Anal. Chem. 1996, 68, 1556-1560
Quantification of Polycyclic Aromatic Hydrocarbons and Polychlorinated Dibenzo-p-dioxins in Human Serum by Combined Micelle-Mediated Extraction (Cloud-Point Extraction) and HPLC Sarath R. Sirimanne,* John R. Barr, and Donald G. Patterson, Jr.
Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention (CDC), Public Health Service, U.S. Department of Health and Human Services, 4770 Buford Highway, NE, Atlanta, Georgia 30341-3724 Li Ma
Department of Chemistry, Emory University, Atlanta, Georgia 30322
Quantification of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) usually requires preconcentration and cleanup prior to analysis. These procedures often involve using large amounts of toxic organic solvents. The sample preparation from serum is even more complex because of the coextraction of lipids and other nonpolar serum components. We describe the unprecedented use of cloud-point extraction to preconcentrate, extract, and clean up PAHs and PCDDs from human serum using the nonionic surfactant Triton X-100. The samples were analyzed by highperformance liquid chromatography with ultraviolet detection. The phase separation was induced by the addition of salt to the micellar serum solutions. The surfactantrich phase was treated with acetonitrile and water to precipitate and remove some of the unwanted substances in the serum sample extract without significantly affecting the recoveries of the analytes. The favorable characteristics of cloud-point extraction discussed here strengthen its potential use as an alternative to other techniques of separation. Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) are ubiquitous environmental pollutants resulting from emissions from sources such as automobile exhaust, waste from paper mills, contaminants of chlorophenols used as starting materials for a series of agricultural and industrial chemicals, and industrial combustion of fossil fuels.1,2 Exposure to PAHs and PCDDs is a potential health concern because of the toxicity, mutagenicity, and carcinogenicity of these substances in animals. In fact, 2,3,7,8-tetrachlorodibenzo-p-dioxin has been found to be one of the most toxic substances ever tested in certain animals.2 PAHs and PCDDs are lipophilic and hence tend to bioaccumulate in the lipid stores of animals and humans. Developing fast, simple, sensitive, and efficient sample preparation and analytical methods is of utmost importance in conducting epidemiologic studies that focus on health issues related to exposure (1) Wild, S. R.; Waterhouse, K. S.; Mcgrath, S. P.; Jones, K. C. Environ. Sci. Technol. 1990, 24, 1706-11. (2) Patterson, D. G., Jr.; Hampton, L.; Lapeza, C. R.. Jr.; Belser, W. T.; Green, V. E.; Alexander, L. R.; Needham, L. L. Anal. Chem. 1987, 59, 2000.
1556 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
of human populations to these environmental toxicants. Methods for quantifying these compounds at trace levels generally involve solvent extraction and an extensive sample cleanup procedure prior to the analysis. The extent of cleanup depends on the complexity of the sample matrix. These solvent extraction methods usually use considerable amounts of organic solvents during sample cleanup, and they generate large quantities of solvent waste. Aqueous solutions of many nonionic surfactants undergo phase separation above a certain temperature that is also known as the cloud-point temperature.3,4 The surfactant-rich phase can be separated by centrifugation. The temperature at which the phase separation occurs depends on the surfactant concentration as well as on the pressure, amount, and type of organic additives. The phase separation is also reversible since the phases merge into a homogeneous phase on cooling. The mechanism by which the separation of these microheterogeneous structures takes place is still being debated.4 These dynamic micellar entities having a nonpolar core possess the capacity to interact with nonpolar species by hydrophobic interactions. During the cloud-point precipitation process, these micellar vesicles aggregate into a surfactant-rich phase, and any bound nonpolar species concentrate in the surfactant-rich phase. The ability of this process to concentrate and separate nonpolar target species from aqueous matrices has been demonstrated in analytical chemistry and separation science.4,5 The cloud-point methodology has recently been applied to the extraction of a wide range of analytes, including polycyclic aromatic hydrocarbons, porphyrins, metalloporphyrins, vitamin A, vitamin E, β-estradiol, estriol, estrone, progesterone, and proteins,6-12 from simple aqueous solutions. (3) Watanabe, H. In Solution Behavior of Surfactants; Nfittal, K. L., Fender, E. J., Eds.; Plenum Press: New York, 1982; pp 1305-16. (4) Hinze, W. L.; Pramauro, E. Crit. Rev. Anal. Chem. 1993, 24, 133-77. (5) McIntire, G. L. Crit. Rev. Anal. Chem. 1990, 21, 257-78. (6) Saitoh, T.; Hinze, W. Anal. Chem. 1991, 63, 2520-25. (7) Horvath, W. J.; Huie, C. W. Talanta 1992, 39, 487-92. (8) Alfred, P. A.; Koslowski, A.; Harris, J. M.; Tjemeld, F. J. Chromatogr. 1994, 659, 289-98. (9) Cordero, B. M.; Pavon, J. L.; Pinto, C. G.; Laespada, M. E. F. Talanta 1993, 40, 1703-10. (10) Garcia, A. L.; Gonzalez, E. B.; Alonzo, J. G.; Sanz-medel, A. Anal. Chim. Acta 1992, 264, 24-48. (11) Bocketen, A.; Niessner, R. Fresenius J. Anal. Chem. 1993, 346, 435-40. 0003-2700/96/0368-1556$12.00/0
© 1996 American Chemical Society
In addition, the analyte/surfactant-rich phase also permits analysis and quantification of the analytes by techniques such as reversed-phase high-performance liquid chromatography (HPLC) without the need for further sample cleanup. Recently, cloudpoint extraction attracted the attention of analytical chemists for these reasons: (a) its ability to concentrate a variety of analytes with high concentration factors; (b) safety and cost benefits (its value as an excellent alternative to conventional sample concentration methods that use large amounts of toxic and flammable organic solvents); (c) easy disposal of the surfactant (it is reportedly6 easily incinerated in the presence of waste acetone or ethanol); (d) the enhancement of the detection due to analyte concentration; (e) the compatibility of the surfactant-rich phase with micellar liquid chromatographic techniques; (f) the preclusion of analyte losses during the evaporation of solvents used in traditional liquid-liquid extraction techniques; and (g) the inhibition by the surfactants of adsorption of nonpolar analytes to glass surfaces.13 In our laboratory, we assess human exposure to environmental toxicants by quantifying the levels of target toxicants in the serum and urine of exposed people. The target analytes are often found in trace levels; hence, preconcentration and cleanup are prerequisites to the analysis of these trace toxicants. We have identified cloud-point extraction as an impressive alternative to conventional solvent extraction because of its environmentally friendly properties and its greater extraction efficiencies. We successfully demonstrated that cloud-point extraction can be used to extract PAHs and PCDDs spiked at concentrations higher than those normally found in humans. In this paper, we report for the first time the extraction of PCDDs and PAHs from human serum using cloud-point technology and analysis by HPLC/UV detection. EXPERIMENTAL SECTION Note: Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services. Instrumentation. The HPLC system used for PAH analysis consisted of a Waters (Waters Associates, Milford, MA) fluidhandling unit with Model 600E system controller, Model 994 programmable photodiode array detector, and Model 5200 printer plotter. The dioxin analysis was performed on an HPLC system equipped with Millennium chromatography manager software, a Waters fluid-handling unit coupled with Model 600E system controller, Model 991 photodiode array detector, and Model 717 plus autosampler (Waters Associates). Both analyses were performed using a 250 mm × 4.6 mm Vydac 218TP54 octadecylsilica (ODS) column (Rainin Instrument Co., Woburn, MA). Incubation and centrifugation of samples were performed by using a Precision Scientific Model 25 reciprocal shaking bath (Precision Scientific, Chicago, IL) and a Model HN-S centrifuge (International Equipment Co., Needham Heights, MA). Reagents. The reduced form of the nonionic surfactant Triton X-100 was obtained from Fluka and used without further purification. HPLC-grade acetonitrile was obtained from Burdick & Jackson (Baxter Healthcare Corp., Muskegon, MI). PAH standards were purchased from AccuStandard (New Haven, CT) and Aldrich Chemical Co. (Milwaukee, WI). We synthesized and characterized dioxin standards in our laboratories. The water used in all studies was prepared using a Milli-Q reagent water system from Millipore Corp. (Milford, MA). (12) Pinto, C. G.; Pavon, J. L. P.; Cordero, B. M. Anal. Chem. 1992, 64, 233438. (13) Pinto, C. G.; Pavon, J. L. P.; Cordero, B. M. Anal. Chem. 1994, 66, 87481.
Cloud-Point Extraction Procedure. An aliquot (0.5 or 5 mL) of human serum spiked with either selected PAHs (250-550 ppb) or dioxins (3.9-4.4 ppm) was pipetted into a 10 mL long tapered centrifuge tube. To this was added an aliquot of Triton X-100 (0.5%-12%) and crystalline sodium chloride (2.5-5.5 M) to the desired concentrations. The contents were mixed well using a Vortex Genie Mixer (Scientific Industries Inc., Bohemia, NY) and then incubated in a thermostated shaking water bath for 15 min at the desired temperature (25-60 °C). The micellar serum sample was then centrifuged at 3500 rpm for 5 min, with the centrifuge rotor heated to the desired temperature just before centrifugation. The clear supernatant was then decanted off to obtain a surfactant-rich phase which adhered to the wall of the tube. The greasy surfactant-rich phase was then diluted with water (0.2 mL), and an internal standard was added. Coextractants such as proteins were then removed from the surfactantrich phase by precipitation with 0.5 mL of acetonitrile and filtration through silanized glass wool that was packed in a Pasteur pipet. The results for recoveries are the averages of duplicate determinations. Liquid Chromatographic Analysis. Samples obtained from cloud-point extraction (50 µL aliquots) were injected into the HPLC system, which was equilibrated with 75% acetonitrile/water at a flow rate of 1.0 mL/min. UV absorbance at 254 nm (used for PAHs) was monitored by using the photodiode array detector, and the results were recorded on a plotter/printer. We analyzed the samples containing dioxins using the same chromatographic column that was equilibrated with a mobile phase containing 90% methanol flowing at a rate of 1.0 mL/min and then recording the absorbance at 232 nm. Data processing and quantification were performed by using the Millennium Chromatography Manager software. RESULTS AND DISCUSSION As described in the Experimental Section, human serum samples containing the target analytes were subjected to cloudpoint extraction using Triton X-100 as the nonionic surfactant. We chose the nonionic detergent, Triton X-100 (a tert-octylphenoxy poly(oxyethylene) ether, critical micellar concentration (cmc) ) 0.29 mmol/L, cloud-point temperature, 63.7 °C) for our studies because of its wide use as a cloud-point surfactant.6,9,12,13 The surfactants belonging to the Triton X series show a strong dependence of the cloud-point temperature on the number of hydrophilic oxyethylene groups attached to the hydrophobic octylphenyl residue. For most members of the Triton X series (n < 7), the cloud-point temperatures are lower than 20 °C. Figure 1 depicts the steps involved in the application of this surfactantmediated extraction to the serum sample matrix. In general, the procedure for the cloud-point extraction of aqueous micellar solutions involves adding the surfactant to a concentration above its critical micelle concentration while maintaining the temperature below the cloud-point of the micellar solution. This procedure is then followed by induction of a phase separation by raising the temperature of the solution. The two phases, aqueous and surfactant-rich, are then usually separated by centrifugation. Our centrifugation experiments performed at different temperatures showed that the temperature at which the centrifugation was performed does not have a significant effect on the recovery of analytes as long as the phases remain separated. This finding implies that the extraction is apparently complete during the temperature-induced phase separation process, and any cooling during the centrifugation does not have an appreciable effect on the final recovery. When human serum was used instead of a pure aqueous matrix, the micellar serum samples failed to undergo temperature-induced phase separation under the same conditions Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
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Figure 2. High-performance liquid chromatogram of a cloud-point extract of normal human serum.
Figure 1. Steps involved in application of the surfactant-mediated extraction to the serum sample matrix.
that we used for water samples. This behavior may be attributed to the chemical components in the serum matrix. To induce a phase separation in aqueous micellar solutions, one can use additives in the micellar solution. Some of the additives that have been used to effect a phase separation with Triton X-100 include urea, sodium chloride, sodium azide, and potassium chloride. In our studies, we used sodium chloride (NaCl) to induce phase separation. Above a NaCl concentration of 2.5 M, the micellar serum samples undergo a phase separation, presumably by a salting-out mechanism. The cloud-point extraction using salting-out conditions has been successfully used for applications such as the extraction of porphyrins and metalloporphyrins from water.13 We found that a minimum concentration of 2.5 M NaCl was necessary to effect phase separation in micellar serum samples. The analyte/surfactant-rich phase separates as a sticky paste at the top of the liquid layer, thus enabling the supernatant to be easily decanted off. During the cloud-point extraction, some other substances, such as serum proteins, are also coextracted from the serum into the surfactant-rich phase. If these samples are injected directly into an HPLC system, the coextractants precipitate on mixing with the mobile phase inside the HPLC injector, causing the injector and the frits to clog. Therefore, we removed the coextractants by precipitation with acetonitrile and filtration through silanized glass wool before we conducted the HPLC analysis. To correct for losses due to adsorption on the precipitate, we added an external standard prior to the precipitation of coextractants. Figure 2 shows the HPLC trace for a typical sample obtained from the cloud-point extraction of human serum. No PAHs were detectable in human serum under the present experimental conditions. Figure 3 shows a typical HPLC/UV analytical trace for a sample obtained by the cloud-point extraction of serum spiked with PAHs (250-550 ppb). As shown in Figure 3, Triton X-100 elutes close to the peak due to acenaphthylene, and the 1558 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
Figure 3. High-performance liquid chromatogram of a cloud-point extract of human serum spiked with 16 PAHs. Peak identification: 1, naphthalene; 2, acenaphthylene; 3, acenaphthene and fluorene; 4, phenanthrene; 5, anthracene; 6, fluoranthene; 7, pyrene; 8, 9,10dimethylanthracene; 9, benz[a]anthracene; 10, chrysene; 11, benzo[b]fluoranthene; 12, benzo[k]fluoranthene; 13, benzo[a]pyrene; 14, dibenz[a,h]anthracene; 15, benzo[ghi]perylene; 16, indeno[1,2,3-cd]pyrene.
peaks corresponding to acenaphthene and fluorene are also not resolved under the present HPLC conditions. The HPLC peaks corresponding to 16 PAHs were identified by coelution with authentic standard, and the peaks are labeled in the chromatogram. The recoveries of individual PAHs were found to be quantitative at 8% Triton X-100, 5 M NaCl, and at 60 °C and are summarized in Table 1. Initially, we found that phase separation takes place at room temperature when the salt concentration in the micellar serum solution is 2.5 M NaCl. However, the recoveries from the cloudpoint extraction of PAHs were found to increase with an increase in temperature. The effect of temperature on the recovery of PAHs in serum is shown in Table 2. When we performed extractions at 60 °C, average recovery from the extractions of PAHs reached a 100% maximum at 5.0 M NaCl and 8% Triton X-100 concentration. At room temperature (25 °C) and 4.5 M NaCl concentration, the average extraction efficiencies were as low as
Table 1. Effect of Triton X-100 Concentration on the Recoveries of Cloud-Point Extraction of PAHs from Human Seruma
Table 3. Effect of NaCl Concentration on the Recoveries of PAHs (250-550 ppb) from Human Serum by Cloud-Point Extractiona
Triton X-100 concn (v/v)
NaCl concn (M)
0.5
2.0
4.0
6.0
8.0
naphthalene fluorene and acenaphthene phenanthrene anthracene fluoranthene pyrene benzo[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene dibenz[a,h]anthracene benzo[ghi]perylene indeno[1,2,3-cd]pyrene
27 18
56 48
50 63
93 46
100 100
42 46 52 50 56 56 59 56 59 65 59 55
73 77 81 81 85 86 85 86 88 87 84 82
85 87 91 83 97 100 98 96 95 100 100 98
93 93 100 94 100 100 100 100 100 100 b 100
100 100 100 100 100 100 100 100 100 100 100 100
average recovery
50
79
89
94
100
a
The NaCl concentration, extraction temperature, and PAH level in serum are 5.0 M, 60 °C, and 250-550 ppb, respectively. b Integration of the peak failed.
Table 2. Effect of Extraction Temperature on the Recoveries from Cloud-Point Extraction of PAHs from Human Seruma temperature (°C) 25
40
50
60
naphthalene fluorene and acenaphthene phenanthrene anthracene fluoranthene pyrene benzo[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene dibenz[a,h]anthracene benzo[ghi]perylene indeno[1,2,3-cd]pyrene
17 21 23 22 26 26 26 24 27 28 25 24 29 30
47 48 63 62 71 70 75 71 76 75 75 84 84 73
34 68 75 84 79 78 85 81 90 89 81 48 85 84
59 75 80 74 86 84 90 85 92 93 85 91 94 89
average recovery
25
70
76
84
a The cloud-point extraction was performed according to the procedure given in the Experimental Section, using 4.5 M NaCl, 2% (v/v) Triton X-100, and 1.0-2.0 ppm PAHs.
25%. When the incubation temperature was increased from 25 °C to 50 °C, the average recoveries tripled with no significant improvement at temperatures above 60 °C. Table 3 summarizes the recoveries at different NaCl concentrations of individual PAHs. The recoveries of most of the PAHs increase as the sodium chloride concentration increases from 3.0 to 5.0 M; at higher concentrations, salt remains undissolved, indicating that the serum/surfactant solution is saturated. The concentration of Triton X-100 has the greatest effect on the recovery of PAHs from serum. The recovery of PAHs increased from 50% to 100% as the Triton X-100 concentration was increased from 0.5% to 8% (w/v), indicating that the extraction efficiency strongly depends on the micellar concentration. The results of the cloud-point extraction of human serum samples spiked at three levels of PAHs are shown in Table 4. The average recovery is around 82% ( 5% (5 M NaCl, 25 °C, and 2% (w/v) Triton X-100
3.0
4.0
5.0
5.5
naphthalene fluorene and acenaphthene phenanthrene anthracene fluoranthene pyrene benzo[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene dibenz[a,h]anthracene benzo[ghi]perylene indeno[1,2,3-cd]pyrene
41 60 63 65 69 64 71 69 71 71 69 76 64 70
49 56 68 70 76 73 78 76 81 79 77 77 82 78
56 48 73 77 81 81 85 86 85 86 88 87 84 82
54 39 68 72 76 b 85 95 83 82 82 82 88 76
average recovery
66
73
79
76
a The extraction conditions are Triton X-100, 2% (v/v), and temperature, 60 °C. b Integration of the peak failed.
Table 4. Efficiency of Cloud-Point Extraction of PAHs from Serum at 1-2 ppm and 250-550 and 120-280 ppb Concentrationsa 1-2 ppm
250-550 ppb
120-280 ppb
naphthalene fluorene and acenaphthene phenanthrene anthracene fluoranthene pyrene benzo[a]anthracene chrysene benzo[b]fluoranthene benzo[k]fluoranthene benzo[a]pyrene dibenz[a,h]anthracene benzo[ghi]perylene indeno[1,2,3-cd]pyrene
58 b 82 78 88 86 91 86 93 95 86 96 93 89
63 b 74 75 81 80 86 88 85 84 85 82 82 80
80 b 71 71 81 71 86 91 88 82 86 91 c 80
average recovery
86
80
82
a The NaCl concentration, Triton X-100 concentration, and extraction temperature are 5.0 M, 2% (w/v), and 60 °C, respectively. b Peaks were not resolved to obtain accurate values. c Integration of the peak failed.
concentration), which is a very good extraction efficiency over a wide range of analyte levels (Table 4). For cloud-point extraction studies of PCCDs, we used a mixture of five tetra- and penta-PCDDs (see chromatogram in Figure 4). Temperature, NaCl concentration, and Triton X-100 exhibited effects on extraction recoveries similar to those observed for the extraction of PAHs from human serum. As seen in Table 5, the average recovery of PCDDs can be doubled to 58% with an increase in NaCl concentration from 2.5 to 4.5 M (2% (w/v) Triton X-100 and 50 °C). The results shown in Table 6 indicate that the recoveries of PCDDs continue to increase up to an incubation temperature of 60 °C. At temperatures above 65 °C, no phase separation occurs; instead, the extraction mixture solidifies in the centrifuge tube. The results summarized in Table 7 show that the detergent concentration is the major contributing factor in achieving the maximum recovery. The results summarized in Table 8 indicate that, at the optimum detergent concentration of 12%, the NaCl concentration above 15% has no significant effect Analytical Chemistry, Vol. 68, No. 9, May 1, 1996
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Table 7. Effect of Triton X-100 Concentration on the Recoveries of Dioxins from Human Seruma Triton X-100 concn (%, w/v) 2.0
4.0
6.0
8.0
12.0
1,4,7,8-TCDD 1,2,3,4-TCDD 1,2,4,6,9-PCDD 1,2,3,4,7-PCDD
63 56 53 54
77 67 67 66
87 83 80 79
93 93 91 96
100 98 98 91
average recovery
57
69
82
93
97
a The NaCl concentration, temperature, and dioxin concentrations were 4.5 M, 50 °C, and 3.9-4.4 ppm, respectively.
Figure 4. High-performance liquid chromatogram of a cloud-point extract of human serum spiked with five dioxins. Table 5. Effect of NaCl Concentration on the Recoveries of the Cloud-Point Extraction of Dioxins from Human Seruma NaCl concn (%, w/v) 15
20
26
30
1,4,7,8-TCDD 1,2,3,4-TCDD 1,2,4,6,9-PCDD 1,2,3,4,7-PCDD
27 24 24 31
63 55 55 55
64 57 54 55
75 62 63 71
average recovery
27
57
58
68
a Triton concentration, temperature, and dioxin level used in the extraction were 2% (w/v), 50 °C, and 3.9-4.4 ppm, respectively.
Table 6. Effect of Extraction Temperature on the Recoveries of Dioxins from Human Seruma temperature (°C) 40
45
50
60
1,4,7,8-TCDD 1,2,3,4-TCDD 1,2,4,6,9-PCDD 1,2,3,4,7-PCDD
72 67 66 68
83 77 75 75
84 79 77 78
86 82 80 80
average recovery
68
78
80
82
a The NaCl, Triton X-100, and dioxin concentrations were 4.5 M, 4% (w/v), and 3.9-4.4 ppm, respectively.
on the recovery. The highest average extraction recovery of 98% was obtained at 12% (w/v) Triton X-100, 4.5 M NaCl, and 50 °C. It is clear from our experiments that the presence of salt is essential for phase separation of the micellar serum solutions. When the salt concentration is increased, the micelle size and the aggregation number are increased, keeping the micellar concentration constant.4 In addition, nonpolar analytes may become less soluble in the matrix at higher salt concentrations and thus contribute to higher recoveries. The increase in recovery at elevated temperatures may be due to an increase in solubility of the analytes in the micellar phase. The results presented in this paper indicate that a proper combination of temperature, salt
Table 8. Effect of NaCl Concentration on the Recoveries of Cloud-Point Extraction of Dioxins from Human Serum at Optimum Triton X-100 Concentrationa NaCl concn (%, w/v) 10
15
20
26
1,4,7,8-TCDD 1,2,3,4-TCDD 1,2,4,6,9-PCDD 1,2,3,4,7-PCDD
88 88 83 82
100 100 98 92
99 99 91 97
100 98 98 91
average recovery
85
98
97
97
a The Triton concentration, extraction temperature, and dioxin level were 12% (w/v), 50 °C, and 3.9-4.4 ppm, respectively.
concentration, and surfactant concentration can produce excellent recoveries of dioxins and PAHs from human serum. The throughput using this procedure can be increased by processing batches of samples, and the costs can be reduced by using relatively inexpensive and nontoxic reagents such as NaCl and Triton X-100. Additionally, the surfactants reportedly help inhibit adsorption of analytes to glass surfaces6,10 and thereby increase analyte recoveries. The cloud-point extraction procedure is applicable to the sample preparation of analytes of varying polarity, including extractions of metal ions as chelates,14 polycyclic aromatic hydrocarbons,6,10,11 steroids,7,8 vitamins,9,12 and pesticides.15 In principle, this technique can be adapted to extract any target analyte, provided that a proper amphiphilic ligand can be found. The appropriate ligand must have a functional group specific for the analyte and a hydrophobic moiety that can bind both the target analyte and the micelles, as is the case, for example, when a combination of nonionic surfactants and metal chelates is being used to extract metal ions from an aqueous solution.14 Currently, analysis of the samples from cloud-point extraction is limited to liquid chromatographic techniques because of the presence of the surfactant. However, attempts are underway, through the removal of the surfactant from the final sample using techniques such as gel permeation chromatography, to expand the applicability of cloud-point extraction to other analytical techniques such as GC/MS. Received for review October 16, 1995. Accepted February 6, 1996.X AC951028+
(14) Watanabe, H.; Tanaka, H. Talanta 1978, 25, 585-89. (15) Pinto, C. G.; Pavo´n, J.; Cordero, B. M. Anal. Chem. 1995, 67, 2606-12.
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Abstract published in Advance ACS Abstracts, March 15, 1996.
