Solute−Solvent Interactions in Micellar Electrokinetic Chromatography

Anal. Chem. 2002, 74, 4447-4455

Solute-Solvent Interactions in Micellar Electrokinetic Chromatography. 6. Optimization of the Selectivity of Lithium Dodecyl Sulfate-Lithium Perfluorooctanesulfonate Mixed Micellar Buffers Elisabet Fuguet,† Clara Ra`fols,† Jose´ Ramo´n Torres-Lapasio´,‡ Marı´a Celia Garcı´a-A Ä lvarez-Coque,‡ † ,† Elisabeth Bosch, and Martı´ Rose´s*

Departament de Quı´mica Analı´tica, Universitat de Barcelona, Diagonal 647, E-08028 Barcelona, Spain, and Departament de Quı´mica Analı´tica, Universitat de Vale` ncia, c/Dr. Moliner 50, E-46100 Burjassot (Vale` ncia), Spain

The optimization of the composition of mixed surfactants used as micellar electrokinetic chromatography (MEKC) pseudostationary phases is proposed as an effective method for the separation of complex mixtures of analytes. The solvation parameter model is used to select two surfactants (lithium dodecyl sulfate, LDS, and lithium perfluorooctanesulfonate, LPFOS) with contrasting solvation properties. Combination of these two surfactants allows variations of the solvation properties of MEKC pseudostationary phase along a wide range. Thus, the convenient variation of the proportion of both surfactants allows an effective control of the selectivity in such systems. An algorithm that predicts the overall resolution of a given mixture of compounds is described and applied to optimize the composition of the mixed surfactant for the separation of the mixture. The algorithm is based on the calculation of peak purities on simulated chromatograms as a function of the composition of the mixed LDS/ LPFOS micellar buffer from data at several micellar buffer compositions. Successful separations were achieved for mixtures containing up to 20 compounds, in less than 12 min.

etc.), but the “pseudostationary phase” is a charged surfactant above its critical micellar concentration in the separation buffer. In contrast with classical liquid chromatography, this pseudostationary phase can be easily changed by changing the surfactant. It is generally agreed that the choice of the surfactant is the most important consideration for optimizing MEKC selectivity.1,2 Optimization of separations in MEKC is largely based on trialand-error experiments assisted by some useful formal general observations.1,3-8 A successful separation depends mainly on the choice of an appropriate surfactant and the establishment of experimental conditions that provide a high efficiency and an acceptable elution window. These conditions are obtained by control of the applied field; buffer composition, ionic strength, and pH; temperature; and type and concentration of additive (complexing agent, organic solvent, etc.). A few years ago, the limited number and homologous character of the commercial surfactants employed in MEKC restricted the variation of selectivity produced by the change of surfactant.9 Nowadays, numerous surfactants of diverse chemical nature are commercially available. The solvation properties of most of these surfactants have been characterized by means of the linear solvation energy relationships,1,2,9-18 or solvation parameter model,

Selectivity of liquid chromatography systems toward a series of analytes depends on the solvation properties of both mobile and stationary phases. Since the properties of the mobile phase can be varied more easily than those of the stationary phase, optimization of the selectivity is usually achieved by changes in the composition of the mobile phase after selection of an appropriate stationary phase. Micellar electrokinetic chromatography (MEKC), or more correctly micellar capillary electrophoresis, is an analytical separation technique that combines features of liquid chromatography and capillary electrophoresis. The mobile phase is an aqueous buffer, whose properties can be changed by addition of appropriate modifiers (organic solvents, cyclodextrins, urea, chiral additives,

(1) Poole, C. F.; Poole, S. K. J. Chromatogr., A 1997, 792, 89-104. (2) Poole, C. F.; Poole, S. K.; Abraham, M. H. J. Chromatogr., A 1998, 798, 207-222. (3) Terabe, S. J. Pharm. Biomed. Anal. 1992, 10, 705-715. (4) Muijselaar, P. G. H. M.; Claessens, H. A.; Cramer, C. A. J. Chromatogr., A 1995, 696, 273-284. (5) Ahuja, E. S.; Foley, L. P. Anal. Chem. 1995, 67, 2315-2324. (6) Corstjens, H. A. H.; Frank, J.; Luyben, K. Ch. A. M. J. Chromatogr., A 1995, 715, 1-11. (7) Pyell, U.; Butehorn, U. Chromatographia 1995, 40, 175-184. (8) Zomeren, P. V.; Hilhorst, M. J.; Coenegracht, P. M. J.; de Jong, G. J. J. Chromatogr., A 2000, 867, 247-259. (9) Poole, S. K.; Poole, C. F. Anal. Commun. 1997, 34, 57-62. (10) Yang, S.; Khaledi, M. G. Anal. Chem. 1995, 67, 499-510. (11) Yang, S.; Bumgarner, J. G.; Kruk, L. F. R.; Khaledi, M. G. J. Chromatogr., A 1996, 721, 323-335. (12) Poole, S. K.; Poole, C. F. J. High Resolut. Chromatogr. 1997, 20, 174-178. (13) Poole, S. K.; Poole, C. F. Analyst 1997, 122, 267-274. (14) Rose´s, M.; Ra`fols, C.; Bosch, E.; Martı´nez, A. M.; Abraham, M. H. J. Chromatogr., A 1999, 845, 217-226. (15) Trone, M. D.; Leonard, M. S.; Khaledi, M. G. Anal. Chem. 2000, 72, 12281235. (16) Trone, M. D.; Khaledi, M. G. J. Chromatogr., A 2000, 886, 245-257.

* To whom correspondence should be addressed. Fax: 34 93 402 12 33. E-mail: [email protected]. † Universitat de Barcelona. ‡ Universitat de Vale`ncia. 10.1021/ac0201530 CCC: $22.00 Published on Web 07/30/2002

© 2002 American Chemical Society

Analytical Chemistry, Vol. 74, No. 17, September 1, 2002 4447

proposed by Abraham, to account for the different solute-solvent interactions that determine distribution of a solute between two phases.19 The tabulated surfactant solvation properties offer a scientific basis to choose the surfactant with the most appropriate solvation properties for the separation problem at hand. However, there is a peculiar possibility of MEKC technique that is seldom considered for systematic optimization of analytical separations. This is the modeling of the properties of the pseudostationary phase by changing the proportions of the components of a mixed surfactant. This procedure has been applied to develop MEKC systems from sodium dodecyl sulfate and Brij-35 mixed micellar buffers, with partition properties that model processes of biological interest, such as octanol-water partition and tadpole narcosis.14 The same procedure can be used to optimize the properties of a MEKC pseudostationary phase for the separation of a particular set of analytes. With an appropriate algorithm, the retention data of the analytes at several mixed surfactant compositions can be used to predict the composition of the micellar phase that produces the best separation. To get a wide variation of the selectivity of the micellar phase, at least two surfactants with very different solvation properties are required to prepare the mixed micellar buffer. A few studies about the systematic optimization of the composition of mixtures of bile salts and sodium dodecyl sulfate as MEKC surfactants for the separation of corticosteroids have been published.20-22 In a recent study,18 the solvation properties of some common MEKC surfactants were characterized. Bile salts (cholate and deoxycholate) and dodecyl sulfate surfactants showed important differences in solvation properties, but a principal component analysis of the solvation properties demonstrated that perfluorooctanesulfonate was the anionic surfactant that presented the behavior most different from that of dodecyl sulfate. In this work, we develop a method for the separation of complex mixtures of neutral analytes by optimizing the composition of lithium perfluorooctanesulfonate (LPFOS) and lithium dodecyl sulfate (LDS) mixed micellar buffers as MEKC pseudostationary phases. Lithium salts have been used because of the low aqueous solubility of sodium and potassium perfluorooctanesulfonates. It has been argued that the mixtures of LPFOS and LDS may form two different types of micelles. One type would be fluorocarbon-rich and the other would be one rich in hydrocarbon surfactant.23,24 The coexistence of two types of micelles would improve the selectivity of the MEKC system, since this will lead to an additional partition process for each solute. In fact, LDS + LPFOS micellar systems have been proved to be very effective for the separation of small peptides.24 The calculation of peak purities of simulated chromatograms as a function of the composition of the mixed LDS/LPFOS micellar (17) Fuguet, E.; Ra`fols, C.; Bosch, E.; Rose´s, M.; Abraham, M. H. J. Chromatogr., A 2001, 907, 257-265. (18) Fuguet, E.; Ra`fols, C.; Bosch, E.; Abraham, M. H.; Rose´s, M. J. Chromatogr., A 2001, 942, 237-248. (19) Abraham, M. H. Chem. Soc. Rev. 1993, 22, 73-83. (20) Bumgarner, J. G.; Khaledi, M. G. Electrophoresis 1994, 15, 1260-1266. (21) Wiedmer, S. K.; Jumppanen, J. H.; Haario, H.; Riekkola, M. L. Electrophoresis 1996, 17, 1931-1937. (22) Wiedmer, S. K.; Riekkola, M. L.; Nyde´n, M.; So ¨derman, O. Anal. Chem. 1997, 69, 1577-1584. (23) Mukerjee, P.; Yang, A. Y. S. J. Phys. Chem. 1976, 80, 1388-1390. (24) Ye, B.; Hadjmohammadi, M.; Khaledi, M. G. J. Chromatogr., A 1995, 692, 291-300.

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buffer from data at several micellar buffer compositions is proposed as optimization algorithm. This algorithm produces resolution maps that allow choosing the micellar composition with the best resolution of the particular mixture of analytes studied. EXPERIMENTAL SECTION Apparatus and Conditions. Separations were done with a Beckman P/ACE System 5500 with a UV diode array detector. The fused-silica separation capillaries were 50 µm i.d. and 47 cm long (effective length 40 cm). The capillaries were activated by the following washing sequence: 5 min of water, 20 min of 1 M LiOH, 10 min of water, and 30 min of separation buffer. Prior to each separation with the same surfactant buffer, the capillaries were flushed with 0.1 M LiOH for 2 min, followed by the separation buffer for 5 min. When the surfactant buffer was changed, the capillary was conditioned for 10 min with water, 10 min with LiOH, 5 min with water, and 20 min with the separation buffer. Retention measurements were made at 25 °C and +15 kV. Detection was usually at 214 nm. The separation buffers were prepared by solving the surfactants in water, adding H3PO4, and neutralizing with LiOH up to pH 7.0. Water was finally added to obtain separation solutions 40 mM in surfactant and 20 mM in buffer. Solutes were solved in methanol (used as electroosmotic flow marker) at ∼2 mg mL-1 and contained ∼2 mg mL-1 dodecanophenone as micellar marker.25 All sample solutions and buffers were filtered through 45-µm nylon syringe filters (Albet). Reagents and Materials. Phosphoric acid (85% in water), lithium hydroxide (98% in water), methanol (for chromatography), and lithium dodecyl sulfate (>99%) were from Merck. Lithium perfluorooctanesulfonate was from Fluka (25% in water). Water was Milli-Q plus (Millipore) with a resistivity of 18.2 MΩ cm. The test solutes were reagent grade or better obtained from several makers. DATA TREATMENT Retention Factor. The retention factor, k, was calculated as follows:

k)

(tR - t0) t0(1 - tR/tm)

(1)

where tR is the solute migration time, t0 the migration time of methanol (electroosmotic flow marker) and tm the migration time of dodecanophenone, which indicates the migration of micelles. Optimization Algorithm. The optimization methodology is based on maps representing a global resolution parameter for the compounds present in the mixture. These maps describe the separation of a given peak from the remaining, for a set of computer-simulated chromatograms predicted for a regular distribution of surfactant buffers. Cubic polynomials, fitted by weighted linear regression, were used to describe the migration behavior of the solutes:

log k ) a0 + a1cLDS + a2cLDS2 + a3cLDS3

(2)

(25) Fuguet, E.; Ra`fols, C.; Bosch, E.; Rose´s, M. Electrophoresis 2002, 23, 5666.

where cLDS is the lithium dodecyl sulfate concentration. Only one factor was modeled, since the other (LPFOS concentration) was in all cases 40 - cLDS (mmol‚L-1) in the experimental design. Peak shape was modeled using a Gaussian-modified function defined as26

[ (

h(t) ) H0 exp -

t - tR

)]

1 2 s0 + s1(t - tR)

2

(3)

where H0 represents the maximal peak height and h(t) the peak height at a given t time. Areas were normalized for searching the optimal separation conditions, but area values obtained from experimental chromatograms were used for comparison purposes in the shown simulations. The peak profile parameters in eq 3 (s0 and s1) are related to the efficiency (N) and asymmetry factor measured at 10% of peak height (B/A, where A and B are the time distance between the maximum and the fronting and tailing edges of the chromatographic peak), as follows:

s0 ) 0.466

x

41.7tR2

N(1.25 + B/A) B/A - 1 11 B/A + 1 1+ B/A

(

B/A - 1 s1 ) 0.466 B/A + 1

)

(4)

(5)

s0 is a measurement of peak width and s1 a parameter that quantifies peak distortion. Due to the strong and often unpredictable variations in efficiencies and asymmetry factors with the composition, difficult to model, local linear equations were applied as predictors of these properties, using the experimental values obtained from the experimental design.26,27 The separation of each peak from the others was calculated as peak purity, defined as

ri ) 1 - w′i/wi

(6)

where wi is the total area of the peak and w′i is the peak area overlapped by the chromatogram obtained for the remaining compounds in the mixture. The overall resolution (R) was then computed: n

R)

∏r

i

(7)

i)1

R varies between 0 (when at least one peak is fully overlapped) and 1 (when all peaks are baseline resolved). The maximal resolution inside the studied composition range denotes the pseudostationary phase that best resolves the mixture.27,28 RESULTS AND DISCUSSION Solvation Properties of LDS and LPFOS. The solvation properties of LDS and LPFOS, characterized in a previous work (26) Torres-Lapasio´, J. R.; Baeza-Baeza, J. J.; Garcı´a-AÄ lvarez-Coque, M. C. Anal. Chem. 1997, 69, 3822-3831. (27) Carda-Broch, S.; Torres-Lapasio´, J. R.; Garcı´a-AÄ lvarez-Coque, M. C. Anal. Chim. Acta 1999, 396, 61-74. (28) Torres-Lapasio´, J. R.; Rose´s, M.; Bosch, E.; Garcı´a-AÄ lvarez-Coque, M. C. J. Chromatogr., A 2000, 886, 31-46.

Table 1. Coefficients of the Solvation Parameter Model (Eq 8) for LDS and LPFOS Pure Pseudostationary Phases coefficients e

s

a

b

statistics v

c

n

r

SD

F

LDS 0.586 -0.595 -0.317 -1.565 2.609 -1.575 63 0.989 0.128 526 (40 mM) LPFOS -0.113 -0.243 -0.876 -0.455 1.966 -1.410 62 0.970 0.190 180 (40 mM)

by means of the application of the solvation parameter model,18 are given in Table 1. The values in the table are the coefficients of the following correlation equation:

log k ) c + eE + sS + aA + bB + vV

(8)

where E, S, A, B, and V are the Abraham solute descriptors. E is an excess molar refraction, S is the solute dipolarity/polarizability, A and B are parameters characterizing the effective hydrogen bond acidity and hydrogen bond basicity, respectively, and V is the McGowan characteristic volume. The values of the coefficients of the correlation reflect the system properties that interact with the corresponding solute property: e depends on the difference in capacity of the buffer and micellar phase to interact with solute n- or π-electrons; s is a measure of the difference in dipolarity/ polarizability between the two phases; a and b are measures of the difference in hydrogen bond basicity and acidity, respectively, between the buffer and micellar phase; and v is a measure of the relative ease of forming a cavity for the solute in the buffer and micellar phase. The intercept of the correlation (c) is related to the micelle/aqueous buffer phase ratio. Its value influences the retention time, but not MEKC selectivity. The values of the coefficients in Table 1 measure the solvation properties of the surfactants in reference to water. The high positive v values show that both surfactants are more lipophilic than water; i.e., it is easier for the solute to create a cavity in the micelle than in water. The higher v value for LDS denotes that LDS is more lipophilic than LPFOS and that a given variation of solute volume (V) will produce a log k variation in LDS system larger than the one in LPFOS system. The positive e value for LDS indicates that this surfactant interacts with solute n- and π-electrons more strongly than water, and the small negative e value for LPFOS means that it interacts slightly less than water. An increase in the number of solute n- and π-electrons (which determine solute polarizability and molar refraction, E) will produce a higher retention in LDS or a slightly lower retention in LPFOS. Since the s, a, and b coefficients are negative in reference to water, both surfactants must have a lower dipolarity (s), hydrogen bond acceptor basicity (a), and hydrogen bond donor acidity (b). An increase in dipolarity (S) of the solute produces a decrease in retention, more noticeable for LDS than for LPFOS. An increase in solute hydrogen bond acidity (A) results in a lower retention, especially for LPFOS. An increase in solute hydrogen bond basicity (B) should produce a large decrease in retention in LDS but only a moderate decrease in LPFOS. In summary, the LDS system is more sensitive to changes in solute volume, dipolarity, polarizability and hydrogen bond basicity Analytical Chemistry, Vol. 74, No. 17, September 1, 2002

4449

Figure 1. Chromatograms of a mixture of the compounds listed in Table 2, obtained in 40 mM LDS and 40 mM LPFOS pure systems. Compound identity is given in Table 3. The signals of the electroosmotic flow (t0) and micellar (tm) markers are shown. Table 2. Solute Descriptors of Test Compounds

benzene toluene ethylbenzene propylbenzene butylbenzene benzonitrile naphthalene acetophenone butyrophenone 2-naphthol

E

S

A

B

V

0.610 0.601 0.613 0.604 0.600 0.742 1.340 0.818 0.797 1.520

0.52 0.52 0.51 0.50 0.51 1.11 0.92 1.01 0.95 1.08

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.61

0.14 0.14 0.15 0.15 0.15 0.33 0.20 0.48 0.51 0.40

0.7164 0.8573 0.9982 1.1391 1.2800 0.8711 1.0854 1.0139 1.2957 1.1441

than LPFOS, whereas LPFOS system is more sensitive than LDS to variations in solute hydrogen bond acidity. Selectivity of LDS and LPFOS Pure Systems. The different solvation properties of LDS and LPFOS MEKC give rise to a different selectivity for a given set of solutes. As explained above, these selectivities will be determined by the solute properties of volume, polarity, and hydrogen bond abilities. To demonstrate the different selectivity of the two MEKC systems, a representative set of solutes with known descriptors was selected. The solutes and their descriptors are given in Table 2. Benzene, toluene, ethylbenzene, propylbenzene, and butylbenzene form an homologous series of alkylbenzenes of variable volume (V). All other descriptors of these compounds remain practically constant. The alkylbenzenes have no hydrogen bond acidity and a very low hydrogen bond basicity (caused by the π-electrons of the aromatic ring). The dipolarity (S) and polarizability (E), which are caused again by the π-electrons of the aromatic ring, are moderate. Benzonitrile is similar to toluene, except for a slightly larger hydrogen bond basicity (B) and polarizability (E) and a much larger dipolarity (S). Naphthalene is similar to benzonitrile except that it has a larger polarizability (E) because of the two aromatic rings. Acetophenone and butyrophenone have hydrogen bond acceptor basicities (B) larger than that of benzonitrile. Volumes of these compounds are comparable to those of ethylbenzene and butylbenzene, respectively. Finally, 2-naphthol is close to naphthalene except that it has a remarkable hydrogen bond acidity (A). Its volume is similar to propylbenzene. Consequently, this set of compounds is repre4450 Analytical Chemistry, Vol. 74, No. 17, September 1, 2002

sentative of changes in solute volume V (alkylbenzenes), dipolarity S (benzonitrile), polarizability E (naphthalene), hydrogen bond basicity B (phenones), and hydrogen bond acidity A (2-naphthol). The chromatograms obtained for the test mixture in LDS and LPFOS pure micellar systems are presented in Figure 1. Retention of the series of alkylbenzenes is mainly governed by volume effects. The same trend is followed in both MEKC systems: the larger the volume of the alkylbenzene, the larger the retention. However, the retention between the first and last members of the series varies more strongly in LDS than in LPFOS, because of the larger selectivity of LDS toward solute volume changes (larger v coefficient in Table 1). According to its volume (Table 2), benzonitrile should elute between toluene and ethylbenzene. This is observed in LPFOS but not in LDS. The larger dipolarity (S) and hydrogen bond basicity (B) of benzonitrile in reference to the alkylbenzenes, and the larger negative values of the corresponding coefficients (s and b) in LDS, determine a large decrease in retention of this analyte in reference to the alkylbenzenes. In LPFOS, the decrease of retention is much lower because of the lower weight of s and b coefficients in this system. Naphthalene has a volume (V) and polarizability (E) larger than those of benzonitrile. The increase in volume increases retention in both systems, especially in LDS (with a larger v value). The increase in polarizability increases retention in LDS but should produce a slight decrease of retention in LPFOS (which has a low negative e value). Thus, the difference in retention between naphthalene and benzonitrile is reduced in LPFOS. In LPFOS, naphthalene and ethylbenzene, which have a similar volume, coelute, whereas these compounds are completely resolved in LDS. The large contribution of the polarizability term to retention in LDS (with a considerable positive e value) determines that naphthalene delays strongly its elution, which is close to that of propylbenzene. Decrease in retention (in reference to alkylbenzenes of similar volume) is expected for phenones in both systems owing to the high hydrogen bond basicity (B) of these compounds. The decrease should be larger in LDS than in LPFOS because of the larger negative b coefficient. Indeed, in LPFOS, butyrophenone coelutes with butylbenzene, but in LDS, it elutes between

Figure 2. Variation of the retention factor of the compounds in Table 2 with the composition of LDS + LPFOS mixed micellar buffers. Compounds: (]) Butylbenzene, (9) propylbenzene, (2) butyrophenone, (/) naphthalene, (b) acetophenone, (4) ethylbenzene, (O) benzonitrile, (0) toluene, (+) 2-naphthol, and ([) benzene.

ethylbenzene and propylbenzene. Acetophenone elutes between ethylbenzene and propylbenzene in LPFOS, but in LDS, the elution is between benzene and toluene. Finally, 2-naphthol should decrease its retention in reference to alkylbenzenes because of its high hydrogen bond acidity (A). This effect should be stronger for LPFOS due to the more negative a coefficient of this surfactant. Actually, in LDS, 2-naphthol elutes after ethylbenzene, whereas in LPFOS, it elutes before toluene. These examples show that the solvation properties and selectivity of the two selected surfactants are very different. The differential behavior can give rise to a suitable method to modify

the selectivity based on a simple combination of the two surfactants, in adequate proportions. Optimization of the Composition of LDS + LPFOS Mixtures for the Separation of Compounds. The changes in selectivity in the mixed systems formed with the binary combinations of the two surfactants (i.e., hybrid pseudostationary phases) were initially explored. Figure 1 shows that the test compounds of Table 2 can be quite well separated in the 40 mM LDS system, although not in the 40 mM LPFOS system. However, some compositions of LDS + LPFOS mixtures may produce a good separation of the test compounds too. The algorithm described in the Data Treatment section was applied to the retention data of the test mixture, to predict the LDS + LPFOS composition that yields the maximal overall resolution R. The procedure starts with measurement of the retention, efficiency, and peak asymmetry data for the test compounds at several LDS + LPFOS compositions. These data were obtained in a previous work for the test compounds, eluted with five pseudostationary phases having an overall surfactant concentration of 40 mM.17 The concentrations (mM) of LDS/LPFOS were 40/0, 30/10, 20/20, 10/30, and 40/0. The compounds were injected separately in the surfactant buffers, and log k values were fitted to eq 2. The k values at any LDS/ LPFOS composition were predicted through this equation and further used to calculate tR through eq 1. For this purpose, t0 and tm values were obtained through a polynomial approximation of the respective experimental values at different mixed surfactant compositions. The individual peaks of all compounds at ∼100 LDS/LPFOS compositions were computer simulated. The required peak profile parameters were calculated with eqs 4 and 5 and the peak purities with eq 6. Finally, the overall resolution R for the test mixture at each LDS/LPFOS composition was obtained through eq 7.

Figure 3. Resolution map for the mixture of the compounds in Table 2 and simulated and experimental chromatograms for the optimal composition of the MEKC pseudostationary phase (9 mM LDS + 31 mM LPFOS). Compound identity is given in Table 3.

Analytical Chemistry, Vol. 74, No. 17, September 1, 2002

4451

Table 3. Fitting Coefficients (Eq 2) and Statistics for the 40 Compounds Studied

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 34 35 36 37 38 39 40 a

compound

a0 × 102

a1 × 102

a2 × 104

a3 × 106

Ra

error (%)b

benzene toluene ethylbenzene propylbenzene butylbenzene acetophenone propiophenone butyrophenone valerophenone benzophenone aniline 2-nitroaniline 3-nitroaniline 4-nitroaniline 3-chloroaniline 4-chloroaniline phenol 4-chlorophenol 2,3-dimethylphenol 2,4-dimethylphenol pyrrole m-cresol resorcinol 2-nitroanisole anisole 4-chloroacetanilide pyrimidine benzamide o-toluidine bromobenzene chlorobenzene benzonitrile nitrobenzene p-xylene furan 2,3-benzofuran benzaldehyde methyl benzoate 2-naphthol naphthalene

-121 -44.9 19.5 93.7 165 49.8 108 173 239 239 -146 -39.5 -103 -120 -129 -127 -207 -156 -71.9 -55.7 -289 -125 -282 65.3 -31.1 -18.9 -162 -108 -89.4 -40.4 -49.1 -9.58 -7.72 28.8 -180 -35.9 -16.1 74.2 -58.6 23.8

3.68 5.40 6.85 8.18 10.4 1.11 1.65 2.04 2.64 3.73 2.25 3.07 3.72 3.50 5.41 6.98 4.07 7.92 6.21 6.81 -1.73 5.31 -0.019 1.32 3.32 -0.59 -1.74 2.84 2.97 7.06 6.79 2.68 3.23 6.50 -1.03 6.15 2.31 4.72 10.9 10.4

0.940 -3.66 -6.37 -6.20 -12.2 -1.62 -0.879 0.933 3.06 -1.81 0.196 -0.542 -4.40 350 8.34 -6.75 -1.21 -5.87 -4.61 -7.80 25.8 -6.31 21.6 -5.00 -3.19 25.9 0.628 -4.51 -0.940 -2.14 -6.74 -8.61 -18.1 -3.38 13.2 -4.62 -6.98 -13.8 -16.8 -14.0

-8.00 -3.45 -1.52 -4.79 3.03 -3.26 -5.86 -10.2 -15.4 -7.27 -2.94 -2.28 3.44 -3.76 -31.6 2.12 -2.27 0.098 -1.95 2.43 -40.8 4.23 -34.8 4.03 -0.542 -37.5 -5.95 -0.11 -2.62 -9.11 0.319 6.64 34.4 -6.98 -23.0 -2.95 6.12 12.1 11.9 5.99

0.9997 0.9998 0.9998 0.9999 0.9998 0.9914 0.9954 0.9968 0.9933 0.9966 0.9980 0.9991 0.9996 0.9998 0.9999 0.9990 0.9999 1.0000 1.0000 0.9997 0.9999 1.0000 0.9999 0.9404 1.0000 0.9863 0.9991 0.9989 0.9982 1.0000 0.9998 0.9941 0.9914 1.0000 0.9962 0.9999 0.9891 0.9929 1.0000 0.9999

0.8 0.8 0.8 0.6 1.1 0.7 0.8 1.0 2.2 1.8 1.4 1.2 0.8 0.7 0.5 2.1 0.6 0.5 0.3 1.0 0.3 0.1 0.6 1.4 0.0 7.6 1.2 0.6 1.5 0.4 0.8 0.8 2.4 0.0 0.8 0.5 1.0 1.7 0.5 0.7

Correlation coefficient. b Error ) ∑(k - kˆ )/∑k × 100, where k and kˆ are the experimental and predicted retention factors.

The variation of log k values with surfactant composition (only LDS concentration is indicated, since cLPFOS ) 40 - cLDS), for the test compounds of Table 2, is depicted in Figure 2. Note that the selectivity changes remarkably with the LDS/LPFOS ratio, which suggests the interest of using hybrid pseudostationary phases to modify the separation, and eventually, enhance the resolution. The resolution map, R versus LDS concentration, is presented in Figure 3. In addition to pure LDS, there is another optimal region of resolution for LDS concentrations close to 9 mM. These two regions show overall resolutions greater than 0.95, which is almost the maximal resolution possible (R ) 1). There are also other regions with overall resolutions close to R ) 0.9: a wide region between 18 and 24 mM LDS and another one between 30 and 32 mM LDS. A mixed surfactant with an LDS concentration in any of these regions should give an acceptable separation of the test mixture. The maximum at ∼5 mM LDS will be unsuitable because a small variation in LDS concentration would produce a large drop of resolution. Figure 3 shows the simulated and experimental chromatograms obtained for 9 mM LDS. This concentration gives a resolution as good as the one obtained for pure LDS (Figure 1), although the selectivity is different. The elution order of the compounds in the 9 mM LDS system is more 4452 Analytical Chemistry, Vol. 74, No. 17, September 1, 2002

similar to the order found for pure LPFOS, which is the major component in this mixed buffer. When changing from pure LPFOS (40 mM) to 9 mM LDS/31 mM LPFOS, only 2-naphthol moves from second to fourth position and acetophenone from seventh to fifth position. Note that the peaks of ethylbenzene/naphthalene and butylbenzene/butyrophenone, which overlap in pure LPFOS, are well separated in the mixed buffer. The algorithm described above to optimize the separation was also tested with more complex mixtures. The aim of this study was to examine whether problems that cannot be resolved using the pure surfactant systems can be resolved using mixed systems. The sets to be separated were also selected among 40 compounds used in a previous work to characterize the solvation properties of LDS + LPFOS mixtures.17 The individual retention data of these compounds were fitted to eq 2. Table 3 reports the model coefficients and some statistics. A first attempt was made to check if the separation of all 40 compounds was possible. The calculated resolution map (Figure 4) showed that the overall resolution for the optimal surfactant composition (35.2 mM in LDS and 4.8 mM in LPFOS) is very poor (R ) 3 × 10-7). However, one should consider that 40

Figure 4. Resolution map for the mixture of the 40 compounds in Table 3 and simulated chromatogram for the optimal composition of the MEKC pseudostationary phase (35.2 mM LDS + 4.8 mM LPFOS).

elementary resolution values (ri) are multiplied to obtain the overall measurement. Figure 4 shows also the predicted chromatogram for the optimal mixed buffer. As observed, some compounds coelute at least partially due to the small separation window. However, 28 peaks are partially or completely resolved, although the experimental chromatogram is expected to show some poorer resolution due to uncertainties in peak position and profile and also to the unruggedness of the resolution maximum. A more realistic maximal number of compounds that could be resolved in this system is about 20-25. The feasibility of the separation of several subsets of compounds was evaluated with the aid of resolution maps. Figures 5-7 present the resolution maps, and the simulated and experimental chromatograms for three successful separations, carried out with the shown methodology. The optimal composition of surfactant (composition with maximal R value) is different for each mixture, although for complex mixtures, it is generally obtained at large LDS concentrations. A general trend of improvement of R is observed in the resolution maps of Figures 4-7 with the increase in LDS concentration, despite some local minimums caused by peak crossing. However, it must be also observed that the pure LDS system (40 mM) does usually not resolve the mixtures.

Figure 5 presents the separation of a subset of 15 compounds with an optimum at 22.6 mM LDS (R ≈ 0.9). Less convenient maximums are observed at 9, 30, and between 34 and 37 mM LDS. Pure LPFOS should also give an acceptable resolution of ∼0.75. A successful separation of up to 18 compounds at high LDS concentrations (28.7 mM) is presented in Figure 6, which gives an overall resolution R of ∼0.9. Pure LDS should also give a good overall resolution (R > 0.8) for this mixture. Finally, the resolution map and chromatograms for the most complex mixture of the studied set of 40 compounds, which was successfully separated (20 compounds plus the micellar marker dodecanophenone), is presented in Figure 7. The resolution of this mixture is very poor for pure LPFOS and pure LDS, but acceptable resolution (R ≈ 0.7) can be achieved for ∼22 mM LDS and between 30 and 33 mM. This second region, with a maximum at 31.7 mM LDS, is preferable due to its greater ruggedness. The simulated and experimental chromatograms for this mixture show that all compounds are almost completely separated. The above examples illustrate that complex mixtures of up to 20 analytes can be successfully resolved by MEKC in less than 12 min, if the composition of the LDS + LPFOS micellar buffer that constitutes the pseudostationary phase is conveniently optimized by an appropriate algorithm, such as the one used here. Analytical Chemistry, Vol. 74, No. 17, September 1, 2002

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Figure 5. Resolution map for a mixture of 15 compounds and simulated and experimental chromatograms for the optimal composition of the MEKC pseudostationary phase (22.6 mM LDS + 17.4 mM LPFOS). Compound identity is given in Table 3.

Figure 6. Resolution map for a mixture of 18 compounds and simulated and experimental chromatograms for the optimal composition of the MEKC pseudostationary phase (28.7 mM LDS + 11.3 mM LPFOS). Compound identity is given in Table 3.

CONCLUSIONS The separation of compounds by MEKC can be optimized by changing the selectivity of the pseudostationary phase. This can be achieved using mixed micellar buffers, whose composition and 4454

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properties can be easily modified. The solvation parameter model proposed by Abraham is a valuable tool to characterize the solvation properties and selectivity of potential surfactants. It provides a scientific basis to select the most appropriate individual

Figure 7. Resolution map for a mixture of 20 compounds and simulated and experimental chromatograms for the optimal composition of the MEKC pseudostationary phase (31.7 mM LDS + 8.3 mM LPFOS). Compound identity is given in Table 3.

surfactants to compose the mixed micellar buffer. Mixtures of lithium dodecyl sulfate and lithium perfluorooctanesulfonate are specially useful, because of their contrasting solvation properties. The composition of the mixed micellar buffer that produces the best separation can be estimated with an appropriate algorithm from retention data at different surfactant compositions. The optimization methodology proposed here is based on the simulation of the peaks in a chromatogram by a modified Gaussian and calculation of the peak purity for each compound. Resolution maps depicting the overall resolution are demonstrated to be very effective to predict accurately the conditions (mixed surfactant composition) for the best separation. The accuracy of the predictions is limited by the difficulty in modeling the variation of efficiency and asymmetry of the solute peaks, which in the examples shown were in the ranges N ) 3700-175 000 and B/A ) 0.12-15, respectively.

The proposed procedure and algorithm has allowed the prediction of the composition of the optimal pseudostationary phase for the separation of complex mixtures in short analysis times. ACKNOWLEDGMENT We are thankful for joint financial support from the MCYT of the Spanish Government (projects BQU2001-3226 and BQU20013047) and from the Catalan (Grant 2001SGR00055) and Valencian (GR01-63) Governments. J.R.T.-L. thanks the MCYT for a Ramo´n y Cajal position.

Received for review March 13, 2002. Accepted June 16, 2002. AC0201530

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