- Email: [email protected]
Reverse-micelle formation: a strategy for enhancing CO2-supercritical fluid extraction of polar analytes
Analytica Chimica Acta 358 (1998) 1±4
Letter
Reverse-micelle formation: a strategy for enhancing CO2-supercritical ¯uid extraction of polar analytes M.M. JimeÂnez-Carmona, M.D. Luque de Castro* Department of Analytical Chemistry, Faculty of Sciences, University of CoÂrdoba, E-14004 CoÂrdoba, Spain Received 11 September 1997; accepted 28 September 1997
Abstract Reverse-micelle formation is revealed as a useful tool for improving the extraction of polar species by a non-polar extractant as supercritical (SC) CO2. As an example, the extraction of a medium polar analyte (cholesterol) by supercritical CO2 is dramatically improved in the presence of a non-ionic reverse micelle-former such as Triton X-100 (recoveries increased more than three times with respect to those obtained in the absence of the surfactant). Quantitation of the target analyte is performed by the Liebermann±Burchard photometric method, which provides a linear determination range between 40 and 500 mg/ml, with precision, expressed as relative standard deviation (R.S.D) of 2.62% for n7. # 1998 Elsevier Science B.V. Keywords: Reverse-micelles; Carbondioxide; Supercritical ¯uid extraction; Polar analytes
1. Introduction Supercritical ¯uid extraction (SFE) has proved to be one of the most signi®cant techniques for solid sample pretreatment in the last few years [1]. The majority of analytical supercritical ¯uids extractions have used supercritical (SC) CO2 as extractant agent because of its prefered critical properties, low toxicity and chemical inertness. Although CO2 is an excellent solvent for non-polar analytes, the main shortcoming of this extractant is its low dielectric constant, which makes the extraction of polar and ionic analytes dif®cult. For instance, analytes such as alcohol phenol ethoxylate [2] and clembuterol [3] are not extracted at all when pure CO2 is used as extractant. *Corresponding auhtor. Fax: +34 (57) 218606. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00573-4
The strategies used to facilitate the SFE of polar and ionic compounds [4] are based on two general principles. The ®rst is the increasing the polarity of the CO2 used as extractant, by means of the change of physical parameters of supercritical CO2, (e.g. the raising of the pressure [5]), by using supercritical ¯uids other than CO2. For example, one of the most promising alternatives in this ®eld is the suband supercritical water that allow even class-selective extractions to be achieved by raising the temperature of water from sub- to supercritical values [6]. Another way of increasing the polarity of the CO2 is the addition of cosolvents, mainly methanol [7]. A second group of strategies used for the enhancing of the SFE of polar and ionic compounds is the one based on reducing the polarity of the analyte to be extracted, namely: Ion-pair formation [3], esteri®cation and analogous reactions [8], organometallic
2
M.M. JimeÂnez-Carmona, M.D. Luque de Castro / Analytica Chimica Acta 358 (1998) 1±4
compound formation [9] and complex formation [10]. Reverse-micelle formation is presented in this paper as a new way of enhancing the solubility of the analyte in SC-CO2 of the analyte. The extraction of cholesterol into SC-CO2 is enhanced by using a non-ionic surfactant as the reverse micelle forming reagent. The reverse-micelle formed is less polar than cholesterol and so more soluble in SC-CO2.
an automated variable-diameter restrictor which provided a constant ¯ow-rate during the extraction process virtually avoiding plugging. In the subsequent rinsing step of the trap, a 2-propanol stream at 0.5 ml/ min ¯ow-rate was pumped through the trap by a syringe pump. The restrictor and trap temperatures were 308C during the extraction and rinsing step.
2. Experimental
Diatomaceous earth (0.5 g) were spiked with 1 ml of cholesterol stock solution. This solid matrix was chosen as an example because it was easy to handle. Once homogenized and evaporated until dryness, a 1 ml aliquot of microemulsion containing Triton X-100 was added. The sample was stirred vigorously with a magnetic stirrer in a glass-vessel, following a 10 min drying stage at 708C, after which it was added to the extraction cell. The extract was collected in vials which had been previously weighed in order to determine the weight of the extract as the difference. The volume of the extract was obtained from the weight of vial containing the extract, expressed in g, divided by the rinse solvent density, expressed in g/ml data, which was more precise than that obtained from the values of the volume dispensed by the extractor. Determination of cholesterol in the extracts was performed using the Liebermann±Burchard reagent and spectrophotometrically measuring the absorbance at 630 nm. Recoveries were calculated from the ratio extracted mg obtained by the spectrophotometric measure meant/ added mg to the extraction chamber.
2.1. Instrumentation A Hewlett-Packard HP 7680A supercritical ¯uid extractor equipped with a 7 ml thimble extraction cell and an analyte trap packed with small stainless steel balls was used in these studies. A Philips PU 8625 UVspectrophotometer was used for the determination of cholesterol in the extracts. 2.2. Materials A standard stock solution of cholesterol (99%, Sigma, St. Louis, MO, USA) of 1.7 g lÿ1 in 2-propanol (Merck, Darmstadt, Germany) was prepared. Diatomaceous earth (Sigma) was used as solid support. SFE/SFC grade CO2 (Air Products, France) and 2-propanol (Merck) were used as the extractant and solvent for rinsing the trap, respectively. Microemulsions of Triton X-100 (Serva, Heidelberg, Germany) 13.3%cyclohexane (Merck) 73.3%1-butanol (Merck) 13.3%; cetyltrimethylammonium bromide, CTAB (Serva) 13.3%cyclohexane (Merck) 73.3%1-butanol (Merck) 13.3% and dodecyl sulfate sodium salt (Merck) 13.1%cyclohexane (Merck) 65.9%1-butanol (Merck) 20.9% were tested as reverse-micelle former reagents. All percentages are expressed as w/w. 2.3. Extraction working conditions The CO2 was delivered from a cylinder supplied with a dip tube, fed by a double piston pump and passed through the 7 ml extraction cell which contained the sample. The extracted cholesterol was delivered to a 0.45 ml stainless-steel bead trap through
2.4. Procedure
3. Results and discussion Preliminary experiments were carried out to determine the extraction ef®ciency of cholesterol with SCCO2 and an extraction time of 10 min. This produced recoveries of 20%. 3.1. Optimization of variables The experimental variables were optimized in order to maximize the recovery of cholesterol in a time interval as short as possible. The univariate method was used for this purpose.
M.M. JimeÂnez-Carmona, M.D. Luque de Castro / Analytica Chimica Acta 358 (1998) 1±4
3
Table 1 Influence of micelle former on the SFE of cholesterol a Drying time c
Percent recovery of cholesterol S.D b (in the presence of) Triton X-100 microemulsion
SDS microemulsion
CTAB microemulsion
0 10
39.91.4 65.52.8
17.53.0 26.32.0
17.02.6 57.21.7
a
Experimental conditions: extraction time, 10 min; CO2 flow-rate, 1 ml/min; temperature, 408C and CO2 density, 0.8 g/ml. Here n3. c Min at 708C. b
3.1.1. Surfactant variables Both the type and the concentration of the surfactant in the microemulsion was studied in order to optime the recovery of cholesterol. Experimental conditions used for this study are listed in Table 1. Microemulsions of non-ionic (Triton X-100), anionic (SDS) and cationic (CTAB) surfactants were tested as reversemicelle forming reagents. Both the recoveries obtained and the effect of a drying stage are summarised in Table 1, where the experimental conditions are also listed. The use of the Triton X-100 microemulsion (with a drying stage prior to extraction) increases the recovery of cholesterol ca. 40%. So, this reagent was selected for further experiments. In order to obtain the optimum surfactant concentration to enhance the extraction of cholesterol, microemulsions between 110ÿ5 and 3.7210ÿ1 M of Triton X-100 were used. The optimum recovery (62.2%) was obtained for a concentration 1.8610ÿ1 M, which coincides with that found by Lee and Biellmann [11] for optimal formation of reverse micelles. Hence, this concentration of surfactant was selected as optimal.
further experiments. The effect of temperature was studied at a constant CO2 density of 0.8 g/ml, which allowed a wider range of temperatures than 0.9 g/ml (because of instrumental pressure limitations) to be studied. The temperature range over which the effect was investigated was 40±838C. Increased temperature resulted in increased recoveries, the recovery at 838C and 0.8 g/ml being 83.0% versus a recovery of 87.5% at 408C and 0.95 g/ml. Thus, a temperature of 408C was selected as optimum. The effect of ¯ow rate was studied under the following working conditions: CO2 density, 0.8 g/ml; extraction time, 10 min and temperature 408C. The recovery increased up to 2 ml/min. Higher ¯ow-rates provided a constant recovery of 74%. So, 2 ml/min was the value chosen as optimum. The effect of extraction time was studied under optimum working conditions in the range 0±20 min and quantitative exctraction of cholesterol was achieved in 18 min. The ranges of experimental variables studied and optimum values of the variables for the SFE of cholesterol from diatomaceous earth are summarised in Table 2.
3.1.2. SFE variables Experimental variables such as CO2 density, temperature of the extraction chamber, extraction time and CO2 ¯ow-rate were optimised in order to maximize the recovery of cholesterol in an extraction time as short as possible. A univariate method was used for this purpose. The effect of CO2 density was studied within the range 0.7±0.95 g/ml (pressures from 115 to 383 bar) under the following conditions: extraction time, 10 min; CO2 ¯ow-rate, 1 ml/min and temperature, 408C. Increased density resulted in increased recovery with a maximum of 87.5% for a density of 0.95 g/ml. So, this value was selected as optimum for
3.2. Features of the method The calibration graph was run by measuring photometrically cholesterol standard solutions. The range of Table 2 Optimization of extraction variables Variables [Triton X-100] (M) CO2 density (g/ml) Temperature (8C) Flow-rate (ml/min) Extraction time (min)
Range studied ÿ5
Optimum value ÿ1
110 ±3.7210 0.7±0.95 40±83 0.5±3 0±20
1.8610ÿ1 0.95 40 2 18
4
M.M. JimeÂnez-Carmona, M.D. Luque de Castro / Analytica Chimica Acta 358 (1998) 1±4
linearity is 40±500 mg/ml of cholesterol, the equation obtained is Y 2:21687 10ÿ3 X 0:0124046 where Y is the absorbance and X is the concentration of cholesterol in mg/ml, and the regression coef®cient is 0.998. The detection and determination limits are 15 and 40 mg/ml, respectively. The precision of the photometric step used for the determination of analyte in the extracts was obtained by reading the absorbance for standard solutions of 100 mg/ml of cholesterol. The R.S.D. (%) was 2.62 for n7. 4. Conclusions and future developments As shown in Table 2, the recovery on the extraction of a medium polar analyte such as cholesterol improved more than three times in the presence of a non-ionic reverse-micelle forming compounds such as Triton X-100. A 18 min extraction time is suf®cient for quantitative removal of cholesterol versus 30 min [12] neccessary in the absence of surfactant. New posibilities from this research are: 1. Use of enzymic reactions in order to improve the derivatization/determination step in terms of sensitivity and selectivity, thus making possible the application of the method to complex samples with low cholesterol content, such as low-cholesterol foods and certi®ed reference materials. 2. A further study of the effect of non-ionic and ionic micelle forming reagents on the extraction of analytes of different polarity.
3. Influence of micelle-formation on the extraction of non-polar analytes by a polar extractant such as supercritical water. Acknowledgements ComisioÂn Interministerial de Ciencia y TecnologõÂa (CICyT) is thanked for ®nancial support (Project PB95-1265) References [1] M.D. Luque de Castro, M. ValcaÂrcel, M.T. Tena, Analytical Supercritical Fluid Extraction, Springer, Heidelberg, 1994. [2] M. Kane, J.R. Dean, S.M. Hitchen, C.J. Dowle, R.L. Tranter, Analyst 120 (1995) 355. [3] M.M. JimeÂnez-Carmona, M.T. Tena, M.D. Luque de Castro, J. Chromatogr. A 711 (1995) 269. [4] M.D. Luque de Castro, M.T. Tena, Strategies for supercritical fluid extraction of polar and ionic compounds, Trends Anal. Chem. 15 (1996) 32±37. [5] S.B. Hawthorne, D.J. Miller, J. Chromatogr. Sci. 24 (1986) 258. [6] S.B. Hawthorne, Y. Yang, D.J. Miller, Anal. Chem. 66 (1994) 2912. [7] Y. Yang, A. Gharaibeh, S.B. Hawthorne, D.J. Miller, Anal. Chem. 67 (1995) 641. [8] S.B. Hawthorne, D.J. Miller, D.E. Nievens, D.C. White, Anal. Chem. 64 (1992) 405. [9] Y. Cai, R. Alzaga, J.M. Bayona, Anal. Chem. 66 (1994) 1161. È vila, M. Alcaraz, Anal. Chem. 66 (1994) [10] Y. Liu, V. LoÂpez-A 3788. [11] K.M. Lee, J.F. Biellmann, Bioorg. Chem. 14 (1986) 262± 273. [12] C.P. Ong, H.M. Ong, S.F.Y. Li, H.K. Lee, J. Microcol. Sep 2 (1990) 69.
Letter
Reverse-micelle formation: a strategy for enhancing CO2-supercritical ¯uid extraction of polar analytes M.M. JimeÂnez-Carmona, M.D. Luque de Castro* Department of Analytical Chemistry, Faculty of Sciences, University of CoÂrdoba, E-14004 CoÂrdoba, Spain Received 11 September 1997; accepted 28 September 1997
Abstract Reverse-micelle formation is revealed as a useful tool for improving the extraction of polar species by a non-polar extractant as supercritical (SC) CO2. As an example, the extraction of a medium polar analyte (cholesterol) by supercritical CO2 is dramatically improved in the presence of a non-ionic reverse micelle-former such as Triton X-100 (recoveries increased more than three times with respect to those obtained in the absence of the surfactant). Quantitation of the target analyte is performed by the Liebermann±Burchard photometric method, which provides a linear determination range between 40 and 500 mg/ml, with precision, expressed as relative standard deviation (R.S.D) of 2.62% for n7. # 1998 Elsevier Science B.V. Keywords: Reverse-micelles; Carbondioxide; Supercritical ¯uid extraction; Polar analytes
1. Introduction Supercritical ¯uid extraction (SFE) has proved to be one of the most signi®cant techniques for solid sample pretreatment in the last few years [1]. The majority of analytical supercritical ¯uids extractions have used supercritical (SC) CO2 as extractant agent because of its prefered critical properties, low toxicity and chemical inertness. Although CO2 is an excellent solvent for non-polar analytes, the main shortcoming of this extractant is its low dielectric constant, which makes the extraction of polar and ionic analytes dif®cult. For instance, analytes such as alcohol phenol ethoxylate [2] and clembuterol [3] are not extracted at all when pure CO2 is used as extractant. *Corresponding auhtor. Fax: +34 (57) 218606. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00573-4
The strategies used to facilitate the SFE of polar and ionic compounds [4] are based on two general principles. The ®rst is the increasing the polarity of the CO2 used as extractant, by means of the change of physical parameters of supercritical CO2, (e.g. the raising of the pressure [5]), by using supercritical ¯uids other than CO2. For example, one of the most promising alternatives in this ®eld is the suband supercritical water that allow even class-selective extractions to be achieved by raising the temperature of water from sub- to supercritical values [6]. Another way of increasing the polarity of the CO2 is the addition of cosolvents, mainly methanol [7]. A second group of strategies used for the enhancing of the SFE of polar and ionic compounds is the one based on reducing the polarity of the analyte to be extracted, namely: Ion-pair formation [3], esteri®cation and analogous reactions [8], organometallic
2
M.M. JimeÂnez-Carmona, M.D. Luque de Castro / Analytica Chimica Acta 358 (1998) 1±4
compound formation [9] and complex formation [10]. Reverse-micelle formation is presented in this paper as a new way of enhancing the solubility of the analyte in SC-CO2 of the analyte. The extraction of cholesterol into SC-CO2 is enhanced by using a non-ionic surfactant as the reverse micelle forming reagent. The reverse-micelle formed is less polar than cholesterol and so more soluble in SC-CO2.
an automated variable-diameter restrictor which provided a constant ¯ow-rate during the extraction process virtually avoiding plugging. In the subsequent rinsing step of the trap, a 2-propanol stream at 0.5 ml/ min ¯ow-rate was pumped through the trap by a syringe pump. The restrictor and trap temperatures were 308C during the extraction and rinsing step.
2. Experimental
Diatomaceous earth (0.5 g) were spiked with 1 ml of cholesterol stock solution. This solid matrix was chosen as an example because it was easy to handle. Once homogenized and evaporated until dryness, a 1 ml aliquot of microemulsion containing Triton X-100 was added. The sample was stirred vigorously with a magnetic stirrer in a glass-vessel, following a 10 min drying stage at 708C, after which it was added to the extraction cell. The extract was collected in vials which had been previously weighed in order to determine the weight of the extract as the difference. The volume of the extract was obtained from the weight of vial containing the extract, expressed in g, divided by the rinse solvent density, expressed in g/ml data, which was more precise than that obtained from the values of the volume dispensed by the extractor. Determination of cholesterol in the extracts was performed using the Liebermann±Burchard reagent and spectrophotometrically measuring the absorbance at 630 nm. Recoveries were calculated from the ratio extracted mg obtained by the spectrophotometric measure meant/ added mg to the extraction chamber.
2.1. Instrumentation A Hewlett-Packard HP 7680A supercritical ¯uid extractor equipped with a 7 ml thimble extraction cell and an analyte trap packed with small stainless steel balls was used in these studies. A Philips PU 8625 UVspectrophotometer was used for the determination of cholesterol in the extracts. 2.2. Materials A standard stock solution of cholesterol (99%, Sigma, St. Louis, MO, USA) of 1.7 g lÿ1 in 2-propanol (Merck, Darmstadt, Germany) was prepared. Diatomaceous earth (Sigma) was used as solid support. SFE/SFC grade CO2 (Air Products, France) and 2-propanol (Merck) were used as the extractant and solvent for rinsing the trap, respectively. Microemulsions of Triton X-100 (Serva, Heidelberg, Germany) 13.3%cyclohexane (Merck) 73.3%1-butanol (Merck) 13.3%; cetyltrimethylammonium bromide, CTAB (Serva) 13.3%cyclohexane (Merck) 73.3%1-butanol (Merck) 13.3% and dodecyl sulfate sodium salt (Merck) 13.1%cyclohexane (Merck) 65.9%1-butanol (Merck) 20.9% were tested as reverse-micelle former reagents. All percentages are expressed as w/w. 2.3. Extraction working conditions The CO2 was delivered from a cylinder supplied with a dip tube, fed by a double piston pump and passed through the 7 ml extraction cell which contained the sample. The extracted cholesterol was delivered to a 0.45 ml stainless-steel bead trap through
2.4. Procedure
3. Results and discussion Preliminary experiments were carried out to determine the extraction ef®ciency of cholesterol with SCCO2 and an extraction time of 10 min. This produced recoveries of 20%. 3.1. Optimization of variables The experimental variables were optimized in order to maximize the recovery of cholesterol in a time interval as short as possible. The univariate method was used for this purpose.
M.M. JimeÂnez-Carmona, M.D. Luque de Castro / Analytica Chimica Acta 358 (1998) 1±4
3
Table 1 Influence of micelle former on the SFE of cholesterol a Drying time c
Percent recovery of cholesterol S.D b (in the presence of) Triton X-100 microemulsion
SDS microemulsion
CTAB microemulsion
0 10
39.91.4 65.52.8
17.53.0 26.32.0
17.02.6 57.21.7
a
Experimental conditions: extraction time, 10 min; CO2 flow-rate, 1 ml/min; temperature, 408C and CO2 density, 0.8 g/ml. Here n3. c Min at 708C. b
3.1.1. Surfactant variables Both the type and the concentration of the surfactant in the microemulsion was studied in order to optime the recovery of cholesterol. Experimental conditions used for this study are listed in Table 1. Microemulsions of non-ionic (Triton X-100), anionic (SDS) and cationic (CTAB) surfactants were tested as reversemicelle forming reagents. Both the recoveries obtained and the effect of a drying stage are summarised in Table 1, where the experimental conditions are also listed. The use of the Triton X-100 microemulsion (with a drying stage prior to extraction) increases the recovery of cholesterol ca. 40%. So, this reagent was selected for further experiments. In order to obtain the optimum surfactant concentration to enhance the extraction of cholesterol, microemulsions between 110ÿ5 and 3.7210ÿ1 M of Triton X-100 were used. The optimum recovery (62.2%) was obtained for a concentration 1.8610ÿ1 M, which coincides with that found by Lee and Biellmann [11] for optimal formation of reverse micelles. Hence, this concentration of surfactant was selected as optimal.
further experiments. The effect of temperature was studied at a constant CO2 density of 0.8 g/ml, which allowed a wider range of temperatures than 0.9 g/ml (because of instrumental pressure limitations) to be studied. The temperature range over which the effect was investigated was 40±838C. Increased temperature resulted in increased recoveries, the recovery at 838C and 0.8 g/ml being 83.0% versus a recovery of 87.5% at 408C and 0.95 g/ml. Thus, a temperature of 408C was selected as optimum. The effect of ¯ow rate was studied under the following working conditions: CO2 density, 0.8 g/ml; extraction time, 10 min and temperature 408C. The recovery increased up to 2 ml/min. Higher ¯ow-rates provided a constant recovery of 74%. So, 2 ml/min was the value chosen as optimum. The effect of extraction time was studied under optimum working conditions in the range 0±20 min and quantitative exctraction of cholesterol was achieved in 18 min. The ranges of experimental variables studied and optimum values of the variables for the SFE of cholesterol from diatomaceous earth are summarised in Table 2.
3.1.2. SFE variables Experimental variables such as CO2 density, temperature of the extraction chamber, extraction time and CO2 ¯ow-rate were optimised in order to maximize the recovery of cholesterol in an extraction time as short as possible. A univariate method was used for this purpose. The effect of CO2 density was studied within the range 0.7±0.95 g/ml (pressures from 115 to 383 bar) under the following conditions: extraction time, 10 min; CO2 ¯ow-rate, 1 ml/min and temperature, 408C. Increased density resulted in increased recovery with a maximum of 87.5% for a density of 0.95 g/ml. So, this value was selected as optimum for
3.2. Features of the method The calibration graph was run by measuring photometrically cholesterol standard solutions. The range of Table 2 Optimization of extraction variables Variables [Triton X-100] (M) CO2 density (g/ml) Temperature (8C) Flow-rate (ml/min) Extraction time (min)
Range studied ÿ5
Optimum value ÿ1
110 ±3.7210 0.7±0.95 40±83 0.5±3 0±20
1.8610ÿ1 0.95 40 2 18
4
M.M. JimeÂnez-Carmona, M.D. Luque de Castro / Analytica Chimica Acta 358 (1998) 1±4
linearity is 40±500 mg/ml of cholesterol, the equation obtained is Y 2:21687 10ÿ3 X 0:0124046 where Y is the absorbance and X is the concentration of cholesterol in mg/ml, and the regression coef®cient is 0.998. The detection and determination limits are 15 and 40 mg/ml, respectively. The precision of the photometric step used for the determination of analyte in the extracts was obtained by reading the absorbance for standard solutions of 100 mg/ml of cholesterol. The R.S.D. (%) was 2.62 for n7. 4. Conclusions and future developments As shown in Table 2, the recovery on the extraction of a medium polar analyte such as cholesterol improved more than three times in the presence of a non-ionic reverse-micelle forming compounds such as Triton X-100. A 18 min extraction time is suf®cient for quantitative removal of cholesterol versus 30 min [12] neccessary in the absence of surfactant. New posibilities from this research are: 1. Use of enzymic reactions in order to improve the derivatization/determination step in terms of sensitivity and selectivity, thus making possible the application of the method to complex samples with low cholesterol content, such as low-cholesterol foods and certi®ed reference materials. 2. A further study of the effect of non-ionic and ionic micelle forming reagents on the extraction of analytes of different polarity.
3. Influence of micelle-formation on the extraction of non-polar analytes by a polar extractant such as supercritical water. Acknowledgements ComisioÂn Interministerial de Ciencia y TecnologõÂa (CICyT) is thanked for ®nancial support (Project PB95-1265) References [1] M.D. Luque de Castro, M. ValcaÂrcel, M.T. Tena, Analytical Supercritical Fluid Extraction, Springer, Heidelberg, 1994. [2] M. Kane, J.R. Dean, S.M. Hitchen, C.J. Dowle, R.L. Tranter, Analyst 120 (1995) 355. [3] M.M. JimeÂnez-Carmona, M.T. Tena, M.D. Luque de Castro, J. Chromatogr. A 711 (1995) 269. [4] M.D. Luque de Castro, M.T. Tena, Strategies for supercritical fluid extraction of polar and ionic compounds, Trends Anal. Chem. 15 (1996) 32±37. [5] S.B. Hawthorne, D.J. Miller, J. Chromatogr. Sci. 24 (1986) 258. [6] S.B. Hawthorne, Y. Yang, D.J. Miller, Anal. Chem. 66 (1994) 2912. [7] Y. Yang, A. Gharaibeh, S.B. Hawthorne, D.J. Miller, Anal. Chem. 67 (1995) 641. [8] S.B. Hawthorne, D.J. Miller, D.E. Nievens, D.C. White, Anal. Chem. 64 (1992) 405. [9] Y. Cai, R. Alzaga, J.M. Bayona, Anal. Chem. 66 (1994) 1161. È vila, M. Alcaraz, Anal. Chem. 66 (1994) [10] Y. Liu, V. LoÂpez-A 3788. [11] K.M. Lee, J.F. Biellmann, Bioorg. Chem. 14 (1986) 262± 273. [12] C.P. Ong, H.M. Ong, S.F.Y. Li, H.K. Lee, J. Microcol. Sep 2 (1990) 69.