Anal. Chem. 1997, 69, 3243-3250
Separation of Priority Pollutant Phenols with Coelectroosmotic Capillary Electrophoresis Andreas Zemann* and Dietmar Volgger
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
Various mixtures of phenolic compounds, most of them being priority pollutant phenols, are separated by coelectroosmotic capillary electrophoresis. To obtain short separation times a codirectional movement of the anionic phenolates and the electroosmotic flow (EOF) is established by adding a polycationic EOF modifier to the alkaline buffer electrolyte. To increase the selectivity of the separation and the resolution between the solutes, organic solvent mixtures are added to the separation buffer. Furthermore, coelectroosmotic MECC of phenols using cetyltrimethylammonium bromide (CTAB) as pseudostationary phase and acetonitrile as organic modifier is performed. The developed methods are used for the fast separation of the 9 cresol and xylenol isomers, as well as mixtures of 18 chlorophenols, 11 phenols (acid extractable mixture, EPA M-625A), and 9 phenols (EPA M-8040B-R). Separation mechanisms are discussed on the basis of solvation effects of the phenolic solutes. In the case of CTAB-MECC the influence of the CTAB concentration and of acetonitrile on the solubilization of the phenols is investigated. Phenolic compounds are of great concern, because they occur in large amounts as natural constituents of plant lignin and as products and byproducts of numerous industrial processes. Lowmolecular weight phenols often cause problems as pollutants with a considerable toxicity because of their frequent utilization and appearance. In particular, chlorophenolic compounds are hazardous as they are accumulated in fat tissue due to their lipophilic character. As a consequence, they are considered as organic priority pollutants by the U.S. Environmental Protection Agency (EPA) as well as by the European Union. Routine analysis of phenols can be carried out using chromatographic methods such as GC,1,2 HPLC,3,4 SFC,5 and ITP.6,7 In recent years, a growing number of reports deal with the * To whom correspondence should be addressed. Tel: +43-512-507-5180. Fax: +43-512-507-2965. E-mail:
[email protected]. (1) Abrahamson, K.; Ekdahl, A. J. Chromatogr. 1993, 643, 239. (2) Cooper, J. F.; Tourte, J.; Gros, P. Chromatographia 1994, 38, 147. (3) Thomson, C. A.; Chesney, D. J. Anal. Chem. 1992, 64, 848. (4) Klampfl, C. W.; Spanos, E. J. Chromatogr. A 1995, 715, 213. (5) Raynor, M. W.; Bartle, K. D.; Clifford, A. A.; Chalmers, J. M.; Katase, K.; Rouse, C. A.; Markides, K. E.; Lee, M. L. J. Chromatogr. 1990, 505, 179. (6) Pfeifer, P. A.; Bonn, G. K.; Bobleter, O. Fresenius J. Anal. Chem. 1983, 315, 205. (7) Praus, P. Anal. Chim. Acta 1995, 302, 39. (8) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111. (9) Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 348, 39. S0003-2700(97)00371-5 CCC: $14.00
© 1997 American Chemical Society
application of capillary electrophoresis (CE) for the separation of phenolic compounds.8-18 In traditional CE, the inherent electrophoretic mobility vectors of organic anions are directed against the cathodic electroosmotic flow (EOF) vector, which may be termed as counterelectroosmotic CE.19 By this means, the anionic analytes pass the detection window after the neutral EOF marker. To reduce the separation time for anionic analytes, high separation voltages, short capillaries, or high observed mobilities are required as expressed in eq 1 where L and l are the total and the effective length of the
t)
Ll µobsU
(1)
capillary, respectively, U is the applied voltage, and µobs is the observed mobility, which is the sum of the inherent electrophoretic mobility µep and the electroosmotic mobility µeof. However, by application of a high separation voltage, the increase of separation speed thus achieved is often counteracted by a loss of separation efficiency due to the production of Joule’s heat. With short effective separation lengths, fast separations are achieved preferably at the expense of resolution. Another attempt to reduce the separation time is to increase the observed mobility of the anionic analytes. This can be accomplished by establishing an anodic EOF, which is achieved by positively coating the inner surface of the silica capillary using long-chained alkylammonium bromides20,21 or polycations13,16,17,22,23 and switching the polarity of the power supply. By this means, anionic species migrate in the same direction as the EOF and pass the detector before a neutral EOF marker (coelectroosmotic CE19). Assuming identical experimental conditions in terms of separation length, applied high voltage, and produced current, the (10) Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1987, 396, 350. (11) Gaitonde, G.; Pathak, P. J. Chromatogr. 1990, 514, 389. (12) Gonnord, M. F.; Collet, J. J. Chromatogr. 1993, 645, 327. (13) Masselter, S. M.; Zemann, A. J.; Bobleter, O. Electrophoresis 1993, 14, 36. (14) Chao, Y.-C.; Whang, C.-W. J. Chromatogr. 1994, 663, 229. (15) Smith, S. C.; Khaledi, M. G. Anal. Chem. 1995, 65, 193. (16) Masselter, S. M.; Zemann, A. J. J. Chromatogr. A 1995, 693, 359. (17) Masselter, S. M.; Zemann, A. J. Anal. Chem. 1995, 67, 1047. (18) Li, G.; Locke, C. J. Chromatogr. 1995, 669, 93. (19) Jandik, P.; Bonn, G. Capillary Electrophoresis of Small Molecules and Ions; VCH Publishers: New York, 1993. (20) Jandik, P.; Jones, W. R.; Weston, A.; Brown, P. R. LC-GC 1991, 9, 634. (21) Jones, W. R.; Jandik, P.; Merion, M. U.S. Patent 5,104,506, 1992. (22) Wiktorowicz, J. E.; Colburn, J. C. Electrophoresis 1990, 11, 769. (23) Volgger, D.; Zemann, A. J.; Bonn, G. K.; Antal, M. J., Jr. J. Chromatogr. A 1997, 758, 263. (24) Jorgenson, J. W. Lukacs, K. D. Anal. Chem. 1981, 53, 1298. (25) Jorgenson, J. W. Lukacs, K. D. Science 1983, 222, 266.
Analytical Chemistry, Vol. 69, No. 16, August 15, 1997 3243
application of this concept bears several advantages. First, the observed mobility µobs is increased, which speeds up the separation. Second, in accordance with eq 2,24,25 high observed mobilities
N)
(µep + µeof)U 2D
(2)
result in an increase of separation efficiency compared to counterelectroosmotic separations. On the contrary the gain in separation efficiency may be paid by a loss in resolution if the electrophoretic mobilities of the analytes remain constant and if the sum of the average mobility of the analytes and the electroosmotic mobility inceases more markedly than the square root of the separation efficiency as expressed in eq 3.24,26 Here Rs is the
Rs )
xN µep(1) - µep(2) 4 µep(av) + µeof
(3)
resolution between two analytes with electrophoretic mobilities µep(1) and µep(2), respectively, where µep(av) denotes the average electrophoretic mobility of the two analytes. Method optimization to improve selectivity and resolution in addition to high separation efficiencies is generally required. Some authors employ organic solvents in order to improve resolution and selectivity of specific analytes,13,17,27-35 and others make use of the different rates of solubilization of the analytes in a charged micellar pseudophase (micellar electrokinetic capillary chromatography, MECC).9,15 With counterelectroosmotic methods the addition of organic solvents usually increases the separation time due to the reduced EOF velocity. MECC methods increase separation times because the solutes are retained as they are solubilized into anionic micelles. Thus, an increase in selectivity with counterelectroosmotic methods often comes on the expense of separation speed. Coelectroosmotic separations, however, compensate for the theoretical loss in resolution according to eq 3 by the increase in selectivity which is achieved by the addition of organic solvents. Furthermore, due to the codirectional movement of analytes and the EOF the contribution of the reduced EOF to the migration time is less marked than with counterelectroosmotic methods. In the present paper, fast separations of mixtures containing priority pollutant phenols are presented by employing coelectroosmotic CE with reversed EOF in fused silica capillaries, which are dynamically coated with a polycationic EOF modifier. In order to optimize selectivity and resolution, mixed organic solvent electrolyte systems as well as cationic micelles are used. EXPERIMENTAL SECTION Instrumentation. The equipment used in this contribution consisted of a capillary electrophoresis system (Waters Quanta (26) Giddings, J. C. Sep. Sci. 1969, 4, 181. (27) Fujiwara, S.; Honda, S. Anal. Chem. 1987, 59, 487. (28) Khaledi, M. G.; Smith, S. C.; Strasters, J. K. Anal. Chem. 1991, 63, 1820. (29) Gorse, J.; Balchunas, A. T.; Swaile, D. F.; Sepaniak, M. J. J. High Resolut. Chromatogr. 1988, 11, 554. (30) Schu ¨ tzner, W.; Kenndler, E. Anal. Chem. 1992, 64, 1991. (31) Sepaniak, M. J.; Swaile, D. F.; Powell, A. C.; Cole, R. O. J. High Resolut. Chromatogr. 1990, 13, 679. (32) Buchberger, W.; Haddad, P. R. J. Chromatogr. 1992, 608, 59. (33) Walbroehl, Y.; Jorgenson, J. W. Anal. Chem. 1986, 58, 479. (34) Sahota, R. S.; Khaledi, M. G. Anal. Chem. 1994, 66, 1141. (35) Salimi-Moosavi, H.; Cassidy, R. M. Anal. Chem. 1995, 68, 293.
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4000) connected to an A/D interface (System Interface Module) and a personal computer equipped with chromatography software (Maxima 820; Waters Chromatography, Milford, MA). Uncoated, narrow-bore silica capillaries (Composite Metal Services, Worchester, U.K.) with a total length of 32 cm, an effective separation length of 24.5 cm, and an inner diameter of 50 µm were used. Hydrostatic injection of the samples was carried out between 3 and 15 s at a height of 10 cm. Direct UV detection was performed at 214 nm. Reagents. All reagents were of analytical grade. Standard solutions were prepared by dissolving the pure phenols (AccuStandard Inc., New Haven, CT; Sigma-Aldrich Handels-GesmbH, Vienna, Austria) in gradient grade methanol (Fluka AG, Buchs, Switzerland) to a final concentration of 100 ppm. Methanol, acetonitrile, 2-butanol, ethylene glycol, toluene, cetyltrimethylammonium bromide (CTAB), and 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (hexadimethrine bromide; polybrene) were obtained from Sigma-Aldrich Handels-GesmbH, Vienna, Austria. In all experiments hexadimethrine bromide was used as EOF modifier at a concentration of 0.001% (w/v), prepared by dilution using a 1% (w/v) stock solution. Buffer electrolyte mixtures were prepared from sodium tetraborate decahydrate (Fluka AG, Buchs, Switzerland) and disodium hydrogen phosphate (Merck, Vienna, Austria) in ultrapure water with a conductivity of 18.2 MΩ (Barnstead/Thermolyne, Dubuque, IA). The pH values were carefully adjusted with 1 M NaOH prior to the addition of organic solvents using a pH meter (WTW GmbH, Weilheim, Germany). All buffer solutions were vacuum degassed and sonicated prior to use.
RESULTS AND DISCUSSION In order to apply the coelectroosmotic separation principle for phenolic compounds, electrolyte buffers with pH values above the respective pKA values of the solutes are used. To a large extent, the pKA values of phenolic compounds depend on the site and degree of substitution, as well as on the electron donor or acceptor properties of the substituents. This results in a wide span of aqueous pKA values for the investigated phenols ranging from a pKA of 4.08 for 2,4-dinitrophenol up to a pKA of 10.63 for 2,6-dimethylphenol. The respective pKA values of the investigated phenols are listed in Table 1.36-40 Mixed Organic Solvent Systems. With hexadimethrine bromide and other EOF modifiers, such as cetyltrimethylammonium bromide (CTAB) the coelectroosmotic CE analysis of hydrophobic aromatic solutes, such as phenols, often results in peak zone broadening. This is due to the affinity of the phenolic compounds, especially of higher chlorinated and alkylated phenols, to the aliphatic chains of the EOF modifier, which causes a retention of the phenols. On the contact of two hydrophobic species, such as a phenol and an EOF modifier molecule containing a long alkyl chain, the entropy of the system increases as water molecules are removed from the hydration shell and are rearranged in the natural water structure.41 With polycationic EOF (36) Li, S.; Paleologou, M.; Purdy, W. C. J. Chromatogr. Sci. 1991, 29, 66. (37) Fiege, H. In Ullmann’s Encyclopedia of Industrial Chemistry; Gephartz, W., Yamamoto, Y. S., Kandy, L., Pfefferkorn, R., Rouncaville, J. F., Eds.; VCH Publishers, Inc.: Weinhein, Germany, 1987. (38) Lange’s Handbook of Chemistry; McGraw-Hill: New York, 1992. (39) Bo ¨hmer, V.; Wamsser, R.; Ka¨mmerer, H. Monatsh. Chem. 1973, 104, 1315. (40) The Merck Index; Merck & Co., Inc.: Rahway, NJ, 1989. (41) Baker, B. R. J. Chem. Educ. 1967, 44, 610.
Table 1. Aqueous Acidity Constants, Migration Times, and Separation Efficiencies of the Investigated Phenolic Compounds compound
pKA
Figure 1f
Figure 2f
2,4-dinitrophenol 4,6-dinitro-2-methylphenol 2-(1-methylpropyl)-4,6-dinitrophenol (Dinoseb) pentachlorophenol 2,3,4,6-tetrachlorophenol 2,3,5,6-tetrachlorophenol 2,3,6-trichlorophenol 2,4,6-trichlorophenol 2,3,5-trichlorophenol 4-nitrophenol 2,6-dichlorophenol 2,4,5-trichlorophenol 2-nitrophenol 2,3,4-trichlorophenol 2,5-dichlorophenol 2,4-dichlorophenol 2,3-dichlorophenol 3,5-dichlorophenol 3,4-dichlorophenol 2-chlorophenol 3-chlorophenol 4-chloro-3-methylphenol 4-chlorophenol phenol 3-methylphenol 3,5-dimethylphenol 4-methylphenol 2-methylphenol 3,4-dimethylphenol 2,5-dimethylphenol 2,3-dimethylphenol 2,4-dimethylphenol 2,6-dimethylphenol migration time reproducibility
4.08c 4.34d 4.62e
1.430 (868)
1.483 (908) 1.553 (944)
No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 a
b
c
4.93a 5.53a 5.76a 6.10a 6.51a 6.92a 7.15c 7.15a 7.20a 7.22c 7.34a 7.69a 8.51a 8.52a 8.54a 8.87a 9.13a 9.53a 9.55c 9.70a 10.09a 10.09b 10.19b 10.27b 10.32b 10.36b 10.41b 10.54b 10.60b 10.63b d
Figure 3f
Figure 4f
Figure 7f 1.455 (1089)
1.653 (452)
2.221 (445) 1.797 (362)
1.605 (688) 1.620 (658)
1.901 (510) 1.823 (645) 1.803 (750) 1.692 (781) 1.755 (696) 1.846 (930)
2.153(124)
1.424 (1022) 1.397 (1044) 1.566 (986)
1.709 (661) 1.923 (865)
1.380 (838) 1.779 (236)
1.372 (948) 1.583 (823)
1.499 (256)
1.528 (767)
2.029 (878) 2.116 (628) 2.531 (644) 2.375 (730) 2.738 (724) 3.171 (815) 3.146 (691) 3.438 (648)
1.293 (868)
2.688 (281) 2.873 (379)
3.535 (563) 3.604 (254)
2.744 (618) 2.828 (724)
2.973 (597)
3.502 (346)
0.46-0.73
0.60-0.99
e
0.18-0.73
1.663 (465) 1.892 (694) 1.782 (732) 1.807 (737) 1.978 (669) 2.030 (794) 2.112 (570) 2.170 (681) 2.143 (598) 0.53-0.97
1.538 (459) 1.638 (445)
1.986 (244) 0.83-1.95
f
Reference 36. Reference 37. Reference 38. Reference 39. Reference 40. Figure columns: Migration times in minutes. Numbers in parentheses: Separation efficiencies × 103 theoretical plates per meter separation length; migration time reproducibility expressed as % relative standard deviation of six injections (lowest and highest value).
modifiers, such as hexadimethrine bromide, these effects cause less problems due to the shorter aliphatic chains and the lower concentration required as compared to CTAB.16 As a consequence, this cationic EOF modifier was used throughout the experiments in this investigation. However, a dramatic improvement concerning selectivity, resolution, and separation efficiency can be reached with both CTAB and HDB as EOF modifier if organic solvents, such as aliphatic alcohols (e.g., methanol, ethanol, propanol) and acetonitrile or mixtures thereof, are added to the carrier electrolyte.13,16,17,42 The positive effects of organic solvents are attributed to a reduction of the interactions of aromatic solutes and the aliphatic chains of the EOF modifier. The suitability of a specific organic solvent for this purpose mainly depends on its solvophobic properties and the ability to solvate the solute. Figure 1 depicts the baseline separation of a mixture of 9 priority pollutant phenols (EPA M-8040B-R) in less than 3 min. Besides phosphate, borate, and hexadimethrine bromide the electrolyte consisted of 18% 2-butanol, 12% ethylene glycol, and 10% acetonitrile. Figure 2 shows the separation of another mixture of priority pollutant phenols (EPA M-625A) containing 11 compounds in a part of the time required for counterelectroosmotic methods.14,18
The separation is achieved using an electrolyte containing 25% 2-butanol, 5% ethylene glycol, and 10% acetonitrile. The high amount of 2-butanol is responsible for the selectivity especially of the fast migrating phenols, which can thus be separated with high resolution. Figure 3 depicts the separation of a mixture of 18 phenolic compounds consisting of 17 chlorophenols and unsubstituted phenol. This separation is obtained with a buffer consisting of 17% 2-butanol, 13% diethylene glycol, and 10% acetonitrile. 2-Butanol ensures a high resolution of the slower migrating phenols, especially for 2-chlorophenol and 3,4-dichlorophenol. If the ethylene glycol concentration is further increased and/or the 2-butanol concentration is decreased, the faster migrating phenols exhibit an even better resolution, but then 2-chlorophenol and 3,4-dichlorophenol comigrate and are not separated. The buffer pH of 8.25 is noteworthy, as the pKA values of the analytes in this mixture span a range from 4.93 (pentachlorophenol) to 10.09 (phenol). In the past the separation of the chlorophenol isomers could not be performed by CZE43 and required long migration times with MECC methods,9,43 respectively. Comparably efficient and fast coelectroosmotic separations of the phenolic compounds as depicted in Figures 1-3 could not be achieved with buffer systems containing only one organic solvent.
(42) Zemann, A. J. J. Capillary Electrophor. 1995, 2, 131.
(43) Crego, A. L., Marina, M. L. J. Liq. Chromatogr. Relat. Technol. 1997, 20, 1.
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3245
Figure 1. Separation of 9 priority pollutant phenols (EPA M-8040BR) by coelectroosmotic CZE using a mixed organic solvent electrolyte system. Electrolyte: 10 mM phopshate, 1.25 mM tetraborate, pH 10.3, 18% 2-butanol, 12% ethylene glycol, 10% acetonitrile, U ) -30 kV, I ) 28.4 µA, T ) 24 °C; injection, 10 s hydrostatically. Peak assignments are as in Table 1.
Figure 2. Separation of 11 priority pollutant phenols (EPA M-625A) by coelectroosmotic CZE using a mixed organic solvent electrolyte system. Electrolyte: 15 mM phopshate, 1.25 mM tetraborate, pH 10.3, 25% 2-butanol, 5% ethylene glycol, 10% acetonitrile, U ) -30 kV, I ) 36 µA, T ) 26 °C; injection, 8 s. Peak assignments are as in Table 1.
However, with mixed organic solvent systems significant changes in the solvation of the analytes can be assumed. A selective solvation is likely to occur with a higher portion of organic solvents in the primary solvation layer compared to the bulk solution.44 Generally, most of the inorganic anions are spherically symmetrical, whereas in unsubstituted phenolate a main part of the charge is allocated on the small hydroxyl group.45 The introduction of electron-directing substituents on a phenolic compound causes a charge redistribution, which further affects the solvation of the solute. The electrokinetic properties of solvated ions can be influenced by the interactions of the analytes with the very adjacent solvent molecules in the primary solvation layer which is affected by the choice of the solvent composition. For example, phenols carrying nitro groups are known to be selectively solvated by acetonitrile rather than by water. On the contrary, other aromatic compounds, such as benzoic acid, are preferentially solvated by water.46 Another specific property of weak acids, such as phenolic compounds, may take effect in electrolytes with a pH value approximately equal to the pKA of the solutes. In protophobic solvents the formation of homoconjugated species of the hydrogen bis(phenolate) type is reported, which stabilizes the phenols by formation of hydrogen bonds. Strong homoconjugation is ob-
served especially for phenols carrying chloro and nitro substituents in the o- and p-positions. This affects the migration behavior of such analytes in electrolyte systems containing specific amounts of protophobic solvents, e.g., acetonitrile. Solvents with low dielectric constants facilitate the formation of ternary aggregates and some hydrogen bis(phenolates) are known to be stable even in protic solvents.47,48 All these mechanisms significantly contribute to the physicochemical characteristics of the analytes and of the buffer, such as effective ionic radii, the ζ-potential of solutes and of the capillary wall, the dielectric constant, and the viscosity of the buffer. In Table 1 it is evident that the order of migration in all electropherograms presented is not consistent with the aqueous pKA values of the phenols. This can partly be explained by the fact that in electrolyte systems which contain high amounts of organic solvents the dissociation constants of weak organic acids, such as substituted phenols, may be considerably different from water. Futhermore, pH ranges in organic solvents can exceed the values known for aqueous solutions by far.49 The optimization of separation speed and zone resolution, however, is currently a subject of experimental trial and error and may be simplified if the electrophoretic behavior of the phenolic compounds can be more reliably attributed to specific mechanisms. A quantitative prediction of the changes caused by the
(44) Strehlow, H.; Knoche, W.; Schneider, H. Ber. Bunsenges. Phys. Chem. 1973, 77, 760. (45) Kebarle, P.; Davidson, W. R.; French, M.; Cumming, J. B.; McMahon, T. B. Faraday Discuss. Chem. Soc. 1977, 64, 220. (46) Benter, G.; Schneider, H. Ber. Bunsenges. Phys. Chem. 1973, 77, 997.
3246 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
(47) Kolthoff, I. M. Anal. Chem. 1974, 46, 1992. (48) Fritz, J. S. Acid-Base Titrations in Non-Aqueous Solvents; Allyn and Bacon: Boston, MA, 1973. (49) Galster, H. pH-Messung; VCH: Weinheim, Germany, 1990.
Figure 3. Separation of 18 chlorophenols by coelectroosmotic CZE using a mixed organic solvent electrolyte system. Electrolyte: 10 mM phopshate, 1.25 mM tetraborate, pH 8.25, 17% 2-butanol, 13% ethylene glycol, 10% acetonitrile, U ) -30 kV, I ) 21.5 µA, T ) 29 °C; injection, 3 s. Peak assignments are as in Table 1.
presence of organic solvents in the electrophoretic separation of phenolic compounds is difficult to perform because the influence of various substituents on electrokinetic properties is hardly assessable. CTAB-MECC. Another approach for the separation of phenolic mixtures uses the fact that phenols can be solubilized in charged micelles. This has been extensively described in counterelectroosmotic methods with sodium dodecyl sulfate (SDS) as a micelle-forming agent.50,51 In this investigation, micellar coelectroosmotic CE of phenolic compounds is performed with cetyltrimethylammonium bromide (CTAB), which is used as a micellar pseudophase. The separation of phenolic compounds carried out coelectroosmotically with cationic micelles bears several advantages. A fast separation is obtained as the analytes still migrate in front of the EOF, and at the same time, selectivity is increased by the interaction of the phenols with CTAB micelles. The use of cationic alkylammonium surfactants for EOF reversal for the separation of anionic analytes has been widely described.52-56 In order to reverse the EOF, the CTAB surfactant is commonly used at concentrations below the critical micelle (50) Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 348, 39. (51) Smith, S. C.; Khaledi, M. G. J. Chromatogr. 1993, 632, 177. (52) Romano, J.; Jandik, P.; Jones, W. R.; Jackson, P. E. J. Chromatogr. 1991, 546, 411. (53) Jones, W. R.; Jandik, P. J. Chromatogr. 1991, 546, 445. (54) Benz, N. J.; Fritz, J. S. J. Chromatogr. 1994, 671, 437. (55) Ro¨der, A.; Ba¨chmann, K. J. Chromatogr. A 1995, 689, 305. (56) Nishi, H.; Tsumagari, N.; Terabe, S. Anal. Chem. 1989, 61, 2434.
concentration (cmc). If the critical hemimicelle concentration is reached, a dilayer is formed consisting of dimeric associates, which are called admicelles or hemimicelles.57-59 After adsorption on the capillary wall, the CTAB hemimicelles establish a positively charged, dynamic coating, which reverses the sign of the ζ-potential. As a consequence, the direction of the EOF is reversed and moves towards the anode. For phenolic compounds, however, simply increasing the CTAB concentration above the cmc and then performing MECC with CTAB acting both as EOF modifier and as micellar pseudophase is not recommendable.60 Although a strong anodic EOF can be achieved with CTAB at concentrations above the cmc, electropherograms of hydrophobic solutes with poor separation efficiencies, resolution, and reproducibility are achieved. Even though the use of organic solvents is possible in order to improve the separation, reproducibility is still poor. This is due to the fact that important properties of both the hemimicelle and of the micelle are altered. Solvent molecules affect the association tendency of the hemimicelle, and if aliphatic alcohols are present, the concentration required to reverse the EOF is reduced by more than 1 order of magnitude.42,54 A suitable way to overcome the problems associated with CTAB as EOF modifier is the use of surfactants, which coat the capillary wall without the formation of hemimicelles. Hexadimethrine bromide was thus used as EOF modifier for the experiments with the mixed organic solvent systems as well as with the CTAB-MECC system. This polycationic surfactant bears the advantage that the positive wall coating is more or less unaffected by most of the commonly used organic solvents. Finally, a system consisting of hexadimethrine bromide as EOF modifier, CTAB as micellar pseudophase, and specific amounts of organic solvents to ensure high separation efficiencies is used for the separation of the phenolic compounds. Figure 4 demonstrates the baseline separation of the 9 cresol and xylenol isomers. A CTAB concentration of 6 mM was found to be suitable in order to achieve both high resolution and short migration times. Acetonitrile was used as organic modifier at a concentration of 25%. The irregular baseline after the EOF is reproducible and becomes constant again after the micelle having passed the detector. The effect of the CTAB concentration on the separation can be discussed on the basis of capacity factors describing the degree of solubilization, which are calculated according to eq 4,28 where
k′ )
µS(CTAB) - µS µCTAB - µS(CTAB)
(4)
µS(CTAB) is the electrophoretic mobility of the solute solubilized in the micelle, µS is the mobility of the free solute, and µCTAB is the mobility of the cationic micelle. The mobility of the free micelle was determined by means of toluene assuming a complete solubilization. Equation 4 differs from the formula introduced by Terabe for the calculation of capacity factors for SDS-MECC50 because in the present discussion the unsolubilized analytes (57) Huang, Z.; Ma, J.; Gu, T. Acta Chim. Sin. 1989, 105; Chem. Abstr. 1989, 111, 202 679z. (58) Lucy, C. A.; Underhill, R. S. Anal. Chem. 1996, 68, 300. (59) Crosby, D.; El Rassi, Z. J. Liquid Chromatogr. 1993, 16, 2161. (60) Bjergegaard, C.; Michaelson, S.; Mortensen, K.; Sorensen, H. J. Chromatogr. 1993, 652, 477.
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Figure 4. Separation of the 9 cresol and xylenol isomers with coelectroosmotic CTAB-MECC. Electrolyte: 10 mM phopshate, 1.25 mM tetraborate, pH 10.7, 25% acetonitrile, 6 mM CTAB; U ) -20 kV, I ) 37 µA, T ) 26 °C; injection, 3 s. Peak assignments are as in Table 1.
of the solutes in the micelle and, as a consequence, no separation is achieved. If the CTAB concentration is increased above 10 mM, the analytes are completely solubilized and migrate with the same speed as the micelle. Generally, dimethylphenols exhibit a higher tendency to solubilize in the micelle than monomethylphenols, which is explained by the higher hydrophobicity due to the second methyl group. Three mechanisms are conceivable for a solubilization of phenols in CTAB micelles. First, the aromatic moiety of the phenol may be inserted into the micelle between the ammonium head groups with the hydoxyl-O being strongly solvated. Second, the phenol may penetrate deeper into the micelle with interacting hydroxyl-O and the ammonium head groups. Third, the molecular axis of the phenol and the micellar surface may be aligned parallel with interactions of the ammonium head groups with both the hydroxyl-O and the aromatic moiety.61 As the palisade layer is the preferred part of the CTAB micelle for solubilization of phenolic solutes, changes in the geometry of this part of the micelle mostly affect the capacity factors. The electrophoretic mobility of the CTAB micelle itself depends on the acetonitrile concentration, which affects size and ionization degree of the micelle due to the solubulization of acetonitrile in the micelle. With higher amounts of acetonitrile in the buffer, the electrophoretic mobility of the CTAB decreases. Intercalation of solvent molecules into the palisade layer of the micelle increases the average distance between the ionic head groups, which causes a reduction of the micelle surface charge density.61,62 In addition, the micellar core is also affected as solvents solubilize into the micelle under formation of mixed solvent-surfactant micelles and, at the same time, decrease the cmc. This is the case especially for hydrophobic solvents and solvents with long alkyl chains.62 Generally, the equlibrium of a solute solubilized in a CTAB micelle can be expressed by eq 561,63
KS )
Figure 5. Dependence of the capacity factors of cresol and xylenol isomers on the CTAB concentration. Acetonitrile concentration: 25% (v/v). Electrophoretic conditions are as in Figure 4.
exhibit an electrophoretic mobility, whereas the formula used for SDS-MECC assumes uncharged analytes without an inherent electrophoretic mobility. Figure 5 depicts the dependence of the capacity factors of the cresol and xylenol isomers on the CTAB concentration at a constant acetonitrile concentration of 25%. If the CTAB concentration is chosen too low, poor micelle formation, no solubilization 3248
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[SM] ([CTAB] - cmc - [SM]) [SS]
(5)
The solubilization constant KS is thus dependent both on the actual CTAB concentration and on the cmc of the surfactant, as well as on the distribution ratio of the solute in the micellar phase [SM] and in the aqueous-solvent phase [SS]. Both the cmc of CTAB and the distribution ratio are subject to alteration when acetonitrile is present. To evaluate the influence of acetonitrile on the micellar solubilization, the capacity factors of the phenols at a CTAB concentration of 6 mM are drawn against the acetonitrile concentration (Figure 6). At low acetonitrile contents the phenols are strongly solubilized in the micelle. This is mainly due to the more hydrophilic character of the bulk solution compared to the interior of the micelle which increases the affinity of the hydrophobic solutes to the micelle. With increasing acetonitrile concentration, the solubilization tendency of the phenols into the micelle is reduced as the hydrophilic properties of the bulk solution are reduced. This facilitates solvation and stabilization of the phenols outside the micelle. As depicted in Figure 6, dimethylphenols experience a higher distribution ratio at aceto(61) Menger, F. M.; Portnoy, C. E. J. Am. Chem. Soc. 1967, 89, 4698. (62) Zana, R.; Yiv, S.; Strazielle, C.; Lianos, P. J. Colloid Interface Sci. 1981, 80, 208. (63) Bunton, C. A.; Cowell, C. P. J. Colloid Interface Sci. 1988, 122, 154.
Figure 6. Dependence of the capacity factors of a mixture of cresols and xylenols on the acetonitrile concentration. CTAB concentration: 6 mM. Electrophoretic conditions are as in Figure 4.
Figure 7. Separation of 9 priority pollutant phenols (EPA M-8040BR) with coelectroosmotic CTAB-MECC. Electrolyte: 10 mM phopshate, 1.25 mM tetraborate, pH 11.15, 30% acetonitrile, 8 mM CTAB; U ) -20 kV, I ) 40 µA, T ) 27 °C; injection, 15 s. Peak assignments are as in Table 1.
nitrile concentrations between 20 and 25% compared to monomethylphenols which is due to the additional methyl group. Figure 7 demonstrates the application of CTAB-MECC for the separation of 9 priority pollutant phenols (EPA M-8040B-R) using 8 mM CTAB and 30% acetonitrile. Compared to the electropherogram in Figure 1 the migration order of the compounds is different
Figure 8. Dependence of the capacity factors of a phenolic mixture (EPA M-8040B-R) on the CTAB concentration. Acetonitrile concentration: 30% (v/v). Electrophoretic conditions are as in Figure 7.
in the CTAB-MECC system. This is due to the different separation mechanisms of MECC compared to zone electrophoresis with mixed organic solvent systems. The separation in the mixed organic solvent system is mainly dependent on the pH value of the buffer electrolyte and the electrokinetic potential of the solutes whereas for CTAB-MECC the hydrophobicity of the solutes and the affinity to the micelle are responsible for the separation. Some advantages of the CTAB system become apparent when the separation in Figure 7 is compared to Figure 1. The resolution between the respective zones is higher, and a more uniform distribution of the peak zones throughout the separation window is observed even though the effective separation window is smaller. The dependence of the capacity factors of the EPA M-8040B-R phenols on the CTAB concentration is shown in Figure 8. Unlike the wide concentration range of SDS commonly suitable for MECC, the concentration range of CTAB appropriate for coelectroosmotic MECC is considerably small. Furthermore, the absolute concentration of the micellar pseudophase is lesser in the CTAB system (below 10 mM) compared to the SDS system (20-100 mM), which results in a lower ionic strength of the buffer itself. As a consequence, the production of current is lower, which in turn reduces the contribution of Joule heat to zone broadening. Various parameters for reproducibility and quantitation purposes for the developed systems were determined. A linear range of 1-40 mg/L (exception: Dinoseb with 2-40 mg/L), limits of detection for the phenols between 0.3 and 0.5 mg/L (Dinoseb 0.8 mg/L), and correlation coefficients (r2) of 0.9976-0.9999 were revealed. Good migration time reproducibilities of less than 1% relative standard deviation (6 injections) for the mixed organic solvent systems are obtained, whereas the values for the CTABMECC system were between 0.53 and 1.95, as depicted in Table 1. For both the mixed organic solvent system and the CTABMECC system a careful adjustment of the buffer pH is indispensible for reproducible results. Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
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CONCLUSIONS It is demonstrated that high resolution and separation efficiencies as well as fast separation times are achieved with coelectroosmotic CE using mixed organic solvent electrolyte systems as well as with coelectroosmotic MECC using CTAB as micellar pseudophase for the separation of priority pollutant phenols. Separation speed, resolution, and selectivity mainly depend on the composition of the electrolyte system in terms of organic solvents. It is shown that the effective CTAB-MECC of phenolic compounds
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requires the presence of an organic solvent, in this particular case acetonitrile.
Received for review April 8, 1997. 1997.X
Accepted May 29,
AC9703717 X
Abstract published in Advance ACS Abstracts, July 1, 1997.
