An Acid-Induced Phase Cloud Point Separation Approach Using

Alkyl surfactants were used to preconcentrate polar PAHs and progesterone .... E. K. Paleologos, S. S. Giannakopoulos, P. D. Zygoura, and M. G. Kontom...
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Anal. Chem. 1999, 71, 4519-4526

An Acid-Induced Phase Cloud Point Separation Approach Using Anionic Surfactants for the Extraction and Preconcentration of Organic Compounds I. Casero, D. Sicilia, S. Rubio, and D. Pe´rez-Bendito*

Analytical Chemistry Division, Faculty of Sciences, University of Co´ rdoba, Avda San Alberto Magno s/n, E-14004 Co´ rdoba, Spain

The acid-induced liquid-liquid phase separation of anionic surfactants in aqueous solutions, and its applicability to cloud point extraction methodology, were examined. The phase diagrams obtained (e.g., [HCl] vs [surfactant]) consisted of three regions: a homogeneous liquid region, two coexisting isotropic phases, and a solid region. The breadth of each region was found to depend on the surfactant structure. The behavior of each phase was also examined in relation to temperature and added salts. The anionic surfactants investigated were sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonic acid (SDBSA), sodium dodecanesulfonic acid (SDSA), and sodium dioctylsulfosuccinate (Aerosol OT). The use of anionic surfactant-mediated phase separations provided very high extraction efficiencies (80-100%) for pyrene in aqueous samples and various PAHs in a certified reference material (dried sewage sludge). The preconcentration factor achieved was found to be a function of both surfactant and acid concentrations. Theoretical preconcentration factors as high as 230 can be reached with Aerosol OT. Alkyl surfactants were used to preconcentrate polar PAHs and progesterone prior to their determination by HPLC. The lack of an aromatic moiety in the structure of the surfactants and their ionic character enables complete resolution of their chromatographic peak from those of the analytes. The ability of anionic surfactants to extract thermally labile compounds was confirmed by extracting vitamin E at 10 °C with recoveries of about 80-85%. Aqueous solutions of nonionic surfactants are well-known for their tendency to undergo clouding and liquid-liquid phase separation at elevated temperatures.1-4 Certain zwitterionic surfactants,5 as well as some cationic surfactants in the presence of * Corresponding author. Fax: 957218606. E-mail: [email protected]. (1) DeGiorgio, V. In Physics of Amphiphiles: Micelles, Vesicles and Microemulsions; DeGiorgio, V., Corti, M., Eds.; North-Holland: Amsterdam, 1985; pp 303-335. (2) DeGiorgio, V.; Piazza, R.; Corti, M.; Minero, C. J. Chem. Phys. 1985, 82, 1025-1031. (3) Goldstein, R. E. J. Phys. Chem. 1986, 84, 3367-3378. (4) Hinze, W. L. In Ordered Media in Chemical Separations; Hinze, W. L., Armstrong, D. W., Eds; American Chemical Society: Washington, DC, 1987; pp 48-55. 10.1021/ac990106g CCC: $18.00 Published on Web 09/11/1999

© 1999 American Chemical Society

high concentrations of added electrolyte (>2 M),6 also exhibit phase separation. The mechanism via which the separation occurs remains obscure. Thus, in nonionic surfactants, the role of oscillations in the critical concentration and of micellar growth as mechanisms for the phase separation is still a controversial issue. Evidence of both small, spherical and large, polydisperse (cylindrical) micelles has been reported in apparent contradiction.7,8 Ionic micelles in concentrated electrolyte solutions which phase separate on heating are believed to be long, flexible cylinders or “giant micelles”; they have been the subject of some study, though not in the same depth as nonionic micelles.6,9,10 Large amounts of added electrolyte screen the repulsive electrostatic effects which usually stabilize the solutions. The cloud point behavior of nonionic surfactants has been utilized to design efficient extraction methods for the separation, preconcentration, or purification of a variety of substances including metal ions11,12 and various organic compounds.13-18 The advantages of using of such nonionic cloud point extraction schemes in separation techniques has been discussed,1,4,19 and factors affecting nonionic surfactant-mediated phase separation have been evaluated.18 The ability of these surfactants to concentrate a variety of analytes of widely varying nature with high concentration factors and their compatibility with micellar and (5) Lang, J. C.; Morgan, R. C. J. Chem. Phys. 1980, 73, 5849-5858. (6) Appell, J.; Porte; G. J. Phys. Lett. 1983, 44, 689-695. (7) Zulauf, M.; Weckstrom, K.; Hayter, J. B.; Corti, M.; DeGiorgio, V. J. Phys. Chem. 1985, 89, 3411-3418. (8) Lum Wam, J. A.; Wam, G. G.; White, L. R.; Grieser, F. Colloid Polym. Sci. 1987, 265, 528-534. (9) Imae, T.; Sasaki, M.; Abe, A.; Ikeda, S. Langmuir 1988, 4, 414-418. (10) Porte, G. J. Phys. Chem. 1983, 87, 3541-3550. (11) Ferna´ndez Laespada, M. E.; Pe´rez Pavo´n, J. L.; Moreno Cordero, B. Analyst (Cambridge, U.K.) 1993, 118, 209-212. (12) Okada, T. Anal. Chem. 1992, 64, 2138-2142. (13) Garcı´a Pinto, C.; Pe´rez Pavo´n, J. L.; Moreno Cordero, B. Anal. Chem. 1994, 66, 874-881. (14) Garcı´a Pinto, C.; Pe´rez Pavo´n, J. L.; Moreno Cordero, B. Anal. Chem. 1992, 64, 2334-2338. (15) Ferrer, R.; Beltra´n, J. L.; Guiteras, J. Anal. Chim. Acta 1996, 330, 199206. (16) Koma´romy-Hiller, G.; von Wandruszka, R. Talanta 1995, 42, 83-88. (17) Moreno Cordero, B.; Pe´rez Pavo´n, J. L.; Garcı´a Pinto, C.; Ferna´ndez Laespada, M. E. Talanta 1993, 40, 1703-1710. (18) Frankewich, R. P.; Hinze, W. L. Anal. Chem. 1994, 66, 944-954. (19) Hinze, W. L. Ann. Chim. 1987, 77, 167-207.

Analytical Chemistry, Vol. 71, No. 20, October 15, 1999 4519

hydroorganic mobile phases have been exploited in the past few years for the extraction and preconcentration of organic compounds prior to their liquid chromatographic determination.13-15,17 However, the nonionic surfactants most frequently employed for analytical purposes, which include the Triton X [polyoxyethylene (n) tert-octylphenyl ethers] and PONPE [polyoxyethylene (n) nonyl phenyl ethers] series, have major drawbacks. For instance, they exhibit high background absorbance in the ultraviolet region and high fluorescence signals when an excitation wavelength above 300 nm is used by virtue of aromatic moiety in their structure. Also, they have long retention times owing to the nonpolar character of surfactant molecules. The result is overlap between the chromatographic peaks for the surfactant and those for the more polar analytes that calls for a cleanup operation if adequate selectivity is to be obtained.15 In addition, thermally labile analytes can undergo degradation at the temperatures required for phase separation of some nonionic surfactants. The use of alkylammoniosulfate zwitterionic surfactants in cloud point extraction methodology overcomes some of the above-described drawbacks;20 however, because they are not commercially available, such surfactants have found limited application so far. To the authors’ knowledge, the cloud point of cationic surfactants in the presence of added electrolytes has never to date been exploited for analytical purposes. In this paper, we propose the use of anionic surfactants in cloud point methodology. We found aqueous micellar solutions of surfactant such as sodium dodecyl sulfate (C12H25OSO3-Na+, SDS), sodium dodecanesulfonic acid (C12H25SO3-Na+, SDSA), sodium dodecylbenzenesulfonic acid (C12H25(C6H5)SO3-Na+, SDBSA), and sodium dioctylsulfosuccinate [(C4H9CH(C2H5)CH2OCO)2CHSO3-Na+, Aerosol OT] to separate into two isotropic phases in an acid medium. This surfactant-mediated liquid-liquid phase separation has seemingly never been reported before. We characterized the phase behavior of anionic surfactants under a variety of experimental conditions and examined the feasibility of utilizing them in extraction and preconcentration schemes. Alkyl linear anionic surfactants are commercially available and have an appropriate ionic group for use in the cloud point preconcentration of the more polar analytes prior to HPLC. To confirm this assumption, the phase separation behavior of alkyl linear anionic surfactants such as SDS and SDSA was investigated with a view to their use for the preconcentration of various organic compounds (e.g., PAHs, steroid hormones, and vitamins) following HPLC separation of the surfactant. Because clouding in anionic surfactants is temperature-independent, thermally labile analytes should be easy to extract. The results obtained were compared with those provided by PONPE 6.0, a nonionic surfactant system. EXPERIMENTAL SECTION Apparatus. The liquid chromatographic system (Waters, Milford, MA) consisted of a model 600S delivery apparatus and a model 616 quaternary pump. A model 996 photodiode array detector was used for UV detection, and a model 470 scanning fluorescence detector was employed for fluorimetric detection. In all experiments, a Rheodyne 7125 injection valve furnished with a 20-µL sample loop and a 15-cm Waters Nova-Pack C18 column of 3.9-mm i.d. were used. A Hitachi U-2000 spectrophotomer, a (20) Saitoh, T.; Hinze, W. L. Anal. Chem. 1991, 63, 2520-2525.

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Table 1. Percentages of Surfactant-Rich Phase and Theoretical Preconcentration Factors for Anionic Surfactants in Aqueous Hydrochloric Acid Solutions anionic surfactant

[HCl] (M)

SDS

[anionic surfactant] (% w/v)

% surfactantrich phase

theoretical preconcentration factor

0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0 0.25 0.5 1.0 2.0

2.2 3.0 6.0 10.0 4.2 5.0 7.5 10.0 15.2 16.3 18.8 23.0 0.6 1.5 3.0 6.9 1.1 2.0 7.0 12.0 0.7 1.4 2.8 5.6 0.2 0.5 1.0 2.0 0.8 1.0 1.7 3.0

30 22 11 7 14 12 8 6 5 5 4 3 117 46 23 10 48 27 8 4 67 33 17 8 232 111 59 29 62 50 29 17

4.0

5.0

SDSA

3.0

4.0

SDBSA

5.5

6.5

Aerosol OT

5.0

6.0

Table 2. Percent Recoveries Obtained in Pyrene Surfactant-Mediated Phase Separation Using SDS and SDSA in Hydrochloric Acida % recoveryb pyrene concentration (µg/mL)

SDS

SDSA

0.1 0.25 0.5 1.0 2.0 2.5

95.0 ( 6.0 99.5 ( 8.1 99.0 ( 7.8 98.3 ( 5.3 86.1 ( 3.5 83.0 ( 3.6

99.3 ( 5.8 95.1 ( 8.3 97.4 ( 6.0 99.0 ( 6.1 96.4 ( 3.8 100.2 ( 4.2

a [SDS] ) [SDSA]) 1% (w/v), [HCl] ) 4.2 M, room temperature; Extraction time, 5 min (in a Vortex). b Mean of three individual determinations.

Heidolph Vortex Shaker and a Selecta Angular 6 centrifuge were also employed. Reagents. All chemicals were of analytical-reagent grade and employed as supplied. The anionic surfactants were purchased from Aldrich (SDS, SDBSA, and Aerosol OT) and Fluka (SDSA). PAHs were obtained from Sigma [pyrene, naphthalene, anthracene, acenaphthene, fluorene, benzo[e]pyrene, and phenanthrene], Aldrich [benzo[b]fluoranthene, benzo[b]naphtho(2,1d)thiophene, benzo[a]pyrene, and benzo[k]fluoranthene], Fluka [benzo[a]anthracene], and BCR [indeno(1,2,3-cd)pyrene]. Progesterone and vitamin E were obtained from Sigma. The sewage

Table 3. Recoveries of PAHs from Dried Sewage Sludge (CRM 088)a concentration found,b µg/g

a

compound

certified value, µg/g

SDS

pyrene benz[a]anthracene benzo[a]pyrene benzo[e]pyrene benzo[b]fluoranthene benzo[k]fluoranthene indeno(1,2,3-cd)pyrene benzo[b]naphtho(2,1-d)-thiophene

2.16 ( 0.09 0.93 ( 0.09 0.91 ( 0.09 1.02 ( 0.07 1.17 ( 0.08 0.57 ( 0.05 0.81 ( 0.06 0.42 ( 0.05

% recovery

SDSA

SDS

SDSA

3.0 ( 0.2 1.47 ( 0.05 1.93 ( 0.08

3.2 ( 0.3 1.5 ( 0.1 1.9 ( 0.1

138 158 100

148 161 98

1.12 ( 0.04 0.53 ( 0.04 0.65 ( 0.08

1.16 ( 0.08 0.52 ( 0.06 0.7 ( 0.1

96 93 80

99 91 86

[SDS] ) [SDSA] ) 1% (w/v), [HCl] ) 4.2 M, room temperature. b Mean of three individual determinations.

sludge, containing eight certified PAHs (CRM 088), was purchased from BCR; its composition is shown in Table 3. Procedures. Phase Diagrams and Phase Volume Ratio Determinations. The phase behavior of each surfactant system was examined under different experimental conditions by mixing appropriate volumes of hydrochloric acid and aqueous surfactant solutions in glass tubes and making a volume of 10 mL with distilled water. After 24 h of standing, the tubes were inspected to check whether one or two phases (isotropic liquid phases, liquid-solid phases) were present. Phase diagrams were obtained from these observations as a function of the hydrochloric acid and surfactant concentrations. Surfactant-rich phase to aqueous phase volume ratios (Vs/Vw) for each system under different conditions were determined in the same way by using calibrated glass tubes. When the surfactantrich phase was too small in volume for reliable measurement, 50 mL of solution was used instead. The tubes were allowed to stand at room temperature for 1 week prior to measurement of the respective phase volumes. The results shown are the average of triplicate measurements. Cloud Point Extraction from Aqueous Samples. Most analytical uses of anionic surfactant-based cloud point methodology were tested by using SDSA. In some experiments, SDS was also employed. In a typical extraction experiment, 0.1 g of SDSA was added to 6.5 mL of the solution containing the analytes, followed by the addition of 3.5 mL of 12 M hydrochloric acid. The mixture was stirred for 5 min in the Vortex. Separation of the two phases was achieved by centrifugation at 1500 rpm for 10 min. Extraction with the SDS-rich phase required about 24 h of equilibration before centrifugation. Three sets of experiments were conducted in order to evaluate recovery in anionic surfactant-mediated phase separations, the use of this approach as a preconcentration technique for HPLC, and its ability to extract thermally labile analytes, respectively. The extent of extraction was evaluated by using pyrene as a model compound. In this experimental series, water samples were spiked with pyrene concentrations between 0.1 and 2.5 µg/mL. Aliquots (100 µL) of the surfactant-rich phase were withdrawn with a micropipet and diluted to 2 mL with distilled water and their absorbance at 240 nm measured in a spectrophotometer. The ability of anionic surfactant cloud point methodology to preconcentrate analytes for HPLC was assessed by using compounds with short chromatographic retention times. Aqueous samples containing 0.25 µg/mL of progesterone or a mixture of PAHs (naphthalene, fluorene, acenaphthene, phenanthrene, an-

Figure 1. Phase diagrams ([HCl] vs [surfactant]) of the anionic surfactants [(A) SDS, (B) SDSA, (C) SDBSA, and (D) Aerosol OT] obtained at room temperature. L, homogeneous liquid region; L-L, two-phase region; S, solid region.

thracene and pyrene, 50 µg/mL each), respectively, were extracted with the anionic surfactant-rich phase. Aliquots (200 µL) of this phase were mixed with 200 µL of methanol, to lower viscosity, and 20 µL of the mixture was injected into the chromatographic system. For comparison, the cloud point extraction of these analytes was also conducted by using a nonionic surfactant system in which the sample was dissolved in a 1% (w/ v) PONPE-6.0 aqueous solution. Phase separation was accomplished by heating the nonionic surfactant solution at 60 °C in a thermostated bath. Vitamin E was selected as the thermally labile analyte. Concentrations in the range 0.1-2 µg mL-1 used were spiked to aqueous samples, and these same concentrations were extracted by using cloud point methodology at a constant temperature of 10 °C and the SDSA solution as extractant. Aliquots of the Analytical Chemistry, Vol. 71, No. 20, October 15, 1999

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Figure 2. Phase diagrams of SDS [5% (w/v)] (A) [HCl] vs temperature, (B) [HCl] vs [NaCl] at 20 °C, and (C) [NaCl] vs temperature in the presence of 4 M HCl. Letters have same meaning as in Figure 1.

surfactant-rich phase were mixed with identical volumes of methanol, and 20 µL of the resulting mixture was injected into the chromatographic system. Cloud Point Extraction of PAHs from Solid Samples. Appropriate amounts of the dried sewage sludge (CRM 088), viz 50 and 20 mg with SDS and SDSA, respectively, as extractant, were poured into 10 mL of surfactant solution (1% w/v of SDS or SDSA) in 4.2 M HCl. The suspension was stirred for 2 h, following which it was centrifuged at 1500 rpm for 30 min. Aliquots of the surfactantrich phase were then withdrawn, diluted 2-fold with methanol, and analyzed chromatographically. With SDS as extractant, equilibration for 24 h was required after centrifugation to ensure phase separation. Liquid Chromatographic Analysis. PAHs were separated by using a mobile phase consisting of 75:25 (v/v) acetonitrile-water (q ) 0.4 mL/min). UV-detection at 220 nm was used for PAHs extracted from aqueous samples and fluorescence detection with excitation-emission wavelength program for those extracted from the sewage sludge. The excitation-emission wavelengths (in nanometers) used for each compound were as follows: 334-384 for pyrene and benz[a]anthracene; 261-357 for benzo[b]naphtho(2,1-d)-thiophene; 300-430 for benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, and benzo[e]pyrene; and 300-500 for indeno(1,2,3-cd)pyrene. Progesterone and vitamin E analyses were performed by using a mobile phase consisting of 80:20 (v/ v) acetonitrile-water (q ) 0.4 mL/min) and 100% acetonitrile (q ) 1.0 mL/min), respectively, with UV detection (257 and 220 nm for progesterone and vitamin E, respectively). No special washing steps were required to remove surfactants from the column. RESULTS AND DISCUSSION Phase Diagrams of Anionic Surfactants in Hydrochloric Acid Aqueous Solutions. Figure 1 shows phase diagrams of hydrochloric acid concentration vs surfactant concentration (SDS, SDSA, SDBSA, and Aerosol OT) at room temperature. The surfactant concentrations studied were those of greater analytical interest. Three different regions were observed, viz a homogeneous liquid region (L), two coexisting isotropic phases (L-L), and a solid region (S); their breadth depended on the surfactant structure. Acid-induced liquid-liquid phase separation was observed with all anionic surfactants tested, so it seems to be a 4522 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999

Figure 3. Effect of (A) temperature and (B) hydrochloric acid concentration on the time required for phase separation of SDS. Experimental conditions: [SDS] ) 1% (w/v), (A) [HCl] ) 4 M, (B) T ) 60 °C.

general property of this type of surfactant. The region encompassed by the two isotropic phases was wide enough for analytical use. These coexisting phases were found to remain unchanged for at least 3 days at room temperature. After 4 days, the surfactantrich phase gradually become cloudy. Hydrochloric acid was taken to be the most suitable medium to obtain the two isotropic phases. The use of oxidizing acids such as nitric or perchloric was avoided because of the likely gradual oxidation of the surfactants in these media. Also, the use of sulfuric acid would have required special precautions so was avoided. At the hydrochloric acid concentrations at which two isotropic phases were obtained, both sulfate and sulfonic groups in the anionic surfactants should be protonated. As a consequence, the acid medium will convert the anionic surfactants into nonionic ones. To determine the hydrogen ion concentration in the surfactant-rich phase, the pH values of solutions prepared by dilution of this surfactant-rich phase (dilutions comprised in the range from 1:103 to 1:106) were measured. The surfactant SDS was selected for this purpose. An SDS aqueous solution was mixed with hydrochloric acid at room temperature (final concentrations 1% SDS and 4 M HCl). The hydrogen ion concentration in the SDS-rich phase was found to be 3.7 ( 0.5 M, which is roughly

Figure 4. Chromatogram for the PAHs [(1) pyrene, (2) benz[a]anthracene, (3)benzo[b]fluoranthene, (4) benzo[k]fluoranthene, (5) benzo[a]pyrene and benzo[e]pyrene, and (6) indeno(1,2,3-cd)pyrene] obtained following injection of the sample into the surfactant-rich phase after 2 h of contact of a 1% (w/v) SDSA + 4.2 M HCl solution with 20 mg of dried sewage sludge. Chromatographic conditions as described in the Experimental Section.

equal to that added to the SDS aqueous solution in order to promote phase separation. Therefore, the hydrogen ion concentration in both phases was similar, and the phases differed only in their surfactant concentration. The phase behavior of the anionic surfactants was also examined as a function of the temperature and ionic strength. Figure 2 illustrates the behavior of a 5% (w/v) SDS solution. Increased temperatures considerably raised the region where the two isotropic phases coexisted (Figure 2A), mainly as a result of the increased solubility of the surfactant and, to a lesser extent, the decreased amount of hydrochloric acid required for liquidliquid phase separation (see the lower boundary in Figure 2A). This behavior was similar for all the anionic surfactants tested. The region of two liquid phases existed throughout the temperature range tested (10-90 °C); this is of analytical interest because it may allow one to perform the anionic phase separation extraction step at the very low temperatures required by thermally labile compounds. On the other hand, if a high temperature is required for extraction from solid matrixes, a clear, homogeneous surfactant micellar solution can be prepared by using a relatively low hydrochloric acid concentration (below 1.5 M for SDS, see Figure 2A), and this acid concentration be increased to convert the single isotropic solution into two isotropic phases. The effect of ionic strength on the phase diagram of the anionic surfactants was examined as this variable is known to influence surfactant cloud points.20 The main effect observed was insolubilization of the surfactant as the salt concentration was raised (e.g.,

Figure 2B for SDS and NaCl); however, the amount of salt that can be added to the system can be increased by raising the temperature (Figure 2C). In the absence of hydrochloric acid, the two isotropic phases were not obtained at any ionic strength (0.02.0 M) or temperature (10-90°C) tested. Therefore, the presence of hydrochloric acid is essential for the two isotropic phases to be formed from anionic surfactant solutions. Phase Volume Ratios of Anionic Surfactants in Aqueous Hydrochloric Acid Solutions. Phase volume ratios of hydrochloric acid solutions of the four anionic surfactants tested (SDS, SDSA, SDBSA, and Aerosol OT) were determined at different concentrations of surfactant and acid. Table 1 shows the percentages of surfactant-rich phase and the theoretical preconcentration factors (maximum possible value at 100% recovery) obtained. The former was calculated as the ratio of the volume of rich phase to the total volume of the solution used for cloud point separation; the latter was obtained as the ratio of the volume of the sample solution used to that of the surfactant-rich phase. Preconcentration factors increased with decreasing surfactant concentration; in this respect, anionic surfactants in hydrochloric acid behaved as nonionic surfactant in aqueous solutions.17 The highest theoretical preconcentration factor (about 230) was achieved with Aerosol OT; the maximum preconcentration factor obtained with SDS was 30. The preconcentration factor can easily be altered by changing the amount of surfactant used; this allows one to design analytical schemes with a given separation factor as a function of the amount of analyte to be determined, the available volume of sample, and the analytical technique to be used. The dependence of the theoretical preconcentration factor on the hydrochloric acid concentration was not so clear. The factors for SDS and Aerosol OT decreased when the hydrochloric acid concentration was raised by one unit; however, an identical increase in the acid concentration resulted in increased preconcentration factors with SDSA and SDBSA. From these results, we can conclude that using anionic surfactants in a hydrochloric acid medium for cloud point extraction provides very high preconcentration factors, especially with Aerosol OT, which enables preconcentration in highly dilute samples. Optimization of the Time for Phase Separation. Aqueous solutions of surfactants with a sulfonic group (SDSA, SDBSA, and Aerosol OT) separated immediately into two isotropic phases upon addition of hydrochloric acid in the corresponding two liquid-phase regions (see Figure 1). Centrifugation at 1500 rpm for 10 min provided perfectly clear, homogeneous phases. On the other hand, the anionic surfactant SDS (with a sulfate group) required waiting for about 24 h before centrifugation for the surfactant-rich phase to be formed. To shorten this time, which was too long for analytical purposes, we investigated the influence of various parameters (centrifugation time and rate, temperature, and hydrochloric acid concentration) on the kinetics of phase separation for this anionic surfactant. The effect of the centrifugation was tested at 20 °C at rates and times over the ranges 500-10 000 rpm and 10 min-3 h, respectively. Centrifugation was found not to influence the rate of separation; in fact, only after these phases separated was centrifugation required in order to obtain clear, homogeneous phases. Analytical Chemistry, Vol. 71, No. 20, October 15, 1999

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Figure 5. (A, B) Chromatograms for the PAHs [(2) naphthalene, (3) fluorene, (4) acenaphthene, (5) phenanthrene, (6) anthracene, and (7) pyrene] obtained following injection of the sample into the surfactant-rich phase after cloud point extraction with (A) SDSA in hydrochloric acid ([HCl] ) 4.2 M) and (B) PONPE 6.0. Peak 1 in chromatogram A is that of the anionic surfactant, SDSA. Arrowheads in chromatogram B indicate the retention times for the PAHs tested [PONPE 6.0] ) [SDSA] ) 1% (w/v). (C, D) Chromatograms for progesterone (peak 2 in chromatogram C) obtained following injection of sample into the surfactant-rich phase after cloud point extraction with (C) SDSA in hydrochloric acid ([HCl] ) 4.2 M) or (D) PONPE 6.0. Arrowheads in chromatogram D indicate the retention time for progesterone. Peak 1 in chromatogram C is that of the anionic surfactant, SDSA. Chromatographic conditions as described in the Experimental Section. Detection wavelengths, 220 and 257 nm for PAHs and progesterone, respectively.

The effect of temperature and the hydrochloric acid concentration on the time required for SDS phase separation is illustrated in Figure 3. As can be seen from Figure 3A, the rate of the process considerably increased with increasing temperature. About 1 h 30 min was required for phase separation when the solution was keep at 60 °C. The kinetics of the process was also influenced by the hydrochloric acid concentration (Figure 3B); however, the time required for separation was only slightly reduced at acid concentrations above 4 M. Therefore, sulfonic anionic surfactants seem to excel those bearing sulfate groups in terms of analytical expeditiousness. This assertion, however, requires confirmation by further studies involving different anionic sulfate surfactants. Anionic Surfactant-Mediated Cloud Point Extractions from Liquid and Solid Samples. The ability of anionic sulfate and sulfonate surfactants to extract analytes from liquid and solid samples was investigated by using SDS and SDSA. Since both surfactants differ only in the ionic group, they allowed us to determine whether this group influences the efficiency of the extraction process in any way. The aim was to determine whether 4524

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the slow kinetics of phase separation of SDS affects the amount of analyte that is extracted. Table 2 shows the percent recoveries achieved for aqueous samples containing variable pyrene concentration. Good recoveries were obtained in both surfactantmediated cloud point extractions. With SDSA, percent recoveries were close to 100, even at the highest pyrene concentrations tested. No attempts were made to optimize the extraction conditions (e.g., extraction time or amount of surfactant) with SDS at pyrene concentration greater than 2 µg/mL with SDS. Preconcentration factors were about 10 and 20 for SDS and SDSA, respectively, under these experimental conditions. Higher preconcentration factors can be obtained by decreasing the surfactant content in the solution or increasing the sample volume used for cloud point extraction. The ability of aqueous acid solutions of anionic surfactants to extract and preconcentrate analytes from solid samples was tested by using a certified reference material (CRM 088, which consists of PAHs in dried sewage sludge). Figure 4 shows the chromatogram obtained for a sample containing 20 mg of sewage sludge that was extracted with 10 mL of a 1%(w/v) aqueous solution of

SDSA in 4.2 M hydrochloric acid for 2 h; after centrifugation for 30 min, an aliquot of the surfactant-rich phase was injected into the chromatographic system. The retention times for the six numbered peaks in the figure correspond to seven of the PAHs studied; in fact, benzo[a]pyrene and benzo[e]pyrene (peak 5) coelute when the Nova-Pack C18 column is used. No separation of the isotropic phases was observed when an amount of dried sewage sludge greater than 25 mg was used for extraction with SDSA. With 10 mL of a 1% (w/v) aqueous solution of SDS in 4.2 M hydrochloric acid as extractant, the largest amount of sample that allowed phase separation was 50 mg. Because of the sample amount permitted, it would be advisable to check the absence of errors due to inhomogenicities when analyzing authentic contaminated sewage sludge samples. Both SDSA and SDS solutions proved effective to extract PAHs from the solid sample (Table 3). Calibration curves for each such PAH were obtained from injections of different standards in the surfactant-rich phase. All the compounds studied appeared to be completely separated from the anionic surfactant in the chromatograms obtained for injections of the standards; therefore, no influence of the anionic micellar medium on the measured fluorescence intensity for these compounds was observed. It was checked that the surfactant-rich phase had no effect on the chromatographic separation achieved for the PAHs tested. Chromatograms obtained in the absence and presence of the surfactant-rich phase were identical. On the other hand, no change in the C18 column efficiency was observed after repeated injections of surfactant. No cleanup step was conducted prior to injection, so recoveries above 100% were obtained for some of the PAHs tested (viz pyrene and benz[a]anthracene). Interfering species extracted in the surfactant-rich phase increased the fluorescence signals obtained at the excitation and emission wavelengths used for these PAHs. No attempt was made to remove the interferents, however. Benzo[b]naphtho(2,1-d)thiophene could not be quantified in the solid sample because of the low sensitivity of fluorimetric detection for this PAH. For the other PAHs tested, recoveries were close to 100% with both SDSA and SDS as extractant. Therefore, sulfate and sulfonate anionic surfactants with the same hydrophobic group are similarly effective in terms of extraction. The most common procedures for the extraction of PAHs from environmental samples are either the use of solid sorbents21-24 or liquid-liquid extraction.25 Neither of these methods is ideal, and both have several significant disadvantages.26 The major disadvantage of liquid-liquid extraction is the use of large volumes of expensive, high-purity organic solvents, which eventually must be disposed of. Other drawbacks include emulsion formation, lack of sensitivity for more volatile analytes, and extremely time-consuming analysis. While solid-phase extraction reduces solvent requirements considerably compared with liquid(21) Brouwer, E. R.; Hermans, A. N. J.; Lingeman, H.; Brinkman, U. A. Th. J. Chromatogr., A 1994, 669, 45-57. (22) Kicinski, H. G.; Adamek, S.; Kettrup, A. Chromatographia 1989, 28, 203208. (23) Krylov, A. I.; Kostyuk, I. O.; Volynets, N. F. J. Anal. Chem. 1995, 50, 543551. (24) Eastwood, D.; Domı´nguez, M. E.; Lidberg, R. L.; Poziomek, E. J. Analusis 1994, 22, 305-310. (25) Villaizan, M. J. L. A.; Falcon, S. G.; Yusty, M. A. L.; Lozano, J. S. J. AOAC Int. 1995, 78, 402-406. (26) Liska, I.; Krupcik, J.; Leclercq J. High Resolut. Chromatogr. 1989, 12, 577590.

Figure 6. Chromatogram for vitamin E obtained following injection of the sample into the surfactant-rich phase after cloud point extraction with SDSA in hydrochloric acid ([HCl] ) 4.2 M). Peaks 1 and 2 in the chromatogram are those of SDSA and vitamin E, respectively. [vitamin E] ) 2 µg/mL. Chromatographic conditions as described in the Experimental Section.

liquid extraction, they are not eliminated. Finally, humic acids interfere in liquid-solid extraction of organics, leading to lower recoveries than those obtained by liquid-liquid extraction.27 Anionic Surfactant-Mediated Cloud Point Extraction as a Preconcentration Technique for HPLC. The principal aim of this experiment was to check whether nonaromatic ionic surfactants have retention times and absorbance or fluorescence signals low enough to allow the chromatographic determination of polar analytes. In such a case, the surfactant need not be removed before sample injection.15 The analytes used as models for this application were those that could not be adequately separated from nonionic surfactants (Triton X or PONPE series).15,20 A mixture of PAHs (naphthalene, fluorene, acenaphthene, phenanthrene, anthracene, and pyrene), and progesterone, were investigated in this respect. Figure 5 A and C show the chromatograms for the selected PAHs and progesterone, respectively. Complete separation of analytes from the surfactant was achieved. Both SDS and SDSA (27) Johnson, W. E.; Fendinger, N. J.; Plimmer, J. R. Anal. Chem. 1991, 63, 3, 1510-1513.

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behaved similarly in this respect. For comparison, the chromatograms obtained with PONPE 6.0 under the same conditions as for anionic surfactants are shown in Figure 5 parts B and D for PAHs and progesterone, respectively. The PONPE 6.0 peak overlapped with those for the analytes. Because anionic surfactants separate into two isotropic phases at temperatures as low as 10 °C, we investigated their usefulness for extracting thermally labile compounds, using SDSA. The use of this cloud point approach was found to minimize vitamin E decomposition; recoveries between 80 and 85% were achieved for analyte concentrations in the range 0.1-2.0 µg/mL. Figure 6 shows a typical chromatogram; complete separation of vitamin E from the surfactant was achieved. Parallel experiments with PONPE 6.0 provided inadequate separation. Vitamin E reportedly decomposes on extraction with PONPE 7.5 at 30 °C.20 In summary, although extensive investigations are required to demonstrate the actual utility of anionic surfactants in cloud point methodology, some of their features make them highly atractive for use in this approach. Thus, they separate under a

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broad range of experimental conditions (acid concentrations, temperature, ionic strength, etc.); also, they are commercially available in a variety of structures. Their ionic character and the absence of aromatic groups from many of the more widely used anionic surfactants make them especially useful for the preconcentration of polar analytes prior to HPLC; in fact, they seemingly provide high preconcentration factors and high recoveries from solid and liquid samples. Finally, they can be used for the separation or preconcentration of temperature-sensitive molecules. In the authors’ opinion, efforts should focus on automating cloud point methodology in order to make it competitive with other extraction alternatives (e.g., liquid-liquid and solid-phase extraction).

Received for review February 1, 1999. Accepted July 21, 1999. AC990106G