Anal. Chem. 1995, 67,1588-1595
Evaluation of Solute Hydrophobicity by Microemulsion Electrokinetic Chromatography Yasushi Ishihama,* Yoshiya Oda, Kiyohiko Uchikawa, and Naoki Asakawa Department of Physical and Analytical Chemistry, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, lbaraki 300-26, Japan
Microemulsion electrokinetic chromatography(MEEKC) was introduced to evaluate the hydrophobicity of various kinds of compounds. Using a separation solution consisting of sodium dodecyl sulfate (SDS), 1-butanol, h e p tane, and a borate-phosphate buffer, the logarithm of the capacity factors, which is proportional to the free energy of transfer into the microemulsion, was highly correlated with the logarithm of the octanoVwater partition coefrather than with that of the SDS micelle/ ficients (log Pow) water partition coefficientsmeasured by micellar electrokinetic chromatography. Even for compounds with hydrogen bond accepting or donating sites, the correlation of the partition coefficients between the microemulsion and the octanoVwater system was established without corrections for hydrogen bond effects. Reproducibilities of both run-to-runand batch-to-batch analyses were drastically improved by the use of the migration index concept, which is analogous to the retention index concept. Furthermore, the thermodynamic behavior of partitioning into the microemulsion was examined. In both the enthalpy and the entropy terms of transfer, the microemulsion system was more similar to a phospholipid vesicle than the octanoVwater and SDS micellar systems. Preliminary results of quantitative migration-activity relationship studies, which are defmed as quantitative structure-activity relationship studies using the electrophoretic migration data of MEEKC, indicated that this hydrophobicparameter provided slightlybetter regression to the toxicity data of phenol derivatives than the conventional log P ,. MEEKC will provide a biologically useful hydrophobic parameter with m i n i " samples, high reproducibility, wider range of measurement, high resolution, and a single, universal scale.
fact that it is criticizable to use two-phase bulk solvents as biochemical models. As for the measurement of log Pow, the conventional shake-flask technique is quite time-consuming, and high-purity of samples and skilled operators are required. Alternative techniques have been developed, such as a reversed-phase HPLC m e t h ~ d . This ~ method, however, is also problematic because the scale of hydrophobicity obtained had a limited range and was dependent on each partition chromatographic systems4 Furthermore, hydrogen bond effects must be considered for hydrogen bond acceptor and donor compounds in correlation with log Pow.S As biochemical model systems, two-phase immiscible solvent systems such as octanol/water were not appropriate in some cases because biomembranes consisting of phospholipids are different from octanol/water systems in terms of their rigid structures, anisotopic and amphiphilic properties, and limited sizes. In this respect, some alternative models were proposed, such as liposomes,6-*cell~,6.~ and micelles.10 However,liposomes and cells were unsuitable for obtaining reproducible results. The purpose of this study is to develop a biologically useful hydrophobic parameter measured by an easy, rapid, and reproducible method. Capillary electrophoresis (CE) has been developed not only as a microseparation technique but also as a powerful tool for determination of some physicochemical properties such as dissociation constantsll and diffusion coefficients12 because of its high efficiency,good reproducibility, automatization, rapid analysis time, and microscale sample amount. In our previous work, micellar electrokinetic chromatography (MEKC), which is a branch of CE, was introduced to evaluate partitioningbehavior of some neutral solutes in micellar systems.13 The separation principle of MEKC is based on the partitioning of the solute between an aqueous phase and a micellar phase.14,15
Because a drug must pass across various biomembranes to reach its site of action, the permeability of biomembranes to the drug is of great importance in the evaluation of the biological effects. Therefore, in developing a new drug, the physicochemical properties such as the hydrophobicity and the dissociation constants must be considered because the membranes have a lipoid nature.' The logarithm of the partition coefficient between 1-octanol and water, log Pow,has been most widely used as a hydrophobic parameter and was applied to quantitative structureactivity relationship (QSAR) studies.2 However, log Powhas two main drawbacks: the tediousness of the measurement and the
(3) Kaliszan, R J. Chromatop. Sci. 1984,22, 362. (4) (a) Kaliszan,R Anal. Chem. 1992,64,619A (b) Kaliszan, R J. Chromatogr. A 1993, 656, 417. (5) Yamagami, C.; Yokota, M.; Takao, N. J. Chromafogr. A 1994,662, 49. (6) Davis, S. S.; James, M. J.; Anderson, N. H. Faradny Discuss. Chem. Soc. 1986,81,313. (7) Katz, Y.; Diamond, J. M. j . Membr. Biol. 1974, 17, 101. (8) Miyoshi, H.; Maeda, H.; Tokutake, N.; Fijita, T. Bull. Chem. Soc. jpn. 1987, 60, 4357. (9) Seeman, P.;Roth, S.; Schneider, H. Biochim, Biophys. Acta. 1971,225,171. (10) (a) Treiner, C. J. Colloid Interfnce Sci. 1983, 93, 33. (b) Treiner, C.; Chattopadhyay, A. K J. Colloid Interface Sci. 1986, 109, 101. (11) Ishihama, Y.; Oda, Y.; Asakawa, N. J. Pharm. Sci. 1994, 83, 1500. (12) Walbroehl, Y.; Jorgenson, J. W. J Microcolumn. Sep. 1989, I , 41. (13) Ishihama, Y.; Oda, Y.; Uchikawa, IC;Asakawa, N. Chem. Pharm. Bull. 1994, 42, 1525. (14) Terabe, S.; Otsuka, IC; Ichikawa, K; Tsuchiya, A; Ando, T. Anal. Chem. 1 9 8 4 , 56, 111.
(1) Goth, A Medical Pharmacology; Mosby: St. Louis, 1981. (2) Leo, A; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71, 525.
1588 Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
0003-2700/95/0367-1588$9.00/0 0 1995 American Chemical Society
For various compounds such as esters, ethers, amides, and so on, it was necessary to shield the surface charges of the micelle in correlation with log Pawbecause electrostatic interactions between polar solutes and ionic micelles were observed. On the other hand, Jinno et al. reported that linear relationships were observed between capacity factors and log Paw for polycyclic aromatic hydrocarbons in cyclodextrin-modi6ed MEKC using DMS0.16 However, this system might be suitable only for congeneric compounds because the molecular length of solute must be considered in partitioning to cyclodextrins, although this system has the advantage of being able to analyze hydrophobic compounds. Microemulsions are transparent liquids that generally consist of a surfactant, a cosurfactant such as a mediumchain alcohol, oil, and water, and the dispersion systems are thermodynamically ~tab1e.l~ These systems have three dispersion forms depending on the composition, i.e., oil in water (o/w), bicontinuous,and water in oil (w/o) forms, and they have unique characteristics such as their high solubilization capacity and their ultralow interfacial tensions. Watarai used o/w microemulsions of water/sodium dodecyl sulfate (SDS)/l-butanol/heptane as a pseudostationary phase in EKC,'* and Terabe et al. compared microemulsion electrokinetic chromatography (MEEKC) with MEKC in terms of the separation selectivity, the efficiency, and the effect of the surfactant fraction.lg In this work, the partitioning behavior of various solutes into microemulsions was evaluated by MEEKC in comparison with the octanol/water or SDS micellar systems. Temperature dependence of the partitioning was also examined, and the thermodynamic differences between microemulsion, micelle, octanol/water and liposome systems are discussed. In addition, preliminary results of QSAR studies using new hydrophobic parameters measured by MEEKC are shown. THEORY
Analogous to MEKC, the capacity factor, k', of a neutral solute in MEEKC can be calculated as follows:
where to, ts,and t, are the migration times of the electroosmotic flow, the solute, and the microemulsion, respectively. When the volume of the microemulsion phase, V , , is constant, k' is proportional to the partition coefficient between the two phases, P,,, as follows:
where V, is the volume of the aqueous phase. Therefore, when the partitioning behavior in both the microemulsion system and the other system is subject to the same properties of the solutes, (15) Terabe, S.; Otsuka, K; Ando, T. Anal. Chem. 1985,57, 834. (16) Jinno, K;Sawada, Y. J Capillary Electrophor. 1994,1, 106. (17) Shmoda, K;Lindman, B. Langmuir 1987.3,135. (18) Watarai, H.Chem. Lett. 1991,391. (19) Terabe, S.;Matsubara, N.; Ishihama, Y.; Okada, Y. J Chromatogr. 1992, 608,23.
the logarithm of k' can be represented by
log k' = a logP+ b
(3)
This linear relationship is based on the change in the free energy in the partitioning process between the two phases. In this work, the migration index (MI) scale, which was introduced by Muijselaar et al. in MEKC,2O was applied to MEEKC with some modifications mentioned below. The MI value of a solute is defined as follows:
MI = c logk'
+d
(4)
where c and d are the slope and the intercept of a calibration line between log k' values of reference solutes such as alkylbenzenes and their carbon number, respectively. MI can be applied for all neutral solutes migrating in the range from to to tm, and this might be independent of the volume of the microemulsion as well as the micelles in MEKC.20 Therefore, the influence of the variation in the microemulsion preparation can be minimized. EXPERIMENTAL SECTION
Apparatus. MEEKC was performed with a Beckman (Palo Alto, CA) P/ACE System 2100. The wavelength of the UV detection was set at 214 nm. An uncoated fused-silica capillary (GL Sciences, Tokyo, Japan) of 50 pm i.d. and 27 cm length (20 cm to the detector) ,which was thermostated by a liquid coolant, was employed in d experiments. A presented chromatogramwas redrawn using Microsoft Excel. Chemicals. All tested samples for MEEKC were obtained from Wako (Osaka, Japan) and Aldrich (Milwaukee,WT). Sodium dodecyl sulfate (electrophoresis reagent) was purchased from Sigma (St. Louis, MO). AO-10-Dodecyl bromide was obtained from Dojin (Kumamoto, Japan). All other chemicals were of analytical reagent grade. All samples for MEEKC were dissolved in the microemulsion solution. Preparation of Microemulsion. The desired amounts of a surfactant, 1-butanol, and heptane were added to the buffer solution (0.05 M phosphate-0.1 M borate, pH 7.0) and mixed by ultrasonifcation in a water bath for 30 min. The transparent solution was then left to stand for 1h at room temperature. The solution was filtered through a 0.45 pm filter prior to use. It was quite important to ultrasonicate the solution adequately because the solution became turbid during standing at room temperature when the mixing step was insufficient.lg However, when the preparation was done in the appropriate manner, the solutions remained transparent for at least 4 months at room temperature. Determination of Critical Micelle Concentrations. The fluorescence intensity of AO-10-dodecyl bromide (3,Cbis(dimethylamino)-lOdodecylacridiniumbromide), which was known as a fluorescence probe for the hydrophobic regions of micelles and membranes, was markedly enhanced in hydrophobic surroundings compared with that in aqueous solutions.21 The determination procedure was performed as follows: 100 pM AO-10-dodecyl bromide was added to various concentrations of SDS solutions, and their fluorescence intensity was measured by a Jasco 820-FP spectrofluorometricdetector (Tokyo, Japan) with some modifica(20) Muijselaar, P.G.H. M.; Claessens, H. A; Cramers, C. AAnal. Chem. 1994, 66,635. (21)Kubota, Y.;Kodama, M.; Miura, M. Bull. Chem. SOC.Jpn. 1973,46, 100.
Analytical Chernistty, Vol. 67,No. 9,May 1, 7995
1589
Table 1. Migration Velocity of Tracers'
tracer Sudan I11 dodecylbenzene
u,(tracer)lu,(calcd)* 0.995c 1.003c
Conditions: separation solution, 1.44%SDS, 6.49%1-butanol,0.82% heptane (wt %) in 0.1 M borate-0.05 M phosphate (PH 7.0). Capillary, 50 pm i.d. x 27 cm (20 cm to the detector); detection, 214 nm; temperature, 25 "C; applied voltage, 7 kV (Sudan III) or 10 kV (dodecylbenzene). v,(traer), velocity of Sudan 111or dodecylbenzene; v,(calcd), velocity calculated by the iteration method using alkylbenzenes. n = 3. 9
2
3
log k' (Irue)
Figure 1. Influence of fm on capacity factors. b and fm are 3 and 15 min, respectively. The values of L(tracer)lf,(true) are given on each line.
tions as described previously.22 The detector was operated at 460 nm for excitation (Adand 525 nm for emission (A,,,). Thus, the critical micelle concentration (cmc) was determined as the concentration at the beginniig of the intensity increase in the curves between the SDS concentration and the fluorescence intensity. ~ i m e -min /
RESULTS AND DISCUSSION
Tracers for Microemulsion Phases. To obtain a true k' in EKC, it is quite important to trace the migration of the pseudostationary phase accurately. When an employed tracer is distrib uted incompletely to the pseudostationary phase, observed k' values are larger than the true ones, especially for solutes with larger k', as shown in Figure 1. Sudan 111, timepidium bromide, and quinine, which have generally been used as tracers for micelles in MEKC, could not be employed as a tracers for microemulsions consisting of SDS or CTAB,1-butanol,heptane, and a borate-phosphate buffer because Sudan I11 gave a small, split, broad peak and both timepidium bromide and quinine migrated faster than anthracene. Other tracers and tracing methods for the microemulsions were sought. An iteration method, which is based on a linear relationship between log k' and the carbon number for alkylben~enes?~ seemed to provide a reasonable value of the migration time of the microemulsions. However, the migration time of dodecylbenzene was larger than the value calculated by the iteration method and those of other hydrophobic compounds such as phenanthrene, fluoranthene, and Sudan I11 (Table 1). Therefore, dodecylbenzene was used as a tracer for the microemulsions in all experiments, while Sudan I11 was used in MEKC. On the other hand, methanol was employed as the tracer for the aqueous phase in both MEEKC and MEKC. As mentioned above, sample solutions must be dissolved not in water but in the separation solution, because samples dissolved in water gave smaller k' values than those in the separation solutions. Correlation with log P ,. As shown in Figure 2, a microemulsion consisting of SDS, 1-butanol, heptane, and a boratephosphate buffer had a high separation efficiency comparable to that of MEKC.lg However, the separation selectivity was quite (22) Ishihama, Y.; Terabe, S.J Liq. Chromatogr. 1993, 16,933. (23) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1989, 61,491.
1590 Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
Figure 2. Electrokinetic chromatogram of some test solutes: (1) methanol, (2) resorcinol, (3)pnitroaniline, (4) benzaldehyde, (5) nitrobenzene, (6) anisole, (7) 2-naphtho1, (8) toluene, (9) naphthalene, and (10) dodecylbenzene. Separation solution: 1.44% SDS, 6.49% 1-butanol, 0.82% heptane (wt %) in 0.1 M borate-0.05 M phosphate (pH 7.0). Capillary, 50pm i.d. x 27 cm (20 cm to the detector); applied voltage, 7.5 kV; detection, 214 nm; temperature, 25 "C.
different from that of MEKC with SDS, whose capacity factors showed correlationswith partition coefficients of the OctanoVwater system.13 However, the microemulsion system was better correlated with the octanol/water system in partitioning behavior F i e 3). For 20 aromatic compounds, the correlationcoefficient between log Powand log k' of MEEKC was 0.995, although that between log Powand log k' of MEKC was 0.952. The increase in the surfactant concentration in the microemulsion did not influence the correlation,although the migration window became wider and the analysis time increased.Ig In the use of CTAB instead of SDS in MEEKC, the correlation with octanol/water systems was poorer than that with the use of SDS. Addition of 50%Brij 35 to the SDS microemulsion solution, which was effective in the case of MEKC,13was not effective in this case because the migration window became too narrow to separate one from the other. Therefore, a 1.44% (50 mM) SDS microemulsion solution was chosen as a separation solution for determination of hydrophobic parameters by MEEKC in this work. Compared with conventional methods, this approach has many advantages as a determination method, i.e., in principle, this can be applied to all solutes having any log Powvalue without changing the analytical conditions. Furthermore, the usual analysis time was less than 20 min, and the necessary sample amounts were at the nanogram level. Because the efficiency of the separation was quite high and comparable to that of MEKC, the purity of the samples might not be considered, and mixed samples having similar hydrophobicity might also be separated from each other and each
Table 3. Reproduclblllty In Different Scales' 0
0
"O
'
MEKC, r-0.952
RSD (%)b
MEEKC, r I0.995
' 3
a
1.0-
0
? a
3
%*
o o
solute
MI
k'
RI
MIc
benzyl alcohol kmethoxyphenol acetophenone propiophenone @chlorophenol pethylphenol
3.92 4.02 4.87 5.83 6.26 6.36 6.75 7.51 8.19
1.66 1.40 2.67 3.53 2.98 3.40 5.32 3.92 6.36
1.26 1.31 0.86 0.63 0.70 0.62 0.30 0.51 0.18
0.57 0.64 0.41 0.36 0.48 0.41 0.14 0.43 0.15
butyrophenone
kpropylphenol naphthalene
.I."
I
1
0
1
a Analytical conditions are as given in Figure 2. n = 4. Reference standards: benzaldehyde/benzene/toluene/ethylbenzene/propyl benzene/butylbenzene.
.
2
3
4
5
log P
Figure 3. Correlation of log Po, with log K of MEEKC and MEKC for 20 test solutes. Samples: resorcinol, benzyl alcohol, acetoanilide, pnitroaniline, phenol, benzaldehyde, benzonitrile, acetophenone, nitrobenzene, anisole, methyl benzoate, benzene, propiophenone, pnitrotoluene, butyrophenone, toluene, 2-naphthol, chlorobenzene, naphthalene, anthracene. Separation solutions: (0) 1.44% SDS, 6.49% 1-butanol, and 0.82% heptane (wt %) in 0.1 M borate-0.05 M phosphate (pH 7.0); (0) 1.44% (w/w) SDS (50 mM) in 0.1 M borate-0.05 M phosphate (pH 7.0). Other conditions are as given in Figure 2. Table 2. Reproducibility in MEEKC'
nitrobenzene batch-tebatch batch I batch I1 batch I11
batch IV batch V av RSD (%) run-to-runc RSD (%)
k'
RIb
1.060 1.252 1.136 1.661 1.588 1.339 20.18
5.100 5.102 5.095 5.134 5.125 5.111 0.33
4.58
0.56
"Analytical conditions are as given in Figure 2. Preparation conditions are as follows: batch I, stored at 25 "C for 15 days after reparation; batch 11, stored at 25 "C for 10 days after preparation; {atch 111, ultrasonicated at 40 "C for 1 h after preparation; batch IV, used just after preparation; batch V, stored at 25 "C for 1 day. Reference standards: benzene/toluene/ethylbenzene/ propylbenzene/butylbenzene. Using batch I, n = 4.
hydrophobicity determined unless the injected samples interact with each other. Reproducibility. In order to determine a single and universal hydrophobic parameter, a highly reproducible determination method, which is independent of the operator, the instruments, and the laboratory, is necessary. In this method, however, k' decreased gradually in run-to-run analysis because 1-butanoland heptane can vaporize. Five batches of the microemulsion solutions were prepared under different conditions and were compared (Table 2). For nitrobenzene, the run-to-run reproducibility in batch I was 4.58%(relative standard deviation (RSD), n = 4),and the batch-to-batch reproducibility was much worse (20.18%). Recently, a retention index 0 scale was introduced to MEKC and was shown to be independent of the surfactant To overcome the poorer stability of the separation solution in this system, the RI scale was also applied to MEEKC using alkylben-
zenes as reference standards. Although the reproducibility for both run-to-run and batch-to-batch analyses was drastically improved, this scale gave relatively worse reproducibility for polar solutes whose indices are out of the range of the reference standards, i.e., less than 6.00. Thus, the RI scale was modfied to the MI scale, in which a solute with its MI value determined previously using alkylbenzenes could be added to a set of the reference standards. Therefore, this MI scale coul'd cover almost the whole range of the migration window, and the index value of the solute migrating together with one of the references might be measured more accurately than with the RI scale (Table 3). In this study, benzene, toluene, ethylbenzene, propylbenzene, butylbenzene, and benzaldehyde (MI = 4.53) were used as the reference standards. Hydrogen Bond Effects of Solutes. It is well known that the partitioning behavior of some solutes is greatly affected by the hydrogen bond abilities of both the solventc and the solutes. Thus, especially for hydrogen acceptors or donors, the correlation of their partition coefficients between different two-phase systems often failed, and some corrections were required for the correlation of their partition coeffi~ients.~~ To investigate the influence of the hydrogen bond effect on this microemulsion system, some heteroaromatic compounds, which contain a ring heteroatom such as nitrogen, oxygen, and sulfur, and their alkyl derivatives were employed. In addition, the effects of their substituents on the heterorings were also examined in comparison with the octanol/ water system. These compounds had been reported in a reversedphase liquid chromatographic system, and the logarithms of the capacity factors were not correlated with log Powwithout taking into account the hydrogen acceptor and hydrogen donor effects5 The relationship between the microemulsion and the octanol/ water systems is shown in Figure 4 and Table 4. The obtained MI values were highly correlated with log Powwithout any correction. The correlation was expressed by
log P = 0.518MI - 0.854
(5)
n = 53, r = 0.996, s = 0.094, F = 6083 where r is the correlation coefficient, s is the standard deviation from regression, and F is the value of the F-ratio between regression and residual variances. In MEKC, electrostatic interac(24) Abraham, M. H.; Chadha, H. S.; Whiting, G. S.; Mitchell, R C. J. Pham. Sci. 1994, 83,1085.
Analytical Chemistry, Vol. 67, No. 9, May 7, 7995
1591
Table 4. MI Values of 53 Test Solutes
4l 31 2
0
2
4
6
8
10
MI
Figure 4. Relationship between log Po, and MI for 53 samples. Samples are listed in Table 3. Other conditions are as given in Figure 2.
tions between ionic micelles and polar solutes such as phenol and 2-naphthol were observed, and shielding of the surface charge of the micelles was necessary to correlate with log In this case, these electrostatic interactions caused by the charge of SDS might be minimized because 1-butanol might adsorb on or penetrate the charged microdroplets, as mentioned below. This approach was found to provide a wider measurement range (-0.40 < log P < 4.45) without changing the analytical conditions, compared with the conventional HPLC m e t h ~ d .Because ~ this method can be applied to all solutes in principle as mentioned above, the measurement range will be wider by optimization of the volume and the composition of the microemulsion phase, the applied voltage, and other analytical conditions. Effect of Butanol and Heptane. To investigate the effects of the cosurfactant and the oil components on the microemulsion, SDS, SDS/methanol, SDS/butanol, and SDS/butanol/heptane systems were employed, and their selectivities were compared for 11 test samples cable 5). Regarding the effects of alcohols, the separation selectivity of the SDS micellar system was not affectedby the addition of methanol, although the k' values of all solutes were reduced. On the other hand, the selectivity in the SDS/butanol systems was apparently different from that in SDS with or without methanol, and the reverse of the migration order was observed for some solutes. Furthermore, Sudan 111, which was used as the tracer for the micelles in both SDS and SDS! methanol systems, migrated faster than dodecylbenzene in the SDS/butanol system. Manabe et al. reported that normal alcohols with alkyl chains shorter than butanol can hardly be distributed to the micellar phase.25 Their volumetric measurements show that 7.7% of added butanol penetrates the micellar phase (not including adsorption on the micelle surface), and the cmc values decreased on the addition of butanol, although they did not take into account the aggregation of micelles. Our results also suggested that added butanol affected not only the aqueous phase but also the micellar phase. Similar observations have been made by Hayase et al.26They concluded that penetration of alcohol into the micellar phase may be attributed to the release of the compact iceberg structure of water molecules around the hydrophobic parts (25) Manabe, M.; Shirahama, IC: Koda, M. Bull. Chem. SOC.Jpn. 1976,49,2904. (26)Hayase, K; Hayano, S. BUN. Chem. SOC.Jpn. 1977,50,83.
1592 Analytical Chemistry, Vol. 67, No. 9, May 7, 1995
log
sample
P(obsd)O
pyrimidine PYhe Cmethylpyrimidine methylpyrazine 4,Wiethylpyrimidine ethylpyrazine Pyrrole resorcinol N-methylbenzamide methyl 2-furoate benzyl alcohol 1-methylpyrrole acetanilide quinoxaline pmethoxyphenol furan pnitroanilie phenol 2,5dimethylpyrrole benzaldehyde ethyl 2-furoate benzonitrile acetophenone thiophene 2-methylfuran nitrobenzene pcresol ocresol mcresol pnitroanisole anisole methyl benzoate benzene indole propiophenone pnitrotoluene $-chlorophenol 2ethylfuran pethylphenol 2-methylindole Smethylindole 1-methylindole butyrophenone benzofuran toluene 2-naphthol chlorobenzene ppropylphenol ethylbenzene naphthalene propylbenzene butylbenzene anthracene
-0.40 -0.26 0.16 0.21 0.62 0.69 0.75 0.80 0.90 1.00 1.10 1.15 1.16 1.30 1.34 1.34 1.39 1.46 1.47 1.48 1.50 1.56 1.58 1.81 1.85 1.86 1.94 1.95 1.96 2.03 2.11 2.12 2.13 2.14 2.20 2.37 2.39 2.40 2.47 2.53 2.60 2.64 2.66 2.67 2.69 2.84 2.84 3.00 3.13 3.37 3.69 4.28 4.45
log MIb P(calcd)c
1.00 1.28 1.90 2.15 2.65 3.17 3.03 3.12 3.67 3.74 3.92 3.95 3.93 4.21 4.02 4.17 4.37 4.29 4.20 4.53 4.67 4.51 4.87 5.23 5.40 5.15 5.17 5.09 5.09 5.54 5.74 5.82 5.93 5.69 5.83 6.03 6.26 6.63 6.36 6.24 6.69 6.68 6.75 6.85 7.03 6.77 7.26 7.51 8.05 8.19 9.10 9.89 9.90
-0.34 -0.19 0.13 0.26 0.52 0.79 0.72 0.76 1.05 1.08 1.18 1.19 1.18 1.33 1.23 1.31 1.41 1.37 1.32 1.49 1.57 1.48 1.67 1.86 1.94 1.81 1.82 1.78 1.78 2.02 2.12 2.16 2.22 2.09 2.17 2.27 2.39 2.58 2.44 2.38 2.61 2.61 2.64 2.70 2.79 2.65 2.91 3.04 3.32 3.39 3.86 4.27 4.28
A log Powd
0.06 0.07 -0.03 0.05 -0.10 0.10 -0.03 -0.04 0.15 0.08 0.08 0.04 0.02 0.03 -0.11 -0.03 0.02 -0.09 -0.15 0.01 0.07 -0.08 0.09 0.05 0.09 -0.05 -0.12 -0.17 -0.18 -0.01 0.01 0.04 0.09 -0.05 -0.03 -0.10 0.00 0.18 -0.03 -0.15 0.01 -0.03 -0.02 0.03 0.10 -0.18 0.07 0.04 0.19 0.02 0.17 -0.01 -0.17
From refs 2, 5, and 20 (except quinoxaline, pethylphenol, and ppropylphenol, whose values were measured in this work. Analytical conditions are as given in Figure 2. Calculated values from eq 5. Calculated from the following equation: A log P = log P(calcd) log P(obsd) . (1
of the alcohol. Some groups reported that a part of the added alcohol penetrates the micellar phase, reduces the charge density, and causes the formation of smaller, more dissociated micelles.n-B Nieuwkoop et al. also measured the cmc for an SDS solution containing 0-3% butanol (~01%) by a conductivity method.B With increasing 1-butanolcontent, the cmc decreases rapidly, although (27) Grieser, F. J. Phys. Chem. 1981,85,928. (28) (a) Lianos, P.:Lang,J.: Strazielle, C.; Zana, R J Phys. Chem. 1982,86, 1019.(b) Malliaris, A; Lang,J.; Strum, J.; Zana, R J. Phys. Chem. 1987, 91, 1475. (29)Nieuwkoop, J. V.; Snoei, G. /. Colloid Interface Sci. 1985,103, 417.
~
~~
Table 5. Capacity Factors of Test Solutes In Dlfferent Systems
Table 6. Enthalpy and Entropy Changes In Partltlonlng of Solutes in Dlfferent Systems
k'
solute
SDS/ MeOHb
SDSa
resorcinol phenol p-nitroaniline nitrobenzene finitroanisole benzene 2-naphthol toluene ethylbenzene propylbenzene butylbenzene tracer
0.244 0.526 1.154 1.330 3.095 1.095 7.898 2.976 7.492 22.448 65.204
vm/veo
0.233
0.205 0.449 0.866 1.038 2.191 0.942 5.578 2.428 5.614 15.832 44.707
0.297 0.626 0.797 1.202 1.838 1.838 5.222 4.488 10.399 25.933 55.245
0.328 0.784 0.874 1.778 2.550 3.517 7.776 9.800 24.955 65.077 137.878
Sudan I11 Sudan I11 DBe
DBe
0.218
0.175
AS
L W
SDS/ SDS/ BuOHC BuOH/heptaned
0.209
1.44%SDS (wt %) in 0.1 M borate-0.05 M phosphate @H 7.0); 1.44wt % of SDS corresponds to 50 mM. 1.44%SDS (wt %) in 0.1 M borate-0.05 M phosphate (PH 7.0) containing 8% methanol (vol %). 1.44% SDS and 6.49% 1-butanol (wt %) in 0.1 M borate-0.05 M Bhosphate (PH 7.0); 6.49 wt % of 1-butanol corresponds to 8 vol %. 1.44%SDS, 6.49% 1-butanol,and 0.8% heptane (wt %) in 0.1 M borate0.05 M phosphate (PH 7.0). e Dodecylbenzene.
solute
ME"
resorcinol
MC*
OC'
LPd ME" MCb OC' LPd
-4.3 -12.58 -15.3 48.4 5.4 -17.4' -18 240 -2.3 169.5 8.6 -17.2 37 644 -4.8 -11.1' -6.4 10.7 -5.48 26 -9.3 -18.5' -3.0 -20.9 22.3 -3.1 -9.7' 8.5' 1.6' 34 247 -8.9 -11.lC -6.9 52.4 3.0 -7.6 88.5 6.3 2.9 31 367 -8.2 -lo& -8.8 -10.9 -7.3 92.0 4.1 3.3e 31 386 -8.3 -15.0 -1.0 78.8 8.0 -3.6 53 335 -14.4 -16.5 -15.9 21.6 -6.9 -10.0 13 155 3.7 -11.9 -13.1 33 261 -9.2 52.8 2.1 -16.5 -22.4c -9.5 -17.9 -12.4 -7.6e 3.9 20.4e 12.3e 42 94 -15.2 -13.1' -8.9 3.9 -1.5
p-methoxyphenLO] -5.0 -15.2 phenol p-nitroaniline nitrobenzene ocresol
m-cresol
p-cresol finitroanisole pchlorophenol
p-ethylphenol
2-naphthol toluene p-propylphenol
a 1.44%SDS, 6.49% 1-butanol, and 0.82% heptane (wt %) in 0.1 M borate-0.05 M hosphate (PH 7.0). 1.44% SDS (wt %) in 0.1 M borate-0.05 M pEosphate (PH7.0). 1-Octanol/water system. Values are from ref 6. DMPC liposome system below the phase transition temperature. Values are from ref 6. e Values are from ref 30.
Table 7. Correlation Coefficients of Thermodynamic Parameters in Different Systems
150000
1
LP e
I
20
AG (25 "C)
AS
MC
ME 0.71 0.32 0.44 (0.30)" (0.22)" MC 0.02 0.06 (0.02)" OC 0.65
S"
.
AH OC
LP
OC
MC LP
OC
MC
0.75 0.16 0.33 0.42 0.98 0.93 (0.20)" (0.24)" (0.98)" (0.97)" 0.44 0.56 0.30 0.94 (0.56)" (0.94)" 0.23 0.33
a For nine sam les (except phenol, p-nitroaniline, nitrobenzene, toluene, and 2-napRtho1).
.
I
30
.
,
40
.
50
SDS conc.(m#)
Figure 5. Determination of the cmc by the fluorescence method. Samples: SDS in 0.1 M borate-0.05 M phosphate buffer containing 0.1 mM AO-10-dodecyl bromide; I,,, 460 nm; I,,, 525 nm.
this method could not be applied to a solution containing more than 4% butanol. In this work, the cmc, which was measured by addition of a fluorescence probe, AO-lcdodecyl bromide, was also reduced from 2.91 to 0.57 mM by the addition of butanol (8 vol %, corresponding to 6.49 wt %). This method for cmc determination might be reliable because the obtained cmc value of the SDS in a borate-phosphate buffer solution (2.91 mM, Figure 5) was consistent with that of the conductivity method (2.9 mM at 25 0C).30 Thus, we concluded that the added butanol was distributed to both the aqueous and the micellar phases and that the change in the water structure might have a negligible effect on the selectivity; whereas, the distribution of the alcohol to the micellar phase might reduce the charge density of the micelle and promote the formation of the micelle. For correlation with an octanol/water system, it might be important for the alcohol to penetrate or adsorb on the micellar phase. 1-Pentanol and 1-hexanol,which penetrate the micellar phase more than 1-butanol, (30) Terabe, S.;Katsura, T.; Okada, Y.; Ishihama, Y.; Otsuka, K]. Microcolumn Sep. 1993,5,23.
were then used instead of 1-butanol. However, the solutions containing 8 vol % of both of these alcohols were turbid or thermodynamically unstable. Because the selectivity was almost constant even if the microemulsion solution contained 2-fold SDS as mentioned above and only a few percent of the added butanol penetrated the micellar phase, the adsorption rather than the penetration might have an important role in the selectivity control, and excess butanol in the aqueous phase might be adsorbed and shield the charged micelle. Regarding the effects of heptane on the micellization of SDS, Malliaris reported that micellar aggregation number increases continuously as heptane is added, whereas the charge density on the micellar interface is not altered by the solubilization of heptane.31 In this case, remarkable changes in the selectivity were not observed as expected from the results of MEKC,I5 in which the selectivity was controlled by the structure of the hydrophilic surface of the micelle rather than that of the hydrophobic core. However, the solubilization capacity of the microemulsion was generally larger than that of other micellar systems, especially for alkylbenzenes. This was an advantage in terms of determination of the hydrophobicity over a wider range. Although heptane was the only oil component in this work, the solubilization capacity may be changed by the replacement of heptane with a more suitable oil component. The electrophoretic mobility of the microdroplet was slower than that of the micelle with 1-butanol owing to swelling of the particle by heptane. (31) Malliaris, A]. Phys. Chem. 1987, 91, 6511. Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
1593
Table 8. Linear Regression Analyses of Toxicities of Para-Substituted Phenols equations log P or MI -log ICs0 = 0.139 log P 0.280pKa 2.848 -log ICs0 = 0.065MI O.276pKa 2.832 enthalpy -log ICs0 = O.O33AH(bg P ) 0.469pKa 4.926 -log IC50 = 0.021 AH(MI) 0.303pKa + 3.118 entropy -log ICs0 = 0.026ASflog p ) 0.836pKa 7.657 -log ICs0 = 0.368AS(MI) 0.018pKa 3.667 enthalpy and entropy -log ICs0 = 0.040AH00g P ) + 0.027AS(log P ) 1.020pKa 9.744 -log ICs0 = O.O89AH(MI) + 0.07lAS(MI) 0.546pKa 4.790
+
+
+
+ + + +
+
+
+
+
+
+
+
+
TemperatureDependence. To obtain further knowledge of the mechanism of the partitioning in the microemulsion system, the temperature dependence was examined and was compared with those of SDS micellar, octanol/water, and dimyristoylphosphatidylcholine (DMPC) liposome systems, in which the thermodynamic aspects of the partitioning have been examined to evaluate the characteristics of these biomembrane model^.^^^"^ Based on the difference in the relative contributions of the enthalpic and entropic portions of the partition properties between the biophase and the model systems, it had been proposed to separate the partition coefficients into their enthalpy and entropy parts.32 In fact, better correlation of the biological activity with the hydrophobic parameter was obtained when enthalpy change was used as a hydrophobic parameter rather than partition ~oefficients.3~However, it was generally tedious and timeconsuming to measure the thermodynamic parameter accurately. If we can develop a membrane model thermodynamically similar to biomembranes, the partition coefficients of the model can be used as more easily obtainable and reasonable hydrophobic parameters of QSAR studies instead of the conventional log Pow Liposomes and cells are apparently more reasonable models of a biomembrane than a two-phase immiscible solvent system such as octanol/water. However, these systems are not suitable for use as a universal model because they do not provide good batchto-batch reproducibility. In Table 6, the obtained values are listed. All values in the microemulsion system and part of the values in the SDS micellar system were measured using the procedure reported previo ~ s l y ,whereas ~ ~ , ~ ~others were cited from two reference^.^,^^ Measurements in MEEKC and MEKC were carried out under conditions where the applied voltage was 2.5, 5.0, 7.5, and 10.0 kV, and the temperature was set at 25, 33, 42, and 50 "C. The obtained capacity factors at each temperature were converted to the partition coefficients using the eq 2. However, in the case of the microemulsion, the volume of the microemulsion phase was unknown, which was different from the case of the SDS micellar phase. Thus, it was assumed that the concentrations of SDS and 1-butanol in the microemulsion phase were the same as those in the case of an SDS micellar system containing 1-butanol. The cmc values of SDS in the systems were found to be 0.57 mM by the fluorescence probe method, and the partial specific volumes at each temperature were estimated from values in ref 30. The enthalpy and entropy changes were then obtained by van't Hoff (32) Fujiwara, H.; Da, Y.; Ito, IC;Takagi, T.; Nishioka, Y. Bull. Chem. SOC. Jpn. 1991,64,3707. (33) Da, Y.: Ito, K; Fujiwara, H. J. Med. Chem. 1992,35,3382.
1594 Analytical Chemisfry, Vol. 67,No. 9,May 7, 7995
r
F
S
0.670 0.683
1.224 1.313
0.198 0.195
0.642 0.642
1.050 1.051
0.205 0.205
0.453 0.576
0.386 0.744
0.238 0.218
0.669 0.680
0.540 0.573
0.243 0.240
plots. The correlation coefficients of these plots ranged from 0.92 to 0.99. It is noted that the absolute values of entropy changes might include some errors due to the assumption mentioned above, although the enthalpy changes were independent of the volume of the microemulsion because the values were obtained from the slope in the van't Hoff plots. Also, the relative values of the entropy changes among solutes were not affected by the volume of the microemulsion because they were expressed as the difference in the intercept values in van't Hoff plots of the solutes. In Table 7, the correlation coefficients of the thermodynamic quantities for four different systems are listed. In general, the absolute values of both enthalpy and entropy changes in the microemulsion system were slightly larger than those in the SDS micellar system. Regarding the free energy changes, the microemulsion, the micellar, and the octanol/water systems were correlated with each other. However, surprisingly, not only entropy changes but also enthalpy changes in these three systems showed little correlation. The microemulsion system was similar to a gel-phase liposome, which was a more reasonable biomembrane model, rather than octanol/water or micellar systems in terms of the correlation of enthalpy and entropy changes, although enthalpy changes in the octanol/water system were also correlated with those in the liposome system. The reason was not clear, but this might be due to the fact that the microemulsion has anisotopic and amphiphilic properties and a limited size as biomembranes have, while the octanol/water system does not have such properties. Additionally, the surface charges of the microdroplet were enough to provide steric shielding, but they were insufficient in the SDS micellar system. These thermodynamic correlations were advantageous because we can obtain more reliable hydrophobic parameters easily without separation of the free energy into enthalpy and entropy as proposed by Fujiwara et al?2333 It was concluded that the partitioning behavior in the microemulsion system was apparently different from that in the octanol/water and the micellar systems and similar to that in the gel-phase liposome system, although the free energy changes of these three systems were correlated with each other in this case because of compensation effects. Quantitat&e Migration-Activity Relationship. To ensure the utility of the new hydrophobic parameter, Le., the MI values, a preliminary study of the quantitative migration-activity relationship (QMAR) was performed. The QMAR approach involves performing QSAR using migration data measured by capillary electrophoresis instead of a conventional hydrophobic parameter such as log Pow.For the toxicity of the para-subsutituted phenols, the results of QMAR were compared with those of conventional
QSAR3 In this work, six phenols, which are almost neutral at pH 7.0, were selected. As shown in Table 8, a two-variable QSAR model gave r = 0.670. On the other hand, the QMAR model provided slightly better results. The same tendency was observed when enthalpy and entropy changes were used instead of log Po,,. or MI. In this case, MI values could be directly applied to QMAR without separation of MIS into their thermodynamic parameters because the microemulsion system might be similar to the biomembrane in terms of the thermodynamic partition mechanism of solutes. In conclusion,it was found that MI values obtained by MEEKC could be used as hydrophobic parameters instead of log Pow In this rapid, reproducible, and microscale technique, purity and stability of the samples need not be considered. In addition, (34) Breyer, E. D.; Strasters, J. K; Khaledi, M.G. Anal. Chem. 1991,63, 828. (35) Sahota,R S.; Khaledi, M. G. Anal. Chem. 1994,66, 2374.
MEEKC provides a single, continuous, and universal scale independent of the operator, the capillary, and the instrument due to the pseudostationary-phase nature of microemulsions. Reliminary QMAR studies might suggest the usefulness of the MI values as biological hydrophobic parameters, although further studies are necessary. Furthermore, results of this work could be used not only to predict bioactivity of drugs but also to predict the migration order or selectivity in MEEKC as the reference scale. Although the samples employed in this work were limited to neutral species, this method will also be applicable to ionic species using a mipation behavior model in MEKC.35 Received for review October 18, 1994. Accepted February 22, 1995.a AC9410169 e Abstract published
in Advance ACS Abstructs, April 1,1995.
Analytical Chemistry, Vol. 67, No. 9, May 1, 1995
1595
