Anal. Chem. 1998, 70, 1394-1403
Electrokinetic Chromatography Using Thermodynamically Stable Vesicles and Mixed Micelles Formed from Oppositely Charged Surfactants Mei Hong, Brian S. Weekley, Sally J. Grieb, and Joe P. Foley*
Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085
The electrokinetic chromatography (EKC) of a novel mixed surfactant system consisting of oppositely charged surfactants, sodium dodecyl sulfate (SDS) and n-dodecyltrimethylammonium bromide (DTAB), was investigated. The chromatographic characteristics of large liposome-like spontaneous vesicles and rodlike mixed micelles formed from the mixture were explored and compared with those of SDS micelles. Separations of a series of n-alkylphenones showed that the spontaneous vesicles provided about a 2 times wider elution window than SDS micelles. Both vesicle and mixed micelle systems were found to provide larger methylene selectivity than SDS. The different elution order of a group of nitrotoluene geometric isomers with DTAB/SDS spontaneous vesicles and SDS micelles pseudostationary phases suggested the possibility of different separation mechanisms with these two systems. Comparisons of polar group selectivity, retention, and efficiency were made between vesicles, mixed micelles, and SDS micelles. The correlation between the logarithms of the retention factors (log k′) and octanolwater partition coefficients (log Pow) for a group of 20 neutral compounds was also studied with DTAB/SDS vesicles. Spontaneous vesicles have great potential as a pseudostationary phase in electrokinetic chromatography. Micellar electrokinetic chromatography (MEKC), which was first introduced by Terabe in 1984,1-3 is a very common mode of capillary electrophoresis for high-efficiency separations. In MEKC, surfactant monomers that form micelles are added to the buffer to act as a pseudostationary phase. This facilitates the separation of neutral molecules on the basis of their hydrophobicity. Also, the partitioning of analytes between the aqueous and micellar phases increases the selectivity and allows the separation of ions with very similar electrophoretic mobilities. A wide variety of successful applications of MEKC, including separation of pharmaceutical compounds,4-8 derivatized amino acids,9-11 water- and fat-soluble vitamins,12-15 and herbicides,16 has shown its significant promise for the analysis of both charged and neutral analytes. (1) Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 348, 39-47. (2) Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 332, 219-26. (3) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113.
1394 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
Among the applications, sodium dodecyl sulfate (SDS) is by far the most popular micellar system in MEKC due to its negative charge state, low cost, availability in high purity, and low UV absorption characteristics. However, a drawback of this popular MEKC system at neutral and alkaline pH is the limited elution range during which solute elution occurs. Efforts have been made to extend the elution window by adjusting buffer pH,17,18 modifying the capillary,19,20 adding tetramethylammonium ion21 or organic solvent22-24 to the running buffer, running in a suppressed-flow environment,25,26 or using a mixed micellar system.27 It has been found that the retention behaviors and chemical selectivity in MEKC are influenced by surfactant type significantly.28,29 Therefore, mixed surfactant systems have great potential to provide different selectivities and chromatographic (4) Croubels, S.; Baeyens, W.; Dewaele, C.; Vanpeteghem, C. J. Chromatogr. A 1994, 673, 267-274. (5) Lukkari, P.; Siren, H.; Pantsar, M.; Riekkola, M. L. J. Chromatogr. 1993, 632, 143-148. (6) Nishi, H.; Tsumagari, N.; Kakimoto, T.; Terabe, S. J. Chromatogr. 1989, 477, 259-70. (7) Nishi, H.; Terabe, S. J. Pharm. Biomed. Anal.1993, 11, 1277-1287. (8) Ong, C. P.; Ng, C. L.; Lee, H. K.; Li, S. F. Y. J. Chromatogr. 1991, 588, 335-339. (9) Little, E. L.; Foley, J. P. J. Microcolumn Sep. 1992, 4, 145-154. (10) Castagnola, M.; Rossetti, D. V.; Cassiano, L.; Rabino, R.; Nocca, G.; Giardina, B. J. Chromatogr. 1993, 638, 327-334. (11) Ong, C. P.; Ng, C. L.; Lee, H. K.; Li, S. F. Y. J. Chromatogr. 1991, 559, 537-545. (12) Fujiwara, S.; Iwase, S.; Honda, S. J. Chromatogr. 1988, 447, 133-40. (13) Nishi, H.; Tsumagari, N.; Kakimoto, T.; Terabe, S. J. Chromatogr. 1989, 465, 331-43. (14) Ong, C. P.; Ng, C. L.; Lee, H. K.; Li, F. Y. J. Chromatogr. 1991, 547, 419428. (15) Profumo, A.; Profumo, V.; Vidali, G. Electrophoresis 1996, 17, 1617-1621. (16) Wu, Q.; Claessens, H. A.; Cramers, C. A. Chromatographia 1992, 34, 2530. (17) Watzig, H.; Lloyd, D. K. Electrophoresis 1995, 16, 57-63. (18) Otsuka, K.; Terabe, S. J. Microcolumn Sep. 1989, 1, 150-4. (19) Balchunas, A. T.; Sepaniak, M. J. Anal. Chem. 1987, 59, 1466-70. (20) Wu, Q.; Claessens, H. A.; Cramers, C. A. Chromatographia 1992, 33, 303308. (21) Nielsen, K. R.; Foley, J. P. J. Microcolumn Sep. 1994, 6, 139-149. (22) Bretnall, A. E.; Clarke, G. S. J. Chromatogr. A 1995, 716, 49-55. (23) Chen, N.; Terabe, S.; Nakagawa, T. Electrophoresis 1995, 16, 1457-1462. (24) Chen, N.; Terabe, S. Electrophoresis 1995, 16, 2100-2103. (25) Janini, G. M.; Muschik, G. M.; Issaq, H. J. Electrophoresis 1996, 17, 15751583. (26) Janini, G. M.; Muschik, G. M.; Issaq, H. J. J. Chromatogr. B 1996, 683, 29-35. (27) Ahuja, E. S.; Little, E. L.; Nielsen, K. R.; Foley, J. P. Anal. Chem. 1995, 67, 26-33. S0003-2700(97)00730-0 CCC: $15.00
© 1998 American Chemical Society Published on Web 03/04/1998
properties compared to regular (single-component) micelles. At present, most mixed micelles reported have been limited to mixtures of similarly charged surfactants30,31 or to mixtures of charged surfactants and “net zero charge” (nonionic or zwitterionic) surfactants.27,32 The use of mixed cationic and anionic surfactants has rarely been examined.21,33 Vesicles are large aggregates (compared to micelles) of monomers that have a spherical structure, formed by a bilayer that surrounds an internal cavity. The hydrophobic part of the bilayer is expected to provide hydrophilic-hydrophobic discrimination power. One type of commonly encountered vesicles is those formed from phospholipid molecules.34 Often termed liposomes, they have been used as model membranes, drug delivery devices, and microreactors. A review by Lundhal and Yang35 illustrated the use of liposomes for separating biomolecules by direct ion-exchange with the bilayer membrane and by affinity interactions using bilayer-immobilized receptors. Recently, it has been suggested that it might be possible to separate other types of solutes using liposomes as pseudostationary phase in capillary electrophoresis (CE).36,37 Roberts et al. demonstrated that the migration time of riboflavin increased in the presence of DiI-doped liposomes (liposomes with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine as cationic membrane probe).36 Zhang et al. also investigated drug- and peptide-liposome interactions by CE.37 Alternatively, spontaneous vesicles can be prepared from oppositely charged surfactants. It has been found that aqueous mixtures of anionic and cationic surfactants exhibit many unique properties due to the strong electrostatic interactions between the oppositely charged headgroups. Interestingly, microstructures not formed by the pure components, such as vesicles and/or rodlike micelles, can be observed upon mixing the two surfactants together.38,39 Compared to liposomes that are assembled from phospholipid molecules, the spontaneous vesicles have the advantages of ease of preparation, controllable size and surface charge, thermodynamic stability, and lower cost. The phase behavior of aqueous mixtures of n-dodecyltrimethylammonium bromide (DTAB) and SDS was investigated and described by Herrington and Kaler40 in 1993. The phase diagram provided by the authors indicated that there was a narrow one-phase vesicle lobe (V) in SDS-rich mixtures and large mixed micelle regions at both sides of the phase diagram. Compared to SDS micelles, the (28) Yang, S. Y.; Khaledi, M. G. Anal. Chem. 1995, 67, 499-510. (29) Yang, S. Y.; Bumgarner, J. G.; Khaledi, M. G. J. Chromatogr. A 1996, 738, 265-274. (30) Yang, S. Y.; Bumgarner, J. G.; Kruk, L. F. R.; Khaledi, M. G. J. Chromatogr. A 1996, 721, 323-335. (31) Wallingford, R. A.; Curry, P. D., Jr.; Ewing, A. G. J. Microcolumn Sep. 1989, 1, 23-7. (32) Ahuja, E. S.; Preston, B. P.; Foley, J. P. J. Chromatogr. 1994, 657, 271284. (33) Ong, C. P.; Ng, C. L.; Lee, H. K.; Li, S. F. Y. Electrophoresis 1994, 15, 12731275. (34) Fendler, J. Membrane Mimetic Chemistry; Wiley: New York, 1983. (35) Lundahl, P.; Yang, Q. J. Chromatogr. 1991, 544, 283-304. (36) Roberts, M. A.; Locasciobrown, L.; MacCrehan, W. A.; Durst, R. A. Anal. Chem. 1996, 68, 3434-3440. (37) Zhang, Y. X.; Zhang, R.; Hjerten, S.; Lundahl, P. Electrophoresis 1995, 16, 1519-1523. (38) Huang, J. B.; Zhao, G. X. Colloid Polym. Sci. 1995, 273, 156-164. (39) Kaler, E. W.; Herrington, K. L.; Murthy, A. K. J. Phys. Chem. 1992, 96, 6698-6707. (40) Herrington, K. L.; Kaler, E. W. J. Phys. Chem. 1993, 97, 13792-13802.
DTAB/SDS vesicles have a much larger diameter (several hundred nanometers) and a different surface charge distribution. These characteristics may explain some of the unique chromatographic properties of this system. Due to the presence of large particles, vesicle solutions appear bluish to the eye. It is anticipated that the spontaneous vesicles can provide potentially better efficiency and selectivity as well as a longer elution window as a new electrokinetic separation media. In this study, we examined the DTAB and SDS mixed cationic/anionic surfactant system since these surfactants are commonly encountered and available at relatively low cost. The mixed micelle region we chose to study was adjacent to the vesicle lobe on the phase diagram. Our purpose was to compare these two systems that have very similar compositions but different microstructures. To the best of our knowledge, we report here the first investigation of spontaneous vesicles as an alternative pseudostationary phase in electrokinetic chromatography. EXPERIMENTAL SECTION Apparatus. A Waters Quanta 4000E capillary electrophoresis system (Waters Inc., Milford, MA) equipped with fixed-wavelength UV detection at either 254 or 214 nm was employed for all the separations performed in this study. Electrokinetic separations were performed in either a 47.5-cm (total length) × 75-µm-i.d. (366-µm-o.d.) or a 37.5-cm × 50-µm-i.d. (368-µm-o.d.) fused-silica capillary (Polymicro Technologies, Tucson, AZ), except for the van Deemter studies, which were performed with a 35-cm × 50µm-i.d. capillary. Injections were made hydrostatically for 2 s (at a height of 9.8 cm). The data were collected at a rate of 5 Hz and processed on an NEC Image 466es computer (Milford, MA) using Millennium 2000 or 2010 software (Waters, Inc.). A Perkin-Elmer Lambda 4B UV/visible spectrophotometer (Perkin-Elmer, Norwark, CT) was employed to obtain the UV-visible absorbance spectra. All experiments were performed at ambient temperature (25 °C). The mean vesicle size was determined by laser-assisted photon correlation spectroscopy (PCS) using a Brookhaven model BT200SM particle size analyzer and a BI9000 autocorrelator. A Horiba LA-910 laser scattering particle size analyzer was employed to analyze the vesicle size distribution. Materials. The n-alkylphenone homologous series was purchased as a kit from Aldrich (Milwaukee, WI). Sodium dodecyl sulfate (SDS) was purchased from Sigma (St. Louis, MO), while n-dodecyltrimethylammonium bromide (DTAB) was purchased from Lancaster (Windham, NH). All surfactants were used as received. The buffers, sodium phosphate and HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) were purchased from J. T. Baker (Phillipsburg, NJ). Neutral test analytes were purchased from Aldrich unless otherwise noted. The neutral test mixture consisted of benzene (J. T. Baker), butylbenzene, benzyl alcohol (EM Science, Gibbstown, NJ), nitrobenzene, nitroethane, anisole, naphthalene, benzaldehyde, methyl benzoate, p-chloronitrobenzene, benzophenone, biphenyl (MCB Reagents, Cincinnati, OH), 2-, 3-, and 4-nitrotoluene (Chem Service, West Chester, PA), toluene (J. T. Baker) bromobenzene (Fisher Scientific, Fair Lawn, NJ), and chlorobenzene (Chem Service). HPLC grade water used for all solutions was obtained from J. T. Baker. Both phosphate buffer and HEPES buffer were examined as electrolytes. Phosphate buffer gave a slightly longer elution range Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
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(10%-15%) due to the higher ionic strength, which results in double-layer compression, decreased ζ potential, and reduced EOF. But since HEPES is a zwitterionic buffer that has much lower conductivity, Joule heating is less significant, and it was used as the electrolyte in all separations reported in this paper. Stock buffer solutions were prepared with HEPES and sodium hydroxide (NaOH) to give a 50 mM HEPES buffer at pH 7.2. A HEPES buffer concentration of 10 mM was used in all the experiments. The SDS (175 mM) micelle stock solution was prepared in distilled water. SDS (35 mM) in HEPES at pH 7.2 running buffer was used for MEKC experiments. The stock solutions of DTAB and SDS for making vesicles or mixed micelles were prepared in the same way as the SDS micelle stock solution. Vesicle and mixed micelle solutions for electrokinetic separations were made by pipetting the two stock solutions and buffer stock into a 25-mL volumetric flask, diluting with distilled water, and then vortex-mixing for several minutes. The vesicle solution had a total surfactant concentration of 1% w/v and a weight ratio of 39.06/60.94 DTAB/SDS. The mixed micelle solution had the same total surfactant concentration as the vesicle solution but a different ratio of 30/70 DTAB/SDS. All vesicle solutions were equilibrated at least overnight in a 25 °C isothermal water bath. Other solutions were kept at room temperature. Electrolytes were filtered through either 0.20-µm (SDS micelle and/or mixed micelle) or 0.45-µm (vesicle) membrane filters obtained from Alltech Associates, Inc. (Deerfield, IL). Sample solutions were made up of 50% acetonitrile and 50% HEPES buffer, with solute concentrations of approximately 0.2 mg/mL. Methods. The capillary was activated by purging with 0.1 M NaOH for 20 min, followed by distilled water for 20 min. The capillary was then purged for 20 min with the operating buffer. Purges with the buffer without any surfactant were performed after each run for 4-8 min to keep the adsorption of surfactant on the capillary wall to a minimum. Ohm’s law plots were prepared to determine the linear voltage range where Joule heating would not cause significant nonuniform temperature gradients and subsequent zone broadening. To keep the analysis time short, the highest operating voltage within the linear part of the Ohm’s plot, 17 kV (75-µm-i.d. capillary) or 21 kV (50-µm-i.d. capillary), was chosen for each set of separation conditions. Immediately prior to each mean particle size and size distribution measurement, the sample was filtered through a Millex-HV 13-µm filter unit with a 0.45-µm nominal pore size. Calculations. The electroosmotic mobility, µeo, was determined for each system from the relationship
µeo )
Ld Lt Vto
(1)
where Ld is the injector-to-detector column length, Lt is the total length of the capillary, V is the applied voltage, and to is the migration time of acetonitrile. The tmc value, which represents the elution time of the pseudostationary phase for each separation, was calculated using alkylphenone homologues with the iterative computation method developed by Bushey and Jorgensen41 and confirmed by using decanophenone as the tmc marker. (41) Bushey, M. M.; Jorgenson, J. W. J. Microcolumn Sep. 1989, 1, 125-130.
1396 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
The electrophoretic mobility of the pseudostationary phase, µep, was calculated from the following relationship:
µep ) µeo - µmc
(2)
where µmc was calculated using eq 1 by substituting tmc for to. The retention factor, k, was calculated using the equation
k)
tR - to to[1 - (tR/tmc)]
(3)
where tR is the migration time of the solute. Methylene selectivity (RCH2) was calculated by measuring the slope of a regression line of log k versus alkyl chain length (number of -CH2- units) of a series of alkylphenones (acetophenone to hexanophenone). The plate-count equation based on W0.1 was used to calculate the efficiency:
N ) 18.42(tR/W0.1)2
(4)
Polar group selectivity (RPG) was calculated from the following relationship:
RPG ) ks/kb
(5)
where ks is the retention factor of the solute and kb is the retention factor of benzene. RESULTS AND DISCUSSION Vesicle size is composition-dependent. When more cationic surfactant was added to the mixture (approaching the equimolar composition), the headgroup repulsion decreased; hence, the curvature decreased, and, correspondingly, the vesicle size increased.42 At the surfactant ratio used in this study, the diameters of the DTAB/SDS vesicles are expected to be around 100 nm, much larger than those of the SDS micelles (several nanometers). The DTAB/SDS mixed micelles are smaller than vesicles but larger than SDS micelles. Optical Characteristics of Surfactant Systems. The absorbance spectra (1-cm path length) of SDS micelles, DTAB/SDS mixed micelles, and DTAB/SDS vesicles are shown in Figure 1. Due to the light-scattering properties of large molecular aggregates, the apparent absorbance of the DTAB/SDS vesicles is large in both the visible and UV regions of the electromagnetic spectrum compared to the SDS micelles and DTAB/SDS mixed micelles. To the eye, the mixed micelle solution looks clear and colorless, while the vesicle solution appears bluish. Nevertheless, the DTAB/SDS vesicles proved to be fully compatible with UV absorbance detection in our study because of the relatively short path lengths across the capillary. Mean Particle Size and Size Distribution. The mean size and distribution of the DTAB/SDS vesicles were measured by laser-assisted photon correlation spectroscopy (PCS) at various (42) Kaler, E. W.; Herrington, K. L.; Zasadzinski, J. A. N. Materials Research Society Symposium Proceedings; Materials Research Society: Pittsburgh, PA, 1992; pp 3-10.
a
b
c
Figure 1. Room-temperature absorbance spectra of (a) SDS micelles, (b) DTAB/SDS mixed micelles, and (c) DTAB/SDS vesicles in water. Solution composition is as described in the Experimental Section.
time intervals. Sometimes, a very small amount of fine crystals was observed floating in the solution after it had aged for approximately 1 day; therefore, the samples for mean particle size and size distribution analyses were filtered through a 0.45-µm membrane filter. This pore size was the same as that of the membrane filters used to filter the electrolytes before a separation. Figure 2 illustrates the time dependence of the vesicle size. It can be seen that, within the first several hours after solution preparation, the mean particle size was relatively small and increased rather quickly (about 80% in the first 8 h). But the growth rate of the vesicles gradually slowed: within the first 2
Figure 2. Time dependence of the average size DTAB/SDS vesicles.
days, the size of the vesicles increased less than 10% to approximately 110-120 nm (day 1 to day 2), and in the next 3 days it increased about 17% to approximately 140 nm (day 2 to day 5). Most of the vesicle solutions used in this study were either 1 day or 2 days old and, thus, nearly if not completely at equilibrium (Figure 2). Importantly, no significant chromatographic differences were observed among such solutions. Moreover, although small amounts of precipitate were occasionally observed, no changes in the chromatographic properties were observed upon filtration and reuse. In terms of vesicle size distribution, no significant differences were observed between day 1 and day 2 for a given solution (distribution measurements were not made after 48 h); the distribution of vesicle sizes (reported as the standard deviation) varied from solution to solution and ranged from 15 to 31 nm. Although a more reproducible distribution of vesicle size could possibly be achieved with still greater care in vesicle preparation, for purposes of separation or partitioning studies, the slight polydispersity in vesicle size is unimportant since, as mentioned earlier, the chomatographic properties (efficiency, selectivity, retention, elution range) of all vesicle solutions aged for at least 24 h were essentially the same. Specific Considerations on Vesicle Preparation. Since the vesicle solution used for the electrokinetic separations was made in buffer instead of pure water, its ionic strength was different from that in the phase behavior study. According to the phase diagram,40 we first made the vesicle solution in the center of the lobe (DTAB/SDS 37.5/62.5 w/v ratio), but the results obtained with this system were not reproducible. Probably, the increase in ionic strength caused a slight shift of the vesicle lobe in the phase diagram. The stable vesicle system that was used in this study was found by slightly modifying the surfactant ratio. Because the size of the vesicles and mixed micelles was composition dependent, solutions that are prepared with insufficient mixing produce widely varying local compositions and a correspondingly wide distribution of initial sizes. In this investigation, the second stock solution was added into the first one slowly while vortex-mixing. After the solution was brought up to volume, it was vortex-mixed for several more minutes. This method of preparation resulted in satisfactory precision without internal standards; the relative standard deviations (RSDs) of migration Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
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times were approximately 1%, while RSDs of absolute peak areas were less than 3%. Phase Ratio. In MEKC, the phase ratio (b) is defined as the volume of the micellar phase divided by the volume of the aqueous phase. It can be written in terms of surfactant concentration [SURF], the critical micelle concentration (cmc), and the partial molar volume (V) of the surfactant:
β)
V([SURF] - cmc) 1 - V([SURF] - cmc)
(6)
By substituting the partial molar volume, 0.246 L/mol, and the cmc, 4.5 × 10-3 M (in buffer) of SDS into the above equation, the phase ratio of 35 mM SDS micellar solution was calculated to be 7.56 × 10-3. When mixtures of two or more surfactants produce mixed micelles, vesicles, or other aggregate structures from the surfactant monomers, the volume of the aggregated phase is the sum of the volumes occupied by the individual surfactants. The phase ratio may, therefore, be estimated from
β)
∑V 1 - ∑V
surf,i(Csurf,i
- χiCAC)
surf,i(Csurf,i
- χiCAC)
b
(7)
where χi is the overall mole fraction of the ith surfactant, CAC is the critical aggregation concentration (total surfactant concentration above which aggregates are observed), and the mole fractions of surfactants in the monomer and aggregated states are assumed to be approximately if not exactly the same. The CAC turned out to be the cmc of the predominant pure component40 (which was SDS). Since the molar ratio of DTAB and SDS was 1:1.67, the cmcis were estimated as cmcdtab ≈ cmctotal {[DTAB]/([DTAB] + [SDS])} and cmcsds ≈ cmctotal {[SDS]/[DTAB] + [SDS])} instead of using the cmcs of pure DTAB and SDS, which would be inappropriate for the vesicle mixture. The partial molar volume of DTAB, 0.325 L/mol, was measured by dissolving a certain amount of surfactant in distilled water and then determining the increased volume from the weight of the expanded amount of water. Using these approaches, the phase ratio of the vesicular solution was 8.14 × 10-3, slightly higher than that of the SDS micelle solution. The phase ratio of DTAB/SDS mixed micelle solution was calculated similarly after changing the DTAB/SDS molar ratio to 1:2.49, and was found to be 7.98 × 10-3. Elution Range and Methylene Selectivity. The separation of a series of alkylphenones (acetophenone to hexanophenone) using DTAB/SDS vesicles in HEPES buffer is shown in Figure 3c. For comparison, a SDS solution (35 mM) and a solution from the SDS-rich mixed micelle region which is very close to the vesicle lobe of the phase diagram (30/70 DTAB/SDS w/v) were prepared. Both solutions were clear and colorless and had the same total concentration of surfactant(s) as the vesicle solution. Electropherograms for the separation of the same alkylphenone series are shown in Figure 3a and b. On examining the electropherograms, it is immediately obvious that the vesicle solution provides a significantly larger elution range, as exemplified by the much larger resolution between peaks 4 and 5 compared to that in either micellar solution. 1398 Analytical Chemistry, Vol. 70, No. 7, April 1, 1998
a
c
Figure 3. Comparison of alkylphenone separation using (a) SDS micelles, (b) DTAB/SDS mixed micelles, and (c) DTAB/SDS vesicles. Conditions: sample and running buffers, 10 mM pH 7.2 HEPES; applied voltage, 17 kV (33-39 µA); capillary dimensions (bare fused silica), 75 µm i.d. × 40 cm (effective length). Solute identification: (1) acetophenone, (2) propiophenone, (3) butyrophenone, (4) valerophenone, and (5) hexanophenone.
Interestingly, the electroosmotic flow was approximately the same in all systems, but the migration time of the most hydrophobic compound was much longer with the vesicle solution. The peak shapes were generally very good, except that hydrophobic peaks were broader for the vesicle solution. The major part of this
broadening was due to the lower migration velocity of the hydrophobic compounds across the detector window. By dividing the width of each peak by its migration time,43 it was found that the normalized peak width of the hydrophobic compounds only increased by 20%-30% per methylene unit. The separation abilities of the three systems were further compared in terms of elution range tmc/to, methylene selectivity RCH2, µeo, µep, and efficiency N. The results are shown in Table 1. Under the investigation conditions, all three aggregates are negatively charged and migrate against the electroosmotic flow (EOF). The electroosmotic mobilities of each solution are very similar indicating that the viscosities and ζ potentials at the double layer of the mixed cationic/anionic solutions are similar to those of the SDS micelle solution. Although the vesicles have much larger size than the micelles, their surface-to-volume ratio (inversely proportional to the radius) is much smaller than that of the micelles. Therefore, the frictional drag per unit volume is actually smaller for these vesicles than for micelles. Furthermore, due to their bilayer structures, the vesicles may have a higher charge density than typical micelles. Hence, the DTAB/SDS vesicles have larger electrophoretic mobilities in the opposite direction of EOF. This gives the vesicle solution a migration window that is about 2 times longer than that of the micellar solutions. Surprisingly, the efficiency of the vesicle solution is somewhat better than that of the micellar solutions. In contrast, a decrease in efficiency was observed with liposomes formed from lipids used as the pseudostationary phase in EKC.36,37 In this study, the highest efficiency achieved was about 260 000 plates using vesicles, without optimization of the sample buffer and running buffer composition, ionic strength, and the detector slit width. Since peak capacity nc ) 1 + N1/2/4 ln(tmc/to), the longer elution range and the higher efficiency give the vesicle solution a significantly larger peak capacity than for either micellar system. The maximum attainable resolution (Rs)max is also related to the efficiency and elution range,44 as shown in the equations below, applicable to neutral solutes:
Rs )
( )( )(
1 - to/tmc xN R - 1 k 4 R 1 + k 1 + k(to/tmc) kopt(max Rs) ) xtmc/to
)
(8) (9)
Assuming an efficiency (N) and selectivity (R) of 150 000 and 1.03, respectively, the maximum attainable resolution of the DTAB/ SDS vesicle solution (tmc/to ) 6.74) would be 1.25, whereas the (Rs)max for the SDS micelle solution (tmc/to ) 3.12) would be 0.78. The higher methylene selectivity of the vesicles and the mixed micelles could possibly be explained by “the degree of water penetration” model that is under further investigation in our research group. This model was conceived after our observation that zwitterionic buffers such as HEPES provided larger methylene selectivity than anionic buffers such as phosphate at the same (43) Hjerte´n, S.; Elenbring, K.; Kilar, F.; Liao, J.-L.; Chen, A. J. C.; Siebert, C. J.; Zhu, M.-D. J. Chromatogr. 1987, 403, 47-61. (44) Foley, J. P.; Ahuja, E. S. In Pharmaceutical and Biomedical Applications of Capillary Electrophoresis; Lunte, S. M., Radzik, D. M., Eds.; Pergamon Press: Tarrytown, NY, 1996; Vol. 2, pp 81-178.
Table 1. Comparison of Electrokinetic and Chromatographic Properties of SDS Micelles, DTAB/ SDS Mixed Micelles, and DTAB/SDS Vesicles
µeo (cm2/(V‚s)) µep (cm2/(V‚s)) tmc/to Npropiophenone Npropiophenone/m Nbutyrophenone Nbutyrophenone/m RCH2
SDS micelle
DTAB/SDS mixed micelle
DTAB/SDS vesicle
6.49 × 10-4 -4.20 × 10-4 3.12 66 600 167 000 41 200 103 000 2.18
6.63 × 10-4 -4.15 × 10-4 2.97 32 600 81 500 27 500 68 800 2.98
6.65 × 10-4 -6.53 × 10-4 6.74 105 000 263 000 59 700 149 000 3.04
pH and ionic strength. This may be due to interactions of the hydrophobic and positively charged regions of HEPES with the hydrophobic and negatively charged regions of SDS, resulting in insertion of the HEPES molecules into the micelle and partially occupying the space between SDS molecules. As a result, fewer water molecules could penetrate into the micelle, and the hydrophobicity of the interior of the micelle increased. Since the hydrophobic interaction between DTAB and SDS molecules is stronger than that between the HEPES and SDS molecules, and the DTAB concentration was higher than the HEPES concentration in the vesicle and mixed micelle solutions, it is more difficult for the water molecules to access the centers of these microstructures. Presumably, it is due to the structures of vesicles, and mixed micelles would be more rigidly packed than SDS micelles in the same buffer. The lower degree of water penetration and higher inherent hydrophobicity of the vesicles and the mixed micelles provided a significantly higher hydrophobic selectivity than that of SDS micelles. Reduction in Analysis Time. Analysis time is another important parameter to evaluate a separation system. To shorten the analysis time of vesicle-mediated separations, we also used a 50-µm-i.d. × 30-cm (injector to detector) capillary in place of the 75-µm-i.d. × 40-cm capillary. Since Joule heating is proportional to the square of the capillary diameter, it will be significantly lower with the smaller inner diameter capillaries; thus, a higher electric field can be employed. Figure 4 shows the separation of the same alkylphenone series as Figure 2 but with a 50-µm-i.d. column. The applied voltage was 21 kV. All of the five analytes eluted within 7.5 min, whereas it took about 18 min for all the peaks to elute from the 75-µm-i.d. capillary. The major shortcoming of the smaller inner diameter column is that the signal-to-noise ratio and the limit of detection are somewhat lower due to the shorter optical path length. However, this can be alleviated by the use of extended path (“bubble”) capillaries or “Z” flow cells that are now commercially available. Polar Group Selectivity. The polar group selectivity is the ratio of the solute retention factor to the retention factor of benzene between the vesicle and the micellar solutions. Table 2 lists the retention factors and polar group selectivities for a number of substituted benzenes. By examining the results in Table 2, it can be shown that, for the relatively hydrophilic compounds (i.e., benzaldehyde, benzyl alcohol, and nitrobenzene), the vesicle solution provided the lowest degree of retention, whereas an equimolar SDS micellar solution provided the highest. Conversely, hydrophobic compounds such as toluene and chlorobenAnalytical Chemistry, Vol. 70, No. 7, April 1, 1998
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Table 2. Comparison of Retention, Relative Retention, and Polar Group Selectivity of Substituted Benzenes between SDS Micelle, DTAB/SDS Mixed Micelle, and Vesicle Solutions
substituted benzenes
SDS micelle k′
benzyl alcohol benzaldehyde benzene nitrobenzene toluene chlorobenzene p-nitrotoluene p-chloronitrobenzene
0.33 0.61 0.65 0.82 1.76 2.32 2.40 2.40
DTAB/SDS mixed micelle k′
k′MM/ k′SDS
k′
k′V/ k′SDS
0.25 0.45 1.08 1.08 1.54 4.95 1.54 1.54
0.76 0.73 1.66 1.32 0.88 2.13 0.64 0.64
0.17 0.32 0.94 0.90 3.21 5.74 2.61 2.71
0.52 0.52 1.45 1.10 1.82 2.47 1.09 1.13
Figure 4. Reduction in the time required to separate a series of alkylphenones using DTAB/SDS vesicles via a reduction in capillary dimensions and an increase in the applied voltage. A 50-µm-i.d. × 30-cm (effective length) bare fused silica capillary was used. Applied voltage, 21 kV; current, 27 µA; other conditions are as in Figure 3.
zene had the largest retention factors with the vesicles and the lowest with SDS micelles as the pseudostationary phase. It is not yet clear if the separation mechanism differs significantly between the vesicle- and micelle-mediated electrokinetic chromatography systems, or if differences in chromatographic properties such as selectivity merely reflect the significant difference in the aggregated structures of vesicles, mixed micelles, and micelles. To make the comparison more explicit, a group of substituted benzenes was separated with the three solutions. The electropherograms are shown in Figures 5. The superior selectivity of the DTAB/SDS vesicle solution is immediately obvious, since the resolution of all eight compounds was achieved. In contrast, compounds 3-5 (methyl benzoate, p-nitrotoluene, and p-nitrochlorobenzene) and compounds 4 and 5 completely coeluted with SDS micelles and DTAB/SDS mixed micelles, respectively. “Shape Selectivity” for Geometrical Isomers. The ability of a stationary phase to separate closely related compounds or structural isomers has been investigated extensively in HPLC (see ref 45 for a review) but much less so in MEKC. In the present study, a set of geometrical isomers (p-, o-, m-nitrotoluene) was employed as probes to compare the shape selectivity among vesicle and micellar solutions; the chromatograms are shown in (45) Sander, L. C.; Wise, S. A. J. Chromatogr. 1993, 656, 335-351.
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DTAB/SDS vesicle
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SDS micelle RPG
DTAB/SDS mixed micelle RPG
DTAB/SDS vesicle RPG
0.51 0.94
0.23 0.41
0.18 0.34
1.27 2.72 3.58 3.70 3.70
1.00 1.43 4.60 1.43 1.43
0.96 3.42 6.12 2.78 2.88
Figure 6. Using DTAB/SDS vesicles as the pseudostationary phase, the three geometric isomers were almost baseline separated, whereas two of them were only partially separated using SDS micelle solution. Using DTAB/SDS mixed micelles, only two partially separated peaks were observed. It is very interesting that the elution order of o-nitrotoluene and p-nitrotoluene was reversed when using the vesicle solution compared to the SDS micelles. Using the DTAB/SDS vesicle, p-nitrotoluene eluted first followed by o-nitrotoluene, whereas with the SDS micellar solution, o-nitrotoluene eluted first, followed by p-nitrotoluene. The elution order for the DTAB/SDS mixed micelles was the same as that for the vesicle solution, but o- and m-nitrotoluene coeluted and were only partially separated from the p-nitrotoluene peak. The change of elution order between the DTAB/SDS vesicle and SDS micelle could be due to the different surface charge of the cationic/anionic mixtures. This implies that there may be some different separation mechanism other than hydrophobic interactions, i.e., electrostatic interactions. If this is true, unique selectivities could be expected with the vesicle pseudostationary phase. Van Deemter Studies of DTAB/SDS Vesicle System. The dependence of plate height on electric field strength is shown in Figure 7 for six test solutes. Generally, relatively hydrophilic compounds (i.e., acetophenone, benzaldehyde, and propiophenone) had better efficiencies (lower plate heights) over the entire field strength range. The optimum field strength for hydrophilic compounds was around 400 V/cm, whereas it was around 600 V/cm for hydrophobic compounds (butyrophenone, valerophenone, and benzophenone). The reason for this apparent trend is not clear and is being investigated. Given power levels of 0.70, 1.89, and 3.76 W/m at field strengths of 400, 600, and 800 V/cm, respectively, and the onset of nonlinearity in the Ohm’s law plot for these vesicles at about 1.2 W/m (50-µm-i.d. capillaries), we would expect Joule heating to play a role, along with resistance to mass transfer. However, it is not possible to deconvolute these effects under the present experimental conditions. Estimating the Hydrophobicity of Neutral Compounds. Hydrophobic interactions play a critical role in many kinds of transmembrane processes. Therefore, a physical-chemical method that can quantitatively measure the hydrophobicity of biologically active substances is of great importance for drug design, toxicology, and other related fields. The octanol-water partition coefficient (Pow), which was first proposed by Fujita et al. in the early 1960s,46 has become the
a
b
c
Figure 5. Comparison of polar group selectivity for the separation of substituted benzenes using (a) SDS micelles, (b) DTAB/SDS mixed micelles, and (c) DTAB/SDS vesicles. Solute identification: (1) benzaldehyde, (2) nitrobenzene, (3) methyl benzoate, (4) p-nitrotoluene, (5) p-chloronitrobenzene, (6) m-nitrotoluene, (7) benzophenone, and (8) biphenyl. Other conditions are as in Figure 3.
standard. The direct determination of Po/w values by the shakeflask technique is very time-consuming and inconvenient. Many (46) Fujita, T.; Iwasa, J.; Hansch, C. J. Am. Chem. Soc. 1964, 86, 5175-5180.
a
b
c
Figure 6. Comparison of shape selectivity via the separation of the geometrical isomers of nitrotoluene using (a) SDS micelles, (b) DTAB/ SDS mixed micelles, and (c) DTAB/SDS vesicles. Solute identification: (1) p-nitrotoluene, (2) o-nitrotoluene, and (3) m-nitrotoluene. Other conditions are as in Figure 3.
attempts have been made to measure the hydrophobicity in a faster, less costly, and theoretically more correct way.47-50 (47) Medinahernandez, M. J.; Sagrado, S. J. Chromatogr. A 1995, 718, 273282.
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Table 3. Data for log k′-log Pow Correlation for 20 Neutral Compounds compounda
log Pb
log k′
compounda
log Pb
log k′
nitroethanea 1-nitropropane benzyl alcohola benzaldehydea acetophenonea nitrobenzenea anisolea benzene propiophenone 4-nitrotoluene
0.18 0.87 0.87 1.48 1.58 1.85 2.11 2.13 2.19 2.37
-1.33 -1.00 -0.77 -0.49 -0.39 -0.035 0.13 -0.018 0.098 0.42
1,4-chloronitrobenzene 1,3-chloronitrobenzenea toluene butyrophenone chlorobenzenea bromobenzenea benzophenonea naphthalenea biphenyla butylbenzenea
2.39 2.46 2.73 2.77 2.89 2.99 3.18 3.37 3.95 4.44
0.43 0.63 0.51 0.52 0.76 0.90 1.02 1.32 1.59 1.89
a
Noncongeneric compounds. b Reference 54.
Figure 7. Van Deemter study of efficiency in vesicular electrokinetic chromatography (VEKC) using six neutral compounds with DTAB/ SDS vesicles.
Although using log Po/w values for explicating the biological partitioning has been suggested,48 there are remarkable mechanism differences between the two processes. Biological membranes consist of phospholipids arranged in anisotropic bilayer structures, and the physical properties vary with distance from the interface. In contrast, for bulk-phase hydrocarbon-water partitioning, the physical properties within each phase are uniform. Studies have shown that membrane-water partitioning is dependent not only on the hydrophobic interaction but also on the surface density of the bilayer chains.51 The higher the surface density, the lower the partitioning through the membrane. Therefore, liposomes, cells, and micelles have been proposed as alternative models. MEKC has been evaluated as a method for the estimation of hydrophobicities52 and a model for biopartitioning.53 In this study, the feasibility of vesicular electrokinetic chromatography (VEKC) as an improved method for the estimation of hydrophobicities was explored. Compared to micelles, spontaneous vesicles, which have a bilayer structure, more closely model biomembranes. The surface density of hydrocarbon chains for these vesicles is variable by selecting different surfactants and/ or changing the composition, and, like in MEKC, the experimental conditions (pH, ionic strength, temperature) can be carefully controlled to mimic physiological conditions. The logarithms of retention factors for a group of neutral solutes were measured and calculated with the DTAB/SDS vesicle as a pseudostationary phase. Since the linear range of a correlation curve is always an important parameter to consider in evaluating a new method, compounds with a log Po/w value ranging from 0.18 to 4.44 were analyzed. The log Po/w data were taken from literature.54 The log k′ and log Po/w data for the 20 solutes are listed in Table 3, and the correlation plot is shown in Figure (48) Lambert, W. J. J Chromatogr 1993, 656, 469-484. (49) Chen, N.; Zhang, Y. K.; Terabe, S.; Nakagawa, T. J. Chromatogr. A 1994, 678, 327-332. (50) Kaliszan, R. Quantitative Structure-Activity Chromatographic Retention Relationships; Wiley: New York, 1987. (51) Rosen, M. J. Surfactants and Interfacial Phenomena; John Wiley and Sons: New York, 1989. (52) Herbert, B. J.; Dorsey, J. G. Anal. Chem. 1995, 67, 744-749. (53) Woodrow, B. N.; Dorsey, J. G. Environ. Sci. Technol. 1997, 31, 28122820.
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Figure 8. Correlation of retention factors in vesicular electrokinetic chromatography (VEKC) with DTAB/SDS vesicles and octanol-water partition coefficients for 20 neutral test compounds, including 13 noncongeneric compounds (squares). Trendline shown is for all 20 compounds. Correlation coefficients (r2) are greater than 0.98 for all compounds and noncongeneric subset as described in text.
8. Linear regression gives the equation
log Po/w ) 0.8019 log k′ - 1.5672 r2 ) 0.9823, n ) 20
To further examine the validity of this correlation with structurally unrelated compounds, 13 noncongeneric solutes out of the 20 were selected and plotted (Figure 8). The linear leastsquares fit equation is
log Po/w ) 0.7920 log k′ - 1.5081 r2 ) 0.9890, n ) 13
Obviously, more compounds could have been included in order to increase the breadth and significance of the correlation. We believe, however, that the results obtained with the 20 compounds of the present study (13 noncongeners) are sufficient to illustrate the potential of vesicles as a model for the estimation of the hydrophobicity and/or biopartitioning of neutral compounds. (54) Hansch, C.; Leo, A. Exploring QSAR: Fundamentals and Applications in Chemistry and Biology; American Chemical Society: Washington, DC, 1995.
Optimization of Retention. In EKC with finite elution range, the optimization of retention can be viewed from two perspectives: (i) the width of the optimum range of retention,44 i.e., what “polarity range” of analytes can be separated optimally with a given pseudostationary phase under a given set of conditions, and (ii) the ease with which retention can be varied with a given pseudostationary phase (e.g., by changing surfactant concentration or organic modifier composition). The optimum range of retention for the best Rs may be defined as the retention factor range over which the resolution is within an arbitrary percentage (e.g., 80% or 90% of its maximum value). As shown in Figure 10 of ref 44, the optimum retention range depends on the elution range, which is about 2.2 times larger for the DTAB/SDS vesicles than for SDS micelles, as dicussed earlier (Table 1). As far as varying retention, SDS micelles are much more adaptable than the DTAB/SDS vesicles. Since the pure vesicle lobe of DTAB/SDS mixture on the phase diagram is quite small, adjusting retention by changing the concentration of the surfactants is difficult due to the restriction on the vesicle phase composition. Also, in limited experiments with organic solvents, which are useful in decreasing retention, precipitation was observed at relatively low percentages (>10%). It has been determined that the large difference in the lengths or branches of the surfactants’ hydrophobic chains helps to stabilize vesicle aggregates.55 The reason for this is that the asymmetric tails cannot pack efficiently into crystalline precipitates that are predominant when oppositely charged surfactants with same length chains are mixed together. Currently, we are studying a cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) mixture that has a much larger vesicle region on the phase diagram.55 Due to the larger region of the vesicle (55) Yatcilla, M. T.; Herrington, K. L.; Brasher, L. L.; Kaler, E. W. J. Phys. Chem. 1996, 100, 5874-5879.
phase, varying retention by changing total surfactant concentration or surfactant ratio or the addition of organic solvent should be possible. CONCLUSION The potential of spontaneous vesicles prepared from oppositely charged surfactants as a pseudostationary phase in electrokinetic chromatography has been demonstrated. The chromatographic properties of the vesicle solution were compared with those of the SDS micelle solution as well as the mixed micelle solution. The spontaneous vesicles had the largest elution window and the greatest selectivity compared to those of both SDS micelles and the mixed micelles. In addition, higher efficiency and larger peak capacity facilitated the resolution of eight substituted benzenes and three geometrical isomers. However, since the pure vesicle lobe of the DTAB/SDS mixture on the phase diagram is quite small, it is somewhat tricky to use. Adjusting retention by changing the total concentration or relative proportions of the surfactants is not feasible. As mentioned before, since the DTAB/ SDS vesicle consists of two of the most commonly used surfactants, it was convenient and inexpensive to use for initial investigations. The drawbacks related to the restriction on the phase composition can be overcome by switching to vesicle systems formed from other combinations of cationic and anionic surfactants. ACKNOWLEDGMENT The authors thank Waters Inc. for loaning the Waters 4000E CE instrument, and Rhoˆne-Poulenc Rorer for partial financial support. Received for review July 7, 1997. Accepted January 23, 1998. AC970730Y
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