Anal. Chem. 2000, 72, 1228-1235
Congeneric Behavior in Estimations of Octanol-Water Partition Coefficients by Micellar Electrokinetic Chromatography Mark D. Trone, Michael S. Leonard, and Morteza G. Khaledi*
North Carolina State University, Department of Chemistry, Campus Box 8204, Raleigh, North Carolina 27695
Linear Solvation Energy Relationships (LSERs) are used to explain the congeneric behavior observed when using Micellar Electrokinetic Chromatography (MEKC) to estimate the octanol-water partition coefficient scale of solute hydrophobicity. Such studies provide useful insights about the nature of solute interactions that are responsible for the sources of congeneric relationships between MEKC retention and log Po/w. It was determined that solute dipolarity/polarizability and hydrogen-bonding character play the most important roles in the congeneric behavior observed for many surfactant systems. The individual dipolarity/polarizability and hydrogen-bonding contributions to the free energy of transfer were also investigated. Hydrophobic interaction plays a significant role in partitioning into lipid bilayers of biomembranes, bioavailability, and activity of many drugs and biologically important compounds. Hansch and Leo first demonstrated the relationship between bioactivity and the logarithm of the n-octanol/water partition coefficient, log Po/w. Since then, log Po/w has become the most widely used scale for solute hydrophobicity.1-3 Direct measurement of log Po/w by the shake-flask method is cumbersome, time-consuming, requires pure compounds, and has a limited dynamic range.4 Although many different techniques have been used to estimate log Po/w values,5 a popular alternative has been indirect determination of log Po/w through its linear relationship with chromatographic retention, k′, as6,7
log Po/w ) y log k′ + z
(1)
Collander suggested that linear free-energy relationships (LFER) between partition coefficients of solutes in different biphasic (aqueous-organic) systems exist as long as the underlying interactions in these systems are the same.8,9 Much work has been done for the “best” chromatographic conditions in order (1) Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis in Chemistry and Biology; Wiley-Interscience: New York, 1979. (2) Hansch, C. In Drug Design; Aries, E., Ed.; Academic Press: New York, 1971; Vol. 1. (3) Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525. (4) Konemann, H.; Zelle, R.; Busser, F.; Hammers, W. E. J. Chromatogr. 1979, 178, 2769. (5) Leo, A. J. Chem. Rev. 1993, 93, 1281. (6) Kaliszan, R. Quantitative Structure - Chromatographic Retention Relationships; Wiley-Interscience: New York, 1987. (7) Dross, K.; Sonntag, C.; Mannhold, R. J. Chromatogr., A 1994, 673, 113.
1228 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
to establish eq 1 for a wide range of compounds. Various chromatographic techniques such as centrifugal partition chromatography,10 micellar liquid chromatography,11,12 and reversedphase LC (RPLC)6,13 have been used for estimation of log Po/w. It is widely accepted that the logarithm of retention factor in RPLC at a purely aqueous mobile phase, log k′w, provides the best correlation with log Po/w.13,14 However, direct measurement of k′w is not possible for many compounds due to the excessive retention, thus the chromatographic retention would have to be determined indirectly as well. The introduction of micellar electrokinetic chromatography (MEKC)15,16 has further enhanced the use of separation methods for the estimation of hydrophobicity. Micelle/water partition coefficients were first correlated with log Po/w by Collet and Koo17 and then more extensively by Treiner.18-20 Takeda and co-workers used MEKC to determine micelle-water partition coefficients (Pm/w) for phthalate esters and found good correlations between log Pm/w and log Po/w.21 Since the retention factor in MEKC is directly related to the micelle/water partition coefficient, it is possible to establish a linear relationship between retention factor (log k′) and log Po/w similar to eq 1. Chen et al. confirmed this by reporting a good relationship between log k′ and log Po/w as well as log Pm/w and log Po/w for a set of aromatic solutes in both SDS and Mg(DS)2.22 In addition to these studies, several other MEKC surfactant systems have been used to evaluate solute hydrophobicity including cationic, anionic, and anionic/nonionic mixed micellar systems,23,24 bile salts,25 SDS,26,27 SDS in 10% 2-propanol,28 cyclodextrins,29 as well as aqueous SDS/butanol/heptane microemulsions.30-32 (8) Collander, R. Acta Chem. Scand. 1950, 4, 1085. (9) Collander, R. Acta Chem. Scand. 1951, 5, 774. (10) Gluck, S. J.; Martin, E. J. J. Liq. Chromatogr. 1990, 13, 2529. (11) Khaledi, M. G.; Breyer, E. D. Anal. Chem. 1989, 61, 1040. (12) Gag, F.; Alvarez-Buil, J.; Elguero, J.; Diez-Masa, J. C. Anal. Chem. 1987, 59, 921. (13) Dorsey, J. G.; Khaledi, M. G. J. Chromatogr. 1993, 656, 485. (14) Hsieh, M. M.; Dorsey, J. G. J. Chromatogr. 1993, 631, 63. (15) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111. (16) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834. (17) Collet, J. L.; Koo, L. J. Pharm. Sci. 1975, 64, 1253. (18) Treiner, C. Can. J. Chem. 1981, 59, 2518. (19) Treiner, C. J. Colloid Interface Sci. 1983, 93, 33. (20) Treiner, C.; Chattopadhyay, A. K. J. Colloid Interface Sci. 1986, 109, 101. (21) Takeda, S.; Wakida, S.; Yamane, M.; Kawahara, A.; Higashi, K. Anal. Chem. 1993, 65, 2489. (22) Chen, N.; Zhang, Y.; Terabe, S.; Nakagawa, T. J. Chromatogr. 1994, 678, 327. 10.1021/ac990852l CCC: $19.00
© 2000 American Chemical Society Published on Web 02/12/2000
Similar to HPLC, congeneric behavior is also observed for certain MEKC systems; that is, different log Po/w - log k′ relationships are observed for various groups of solutes. The main source of congenerity is the differences in the nature of interactions in octanol-water as compared with that in micelle-water systems. Yang and co-workers reported the congeneric behavior in MEKC retentionslog Po/w relationships.24 For anionic SDS and cationic CTAB micelles they recognized that the congenerity pattern can be mainly described by the hydrogen-bonding characteristics of solutes that were categorized as non-hydrogenbond donors (NHB), hydrogen-bond acceptors (HBA), and hydrogen-bond donors (HBD). A powerful strategy to investigate the similarities and differences in the interactive nature of various phases is linear solvation energy relationships (LSER) as:33,34
log SP ) c + mVx + sπ*2 + rR2 + bΣβ2 + aΣR2
(2)
In this equation, the dependent variable (SP) is the octanolwater partition coefficient (log Po/w) or MEKC retention (log k′) and is regressed against known solute parameters (Vx, R2, π*2, Σβ2, ΣR2). Vx is the McGowan characteristic volume of the solute (divided by 100 to bring it to scale with the other parameters)35 and R2 is the excess molar refraction (divided by 10 to bring it to scale with the other parameters). π*2 represents the solute dipolarity/polarizability. The solute hydrogen-bonding terms are given by Σβ2 and ΣR2, where Σβ2 describes the solute hydrogen bond accepting characteristics and ΣR2 describes the solute hydrogen bond donating properties. The coefficients of these parameters (m, s, r, b, and a) represent a relative measure of the cohesiveness, polarizability, polarity/polarizability, hydrogen-bond donating, and hydrogen-bond accepting strengths of the organic phase, respectively. Although the mVx term is a measure of hydrophobic interaction, it is also related to the cohesiveness and dispersive properties of the organic phase. In this report, the interactive nature of MEKC pseudostationary phases is compared with that of octanol-water using LSER modeling. It is shown that the LSER methodology is of great value in identifying pseudo-phases that provide the best correlations between log k′ and log Po/w as well as the probable sources of congenerity in the relationships. An extensive list of conventional MEKC surfactants (anionic, cationic, bile salt surfactants), novel phases (e.g., uni - molecular polymers), mixed micellar (23) Ishihama, Y.; Oda, Y.; Uchikawa, K.; Asakawa, N. Chem. Pharm. Bull. 1994, 42, 1525. (24) Yang, S.; Bumgarner, J. G.; Kruk, L. F. R.; Khaledi, M. G. J. Chromatogr., A 1996, 721, 323. (25) Adlard, M.; Okafo, G.; Meenan, E.; Camilleri, P. J. Chem. Soc., Chem. Commun. 1995, 2241. (26) Herbert, B. J.; Dorsey, J. G. Anal. Chem. 1995, 67, 744. (27) Muijselaar, P. G. H. M.; Claessens, H. A.; Cramers, C. A. Anal. Chem. 1994, 66, 635. (28) Smith, J. T.; Vinjamoori, D. V. J. Chromatogr. 1995, 669, 59. (29) Jinno, K.; Sawada, Y. J. Liq. Chromatogr. 1995, 18, 3719. (30) Ishihama, Y.; Oda, Y.; Uchikawa, K.; Asakawa, N. Anal. Chem. 1995, 67, 1588. (31) Ishihama, Y.; Oda, Y.; Asakawa, N. Anal. Chem. 1996, 68, 4281. (32) Ishihama, Y.; Oda, Y.; Asakawa, N. Anal. Chem. 1996, 68, 1028. (33) Abraham, M. H.; Chadha, H. S.; Whiting, G. S.; Mitchell, R. C. J. Pharm. Sci. 1994, 83, 1085. (34) Abraham, M. H. Chem. Soc. Rev. 1993, 73. (35) Abraham, M. H.; McGowan, J. C. Chromatographia 1987, 23, 243.
phases, as well as SDS/organic modifier mixed systems are examined. The single component surfactant systems include sodium dodecyl sulfate (SDS), lithium perfluorooctane sulfonate (LiPFOS), tetradecylammonium bromide (TTAB), sodium cholate (SC), sodium (S)-N-dodecoxycarbonyl valine (SDCV), sodium laurylsulfoacetate (LSA), and the block copolymer phase of Elvacite 2669. The mixed surfactant systems investigated include lithium dodecyl sulfate (LiDS)/LiPFOS and SDS and sodium deoxycholate (SDC), SDS/SDC. The effects of adding various concentrations of aliphatic alcohol modifiers from methanol (MeOH) to octanol (OctOH) in 40 mM SDS solutions has also been examined. EXPERIMENTAL SECTION All of the MEKC experiments were run at 25 °C and pH 7 unless otherwise noted. The details of the MEKC solutions and conditions for all of the separation systems have been described elsewhere.36-39 A test set of 36 solutes with a wide range of LSER parameters and log Po/w values is used in the analysis (Table 1).33,34 Although the solutes exhibit a wide range of sizes and polarities, it is useful to roughly categorize them by their hydrogen-bonding characteristics. There are 12 solutes that have weak hydrogen bond accepting characteristics (Σβ2 e 0.22) and are considered to be nonhydrogen bond donating solutes (NHB). The test set also consists of 12 strong hydrogen-bond acceptors (HBA) and 12 strong hydrogen-bond donors (HBD). The retention factor for the test solutes was measured for each surfactant system using eq 3.12
k′ )
(tr - teo) teo(1 - tr/tmc)
(3)
In this equation, teo is the migration time of an unretained solute, tr is the solute retention time, and tmc represents the migration time of the micelle. Solutes were detected at 214 nm. Methanol was used as the electroosmotic flow (teo) marker and was measured from the time of injection to the first deviation from the baseline. The migration time of the micelle (tmc) was determined using n-dodecanophenone as a marker. The same test solutes are used for log Po/w vs log k′ correlations and in the LSER modeling of MEKC retention as well as log Po/w in order to eliminate any potential discrepancy on the basis of different solute sets. RESULTS AND DISCUSSION Linear Solvation Energy Relationships. The LSER results for single surfactant systems, mixed surfactant systems, and SDS/ alcohol mixed systems are listed in Table 2(A)-(C). The LSER results for some of these pseudo-stationary phases have been reported previously.36-39 However, a different set of solutes and/ or different separation conditions were used in those studies, therefore they have been included here for a more comprehensive (36) Yang, S.; Khaledi, M. G. Anal. Chem. 1995, 67, 499. (37) Trone, M. D.; Khaledi, M. G. Anal. Chem. 1999, 71, 1270. (38) Leonard, M. S.; Khaledi, M. G., submitted to J. Chromotogr., A. (39) Khaledi, M. G.; Bumgarner, J. G.; Hadjmohammadi, M. J. Chromatogr., A 1998, 802, 35.
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Table 1. Test Solutes and Their Solvation Descriptors and log Po/w Valuesa
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 a
solutes
Vx
π2*
R2
benzene toluene ethylbenzene propylbenzene p-xylene chlorobenzene bromobenzene iodobenzene 4-chlorotoluene biphenyl naphthalene 1-methylnaphthalene acetophenone benzonitrile nitrobenzene methyl benzoate ethyl benzoate 4-chloroanisole 4-nitrotoluene 4-chloroacetophenone methyl 2-methylbenzoate phenyl acetate 3-methylbenzyl alcohol phenethyl alcohol benzyl alcohol phenol 4-methylphenol 4-ethylphenol 4-fluorophenol 4-chlorophenol 4-bromophenol 4-chloroaniline 3-chlorophenol 3-methylphenol 3-bromophenol 3,5-dimethylphenol
0.716 0.857 0.998 1.139 0.998 0.839 0.891 0.975 0.980 1.324 1.085 1.226 1.014 0.871 0.891 1.073 1.214 1.038 1.032 1.136 1.214 1.073 1.057 1.057 0.916 0.775 0.916 1.057 0.793 0.898 0.950 0.939 0.898 0.916 0.950 1.057
0.52 0.52 0.51 0.50 0.52 0.65 0.73 0.82 0.67 0.99 0.92 0.90 1.01 1.11 1.11 0.85 0.85 0.86 1.11 1.09 0.87 1.13 0.90 0.83 0.87 0.89 0.87 0.90 0.97 1.08 1.17 1.13 1.06 0.88 1.15 0.84
0.610 0.601 0.613 0.604 0.613 0.718 0.882 1.188 0.705 1.360 1.360 1.344 0.818 0.742 0.871 0.733 0.689 0.838 0.870 0.955 0.772 0.661 0.815 0.784 0.803 0.805 0.820 0.800 0.670 0.915 1.080 1.060 0.909 0.822 1.060 0.820
Σβ2 ΣR2 log Po/w 0.14 0.14 0.15 0.15 0.16 0.07 0.09 0.12 0.07 0.22 0.20 0.20 0.48 0.33 0.28 0.46 0.46 0.24 0.28 0.44 0.43 0.54 0.59 0.66 0.56 0.30 0.31 0.36 0.23 0.20 0.20 0.31 0.15 0.34 0.16 0.36
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.33 0.30 0.33 0.60 0.57 0.55 0.63 0.67 0.67 0.30 0.69 0.57 0.70 0.57
2.13 2.73 3.15 3.72 3.15 2.89 2.99 3.25 3.33 4.06 3.30 3.87 1.58 1.56 1.85 2.12 2.64 2.78 2.37 2.32 2.75 1.49 1.60 1.42 1.10 1.46 1.97 2.58 1.77 2.40 2.59 1.83 2.50 1.98 2.63 2.35
Solute descriptors from ref 28.
discussion. The LSER model for log Po/w is also listed in Table 2(A) for the same set of test solutes. Cohesiveness and Dispersion Interactions. All of the phases have large, positive m values, which shows hydrophobic molecules preferentially partition into the micellar and/or octanol phases. The octanol-water system has the largest m coefficient of all of the systems in the study which shows that octanol is the least cohesive phase. This is not surprising since octanol is an isotropic organic solvent, while the pseudo-phases are heterogeneous organized assemblies. However, it is still readily apparent from Table 2 that the most important factor that influences partitioning in micelles and, therefore, retention in MEKC is the solute size. Most of the surfactant systems in Table 2 have similar m coefficients, which suggests that they have similar cohesiveness and dispersive interactions with solute molecules. The fluorinated surfactant (LiPFOS) and the block copolymer (Elvacite 2669), however, show significantly smaller hydrophobic interactions. Hydrogen Bonding. The large b coefficient for all of the systems shows that the hydrogen bond donating ability is also an important factor when determining solute partitioning. The fact that it is negative shows that all of the systems have poor hydrogen bond donating characteristics (relative to the bulk aqueous phase) and that the solute hydrogen bond accepting ability opposes its partitioning into the organic phase. The large negative b coefficient 1230
Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
Table 2. Linear Solvation Energy Relationships for Various MEKC Pseudo-Stationary Phases A. Micellar, Bile Salt, Liposome, and Polymer Surfactants surfactant c m s r b a 40 mM SDS
-1.86
40 mM LiPFOS
-2.01
10 mM TTAB
-2.26
60 mM SC
-1.82
40 mM SDCV
-1.61
40 mM LSA
-1.88
2% Elvacite 2669 -1.67 log Po/w
surfactants
0.07
c
2.98 (0.09) 2.36 (0.16) 2.99 (0.11) 2.75 (0.12) 3.07 (0.12) 2.98 (0.15) 2.05 (0.16) 3.94 (0.13)
-0.30 0.24 (0.07) (0.06) 0.46 -0.68 (0.12) (0.11) -0.21 0.30 (0.08) (0.08) -0.65 0.55 (0.08) (0.08) -0.59 0.42 (0.09) (0.09) -0.38 0.56 (0.11) (0.11) -0.19 0.36 (0.12) (0.11) -0.92 0.41 (0.10) (0.09)
-1.85 -0.18 (0.08) (0.04) -0.61 -0.80 (0.14) (0.07) -2.71 0.87 (0.10) (0.05) -2.51 0.08 (0.10) (0.05) -2.53 0.14 (0.11) (0.05) -2.49 0.15 (0.13) (0.06) -1.88 0.07 (0.14) (0.07) -3.77 0.09 (0.11) (0.06)
B. Mixed Micellar Systems m s r
b
a
R2 0.989 0.954 0.985 0.986 0.983 0.976 0.947
R2
20/20 mM -1.92 2.85 -0.05 -0.11 -1.32 -0.51 0.986 LiDS/LiPFOS (0.10) (0.07) (0.07) (0.08) (0.04) 30/30 mM -1.67 2.67 -0.51 0.45 -2.24 0.08 0.975 SDS/SC (0.14) (0.10) (0.10) (0.12) (0.06) 30/30 mM -1.64 2.65 -0.59 0.49 -1.99 0.02 0.980 SDS/SDC (0.12) (0.09) (0.09) (0.10) (0.05) (pH ) 9.0)
Alcohol
C. 40 mM SDS with Alcohol Modifiers c m s r b
3.0 M MeOH 2.0 M EtOH 1.5 M n-PrOH
-2.00
200 mM BuOH 60 mM PeOH 92.4 mM PeOH 200 mM PeOH 400 mM PeOH 120 mM HexOH 35 mM HeptOH 16 mM OctOH
-1.87
-1.81 -1.66
-1.80 -1.78 -1.79 -1.42 -1.58 -1.89 -1.81
2.79 (0.20) 2.69 (0.12) 2.68 (0.12) 2.87 (0.12) 2.92 (0.14) 2.98 (0.13) 2.94 (0.14) 3.07 (0.17) 3.08 (0.14) 3.05 (0.14) 3.04 (0.12)
-0.40 (0.14) -0.33 (0.09) -0.47 (0.08) -0.47 (0.09) -0.47 (0.10) -0.58 (0.09) -0.59 (0.10) -0.68 (0.12) -0.77 (0.10) -0.59 (0.10) -0.50 (0.08)
0.37 (0.14) 0.24 (0.09) 0.32 (0.08) 0.41 (0.09) 0.38 (0.10) 0.41 (0.09) 0.49 (0.10) 0.33 (0.12) 0.44 (0.10) 0.51 (0.10) 0.37 (0.08)
-1.89 (0.17) -2.07 (0.10) -2.23 (0.10) -2.07 (0.10) -2.07 (0.12) -2.19 (0.11) -2.32 (0.12) -2.63 (0.14) -2.46 (0.12) -2.23 (0.12) -2.05 (0.10)
a
R2
-0.01 (0.08) -0.01 (0.05) 0.02 (0.05) 0.01 (0.05) -0.07 (0.06) -0.04 (0.05) -0.03 (0.06) 0.01 (0.07) -0.05 (0.06) -0.07 (0.06) -0.10 (0.05)
0.947 0.978 0.982 0.982 0.976 0.982 0.979 0.974 0.981 0.981 0.984
of octanol indicates that it is the weakest proton-donating phase. The surfactant systems show a significant range of hydrogen bond donating ability depending on the surfactant structure and the presence of additives. The reader is referred to the literature for a more detailed discussion of the structural and additive effects in MEKC.37,38 As mentioned earlier, the a constant describes the hydrogen bond accepting ability of the organic phase. For octanol and most of the pseudo-stationary phases, the a constant is statistically insignificant, suggesting that their hydrogen bond accepting ability is similar to the bulk aqueous phase and has little influence on solute partitioning. However, TTAB, DHP, and DHAB should be noted for being very strong hydrogen bond acceptors, and LiPFOS should be noted for being the weakest.
Figure 1. MEKC estimation of solute hydrophobicity using SDS. Three distinct trends can be observed. Nonpolar, nonhydrogen bonding solutes (9); polar, hydrogen bond accepting solutes (1); polar, hydrogen bond donating solutes (×). The regression equation for the nonpolar, nonhydrogen bonding solutes is: log Po/w ) 1.13(log k′) + 2.23; R2 ) 0.988.
Partitioning of hydrogen-bond donors into these phases is significantly influenced by these characteristics. Dipolarity and Polarizability. The importance of polarity and polarizability (s and r) on solute partitioning varies depending on the surfactant. The differences between surfactants have been shown to be significant and responsible for selectivity differences. The more negative s coefficient of the octanol-water system shows it is the least polar phase in this study. Although most of the r values are fairly large and positive, all the values are similar, suggesting that all of the phases have a similar capacity to interact with solute n and π electrons. The only exception to this trend is the fluorocarbon chains of LiPFOS, which cause this micellar phase to have much weaker interactions with lone-pair electrons. An important final note on the LSER results is how the octanol-water system is a significantly weaker hydrogen bond donor and less polar phase than the MEKC micellar phases. This is in spite of the fact that the concentration of water in octanol is quite large, around 2 mol/L. The proton-donor ability and dipolarity of octanol and micelles arise mainly from the water molecules that hydrate the organic solvent and the pseudo-phases. However, the water molecules in octanol are tightly bound to the hydroxyl group of octanol and thus have significantly weaker hydrogen bonding and dipolarity characteristics than those in a bulk aqueous phase. In fact, the rather large and unusually high solubility of water in octanol can only be justified by the significant hydration of the hydroxyl groups of octanol through hydrogen bonding. Congenerity. Collander’s theory is dependent on the assumption that the nature of the interactions is similar for both phases. From Figures 1-8, it can be seen that this assumption does not always hold for a wide range of solutes when using retention in MEKC. Comparing SDS and SC (Figures 1 and 2) shows how varied the congenerity can be. Similar plots for SDS and SC have been published in an earlier report for a different data set; however, these plots are included here for a more complete comparison.24 Three distinct congeneric lines can be observed for solutes using SDS micellar pseudo-stationary phase (Figure 1). This indicates that the factors that influence retention in SDS media are significantly different from those that influence octanol-
Figure 2. The relationship between solute hydrophobicity and MEKC retention using the bile salt, sodium cholate (SC). Solute identification is given in Figure 1. The regression equation for nonpolar solutes is as follows: log Po/w ) 1.17(log k′) + 2.50; R2 ) 0.983.
water partitioning. Their existence also suggests that SDS micelles have a significantly stronger interaction with polar/hydrogenbonding molecules than with octanol. Using SC micelles as the pseudophase yields notable improvement in this correlation (Figure 2). Aggregates of the bile surfactant, SC, provide a better model of octanol-water partition coefficients for polar solutes. To better understand the origins of this type of congeneric behavior for certain surfactant systems, it is useful to compare the LSER results. Abraham et al. have used this equation to explain the partitioning mechanism of solutes in biphasic systems including the octanol-water system.33,40 Consistent with those papers, these results show that the solute polarizability (R2) and volume (Vx) enhance log Po/w, while the solute dipolarity (π*2) and hydrogen bond accepting ability (Σβ2) inhibit it. The solute hydrogen bond donating ability (ΣR2) was found to have no significant influence on log Po/w values. In addition to the bulk phase systems, Abraham et al. have also shown the utility of LSER in explaining the observed correlation (or lack thereof) between log Po/w and solute retention in several RP-HPLC stationary-phase and mobile-phase conditions41-43sreversed phase thin-layer chromatography44 and microemulsion electrokinetic chromatography (MEEKC).45 In that same report, Abraham has suggested that the absolute value of the LSER coefficients is not as descriptive as their relative values. Since hydrophobic interaction is the primary driving force in partitioning, the r, s, b, and a coefficients have been normalized against m for octanol-water and all of the MEKC systems in this study. Table 3 contains these LSER coefficient ratios for all of the MEKC systems in this study. The last column in Table 3 contains the coefficient of determination for the log Po/w - log k′ correlation (40) Abraham, M. H.; Chadha, H. S.; Dixon, J. P.; Leo, A. J. J. Phys. Org. Chem. 1994, 7, 712. (41) Abraham, M. H.; Chadha, H. S.; Leitao, A. E.; Mitchell, R. C.; Lambert, W. J.; Kaliszan, R.; Nasai, A.; Haber, P. J. Chromatogr., A 1997, 766, 35. (42) Abraham, M. H.; Chadha, H. S.; Leo, A. J. J. Chromatogr., A 1994, 685, 203. (43) Kamlet, M. J.; Abraham, M. H.; Carr, P. W.; Doherty, R. M.; Taft, R. W. J. Chem. Soc., Perkin Trans. 2 1998, 2087. (44) Abraham, M. H.; Poole, C. F.; Poole, S. K. J. Chromatogr., A 1996, 749, 201. (45) Abraham, M. H.; Treiner, C.; Roses, M.; Rafols, C.; Ishihama, Y. J. Chromatogr., A 1996, 752, 243.
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Table 3. LSER Coefficient Ratios, log Po/w Constants, and Correlation Coefficients from Eq 2 for Various MEKC Pseudo-Stationary Phases
log Po/w ) y log k′ + z
R2o/w
A. Micellar, Bile Salt, and Polymer Surfactants surfactant s/m r/m b/m a/m y z 40 mM SDS 40 mM LiPFOS 10 mM TTAB 60 mM SC 40 mM SDCV 40 mM LSA 2% Elvacite 2669 log Po/w
surfactants 20/20 mM LiDS/LiPFOS 30/30 mM SDS/SC 30/30 mM SDS/SDC (pH ) 9.0)
alcohol 3.0 M MeOH 2.0 M EtOH 1.5 M n-PrOH 200 mM BuOH 60 mM PeOH 92.4 mM PeOH 200 mM PeOH 400 mM PeOH 120 mM HexOH 35 mM HeptOH 16 mM OctOH
R2o/w
-0.10 0.20 -0.07 -0.24 -0.19 -0.13 -0.09
0.08 -0.29 0.10 0.20 0.14 0.19 0.18
-0.62 -0.25 -0.91 -0.92 -0.83 -0.84 -0.92
-0.06 -0.34 0.29 0.03 0.05 0.05 0.04
1.47 0.89 1.28 1.39 1.37 1.35 1.84
1.77 2.61 2.22 2.33 1.66 1.75 2.50
0.877 0.243 0.737 0.979 0.970 0.927 0.893
-0.23
0.10
-0.96
0.02
1.00
0.00
1.00
B. Mixed Micellar Systems s/m r/m b/m a/m
Y
z
R2o/w
-0.02
-0.03
-0.46
-0.18
1.35
2.06
0.662
-0.19
0.18
-0.84
0.03
1.51
2.01
0.957
-0.22
0.18
-0.75
0.08
1.54
1.96
0.949
z
R2o/w
C. 40 mM SDS with Alcohol Modifiers s/m r/m b/m a/m Y -0.14
0.13
-0.68
0.00
1.50
2.17
0.878
-0.12
0.09
-0.77
0.00
1.62
2.16
0.947
-0.17 -0.18
0.12 0.14
-0.83 -0.72
0.01 0.00
1.56 1.48
2.10 1.98
0.970 0.928
-0.16
0.13
-0.71
-0.02
1.45
1.88
0.926
-0.19
0.14
-0.73
-0.01
1.37
1.97
0.946
-0.20
0.17
-0.79
-0.01
1.38
1.91
0.963
-0.22
0.11
-0.86
0.00
1.32
1.73
0.972
-0.25
0.14
-0.80
-0.02
1.30
1.85
0.965
-0.19
0.17
-0.73
-0.02
1.34
1.91
0.938
-0.17
0.12
-0.67
-0.03
1.40
1.78
0.919
(R2o/w). It is evident that the systems that provide the best relationship with log Po/w are those that have LSER ratios similar to log Po/w. The LSER comparisons show that the factors that influence solute retention in these separation media are very similar to the factors that influence solute partitioning in octanol/ water systems. They also provide important insight on the sources of the observed congeneric behavior in log Po/w - log k′ correlations. Looking at the SDS (Figure 1), the results in Table 3 show that the LSER s/m and b/m ratios for SDS are much less negative than those for log Po/w. The differences in SDS are great enough that the dipolar/hydrogen-bonding and nonpolar molecules form distinctly different lines. In addition, the hydrogen bond accepting and hydrogen bond donating solutes can be differentiated from one another. The sodium cholate MEKC 1232 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
Figure 3. MEKC estimation of solute hydrophobicity using LSA. Solute identification is given in Figure 1. The regression equation for nonpolar solutes is as follows: log Po/w ) 1.00(log k′) + 2.25; R2 ) 0.958.
Figure 4. MEKC estimation of solute hydrophobicity using SDCV. Solute identification is given in Figure 1. The regression equation for nonpolar solutes is as follows: log Po/w ) 1.00(log k′) + 2.05; R2 ) 0.990.
system, however, provides a medium very similar to that in octanol-water mixed systems. Most importantly, Table 3 shows that s/m and b/m are almost identical for SC and log Po/w. The r/m ratios for both are higher than that for log Po/w, but this term does not appear to have a significant effect on the correlation. The results for SDCV and LSA are shown in Figures 3 and 4. The two surfactants consist of the same twelve-member hydrocarbon chain as SDS, but both have a bulkier headgroup with organic moieties. The headgroup on SDCV contains a carbonyl valine [OC(O)NHCH(CH(CH3)2)CO2-], while LSA has a sulfoacetate [OC(O)CH2SO3-] headgroup. Recently, we demonstrated that the surfactant headgroup can have a dramatic effect on retention and selectivity in MEKC.37 These differences are also seen when extended to log Po/w correlations. It is apparent that congenerity is not as present in log Po/w - log k′ correlations using LSA (Figure 3) and SDCV (Figure 4) pseudo-phases (relative to SDS). The LSER coefficient ratios in Table 3 show that the hydrogen-bonding abilities of both of these surfactants match log Po/w well. However, congenerity is still observed because both LSA and SDCV are more dipolar than octanol-water systems (s/m in Table 3). There is no differentiation between hydrogen-bonding
Figure 5. When mixed SDS-bile salt phases are used to estimate solute hydrophobicity different trends for polar and nonpolar solutes are still shown. The MEKC running solution was 30 mM SDS/30 mM SC at pH ) 7. Solute identification is given in Figure 1. The regression equation for nonpolar solutes is as follows: log Po/w ) 1.23(log k′) 2.30; R2 ) 0.982.
solutes, but there is still clear distinction between dipolar and nonhydrogen-bonding solutes. These LSER results show that, in addition to solute hydrogen bonding, solute dipolarity also plays an important role in determining congeneric behavior. As predicted by the s/m ratio in Table 3, it is also apparent that the polar, hydrogen-bonding solutes in figures 3 and 4 do not deviate as far from the nonpolar solutes when SDCV or LSA are used. It is also important to note that for both of these systems the slope of the regression line for nonhydrogen-bonding solutes is close to unity. This is not true for any of the other surfactant systems and suggests that these surfactants and octanol-water systems possess very similar partitioning mechanisms for these solutes. Modifying the SDS pseudo phases with either a second surfactant or an organic cosolvent was also investigated. The mixed micelles seem to exhibit log Po/w correlations that are intermediate to those of the individual components. Figure 5 shows that the SDS/SC mixed micellar phase also suffers from congenerity based on the polarity/polarizability of solutes. This mixed system provides a much better correlation than SDS alone, but it is clearly an inferior model relative to SC micelle alone. The influence on the log Po/w - log k′ relationships of adding an aliphatic alcohol to SDS micelles is shown in Figures 6-8. Recently the effect of organic modifiers on the chemical selectivity in MEKC was systematically investigated.38 The addition of shortchain alcohols (e.g., eC3) only offers modest improvement in congenerity since they influence both the micellar microenvironment and the bulk aqueous phase. The addition of methanol reduces the congener retention behavior between hydrogen bond accepting and donating solutes, but there is still a clear differentiation between these and nonhydrogen-bonding solutes (Figure 6). The intermediate-length alcohols (e.g., butanol, pentanol, and hexanol), however, can be used to dramatically reduce log Po/w congenerity. At lower concentrations of pentanol (PeOH), there is only marginal improvement over that obtained for SDS alone (Figure 7). The dipolar/hydrogen-bonding solutes can no longer be differentiated, but they still have a different partition mechanism than nonpolar solutes. By increasing the pentanol concentration, the congenerity problems are almost eliminated (Figure 8). The MEKC separation buffer containing SDS/400 mM PeOH was further investigated using an extended list of solutes. In addition to the solutes listed in Table 1, this analysis included pyridine,
Figure 6. The addition of a short-chain alcohol to SDS helps to remove some of the congenerity between polar solutes. The running buffer was comprised of 40 mM SDS and 3.0 M methanol (MeOH). Solute identification is given in Figure 1. The regression equation for nonpolar solutes is as follows: log Po/w ) 1.18(log k′) + 2.54; R2 ) 0.980.
Figure 7. Adding an intermediate chain length alcohol at low concentration has a similar effect as adding short-chain alcohols. The running buffer was comprised of 40 mM SDS and 60 mM pentanol (PeOH). Solute identification is given in Figure 1. The regression equation for nonpolar solutes is as follows: log Po/w ) 1.09(log k′) + 2.29; R2 ) 0.985.
aniline, nitrobenzene, and phenol derivatives. Figure 9 shows that, even after extending the solutes from 36 to 56, this system still provides a good model of solute hydrophobicity (R2o/w ) 0.97). Therefore, it seems possible to use SDS/alcohol mixed MEKC systems in order to provide better mimics of the octanol-water system. Another advantage of SDS/alcohol systems is also worth noting. The existence of a limited elution window in MEKC is a major limitation of the technique that has an adverse effect on the separation of highly hydrophobic solutes. The size of the elution window increases with alcohol concentration and this leads to a dramatic enhancement in the range of log Po/w values that can be modeled by these systems. Unfortunately, the use of longer chain alcohols is limited. The low solubility of long-chain alcohols (e.g., gC7) results in limited improvement in log Po/w correlation. However, as with other MEKC conditions, all of these observations can be explained by the LSER coefficient ratios in Table 3. Free Energy of Transfer Differences. The congenerity can also be investigated by comparing the free energy of transfer for polar and nonpolar solutes in different surfactants and octanolAnalytical Chemistry, Vol. 72, No. 6, March 15, 2000
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Table 4. Contributions to ∆∆Ga in MEKC and Octanol-Water Systems A. log Po/w ∆∆G (kJ/mol)
Figure 8. Intermediate chain length alcohols at higher concentration dramatically reduce the congenerity. The running buffer was comprised of 40 mM SDS and 0.40 M pentanol (PeOH). Solute identification is given in Figure 1. The regression equation for nonpolar solutes is as follows: log Po/w ) 1.09(log k′) + 2.29; R2 ) 0.985.
functional group
m∆(Vx)
CH3 CH2CH3 CH2CH2CH3 CN NO2 COCH3 COOCH2CH3 Cl OH
-3.17 -6.34 -9.51 -3.48 -3.93 -6.70 -11.2 -2.76 -1.33
s∆(π*2)
r∆(R2)
-2.303RT 0 0.02 -0.05 -0.01 -0.11 0.01 3.10 -0.31 3.10 -0.61 2.57 -0.49 1.73 -0.18 0.68 -0.25 1.94 -0.45
b∆(Σβ2)
a∆(ΣR2)
% s∆(π*2)b
0 0.21 0.21 4.08 3.00 7.31 6.88 -1.50 3.44
0 0 0 0 0 0 0 0 -0.31
0 0.8 1.1 28.2 29.1 15.1 8.7 13.1 26.0
b∆(Σβ2)
a∆(ΣR2)
% s∆(π*2)b
0 0.11 0.11 1.99 1.48 3.59 3.38 -0.74 1.69
0 0 0 0 0 0 0 0 0.62
0 0.2 0.4 17.5 17.7 8.6 4.5 6.9 15.5
b∆(Σβ2)
a∆(ΣR2)
% s∆(π*2)b
0 0.14 0.14 2.70 1.99 4.84 4.55 -1.00 2.28
0 0 0 0 0 0 0 0 -0.51
0 0.4 0.6 18.3 18.2 9.2 5.2 7.7 15.5
b∆(Σβ2)
a∆(ΣR2)
% s∆(π*2)b
0 0.15 0.15 2.86 2.10 5.10 4.81 -1.05 2.40
0 0 0 0 0 0 0 0 -0.05
0 0.8 1.0 28.2 28.8 15.0 8.6 12.8 27.1
B. SDS ∆∆G (kJ/mol) functiona group
m∆(Vx)
CH3 CH2CH3 CH2CH2CH3 CN NO2 COCH3 COOCH2CH3 Cl OH
-2.40 -4.79 -7.19 -2.64 -2.97 -5.07 -8.46 -2.09 -1.00
s∆(π*2)
r∆(R2)
-2.303RT 0 0.01 -0.02 -0.01 -0.03 0.01 1.01 -0.14 1.01 -0.36 0.84 -0.28 0.56 -0.11 0.22 -0.15 0.63 -0.17
C. LSA ∆∆G (kJ/mol) Figure 9. Solute octanol-water partition coefficients vs retention in 40 mM SDS/400 mM PeOH for an extended set of solutes.
water. The overall free energy of transfer relationship is given in eq 4.
∆∆G ) -RT ln[k′(Bz - R)/k′(Bz - H)]
(4)
In this equation, the term in brackets describes the functionalgroup selectivity where k′(Bz -R) is the MEKC retention factor of a singly substituted benzene and k′(Bz - H) is the retention factor for benzene. The change in free energy of transfer of functional groups from water to octanol can also be determined in the same manner. Rearranging eq 4 provides an estimate for the individual contributions to partitioning.
∆∆G ) (-2.303RT) [m∆(Vx) + s∆(π*2) + r∆(R2) + b∆(Σβ2) + a∆(ΣR2)] (5) This expression is identical to that in eq 4 except that ∆∆G is now determined by the MEKC systems’ LSER coefficient (e.g., SDS, LSA, or octanol-water) and the difference in solute descriptor values between a substituted benzene and benzene. Table 4 shows the results from this analysis. These data clearly reveal the factors that favor partitioning (∆∆G < 0) and those that inhibit partitioning (∆∆G > 0) into octanol and surfactant systems. In many cases, the congenerity is a result of the solutes’ polarity and polarizability (Figures 1-8), which impedes partition1234 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
functional group
m∆(Vx)
CH3 CH2CH3 CH2CH2CH3 CN NO2 COCH3 COOCH2CH3 Cl OH
-2.39 -4.79 -7.18 -2.63 -2.97 -5.06 -8.45 -2.09 -1.00
s∆(π*2)
r∆(R2)
-2.303RT 0 0.03 -0.02 -0.01 -0.04 0.02 1.29 -0.42 1.29 -0.84 1.07 -0.67 0.72 -0.25 0.28 -0.34 0.81 -0.62
D. SDS/400 mM PeOH ∆∆G (kJ/mol) functional group
m∆(Vx)
CH3 CH2CH3 CH2CH2CH3 CN NO2 COCH3 COOCH2CH3 Cl OH
-2.47 -4.94 -7.41 -2.71 -3.07 -5.22 -8.72 -2.15 -1.03
s∆(π*2)
r∆(R2)
-2.303RT 0 0.02 -0.04 -0.01 -0.08 0.01 2.29 -0.25 2.29 -0.49 1.90 -0.39 1.28 -0.14 0.50 -0.20 1.43 -0.37
a ∆∆G(cavity) ) -2.303RT(m∆(V )); ∆∆G(dipolarity) ) -2.303RTx (S∆(π*2)); etc. b %S∆(π*2) ) ∆∆G(dipolarity)/∆∆G(total).
ing into nonaqueous phases. The relative importance of solute dipolarity on solute interactions with nonaqueous media are also
listed in the last column in Table 4. This value was determined by summing the absolute values for all of the ∆∆G terms and then calculating the percent contribution of ∆∆G(dipolarity). As one might expect, the polarity and polarizability contribution to partitioning for nonpolar solutes (nos. 1-3 in Table 4) is very small in all of the phases. However, the importance of this term can be seen when looking at its contribution for polar solutes. Solute polarity has a rather significant contribution toward the overall partitioning in octanol-water systems. There is a marked decrease in its importance in SDS phases. As mentioned previously, the LSER b/m coefficient ratio suggests that the hydrogen-bonding properties of LSA and log Po/w are similar, yet congenerity is still observed. Table 4 provides further evidence that these differences are based on the polarity of solutes. It can also be seen that the reason no congenerity is observed between log Po/w and SDS/ 400 mM PeOH systems is that both have very similar contributions from the solute dipolarity/polarizability and hydrogen bonding. Solute Selection. As a result of the observed congenerity, significant consideration must be taken when determining the proper set of test solutes. For some systems that provide reliable estimates (e.g., SC and SDS/400 mM PeOH), solute selection does not seem critical. However, all of these results show that many of the MEKC surfactant systems cannot model log Po/w for a wide range of solutes. This does not mean that they are ineffective for smaller, more specific groups of solutes, but it does suggest that solute selection is important. This is also evident in Figures 1-8, which show that many of the surfactants provide good “partial” models for different groups of solutes (e.g., model nonpolar solutes separately from polar solutes). Table 5 uses TTAB to show the importance of solute selection for these types of systems. TTAB shows high correlation with log Po/w for each subset of solutes when they are separated on the basis of their hydrogen-bonding properties. However, a model based on this surfactant begins to break down as soon as two or more of these subsets are combined to provide a more “global” estimate. This is particularly true when polar, hydrogen bond donating solutes are included in the solute
Table 5. Effect of the Solute Set on Hydrophobic Modeling Using TTAB Micelles in MEKC solutes
y
z
R2o/w
n
nonpolar PIa PIIb nonpolar/PI nonpolar/PII all
1.13 1.54 1.06 1.49 1.31 1.28
2.69 2.34 1.81 2.42 2.17 2.22
0.968 0.963 0.976 0.950 0.638 0.737
12 12 12 24 24 36
a
PI ) polar; HBA. b PII ) polar; HBD.
set. As mentioned previously, TTAB micelle is a strong hydrogenbond acceptor. Therefore, the addition of these solutes results in large deviations from nonpolar and polar, hydrogen bond accepting solutes showing the importance of the a/m ratio as well as the s/m ratio. CONCLUSIONS Linear solvation energy relationships (LSERs) can be very useful in describing MEKC surfactant systems. In addition, LSERs provide important insight into the sources of congeneric behavior when using MEKC to estimate octanol-water partition coefficients even for structurally similar solutes. From this analysis, it is apparent that the solute dipolarity and hydrogen bonding are all major contributors to these observed deviations. ACKNOWLEDGMENT We gratefully acknowledge a research grant from the U.S. National Institute of Health (GM 38738). We also thank Waters Corporation for the donation of SDCV, the 3M Co. for the donation of LiPFOS, Dr. S. Takeda at the Osaka National Research Institute for the donation of LSA, and ICI Acrylics for the donation of Elvacite 2669. Received for review August 2, 1999. Accepted October 18, 1999. AC990852L
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