GC

Anal. Chem. 1994,66, 888-892

Use of a Programmed Temperature Vaporizer for SFE/GC Analysis in Food Composition Studies Gracla P. Blanch, Elena Ibhfier, Marta Herralz, and Gulllermo Reglero' Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain

A simple procedure is proposed for off-line supercritical fluid extraction and capillary gas chromatography using a programmed temperature vaporizer (PTV)injector. The independent control of flow and temperature of the supercritical fluid is achieved by means of a variable restrictor (i.e., nozzle). The described system combinessome advantagesof the on-line coupling SFE/GC (high sensitivity and reduced sample handling) and the off-line approach (simpler performance and depressurization outside the chromatographic column). The proposed procedure and the solvent recovery method used to collect the extracted analytes are applied to the analysis of the essential oil obtained from Rosmarinus otZcinaalis L. Also, a comparative study with the extracts obtained by using the simultaneous distillation extraction technique (SDE) is presented.

The reliable analysis of foods usually requires maximum performance from the sample preparation step since some of the constituents possess low thermal stabilities or high reactivity. Also losses of trace volatile compounds can occur. Several isolation and concentration methods have been already reported, although none of them can be considered as a universal procedure. The simultaneous distillation-solvent extraction (SDE) 1-3 has proved its usefulness for extracting volatiles of various types of food samples without a subsequent concentration step,k8 including plant volatiles used frequently as raw material for flavoring food.9 Recently, we proposed a new SDE version that uses a more efficient cooling surface and allows the operation with solvents denser or lighter than water with only one configuration.1° On the other hand, the potentially wide applicability of supercritical fluids for the extraction of organic compounds from a great variety of matrices is widely recognized. The low viscosity and high diffusivity of supercritical fluids facilitates the mass transfer of solutes, thus allowing improved extraction efficiencies within shorter times. A further advantage is the fact that the solvent strength of supercritical ~~

~~

(1) Likens, S.T.; Nickerson, G. B. Proc. Am. Soc. Brew. Cfiem. 1964.22, 5-13. (2) Nickerson, G. B.; Likens, S.T.J . Cfiromatogr. 1966, 21, 1-5. (3) Godefroot, M.; Sandra, P.; Verzele, M. J. Cfiromarogr. 1981, 203, 325-35. (4) Nuflez, A. J.; Bemelmans, J. M. H.; Maarse, H. Cfiromatograpfiia 1984,18,

153-8. ( 5 ) Nuflez, A. J.; Bemelmans, J. M. H. J . Chromatogr. 1984, 294, 361-5. (6) de Frutos, M.; Sanz, J.; Martlnez-Castro, I. Cfiromatograpfiia 1988,25,861-

4. (7) Blanch. G. P.; Reglero, G.; Herraiz, M.; Tabcra, J. J . Cfiromatogr.Sci. 1991, 29, 11-5. (8) Blanch, G. P.; Tabera, J.; Sanz, J.; Herraiz, M.; Reglero, G. J. Agric. Food

Cfiem. 1992, 40, 1046-9. (9) Reglero, G.; Hcrraiz, M.; Hcrraiz, T.; Sanz, J. J . Cfiromarogr. 1989, 483, 43-50. (10) Blanch, G. P.; Tabera, J.; Herraiz, M.; Rcglero, G. J. Cfiromarogr. 1993,628, 261-8.

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Analytical Chemistty, Vol. 66,No. 6,March 15, 1994

fluids can be controlled just by modifying either its pressure or its temperature." SFE has been applied in food analysis for the determination of the natural chemical composition of different products and the analysis of added ingredients and food contaminants.12 Several reports have been already published regarding different methods for trapping analytes after the SFE depressurization step, mainly including use of an organic solvent as a collection medium,13J4 collection on a solid support,15-19collection on cryogenically cooled surfa~es,2"-~~ and on-line methods coupled to various chromatographic in~trumentation.~"~3 Supercritical fluid extraction (SFE) can be performed either purely for sample preparation (off-line approach) or for replacing the normal sample injection process into the chromatograph (on-line or "coupledn approach). Several advantages and disadvantages of both approaches have been already described.12 Off-line SFE is generally simpler to perform whereas on-line SFE allows maximum sensitivity to be achieved. According to several authors, the off-line approach should be the choice if a new sample preparation method must be developed since off-line optimization is more versatile than on-line o p t i m i ~ a t i o n . ~ ~ (1 1) Smith,R. M.,Ed.SuperctiticalFluidChromorograpfiy;RSCChromatography Monographs; RSC: London, 1988. (12) Lee, M. L.; Markidcs, K. E., Eds. AnolyricaiSupercriticol Fluid Chromatography and Exfraction; Chromatography Conferences, Inc.: Provo, UT, 1990. (13) Lee, H.-B.; Peart, T.E. J. Chromatogr. 1992,594, 309-15. (14) Verschuere, M.; Sandra, P.; David, F. J. Cfiromatogr. Sci. 1992,30,388-91. (15) Miller Schantz, M.; Cheder, S.N. J . Chromatogr. 1986, 363, 397401. (16) Schneiderman,M. A.; Sharma, A. K.; Lock, D. C. J . Chromatogr. 1987,409,

343-53.

(17) Saito, M.; Yamauchi, Y.; Inomata, K.; Kottkamp, W. J . Cfiromatogr. Sci. 1989,27, 79-85. (18) Hedrick, J. L.; Taylor, L. T.J . High Resolur. Chromatogr. 1990,13,312-6. (19) Squeira, A. J.; Taylor, L. T.J . Cfiromatogr. Sci. 1992, 30, 405-8. (20) Wright, B. W.; Wright, C. W.; Gale, R. W.; Smith, R. D. A w l . Chem. 1987, 59, 3 8 4 4 . (21) Licbman, S. A.; Levy, E. J.; Lurcott, S.;ONeil, S.;Guthrie, J.; Ryan, T.; Yocklovich, S. J . Cfiromatogr. Sci. 1989, 27, 118-26. (22) Anderson, M. R.; Swanson, J. T.; Porter, N. L.; Richter, B. E. J . Cfiromatogr. Sci. 1989, 27, 371-7. (23) Smith, R. M.;Burford, M. D. J . Cfiromatogr. 1992, 600, 175-81. (24) Hawthorne, S.B.; Miller, D. J. J . Cfiromarogr. 1987, 403, 63-76. (25) Wright, B. W.; Frye, S.R.; McMinn, D. G.; Smith, R. D. Anal. Cfiem. 1987, 59, 640-4.

(26) Levy, J. M.; Guzowski. J. P.; Huhak, W. E. H R C & CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1987. 10, 337. (27) Hawthorne, S. B.; Miller, D. J.; Krieger, M. S.Fresenius Z. Anal. Cfiem. 1988, 330, 21 1-5. (28) Hawthorne, S.B.; Krieger, M. S.; Miller, D. J. Anal. Cfiem. 1988,60,472-7. (29) Schmidt, S.;Blombcrg, L.; WHnnman, T.Cfiromotographio 1989,28,400-4. (30) Hawthorne, S. B.; Miller, D. J.; Krieger, M. S.J . Cfiromatogr. Sci. 1989, 27,

347-54.

(31) Hawthorne, S.B.; Miller, D. J.; Krieger, M. S.J. High Resolur. Cfiromatogr. 1989, 12, 714-20. (32) Levy, J. M.; Cavalier, R. A.; Basch, T. N.; Rynaski, A. F.; Huhak, W. E. J. Cfiromafogr. Sci. 1989, 27, 341-6. (33) Hawthorne, S.B.; Miller, D. J.; Langcnfeld, J. J. J . Cfiromarogr. Sci. 1990, 28, 2-8.

0003-2700/94/038&0888$04.50/0

0 1994 American Chemical Society

Most of the work performed on directly coupled SFE/GC employs an on-columninjection port,24*25*28-30,35 whereas other approaches involve the use of a hot split-splitless i n j e c t ~ r . ~ f ~ ~ , ~ ~ Also, the extract recovery can be accomplished by using direct interfacing to a temperature-programmable vaporizer (PTV).38J9 With respect to this method, the scarce investigation performed so far has involved the use of a fixed restrictor, which was directly inserted into the cooled liner of the PTV injector. Consequently, several problems derived from the use of such a type of restrictor (Le., risks of plugging, difficult replacement, incomplete transfer of labile or nonvolatile solutes without pyrolysis, or formation of analyte particles, etc.) may eventually affect the extraction efficiency and clearly limit the application of the method. The aim of this work was to evaluate the applicability of a PTV for collecting analytesextracted from complex mixtures by using the HP 7680A supercritical fluid extractor, which involves a variable restrictor to achieve the independentcontrol of flow and pressure. The proposed procedure and the solvent recovery method used to collect the extracted analytes were applied to the analysis of the essential oil obtained from Rosmarinus officinalis L. Also, a comparative study with the extracts obtained by using the simultaneous distillation extraction technique (SDE) was carried out.

C02GAS VENT

\\I/

IU

quartz wool

U

EXPERIMENTAL SECTION Plant Material. Leaves from plants of R. officinalis L. were collected and after being dried and milled were used for supercritical fluid extraction. Different quantities of plant material were used, as explained below, depending on the different procedures that have been investigated. Supercritical Fluid Extractions. SFE extractions were performed with a Hewlett Packard 7680A extraction module provided with a nozzle/trap assembly, which acts as a controllable variable restrictor, thus allowing instant depressurization of the supercritical fluid as well as the decoupling of flow and pressure. The extractionsystem is fully automated and includes a self-sealingextractioncell (a 7-mL thick-walled stainless steel thimble). During the extraction step, the obtained analytes were deposited on an internal trap where the supercritical fluid evaporates and leaves the system. Subsequently, a suitable solvent is pumped through the mentioned trap so that the analytes are rinsed off into a vial. As an alternative, we propose a modification of the SFE module configurationdescribed above in such a way that the quartz liner of a programmed temperature vaporizer (PTV) (100 mm X 1 mm id.) can be located in place of the analyte trap through an adequate steel fitting and sealed by a Teflon ferrule (Figure 1). The extracted analytes are spread out over a large surface area (i.e., the packing material used in the quartz liner) but rinsing with organic solvents is not (34) Sandra, P.; David, F.; Stottmeistcr, E. J. High Resolut. Chromutogr. 1990, 13,284-6. (35) Lohleit, M.; Biichmann, K. J. Chromatogr. 1990,505,227-35. (36) Levy, J. M.;Rosselli,A.C.;Boyer,D.S.;Cross,K. J.High Resolut.Chmmutogr. 1990,13,418-21. (37) Levy, J. M.; Storozynsky, E.; Ravey, R. M. J. High Resolut. Chromutogr. 1991,14,661-6. (38) Houben, R. J.; Janssen, H. M.; Lcclercq. P. A.; Rijks, J. A.; Cramers, C. A. J . High Resolut. Chromatogr. 1990, 13,669-73. (39) Huston, C. K.; Ji. H. J. Agric. Food Chem. 1991.39, 1229-33.

SF EXTRACT -re

1. Scheme of the noule-quartz liner assembly.

necessary since the subsequent gas chromatographic analysis is performed just by placing the quartz liner in the PTV body of a gas chromatograph. The thermal desorption of the analytes retained in the packing material is achieved by increasing the PTV temperature. In this work, SFE operations were performed in the two modes described above: (a) working with the original configuration of the HP 7680A SFE and (b) operating with the modification proposed consisting of the replacement of the ODS trap by the quartz liner of a PTV packed with a suitable material. Experimentation in both approaches was performed by considering different values for the involved variables. The investigated values were as follows: Approach a (SFEIODSTrapping). Supercritical CO2 flow: 0.5, 1, 2, 3, and 4 mL/min. Sample weight in the cartridge: 250 and 1000 mg. Extraction time: 2,4,6,8, 10, 20,30, and 110min. COzdensity: 0.25,0.50, and 0.95 g/mL. Trap temperature: 10and 30 OC. The analyte trap was packed with Hypersil octadecylsilica (ODS) (30-40 pm diameter) and a void volume of about 540 pL/trap. The effectiveness of the modifiers was tested by adding water or methanol (10 pL each) on the top of the sample in the extraction cartridge. After extraction, the trap was rinsed with 1 mL of dichloromethane at a rate of 1 mL/min. A 2-pL injection volume was used for the subsequent gas chromatographic analysis. Approach b (SFEIPTVIGC). Supercritical CO2 flow: 2,3, and 4 mL/min. Sample weight in the cartridge: 10,20, and 250 mg. Extraction time: 20, 26.6, and 40 min were established depending on whether the supercriticalC02 flow was respectively 4,3, or 2 mL/min, so that a constant volume AnalytcalChemkby, Vol. 66, No. 6, March 15, 1994

009

of C02 (80 mL) was passed through the extraction cell in eachcase. Modifiers: Water or ethanol (1OpL each). Length of the adsorbent in the quartz liner: 1,3, and 5 cm. Packing material: Glass beads, GasChrom 220, Tenax TA, Thermotrap TA, and Volaspher A-2 silanized (all of them 80/ 100 mesh). Steam DistillationSolvent Extraction (SDE). SDE extracts were achieved from 10 g of leaves of R. officinalis L. and 200 mL of water purified in a Milli-Q system (Millipore). As the extraction solvent, a 2-mL volume of doubly distilled dichloromethane was employed. The SDE apparatus used, which has been described in a previous paper,'O was constructed in our laboratory by modifying the device formerly designed by Godefroot et al.3 and allows operation at both normal pressure and reduced pressure as well as the simultaneous distillation and extraction of the headspace of the sample to be analy~ed.~O In this work, the analyses of the plant material were carried out by operating the SDE apparatus at normal pressure. Experimental conditions were established according to the values resulting from the optimization procedure previously performedl0 by using the sequential Simplex method.41 As a result, the following experimental conditions were used: sample heating bath temperature, 148.7 OC; solvent heating bath temperature, 66.8 OC;coolant temperature, 3.4 OC; and extraction time, 56.3 min. Cleaned boiling chips were added to the sample and solvent flasks, and the SDE device was cleaned between successive runs by rinsing it with acetone and Milli-Q purified water. After completion of the SDE operation, further concentration was not required and a 2-pL volume of the extract was subsequently analyzed by capillary GC . Gas Chromatographic/Mass Spectrometric Analysis. All the GC analyses were performed on a Perkin Elmer Model 8500 gas chromatograph equipped with a PTV Perkin Elmer injector and a FID. A Model 2600 chromatography software system (Nelson Analytical) was used. Detector temperature was maintained at 250 OC, and helium was used as the carrier gas. A 30 m X 250 pm i.d. capillary column (Quadrex) with a 0.25-pm film of Carbowax 20 M was employed for the analysis of the SFE extracts. The column oven program consisted of a 5-min initial hold at 50 OC, a first ramp to 90 OC at 5 "C/min, and a second ramp to 180 OC at 3 OC/min after a hold time of 10 min at 90 O C . Injections of the SFE/ ODS extracts were carried out in the split mode (splitting ratio 1O:l). In the SFE/PTV/GC approach, the splitting ratio was 30: 1. In both cases the vaporizer was maintained at 30 OC upon sample introduction; this temperature was increased at 14 OC/s to 300 OC and held at the final temperature for 5 min. The gas chromatographic analysis of the SDE extracts was performed by using a 50 m X 220 pm i.d. capillary column coated with 0.25 pm BP-21 (SGE). The column was temperature programmed from 40 OC (4 min) to 180 O C (8 min) at 5 OC/min. The injector was held at 30 OC upon injection, and a split ratio of 10:1 was established; subsequently (40) Blanch, G. P.; Reglcro, G.; Tabcra, J.; Hcrraiz, M. J . Chromarogr.1993,655, 141-9. (41) Hcndrix, C. CHEMTECH 1980, 488-97.

890

Anelytlcal Chemistty, Vol. 66, No. 6,Mrch 15, 1994

Table 1. Reldlve SmmlWvtty Achkvablo by Using the SFE/ODS, SFE/PN, and $DE Procedurrr

SFE/ODS SFE/PTV s u m of areasa sample weight (mg) s u m of areas/sample weight

236 272 lo00 236.3

858 353

20 42 917.6

SDE 1 439 443 10 OOO 143.9

OAver e value (n = 5) of the s u m of the absolute peak areas corresposing to the compounds listed in Table 2.

the PTV was ballistically heated to 300 OC, and this temperature was maintained for 5 min. To confirm peak identification, the gas chromatograph was linked to a Perkin Elmer ITD-50 ion trap detector (E1 70 eV). Compounds were identified by comparing the spectra with those of the NBS (National Bureau of Standards) library and by matching their spectral data with those corresponding to pure standards analyzed under identical conditions. Safety Considerations. Special care must be taken to ensure that the fittings used in SFE operations are capable of withstanding the required extraction pressures.

RESULTS AND DISCUSSION Data obtained by performing the experimentation in the different conditions given in the Experimental Section were compared for the qualitative and quantitative SFE analysis of R. officinalis L. Theconditions yielding the highest recovery were as follows: Approach a (SFEIODS trapping). CO2 flow, 4 mL/min; sample weight, 1000mg; extraction time, 20 min; C02 density, 0.95 g/mL (carbon dioxide conditions, T = 40 OC; P = 383 atm); and trap temperature, 30 OC. Approach b (SFEIPTVIGC). COz flow, 3 mL/min; sample weight, 20 mg; extraction time, 26.6 min; C02 density, 0.95 g/mL (carbon dioxide conditions, T = 40 O C ; P = 383 atm); modifier, 10 p L of ethanol; length of the adsorbent in the quartz liner, 5 cm; sorbent in the collector trap, 15 mg; and packing material, GasChrom 220. As to the sample weight used in conjunction with the PTV trap, it should be mentioned that sample amountsfar exceeding the selected value (20 mg) are also feasible although column overloading can be eventually observed. Figures 2 and 3 show, respectively, the chromatograms obtained from the SFE extracts of R. officinalis L. leaves resulting from both the ODS trapping and the SFE/PTV operation modes in the experimental conditions mentioned above. As far as the SDE procedure of sample preparation is concerned, Figure 4 depicts the chromatogram resulting from the obtained extract. It should be emphasized that the chromatograms are recorded at the same sensitivity so that they are illustrative of the benefits in sensitivity of the use of a PTV for the SFE/GC analysis, although it should be taken into account that the different chromatographic conditions used may have slightly affected the data. Table 1 gives the relative sensitivity of the SFE/ODS, SFE/PTV, and SDE analysis. The highest sum of areas corresponds to the SDE although it must be considered that the sample weight used in this procedure is 10 g, whereas 1 g of the sample was required to carry out the SFE extraction of R. officinalis L. by working with the original configuration of the extractor and only 20 mg was needed when the SFE/

1 2

1'

7

3 4 6

7

1112

2

10

9

6

5

t 10 20 30 min. 0 Flguro 2. Chromatogram of 2 NL of an SFEIODS extract from Ro8mefinus ofWne/Is L. leaves injected in the spill mode (spilt ratio, 10:1; column b a d pressure, 22 psig). See text for fwther condltlons. Peak identlficetbn as In Table 2.

Tabh 2. CoMcknb of VIllrHon of Robtlvo Poak Aroar Obtainad trom 8FE Extract of RamMknrr m W 8 L. by Udng an ODs trap

peak no.

compound a-pinene camphene limonene l,&cineole y-terpinene pcymene camphor linalool bomyl acetate caryophyllene a-terpineol bomeol

1 2 3 4 5 6 7 8 9 10 11 12

cv

(%I4

10.3 11.2 8.9 10.1 7.5 8.3 4.6 3.5 1.8 2.8 3.0

cv ( % ) b 10.8 5.5 10.1 4.6 7.5 10.1 7.9 9.8 7.4 9.6 7.8 8.4

-

a Coefficient of variation (n = 5) for the ratio of the absolutepeak areas of each peak to that for standard (camphor). Coefficient of variation (n 5) of normalized peak areas.

PTV/GC analysis was carried out. In terms of relative sensitivity (sum of areas divided by the sample weight), the last method resulted clearly superior in spite of the fact that the corresponding chromatographic analysis was performed at the highest split ratio (301). It should be emphasized, however, that increased sensitivity for both the ODS and the SDE procedures could have been obtained by concentration of the extracts. Tables 2-4 show the coefficientsof variation obtained from five analyses performed, respectively, with the ODS trap, the FTV collector trap, and the SDE apparatus. Each table includes data obtained from relative (normalized)peak areas

30 min 0 10 20 Flguro 3. Chromatogram obtained by the thermal desorption of the SFE extract from R m f i n u s offfchlk L. leaves adsorbed in the glass liner of a proorammed temperaturevaporizer (spill ratb, 3 0 1; d u m n head pressure, 30 p a ) . Sea text for further conditions. Peak Identiflcatkn as in Tabk 2.

Table 3. C o d f k h b of Varlatkn of Rolrtlvo Poak koas Obtalnod from WE Extract ot R e " # Mkhah L. by Uslng Qlrrrr LInw of a PN for co(kcung th. Extractod Andytn

peak no. 1 2 3 4 6 6 7 8 9 10 11 12

compound a-pinene camphene limonene l,&cineole y-terpinene p-cymene camphor linalool bomyl acetate caryophyllene a-terpineol bomeol

cv

(%)4

4.6 6.3 6.2 3.3 8.9 7.1 5.4 10.3 10.3 1.8 1.6

cv ( % ) b 3.9 2.0 7.1 7.1 8.8 9.7 5.7 4.0 3.9 8.8 4.7 5.3

4 Coefficient of variation (n= 5) for the ratio of the absolute peak arm of each peak to that for standard (camphor). Coefficient of variation (n = 5) of normahzed peak areas.

as well as data resulting from considering the ratios of the absolute peak areas of every peak to the one selected as standard (camphor). As can be seen, values obtained when the PTV is used to collect the extracted analytes can be considered, in general, as acceptable, especially if we bear in mind the high sensitivity of the overall analysis. On the other hand, it is clear that further optimization of the proposed technique, mainly concerning the type and characteristics of the packed material used in the glass liner, might significantly enlarge the reliability and applicability of the analysis. Table 5 gives the composition of volatile compounds determined by the three investigated methods from the normalized peak areas obtained, assuming the same response Analytical chemlstty, Vol. 66, No. 6, March 15, 1994

891

7

Table 5. ComporHionaof VoktHo CompOrmdr R o k r r r d from Roamad” o#kh&b L. Loavoo: Compatbon Ot Data ReautHq from Uahg SFEIODS, SFWPN, and SDE Pr0codur.r

1

6

no.

compound

SFE/ODS

SFE/PTV

SDE

1 2 3 4 5 6 7 8 9 10 11 12

a-pinene camphene limonene l,&cineole gamma-terpinene p-cymene camphor (ref.) linalool bomyl acetate caryophyllene a-terpineol borneol

16.1 1.3 0.4 14.0 0.3 0.6 28.3 1.3 2.4 3.8 12.2 19.1

14.6 6.1 1.2 24.6 0.6 1.6 21.5 1.2 2.0 2.7 10.1 14.1

10.4 5.4 1.0 26.8 0.4 1.3 23.4 0.7 0.2 1.9 6.4 22.1

Rslative amount (%) obtained from normalized peak areas.

I,

-fc11 0

10

min

20

.

Fi@um4. Chromatogramof 1MLof an SDE extract from Rosmrlnus oMm/bL. leaves InJectedIn the spm mode (split ratlo, 1 01: column head pressure, 40 pslg). See text for further conditions. Peak ldentiflcatlon as In Table 2. Tabk 4. CoMdonir of Variation of Rdrtlvo Po& Arom Obtalnd by Anatyzlng Ro#na~MwMchahb L. by SDE

peak no.

compound

1 2 3 4 5 6 7 8 9 10 11 12

a-pinene camphene limonene l,&cineole y -terpinene p-cymene camphor linalool bornyl acetate caryophyllene a-terpineol bomeol

cv (%)a 7.3 7.7 6.1 2.8 8.7 3.5 7.1 6.2 2.4 11.8 7.4

cv (%)* 5.2 5.2 7.7 3.2 8.7

4.7 3.0 2.1 12.8 1.2 10.1 5.6

a Coefficient of variation (n = 6)for the ratio of the abeolu+ peak areas of each peak to that for standard (camphor). b Coefficient of variation (n = 5) of normalized wak areas.

factor for each compound. As can be seen, results of SFE/ PTV/GC are similar to those obtained by SDE and SFE/ ODS,with a-pinene, 1,8-cineole, camphor, a-terpineol, and borneol being the main contributors to the essential oil composition. A significant feature of the procedure we propose is that it is inherently simpler to perform than on-line SFE since extraction and analyte collection steps and the chromatographic separation conditionscan be optimized independently. Moreover, the approach we have investigated also allows us to benefit from the main advantage of on-line SFE,Le., the ability to quantitatively transfer all of the extracted analytes to the chromatographic system. This also results in the

reduction of the potential for analyte loss and degradation. On the other hand, the sample handling between extraction and chromatographic separation is quite simple and fast as it only requires the removal of the glass liner from the SFE extractor and the insertion of it into the body injector assembly of the gas chromatograph. The overall procedure involving SFE extraction and GC analysis can be performed in less than 2 h. Obviously,the dilution of the extracted analytes to remove them from the trap, as required in the commercial configuration of the extractor, decreases the overall sensitivity attainable with the analysis so that the alternative which we propose may be an interesting choice for analyzing trace compoundsby combining the benefits of SFE concerningmass transfer with the high resolution achieved with GC capillary columns and the high sensitivity provided by the direct introduction of the SFE extracted compounds into the chromatographic column, although a possible disadvantage of the method is that no multiple analysis can be performed from a single extract. The proposed system allows the SFE operation at very different COz flows due to the use of the above-described variable restrictor. In this respect, it should be emphasized that the previous use of a PTV for SFE/GC interface did not involve this possibility. Also, the very small sample sizes required if the PTV collector trap is used may be of great interest for chemotaxonomic studies of rare or expensive plants.

ACKNOWLEDGMENT This work was made possible by financial assistance from the Comisi6n Interministerial de Ciencia y Tecnologh Project AL191-0621. G.P.B. and E.I. thank the Comunidad de Madrid and the Ministerio de Educaci6n y Ciencia, respectively, for their grants. The authors are also grateful to Hewlett-Packard (Madrid, Spain) for the generous loan of the H P 7680A supercritical fluid extractor module and also to Mr. Josd M. Campos and Mr. Josd Martln from Centro Nacional de Qulmica OrgAnica (CSIC) for their skillful assistance with the nozzle-glass liner assembly. Recehred for revlew August 5, 1003. Accepted December 6, 1003.@ Abstract published in Aduancr ACS Abstructs. January IS. 1994.