using super-
critical carbon dioxide at 100 bar and 313 K in the presence
of water. They observed that the addition of water as a mod-
ifier resulted in an increase in the extraction of essential oil.
For extraction where water was added discontinuously, a large
increase in the extraction of the essential oils was recorded.
The extraction degree increased with increasing w/w% water
added to the bed, reaching an optimum at 80% (w/w). At these
water concentrations, a low but finite yield of waxy material also
resulted. Such a result could be beneficial to attain an essen-
tial oil rich product. In this study, the continuous addition of
water also resulted in an increase in the degree of extraction
Fig. 15. Effect of tomato sample particle size on the yields of supercritical fluid of essential oils, but not necessary an increased yield of waxy
extraction (yields of lipids, lycopene, and carotenes obtained at 300 bar, 60 ◦ C, material. For tests where a higher w/w% was added, the removal
and the CO2 flow rate of 0.792 kg/h from mixtures of tomato skins and seeds
with two different particle sizes). (From ref. [83] with permission.)
of water would have had little effect on the extraction efficiency
of essential oils, even at prolonged extraction times, because
of its abundance. Finally, they showed that their discontinuous
particles lead to a distinct, diffusion-dominated extraction and method is sufficient to influence the extraction degree of the
long processing times. For example, decreasing the particle size essential oils and on a commercial scale, further equipment for
of ground peanuts from a range of 3.35–4.75 mm to a range of continuously humidifying supercritical carbon dioxide would
0.86–1.19 mm was found to increase the total oil recovery from be avoided, providing that the extraction process was completed
36 to 82%. However, particle sizes that are too small can result within a suitable timescale.
in inhomogeneous extractions due to fluid channeling effects in
the fixed bed. In this study Sabio et al. verified an inhomoge- 5.7. Drying effect
neous color distribution in the 80 ␮m particle size solid after
the extraction via post-extraction examination of the substrate, Drying of plant samples, a major preservation process for
indicating an uneven extraction due to the small particle size. spices, can be carried out conventionally by air-drying (with
or without heat) or by freeze-drying. It is obvious that drying
5.6. Effect of water in supercritical fluid extraction and the drying process may have an influence on the content of
aroma compounds. Literature data indicate that the changes of
Water in the sample often affects SFE. There have been appli- aroma compounds during drying depend on the drying method
cations of direct SFE of aqueous samples [84], but precautions as well as the type of the spice. Ibanez and co-workers [87]
must be taken to avoid damaging or destructive interaction of proposed a two-step supercritical fluid extraction of rosemary
sample contents with water; the water must be removed or con- leaves at selected conditions of pressure and temperature to
trolled before performing SFE. Water can aid in the extraction divide the oleoresin into two fractions with different antiox-
process, or be detrimental, depending on water can open pores, idant activities and essential oil compositions. They showed
swell the matrix, thereby allowing the fluid better access to ana- that, there are two main factors that have to be considered: the
lytes, and aid in flow through the matrix. Also, even though drying process, which influences the essential oil composition
water is only ≈0.3% soluble in supercritical CO2 [84], it serves and, therefore extract quality, and the effect of the drying pro-
to increase the polarity of the fluid and enable higher recoveries cess on the plant cells. Damage to plant cell walls can result
of relatively polar species. However, if excess water remains in in compounds being more easily extracted at the quoted SFE
the vessel, a highly water soluble analyte will prefer to partition conditions. Rosemary leaves obtained by different methods of
into the aqueous phase and its SFE recovery will be low. Semi- drying have been extracted and evaluated in terms of antioxidant
polar analytes will dissolve in the aqueous phase, but readily activity and essential oil yield and composition. By analyzing
partition into the supercritical CO2 , and yield high recoveries. the results for three samples of dried rosemary by using differ-
For analytes that are insoluble in water, the analytes precipitate ent procedures, it was shown that the treatment that provided
onto matrix surfaces, and even though the analyte may be highly the highest quantity of rosemary essential oil was freeze-drying
soluble in the extraction fluid, the excess water in the sample acts followed by drying in oven at 45 ◦ C, and then vacuum rotary
as a barrier in transfer of the analyte to the fluid [84]. The sol- evaporation. Freeze-drying is the mildest temperature treatment.
ubility of water in CO2 (0.3%) can cause restrictor plugging Therefore, less aroma loss is expected to be obtained. Drying
upon the fluid depressurization, including the pressure of water in oven at 45 ◦ C implied a higher temperature treatment than
in the collection system. Removal of water is usually done by rotary evaporation (35 ◦ C), but the first method is faster than
freeze-drying the sample matrix, as oven drying may result in the second and is performed in the absence of light. The vac-
solute volatilization. Alternatively, addition of drying agents to uum treatment was a slow process conducted in daylight, and
the sample may be used. This sample treatment is attractive as it artifacts could easily be formed. In fact, olfactory tests showed
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 15

development of a non-characteristic rosemary aroma in this


study.

6. Collection of extracted analyte

The development of appropriate collection system for tar-


get analytes after SFE is often ignored by new users, despite
the obvious fact quantitative extraction conditions cannot be
developed and evaluated unless the collection step is efficient.
Thus, the first job of the analyst is to optimize the collection sys-
tem and determine its efficiency for the target analytes. Apart
from the extraction process, the single most important process in
SFE is the efficient trapping of extracted material. Two different
approaches are commonly used for off-line SFE, liquid solvent
collection and solid-phase trapping. Both systems have their Fig. 16. Schematic representation of the liquid trapping process involving
advantages and disadvantages with respect to ease of handling, immersion of the restrictor into a liquid solvent.
choice of restrictor type, maximum gas flow, and compatibil-
ity with the various types of supercritical fluids, modifiers and of a modifier to the extraction fluid caused a decrease in trap-
analytes [88–90]. ping efficiencies. Eckard and Taylor [96] found that solid-phase
trapping has an additional drawback in that the sorbent trap has
6.1. Solvent collection a finite capacity. They found a 50/50 mixture of Porapak Q and
glass beads exhibited the highest sample capacity and was the
Liquid solvent collection is mechanically simple and has been most effective trap for a wide range of analyte types. In another
the most widely used approach for natural samples. Two com- study, Moore and Taylor [97] found that stainless steel ball trap
mon approaches have been used. In the first approach, the end of efficiency is greatly affected by the addition of a modifier to the
the flow restrictor is placed directly into the collection solvent, supercritical fluids. They found that it was necessary to raise the
and CO2 –analyte mixture is depressurized directly in contact trap temperature in order to achieve quantitative recoveries of
with the solvent. In the second approach, the CO2 –analyte efflu- digitalis glycosides. Solid-phase trapping is normally performed
ent is first depressurized to the gas phase in a glass transfer tube by depressurizing the CO2 and the analytes prior to the trap and
before contacting the solvent. For this system, efficient collec- collecting the analytes from the gas (or aerosol) phase directly
tion appears to depend on efficient transfer of the analytes from onto sorbents such as silica gel, Florisil, or bonded phase pack-
the gas phase to the collection solvent. Somewhat surprisingly, ing or onto insert surfaces such as glass or stainless steel beads.
the first approach has been shown to yield better collection effi- After SFE, the trap is eluted with liquid solvents for subsequent
ciencies of more volatile components, and efficient collection analysis. Both cryogenic and adsorption mechanism are active
using the second approach has often required the addition of a in solid-phase trapping, however, cryogenic trapping on inert
second solid-phase trap [91,92] or a glass wool insert in the glass materials (glass or stainless steel beads) is largely unsuccessful
liner [93]. for analytes with even a small vapor pressure. The use of sorbent
Various articles in the literature report the use of “liquid trap- phases allows adsorption to be used to increase the collection
ping.” One version of liquid trapping involves immersion of the
restrictor into a liquid, as illustrated in Fig. 16, while a second
version concerns an inert solid surface in tandem with a liquid
trap. In the Dionex (Sunnyvale, CA, USA) 703 instrumenta-
tion for example, non-volatile analytes are thought to deposit on
a hang-down tube (solid surface) while the more volatile ana-
lytes partition into the collection liquid after decompression. A
schematic of this type of trapping device is shown in Fig. 17.

6.2. Solid-phase collection

Several different solid-phase trapping methods are used in


SFE. Mulcahey and Taylor [94] conducted a study with non-
modified CO2 to determine the best solid-phase trap composition
and trapping conditions for a test mixture of analytes represent-
ing a wide range of polarities. Mulcahey et al. [95] continued
their studies with a wider assortment of solid-phase sorbent
traps. They found that a single trap composition may not effec- Fig. 17. Schematic representation of the liquid collection systems used in the
tively trap a wide range of analytes. Furthermore, the addition Dionex extractors.
16 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

efficiencies, and the selectivity of the adsorption mechanism can


be used to gain a degree of compound-class fractionation dur-
ing the SFE collection step. In addition, the choice of rinsing
solvent(s) can be used to selectively elute different compound
classes from the trapping system. This degree of selectivity based
on the elution of the trap is a particular advantage of sorbent col-
lection over solvent collection systems. However, solid-phase
trapping can be more trapping material, the trap temperature,
and the rinsing solvent. Some articles may be found in the liter-
ature about investigation on the effect of solid-phase collection
on the composition of supercritical fluid extracted plant oil.
Dugo et al. [98] reported a method for the deterpenation of
the citrus essential oils with supercritical CO2 using adsorption
on silica gel to enhance the selectivity of the separation between
the hydrocarbons and the oxygenated compounds. They showed
that the silica gel retains the oxygenated, more polar compounds,
thus allowing at low temperature and pressure, the extraction of
the non-polar terpene hydrocarbons. By increasing the pressure
and the temperature after a defined time, it is possible to elute the
oxygenated components the volatile fraction. Araujo et al. [99]
extracted Pupunha (Guilielna speciosa) oil in a fixed bed using
carbon dioxide. In their study, the supercritical carbon dioxide
containing the solubilized oil was expanded in the small stain-
less steel tube the precipitated extract was collected in a glass
tube placed inside the separator. Blanch et al. [100] in 1994
proposed a simple procedure for off-line SFE and capillary gas
chromatography analysis of the essential oil obtained from Ros-
Fig. 18. Scheme of the trap assembly. (From ref. [100] with permission.)
marinus officinalis L. During the extraction step, they deposited
the obtained analytes on an internal trap where the supercriti-
cal fluid evaporates. Subsequently, a suitable solvent is pumped tion of lemongrass, orange and nutmeg essential oils at 12 MPa
through the trap so that the analytes are rinsed off into a vial. and 40 ◦ C. The results of their work demonstrate the potential
Fig. 18 shows scheme of their assembly. They used different for use of a commercial reverse osmosis membrane in the sep-
sorbent for trapping extracted essential oils, such as Hypersil aration of lemongrass, orange and nutmeg essential oils from
octadecylsilica (ODS), glass beads, GasChrom 220, Tenax TA, supercritical mixtures with CO2 . All the membranes exhibited
Thermotrap TA, and Volaspher A-2 silanized sorbents. good resistance to the severe pressure conditions employed.
The oil retention index was reduced with the increase in trans-
6.3. Collection in empty vessels membrane pressure from 1 to 4 MPa. The best retention results
were obtained with the SG membrane, which retained up to 90%
Collection in an empty vial or vessel has been success- of all the essential oils tested. However, at the same time, this
fully practiced by a number of investigators and is particularly membrane allowed the lowest CO2 permeate fluxes with values
appropriate for bulk extraction of fat and similar exhaustive up to 8.75 kg h−1 m−2 at a pressure difference of 1 MPa. The
extractions. It is also applicable however, for the extraction of experimental results indicated the occurrence of fouling for all
trace levels of analytes, such as pesticides [101], but larger col- the membrane models after permeation of lemongrass essential
lection vessels are required for capturing such trace analytes in oil. Spricigo et al. [103] showed that the association of mem-
order to minimize their loss. Avoidance of entrainment of ana- brane separation processes to the supercritical fluid extraction
lytes in the escaping fluid stream can be minimized by adding of essential oils from vegetable matrices can be an alternative to
a glass, steel wool, or ball packing to the empty container. The the reduction of recompression costs derived from the depressur-
chosen material should be chemically inert, provide a high sur- ization step necessary for the recovering of the extracts. In their
face for condensing the analyte from the rapidly expanding fluid, work, a cellulose acetate reverse osmosis membrane was applied
but allow ready desorption of the analyte after completion of the to perform the separation of nutmeg essential oil and dense
extraction. carbon dioxide. The effects of feed stream essential oil concen-
tration, temperature and trans-membrane pressure on essential
6.4. Novel collection methods oil retention and CO2 permeability were investigated. The aver-
age retention of essential oil by the membrane was of 96.4% and
In 2004, Sarmento et al. [102] studied the performance of it was not affected significantly by any of the process variables.
three commercial reverse osmosis membranes: SG, CG and AG The CO2 flux was linearly proportional to the trans-membrane
regarding the permeability to supercritical CO2 and the reten- pressure applied and decreased as the essential oil concentration
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 17

in the feed stream increased. The membrane presented good CO2 ally require cryogenic refocusing of the analytes on the front
permeability and resisted well to the severe pressure conditions of the capillary column prior to analysis to obtain good peak
applied. shapes.

6.5. On-line coupling of supercritical fluid extraction with The first time SFE was coupled to another separation tech-
chromatographic techniques nique (i.e. SFC) was in extracting caffeine from roaste coffee
beans [105]. SFE–GC with mass spectrometry (MS) or flame
Sometimes SFE has been directly coupled to chromato- ionization detection (FID) has been demonstrated for flavor
graphic detection systems as this offers some advantages over compounds in spices, chewing gum, orange peel, spruce nee-
off-line SFE. In on-line SFE the entire extracted sample is intro- dles and cedar wood [106], from lime, lime peel, eucalyptus
duced into the system and therefore greater detection sensitivity [107,108], basil [107,109], grapefruit oil [110,111], thyme
can be obtained. However, smaller sample sizes are often used in [109,112], orange juice [108] and chamomile [109]. Many of
on-line SFE to prevent overloading of the chromatographic col- the earlier applications were of qualitative nature. In compar-
umn. There is also less chance of sample contamination because ison between SFE–GC and SFE–SFC, the latter was given an
there is no intermediate handling of the extracted analytes. advantage due to higher yields of oxygenated terpenes and no
Initial studies have focused on coupling SFE to supercritical need for derivation [109,113]. Sato et al. [114] used direct con-
chromatography (SFC), as carbon dioxide is used as both the nection of supercritical fluid extraction and supercritical fluid
extraction fluid and the chromatographic mobile phase, although chromatography as a rapid quantitative method for capsaicinoids
much research into coupling SFE to GC has been carried out. in placentas of Capsicum annuum L. They compared this method
Andersen et al. [104] discussed some theoretical considerations with usual extraction-HPLC method. Their finding showed that
involved in using SFE as a method for sample introduction in the SFE/SFC method has the advantages of no need for pretreat-
chromatography. An SFE–SFC protocol was used to extract ment and no (or minimal) need for organic solvents. Also, this
and determine the herbicides linuron and diuron from a sandy method is useful as a rapid (20 min) and safe screening test for
loam soil and wheat. After initial SFE optimization, carried the pungency of various Capsicum fruits.
out off-line, the two techniques were coupled with flame ion-
ization being used for detection. A modifier was required to 7. Extraction of oxygenated compounds from plant
achieve quantitative extraction, which eluted as a solvent front materials by supercritical fluid extraction
on the chromatogram. Both capillary and microbore columns
were investigated, with better sensitivity being obtained with a Today, antioxidants from natural resources are associated
microbore column and larger sample loops. with health benefits since oxygenated compounds are related
The most common technique used in on-line SFE of natural to a positive action against heart diseases, malaria, neuro-
samples is coupled SFE–GC. This coupling technique is of par- degenerative diseases, AIDS, cancer and longevity [115]. On
ticular important for non-polar analytes, which do not require the other hand, some of the artificial antioxidants are related to
derivation either in situ or prior to GC separation. A variety of health damage, such as kidney edema [116]. For these reasons,
approaches have been used to couple SFE extraction with cap- the market for natural antioxidant should rapidly increase.
illary GC, which can be roughly categorized into three main Several methods have been used to extract antioxidants from
groups: aromatic plants, such as solid–liquid extraction, aqueous alka-
line extraction, extraction with aqueous solutions [117,118], and
(a) An external loop, through which the extract passes, is used supercritical fluid extraction [119,120]. Products obtained by
to introduce the analytes into the GC column. The analytes SFE from different plants, in general, have a higher antioxi-
are transferred into the GC column by sweeping the heated dant activity than extracts obtained by using solvent extraction
loop with carrier gas. This has the advantage of allowing off- with organic solvents [121,122], probably due to a differ-
line collection to be carried out simultaneously, although it ence in composition deriving from the extraction conditions
also suffers from a decrease in sensitivity as only a fraction applied. In 2004, Hu et al. [123] investigated the influences
of the sample is passed to the chromatograph. of extracting pressure, temperature, and flow rate on the
(b) The extract is transferred into a sorbent trap external to the yield of sesame seed extract and the antioxidant activity of
gas chromatograph that may be cryogenically cooled. Sub- extracts by supercritical carbon dioxide and solvent from black
sequent heating of the trap and purging with carrier gas allow sesame seed as compared to ␣-tocopherol, Trolox, and buty-
the extracts to enter the chromatograph. The addition of the lated hydroxytoluene (BHT). They found the highest extracted
Tenax-GC trap gave better peak shapes than direct SFE–GC yield was achieved at 35 ◦ C, 40 MPa, and a CO2 flow rate
because of the refocusing effect. It also allowed the methanol of 205 ml min−1 . Results for the linoleic acid system showed
modifier, used in the extraction, to be removed before flush- that the antioxidant activity follows the following order: extract
ing the analytes on to the column, which previously caused at 35 ◦ C, 20 MPa > BHT > extract at 55 ◦ C, 40 MPa > extract
a “hump” in the baseline in conventional SFE–GC. at 55 ◦ C, 30 MPa > Trolox > solvent extraction > ␣-tocopherol.
(c) The simplest approach utilizes a conventional GC injec- The supercritical carbon dioxide extracts exhibited significantly
tion port to couple SFE with GC with both on-column and higher antioxidant activities comparable to those obtained by
split–splitless ports being used. All three techniques gener- n-hexane extraction.
18 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

The volatile components of Terminalia catappa (green, yel-


low and red fallen) leaves were extracted using supercritical
carbon dioxide at various pressures [124]. The extracts from
yellow and red fallen leaves exhibited higher inhibition of pre-
oxidation that those from green leaves. On the other hand,
supercritical carbon dioxide extraction at 133 atm and 40 ◦ C
resulted in the extracts with better antioxidant activity whereas
lower inhibition of pre-oxidation was observed with extracts
prepared from the extraction at 267 atm and 40 ◦ C. Also, Zan-
can et al. [125] studied the influence of the use of co-solvent in
the kinetics of SFE of ginger oleoresin, in the chemical com-
position of the extracts and in their antioxidant action. They
showed that the major substances present in the ginger extracts
were ␣-zingiberene, gingerols and shogaols the amounts of these Fig. 19. Graph representing the percent of total area of the identified compounds
vs. solvent-to-feed ratio. (From ref. [127] with permission.)
compounds were significantly affected by temperature, pressure
and co-solvent. Nonetheless, the antioxidant activity of the gin-
ger extracts remained constant at 80% and decreased to 60% in
the absence of gingerols and shogaols.
In 2003, the selectivity of subcritical water extraction several
temperatures to extract antioxidant compounds from rosemary
leaves was investigated [126]. Results indicate high selectivity
of subcritical water toward the most active compounds of rose-
mary. The antioxidant activity of fractions obtained by extraction
at different water temperatures was high comparable to those
achieved by SFE of rosemary leaves. In this study, Ibanez et al.
demonstrated the possibility of tuning the selectivity for antiox-
idant extraction by a small change in extraction temperature.
Senorans et al. [127] isolated antioxidants from orange
juice by the use of countercurrent supercritical fluid extraction Fig. 20. Graph representing the log (enrichment) as a function of the S/F ratio
(CC-SFE) and characterized by reversed-phase liquid chro- (a selective enrichment can be observed toward the extracts or the raffinate as a
matography (RPLC) coupled to mass spectrometry (MS) and function of the S/F ratios selected). (From ref. [127] with permission.)
diode-array detection (DAD). They employed a pilot-scale SFE
plant equipped with a packed column for countercurrent extrac- ering both separators together) are also presented. They obtained
tion and fractionation of raw orange juice with carbon dioxide. a high correlation (96%) for log (total enrichment) versus S/F
Several experiments have been performed in order to study the using a linear regression y = 0.3365x + 2.4304r2 = 0.963.
effect of the countercurrent conditions on the content of antiox- In 2002, Simoa et al. [128] determined antioxidants from
idative compounds. In their study, the main variable that has orange juice by the combined use of CC-SFE prior to RPLC
been considered was the solvent-to-feed ratio (S/F) because or micellar electrokinetic chromatography (MEKC). They
it plays an essential role in the extraction efficiency. The val- achieved separation of antioxidants found in the SFE fractions
ues tested covered a wide range of sample and solvent (CO2 ) by using a new MEKC method and a published LC procedure,
flow rates. They obtained three different products after extrac- both using diode-array detection. In the same year, Yepez et al.
tion and fractionation of the orange juice: those in separators [129] obtained fractions from seeds of coriander (Coriandrum
1 (F1) and 2 (F2) and the raffinate (R) which is the byproduct sativum) by extraction with supercritical carbon dioxide in a
of the extracted samples collected at the bottom of the column. semi-continuous lab-scale equipment, and were tested for their
Fig. 19 shows the percent of total area of the identified com- antioxidant activity. Fractions from coriander seeds obtained
pounds versus solvent-to-feed ratio. When low S/F ratios are by SFE exhibited a significant antioxidant activity, as deter-
used, a maximum extraction of flavonoids is obtained, with a mined by removal of DPPH free radicals larger than 50% after
low percentage recovered in the raffinate. The opposite is found 300 min. In addition, high extraction yields, close to 2% (w/w),
at S/F equal to 11 where almost 96% of the compounds iden- were achieved using SFE conditions in the range of 183–326 K,
tified are found in the raffinate. In each experimental run, two corresponding to a CO2 density in the range of 0.74 g/ml.
different extracted fractions and the residual non-extracted juice
were obtained and characterized. Different flavonoids have been 8. Extraction of terpenes and sesquiterpenes by
identified in the fractions obtained after CC-SFE. Also, they supercritical fluid extraction
discussed possibility of using this process for antioxidant com-
pounds enrichment. Enrichment results are shown in Fig. 20 as Goto and co-workers [130] used supercritical CO2 to sepa-
a function of the S/F ratio data corresponding to the enrichment rate oxygenated compounds from essential oils. This technique
achieved in separators 1 and 2 individually and the total (consid- still cannot replace vacuum distillation as an industrial process
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 19

because of low recoveries and inconsistent results. Vacuum dis- sabinene. However, in virgin olive oil, the major constituents
tillation and supercritical CO2 are complementary processes of the above analytical classes were, respectively, oleic acid,
for producing high quality oxygenated compounds with high trilinolein, waxes C36 , unsaturated volatile C6 aldehydes (trans-
recovery rates. The former is more suitable for removing 2-hexenal most markedly), and the same prominent sterols and
monoterpenes at low fraction temperatures (<308 K), and the lat- superior alcohols found in supercritical fluid extract. In commer-
ter is more suitable for separating oxygenated compounds from cial carrot oil, which also contained a proportion of unknown
pigments and waxes. Consequently, the two methods can be used plant oil, several components showed magnitudes that were
in tandem. Sonsuzer et al. [131] successfully applied response lower compared to supercritical oil but higher with respect to
surface methodology for optimization of parameters in SFE of virgin olive oil. The last had the aliphatic and triterpene alco-
Thymbra spicata oil. The high regression coefficients of sec- hol concentration higher compared to that of both supercritical
ond order polynomial of the responses showed that model fitted fluid extract and commercial carrot oil. Rodrigues et al. [135]
to data well. The optimum condition was applied to minimize used sub- and supercritical CO2 to obtain extracts from two ori-
monoterpene hydrocarbons content and maximize oxygenated ganum samples, one commercial, and another cultivated under
compounds and yield. Pressure was the most significant fac- agronomic control. Their results show that the commercial sam-
tor affecting yield in SFE. An adverse effect of temperature on ple provides a higher yield of extract if compared to the other
yield was observed. Monoterpenes which have higher volatil- sample. Chemical analyses allowed the identification of around
ity, lower molecular-weight and lower polarity were extracted 24 compounds for both commercial and cultivated origanum
at the beginning of the process. However, sesquiterpenes and samples. It was also found that the distribution of chemical com-
oxygenated compounds were extracted later due to their higher ponents in the extracts was a function of extraction time and
molecular-weight and polarity, respectively. differed appreciably between the origanum species. The chro-
Carlson et al. [132] extracted lemongrass (Cymbopogon cit- matographic analysis permitted the identification of thymol and
ratus) essential oil with dense carbon dioxide at 23–50 ◦ C and cis-sabinene hydrate as the most prominent compounds present
85–120 bar. The compounds present in larger quantities in the in commercial oregano sample and carvacrol and cis-sabinene
lemongrass essential oil were neral, geranial and myrcene. Their hydrate in the cultivated sample.
results showed that the changes in temperature and pressure Diaz-Maroto et al. [136] obtained volatile oil extracts of fen-
conditions had a significant effect on the composition of the nel seeds (Foeniculum vulgare Mill.) and thyme leaves (Thymus
extracts. Co-extraction of waxes was observed under all the vulgaris L.) by simultaneous distillation-extraction (SDE) and
extraction conditions but supercritical extracts had lower content SFE. They showed that the fennel oil extracted by SDE and SFE
of these compounds than liquid CO2 extracts. Along the extrac- has similar compositions, with trans-anethole, estragole, and
tion experiments, changes in the composition of the extracts were fenchone as the main components. Thymol and p-cymene, the
observed, with higher-molecular-weight compounds extracted most abundant compounds in thyme leaves, showed big differ-
in larger quantities at the end of the process. The optimized ences, with generally higher amounts of monoterpenes obtained
extraction conditions from their tests were 120 bar and 40 ◦ C. by SDE. Key odorants in fennel seeds were determined by
Regina et al. [133] investigated the influence of temper- gas chromatography-olfactometery (GC-O) showed similar elu-
ature and pressure on the characteristics of the essential oil tion patterns when SDE and SFE were utilized. trans-Anethole
obtained from high-pressure carbon dioxide extraction of mar- (anise, licorice), estragole (anise, licorice, sweet), fenchone
joram. Their experiments were performed in a laboratory-scale (mint, camphor, warm), and 1-octen-3-ol (mushroom) were the
unit using the dynamic method in the temperature range of most intense odor compounds detected in fennel extracts. Eller
293.15–313.15 K, from 100 to 200 bar in pressure. Results et al. [137] investigated the extraction of cedarwood oil (CWO)
showed that an increase in temperature leads to a rise in using liquid carbon dioxide and compared it to traditional SFE,
the extract yield despite large changes in solvent density. including the effects of extraction pressure and length of extrac-
Chromatographic analyses permitted the identification of cis- tion. Their finding showed that the cumulative yields of CWO
sabinene hydrate, terpineol-4, R-terpineol, and cis-sabinene from cedarwood chips using 80 l of carbon dioxide varied little
hydrate acetate as the main volatile compounds present in both treatment to treatment, with all temperature/pressure combi-
commercial and cultivated samples. nations yielding between 3.55 and 3.88% CWO, including
Ranalli et al. [134] obtained carrot root oil, by supercritical cumulative yields. Also, the rate of extraction was highest under
fluid carbon dioxide extraction, and compared to commercial the supercritical extraction conditions (i.e. 100 ◦ C and 400 bar)
carrot oil and a virgin olive oil. Their results showed that carrot and under the liquid CO2 conditions (i.e. 25 ◦ C), the extraction
root oil obtained by SFE has much higher content of carotenes, rates did not vary significantly with extraction pressure. How-
phenolics, waxes, phytosterols, and sesquiterpene and monoter- ever, they observed differences in the chemical composition of
pene volatiles. In the SFE oil the most prominent components the collected CWO. For example, extractions at 100 ◦ C gave a
present in the fully investigated analytical fractions (fatty acids, much lower ratio of cedrol/cedrene than extractions at 25 ◦ C,
triglycerides, waxes, phytosterols, long-chain aliphatic alcohols, but the highest ratio of cedrol/cedrene was obtained using 25 ◦ C
superior triterpene alcohols, and volatiles) were, respectively, and 100 bar.
linolenic acid, trilinolein, waxes C38 , ␤-sitosterol, campes- Vági et al. [138] investigated extraction of pigments (chloro-
terol and stigmasterol, 1-hexacosanol, 24-methylencycloartanol phylls and carotenoids) from marjoram (Origanum majorana
and cycloartenol, ␤-caryophyllene, ␣-humulene, ␣-pinene, and L.) with supercritical carbon dioxide. The aim of their study
20 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

was to map the effects of extraction pressure and temperature subsequent analysis are critical steps in the overall sample prepa-
on the yield of coloring materials by applying a 32 full factorial ration procedure. Considerably less organic solvent usage can be
design with three repeated tests in the center of the design. For expected here as compared with Soxhlet extraction, although the
comparison, they carried out laboratory and pilot plant Soxhlet cost of SPE cartridges cannot be discounted. Given sufficient car-
extractions using ethanol and n-hexane solvents and determined tridge, multiple extractions can be performed simultaneously. A
the compositions of pigments in marjoram extracts by HPLC. more recent development in this field is the introduction of filter
Similar amounts of carotenoids, in addition to 40% of chloro- disks which contain solid-sorbent trapped within polytetrafluo-
phylls and their derivatives, were recovered from the SFE when roethylene fibrils. Currently, only C8 and C18 bonded silica is
compared to the ethanol Soxhlet extraction. available. These disks (25 and 47 mm in diameter) enable the
flow of liquid phase analyte plus matrix to be faster than the
9. Comparison of supercritical fluid extraction to SPE cartridges. Organic solvent or supercritical fluid has been
conventional methods employed to release the trapped analyte for subsequent analysis
[139].
One of the oldest and most commonly used extraction pro- Piggott et al. [140] used steam distillation, solvent extrac-
cedures employs a Soxhlet apparatus. It has been the standard tion, supercritical fluid extraction and liquid CO2 extraction
extraction procedure over 100 years even though it fails to exhibit to obtain the volatile oil from Western Australian sandalwood.
many of the ideal extraction criteria. The time for an aver- SFE afforded the highest yields of extractable material and total
age Soxhlet extraction ranges from 1 to 72 h. The completed volatile. Also, volatile secondary metabolites have been isolated
extraction produces a high volume, dilute solution which usually from flowers, leaves and stems of Spilanthes americana [141]
needs to be concentrated prior to analysis. In many cases, Soxh- by simultaneous distillation–solvent extraction and supercriti-
let extraction is not selective, because interfering compounds cal carbon dioxide extraction. In this study, the plant material
may also be extracted by the heated solvent of choice which (flowers, stems, leaves) affected the composition of the extracts.
may further complicate the assay of the analyte(s) of interest. SFE extracts from stems were rich (>40%) in sesquiterpenes,
The choice of solvent obviously controls the solvating power while those from leaves and flowers were abundant in nitro-
as well as the temperature of the extraction. Under these con- genated and oxygenated compounds. However, SDE extracts
ditions the integrity of the analyte may not be maintained in from stems, leaves, and flowers of the plant contained sesquiter-
that thermal decomposition or reaction with the solvent may pene levels of 32, 28 and 20%, and a generally higher proportion
occur. Perhaps the greatest disadvantage of using the Soxhlet of oxygenated compounds and monoterpenes than the SFE coun-
method for extractions is its utilization of expensive, high purity terparts. Also, some heavy hydrocarbons originated from flower
organic solvents such as acetone and methylene chloride. While pigments and waxes were isolated by SFE but not by SDE. It was
automation is seldom attempted, it does have the advantage that found that SFE is both selective and highly efficient in the iso-
numerous Soxhlet extractions can be simultaneously performed. lation of sesquiterpenes, heavy hydrocarbons and nitrogenated
Furthermore, hardware for Soxhlet is fairly simple however, for compounds in this particular case.
quantitative work extreme care must be taken to avoid introduc- Vilegas and Lancas [142] used supercritical CO2 extraction to
tion of contaminants and to minimize losses in sample transfer compare chemical composition of the essential oils obtained by
and solvent exchange. steam distillation procedure from two lauraceae (Ocotea caesia
Liquid–liquid and liquid–solid extractions normally per- Mez.). Their study showed that the supercritical carbon dioxide
formed at room temperature. Large volumes of organic solvents, extractions had lower yields than those from steam distillation,
which must be concentrated and disposed of, are required. Some but the extracts obtained were similar in both species studied.
mixing time is usually necessary for efficient phase exchange. In 1999, Eikani et al. [143] compared the extraction obtained
Multiple extractions are also usually mandatory if quantita- from Cuminum cyminum L. by supercritical carbon dioxide
tive removal is desired. Sample transfer can become a problem with cumin essential oil obtained by conventional steam distil-
especially if phase emulsions are produced. The simultaneous lation. They showed that the physicochemical properties (such
extraction of multiple samples is usually possible if sufficient as refractive index, specific gravity and optical rotation) of the
glassware is available. oils extracted by supercritical CO2 and steam distillation were
Solid-phase extraction (SPE) has become increasingly pop- different. The results of the GC–MS analysis showed that the
ular during the past several years. It appears to be applicable most noticeable difference between the two methods is in the p-
to both non-polar and polar analytes. The analyte and matrix mentha-1,3-dien-7-al and p-mentha-1,4-dien-7-al composition.
must, however, be in the liquid state. Currently, SPE serves as Hydrodistillation and solvent extraction using pentane,
an attractive alternative to liquid–liquid extraction. A wide vari- ethanol and supercritical carbon dioxide were used to isolate
ety of solid–sorbents exhibiting a wide variety of chemistries essential oils from Grapefruit flavedo [144]. The compositions
is available for selective trapping from numerous vendors. The of different extractions were compared. Monoterpene hydrocar-
sorbents are large particle size (40 ␮m I.D.) bonded phase silica bons decrease in supercritical carbon dioxide extracts at 87–90%
in most cases although phases exhibiting new chemistries are with respect to their quantity in pentane extracts (95%) and in
rapidly being introduced. Proper activation of the phase prior to hydrodistillate (97%) these levels in monoterpene hydrocarbons
sample introduction and selective rinsing of the phase in order were related to the limonene content, the most abundant com-
to recover the extracted analyte free of matrix components for pound in grapefruit essence. Sesquiterpenes, aldehydes, alcohols
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 21

Table 5 Table 6
Comparison of the overall chromatographic area percentages for the four main Percent yield and cumulative percent yield of the Laurel oil at different stages
classes of hydrocarbon monoterpenes (HMs), oxygenated monoterpenes (OMs), of the supercritical extraction (60–240 min), the overall run (SFE-T), and the
hydrocarbon sesquiterpenes (HSs), and oxygenated sesquiterpenes (OSs) (at hydrodistillation [146]
different stages of supercritical fluid extraction) in the constituents of the Laurel
Quantity 60 min 120 min 180 min 240 min SFE-T HD
oil [146]
Class 60 min 120 min 180 min 240 min SFE-T HD Yield (%) 0.27 0.28 0.22 0.05 0.82 0.90
Cumulative 0.27 0.56 0.77 0.82 0.82 0.90
HMs 12.67 2.74 1.75 0.31 13.65 15.51 yield (%)
OMs 79.43 77.62 76.94 66.68 70.86 70.28 ms /m0 5.36 10.72 16.08 21.44 21.44 –
HSs 7.45 14.91 14.11 15.52 11.53 7.19
OSs 0.5 4.17 6.92 17.07 3.68 7.03 Columns SFE-1 to SFE-4 refer to the oil fractions collected after each hour of
the supercritical extraction. SFE-T is the overall essential oil obtained by SFE.
Columns SFE-1 to SFE-4 refer to the oil fractions collected after each hour of
the supercritical extraction. SFE-T is the overall essential oil obtained by SFE.
L. leaves using carbon dioxide was carried out under differ-
ent conditions of pressure, temperature, modifier content and
and esters increased their GC area percentage in supercritical dynamic extraction time. Then, they compared proposed extrac-
carbon dioxide extracts obtained at a high fluid density relative tion method with hydrodistillation. A total 22 compounds have
to hydrodistillate and the pentane extracts. been determined in SFE extracts while in the hydrodistilled
In 2000, Cassel et al. [145] compared results of hydrodistilla- oil only 11 components were identified and quantified. SFE
tion and supercritical carbon dioxide methods for the extraction products were found to be markedly different from the corre-
of Baccharis leave oil. They observed the non-oxygenated sponding hydrodistilled oil. A large amount of ␤-phellanderene
monoterpenes, present in the hydrodistilled oil in high con- was present in the hydrodistilled essential oil, also the ratio of ␣-
tents (␤-pinene 28.2% and limonene 10.6%), were not detected pinene and 3-carene in distilled oil were high in comparison with
in the supercritical carbon dioxide extracts. The selectivity of the supercritical carbon dioxide extracts. Their results showed
supercritical carbon dioxide allowed one to maximize the con- that under pressure 200 atm, temperature 45 ◦ C and dynamic
centration of oxygenated compounds. Also, Caredda et al. [146] extraction time of 30 min, SFE of limonene was more selective
studied supercritical carbon dioxide extraction of essential oil and under pressure 350 atm, temperature 45 ◦ C and dynamic
from Laurus nobilis. Extraction conditions were as follows: extraction time of 20 min, extraction was more selective for the
pressure 90 bar, temperature 50 ◦ C and carbon dioxide flow ␣-thoujone, which was not found in the hydrodistilled oil. Pour-
1.0 kg/h. Extracted waxes were trapped in the first separator mortazavi et al. [147] also showed that the composition of the
set at 90 bar and −10 ◦ C. The oil was recovered in the sec- SFE products and the hydrodistilled black cumin essential oils
ond separator held at 15 bar and 10 ◦ C. The main components is significantly different. They compared the composition of the
were 1,8-cineole (22.8%), linalool (12.5%), R-terpinyl acetate SFE product with hydrodistilled oil and found a higher level
(11.4%), and methyleugenol (8.1%). Four classes, hydrocar- of the ␥-terpinene and cuminaldehyde in the hydrodistilled oil.
bon monoterpenes (HMs), oxygenated monoterpenes (OMs), Table 7 shows the p-cymene content of the distilled oils is con-
hydrocarbon sesquiterpenes (HSs), and oxygenated sesquiter- siderable, however, this compound was not found in the SFE
penes (OSs), on the basis of their chemical structure or retention extracts. The method contributes to the automation of pharma-
time were reported. The area percentages relative to each class ceutical industry. Ebrahimzadeh et al. [148] isolated essential oil
are shown in Table 5. In general, volatile compounds (HMs) are of Zataria multiflora Boiss, cultivated in Iran, by steam distil-
extracted almost completely during the first hour of extraction lation and compared with supercritical fluid CO2 extracts. The
(12.67, against 0.31% in the fourth hour). The OMs decreased to extracts obtained by SFE at different conditions were compo-
a minor extent from 79.43 to 66.68%. HSs and OSs are present sitions similar to that of the oil obtained by steam distillation.
at, respectively, 15.52 and 17.07% in the fraction obtained after
180–240 min and at 7.45 and 0.50 in the first hour sample. These
results confirm that a long time run is necessary to obtain oil with Table 7
Comparison of the main components of Iranian black cumin oils obtained by
a stable composition. Comparison with the hydrodistilled oil did SFE (under 200 atm pressure and 45 ◦ C temperature for 15 min static followed
not reveal any significant difference. The yields of each fraction by 20 min dynamic) and hydrodistillation [147]
of the supercritical extraction and hydrodistillation as w/w%,
Compound SFE HD
with respect to the charged material, are reported in Table 6.
In the same table the amount of CO2 consumed in the process, ␣-Pinene 0.8 2.8
expressed as the specific mass of solvent, ms /m0 (m0 is the mass ␤-Pinene 1.5 3.7
Myrcene 0.6 1.0
of leaves charged in the extractor) is specified. The overall yield p-Cymene – 5.6
of the supercritical extraction was 0.82%; 1,8-cineole (22.84%) o-Cymene 7.8 0.1
was the major component. Limonene 6.8 10.6
In 2004, Pourmortazavi et al. [77] showed that differ- ␥-Terpinene 38.0 45.7
ent extraction compositions could be obtained by different Cuminaldehyde 11.5 12.7
Cuminyl alcohol – 6.4
extraction methods applied to natural products. They studied ␣-Methyl-benzenemethanol 25.6 3.5
supercritical fluid extraction of volatile oil from J. communis
22 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

However, the quantitative compositions of the two products were (2.2, 5.0 and 6.4%), and p-cymene (2.2, 4.7 and 4.4%),
quite different; indeed, at higher pressures, and temperatures respectively.
(30.4 MPa and 55 ◦ C) thymol and carvacrol are more soluble in
supercritical carbon dioxide. In all obtained extracts, ␥-terpinene 10. Conclusion
and carvacrol appear to be the major components, the extracts
being richer in thymol. Moreover, the recovery of thymol in SFE This article summarizes research finding involving the super-
is better than that steam distillation. critical fluid extraction of volatile components from plant
Aghel et al. [149] used orthogonal array design in order to materials. Emphasis is placed on optimization of extraction
optimize SFE of Mentha pulegium L. essential oil and com- parameters for complete recovery of analytes from their matri-
pared their SFE results with hydrodistilled oil. They identified ces. The future of supercritical fluid extraction for the extraction
22 compounds consisting over than 91% of the total hydrodis- of volatile components from plants looks bright based on a
tillation essential oil. The major components were pulegone number of considerations. First, SFE has a wide application
(37.8%), menthone (20.3%), piperitenone (6.8%) and para- area. It is capable of extracting a wide range of diverse com-
mentha-1,8-dien-2-one (5.1%). Over 81% of these compounds pounds from variety of sample matrices. Many non-polar to
were monoterpenes and 83.0% were oxygenated terpeneoids. moderately polar compounds can be extracted with carbon diox-
The results of GC/MS analysis of nine supercritical fluid ide while more polar compounds can be extracted with other
extractions showed considerable differences between SFE and fluids or modified carbon dioxide. Secondly, supercritical flu-
hydrodistillation. The concentration of limonene in supercritical ids offer extraction selectivity unsurpassed by solvent polarity.
fluid extract at optimum condition was high (14.6%) in compari- Thirdly, the environmental friendliness of this technique can
son with hydrodistillation (trace). On the other hand, they have a never be disputed, since non-toxic fluids such as carbon dioxide
selective extraction at optimum condition for SFE and only four or this compound modified with up to 20% organic solvents
compounds (menthone, pulegone and limonene make up 96.9% are most commonly employed. Supercritical fluid extraction
of its constituents and the fourth compound was a methoxy phe- has faced a growing interest in the two past decades due to its
nol derivative by 3.1) were extracted from M. pulegium L. In numerous advantages over classical liquid solvent extractions
2004, Stashenko et al. [150] employed hydrodistillation (HD), (mainly rapidity, selectivity, cleanliness, possibility of manipu-
simultaneous distillation solvent extraction, microwave-assisted lating the composition of the extract and low solvent volumes
hydrodistillation (MWHD), and supercritical fluid (CO2 ) extrac- required). On the other hand, coupling established analytical sys-
tion isolate volatile secondary metabolites from fresh leaves tems such as gas liquid chromatography with a comparatively
and stems of Colombian Lippia alba Mill. They identified new technique has led to the development of complex, if not
around 40 components in the various volatile fractions. Car- novel, sample extraction–preparation–analysis–detection super
vone (40–57%) was the most abundant component, followed schemes to cope with difficult matrices in which essential oil of
by limonene (24–37%), bicyclosesquiphellandrene (5–22%), plants, in particular, are inevitably found.
piperitenone (1–2%), piperitone (ca. 1.0%), and ␤-bourbonene
(0.6–1.5%), in the HD, SDE, MWHD, and SFE volatile frac- References
tions. Static headspace (S-HS), simultaneous purge-and-trap
(P&T) in solvent (CH2 Cl2 ), and headspace solid-phase microex- [1] G. Anitescu, C. Doneanu, V. Radulescu, Flavour Fragr. J. 12 (1997) 173.
traction (HS-SPME) were used to sample volatiles from fresh [2] V. Illes, H.G. Daood, S. Perneczki, L. Szokonya, M. Then, J. Supercrit.
L. alba stems and leaves. The main components that they Fluids 17 (2000) 177.
isolated from the headspace of the fresh plant material were [3] M.H. Eikani, I. Goodarznia, M. Mirza, Flavour Fragr. J. 14 (1999) 29.
[4] V.M. Rodrigues, E.M.B.D. Sousa, A.R. Monteiro, O. Chiavone Filho,
limonene (27–77%), carvone (14–30%), piperitone (0.3–0.5%), M.O.M.A.M. Marques, A.A.M. Meireles, J. Supercrit. Fluids 22 (2002)
piperitenone (ca. 0.4%), and ␤-bourbonene (0.5–6.5%). In 21.
another study [151], HD, simultaneous distillation–solvent [5] N.L. Rozzi, R.K. Singh, Comp. Rev. Food Sci. Food Safety 1 (2002) 33.
extraction, MWHD, and supercritical fluid (CO2 ) extraction [6] S.R.S. Ferreira, Z.L. Nikolov, L.K. Doraiswamy, M. Angela, A. Meireles,
A.J. Petenate, J. Supercrit. Fluids 14 (1999) 235.
were used to isolate volatile secondary metabolites from Colom-
[7] S.M. Pourmortazavi, S.S. Hajimirsadeghi, I. Kohsari, S.G. Hosseini, J.
bian Xylopia aromatica (Lamarck) fruits. Static headspace, Chem. Eng. Data 49 (2004) 1530.
simultaneous purge and trap in solvent (CH2 Cl2 ), and headspace [8] C.M. Wai, S. Wang, J. Chromatogr. A 785 (1997) 369.
solid-phase microextraction were utilized to obtain volatile [9] A.I. Cooper, J. Mater. Chem. 10 (2000) 207.
fractions from fruits of aromatica trees, which grow wild in [10] S.M. Pourmortazavi, S.S. Hajimirsadeghi, Ind. Eng. Chem. Res. 44 (2005)
6523.
Central and South America, and are abundant in Colombia. ␤-
[11] S.M. Pourmortazavi, S.G. Hosseini, M. Fathollahi, Presented at the Inter-
Phellandrene was the main component found in the HD and national Autumn Seminar on Propellant Explosives and Pyrotechnics,
MWHD essential oils, SDE and SFE extracts (61, 65, 57, and Guilin, China, 15–18 October 2003, 2003.
40%, respectively), followed by ␤-myrcene (9.1, 9.3, 8.2 and [12] S.M. Wang, Y.C. Ling, Y.S. Giang, Forensic Sci. J. 2 (2003) 5.
5.1%), and ␣-pinene (8.1, 7.3, 8.1 and 5.9%). The main compo- [13] N. Alexandrou, M.J. Lawrence, J. Pawliszyn, Anal. Chem. 64 (1992) 301.
[14] D.E. Raynie, Anal. Chem. 65 (1993) 3127.
nents present in the volatile fractions of the aromatica fruits,
[15] J.M. Levy, E. Storozynsky, R.M. Ravey, J. High Resolut. Chromatogr. 14
isolated by S-HS, P&T and HS-SPME were ␤-phellandrene (1991) 661.
(53.8, 35.7 and 39%), ␤-myrcene (13.3, 12.3 and 10.1%), [16] R.W. Shaw, T.B. Brill, A.A. Clifford, C.A. Eckert, E.U. Franck, Chem.
p-mentha-1(7),8-diene (7.1, 10.6 and 10.4%), ␣-phellandrene Eng. News 23 (1991) 26.
S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24 23

[17] G. Gamiz-Garcia, M.D. Luque de Castro, Talanta 51 (2000) 1179. [58] P. Ambrosino, R. Fresa, V. Fogliano, S.M. Monti, A. Ritieni, J. Agric.
[18] F. Favati, J.W. King, M. Mazzanti, J. Am. Oil Chem. Soc. 68 (1991) 422. Food Chem. 47 (1999) 5252.
[19] V. Illes, O. Szalai, M. Then, H.G. Daood, S. Perneczki, J. Supercrit. Fluids [59] L.M.A.S. Campos, E.M.Z. Michielin, L. Danielski, S.R.S. Ferreira, J.
10 (1997) 209. Supercrit. Fluids 34 (2005) 163.
[20] O.J. Catchpole, J.B. Grey, N.B. Perry, E.J. Burgess, W.A. Redmond, N.G. [60] F. Gaspar, J. Agric. Food Chem. 51 (2003) 6604.
Porter, J. Agric. Food Chem. 51 (2003) 4853. [61] A.P.R.F. Canela, P.T.V. Rosa, M.O.M. Marques, M.A.A. Meireles, Ind.
[21] R.S. Mohamed, M.D.A. Saldaña, P. Mazzafera, Ind. Eng. Chem. Res. 41 Eng. Chem. Res. 41 (2002) 3012.
(2002) 6751. [62] J.M. Walsh, G.D. Ikonomou, M.D. Donohue, Fluid Phase Equilib. 33
[22] I.J. Branabas, J.R. Dean, Analyst 119 (1994) 2381. (1987) 295.
[23] A.A. Clifford, in: S.A. Westwood (Ed.), Supercritical Fluid Extraction [63] G.S. Gurdial, N.R. Foster, J.S.L Yun, in: M.L. Lee, K.E. Markides. (Eds.),
and its Use in Chromatographic Sample Preparation, Chapman & Hall, Chromatography Conferences, Provo, UT, 1991, p. 66.
London, 1993, p. 1. [64] J.B. Crowther, J.D. Henion, Anal. Chem. 57 (1985) 2711.
[24] S. Ashraf, K.D. Bartle, A.A. Clifford, R. Moulder, M.W. Raynor, G.F. [65] M. Taguchi, T. Hobo, J.J. Maeda, J. High Resolut. Chromatogr. 14 (1991)
Shilstone, Analyst 117 (1992) 1697. 140.
[25] K.D. Bartle, A.A. Clifford, G.F. Shilstone, J. Supercrit. Fluids 5 (1992) [66] J.R. Wheeler, M.E. McNally, J. Chromatogr. Sci. 27 (1989) 534.
220. [67] M. Kohler, W. Haerdi, P. Christen, J.L. Veuthey, J. Chromatogr. A 785
[26] M. Kane, J. Dean, S.M. Hitchen, R.L. Tranter, C.J. Dowle, Analyst 118 (1998) 353.
(1993) 1261. [68] Y.H. Choi, Y.W. Chin, J. Kim, S.H. Jeon, K.P. Yoo, J. Chromatogr. A 863
[27] J.N. Jaubert, M.M. Goncalves, D.A. Barth, Ind. Eng. Chem. Res. 39 (1999) 47.
(2000) 4991. [69] A. Pasquel, M.A.A. Meireles, M.O.M. Marques, A.J. Pentenate, Braz. J.
[28] W. Abdoul, E. Rauzy, A. Peı̌neloux, Fluid Phase Equilib. 68 (1992) 47. Chem. Eng. 17 (2000) 271.
[29] F. Gaspar, T. Lu, R. Marriott, S. Mellor, C. Watkinson, B.A. Duri, R. [70] A.R. Monteiro, M.A.A. Meireles, M.O.M. Marques, A.J. Pentenate, J.
Santos, J. Seville, J. Chem. Eng. Data 48 (2003) 107. Supercrit. Fluids 11 (1997) 91.
[30] A. Berna, A. Chafer, J.B. Monton, J. Chem. Eng. Data 45 (2000) 724. [71] M.J. Cocero, J. Garcia, J. Supercrit. Fluids 20 (2001) 229.
[31] B. Mira, M. Blasco, M. Subirats, A. Berna, J. Supercrit. Fluids 9 (1996) [72] K.M. Kerrola, H.P. Kallio, J. Agric. Food Chem. 42 (1994) 2235.
238. [73] M. Palma, L.T. Taylor, R.M. Varela, S.J. Cutler, H.G. Cutler, J. Agric.
[32] H. Sovova, R.P. Stateva, A.A. Galushko, J. Supercrit. Fluids 20 (2001) Food Chem. 47 (1999) 5044.
113. [74] S.M. Pourmortazavi, F. Sefidkon, S.G. Hosseini, J. Agric. Food Chem.
[33] O.J. Catchpole, K. Proells, Ind. Eng. Chem. Res. 40 (2001) 965. 51 (2003) 5414.
[34] E.M.B.D. Sousa, O. Chiavone-Filho, M.T. Moreno, D.N. Silva, M.O.M. [75] E. Stahl, K.W. Quirin, D. Gerard, Dense Gases for Extraction and Refin-
Marques, M.A.A. Meireles, Braz. J. Chem. Eng. 19 (2002) 229. ing, Springer, Berlin, Heidelberg, 1988.
[35] R.T. Kurnik, R.C. Reid, AIChE J. 27 (1981) 861. [76] Y. Cui, C.Y.W. Ang, J. Agric. Food Chem. 50 (2002) 2755.
[36] S.R.S. Ferreira, Z.L. Nikolov, L.K. Doraiswamy, M.A.A. Meireles, A.J. [77] S.M. Pourmortazavi, P. Baghaee, M. Mirhosseini, Flavour Fragr. J. 5
Petenate, J. Supercrit. Fluids 14 (1999) 235. (2004) 417.
[37] G.B. Lim, G.D. Holder, Y.T. Shah, in: K.P. Johnston, J.M.L. Penninger [78] Y. Yamini, F. Sefidkon, S.M. Pourmortazavi, Flavour Fragr. J. 17 (2002)
(Eds.), Supercritical Fluid Science and Technology, American Chemical 345.
Society, Washington, D.C., 1989, p. 379. [79] S.B. Hawthorne, D.J. Miller, D.D. Walker, D.E. Whittington, B.L. Moore,
[38] S.R.S. Ferreira, M.A.A. Meireles, J. Food Eng. 54 (2002) 263. J. Chromatogr. 541 (1991) 185.
[39] H. Sovova, Chem. Eng. Sci. 49 (1994) 409. [80] I. Papamichail, V. Louli, K.J. Magoulas, J. Supercrit. Fluids 18 (2000)
[40] J.C. Germain, J.M. del Valle, J.C. de La Fuente, Ind. Eng. Chem. Res. 44 213.
(2005) 2879. [81] S. Bowadt, S.B. Hawthorne, J. Chromatogr. A 703 (1995) 549.
[41] V. Camel, Trends Anal. Chem. 16 (1997) 351. [82] V. Louli, G. Folas, E. Voutsas, K. Magoulas, J. Supercrit. Fluids 30 (2004)
[42] M.E.P. McNally, J. AOAC Int. 79 (1996) 380. 163.
[43] S.B. Hawthorne, D.J. Miller, M.D. Burford, J.J. Langenfeld, S. Eckert- [83] E. Sabio, M. Lozano, V.M. de Espinosa, R.L. Mendes, A.P. Pereira, A.F.
Tilotta, P.K. Louie, J. Chromatogr. 642 (1993) 301. Palavra, J.A. Coelho, Ind. Eng. Chem. Res. 42 (2003) 6641.
[44] Â.A. Esmelindro, J.D.S. Girardi, A. Mossi, R.A. Jacques, C. Dariva, J. [84] S.J. Lehotay, J. Chromatogr. A 785 (1997) 289.
Agric. Food Chem. 52 (2004) 1990. [85] V. Camel, Anal. Mag. 26 (1998) 99.
[45] J.W. King, J. Chromatogr. Sci. 27 (1989) 355. [86] G. Leeke, F. Gaspar, R. Santos, Ind. Eng. Chem. Res. 41 (2002) 2033.
[46] M.R. Andersen, J.W. King, S.B. Hawthorne, in: M.L. Lee, K.E. Markides [87] E. Oca, A. Ibanez, G. Murga, S.L.d. Sebastian, J. Tabera, G. Reglero, J.
(Eds.), Chromatography Conferences, Provo, UT, 1990, p. 313. Agric. Food Chem. 47 (1999) 1400.
[47] J.C. Giddings, M.N. Myers, J.W. King, J. Chromatogr. Sci. 7 (1969) 276. [88] K. Li, C.P. Ong, S.F.Y. Li, J. Chromatogr. Sci. 32 (1994) 53.
[48] J.C. Giddings, M.N. Myers, L. McLaren, R.A. Keller, Science 162 (1968) [89] M.K.L. Bicking, J. Chromatogr. Sci. 30 (1992) 358.
67. [90] A. Meyer, W. Kleiböhmer, J. Chromatogr. A 657 (1993) 327.
[49] M.E. McNally, J.R. Wheeler, J. Chromatogr. 447 (1988) 53. [91] A.A. Clifford, M.D. Burford, S.B. Hawthorne, J.J. Langenfeld, D.J.
[50] R.K. Roop, A. Akgerman, B.J. Dexter, T.R. Irvin, J. Supercrit. Fluids 2 Miller, unpublished results.
(1989) 51. [92] A. Meyer, W. Kleibohmer, J. Chromatogr. A 657 (1993) 327.
[51] M. Caude, D. Thiebaut (Eds.), Practical Supercritical Fluid Chromatog- [93] S. Bowadt, F. Peluio, L. Montanarella, B. Larsen, S. Kaoila, J. Trace
raphy and Extraction, Harwood, Amsterdam, 1999. Microprobe T. 11 (1993) 117.
[52] T. Baysal, D.A.J. Starmens, J. Supercrit. Fluids 14 (1999) 225. [94] L.J. Mulcahey, L.T. Taylor, Anal. Chem. 64 (1992) 2352.
[53] M. Careri, L. Furlattini, A. Mangia, M. Musci, E. Anklam, A. Thepbald, [95] L.J. Mulcahey, J.L. Hedrick, L.T. Taylor, Anal. Chem. 63 (1991) 2225.
C.V. Holst, J. Chromatogr. A 912 (2001) 61. [96] P.R. Eckard, L.T. Taylor, J. High Resolut. Chromatogr. 19 (1996) 117.
[54] E. Reverchon, G.D. Porta, D. Gorgoglione, Flavour Fragr. J. 12 (1997) [97] W.N. Moore, L.T. Taylor, Anal. Chem. 67 (1995) 2030.
37. [98] P. Dugo, L. Mondello, K.D. Bartle, A.A. Clifford, D.G.P.A. Breen, G.
[55] M. Hamburger, D. Baumann, S. Adler, Phytochem. Anal. 15 (2004) 46. Dugo, Flavour Fragr. J. 10 (2006) 51.
[56] A. Brachet, S. Cherkaoui, P. Christen, J.Y. Gauvrit, P. Lanteri, J.L. [99] M.E. Araujo, N.T. Machado, L.F. Franca, M.A.A. Meireles, Braz. J.
Veuthey, Analusis 27 (1999) 772. Chem. Eng. 17 (2000) 297.
[57] M.S. Gmez-Prieto, M.M. Caja, M. Herraiz, G.S. Mariaa, J. Agric. Food [100] G.P. Blanch, E. Ibanez, M. Herraiz, G. Reglero, Anal. Chem. 66 (1994)
Chem. 51 (2003) 3. 888.
24 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

[101] M.L. Hopper, J.W. King, J. Assoc. Off. Anal. Chem. 74 (1991) 661. [126] E. Ibanez, A. Kubatova, F.J. Senorans, S. Cavero, G. Reglero, S.B.
[102] L.A.V. Sarmento, C.B. Spricigo, J.C.C. Petrus, L.H.C. Carlson, R.A.F. Hawthorne, J. Agric. Food Chem. 51 (2003) 375.
Machadoc, J. Membr. Sci. 237 (2004) 71. [127] F.J. Senorans, A. Rodriguez, R. Cavero, S. Cifuentes, A.E. Ibanez, G.
[103] C.B. Spricigo, A. Bolzan, R.A.F. Machado, L.H.C. Carlson, J.C.C. Petrus, Reglero, J. Agric. Food Chem. 49 (2001) 6039.
J. Membr. Sci. 54 (2001) 1. [128] C. Simoa, E. Ibanez, F.J. Senorans, C. Barbas, G. Reglero, A. Cifuentes,
[104] M.R. Anderson, J.T. Swanson, N.L. Porter, B.E. Richter, J. Chromatogr. J. Agric. Food Chem. 50 (2002) 6648.
Sci. 27 (1989) 371. [129] B. Yepez, M. Espinosa, S. López, G. Bolaños, Fluid Phase Equilib.
[105] K. Sugiyama, M. Saito, T. Hondo, M. Senda, J. Chromatogr. 332 (1985) 194–197 (2002) 879.
107. [130] T. Fang, M. Goto, M. Sasaki, T. Hirose, J. Agric. Food Chem. 52 (2004)
[106] S.B. Hawthorne, M.S. Krieger, D.J. Miller, Anal. Chem. 60 (1988) 472. 5162.
[107] S.B. Hawthorne, D.J. Miller, M.S. Krieger, J. Chromatogr. Sci. 27 (1989) [131] S. Sonsuzer, S. Sahin, L. Yilmaz, J. Supercrit. Fluids 30 (2004) 189.
347. [132] L.H.C. Carlson, R.A.F. Machado, C.B. Spricigo, L.K. Pereira, A. Bolzan,
[108] J.L. Levy, M. Ashraf-Khorassani, Hyphenated techniques in supercriti- J. Supercrit. Fluids 21 (2001) 33.
cal fluid chromatography and extraction, in: K. Jinno (Ed.), Journal of [133] M.R.A. Regina, E.B. Caramão, G.J. Dos Santos, C. Dariva, Z.V. Oliveira,
Chromatography Library, vol. 53, Elsevier, Amsterdam, 1992, p. 197. J. Agric. Food Chem. 51 (2003) 453.
[109] M.L. Riekkola, P. Manninen, K. Hartonen, Hyphenated techniques in [134] A. Ranalli, S. Contento, L. Lucera, G. Pavone, G. Di Giacomo, L. Aloisio,
supercritical fluid chromatography and extraction, in: K. Jinno (Ed.), Jour- L. Di Gregorio, A. Mucci, I. Kourtikakis, J. Agric. Food Chem. 52 (2004)
nal of Chromatography Library, vol. 53, Elsevier, Amsterdam, 1992, p. 4795.
275. [135] M.R.A. Rodrigues, E.B. Caramao, J.G.D. Santos, C. Dariva, J.V. Oliveira,
[110] M. Ashraf-Khorassani, M.L. Kumar, D.J. Koebler, G.P. Williams, J. Chro- J. Agric. Food Chem. 51 (2003) 453.
matogr. Sci. 28 (1990) 599. [136] M.C.D. Diaz-Maroto, I.J.D.M. Hidalgo, E.S. Palomo, M.S.P. Coello, J.
[111] M.R. Andersen, J.T. Swanson, N.L. Porter, B.E. Richter, J. Chromatogr. Agric. Food Chem. 53 (2005) 5385.
Sci. 27 (1989) 317. [137] F.J. Eller, S.L. Taylor, J. Agric. Food Chem. 52 (2004) 2335.
[112] K. Hartonen, M. Jussila, P. Manninen, M.L. Riekkola, J. Microcol. Sep. [138] E. Vági, B. Simándi, H.G. Daood, A. Deák, J. Sawinsky, J. Agric. Food
4 (1992) 3. Chem. 50 (2002) 2297.
[113] T. Greibrokk, J. Chromatogr. A 703 (1995) 523. [139] J.L. Hedrick, L.J. Mulcahey, L.T. Taylor, Mikrochim. Acta 108 (1992)
[114] K. Sato, S.S. Sasaki, Y. Goda, T. Yamada, O. Nunomura, K. Ishikawa, T. 115.
Maitani, J. Agric. Food Chem. 47 (1999) 4665. [140] M.J. Piggott, E.L. Ghisalbeti, R.D. Trengove, Flavour Fragr. J. 12 (1997)
[115] O.I. Aruoma, J.P.E. Spencer, D. Warren, P. Jenner, J. Butler, B. Halliwell, 43.
Food Chem. 60 (1995) 149. [141] E.E. Stashenko, M.A. Puertas, M.Y. Combariza, J. Chromatogr. A 752
[116] H. Kikuzaki, N. Nakatani, J. Food Sci. 58 (1993) 1407. (1996) 223.
[117] K. Teiantaphyllou, G. Blekas, D. Boskou, Int. J. Food Sci. Nutr. 52 (2001) [142] J.H.Y. Vilegas, F.M. Lancas, Flavour Fragr. J. 9 (1994) 39.
313. [143] M. Eikani, I. Goodarznia, M. Mirza, Flavour Fragr. J. 14 (1999) 29.
[118] M. Bergman, L. Varshavsky, H.E. Gottlieb, S. Grossman, Phytochemistry [144] M. Poiana, V. Sicari, B. Mincione, Flavour Fragr. J. 13 (1998) 125.
58 (2001) 143. [145] E. Cassel, C.D. Frizzo, R. Vanderlinde, L.A. Serafini, D. Lorenzo, E.
[119] Z. Djarmati, R.M. Jankov, E. Schwirtlich, B. Djulinac, A. Djordjevic, J. Dellacassa, Ind. Eng. Chem. Res. 39 (2000) 4803.
Am. Oil. Chem. Soc. 6 (1991) 37. [146] A. Caredda, B. Marongiu, S. Prcedda, C. Soro, J. Agric. Food Chem. 50
[120] U. Nguyen, G. Evans, G. Frakman, in: S.S.H. Rizvi (Ed.), Supercritical (2002) 1492.
Fluid Processing of Food and Biomaterials, Blackie, Glasgow, 1994, p. [147] S.M. Pourmortazavi, M. Ghadiri, S.S. Hajimisadeghi, J. Food Compos.
103. Anal. 18 (2005) 439.
[121] F.R.J. Snorans, E. Ibanez, S. Cavero, J. Tabera, G. Reglero, J. Chromatogr. [148] H. Ebrahimzadeh, Y. Yamini, F. Sefidkon, M. Chaloosi, S.M. Pourmor-
A 870 (2000) 491. tazavi, Food Chem. 83 (2003) 357.
[122] E. Dauksas, P.R. Venskutonis, V. Povilaityte, B. Sivik, Nahrung 45 (2001) [149] N. Aghel, Y. Yamini, A. Hadjiakhoondi, S.M. Pourmortazavi, Talanta 62
338. (2004) 407.
[123] Q. Hu, J. Xu, S. Chen, F. Yang, J. Agric. Food Chem. 52 (2004) 943. [150] E.E. Stashenko, B.E. Jaramillo, J.R. Martinez, J. Chromatogr. A 1025
[124] J.L. Mau, P.T. Ko, C.C. Chyau, Food Res. Int. 36 (2003) 97. (2004) 93.
[125] K.C. Zancan, M.O.M. Marques, A.J. Pentenate, M.A.A. Meireles, J. [151] E.E. Stashenko, B.E. Jaramillo, J.R. Martinez, J. Chromatogr. A 1025
Supercrit. Fluids 24 (2002) 57. (2004) 105.