Super Critical Fluid Extraction in Plant Essential and Volatile Oil Analysis

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

Review

Supercritical fluid extraction in plant essential and volatile oil analysis

Seied Mahdi Pourmortazavi ∗, Seiedeh Somayyeh HajimirsadeghiFaculty 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 2007Available online 17 June 2007

bstract

The use of supercritical fluids, especially carbon dioxide, in the extraction of plant volatile components has increased during two last decadesue to the expected advantages of the supercritical extraction process. Supercritical fluid extraction (SFE) is a rapid, selective and convenientethod for sample preparation prior to the analysis of compounds in the volatile product of plant matrices. Also, SFE is a simple, inexpensive, fast,

ffective and virtually solvent-free sample pretreatment technique. This review provides a detailed and updated discussion of the developments,odes and applications of SFE in the isolation of essential oils from plant matrices. SFE is usually performed with pure or modified carbon

ioxide, which facilitates off-line collection of extracts and on-line coupling with other analytical methods such as gas, liquid and supercritical
uid chromatography. In this review, we showed that a number of factors influence extraction yields, these being solubility of the solute in the fluid,iffusion through the matrix and collection process. Finally, SFE has been compared with conventional extraction methods in terms of selectivity,apidity, cleanliness and possibility of manipulating the composition of the extract.
2007 Elsevier B.V. All rights reserved.

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

ontents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32. Introduction to supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33. Solubility and mass-transfer rate of plant oils in supercritical fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44. Effect of matrix on supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75. Effect of extraction parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

5.1. Effect of pressure and temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75.2. Effect of modifiers on supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105.3. Effect of extraction time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.4. Effect of flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 5.5. Sample particle size and packing density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135.6. Effect of water in supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145.7. Drying effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Abbreviations: AHF, adhyperforin; AIDS, acquired immunodeficiency syndrome; AZA-A, azadirachtin A; BHT, butylated hydroxytoluene; CER, constantextraction rate; CWO, cedarwood oil; YCER, concentration of the oil in the supercritical phase; CC-SFE, countercurrent supercritical fluid extraction; DAD, diode-arraydetection; 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, microwaveassisted hydrodistillation; ODS, octadecylsilica; OM, oxygenated monoterpene; OS, oxygenated sesquiterpene; Q, solvent flow rate; RPLC, reversed-phase liquid
hromatography; Re, Reynolds number; SDE, simultaneous distillation–extraction; SFC, supercritical fluid chromatography; SFE, supercritical fluid extraction;JW, 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*,olubility; 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: [email protected] (S.M. Pourmortazavi).

021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.06.021

mailto:[email protected]

dx.doi.org/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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.1. Solvent collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.2. Solid-phase collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.3. Collection in empty vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166.4. Novel collection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166.5. On-line coupling of supercritical fluid extraction with chromatographic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

7. Extraction of oxygenated compounds from plant materials by supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178. Extraction of terpenes and sesquiterpenes by supercritical fluid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189. Comparison of supercritical fluid extraction to conventional methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2010. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Essential oils represent a small fraction of a plant’s compo-ition but confer the characteristic for which aromatic plantsre used in the pharmaceutical, food and fragrance industries.ssential oils have a complex composition, containing from a

ew dozen to several hundred constituents, especially hydrocar-ons (terpenes and sesquiterpenes) and oxygenated compoundsalcohols, aldehydes, ketones, acids, phenols, oxides, lactones,cetalse, ethers and esters). Both hydrocarbons and oxygenatedompounds are responsible for the characteristic odors andavors. The proportion of individual compounds in the oil com-osition is different from trace levels to over 90% (δ-limonenen orange oil). The aroma’s oil is the result of the combination ofhe aromas of all components. Trace components are important,ince they give the oil a characteristic and natural odor. Thus,t is important that the natural proportion of the components is

aintained during extraction of the essential oils from plants byny procedure [1].

Steam distillation has traditionally been applied for essentialils recovery from plant materials. One of the disadvantagesf the hydrodistillation methods is that essential oils undergohemical alteration and the heat-sensitive compounds can easilye destroyed. Therefore, the quality of the essential oil extractss extremely impaired [2].

The extraction of essential oil components using solvents atigh pressure, or supercritical fluids, has received much atten-ion in the past several years, especially in food, pharmaceuticalnd cosmetic industries, because it presents an alternative toonventional processes such as organic solvent extraction andteam distillation [3].

The increasing use of vegetable extracts by the food, cos-etic, and pharmaceutical industries can make the extraction

f essential oils using supercritical carbon dioxide an attractiveechnology compared to conventional processes with respect tohe product quality [4–6]. The knowledge of the mass-transfer

echanism, the kinetics parameters and the thermodynamicsestrictions of the extraction conducted in a bed of vegetableaterial can be used to economically evaluate the extrac-

ion process. This requires information on the thermodynamic
estrictions of the system vegetable material/CO2. On the otherand, the understanding of the various process variables andow they can be connected to a theoretical model to describehe extraction kinetics are also desirable.
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. Introduction to supercritical fluid extraction

Supercritical fluids have been used as solvents for a wideariety of applications such as essential oil extraction [3], metalation extraction [7,8], polymer synthesis [9] and particle nucle-tion [10,11]. In practice, more than 90% of all analyticalupercritical fluid extraction (SFE) is performed with carbonioxide (CO2) for several practical reasons. Apart from havingelatively low critical pressure (74 bar) and temperature (32 ◦C),O2 is relatively non-toxic, non-flammable, available in highurity at relatively low cost, and is easily removed from thextract. In the supercritical state, CO2 has a polarity compara-le to liquid pentane and is, therefore, best suited for lipophilicompounds. The main drawback of CO2 is its lack of polarityor the extraction of polar analytes [12].

In the 1990s, some reports were published about the choice of2O as extraction fluid for analytical SFE [13,14]. This fluid was

onsidered better suited for polar compounds because of its per-anent dipole moment. One of the applications where N2O has

hown significant improvements when compared to CO2 is forxample the extraction of polychlorinated dibenzodioxins fromy ash [13]. Unfortunately, this fluid has been shown to causeiolent explosions when used for samples having high organicontent and should, therefore, be used only when absolutelyecessary [13,14].

Other more exotic supercritical fluids which have been usedor environmental SFE are SF6 and freons. SF6 is a non-polarolecule (although easy polarizable) and as a supercritical fluid,

t has been shown to selectively extract aliphatic hydrocarbonsp to around C-24 from a mixture containing both aliphatic andromatic hydrocarbons. Freons, especially CHClF2 (Freon-22),as on several occasions been shown to increase the extractionfficiency compared to conducting extractions with CO2 [15].

Although supercritical H2O has often been used for theestruction of hazardous organics, the high temperature andressure needed (T > 374 ◦C and P > 221 bar) together with theorrosive nature of H2O at these conditions, has limited the pos-ible practical applications in plant oil analysis [16]. H2O atubcritical conditions has, however, been shown to be an effec-ive fluid for the extraction of several classes of essential oil. In
000, Gamiz-Garcia et al. [17] tested a static–dynamic subcrit-cal water extraction for the isolation of fennel oil. Their resultshowed that, subcritical water extraction is an efficient methodn terms of quantitative composition of the extract.

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ledttotclow(samitraAiit(opbtaCbetween the logarithm of solubility and the logarithm ofsolvent density was obtained. Average deviation between theirmeasured and calculated solubility did not exceed 14%.

Table 1Results of experimental study of solubility for seed oils (C∗

exp) in compressedCO2 [29]

P (bar) T (◦C) ρ (kg/m3) C∗exp (kg/m3)

Echium Borage Lunaria

60 10 883.8 3.17 1.90 0.4925 190.5 0.1140 149.2 0.0055 129.6 0.00

100 10 921.9 4.78 2.80 0.6425 819.5 3.01 1.75 0.5240 629.3 0.81 0.26 0.0955 327.1 0.02 0.13 0.00

200 10 980.8 7.72 5.07 1.1025 915.2 8.61 5.77 1.7040 840.8 7.31 4.39 1.2155 755.5 5.27 2.05 0.65

S.M. Pourmortazavi, S.S. Hajimirsad

Ethane, propane, ethylene, dimethyl ether, etc. have also beenecommended as solvents under sub- and supercritical condi-ions for extraction [18,19].

Catchpole et al. [20] extracted ginger, black pepper, andhilli powder using near-critical carbon dioxide, propane, andimethyl ether on a laboratory scale to determine the overallield and extraction efficiency for selected pungent components.hey also determined the volatile contents of ginger and blackepper extracts. Extraction of all spice types was carried outith acetone to compare overall yields. Their results showed

hat subcritical dimethyl ether was as effective at extracting theungent principles from the spices as supercritical carbon diox-de, although a substantial amount of water was also extracted.ubcritical propane was the least effective solvent. All sol-ents quantitatively extracted the gingerols from ginger. Theields of capsaicins obtained by supercritical CO2 and dimethylther were similar and approximately double that extracted byropane. The yield of piperines obtained by propane extractionf black pepper was low at ∼10% of that achieved with dimethylther and CO2, but improved with increasing extraction tempera-ure. Mohamed et al. [21] explored supercritical extraction usingthane and CO2 for the recovery of the methylxanthines caf-eine and theobromine and cocoa butter from cocoa beans usinghigh-pressure apparatus. Their finding showed that the extrac-

ion yields of cocoa butter obtained with ethane are much higherhan those obtained with CO2 because of the higher solubility ofhis fat in ethane. A pronounced effect of pressure on the extrac-ion of methylxanthines and cocoa butter was observed for botholvents. The methylxanthines in cocoa beans were slightly moreoluble in ethane than in CO2 probably because of co-solvencyffects of cocoa butter, which was extracted more easily usingupercritical ethane. Despite the higher cost of ethane, its criticalressure is lower than that of CO2, and the higher butter solu-ility could render ethane a viable solvent through lower energyosts.

. Solubility and mass-transfer rate of plant oils inupercritical fluid

There are many variables to be considered in SFE andethod development can seem a daunting task. The choice

f extraction conditions has largely been determined empir-cally, which is time consuming. One initial area that muste assessed is the solubility of the analyte to be extractedn the supercritical extracting fluid. This can be investigatedy spiking an inert medium, usually celite or sand, with thenalyte of interest. In addition to providing an indication ofhe solubility of the analyte in the supercritical fluid, addi-ional information is also obtained relating to the efficiencyf collection of the analyte after depressurization. The timeonsuming nature of even these simple experiments has ledeveral groups of workers to propose techniques for modelingnalyte solubility [22]. Fundamentals, theory and equations of
tates for estimating the solubility of various compounds cane found in the literature [23–26]. In this review we will focusn the investigations involving the solubility of plant volatileomponents.
J. Chromatogr. A 1163 (2007) 2–24

Jaubert et al. [27] chose the ternary system CO2–imonene–citral as a model system in order to study thextraction of terpenes from lemon oil using supercritical carbonioxide. Extractions were performed at several pressures andemperatures to evaluate the influence of these parameters onhe separation efficiency. They used a theoretical model, basedn a modified Peng–Robinson equation of state to understandhe thermodynamic and mass-transfer aspects of the extractionolumn. The critical parameters and the acentric factors ofimonene and citral were estimated by group contribution meth-ds. They applied the method developed by Abdoul et al. [28],hich allows the calculation of binary interaction parameters

kij) to terpenic compounds. The extraction experiments wereimulated using this model, and the extraction profiles wereccurately reproduced. On the other hand, Gaspar et al. [29]easured the solubility of borage, echium, and lunaria oils

n compressed CO2 using the dynamic method. Pressure andemperature were varied from 60 to 300 bar and 10 to 55 ◦C,espectively. Their measured solubilities of echium, borage,nd lunaria oils in compressed CO2 are presented in Table 1.s shown in this table, at a given temperature, the solubility

ncreases with the increase of pressure, as a direct result of thencreased solvent density. They showed that the effect of extrac-ion temperature is also similar for all oils. At low pressures60 and 100 bar), an increase of temperature leads to a decreasef solubility, and the opposite is observed at the highest testedressure (300 bar). At 200 bar, there is an improvement in solu-ility when increasing the temperature from 10 to 25 ◦C. Also,hey compared the solubilities to those of other vegetable oilsnd were correlated using the density-based model proposed byhrastil [29]. They predicted by the model, a linear relationship

300 10 1020.2 10.20 6.93 1.4025 966.8 13.44 9.87 2.8840 910.3 14.56 9.90 2.8455 850.6 16.35 9.96 3.23

S.M. Pourmortazavi, S.S. Hajimirsadeghi /

Fig. 1. Comparison of solubility values for the linalool in supercritical CO2

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btained by different studies (values obtained by Berna et al.: (�) at 318.15 K,�) at 328.15 K. Values obtained from Iwai work: (�) at 313.15 K, (©) at23.15 K, (�) at 333.15 K). (From ref. [30] with permission.)

Berna et al. [30] measured the solubilities of essential oil com-onents of orange in supercritical carbon dioxide. In their study,he solubilities of pure limonene and linalool in compressed car-on dioxide were measured using a flow apparatus at 318.2 and28.2 K and pressures ranging from 69 to 111 bar. The obtainedalues of these solubilities are shown in Figs. 1 and 2. From thealues that were obtained, Berna et al. showed that the solubil-ty of limonene in supercritical carbon dioxide is higher than theolubility of linalool at the same conditions but that at higherressures they approach each other. Also, both systems showsudden increase in the solubility at pressures up to approxi-ately 80 bar. For the linalool–CO2 system, it can be noticed

hat when the temperature rises at pressures under 80 bar, theolubility increases but at pressures over 80 bar, the solubilityecreases.

The comparison between their results and previous studies for
he system limonene + CO2 showed that the solubility increaseshen the temperature rises at pressures under 80 bar but at pres-
ures over 80 bar, it decreases. Also, it can be observed that this

ig. 2. Comparison of solubility values for the limonene in supercritical carbonioxide obtained at different studies (values from Berna et al.: (�) at 318.15 K.alues from Iwai et al.: (�) at 313.15 K, (©) at 323.15 K, (�) at 333.15 K.alues from Matos et al.: (�) at 323.15 K. Values from Di Giacomo et al.: (�)t 323.15 K). (From ref. [30] with permission.)

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J. Chromatogr. A 1163 (2007) 2–24 5

ehavior is similar to that of the system linalool + CO2. Thisehavior shows that the best conditions for supercritical extrac-ions in both systems will be at pressures >90–100 bar and aemperature near the critical temperature of carbon dioxide inrder to obtain the maximum amount of product. Their resultsere in agreement with those of kinetic studies which fixed

he best pressure for the extraction or deterpenation of citrusil peel at about 200 bar [31]. Also, they successfully mod-led the solubilities using equations of state (Peng–Robinson,oave–Redlich–Kwong, 3P1T, Dohrn and Prausnitz non-polar)nd a semi-empirical equation (Chrastil model). They obtainedeneralized parameters for the Peng–Robinson equation of stateor each system. These parameters were independent of temper-ture, and they reproduce successfully all data available in theiterature. The results showed that the solubility of limonene inupercritical carbon dioxide was higher than the solubility ofinalool.

On the other hand, Sovova et al. [32] investigated fatty oilnfluence on the solubility of limonene in CO2 under pressures–12 MPa at 313.2 K. They measured solubility in CO2 usinghe dynamic method both for limonene and for the mixture ofimonene and blackcurrant seed oil. The concentration of fattyil in the vapor phase was found to be negligible in comparisonith the concentration of limonene. Limonene was distributedetween the liquid phase, rich in fatty oil, and the vapor phase,ich in CO2, and its equilibrium concentration in the latterecreases with the diminishing limonene-to-oil ratio in the satu-ator. Also, there was a steep increase of the limonene partitionoefficient with pressure between 8 and 10 MPa, near the criti-al pressure of the binary mixture of limonene and CO2. Theirpplied thermodynamic model was the Soave–Redlich–Kwongubic equation of state with either the one fluid linear van deraals mixing rule or with the MHV2 mixing rule. Extraction

ressures should be approximately 20% larger than the criti-al pressure of the essential oil + CO2 binary mixture and ratheright packing of the ground seed in the bed should be applied.

Catchpole and Proells [33] measured the solubilities of lipidsypically found in marine oils and seed oil refining byproducts inubcritical R134a to determine whether R134a could be a viable,ow-pressure alternative to supercritical CO2. The measured sol-bilities of squalene, oleic acid, soya oil, and deep sea sharkiver oil in subcritical R134a in a countercurrent packed columnpparatus over the temperature range 303–353 K at 60 bar. Sol-bility measurements were also made over the pressure range0–200 bar at 343 K for shark liver oil and oleic acid. Theiresults indicated that, the solubilities of all solutes in R134are low, ranging from 0.8 to 10 g solute/kg solvent. The sol-bilities increased almost linearly with increasing temperaturet fixed pressure and increased logarithmically with increasingressure at fixed temperature. The recorded strong temperatureependence of the solubility allows for two-stage fractionationf extracts. They used a linear solvation energy relationshippproach to correlate the enhancement factors of the solutes
s a function of the solvent polarity/dipolarizability factor andbtained linear relationships. However, the dependence of thenhancement factor on other solute–solvent parameters couldot be determined.

6 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

Table 2Spline 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 1.471.08 93 2.05 18.9 1.03 1.731.50 106 2.922.15 75 3.053.16 60 3.77

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tTcvdoofstss2i242%. Therefore, in the first case, the dominant effect was solutevapor pressure, while in the second it was density. The effects ofboth temperature and pressure on solute solubility can be shownin Fig. 4. The overall saturation curves at 66.7 bar and various

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ig. 3. The effect of solvent flow rate on the mass ratio of solute in the fluidhase at the bed outlet at T = 288.15 K and P = 66.7 bar. (From ref. [34] withermission.)

Sousa et al. [34] measured the solubility of the essential oilresent in L. sidoides Cham. in liquid CO2 by the dynamicethod, in which the solvent is saturated by the solute as itows through the bed of solids at a predetermined constant flowate. Table 2 reports their results and Fig. 3 shows the effectf solute in the supercritical phase at the measuring cell out-et. Their finding showed that for experiments accomplished atxtremely low solvent flow rates, the effects of axial dispersionere important, resulting in a smaller value of YCER (the con-

entration of the oil in the supercritical phase at the outlet of theolumn). At high solvent flow rates, smaller values of YCER werebtained due to shorter residence times. As shown in Fig. 3, sol-ent flow rates in the vicinity of 1.5 × 10−5 kg/s is appropriate toe used for measuring solubility, using the dynamic method forhe L. sidoides + CO2 system. The variation in the thermophys-cal properties of the solute and the solvent was relatively small
ue to the narrow interval of both temperature and pressure inhis study. Based on this, the solubility for the L. sidoides + CO2ystem was measured at solvent flow rates in the vicinity of.5 × 10−5 kg/s. Table 3 shows the measured solubility. In this
F(

able 3olubility measured by the dynamic method for the pseudo-ternary system [34]

(K) P (bar) Q (×10−5 kg/s) Y* (

83.15 66.7 1.60 13.88.15 66.7 1.50 19.88.15 78.5 1.60 17.93.15 66.7 1.53 22.93.15 78.5 1.60 20.95.65 66.7 1.53 19.98.15 66.7 1.57 13.

19.5 1.64 3.1118.3 1.45 2.2411.9 1.44 1.96

able, the amount of solute collected up to the end of the CEReriod (YieldCER) along with the total amount of solute collectedor the entire measuring time (YieldTOTAL) are shown.

The effect of temperature on solubility is complex, due tohe combination of two variables, density and vapor pressure.he vapor pressure of the solute increases with temperature,ausing an elevation in solubility. However, decreasing of sol-ent density may cause decreases of the solute solubility. Theominant effect will depend on the magnitude of each effectn the others for each system. Higher solubility values werebtained at 66.7 bar in the range of 288.15–293.15 K. There-ore, the increase in the solubility for this range of temperatureshould mainly be due to the increase in the vapor pressure ofhe solute. For essential oils the vapor pressure is low, however,mall changes in temperature can cause significant changes inolubility. For example, a 5◦ increase in the temperature (from88.015 to 293.015 K) at 66.7 bar caused an increase in solubil-ty of 14%. However, the same increase in temperature, but from93.15 to 298.15 K, resulted in a reduction in solute solubility of

ig. 4. The influence of temperature on the overall saturation curves at 66.7 bar.From ref. [34] with permission.)

×10−3 kg/kg) YieldCER (%) YieldTOTAL (%)

4 1.34 2.195 1.64 3.118 1.63 3.037 1.76 3.211 1.45 2.750 1.55 3.292 1.16 2.82

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emperatures (Fig. 4) reflect, as expected, the behavior of solu-ility, since the slope of each overall saturation curve is directlyroportional to solubility. Also, investigations on the effect ofressure on solubility for temperatures of 288.15 and 293.15 Khowed that, solubility decreases by enhancing pressure from6.7 to 78.5 bar. The behavior is consistent with the literature onolid–fluid equilibria [35].

From the industrial point of view, optimization of the pro-esses that employ supercritical fluids not only requires thenowledge of thermodynamic parameters (solubility and selec-ivity) but also requires consideration of mass-transfer rate36,37].

Ferreira and Meireles [38] modeled the mass-transfer mech-nism for extraction of black pepper essential oil usingupercritical carbon dioxide by the extended Lack’s plug flowodel developed by Sovova (Sovova’s model) [39]. Their pro-

edure was used to evaluate the parameters of the Sovova’sodel from experimental data—it quantitatively described the

xperimental data for the majority of conditions analyzed. Theirnding indicated that temperature and pressure levels used in

heir work did not change mass-transfer coefficient values ands a function of the bed characteristic and initial amounts ofolute. Germain et al. [40] showed that, the external mass-ransfer coefficients (kf) during supercritical fluid extraction ofigh-solubility solutes, under solvent up-flow conditions andow superficial velocities, can be small because of the nega-ive influence of natural convection phenomena. They used ahrinking-core model for mass transfer to estimate best-fit val-es of kf for data on SFE of lipids from pre-pressed rape seeds.alues of kf at a high Reynolds number (Re = 14.1) were sim-

lar when using solvent up-flow or down-flow, but kf at lowere (1.57) was 3.6 times smaller when using solvent up-flow

han that predicted from a literature correlation for down-flowonditions. These kf values are consistent with values estimatedy fitting literature data, or gathered from various sources underimilar, non-adequate conditions (solvent up-flow under low Re)or the extraction of both fatty and essential oils.

. Effect of matrix on supercritical fluid extraction

Different factors such as the particle size, shape, surface area,orosity, moisture, level of extractable solutes and the nature ofhe matrix will affect the supercritical fluid extraction results.imilarly, the interactions between solutes and active sites of

he matrix can necessitate strict extraction conditions. The suc-ess of a SFE method not only depends on the extraction steptself (i.e. nature of the supercritical fluid and choice of extractionarameters) but also on the matrix considered (a pretreatmentay be recommended) as well as on the analyte trapping system

41–43]. Consequently, SFE must be regarded as a four-stagerocess: (1) desorption of the compound from the matrix with2) subsequent diffusion into the matrix, (3) solubilization ofhe analyte by the supercritical fluid, and (4) sweeping out of
he extraction cell by the fluid. Each part of the process has toe carefully optimized in order to obtain quantitative and repro-ucible recoveries. Most of the time, the first step remains mostifficult to control, as solute–matrix interactions are difficult to
f3oo

J. Chromatogr. A 1163 (2007) 2–24 7

redict. The physical structure of the matrix is of critical impor-ance, as the extraction efficiency is related to the ability of theupercritical fluid to diffuse within the matrix. For that reason,he extraction conditions of the same group of oils may differrom one matrix to another.

As a general rule, decreasing the particle size of solid matriceseads to a higher surface area, making extraction more efficient.et, excessive grinding may hinder the extraction due to re-dsorption of the analytes onto matrix surfaces (this could bevoided by increasing the flow rate) and pressure drop insidehe extraction chamber. On the other hand, environmental agents

ay affect the composition and essential oil contents of matrix.smelindro et al. [44] assess the influence of light intensity

plants exposed to direct sun and in controlled lighting condi-ions), and the age of leaves (6–24 months) on the characteristicsf the extracts of mate tea leaves obtained from carbon diox-de at high pressures. Samples of mate were collected in anxperiment conducted under agronomic control at Industria eomercio de Erva-Mate Barao, Brazil. Quantitative analysis ofaffeine, theobromine, phytol, vitamin E, squalene, and stigmas-erol was performed, and the results showed that field variablesxert a strong influence on the liquid yield and on the chemicalistribution of the extracts.

. Effect of extraction parameters

.1. Effect of pressure and temperature

Four parameters are extremely helpful in the understandingf solute behavior in supercritical media, and thus in executinguccessful analytical supercritical fluid extractions [45,46]: (i)he miscibility or threshold pressure [47,48], which correspondso the pressure at which the solute partitions into the supercriticaluid, (ii) the pressure at which the solute reaches its maximumolubility, (iii) the fractionation pressure range, which is theressure region between the miscibility and solubility maximumressures (in this interval it is possible to extract selectivity oneolute by choosing the correct pressure) and (iv) a knowledgef the physical properties of the solute, particularly its meltingoint (in fact most solutes dissolve better when they are in theiriquid state, i.e. above their melting point).

To illustrate the difference between the threshold pressure andhe solubility maximum pressure, the solubility–pressure curvef naphthalene is given in Fig. 5 [46]. This solute is slightly solu-le in CO2 at 75 bar (threshold pressure) as the pressure increaseshe solubility rises, especially around 90 bar, up to its maximumalue. The fluid pressure is the main parameter that influences thextraction efficiency. An elevation of this pressure at a given tem-erature results in an increase in the fluid density (Fig. 6), whicheans an enhanced solubility of the solutes. Consequently, the

igher the extraction pressure, the smaller is the volume of fluidecessary for a given extraction. For example, one needs to dou-le the volume of CO2 in order to extract 70% of diuron herbicide
rom a contaminated soil when working at 110 bar instead of38 bar (Fig. 7) [49]. However, high pressure is not always rec-mmended for complex matrices owing to the higher solubilityf solutes when the pressure is elevated, resulting in complex

8 S.M. Pourmortazavi, S.S. Hajimirsadeghi / J. Chromatogr. A 1163 (2007) 2–24

Fi

ebmaptttdaoothu

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Fig. 7. Variation of the extraction yield of diuron from a polluted soil vs. the

volume of CO2 percolated at different pressures (( ) 110, (�) 235, (�) 338 bar).

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ig. 5. Variation of the solubility of naphthalene with the pressure of supercrit-cal CO2 at 45 ◦C. (From ref. [46] with permission.)

xtracts and difficult analysis. On the other hand, it must beorne in mind that the presence of co-extracted solutes can dra-atically change the solubility level of the solute of interest. Atconstant pressure, the density of CO2 decreases when the tem-erature is increased. This effect becomes more pronounced ashe fluid compressibility increases, as shown in Fig. 6. Tempera-ure also affects the volatility of the solute. Hence, the effect of aemperature elevation is difficult to predict because of its depen-ence on the nature of the sample. For a non-volatile solute,higher temperature would result in lower extraction recoverywing to a decrease in solubility thus the distribution coefficient
f phenol between water and supercritical CO2 decreases whenhe fluid temperature rises from 25 to 30 ◦C [50]. On the otherand, for a volatile solute, there is a competition between its sol-bility in CO2 (which decreases as the temperature increases)
ig. 6. Pressure–density diagram for carbon dioxide. The shaded area cor-esponds to the experimental domain of supercritical phase extraction andhromatography. (From ref. [46] with permission.)

ahoeet

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xtraction conditions: extractant, CO2–CH3CN (90:10, v/v), extraction cell,5 cm × 4.6 mm I.D.; temperature, 100 ◦C; flow rate of liquid CO2, 16.5 ml/min.From ref. [49] with permission.)

nd its volatility (which rises with increasing temperature). Forxample, when the temperature increases from 80 to 120 ◦C, thextraction recovery of diuron from soil with methanol-modifiedO2 is enhanced from 75 to 99% [51].

Baysal and Starmens [52] studies supercritical carbon diox-de of carvone and limonene from caraway seed. Their resultshowed that pressure and temperature have main effects onhe extraction efficiency. They showed that at moderate tem-eratures just above the critical temperature of CO2 (31.1 ◦C),xtraction yield for limonene is considerable at pressures justbove the critical pressure of CO2 (73.8 bar). Below this value,ardly any limonene is obtained from the seed matrix, regardlessf the temperature applied during the extraction procedure. Atlevated temperatures, a pressure of up to 125 bar is required toxtract limonene in only small quantities. Further increasing ofhe pressure yield resembles those found for limonene.

Careri et al. [53] used a chemometric approach to inves-igate the effects of different parameters on the supercriticalxtraction of carotenoids from spirulina Pacifica algae. Theiresults showed that the temperature of the supercritical fluidid not influence extraction efficiency. However, the pressuref the supercritical fluid plays an important role in the SFEf carotenoids from Spirulina pacifica algae. Reverchon et al.54] extracted volatile oil from rose concrete, using differentressures and temperatures of supercritical fluids. The resultshowed at the highest extraction densities (for example 100 barnd 40 ◦C) that large quantities of paraffins and steroptens wereo-extracted with the rose volatile oil. Therefore, lower pres-ure and temperature were used for SFE of the rose concrete.amburger et al. [55] studied the effect of supercritical pressuren yield of extracted substances from three medicinal plantsmarigold, hawthorn and chamomile). They reported that, atressures above 300 bar, the yields of total extract increased.
ncreased yield of non-volatile lipophilic compounds, such asaradiol esters, are achieved at pressure above 300 bar. Brachett al. [56] used central composite designs in the study of threeropane alkaloids: hyoscyamine and scopolamine from Datura


Fig. 8. All-trans-lycopene and cis-lycopene contents (expressed as percentageo0u

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f the total lycopene) in the tomato supercritical fluid extracts performed in the.55–0.90 g/ml density range. Error bars are indicated at each density of CO2

sed. (From ref. [57] with permission.)

rborea hairy roots and cocaine from coca leaves. They showedhat for cocaine extraction, pressure had a weak influence onocaine recovery. The influence of pressure and temperature onxtraction yields is fairly identical for hyoscyamine and scopo-amine. At higher pressure, increasing the extraction temperaturerom 40 to 100 ◦C yielded significant increases in hyoscyaminend scopolamine recoveries, despite the CO2 density decrease.t low pressure, the effect of temperature is less pronounced.hese results confirm that SFE is governed by the volatility ofnalytes as well as by fluid density.

Gmez-Prieto et al. [57] proposed a procedure for the SFE ofll-trans-lycopene from tomato using carbon dioxide at 40 ◦Cithout modifier. Their method minimizes the risk of degra-ation via isomerization and oxidation of health-promotingngredients, such as lycopene. The effects of different experi-

ental variables on the solvating power of the supercritical fluidere evaluated in terms of both the selectivity for and the yieldf the extraction of all-trans-lycopene. Fig. 8 gives the relativemounts of the cis and all-trans forms of lycopene obtained at theifferent densities investigated in the range over which lycopeneas extracted (i.e. 0.55–0.90 g/ml). The amount of the trans-

ycopene extracted increases (and the corresponding cis formontent decreases) if the extraction pressure increases. Fromhese results, it seems clear that the enhancement of the fraction-tion of trans-lycopene requires a proper choice of CO2 density.n extract 88% all-trans-lycopene and 12% cis-lycopene coulde produced.

Ambrosino et al. [58] applied a new supercritical extractionethodology to extract azadirachtin A (AZA-A) from neem

eed kernels. They used supercritical and liquid carbon dioxideCO2) as extractive agents in a three-separation-stage supercrit-cal pilot plant. They carried out comparisons by calculating thefficiency of the pilot plant with respect to the milligrams perilogram of seeds (ms/m0) of extracted AZA-A. Conventionalressure extraction on raw seeds (320 MPa) led to a low yield inil (8%) together with the lowest concentration of AZA-A perilogram of oil and also to the lowest concentration of AZA-A
er kilogram of seeds (44 mg/kg of seeds). Compared to con-entional pressure extraction, both supercritical and subcriticalressures gave rise to a greater enrichment of AZA-A. Signif-cant differences were evident by comparison of supercritical
cpse

ig. 9. Overall extraction curve for marigold extraction with near-critical CO2

t various conditions. (From ref. [59] with permission.)

nd subcritical extractions at ms/m0 = 64 and 119 mg/kg of seeds.he most convenient extraction was gained using an ms/m0 ratiof 119 rather than 64. For supercritical extraction, a separationf cuticular waxes from oil was performed in the pilot plant.

In 2005, Campos et al. [59] investigated extraction ofarigold (Calendula officinalis) oleoresin with supercritical car-

on dioxide. They showed that, the temperature effect in therocess yield is complex due to the combined effect of solventensity and solute vapor pressure. These opposite effects must bevaluated to observe crossing of the yield isotherms. In Fig. 9, aonstant yield at 15 MPa with increasing temperature (from 303o 313 K) is observed, while at 20 MPa the extract yield increasesith temperature going from 299 to 313 K. This behavior is an

ndication that the solute vapor pressure is the dominant effect at0 MPa, while at 15 MPa the difficulty to observe the dominantffect is a suggestion that the crossover region (yield isotherms)s close to 15 MPa, for the temperatures studied.

Gaspar [60] studied the effect of the extraction pressurend temperature on the extraction of essential oils and othero-extracted components (cuticular waxes) from oregano (Ori-anum virens L.) bracts by compressed CO2 from 50 to 300 barnd 300 to 320 K, respectively. Moderate conditions, using sol-ent densities between 0.3 and 0.5 kg m−3, were found to beufficient for efficient extraction of essential oils. The use ofigher pressures and temperatures, despite slight advantagesor the rate of extraction and yields of essential oils extraction,ed to significant co-extraction of waxes and, consequently, toxtracts with lower essential oil content. For CO2 densities below.25 kg m−3, selective extraction of individual essential oils wasttained. At these low-density conditions, the lighter and moreolatile hydrocarbons were preferentially extracted.

Canela et al. [61] studied the supercritical fluid extractionf fatty acids and carotenoids from the Microalgae spirulinaaxima with carbon dioxide, assessing the effect of pressure

nd temperature on the yield and chemical composition of thextracts. Their experiments were conducted at temperaturesf 20–70 ◦C and pressures of 15–180 bar. Statistical analysishowed that neither the temperature nor the pressure signifi-
antly affected the total yield, but both the temperature and theressure affected the extraction rate, and temperature was moreignificant than pressure upon the SFE. The extracts were rich inssential fatty acids and carotenes, and at 100 bar and 45 ◦C the

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xtract contained no carotenes. Also, temperatures larger than0 ◦C degraded the carotenes, as expected.

.2. Effect of modifiers on supercritical fluid extraction

The nature of the modifier depends on the nature of the soluteo be extracted [62]. For example, the extraction of diuron isonsiderably enhanced with methanol instead of acetonitrile asmodifier [63]. A reasonable starting point consists of selectingmodifier that is a good solvent in its liquid state for the target

nalyte. It should be noted that the addition of large amounts ofodifier will change the critical parameters of the mixture [64],

s shown in Fig. 10 for methanol–carbon dioxide mixtures [64].s a result, binary mixtures of carbon dioxide and an organic

olvent are often used in a subcritical state, where the diffusionoefficients are smaller than in a supercritical state. Modifiersan be introduced as mixed fluids in the pumping system with a
econd pump and mixing chamber [65], or by simply injectinghe modifier as a liquid into sample before extraction [66] (theater way being less successful because it leads to concentrationradients within the matrix). Alternatively, one may use directly
ig. 10. Variations of the critical pressure and temperature of CO2–CH3OHixtures with the molar fraction of methanol. (From ref. [64] with permission.)

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J. Chromatogr. A 1163 (2007) 2–24

cylinder tank of modified CO2, but this is much more expensivend, as the tank is emptied, the content of modifier tends toncrease.

Kohler et al. [67] investigate the effects of modifier concentra-ion and its nature on the supercritical carbon dioxide extractionf artemisinin and artemisinic acid from Artemisia annua L.hey selected methanol, ethanol, methanol–water (50/50, v/v)nd toluene as modifiers based on preliminary tests and of theirolvating power for artemisinin. Methanol, ethanol and tolueneave similar results, however, the use of toluene presents a majorrawback due to its high boiling point and concomitant longervaporation times. Methanol–water gave unsuccessful resultsased on the slopes of extraction kinetic curves. This result cane explained by the low solvating power of water for artemisinin.

In order to improving supercritical CO2 extractability ofyoscyamine and scopolamine hydrochloride salts, Choi et al.68] investigate the effect of methanol and water on the extrac-ion yields. It was found that addition of methanol drasticallyncreased the extraction yield of hyoscyamine and scopolamine.owever, water did not show any significant influence on the

xtractabilities of hyoscyamin, although it slightly increasedn the case of scopolamine. The poorer result for water rela-ive to methanol may be due to the fact that water could notufficiently improve the polarity of CO2 as much as methanol,ince only 0.3% (v/v) of water can be completely miscible withO2. They also studied the effects of basified modifiers (diethy-

amine) added to methanol and water on analyte extractability.he addition of 10% diethylamine to methanol or water dramat-

cally enhanced the extraction of hyoscyamine and scopolamineydrochloride compared to using pure methanol or water. Thisesult may be due to the fact that the salts are changed toree bases by minor addition of methanol or with the diethy-amine, allowing supercritical CO2 to more easily dissolve theree bases. Ethanol, water and an equimolar mixture of thesewo solvents were chosen as co-solvents for supercritical car-on dioxide extraction of stevia glycosides by Pasquel et al.69]. The results indicated that due to the high polarity of watern comparison with ethanol, an increase in glycosides solubil-ty resulted. Comparison of the yields for the experiments usingater as co-solvent showed that regardless of whether it wassed with or without ethanol, water increased the solubility ofhe glycosides. Monteiro et al. [70] investigated the CO2 extrac-ion of bacuri fruit. They showed that the co-solvent influencedhe extraction yield of soluble material from shells of the fruitn two different ways. First, owing to its polarity, the co-solventavored dissolution of the polar substances present in the bacurihells. Second, the co-solvent diluted the extract, diminishingts viscosity, thereby enhancing the flow of the extract throughhe extractor.

Cocero and Garcia [71] studied supercritical fluid extractionf sunflower oil with carbon dioxide in a pilot plant at 30.0 MPand 40 ◦C, using different amount of methanol, ethanol, butanolnd hexanol as co-solvent. Comparing the co-solvent extraction
xperimental data with SFE using neat CO2, it was found thathe use of 10% (w/w) of a co-solvent increases oil solubility 10-old. They used a mathematical model to investigate the effectsf co-solvents on two adjustable parameters, i.e. equilibrium

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oefficient and mass-transfer parameter. Their results showedhat equilibrium parameter increases with co-solvent concentra-ion. Butanol gives the higher values which are exactly the sameesult obtained comparing oil solubility. Solute recovery valuessing methanol values are higher than when using hexanol andthanol. This is explained by comparing the mass-transfer val-es, methanol values being the lowest. Also, they showed thatass transfer increases with co-solvent concentration depend-

ng on the nature of co-solvent. This variation is probably causedy the change of physical properties in the supercritical mix-ure. Kerrola and Kallio [72] investigated the effect of addingarious quantities of water and a mixture of ethanol and water1:1, v/v) to the carbon dioxide extraction system on the rel-tive amounts of the volatile components of angelica roots.heir results showed that the yields of recovered components

ncreased when an aliquot of water was added to the system.fter the extraction had completed and carbon dioxide released,

he water co-solvent formed a separate phase. The increase inolume was considered as a positive factor. The co-solvent wasuggested to act as the primary solvent, thus enhancing the dif-usivity within the matrix. The mixture of ethanol and water
ppeared to increase the proportion of monoterpene hydrocar-ons when compared with water alone used as modifier andure liquid CO2 without a modifier. The effect of the co-solventdded on extractability of a compound varied considerably. �-
(woo

ig. 11. Effects of different volume of modifiers [(a) methanol, (b) ethanol, (c) dichlenth. essential oil these main compounds are: (1) �-pinene; (2) �-3-carene; (3) 1,nknown compounds with large retention number (the extraction pressure was 100 at

J. Chromatogr. A 1163 (2007) 2–24 11

hellandrene was the most prominent compound in all extractsbtained with the mixture of ethanol and water as modifier, buthe relative abundance varied from 9.2 to 30.7% depending onxtraction conditions. The relative proportion of both sesquiter-ene hydrocarbons and oxygenated sesquiterpenes decreasedhen the polarity of the solvent was increased by the mixture of

thanol and water in comparison to liquid CO2 extracts obtainedithout any modifier.Palma et al. [73] extracted white grape seeds by sequential

upercritical fluid extraction. Their results showed that, modifieras a main effect on the SFE process and by increasing theolarity of the supercritical fluid using methanol as a modifierf CO2, it was possible to fractionate the extracted compounds.hey obtained two fractions: the first, which was obtained withure CO2, contained mainly fatty acids, aliphatic aldehydes, andterols. The second fraction, obtained with methanol-modifiedO2, had phenolic compounds, mainly catechin, epicatechin,nd gallic acid.

Pourmortazavi et al. [74] studied the supercritical fluid extrac-ion of aerial parts of Perovskia atriplicifolia Benth. In thisesearch, the effect of different modifiers at a constant pressure

◦
100 atm) and temperature (35 C) on the extraction efficiencyas also evaluated. Fig. 11 shows the effect of different volumesf modifiers on the main contents of P. atriplicifolia essentialil. Fig. 11a shows the effect of methanol (1 and 5%, v/v) on the
oromethane and (d) n-hexane] on the main contents of Perovskia atriplicifolia8-cineole + limonene; (4) camphor; (5) �-caryophyllene; (6) �-humulene; (7)m and extraction temperature was 35 ◦C). (From ref. [74] with permission.)

1 eghi / J. Chromatogr. A 1163 (2007) 2–24

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Table 4Effect of dynamic extraction time on the composition of fennel oils obtained bySFE (at static time of 25 min and pressure 200 and 350 bar) [78]

Pressure (bar) 200 200 350 350Temperature (◦C) 45 45 45 45Dynamic time (min) 30 45 30 45

Compound Run1 Run2 Run3 Run4

�-Pinene 1.32 0.89 1.05 1.00Sabinene 0.89 – – –Limonene 9.34 7.94 7.26 7.17(z)-�-Ocimene 1.39 0.79 1.01 –�-Terpinene 0.77 0.83 – –Fenchone 9.25 8.45 9.93 8.36Estragole 2.94 3.07 3.09 2.81(G

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omposition of P. atriplicifolia essential oil. Methanol decreasedhe number of extracted compounds in comparison with thextraction by pure supercritical CO2 from 22 compounds to1 compounds. Also, using 1% methanol as modifier increaseontent of �-pinene, �-pinene, �-caryophyllene, �-3-carene, �-umulene and decrease the percent of 1,8-cineole + limonenend camphor in the extracted essential oil. Methanol increasedhe percent of co-extracted compounds with large retention num-er (unknown compounds) in the oil composition. Ethanol waslso tested for extraction of essential oil from P. atriplicifolia.esults showed that addition of ethanol (Fig. 11b) enhanced

he concentration of �-pinene, camphene, �-pinene, myrcene,-3-carene, and decrease the content of 1,8-cineole + limonene,amphor and �-caryophyllene in the plant oil. However, theseesults showed that addition of %1 ethanol, as modifier wasore effective than 5% ethanol. Dichloromethane as modi-er (Fig. 11c) decreased the number of extracted compoundsrom 22 with pure supercritical CO2 to 8 and 9 compounds inhe presence of 1 and 5% (v/v) dichloromethane, respectively.ichloromethane also increased the concentration of �-pinene,-pinene, �-3-carene, 1,8-cineole + limonene, �-humulene and

educed the concentration of camphor in the P. atriplicifpliassential oil composition. However, dichloromethane increasedhe content of co-extracted compounds with large retentionndices (unknown compounds) in the essential oil composition.urthermore, it was found that dichloromethane was a selec-

ive modifier for the extraction of 1,8-cineole + limonene and-3-carene from P. atriplicifolia. By using hexane as modifierFig. 11d), the number of identified compounds decreased from2 to 12 and 7 compounds (hexane volumes were 1 and 5%,/v, respectively). In the presence of hexane as modifier theercent of �-pinene, �-pinene, �-3-carene, �-terpenyl acetatencreased and the percent of 1,8-cineole + limonene and cam-hor decreased in comparison with extraction by pure carbonioxide. The result of these studies showed that changing modi-er type and identity could significantly affect the selectivity of

he extraction process.

.3. Effect of extraction time

It is important to maximize the contact of the supercriti-al fluid solvent with the sample material in order to enhancehe efficiency of SFE. Several variables that influence the sol-ent contact with sample material include flow rate, SFE time,nd SFE mode (static with no follow-through or dynamic withollow-through). It was reported [75] that, a 10–20 min staticxtraction prior to dynamic extraction improved the extractecoveries in SFE extraction of aflatoxins. Cui and Ang [76]eveloped a small-scale supercritical fluid extraction system forhe selective extraction of phloroglucinols from St. John’s WortSJW) leaf/flower mixtures using carbon dioxide. The extractionfficiency was investigated as influenced by pressure, temper-ture, time, and modifier. They optimized condition of SFE at
67 bar and 50 ◦C. Samples were held in static extraction for0 min, followed by a dynamic extraction for 90 min at the flowate of 1 ml/min. In this study, they showed static extractiononger than 10 min did not increase extraction efficiency. At the
itce

E)-Anethol 72.30 76.64 77.67 72.70ermacrene D 1.78 1.38 – 1.61

ow rate 1 ml/min (60 ◦C and at 367 bar), about 83.2% of theotal extractable hyperforin (HF) and 88.3% extractable adhy-erforin (AHF) were extracted in the first hour, and about 95.3%xtractable HF and 97.2% extractable AHF were extracted in therst 1.5 h. No more than 4% HF and AHF were extracted withinnother hour after the 1.5 h dynamic extraction. Thus, they used0 min static extraction followed by 1.5-h dynamic extractionor all samples.

Pourmortazavi et al. [77] showed the influence of the dynamicxtraction time on the composition of the essential oil ofuniperus communis L. leaves that was studied by perform-ng extraction with supercritical carbon dioxide this consistsf a static extraction of 25 min, followed by 20 and 30 minf dynamic extraction time. Results showed that increasingynamic extraction time at constant pressure 350 atm, enhanceshe content of heavy compounds with large retention indices inhe plant oil. They also tested a static–dynamic SFE approachor the isolation of fennel oil [78]. A static extraction period (fornhancing sample–extractant contact thus favoring the attain-ent of the portion equilibrium) was carried out, followed

y a dynamic extraction period in which extractant passedontinuously through the extraction chamber. The influencef the dynamic extraction time on the composition of thessential oil was studied by performing extraction with super-ritical carbon dioxide consisted of a static extraction step of5 min, followed by 30 and 45 min of dynamic extraction.he results are shown in Table 4. It was found that increasedynamic extraction time enhance the extraction of most of theompounds.

.4. Effect of flow rate

The speed of the supercritical fluid flowing through theell has a strong influence on the extraction efficiencies. Thelower the fluid velocity, the deeper it penetrates the matrix.he fluid speed can be expressed by the linear velocity, which

s strongly dependent on the flow rate and the cell geome-ry. For a given extraction cell, the flow rate can be easilyhanged by using a new restrictor with a different inside diam-ter. Decreasing the flow rate resulted in a lower linear velocity


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we60 ◦C (and a flow rate of 0.792 kg/h) for two different parti-cle sizes are shown in Fig. 15. From this figure, it is evidentthat the smaller particle size reduces the yield obtained in theextractions. Because of their small specific surface area, large

ig. 12. Effect of solvent flow rate on the extraction yield vs. extraction time at50 bar, 45 ◦C. (From ref. [80] with permission.)

nd usually in increased extraction. For example, 14C-labeledinear alkylbenzenesulphonates were better extracted by super-ritical CO2 modified with methanol (40 mol%) (at 380 bar and25 ◦C) from a sludge-amended soil with a liquid CO2 flowate of 0.45 ml min−1 (mean recovery 90.8 ± 1.3%) instead of.2 ml min−1 (mean recovery 75.6 ± 1.1%), the same volumef fluid being used in each instance [79]. Higher flow ratesesult in a decrease in the recovery either by using an ele-ated pressure drop though the extraction cell this phenomenonrobably occurred during the extraction of diuron from a con-aminated soil with a CO2–methanol (90:10, v/v) mixture, ory increasing analyte loss during decompression of the fluid.apamichail et al. [80] studied the SFE of oil from milled celeryeeds using CO2 as a solvent. They investigated the effect ofow rate of CO2 on the extraction rate of celery seeds. Theyhowed that the increase of the solvent flow rate leads to thencrease of the amount of oil extracted versus extraction timeFig. 12). The amount of the extracted oil per kilogram of CO2sed is higher for the lower flow rate due to the intra-particleiffusion resistance. This, actually, has as a result the smallerlope of the extraction curve in Fig. 13 for the higher flowate.

.5. Sample particle size and packing density

In general, decreasing particle size in SFE creates more sur-ace area and benefits extraction, but it also may hinder extractionf the analytes re-adsorb on matrix surfaces. Hawthorne and co-orker [81] discussed elution of the analytes from the matrix in

he vessel, to determine the rate-limiting step in SFE. A higher
ow rate can help reduce partitioning back onto matrix sites
f this is the limiting factor (otherwise, solubility factors areimiting). Larger particles, decreased packing density, smallerample size, and a wider extraction vessel reduce potential

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

atrix effects. Sample particle size and vessel packing densityniformity in day-to-day operations may be a factor in someFE applications that achieve variable results [81].

Louli et al. [82] extracted parsley seed oil with supercriticalarbon dioxide at different conditions. Fig. 14 presents theiresults about the effect of particle size on the extraction rate. Ast was expected, the extraction rate increases by decreasing theize of the seeds. This is due to the higher amount of oil releaseds the seed cells are destroyed by milling. Moreover, after millinghe diffusion paths in the solid matrix become shorter resultingn a smaller intra-particle resistance to solute diffusion.

Sabio et al. [83] extracted tomato skins and their mixturesith seeds by supercritical CO2 extraction. The results of their

xperimental study at a pressure of 300 bar and temperature of

ig. 14. Effect of particle size of Parsley seed samples on the extraction ratedata are presented as the extraction yield vs. the specific amount of solvent (Q)t 10 MPa, 318 K, and a solvent flow rate of 1.1 kg CO2/h). (From ref. [82] withermission.)

14 S.M. Pourmortazavi, S.S. Hajimirsadeghi /

Fig. 15. Effect of tomato sample particle size on the yields of supercritical fluideaw

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xtraction (yields of lipids, lycopene, and carotenes obtained at 300 bar, 60 ◦C,nd the CO2 flow rate of 0.792 kg/h from mixtures of tomato skins and seedsith two different particle sizes). (From ref. [83] with permission.)

articles lead to a distinct, diffusion-dominated extraction andong processing times. For example, decreasing the particle sizef ground peanuts from a range of 3.35–4.75 mm to a range of.86–1.19 mm was found to increase the total oil recovery from6 to 82%. However, particle sizes that are too small can resultn inhomogeneous extractions due to fluid channeling effects inhe fixed bed. In this study Sabio et al. verified an inhomoge-eous color distribution in the 80 �m particle size solid afterhe extraction via post-extraction examination of the substrate,ndicating an uneven extraction due to the small particle size.

.6. Effect of water in supercritical fluid extraction

Water in the sample often affects SFE. There have been appli-ations of direct SFE of aqueous samples [84], but precautionsust be taken to avoid damaging or destructive interaction of

ample contents with water; the water must be removed or con-rolled before performing SFE. Water can aid in the extractionrocess, or be detrimental, depending on water can open pores,well the matrix, thereby allowing the fluid better access to ana-ytes, and aid in flow through the matrix. Also, even thoughater is only ≈0.3% soluble in supercritical CO2 [84], it serves

o increase the polarity of the fluid and enable higher recoveriesf relatively polar species. However, if excess water remains inhe vessel, a highly water soluble analyte will prefer to partitionnto the aqueous phase and its SFE recovery will be low. Semi-olar analytes will dissolve in the aqueous phase, but readilyartition into the supercritical CO2, and yield high recoveries.or analytes that are insoluble in water, the analytes precipitatento matrix surfaces, and even though the analyte may be highlyoluble in the extraction fluid, the excess water in the sample actss a barrier in transfer of the analyte to the fluid [84]. The sol-bility of water in CO2 (0.3%) can cause restrictor pluggingpon the fluid depressurization, including the pressure of water
n the collection system. Removal of water is usually done byreeze-drying the sample matrix, as oven drying may result inolute volatilization. Alternatively, addition of drying agents tohe sample may be used. This sample treatment is attractive as it
rtua

J. Chromatogr. A 1163 (2007) 2–24

avors the dispersion of the analytes in the matrix and the sampleomogenization [85].

Leeke et al. [86] extracted Origanum vulgare L. using super-ritical carbon dioxide at 100 bar and 313 K in the presencef water. They observed that the addition of water as a mod-fier resulted in an increase in the extraction of essential oil.or extraction where water was added discontinuously, a large

ncrease in the extraction of the essential oils was recorded.he extraction degree increased with increasing w/w% waterdded to the bed, reaching an optimum at 80% (w/w). At theseater concentrations, a low but finite yield of waxy material also

esulted. Such a result could be beneficial to attain an essen-ial oil rich product. In this study, the continuous addition ofater also resulted in an increase in the degree of extractionf essential oils, but not necessary an increased yield of waxyaterial. For tests where a higher w/w% was added, the removal

f water would have had little effect on the extraction efficiencyf essential oils, even at prolonged extraction times, becausef its abundance. Finally, they showed that their discontinuousethod is sufficient to influence the extraction degree of the

ssential oils and on a commercial scale, further equipment forontinuously humidifying supercritical carbon dioxide woulde avoided, providing that the extraction process was completedithin a suitable timescale.

.7. Drying effect

Drying of plant samples, a major preservation process forpices, can be carried out conventionally by air-drying (withr without heat) or by freeze-drying. It is obvious that dryingnd the drying process may have an influence on the content ofroma compounds. Literature data indicate that the changes ofroma compounds during drying depend on the drying methods well as the type of the spice. Ibanez and co-workers [87]roposed a two-step supercritical fluid extraction of rosemaryeaves at selected conditions of pressure and temperature toivide the oleoresin into two fractions with different antiox-dant activities and essential oil compositions. They showedhat, there are two main factors that have to be considered: therying process, which influences the essential oil compositionnd, therefore extract quality, and the effect of the drying pro-ess on the plant cells. Damage to plant cell walls can resultn compounds being more easily extracted at the quoted SFEonditions. Rosemary leaves obtained by different methods ofrying have been extracted and evaluated in terms of antioxidantctivity and essential oil yield and composition. By analyzinghe results for three samples of dried rosemary by using differ-nt procedures, it was shown that the treatment that providedhe highest quantity of rosemary essential oil was freeze-dryingollowed by drying in oven at 45 ◦C, and then vacuum rotaryvaporation. Freeze-drying is the mildest temperature treatment.herefore, less aroma loss is expected to be obtained. Drying

n oven at 45 ◦C implied a higher temperature treatment than
otary evaporation (35 ◦C), but the first method is faster thanhe second and is performed in the absence of light. The vac-um treatment was a slow process conducted in daylight, andrtifacts could easily be formed. In fact, olfactory tests showed

eghi / J. Chromatogr. A 1163 (2007) 2–24 15

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optagmsestdbcoiAain solid-phase trapping, however, cryogenic trapping on inertmaterials (glass or stainless steel beads) is largely unsuccessfulfor analytes with even a small vapor pressure. The use of sorbentphases allows adsorption to be used to increase the collection


evelopment of a non-characteristic rosemary aroma in thistudy.

. Collection of extracted analyte

The development of appropriate collection system for tar-et analytes after SFE is often ignored by new users, despitehe obvious fact quantitative extraction conditions cannot beeveloped and evaluated unless the collection step is efficient.hus, the first job of the analyst is to optimize the collection sys-

em and determine its efficiency for the target analytes. Apartrom the extraction process, the single most important process inFE is the efficient trapping of extracted material. Two differentpproaches are commonly used for off-line SFE, liquid solventollection and solid-phase trapping. Both systems have theirdvantages and disadvantages with respect to ease of handling,hoice of restrictor type, maximum gas flow, and compatibil-ty with the various types of supercritical fluids, modifiers andnalytes [88–90].

.1. Solvent collection

Liquid solvent collection is mechanically simple and has beenhe most widely used approach for natural samples. Two com-

on approaches have been used. In the first approach, the end ofhe flow restrictor is placed directly into the collection solvent,nd CO2–analyte mixture is depressurized directly in contactith the solvent. In the second approach, the CO2–analyte efflu-

nt is first depressurized to the gas phase in a glass transfer tubeefore contacting the solvent. For this system, efficient collec-ion appears to depend on efficient transfer of the analytes fromhe gas phase to the collection solvent. Somewhat surprisingly,he first approach has been shown to yield better collection effi-iencies of more volatile components, and efficient collectionsing the second approach has often required the addition of aecond solid-phase trap [91,92] or a glass wool insert in the glassiner [93].

Various articles in the literature report the use of “liquid trap-ing.” One version of liquid trapping involves immersion of theestrictor into a liquid, as illustrated in Fig. 16, while a secondersion concerns an inert solid surface in tandem with a liquidrap. In the Dionex (Sunnyvale, CA, USA) 703 instrumenta-ion for example, non-volatile analytes are thought to deposit onhang-down tube (solid surface) while the more volatile ana-

ytes partition into the collection liquid after decompression. Achematic of this type of trapping device is shown in Fig. 17.

.2. Solid-phase collection

Several different solid-phase trapping methods are used inFE. Mulcahey and Taylor [94] conducted a study with non-odified CO2 to determine the best solid-phase trap composition

nd trapping conditions for a test mixture of analytes represent-
ng a wide range of polarities. Mulcahey et al. [95] continuedheir studies with a wider assortment of solid-phase sorbentraps. They found that a single trap composition may not effec-ively trap a wide range of analytes. Furthermore, the addition
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ig. 16. Schematic representation of the liquid trapping process involvingmmersion of the restrictor into a liquid solvent.

f a modifier to the extraction fluid caused a decrease in trap-ing efficiencies. Eckard and Taylor [96] found that solid-phaserapping has an additional drawback in that the sorbent trap hasfinite capacity. They found a 50/50 mixture of Porapak Q andlass beads exhibited the highest sample capacity and was theost effective trap for a wide range of analyte types. In another

tudy, Moore and Taylor [97] found that stainless steel ball trapfficiency is greatly affected by the addition of a modifier to theupercritical fluids. They found that it was necessary to raise therap temperature in order to achieve quantitative recoveries ofigitalis glycosides. Solid-phase trapping is normally performedy depressurizing the CO2 and the analytes prior to the trap andollecting the analytes from the gas (or aerosol) phase directlynto sorbents such as silica gel, Florisil, or bonded phase pack-ng or onto insert surfaces such as glass or stainless steel beads.fter SFE, the trap is eluted with liquid solvents for subsequent

nalysis. Both cryogenic and adsorption mechanism are active

ig. 17. Schematic representation of the liquid collection systems used in theionex extractors.


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fficiencies, and the selectivity of the adsorption mechanism cane used to gain a degree of compound-class fractionation dur-ng the SFE collection step. In addition, the choice of rinsingolvent(s) can be used to selectively elute different compoundlasses from the trapping system. This degree of selectivity basedn the elution of the trap is a particular advantage of sorbent col-ection over solvent collection systems. However, solid-phaserapping can be more trapping material, the trap temperature,nd the rinsing solvent. Some articles may be found in the liter-ture about investigation on the effect of solid-phase collectionn the composition of supercritical fluid extracted plant oil.

Dugo et al. [98] reported a method for the deterpenation ofhe citrus essential oils with supercritical CO2 using adsorptionn silica gel to enhance the selectivity of the separation betweenhe hydrocarbons and the oxygenated compounds. They showedhat the silica gel retains the oxygenated, more polar compounds,hus allowing at low temperature and pressure, the extraction ofhe non-polar terpene hydrocarbons. By increasing the pressurend the temperature after a defined time, it is possible to elute thexygenated components the volatile fraction. Araujo et al. [99]xtracted Pupunha (Guilielna speciosa) oil in a fixed bed usingarbon dioxide. In their study, the supercritical carbon dioxideontaining the solubilized oil was expanded in the small stain-ess steel tube the precipitated extract was collected in a glassube placed inside the separator. Blanch et al. [100] in 1994roposed a simple procedure for off-line SFE and capillary gashromatography analysis of the essential oil obtained from Ros-arinus officinalis L. During the extraction step, they deposited

he obtained analytes on an internal trap where the supercriti-al fluid evaporates. Subsequently, a suitable solvent is pumpedhrough the trap so that the analytes are rinsed off into a vial.ig. 18 shows scheme of their assembly. They used differentorbent for trapping extracted essential oils, such as Hypersilctadecylsilica (ODS), glass beads, GasChrom 220, Tenax TA,hermotrap TA, and Volaspher A-2 silanized sorbents.

.3. Collection in empty vessels

Collection in an empty vial or vessel has been success-ully practiced by a number of investigators and is particularlyppropriate for bulk extraction of fat and similar exhaustivextractions. It is also applicable however, for the extraction ofrace levels of analytes, such as pesticides [101], but larger col-ection vessels are required for capturing such trace analytes inrder to minimize their loss. Avoidance of entrainment of ana-ytes in the escaping fluid stream can be minimized by addingglass, steel wool, or ball packing to the empty container. The

hosen material should be chemically inert, provide a high sur-ace for condensing the analyte from the rapidly expanding fluid,ut allow ready desorption of the analyte after completion of thextraction.

.4. Novel collection methods

In 2004, Sarmento et al. [102] studied the performance ofhree commercial reverse osmosis membranes: SG, CG and AGegarding the permeability to supercritical CO2 and the reten-

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ig. 18. Scheme of the trap assembly. (From ref. [100] with permission.)

ion of lemongrass, orange and nutmeg essential oils at 12 MPand 40 ◦C. The results of their work demonstrate the potentialor use of a commercial reverse osmosis membrane in the sep-ration of lemongrass, orange and nutmeg essential oils fromupercritical mixtures with CO2. All the membranes exhibitedood resistance to the severe pressure conditions employed.he oil retention index was reduced with the increase in trans-embrane pressure from 1 to 4 MPa. The best retention resultsere obtained with the SG membrane, which retained up to 90%f all the essential oils tested. However, at the same time, thisembrane allowed the lowest CO2 permeate fluxes with values

p to 8.75 kg h−1 m−2 at a pressure difference of 1 MPa. Thexperimental results indicated the occurrence of fouling for allhe membrane models after permeation of lemongrass essentialil. Spricigo et al. [103] showed that the association of mem-rane separation processes to the supercritical fluid extractionf essential oils from vegetable matrices can be an alternative tohe reduction of recompression costs derived from the depressur-zation step necessary for the recovering of the extracts. In theirork, a cellulose acetate reverse osmosis membrane was applied

o perform the separation of nutmeg essential oil and densearbon dioxide. The effects of feed stream essential oil concen-ration, temperature and trans-membrane pressure on essentialil retention and CO2 permeability were investigated. The aver-
ge retention of essential oil by the membrane was of 96.4% andt was not affected significantly by any of the process variables.he CO2 flux was linearly proportional to the trans-membraneressure applied and decreased as the essential oil concentration

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n the feed stream increased. The membrane presented good CO2ermeability and resisted well to the severe pressure conditionspplied.

.5. On-line coupling of supercritical fluid extraction withhromatographic techniques

Sometimes SFE has been directly coupled to chromato-raphic detection systems as this offers some advantages overff-line SFE. In on-line SFE the entire extracted sample is intro-uced into the system and therefore greater detection sensitivityan be obtained. However, smaller sample sizes are often used inn-line SFE to prevent overloading of the chromatographic col-mn. There is also less chance of sample contamination becausehere is no intermediate handling of the extracted analytes.nitial studies have focused on coupling SFE to supercriticalhromatography (SFC), as carbon dioxide is used as both thextraction fluid and the chromatographic mobile phase, althoughuch research into coupling SFE to GC has been carried out.ndersen et al. [104] discussed some theoretical considerations

nvolved in using SFE as a method for sample introduction inhromatography. An SFE–SFC protocol was used to extractnd determine the herbicides linuron and diuron from a sandyoam soil and wheat. After initial SFE optimization, carriedut off-line, the two techniques were coupled with flame ion-zation being used for detection. A modifier was required tochieve quantitative extraction, which eluted as a solvent frontn the chromatogram. Both capillary and microbore columnsere investigated, with better sensitivity being obtained with aicrobore column and larger sample loops.The most common technique used in on-line SFE of natural

amples is coupled SFE–GC. This coupling technique is of par-icular important for non-polar analytes, which do not requireerivation either in situ or prior to GC separation. A variety ofpproaches have been used to couple SFE extraction with cap-llary GC, which can be roughly categorized into three mainroups:

a) An external loop, through which the extract passes, is usedto introduce the analytes into the GC column. The analytesare transferred into the GC column by sweeping the heatedloop with carrier gas. This has the advantage of allowing off-line collection to be carried out simultaneously, although italso suffers from a decrease in sensitivity as only a fractionof the sample is passed to the chromatograph.

b) The extract is transferred into a sorbent trap external to thegas chromatograph that may be cryogenically cooled. Sub-sequent heating of the trap and purging with carrier gas allowthe extracts to enter the chromatograph. The addition of theTenax-GC trap gave better peak shapes than direct SFE–GCbecause of the refocusing effect. It also allowed the methanolmodifier, used in the extraction, to be removed before flush-ing the analytes on to the column, which previously caused
a “hump” in the baseline in conventional SFE–GC.
c) The simplest approach utilizes a conventional GC injec-tion port to couple SFE with GC with both on-column andsplit–splitless ports being used. All three techniques gener-

aThn

J. Chromatogr. A 1163 (2007) 2–24 17

ally require cryogenic refocusing of the analytes on the frontof the capillary column prior to analysis to obtain good peakshapes.

The first time SFE was coupled to another separation tech-ique (i.e. SFC) was in extracting caffeine from roaste coffeeeans [105]. SFE–GC with mass spectrometry (MS) or flameonization detection (FID) has been demonstrated for flavorompounds in spices, chewing gum, orange peel, spruce nee-les and cedar wood [106], from lime, lime peel, eucalyptus107,108], basil [107,109], grapefruit oil [110,111], thyme109,112], orange juice [108] and chamomile [109]. Many ofhe earlier applications were of qualitative nature. In compar-son between SFE–GC and SFE–SFC, the latter was given andvantage due to higher yields of oxygenated terpenes and noeed for derivation [109,113]. Sato et al. [114] used direct con-ection of supercritical fluid extraction and supercritical fluidhromatography as a rapid quantitative method for capsaicinoidsn placentas of Capsicum annuum L. They compared this methodith usual extraction-HPLC method. Their finding showed that

he SFE/SFC method has the advantages of no need for pretreat-ent and no (or minimal) need for organic solvents. Also, thisethod is useful as a rapid (20 min) and safe screening test for

he pungency of various Capsicum fruits.

. Extraction of oxygenated compounds from plantaterials by supercritical fluid extraction

Today, antioxidants from natural resources are associatedith health benefits since oxygenated compounds are related

o a positive action against heart diseases, malaria, neuro-egenerative diseases, AIDS, cancer and longevity [115]. Onhe other hand, some of the artificial antioxidants are related toealth damage, such as kidney edema [116]. For these reasons,he market for natural antioxidant should rapidly increase.

Several methods have been used to extract antioxidants fromromatic plants, such as solid–liquid extraction, aqueous alka-ine extraction, extraction with aqueous solutions [117,118], andupercritical fluid extraction [119,120]. Products obtained byFE from different plants, in general, have a higher antioxi-ant activity than extracts obtained by using solvent extractionith organic solvents [121,122], probably due to a differ-

nce in composition deriving from the extraction conditionspplied. In 2004, Hu et al. [123] investigated the influencesf extracting pressure, temperature, and flow rate on theield of sesame seed extract and the antioxidant activity ofxtracts by supercritical carbon dioxide and solvent from blackesame seed as compared to �-tocopherol, Trolox, and buty-ated hydroxytoluene (BHT). They found the highest extractedield was achieved at 35 ◦C, 40 MPa, and a CO2 flow ratef 205 ml min−1. Results for the linoleic acid system showedhat the antioxidant activity follows the following order: extractt 35 ◦C, 20 MPa > BHT > extract at 55 ◦C, 40 MPa > extract
t 55 ◦C, 30 MPa > Trolox > solvent extraction > �-tocopherol.he supercritical carbon dioxide extracts exhibited significantlyigher antioxidant activities comparable to those obtained by-hexane extraction.


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The volatile components of Terminalia catappa (green, yel-ow and red fallen) leaves were extracted using supercriticalarbon dioxide at various pressures [124]. The extracts fromellow and red fallen leaves exhibited higher inhibition of pre-xidation that those from green leaves. On the other hand,upercritical carbon dioxide extraction at 133 atm and 40 ◦Cesulted in the extracts with better antioxidant activity whereasower inhibition of pre-oxidation was observed with extractsrepared from the extraction at 267 atm and 40 ◦C. Also, Zan-an et al. [125] studied the influence of the use of co-solvent inhe kinetics of SFE of ginger oleoresin, in the chemical com-osition of the extracts and in their antioxidant action. Theyhowed that the major substances present in the ginger extractsere �-zingiberene, gingerols and shogaols the amounts of these

ompounds were significantly affected by temperature, pressurend co-solvent. Nonetheless, the antioxidant activity of the gin-er extracts remained constant at 80% and decreased to 60% inhe absence of gingerols and shogaols.

In 2003, the selectivity of subcritical water extraction severalemperatures to extract antioxidant compounds from rosemaryeaves was investigated [126]. Results indicate high selectivityf subcritical water toward the most active compounds of rose-ary. The antioxidant activity of fractions obtained by extraction

t different water temperatures was high comparable to thosechieved by SFE of rosemary leaves. In this study, Ibanez et al.emonstrated the possibility of tuning the selectivity for antiox-dant extraction by a small change in extraction temperature.

Senorans et al. [127] isolated antioxidants from orangeuice by the use of countercurrent supercritical fluid extractionCC-SFE) and characterized by reversed-phase liquid chro-atography (RPLC) coupled to mass spectrometry (MS) and

iode-array detection (DAD). They employed a pilot-scale SFElant equipped with a packed column for countercurrent extrac-ion and fractionation of raw orange juice with carbon dioxide.everal experiments have been performed in order to study theffect of the countercurrent conditions on the content of antiox-dative compounds. In their study, the main variable that haseen considered was the solvent-to-feed ratio (S/F) becauset plays an essential role in the extraction efficiency. The val-es tested covered a wide range of sample and solvent (CO2)ow rates. They obtained three different products after extrac-

ion and fractionation of the orange juice: those in separators(F1) and 2 (F2) and the raffinate (R) which is the byproduct

f the extracted samples collected at the bottom of the column.ig. 19 shows the percent of total area of the identified com-ounds versus solvent-to-feed ratio. When low S/F ratios aresed, a maximum extraction of flavonoids is obtained, with aow percentage recovered in the raffinate. The opposite is foundt S/F equal to 11 where almost 96% of the compounds iden-ified are found in the raffinate. In each experimental run, twoifferent extracted fractions and the residual non-extracted juiceere obtained and characterized. Different flavonoids have been

dentified in the fractions obtained after CC-SFE. Also, they
iscussed possibility of using this process for antioxidant com-ounds enrichment. Enrichment results are shown in Fig. 20 asfunction of the S/F ratio data corresponding to the enrichmentchieved in separators 1 and 2 individually and the total (consid-
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ig. 20. Graph representing the log (enrichment) as a function of the S/F ratioa selective enrichment can be observed toward the extracts or the raffinate as aunction of the S/F ratios selected). (From ref. [127] with permission.)

ring both separators together) are also presented. They obtainedhigh correlation (96%) for log (total enrichment) versus S/F

sing a linear regression y = 0.3365x + 2.4304r2 = 0.963.In 2002, Simoa et al. [128] determined antioxidants from

range juice by the combined use of CC-SFE prior to RPLCr micellar electrokinetic chromatography (MEKC). Theychieved separation of antioxidants found in the SFE fractionsy using a new MEKC method and a published LC procedure,oth using diode-array detection. In the same year, Yepez et al.129] obtained fractions from seeds of coriander (Coriandrumativum) by extraction with supercritical carbon dioxide in aemi-continuous lab-scale equipment, and were tested for theirntioxidant activity. Fractions from coriander seeds obtainedy SFE exhibited a significant antioxidant activity, as deter-ined by removal of DPPH free radicals larger than 50% after

00 min. In addition, high extraction yields, close to 2% (w/w),ere achieved using SFE conditions in the range of 183–326 K,

orresponding to a CO2 density in the range of 0.74 g/ml.

. Extraction of terpenes and sesquiterpenes byupercritical fluid extraction

Goto and co-workers [130] used supercritical CO2 to sepa-ate oxygenated compounds from essential oils. This techniquetill cannot replace vacuum distillation as an industrial process

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ecause of low recoveries and inconsistent results. Vacuum dis-illation and supercritical CO2 are complementary processesor producing high quality oxygenated compounds with highecovery rates. The former is more suitable for removingonoterpenes at low fraction temperatures (<308 K), and the lat-

er is more suitable for separating oxygenated compounds fromigments and waxes. Consequently, the two methods can be usedn tandem. Sonsuzer et al. [131] successfully applied responseurface methodology for optimization of parameters in SFE ofhymbra spicata oil. The high regression coefficients of sec-nd order polynomial of the responses showed that model fittedo data well. The optimum condition was applied to minimizeonoterpene hydrocarbons content and maximize oxygenated

ompounds and yield. Pressure was the most significant fac-or affecting yield in SFE. An adverse effect of temperature onield was observed. Monoterpenes which have higher volatil-ty, lower molecular-weight and lower polarity were extractedt the beginning of the process. However, sesquiterpenes andxygenated compounds were extracted later due to their higherolecular-weight and polarity, respectively.Carlson et al. [132] extracted lemongrass (Cymbopogon cit-

atus) essential oil with dense carbon dioxide at 23–50 ◦C and5–120 bar. The compounds present in larger quantities in theemongrass essential oil were neral, geranial and myrcene. Theiresults showed that the changes in temperature and pressureonditions had a significant effect on the composition of thextracts. Co-extraction of waxes was observed under all thextraction conditions but supercritical extracts had lower contentf these compounds than liquid CO2 extracts. Along the extrac-ion experiments, changes in the composition of the extracts werebserved, with higher-molecular-weight compounds extractedn larger quantities at the end of the process. The optimizedxtraction conditions from their tests were 120 bar and 40 ◦C.

Regina et al. [133] investigated the influence of temper-ture and pressure on the characteristics of the essential oilbtained from high-pressure carbon dioxide extraction of mar-oram. Their experiments were performed in a laboratory-scalenit using the dynamic method in the temperature range of93.15–313.15 K, from 100 to 200 bar in pressure. Resultshowed that an increase in temperature leads to a rise inhe extract yield despite large changes in solvent density.hromatographic analyses permitted the identification of cis-

abinene hydrate, terpineol-4, R-terpineol, and cis-sabineneydrate acetate as the main volatile compounds present in bothommercial and cultivated samples.

Ranalli et al. [134] obtained carrot root oil, by supercriticaluid carbon dioxide extraction, and compared to commercialarrot oil and a virgin olive oil. Their results showed that carrotoot oil obtained by SFE has much higher content of carotenes,henolics, waxes, phytosterols, and sesquiterpene and monoter-ene volatiles. In the SFE oil the most prominent componentsresent in the fully investigated analytical fractions (fatty acids,riglycerides, waxes, phytosterols, long-chain aliphatic alcohols,
uperior triterpene alcohols, and volatiles) were, respectively,inolenic acid, trilinolein, waxes C38, �-sitosterol, campes-erol and stigmasterol, 1-hexacosanol, 24-methylencycloartanolnd cycloartenol, �-caryophyllene, �-humulene, �-pinene, and
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J. Chromatogr. A 1163 (2007) 2–24 19

abinene. However, in virgin olive oil, the major constituentsf the above analytical classes were, respectively, oleic acid,rilinolein, waxes C36, unsaturated volatile C6 aldehydes (trans--hexenal most markedly), and the same prominent sterols anduperior alcohols found in supercritical fluid extract. In commer-ial carrot oil, which also contained a proportion of unknownlant oil, several components showed magnitudes that wereower compared to supercritical oil but higher with respect toirgin olive oil. The last had the aliphatic and triterpene alco-ol concentration higher compared to that of both supercriticaluid extract and commercial carrot oil. Rodrigues et al. [135]sed sub- and supercritical CO2 to obtain extracts from two ori-anum samples, one commercial, and another cultivated undergronomic control. Their results show that the commercial sam-le provides a higher yield of extract if compared to the otherample. Chemical analyses allowed the identification of around4 compounds for both commercial and cultivated origanumamples. It was also found that the distribution of chemical com-onents in the extracts was a function of extraction time andiffered appreciably between the origanum species. The chro-atographic analysis permitted the identification of thymol and

is-sabinene hydrate as the most prominent compounds presentn commercial oregano sample and carvacrol and cis-sabineneydrate in the cultivated sample.

Diaz-Maroto et al. [136] obtained volatile oil extracts of fen-el seeds (Foeniculum vulgare Mill.) and thyme leaves (Thymusulgaris L.) by simultaneous distillation-extraction (SDE) andFE. They showed that the fennel oil extracted by SDE and SFEas similar compositions, with trans-anethole, estragole, andenchone as the main components. Thymol and p-cymene, theost abundant compounds in thyme leaves, showed big differ-

nces, with generally higher amounts of monoterpenes obtainedy SDE. Key odorants in fennel seeds were determined byas chromatography-olfactometery (GC-O) showed similar elu-ion patterns when SDE and SFE were utilized. trans-Anetholeanise, licorice), estragole (anise, licorice, sweet), fenchonemint, camphor, warm), and 1-octen-3-ol (mushroom) were theost intense odor compounds detected in fennel extracts. Eller

t al. [137] investigated the extraction of cedarwood oil (CWO)sing liquid carbon dioxide and compared it to traditional SFE,ncluding the effects of extraction pressure and length of extrac-ion. Their finding showed that the cumulative yields of CWOrom cedarwood chips using 80 l of carbon dioxide varied littlereatment to treatment, with all temperature/pressure combi-ations yielding between 3.55 and 3.88% CWO, includingumulative yields. Also, the rate of extraction was highest underhe supercritical extraction conditions (i.e. 100 ◦C and 400 bar)nd under the liquid CO2 conditions (i.e. 25 ◦C), the extractionates did not vary significantly with extraction pressure. How-ver, they observed differences in the chemical composition ofhe collected CWO. For example, extractions at 100 ◦C gave a

uch lower ratio of cedrol/cedrene than extractions at 25 ◦C,ut the highest ratio of cedrol/cedrene was obtained using 25 ◦C
nd 100 bar.
Vagi et al. [138] investigated extraction of pigments (chloro-hylls and carotenoids) from marjoram (Origanum majorana.) with supercritical carbon dioxide. The aim of their study

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as to map the effects of extraction pressure and temperaturen the yield of coloring materials by applying a 32 full factorialesign with three repeated tests in the center of the design. Foromparison, they carried out laboratory and pilot plant Soxhletxtractions using ethanol and n-hexane solvents and determinedhe compositions of pigments in marjoram extracts by HPLC.imilar amounts of carotenoids, in addition to 40% of chloro-hylls and their derivatives, were recovered from the SFE whenompared to the ethanol Soxhlet extraction.

. Comparison of supercritical fluid extraction toonventional methods

One of the oldest and most commonly used extraction pro-edures employs a Soxhlet apparatus. It has been the standardxtraction procedure over 100 years even though it fails to exhibitany of the ideal extraction criteria. The time for an aver-

ge Soxhlet extraction ranges from 1 to 72 h. The completedxtraction produces a high volume, dilute solution which usuallyeeds to be concentrated prior to analysis. In many cases, Soxh-et extraction is not selective, because interfering compounds

ay also be extracted by the heated solvent of choice whichay further complicate the assay of the analyte(s) of interest.he choice of solvent obviously controls the solvating powers well as the temperature of the extraction. Under these con-itions the integrity of the analyte may not be maintained inhat thermal decomposition or reaction with the solvent mayccur. Perhaps the greatest disadvantage of using the Soxhletethod for extractions is its utilization of expensive, high purity

rganic solvents such as acetone and methylene chloride. Whileutomation is seldom attempted, it does have the advantage thatumerous Soxhlet extractions can be simultaneously performed.urthermore, hardware for Soxhlet is fairly simple however, foruantitative work extreme care must be taken to avoid introduc-ion of contaminants and to minimize losses in sample transfernd solvent exchange.

Liquid–liquid and liquid–solid extractions normally per-ormed at room temperature. Large volumes of organic solvents,hich must be concentrated and disposed of, are required. Someixing time is usually necessary for efficient phase exchange.ultiple extractions are also usually mandatory if quantita-

ive removal is desired. Sample transfer can become a problemspecially if phase emulsions are produced. The simultaneousxtraction of multiple samples is usually possible if sufficientlassware is available.

Solid-phase extraction (SPE) has become increasingly pop-lar during the past several years. It appears to be applicableo both non-polar and polar analytes. The analyte and matrix

ust, however, be in the liquid state. Currently, SPE serves asn attractive alternative to liquid–liquid extraction. A wide vari-ty of solid–sorbents exhibiting a wide variety of chemistriess available for selective trapping from numerous vendors. Theorbents are large particle size (40 �m I.D.) bonded phase silica
n most cases although phases exhibiting new chemistries areapidly being introduced. Proper activation of the phase prior toample introduction and selective rinsing of the phase in ordero recover the extracted analyte free of matrix components for
whwp

J. Chromatogr. A 1163 (2007) 2–24

ubsequent analysis are critical steps in the overall sample prepa-ation procedure. Considerably less organic solvent usage can bexpected here as compared with Soxhlet extraction, although theost of SPE cartridges cannot be discounted. Given sufficient car-ridge, multiple extractions can be performed simultaneously. A

ore recent development in this field is the introduction of filterisks which contain solid-sorbent trapped within polytetrafluo-oethylene fibrils. Currently, only C8 and C18 bonded silica isvailable. These disks (25 and 47 mm in diameter) enable theow of liquid phase analyte plus matrix to be faster than thePE cartridges. Organic solvent or supercritical fluid has beenmployed to release the trapped analyte for subsequent analysis139].

Piggott et al. [140] used steam distillation, solvent extrac-ion, supercritical fluid extraction and liquid CO2 extractiono obtain the volatile oil from Western Australian sandalwood.FE afforded the highest yields of extractable material and totalolatile. Also, volatile secondary metabolites have been isolatedrom flowers, leaves and stems of Spilanthes americana [141]y simultaneous distillation–solvent extraction and supercriti-al carbon dioxide extraction. In this study, the plant materialflowers, stems, leaves) affected the composition of the extracts.FE extracts from stems were rich (>40%) in sesquiterpenes,hile those from leaves and flowers were abundant in nitro-enated and oxygenated compounds. However, SDE extractsrom stems, leaves, and flowers of the plant contained sesquiter-ene levels of 32, 28 and 20%, and a generally higher proportionf oxygenated compounds and monoterpenes than the SFE coun-erparts. Also, some heavy hydrocarbons originated from flowerigments and waxes were isolated by SFE but not by SDE. It wasound that SFE is both selective and highly efficient in the iso-ation of sesquiterpenes, heavy hydrocarbons and nitrogenatedompounds in this particular case.

Vilegas and Lancas [142] used supercritical CO2 extraction toompare chemical composition of the essential oils obtained byteam distillation procedure from two lauraceae (Ocotea caesia

ez.). Their study showed that the supercritical carbon dioxidextractions had lower yields than those from steam distillation,ut the extracts obtained were similar in both species studied.n 1999, Eikani et al. [143] compared the extraction obtainedrom Cuminum cyminum L. by supercritical carbon dioxideith cumin essential oil obtained by conventional steam distil-

ation. They showed that the physicochemical properties (suchs refractive index, specific gravity and optical rotation) of theils extracted by supercritical CO2 and steam distillation wereifferent. The results of the GC–MS analysis showed that theost noticeable difference between the two methods is in the p-entha-1,3-dien-7-al and p-mentha-1,4-dien-7-al composition.Hydrodistillation and solvent extraction using pentane,

thanol and supercritical carbon dioxide were used to isolatessential oils from Grapefruit flavedo [144]. The compositionsf different extractions were compared. Monoterpene hydrocar-ons decrease in supercritical carbon dioxide extracts at 87–90%
ith respect to their quantity in pentane extracts (95%) and inydrodistillate (97%) these levels in monoterpene hydrocarbonsere related to the limonene content, the most abundant com-ound in grapefruit essence. Sesquiterpenes, aldehydes, alcohols


Table 5Comparison of the overall chromatographic area percentages for the four mainclasses of hydrocarbon monoterpenes (HMs), oxygenated monoterpenes (OMs),hydrocarbon sesquiterpenes (HSs), and oxygenated sesquiterpenes (OSs) (atdifferent stages of supercritical fluid extraction) in the constituents of the Laureloil [146]

Class 60 min 120 min 180 min 240 min SFE-T HD

HMs 12.67 2.74 1.75 0.31 13.65 15.51OMs 79.43 77.62 76.94 66.68 70.86 70.28HSs 7.45 14.91 14.11 15.52 11.53 7.19OSs 0.5 4.17 6.92 17.07 3.68 7.03

Ct

act

tomtiscsfp1sow(bhptae(aa1ranowIeoow

ees

Table 6Percent yield and cumulative percent yield of the Laurel oil at different stagesof the supercritical extraction (60–240 min), the overall run (SFE-T), and thehydrodistillation [146]

Quantity 60 min 120 min 180 min 240 min SFE-T HD

Yield (%) 0.27 0.28 0.22 0.05 0.82 0.90Cumulative

yield (%)0.27 0.56 0.77 0.82 0.82 0.90

ms/m0 5.36 10.72 16.08 21.44 21.44 –

Ct

Ledtbopswptteae�mSiSoTsecolation and compared with supercritical fluid CO2 extracts. Theextracts obtained by SFE at different conditions were compo-sitions similar to that of the oil obtained by steam distillation.

Table 7Comparison of the main components of Iranian black cumin oils obtained bySFE (under 200 atm pressure and 45 ◦C temperature for 15 min static followedby 20 min dynamic) and hydrodistillation [147]

Compound SFE HD

�-Pinene 0.8 2.8�-Pinene 1.5 3.7Myrcene 0.6 1.0p-Cymene – 5.6o-Cymene 7.8 0.1Limonene 6.8 10.6

olumns SFE-1 to SFE-4 refer to the oil fractions collected after each hour ofhe supercritical extraction. SFE-T is the overall essential oil obtained by SFE.

nd esters increased their GC area percentage in supercriticalarbon dioxide extracts obtained at a high fluid density relativeo hydrodistillate and the pentane extracts.

In 2000, Cassel et al. [145] compared results of hydrodistilla-ion and supercritical carbon dioxide methods for the extractionf Baccharis leave oil. They observed the non-oxygenatedonoterpenes, present in the hydrodistilled oil in high con-

ents (�-pinene 28.2% and limonene 10.6%), were not detectedn the supercritical carbon dioxide extracts. The selectivity ofupercritical carbon dioxide allowed one to maximize the con-entration of oxygenated compounds. Also, Caredda et al. [146]tudied supercritical carbon dioxide extraction of essential oilrom Laurus nobilis. Extraction conditions were as follows:ressure 90 bar, temperature 50 ◦C and carbon dioxide flow.0 kg/h. Extracted waxes were trapped in the first separatoret at 90 bar and −10 ◦C. The oil was recovered in the sec-nd separator held at 15 bar and 10 ◦C. The main componentsere 1,8-cineole (22.8%), linalool (12.5%), R-terpinyl acetate

11.4%), and methyleugenol (8.1%). Four classes, hydrocar-on monoterpenes (HMs), oxygenated monoterpenes (OMs),ydrocarbon sesquiterpenes (HSs), and oxygenated sesquiter-enes (OSs), on the basis of their chemical structure or retentionime were reported. The area percentages relative to each classre shown in Table 5. In general, volatile compounds (HMs) arextracted almost completely during the first hour of extraction12.67, against 0.31% in the fourth hour). The OMs decreased tominor extent from 79.43 to 66.68%. HSs and OSs are presentt, respectively, 15.52 and 17.07% in the fraction obtained after80–240 min and at 7.45 and 0.50 in the first hour sample. Theseesults confirm that a long time run is necessary to obtain oil withstable composition. Comparison with the hydrodistilled oil didot reveal any significant difference. The yields of each fractionf the supercritical extraction and hydrodistillation as w/w%,ith respect to the charged material, are reported in Table 6.

n the same table the amount of CO2 consumed in the process,xpressed as the specific mass of solvent, ms/m0 (m0 is the massf leaves charged in the extractor) is specified. The overall yieldf the supercritical extraction was 0.82%; 1,8-cineole (22.84%)as the major component.
In 2004, Pourmortazavi et al. [77] showed that differ-
nt extraction compositions could be obtained by differentxtraction methods applied to natural products. They studiedupercritical fluid extraction of volatile oil from J. communis

�

CC�

olumns SFE-1 to SFE-4 refer to the oil fractions collected after each hour ofhe supercritical extraction. SFE-T is the overall essential oil obtained by SFE.

. leaves using carbon dioxide was carried out under differ-nt conditions of pressure, temperature, modifier content andynamic extraction time. Then, they compared proposed extrac-ion method with hydrodistillation. A total 22 compounds haveeen determined in SFE extracts while in the hydrodistilledil only 11 components were identified and quantified. SFEroducts were found to be markedly different from the corre-ponding hydrodistilled oil. A large amount of �-phellandereneas present in the hydrodistilled essential oil, also the ratio of �-inene and 3-carene in distilled oil were high in comparison withhe supercritical carbon dioxide extracts. Their results showedhat under pressure 200 atm, temperature 45 ◦C and dynamicxtraction time of 30 min, SFE of limonene was more selectivend under pressure 350 atm, temperature 45 ◦C and dynamicxtraction time of 20 min, extraction was more selective for the-thoujone, which was not found in the hydrodistilled oil. Pour-ortazavi et al. [147] also showed that the composition of theFE products and the hydrodistilled black cumin essential oils

s significantly different. They compared the composition of theFE product with hydrodistilled oil and found a higher levelf the �-terpinene and cuminaldehyde in the hydrodistilled oil.able 7 shows the p-cymene content of the distilled oils is con-iderable, however, this compound was not found in the SFExtracts. The method contributes to the automation of pharma-eutical industry. Ebrahimzadeh et al. [148] isolated essential oilf Zataria multiflora Boiss, cultivated in Iran, by steam distil-

-Terpinene 38.0 45.7uminaldehyde 11.5 12.7uminyl alcohol – 6.4-Methyl-benzenemethanol 25.6 3.5

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owever, the quantitative compositions of the two products wereuite different; indeed, at higher pressures, and temperatures30.4 MPa and 55 ◦C) thymol and carvacrol are more soluble inupercritical carbon dioxide. In all obtained extracts, �-terpinenend carvacrol appear to be the major components, the extractseing richer in thymol. Moreover, the recovery of thymol in SFEs better than that steam distillation.

Aghel et al. [149] used orthogonal array design in order toptimize SFE of Mentha pulegium L. essential oil and com-ared their SFE results with hydrodistilled oil. They identified2 compounds consisting over than 91% of the total hydrodis-illation essential oil. The major components were pulegone37.8%), menthone (20.3%), piperitenone (6.8%) and para-entha-1,8-dien-2-one (5.1%). Over 81% of these compoundsere monoterpenes and 83.0% were oxygenated terpeneoids.he results of GC/MS analysis of nine supercritical fluidxtractions showed considerable differences between SFE andydrodistillation. The concentration of limonene in supercriticaluid extract at optimum condition was high (14.6%) in compari-on with hydrodistillation (trace). On the other hand, they have aelective extraction at optimum condition for SFE and only fourompounds (menthone, pulegone and limonene make up 96.9%f its constituents and the fourth compound was a methoxy phe-ol derivative by 3.1) were extracted from M. pulegium L. In004, Stashenko et al. [150] employed hydrodistillation (HD),imultaneous distillation solvent extraction, microwave-assistedydrodistillation (MWHD), and supercritical fluid (CO2) extrac-ion isolate volatile secondary metabolites from fresh leavesnd stems of Colombian Lippia alba Mill. They identifiedround 40 components in the various volatile fractions. Car-one (40–57%) was the most abundant component, followedy limonene (24–37%), bicyclosesquiphellandrene (5–22%),iperitenone (1–2%), piperitone (ca. 1.0%), and �-bourbonene0.6–1.5%), in the HD, SDE, MWHD, and SFE volatile frac-ions. Static headspace (S-HS), simultaneous purge-and-trapP&T) in solvent (CH2Cl2), and headspace solid-phase microex-raction (HS-SPME) were used to sample volatiles from fresh. alba stems and leaves. The main components that they

solated from the headspace of the fresh plant material wereimonene (27–77%), carvone (14–30%), piperitone (0.3–0.5%),iperitenone (ca. 0.4%), and �-bourbonene (0.5–6.5%). Innother study [151], HD, simultaneous distillation–solventxtraction, MWHD, and supercritical fluid (CO2) extractionere used to isolate volatile secondary metabolites from Colom-ian Xylopia aromatica (Lamarck) fruits. Static headspace,imultaneous purge and trap in solvent (CH2Cl2), and headspaceolid-phase microextraction were utilized to obtain volatileractions from fruits of aromatica trees, which grow wild inentral and South America, and are abundant in Colombia. �-hellandrene was the main component found in the HD andWHD essential oils, SDE and SFE extracts (61, 65, 57, and

0%, respectively), followed by �-myrcene (9.1, 9.3, 8.2 and.1%), and �-pinene (8.1, 7.3, 8.1 and 5.9%). The main compo-
ents present in the volatile fractions of the aromatica fruits,solated by S-HS, P&T and HS-SPME were �-phellandrene53.8, 35.7 and 39%), �-myrcene (13.3, 12.3 and 10.1%),-mentha-1(7),8-diene (7.1, 10.6 and 10.4%), �-phellandrene
J. Chromatogr. A 1163 (2007) 2–24

2.2, 5.0 and 6.4%), and p-cymene (2.2, 4.7 and 4.4%),espectively.

0. Conclusion

This article summarizes research finding involving the super-ritical fluid extraction of volatile components from plantaterials. Emphasis is placed on optimization of extraction

arameters for complete recovery of analytes from their matri-es. The future of supercritical fluid extraction for the extractionf volatile components from plants looks bright based on aumber of considerations. First, SFE has a wide applicationrea. It is capable of extracting a wide range of diverse com-ounds from variety of sample matrices. Many non-polar tooderately polar compounds can be extracted with carbon diox-

de while more polar compounds can be extracted with otheruids or modified carbon dioxide. Secondly, supercritical flu-

ds offer extraction selectivity unsurpassed by solvent polarity.hirdly, the environmental friendliness of this technique canever be disputed, since non-toxic fluids such as carbon dioxider this compound modified with up to 20% organic solventsre most commonly employed. Supercritical fluid extractionas faced a growing interest in the two past decades due to itsumerous advantages over classical liquid solvent extractionsmainly rapidity, selectivity, cleanliness, possibility of manipu-ating the composition of the extract and low solvent volumesequired). On the other hand, coupling established analytical sys-ems such as gas liquid chromatography with a comparativelyew technique has led to the development of complex, if notovel, sample extraction–preparation–analysis–detection superchemes to cope with difficult matrices in which essential oil oflants, in particular, are inevitably found.

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