Download - Characterization of surfactant and phospholipid vesicles for use as pseudostationary phases in electrokinetic chromatography

Robert J. Pascoe1

Joe P. Foley2

1RAS-Analytical,Merck & Co. Inc.,West Point, PA, USA

2Department of Chemistry,Drexel University,Philadelphia, PA, USA

Characterization of surfactant and phospholipidvesicles for use as pseudostationary phases inelectrokinetic chromatography

The physical, electrophoretic and chromatographic properties (mean diameter, elec-troosmotic flow, electrophoretic mobility, elution range, efficiency, retention, andhydrophobic, shape, and chemical selectivity) of three surfactant vesicles and onephospholipid vesicle were investigated and compared to a conventional micellar pseu-dostationary phase comprised of sodium dodecyl sulfate (SDS). Chemical selectivity(solute-pseudostationary phase interactions) was discussed from the perspective oflinear solvation energy relationship (LSER) analysis. Two of the surfactant vesicleswere formulated from nonstoichiometric aqueous mixtures of oppositely charged, sin-gle-tailed surfactants, either cetyltrimethylammonium bromide (CTAB) and sodiumoctyl sulfate (SOS) in a 3:7 mole ratio or octyltrimethylammonium bromide (OTAB) andSDS in a 7:3 mole ratio. The remaining surfactant vesicle was comprised solely ofbis(2-ethylhexyl)sodium sulfosuccinate (AOT) in 10% v/v methanol, and the phospho-lipid vesicle consisted of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)and phosphatidyl serine (PS) in 8:2 mole ratio. The mean diameters of the vesicleswere 76.3 nm (AOT), 86.9 nm (CTAB/SOS), 90.1 nm (OTAB/SDS), and 108 nm (POPC/PS). Whereas the coefficient of electroosmotic flow (1024 cm2 V21 s21) varied consider-ably (1.72 (OTAB/SDS), 3.77 (CTAB/SOS), 4.05 (AOT), 5.26 (POPC/PS), 5.31 (SDS)), theelectrophoretic mobility was fairly consistent (23.33 to 23.8761024 cm2 V21 s21),except for the OTAB/SDS vesicles (21.68). This resulted in elution ranges that wereslightly to significantly larger than that observed for SDS (3.12): 3.85 (POPC/PS), 8.6(CTAB/SOS), 10.1 (AOT), 15.2 (OTAB/SDS). Significant differences were also noted inthe efficiency (using propiophenone) and hydrophobic selectivity; the plate countswere lower with the OTAB/SDS and POPC/PS vesicles than the other pseudostation-ary phases (� 75 000/m vs. . 105 000/m), and the methylene selectivity was consider-ably higher with the CTAB/SOS and OTAB/SDS vesicles compared to the others (ca.3.10 vs. � 2.6). In terms of shape selectivity, only the CTAB/SOS vesicles were able toseparate all three positional isomers of nitrotoluene with near-baseline resolution.Finally, through LSER analysis, it was determined that the cohesiveness and hydrogenbond acidity of these pseudostationary phases have the greatest effect on soluteretention and selectivity.

Keywords: Capillary electrophoresis / Electrokinetic chromatography / Linear solvation energyrelationship / Liposome / Vesicle DOI 10.1002/elps.200305655

1 Introduction

Liposomes and vesicles made from natural and syntheticsurfactants, including phospholipids, have been used asmodel membranes to gain a better understanding of thetransport mechanisms of biological systems. The bilayerstructure makes vesicles attractive for use as modelmembranes, drug delivery devices, and microreactors[1–3]. Liposomes have been investigated for use as apseudostationary phase in electrokinetic chromatogra-phy (EKC) [4–6]. Unfortunately, the unilamellar state isnot the most thermodynamically stable form of the phos-

Correspondence: Dr. Joe P. Foley, Department of Chemistry,Drexel University, 32nd and Chestnut Streets, Philadelphia, PA19104, USAE-mail: [email protected]: 1215-895-1265

Abbreviations: AOT, 1,2-bis(ethylhexyl)sodium sulfosuccinate;DTAB, dodecyltrimethylammonium bromide; HBD, hydrogenbond donating; LSER, linear solvation energy relationship;OTAB, octyltrimethyl ammonium bromide; POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine; PS, phosphatidyl serine;SOS, sodium octyl sulfate; VEKC, vesicle electrokinetic chroma-tography

Electrophoresis 2003, 24, 4227–4240 4227

CE

and

CE

C

2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4228 R. J. Pascoe and J. P. Foley Electrophoresis 2003, 24, 4227–4240

pholipid aggregates. Alternatively, nonstoichiometric mix-tures of single-tailed cationic and anionic surfactants aswell as double-tailed surfactants spontaneously form uni-lamellar vesicles [7–13]. Hong et al. [9] first used surfac-tant vesicles as a pseudostationary phase in EKC usingthe cationic surfactant dodecyltrimethylammonium bro-mide (DTAB), and the anionic surfactant sodium dodecylsulfate (SDS). This system demonstrated higher effi-ciency, greater shape selectivity, a larger elution window,and an alternative hydrophobic-hydrophilic selectivitycompared to conventional micellar pseudostationaryphases. More recently, Pascoe and Foley [14] observedsimilar advantages and described the effects of class Iand II modifiers on a potentially more robust vesicle sys-tem based on cetyltrimethylammonium bromide (CTAB)and sodium octyl sulfate (SOS). Similarly, Klotz et al. [15]showed that CTAB/SOS vesicles could be used for therapid estimation of the octanol-water partition coeffi-cients (log Pow) of both typical organic compounds and adiverse set of pesticides. Given these results, vesicleelectrokinetic chromatography (VEKC) clearly has poten-tial both as a separation technique and as a model for en-vironmental and biological partitioning (membrane trans-port, permeability, diffusion, etc.), and merits furtherexploration as new surfactant bilayer aggregates areintroduced.

In this investigation, four vesicle systems were studied bycomparing chromatographic, electrophoretic and physi-cal properties (retention, selectivity, elution range, elec-troosmotic flow (EOF), electrophoretic mobility, mean di-ameter) to a conventional SDS micellar system. TheVEKC systems were further characterized through theuse of linear solvation energy relationships analysis(LSER) to quantitatively gain an understanding of solute/solvent/vesicle interactions. The four vesicle systemsstudied include three synthetic surfactant-based aggre-gates as well as one phospholipid vesicle (liposome).The synthetic surfactant vesicles can be categorizedaccording to their composition: nonstoichiometric mix-tures of oppositely charged, single-tailed surfactants or asolution of double-tailed surfactants alone. The vesiclesformed from single-tailed surfactants were composed ofeither CTAB and SOS, illustrated in Fig. 1, or octyltri-methylammonium bromide (OTAB) and SDS. Kaler et al.[10, 11] previously developed phase diagrams for theDTAB/SDS and CTAB/SOS systems, with the major ad-vantage of the CTAB/SOS vesicles being the much largerstable vesicle region. Although manipulating the vesiclecomposition was not investigated in this study, the largervesicle lobe would allow for manipulation of surfactantcomposition resulting in changes in electrophoretic andchromatographic parameters of the system. Whereas theCTAB/SOS vesicle formulation utilized an excess of the

Figure 1. Cross section of a synthetic surfactant vesiclecomposed of the oppositely charged single-tailed surfac-tants CTAB and SOS.

anionic surfactant SOS, the OTAB/SDS vesicles vesicleformulation utilized an excess of the cationic surfactantOTAB, presumably forming a cationic vesicle. The dou-ble-tailed synthetic vesicle was comprised of the anionicbis(2-ethylhexyl)sodium sulfosuccinate (AOT).

In order to compare chromatographic and electrophoreticproperties between synthetic surfactant and phospholi-pid vesicles, one phospholipid system was investigated,1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine/phos-phatidyl serine (POP/PS). Although the surfactant sys-tems are easier to prepare and have a greater shelf life, itis important to understand if these advantages come atthe expense of chromatographic and electrophoretic pa-rameters. This is of great interest with respect to the useof vesicles as membrane mimetic systems, due to thebiological nature of the phospholipid aggregates.

2 Materials and methods

2.1 Instrumentation

The Agilent 3DCE (Agilent Technologies, Waldbourn, Ger-many) capillary electrophoresis system equipped withdiode array UV detection and temperature control wasemployed for all vesicle and micellar separations. Due tothe large size of the vesicles compared to micelles, thedetection wavelength was � 214 nm to reduce the noisecaused by light scattering. All separations were per-


Electrophoresis 2003, 24, 4227–4240 Surfactant and phospholipid vesicles in EKC 4229

formed on 50 mm ID, 365 mm OD bare fused-silica capil-laries (Polymicro Technologies, Phoenix, AZ, USA) with atotal length of 30–35 cm. Due to the capillary cartridge ofthe Agilent system, the length to the detector was always8.5 cm less than the total length. The applied voltage was16 kV for all separations, resulting in an electric field of457–533 V/cm depending on the capillary length, and aJoule heating level of no more than 1.7 W/m. Sampleintroduction was achieved hydrodynamically using 25mbar for 2 s, and the temperature for all separations washeld constant at 257C. Data from the Agilent 3DCE werecollected at 5 Hz with a detector response time of 0.3 sand processed on a Hewlett Packard Kayak XA system(Agilent Technologies) using Chemstation software. Parti-cle size analysis was accomplished using the Horiba LB-500 particle size analyzer (Horiba Corporation, Irvine, CA,USA) which utilizes the shift in frequency of back-scat-tered light caused by the Brownian motion of particles insolution to determine mean diameter. In addition, theAvanti Mini-extruder incorporating a 0.1 mm polycarbo-nate membrane was used to filter the phospholipid sus-pensions in order to produce unilamellar phospholipidvesicles (Avanti Polar Lipids, Alabaster, AL, USA).

2.2 Materials

The single-tailed anionic surfactant SOS was purchasedfrom Lancaster Synthesis (Pelham, NH, USA). CTAB,SDS, AOT, 4-(2-hydroxyethyl)-1-piperazineethanesulfo-nic acid (HEPES), and all commercial solutes were pur-chased from the suppliers Aldrich (Milwaukee, WI, USA)and Sigma (St. Louis, MO, USA). The phospholipidsPOPC and PS were purchased from Avanti Polar Lipids.Surfactant and phospholipid structures are shown inFig. 2.

2.3 Methods

All sample solutions were prepared by dissolving 10 mg ofsolute in 10 mL of methanol for a stock solution and dilut-ing this to a concentration of 0.4–0.6 mg/mL with a solu-tion of methanol:buffer (40:60) for analysis. All micellarand vesicle studies were performed using a 10 mM

HEPES buffer and were prepared at pH 7.2 through theaddition of 1 M lithium hydroxide (LiOH). Surfactant vesi-cle solutions were prepared by mixing surfactant andstock buffer solution in the proper ratio, diluting withHPLC water and vortex mixing. Table 1 shows the con-centration, the ratio of surfactants, the phase ratio andmean aggregate diameter for each vesicle system. TheCTAB/SOS vesicles consisted of 1.8% w/v total surfac-tant with a 3:7 mole ratio (SOS in excess, Fig. 2). Theweight percentage and mole ratio for the CTAB/SOS vesi-

Table 1. Comparison of aggregate composition andmean diameter

Aggregate Mono-mer

Molarity(mM)

Phaseratio (b)

Meandiameter(nm)

SDS(1.5% w/v)

SDS 52.0 1.18E-02 , 8.0

CTAB/SOS CTAB 14.8 1.60E-02 86.9 6 1.1(1.8% w/v, 3:7) SOS 54.2

OTAB/SDS OTAB 27.8 7.05E-03 90.1 6 1.5(1.0% w/v, 7:3) SDS 10.4

AOT AOT 40.0 1.40E-02 76.3 6 1.2(1% w/v,10% MeOH)

POPC/PS POPC 2.4 1.50E-03 108.0 6 1.2(0.5% w/v, 8:2) PS 0.6

Figure 2. Structures of synthetic and phospholipid sur-factants.



cles was chosen based on previous results indicatingan optimum balance between chromatographic param-eters and analysis time [14]. In an effort to synthesize acationic vesicle, an OTAB/SDS aggregate was devel-oped. The OTAB/SDS vesicles were composed of 1%w/v total surfactant with the same 3:7 mole ratio, butwith excess cationic surfactant (Fig. 2). The optimumsurfactant concentration and mole ratio for OTAB/SDSwas determined via trial and error utilizing the phasediagram of the CTAB/SOS vesicle system as a guide.The CTAB/SOS system was used as a reference dueto the similarities in chain length of the surfactantmonomers in each aggregate. The only double-tailedsurfactant vesicle studied consisted of 40 mM AOT(Fig. 2) in 10% v/v methanol, the organic solvent beingrequired because of this anionic surfactant’s insolubilityin purely aqueous solutions. The optimum concentra-tion of surfactant and organic modifier was determinedvia trial and error, based on the observed chromato-graphic and electrophoretic parameters [15]. All sur-factant vesicle solutions were allowed to stand for12 h after preparation and were filtered using a 0.22 mmnylon syringe filter prior to analysis. The solutions re-mained stable for 3–4 weeks. Phospholipid vesicleswere prepared by dissolving the appropriate amount oflipid in chloroform and then using rotary evaporation tocreate a thin lipid film. The lipid film was reconstituted inthe proper buffer and heated to 607C for 30 min whileintermittently vortex mixing. The resulting solution pro-duced multilamellar vesicles and was then forcedthrough an extruder with a 0.1 mm polycarbonate mem-brane a minimum of 15 times (Avanti Polar Lipids) inorder to obtain unilamellar vesicles. The phospholipidvesicle solutions were allowed to stand for 12 h afterpreparation and filtered using a 0.22 mm nylon syringefilter prior to analysis. The phospholipid aggregate solu-tions remained stable for 2–3 days. The POPC/PSaggregates were composed of 3 mM total phospholipidwith an 80:20 mole ratio of POPC/PS (Fig. 2). The anio-nic PS was incorporated to give the vesicle a net nega-tive electrophoretic mobility due to the zwitterionic na-ture of POPC under the buffer conditions utilized. Thecomposition of the phospholipid system was based onthe low transition temperature of the liposomes (47C),availability, as well as previous studies investigatingphospholipid aggregates [5, 6]. Above the phase transi-tion temperature (47C), the phospholipid mixtures con-sist of a liquid phase. However, below 47C they becomegelatinous, which would be problematic in CE due tothe high solution viscosity. The viscosity (Z) of eachEKC system was determined by measuring the timefor a sample plug to travel hydrodynamically throughthe capillary in relation to the travel time of a solutionof known viscosity.

2.4 Calculations

In EKC, the phase ratio (b) is defined as the volume of thepseudostationary phase divided by the volume of theaqueous phase. The phase ratio is related to the retentionfactor by the following equation:

k ¼ bKeq (1)

where b is the phase ratio and Keq is the thermodynamicsof distribution between the aqueous phase and pseudo-stationary phase. Thus, the phase ratio of each EKC sys-tem investigated was estimated due to its significanteffect on the retention properties of a pseudostationaryphase. For a micellar system, the phase ratio can bedescribed in terms of the concentration of surfactant [S],the critical micelles concentration (cmc) and partial molarvolume of the surfactant (V ):

b ¼ Vð½S� � cmcÞ1 � Vð½S� � cmcÞ

(2)

The calculation of mixed surfactant systems is more com-plex due to the need to account for each surfactant’s con-tribution, and can be expressed as [9]:

b ¼P

V ið½S�i � wicaciÞ1 �

PV ið½S�i � wicaciÞ

(3)

where wi is the mole fraction, caci is the critical aggrega-tion concentration V i is the partial molar volume of thesurfactant, and [S]i is the concentration of surfactant.The partial molar volumes of all surfactants (syntheticand phospholipid) in this study were measure by dissolv-ing a certain amount of surfactant in the appropriate sol-vent and determining the increased volume from theweight of the expanded solvent. Included in Table 1 arethe phase ratios for all investigated aggregate systems.Through substitution of the partial molar volume (0.246L/mol), surfactant concentration (0.052 M) and cmc(4.561023 M in buffer) of SDS into Eq. (2), the phase ratiowas calculated to be 1.1861022. The cac for the CTAB/SOS vesicles was previously determined to be approxi-mately 461025 M, which is much smaller than the con-centration of surfactant and has a minimal effect on thecalculated phase ratio [11]. Through utilization of Eq. (3),the phase ratio for the CTAB/SOS vesicles was estimatedto be 1.661022. Based on the behavior of the CTAB/SOSas well as other documented vesicles, it would beexpected that the cac should be at least 10-fold lessthan the cmc of the predominant surfactant of the aggre-gate. The cac values for the OTAB/SDS and POPC/PSvesicles were then estimated to be 4.561025 M and161026 M, respectively. Again utilizing Eq. (3), the phaseratio for each system was estimated as 7.0561023 for theOTAB/SDS vesicles and 1.561023 for the POPC/PS lipo-somes. The phase ratio for the AOT vesicles was deter-mined to be 1.461022 using Eq. (2) employed for single-



surfactant systems with a cmc of 261023 M, a partialmolar volume of 0.41 L/mol and a surfactant concentra-tion of 0.040 M. Thus, in terms of phase ratio, the order ofaggregates is CTAB/SOS . AOT . SDS . OTAB/SDS .

POPC/PS. The wide variations in concentration used foreach system resulted from factors such as desired aggre-gate formation, Joule heat, and cost.

The migration time (tpsp) and electrophoretic mobility(mep,psp) of the pseudostationary phase were estimatedusing an iterative computational method based on maxi-mizing the correlation coefficient (r2) of a regression line oflog k vs. alkyl chain length of an alkylphenones series. Theelectroosmotic mobilities (EOF or meo) as well as the reten-tion factors (k) of solutes were calculated as previouslyreported [14, 16]. The shape selectivity (ashape) was calcu-lated via a = k2/k1, where k2 and k1 are the later and firsteluting isomers. Methylene or hydrophobic selectivity,aCH2, was obtained from the antilogarithm of the slope ofa regression line of log k vs. alkyl chain length for a seriesof alkyphenones (acetophenone to valerophenone). Effi-ciencies were based on the statistical moments method(N = M1

2/M2, where M1 and M2 are the first and secondstatistical moments, respectively) and were obtainedusing the Agilent ChemStation software.

3 Results and discussion

3.1 Vesicle size distribution

The CTAB/SOS vesicle system was measured by dynam-ic light scattering (DLS) at various time intervals (Fig. 3). Allsolutions were filtered using a 0.22 mm nylon filter prior toanalysis, and refiltered prior to subsequent analysis if anoccasional small amount of precipitate was observed [9].As shown in Fig. 3, the vesicles reach a relatively stablesize after approximately 14 h. The fastest growth wasobserved over the first 3 h, during which the vesicles’mean diameter increases by 75%. Although only theCTAB/SOS vesicles were investigated as a function of

Figure 3. Kinetics of CTAB/SOS vesicle growth.

time, due to the similar nature of all aggregates, all vesi-cle solutions used in this study were at least one day old(24 h) to ensure that they were at or very near equilibri-um. Size stability of the pseudostationary phase is animportant parameter in EKC due to the fact that the mo-bility of the aggregate is dependent on the coefficient offrictional drag and the charge/size ratio. Thus, changesin size can affect the electrophoretic mobility (mep,ves) ofthe vesicles resulting in variability in chromatographicproperties such as elution range, retention and resolu-tion.

The diameter of each vesicle after 24 h of equilibrationwas measured via DLS in order to gain insight into thestructure and packing efficiency of the aggregates, andthe results are given in Table 1. The vesicles ranged froma low of 76 nm for the double-tailed anionic surfactantAOT to a maximum of 108 nm for the phospholipidPOPC/PS system. As a function of the large size of theaggregates (.70 nm), all vesicle solutions appeared witha bluish-white translucent hue to the naked eye. This is incontrast to micelles or microemulsions which are smallerthan 10 nm and appear as clear, colorless liquids. Pre-vious studies have indicated that the length of the surfac-tant monomer carbon chain has a direct effect on theradius of curvature of the vesicle, in turn influencingaggregate size [17]. Therefore, it is not surprising that thesmallest surfactant, AOT, as well as the largest mono-mers, POPC and PS, were observed to form the smallestand largest vesicles, respectively. An additional factormay lie in the packing of the vesicle structure, i.e., tightlypacked vesicles possessing a smaller diameter, while“loosely” packed aggregates are larger. Although themonomers in the POPC/PS system contain long carbonchains (17 and 15 carbons) and might be expected topack tightly, the large polar head groups may allow a sig-nificant amount of water penetration, resulting in a swel-ling of the vesicle structure. In following sections, LSERanalysis will help to further investigate the packing ofmonomers in the vesicle structure.

3.2 Electrophoretic and chromatographicparameters

As a result of the promising separation capabilities of theDTAB/SDS vesicles, it is of significant importance from ananalytical separations perspective to chromatographical-ly characterize the four aggregates in this study. This wasaccomplished through evaluation of electrophoretic (EOF,electrophoretic mobility) and chromatographic param-eters (retention, selectivity, elution range, efficiency). Allstudies were compared to 1.5% w/v SDS (52.2 mM), todemonstrate how values of a typical micellar systemcompare to the new vesicle pseudostationary phases



being characterized. The anionic surfactant SDS was uti-lized due to its commonality and wide applicability tomany EKC separations.

3.2.1 Electroosmotic flow

Table 2 shows the EOF observed with each surfactantaggregate, which can be affected by both the mobilephases and pseudostationary phases utilized. Compara-tively, all vesicle systems demonstrated a decrease inEOF compared to SDS micelles except for the phospho-lipid (POPC/PS) system. These observations can beexplained by looking at both the aggregate as well as themobile phase employed. With respect to the CTAB/SOSand OTAB/SDS vesicles, the observed decrease in EOF isthe result of free cationic surfactant monomers in solutionadsorbed to the negatively charged silanol groups on thecapillary wall. Cationic surfactants such as CTAB are fre-quently employed as EOF suppression and/or reversalagents in CE analyses. The observed decrease in EOFfor the OTAB/SDS vesicles (1.6561024 cm2 V21 s21) wasmuch more pronounced than that of even the CTAB/SOSsystem (3.6661024 cm2 V21 s21), indicating that the con-centration of free OTAB monomers in solution is muchgreater. The OTAB/SDS system contains an excess ofcationic surfactant compared to the excess anionic sur-factant in CTAB/SOS, which may account for the greaterreduction in EOF observed with the OTAB/SDS. The con-centration of OTAB monomers in solution will be dis-cussed further when examining the electrophoretic mobil-ity of the vesicles.

Table 2. Comparison of electrophoretic parameters andelution range

Aggregate meoa) Zmeo mep,psp

a) Zmep,psp Elutionrange

SDS micelles 5.31 4.73 23.61 23.21 3.12CTAB/SOS vesicles 3.77 3.66 23.33 23.23 8.60OTAB/SDS vesicles 1.72 1.65 21.68 21.61 15.20AOT vesicles 4.05 3.89 23.65 23.50 10.14POPC/PS vesicles 5.26 4.94 23.87 23.64 3.85

a) 61024 cm2 V21 s21

In contrast, the decrease in EOF observed with the AOTsurfactant aggregate is probably not due to AOT mono-mer interaction with the capillary wall, but rather to thecomposition of the mobile phase. AOT is not readily solu-ble in purely aqueous solutions, therefore the mobilephase contains 10% v/v methanol to aid in solubilization.It is well documented that addition of organic solvent to

CE systems results in a decrease in EOF due to anincrease in viscosity of the mobile phase as well as a de-crease in the dielectric constant at the capillary wall,where EOF is generated [18]. The POPC/PS vesicle sys-tem possessed an EOF very similar to that of the micellarmethod as a result of the lack of cationic monomers in thepseudostationary phase or organic modifier in the mobilephase. Although a decrease in EOF results in an increasein analysis time, an advantage is the enhancement of elu-tion range of an EKC system, which will be discussed inlater sections.

3.2.2 Electrophoretic mobility

Table 2 also shows the observed electrophoretic mobility(mep,ves) for each of the vesicles compared to commonSDS micelles. For almost all vesicles (CTAB/SOS, AOT,POPC/PS), the electrophoretic mobilities, taking intoaccount differences in viscosity, were greater than orequal to that of the SDS micelles. Although the vesiclesare much larger than micelles (.70 nm vs. , 10 nm) andpossess a larger coefficient of frictional drag, their mobil-ity remains relatively high as a result of their highlycharged aggregate structure. The bilayer structure ofvesicles allows for a greater charge density compared tomicelles, due to the charged monomer head groups onboth the interior and exterior of the aggregate. The slightlylower electrophoretic mobility compared to all other vesi-cles for the POPC/PS aggregate can be rationalized dueto its larger mean diameter. The larger size results in agreater coefficient of frictional drag and an overall les-sened electrophoretic mobility.

One unexpected observation was that although theOTAB/SDS vesicles contained an excess of cationic sur-factant (in the hopes of creating a cationic vesicle), the netelectrophoretic mobility of the OTAB/SDS vesicles wasnegative. There are two possible scenarios to accountfor this behavior: (i) exclusion of OTAB monomer from theaggregate and (ii) charge shielding effects. In the eventthat a significant amount of OTAB monomers was ex-cluded from the vesicle structure, the free OTAB in solu-tion would thus result in a relatively large decrease in EOF,which was in fact observed for this system (Table 2). Alter-natively, although a large number of OTAB monomersmaybe included in the aggregate structure, they could bepositioned in the interior of the vesicle bilayer, with thehydrophobic layer shielding the positive charge. It is diffi-cult to discern, however, whether the negative mobility isa result of only one scenario or a combination of both.Overall, some interesting trends in the EOF and electro-phoretic mobility were observed in EKC using the CTAB/SOS, OTAB/SDS, AOT, and POPC/PS vesicles. It is now



important to consider if and how these differences willaffect the chromatographic properties of the pseudosta-tionary phases.

3.2.3 Elution range

In EKC, the retentive phase in the separation system isfree flowing and in opposition to the bulk flow within thecapillary. This is in direct contrast to HPLC or GC, wherethe retentive phase is bonded to the support media and istermed the stationary phase. In EKC separations wherethe pseudostationary phase cannot fully oppose theEOF, there is a finite window in which compounds canelute. This is known as the elution range or elution win-dow, which is defined as the time of elution of the pseu-dostationary phase (tpsp) divided by the elution time of anonretained neutral compound (to). The elution range canbe optimized by accomplishing one or both of following:reducing the EOF (meo) or increasing the electrophoreticmobility of the pseudostationary phase (mep,psp) in opposi-tion to the bulk flow. When the elution range is increased,chromatographic parameters such as peak capacity andresolution (keys to chromatographic analysis) are alsoincreased. To achieve an infinite elution range (obtainedin HPLC and GC due to the bonded retentive phase),mep,psp needs to be of equal magnitude and in direct oppo-sition to meo. Because analysis time increases with elutionrange, an infinite elution range is often not the ultimategoal; an order of magnitude increase from the typicalvalues of 2.5–4 is more than sufficient. The elution rangefor each vesicle system was determined through theseparation of a homologous series as outlined in theSection 2.3 and compared in Table 2 to EKC incorporatingSDS micelles, hereafter referred to as SDS-MEKC.Figure 4 shows the separation of a series of alkylphe-nones using SDS-MEKC, CTAB/SOS, OTAB/SDS, AOT,and POPC/PS vesicles as pseudostationary phases inEKC.

The elution range data in Table 2 show that all vesiclesystems, surfactant or phospholipid-based, possess agreater elution range than SDS-MEKC. As describedbelow, the reasons for the larger elution range vary some-what for each type of vesicle. The vesicles with the largestelution range, 15.2, were composed of the oppositelycharged single-tailed surfactants OTAB and SDS. As sta-ted previously, although the solution contained an excessof cationic surfactant, a net negative electrophoretic mo-bility was observed. The large observed elution window isthe result of the alteration of both meo and mep,ves. Thehigher charge density of the vesicle lends itself to a mod-erate mep,ves despite the large aggregate size, as well asthe reduction in meo due to free OTAB monomers in solu-

Figure 4. Separation of acetophenone, propiophenone,butyrophenone, and valerophenone using (A) SDSmicelles, (B) CTAB/SOS vesicles, (C) OTAB/SDS vesicles,(D) AOT vesicles, and (E) POPC/PS phospholipid vesicles.

tion. These phenomena allow the EOF to be more fullyopposed by the pseudostationary phase, resulting in thelarge observed elution range. For similar reasons, theCTAB/SOS vesicles also showed a large increase in elu-tion range (8.6) compared to SDS micelles (3.12) althoughnot to the extent of the OTAB/SDS aggregates (15.2). As aresult of the free cationic monomers in solution as well asthe high charge density associated with the bilayer struc-ture, the electrophoretic mobility of the vesicle once againcomes closer to fully opposing to the bulk EOF.

The AOT vesicles showed a similar increase in elutionwindow to the CTAB/SOS system, not as a result of sur-factant monomers, but due to the composition of themobile phase. The anionic double-tailed surfactant AOTis not readily soluble in purely aqueous solutions; there-fore the buffer consisted of 10% v/v methanol. Due tothe decrease in EOF as a result of the addition of metha-nol and the vesicle’s bilayer structure, the AOT systemachieved an elution range of 3.25 times that of SDS micel-lar EKC. The only phospholipid aggregate investigated,POPC/PS, possessed a smaller elution window (3.85)than the other VEKC systems. This is the result of theslower electrophoretic mobility of the vesicles due to theincreased size (108 nm) as well as the lack of a mechan-ism for the reduction of EOF.



3.2.4 Efficiency

The efficiency of each vesicle system was evaluated bymeasuring the plate count (N) of the moderately retained,neutral test compound propiophenone. The statisticalmoment method was utilized to measure plate count dueto the asymmetrical peak shape occasionally observed.Table 3 shows the efficiency per unit length (N/m) forSDS micelles and the surfactant and phospholipid vesi-cles.

Table 3. Efficiency values for propiophenone for eachEKC systema)

Aggregate N/m

SDS Micelles 131 200CTAB/SOS 105 300OTAB/SDS 60 000AOT 107 200POPC/PS 75 100

a) Efficiencies (N) calculated using the statistical momentsmethod.

In order of decreasing efficiency, the results were SDS .

AOT . CTAB/SOS . POPC/PS . OTAB/SDS, which issomewhat surprising based on previous studies usingmicellar and vesicular media. Surfactant vesicles, suchas those composed of DTAB and SDS, have been shownto provide enhanced efficiency compared to micellarmedia [9]. Under the conditions of the present study,however, the CTAB/SOS and AOT vesicles providedabout 106 000 plates/m, slightly lower than those pro-vided by the SDS micelles (131 000/m). In contrast, theEKC results for the liposomes were not surprising, asthey have shown to yield lower efficiencies than micellesdue to the high level of polydispersity encountered [4];our data for POPC/PS (N/m = 75 100) are consistentwith this report.

With respect to the OTAB/SDS vesicles, in addition to thelow efficiency observed (N/m = 60 000), there was also ahigher degree of peak asymmetry observed. When con-sidering the lower efficiency values of this vesicle system,zone broadening contributions from sample introduction,excessive Joule heat, electromigration dispersion, mobilephase mass transfer, and detection can all be dismissedbecause the experimental conditions associated withthese phenomena were the same (or very similar) for allthe pseudostationary phases studied. Instead, factorssuch as pseudostationary phase mass transfer and thepolydispersity of the vesicles are likely contributors tothe lower plate counts observed, especially for theOTAB/SDS and POPC/PS vesicles.

In the event that the negative electrophoretic mobilityencountered for the OTAB/SDS vesicles is in fact due toexclusion of OTAB monomers, secondary chemical equi-librium may be of significance. Although it is unlikely thatthe free OTAB monomers form micelles due to their highcmc (140 mM), solute interactions with monomers alongthe capillary wall are possible. In addition, a lack of homo-geneity of the OTAB wall coating could lead to furtherband broadening due to the variation in electroosmoticpressure in different portions of the capillary, giving riseto zone-spreading eddy currents.

3.2.5 Hydrophobic (methylene) selectivity

Again utilizing the separation of a series of alkylphenones,the hydrophobic selectivity of each vesicle system wascalculated and evaluated (Table 4). Hydrophobic selectiv-ity is a valuable measure of nonspecific hydrophobicinteractions between the solute and pseudostationaryphase in an EKC system, and can be used to gauge howwell a given pseudostationary phase can separate com-pounds with slight differences in hydrophobicity.

Table 4. Comparison of hydrophobic selectivity in EKC

Aggregate Hydrophobicselectivity, aCH2

SDS micelles 2.11 6 0.06CTAB/SOS vesicles 3.10 6 0.03OTAB/SDS vesicles 3.09 6 0.04AOT vesicles 2.28 6 0.09POPC/PS vesicles 2.56 6 0.02

Results are an average of three independent measure-ments on different days.

The two vesicles composed of oppositely charged single-tailed surfactants CTAB/SOS and OTAB/SDS showed thegreatest hydrophobic selectivity, 3.10 and 3.09, respec-tively. This indicates that these vesicles possess the great-est hydrophobic environment for solute partitioning com-pared to all other aggregates investigated. The POPC/PSvesicles also showed an increase in hydrophobic selectiv-ity compared to SDS micelles, but not to the degree ofOTAB/SDS and CTAB/SOS. The lower hydrophobic selec-tivity for the AOTsystem is due to the 10% methanol in themobile phase. The introduction of organic modifier de-creased the polarity difference between the mobile phaseand pseudostationary phase, thus decreasing the solutes’differential preference for the surfactant aggregate.

The extent of the hydrophobic environment created bythe vesicles is also a function of the degree of water pene-tration in and around the aggregate [36, 37]. A tightly



packed aggregate, such as CTAB/SOS, has less waterpenetration, which results in greater hydrophobic selec-tivity. Conversely, the relatively large size of the POPC/PS vesicles and the significant polar head groups of themonomers allow for greater water solvation and penetra-tion compared to the synthetic surfactant vesicles. Thus,there is a decrease in the polarity difference betweenmobile phase and pseudostationary phase, resulting inthe observed decline in methylene selectivity.

3.2.6 Shape selectivity

In HPLC, the ability of a stationary phase to separateclosely related compounds or even isomers has beenresearched extensively [19–24], however the same can-not be said for EKC [25]. Vesicles composed of the sin-gle-tailed oppositely charged surfactants DTAB andSDS, have proven to possess enhanced shape selectivitycompared to micellar systems through the separation ofpositional isomers. In addition to the enhanced separa-tion capabilities compared to SDS micelles, the elutionorder of some test compounds was reversed, indicatinga change in the separation mechanism between MEKCand VEKC. In the present study, the shape selectivity ofall four investigated vesicle systems was compared toSDS micelles through the separation of o-, m-, and p-nitrotoluene. Figure 5 shows the separation of o-, m-,and p-nitrotoluene for the SDS micellar and CTAB/SOS,OTAB/SDS, AOT, and POPC/PS vesicle systems.

Figure 5. Separation of o-, m-, p-nitrotoluene via (A) SDSmicelles, (B) CTAB/SOS vesicles, (C) OTAB/SDS vesicles,(D) AOT vesicles, and (E) POPC/PS phospholipid vesicles.

The CTAB/SOS and OTAB/SDS vesicles achieved nearresolution of all three geometrical isomers, whereas onlytwo of the three were resolved with the AOT, POPC/PSand SDS aggregates (Table 5 and Fig. 5). It is no coinci-dence that the vesicles achieving high hydrophobicselectivity also demonstrated enhanced shape selectivity.Vesicles with a tightly packed structure keep water pene-tration to a minimum allowing the solute the greatesthydrophobic environment for partitioning. This highlyhydrophobic environment offers discrimination power inthe free energy required to create a cavity for solutes dif-fering to a very small degree in orientation and shape. Thehydrophobic selectivity of the POPC/PS vesicles as wellas SDS micelles was quite low, indicating a high degree ofwater penetration that resulted in poorer shape selectivityas well. The lesser shape selectivity of the AOT vesicles islikely due to the presence of organic modifier, which notonly decreases the polarity difference between the mobilephase and pseudostationary phase but may also loosenthe AOT vesicles by reducing surfactant-surfactant inter-actions.

Table 5. Shape selectivitya) provided by all aggregates

Modifier Nitrotoluene isomers aShape

SDS o ,, p , m m/p 1.00micelles m/o 1.12

CTAB/SOS p ,, o ,, m m/o 1.08vesicles m/p 1.14

OTAB/SDS p ,, o ,, m m/o 1.05vesicles m/p 1.13

AOT p , o ,, m m/o 1.10vesicles m/p 1.10

POPC/PS p , o ,, m m/o 1.08vesicles m/p 1.08

a) aShape = kj/ki, where j represents the last eluting posi-tional isomer and i, the previous eluting isomer.

In addition to the obvious differences in resolution of o-,m-, and p-nitrotoluene between vesicles and micelles, achange in elution order was also observed. The elutionorder employing SDS micelles was o-, p-/m-nitrotoluene,while for the vesicle systems, elution order was observedto be p-, o-, and m-nitrotoluene. The change in elutionorder is an indication of an additional separation mechan-ism other than hydrophobic interactions, possibly basedon a change in dipolar interactions and/or hydrogenbonding. Comparing the three systems achieving resolu-tion of only two of the three isomers, it was interesting toobserve the difference in coelution. Using SDS micellesas the pseudostationary phase resulted in the coelution



of p- and m-nitrotoluene while the use of AOTand POPC/PS vesicles resulted in the coelution of p- and o-nitroto-luene.

3.3 Linear solvation energy relationships analy-sis

In order to gain a further understanding of the effects ofvesicle composition and structure on retention and selec-tivity, the LSER model was used to quantitatively investi-gate the various solute-solvent interactions involved inthe partitioning process. Previous studies have appliedthe LSER methodology to systems for GC, HPLC, andEKC [26–31]. The solvation equation developed by Abra-ham et al. [32, 33] is as follows:

log k ¼ C þ vV2 þ sp�2 þ aSaH2 þ bSbH

2 þ rR2 (4)

The independent variable, which can generally be a sol-vent or solubility related property of a fixed system, is thelogarithm of retention factor, k, for a set of solutes in thegiven pseudostationary and mobile phase system. There-fore, for the VEKC system, the logarithm of the retentionfactor is divided into several molecular interaction termsfrom the equation above. Terms designated with the sub-script 2 are structural descriptors of the solute thatinclude the solute dipolarity (p*), hydrogen bond acidity(SaH), hydrogen bond basicity (SbH), excess molar refrac-tivity (R, polarizability/10 to bring to scale), the McGowanmolar volume (V, divided by 100 to bring to scale).

Retention data and the solute descriptors are substitutedinto the previous equation and through multiple linearregression the coefficients are calculated. These coeffi-cients are designated C, v, s, a, b, and r. C is a constantthat depends on the phase ratio of the system, vdescribes the cohesiveness of the pseudostationaryphase or the ability of cavity formation of the vesicle forthe solute. The s coefficient measures the dipolarity/po-larizability of the solvent while r is the ability of the solventto interact with solutes’ Z or p electrons (London disper-sion forces). The hydrogen bonding acidity and basicityare measured by the b and a terms, respectively [19, 25].These coefficients relate to the differences in a given char-acteristic of the pseudostationary phase (B) relative to theaqueous buffer (A):

v ¼ Vðd2A � d2

BÞ (5)

b ¼ CðaB � aAÞ (6)

a ¼ DðbB � bAÞ (7)

s ¼ Sðp�B � p�AÞ (8)

r ¼ MðRB � RAÞ (9)

where d is the Hildebrand solubility parameter, a is thehydrogen bond acidity, b is the hydrogen bond basicity,p* is the dipolarity/polarizability, and R is the molar refrac-tivity [32, 33]. The subscripts B and A denote the vesicleand aqueous phases respectively, while V, S, C, and D areproportionality constants. From these coefficients onecan determine the interactions that govern retention andselectivity in the vesicle system under investigation.

A general approach to successfully utilizing LSER forMEKC system was developed by Poole et al. [25]. Thefirst requirement is that a sufficient number of compoundsmust be used in order to have a statistically valid study.The second is that a diverse set of solutes must be usedencompassing a wide range of interactions. In addition, itis also important that there is not a high correlation ofsolute descriptors in the test compound set. The cross-correlation matrix for the compound set in this study iscomparable to previous LSER studies of micellar systems[25, 32–34]. Following these criteria will lead to an LSERanalysis with a high correlation coefficient of linear regres-sion as well as small standard error.

In addition to following the above criteria for successfulLSER analysis, outliers were checked through the com-parison of observed and predicted values of log k. Thepredicted value of k is based on the polynomial equationresulting from the regression where the solute descriptorswere plugged into the equation to solve for the predictedlog k. In the event that outliers were present, all were dis-carded unless they were from the same class of com-pound [25, 35]. In this study, no outliers were rejectedfrom the set of 26 compounds and by following the abovecriteria, regression coefficients were . 0.96 for all sys-tems investigated. The following F-test:

F ¼ r2ðN � n � 1Þð1 � r2Þn (10)

where r2 is the correlation coefficient, n is the number ofvariables and N is the number of data points, was used todetermine if the high correlation coefficient occurred bychance. For all EKC systems, Fobserved was greater thanFcritical (3.29, double-tailed, a = 0.05, df = 20), showingthat statistically there is a relationship between the de-pendent and independent variables and each regressionmodel as a whole is significant. The following t-test wasalso used in order to determine if each slope coefficientwas statistically significant (different from zero) in theoverall model:

t ¼ mn

sen(11)

where mn is the slope variable and sen is the standarderror for that coefficient. In all of the investigated EKC sys-tems, the tobserved. tcritical (2.04, double-tailed, a = 0.05, df



= 20) for each coefficient, thus, the null hypothesis wasrejected, indicating the importance of each variable inthe overall model.

3.3.1 LSER interpretation

Understanding the solute-solvent interactions involved inthe EKC partitioning process, can give insight into theobserved differences in retention and selectivity encoun-tered with the investigated systems. In order to furtherinvestigate the novel vesicle systems, the solvation pa-rameter model was incorporated. Table 6 is a list of allsolutes and solvent descriptors used to determine asolvation model for the systems being studied [25, 28].Table 7 compares the results from LSER analysis using astandard SDS micellar system to the CTAB/SOS, OTAB/SDS, AOT, and POPC/PS vesicles.

3.3.2 Cohesiveness

In all EKC systems, the v coefficient or cohesiveness,was positive and had the greatest magnitude, thus indi-cating the greatest influence on solute retention. Forinterpretation purposes, it is important to understandthat the v coefficient is in fact the difference in cohesive-ness between the mobile phase and stationary phase(v = V(d2

A-d2B)), where di is the solubility parameter of the

phase. Water, being very cohesive, has a high value ofd2

A, while the aggregate structures are relatively muchless cohesive, therefore a positive value of v wasobtained. The differences in overall cohesiveness (v) be-tween the micelles and vesicles as well as each vesiclesystem result from properties of both the pseudostation-ary phases and mobile phases. As will be seen throughthe analysis of the vesicle systems using LSER, one ofthe most important factors involved is the degree ofwater penetration in the aggregate structure [36, 37].The vesicle system composed of the single-tailed op-positely charged surfactants CTAB/SOS exhibited thehighest value of v, 3.37. This indicates that these vesi-cles are the least cohesive, resulting from minimal water

Table 6. Test solutes and solvent descriptors for LSERanalysis [30–32]

Solute V2 p2 Sa2 Sb2 R2

Nitroethane 0.56 0.95 0.20 0.33 0.271-Nitropropane 0.71 0.95 0.00 0.31 0.24Benzyl alcohol 0.92 0.87 0.33 0.56 0.80Aniline 0.82 0.96 0.26 0.50 0.96m-Nitroaniline 0.99 1.71 0.40 0.35 1.20Benzaldehyde 0.87 1.00 0.00 0.39 0.82Phenol 0.78 0.89 0.60 0.31 0.81Benzonitrile 0.87 1.11 0.00 0.33 0.74Acetophenone 1.01 1.01 0.00 0.48 0.82Nitrobenzene 0.89 1.11 0.00 0.28 0.87m-Cresol 0.92 0.88 0.57 0.34 0.82p-Cresol 0.92 0.87 0.57 0.31 0.82Anisole 0.92 0.75 0.00 0.29 0.71Benzene 0.72 0.52 0.00 0.14 0.61Propiophenone 1.15 0.95 0.00 0.51 0.80Methyl benzoate 1.07 0.85 0.00 0.46 0.734-Nitrotoluene 1.03 1.11 0.00 0.28 0.87Toluene 0.86 0.52 0.00 0.14 0.60Butyrophenone 1.30 0.95 0.00 0.51 0.80Chlorobenzene 0.84 0.65 0.00 0.07 0.72Bromobenzene 0.89 0.73 0.00 0.09 0.88p-Xylene 1.00 0.52 0.00 0.16 0.61Benzophenone 1.48 1.50 0.00 0.50 1.45Naphthalene 1.08 0.92 0.00 0.20 1.34Biphenyl 1.32 0.99 0.00 0.26 1.36Butylbenzene 1.28 0.51 0.00 0.20 0.60

penetration due to the tightly packed nature of the aggre-gate. They offered the greatest hydrophobic environmentfor solute partitioning, which in turn resulted in the highretention and hydrophobic selectivity achieved with thesesystems. The long hydrocarbon tails as well as the elec-trostatic attractions of the oppositely charged surfactanthead groups are the main reasons for the “tightly packedstructure” and water excluding properties of the CTAB/SOS vesicles. The OTAB/SDS and AOT vesicles alsodemonstrated a higher v coefficient compared to SDSmicelles. However, differentiation between these twopseudostationary phases (PSPs) was not possible due to

Table 7. Comparison of LSER coefficients

Aggregate v s b a r C r2, n = 26

SDS (1.5% w/v) 2.76 20.45 21.99 20.13 0.40 21.36 0.98CTAB/SOS (1.8% w/v, 3:7) 3.37 20.56 23.66 0.75 0.48 21.58 0.97OTAB/SDS (1.0% w/v, 7:3) 3.01 20.84 22.98 0.65 0.54 21.70 0.97AOT (40 mM, 10% v/v MeOH) 2.85 20.43 23.02 20.13 0.68 21.62 0.98POPC/PS (3 mM, 8:2) 2.68 20.54 22.90 0.02 0.70 22.04 0.97Avg. standard error 0.25 0.18 0.30 0.17 0.22 0.24



the fact that they were deemed statistically similarthrough t-test verification. In contrast to these three vesi-cle systems (CTAB/SOS, OTAB/SDS, and AOT), chargedmicelles, due to their formation from single-tailed mono-mers and electrostatic head group repulsion, provide anenvironment where water molecules can penetrate theaggregate structure. The result is a more cohesive aggre-gate in which a less hydrophobic environment is availablefor solute partitioning, causing the observed decreases inchromatographic parameters such as retention andselectivity.

The lower v coefficient for the AOT vesicles compared tothe CTAB/SOS system is not simply based on the degreeof water penetration in the vesicles, but in fact results fromalteration of the mobile phase as well. The introductionof 10% v/v methanol decreases the cohesiveness of thebulk aqueous phase (d2

A), which also contributes to theobserved decrease in the v coefficient. A subtle secondarycontribution may also be due to organic solvent. As modi-fier is added, the difference in polarity between the mobilephase and pseudostationary phase is lessened, which candecrease the hydrophobic effect responsible for aggre-gate formation. The reduction of the hydrophobic effectmay loosen the vesicle structure, allowing for a greaterdegree of water penetration and an increase in d2

B.

Compared to all studied vesicle systems, the POPC/PSbilayer aggregate had the lowest v coefficient (v = 2.68),statistically similar to the SDS-MEKC. This is indicative ofa higher cohesiveness value (d2

B) as the result of a greaterdegree of water penetration into the aggregate structure.This high level of water penetration can be attributed tothe large overall vesicle size as well as the large polarmonomer head groups associated with both POPC andPS. These large polar head groups along with the longsurfactant chains promote a large radius of curvaturewith an increase in water penetration at the vesicle/waterinterface, thus creating an aggregate with a relatively highdegree of cohesion. It is no coincidence that the POPC/PS vesicles showed decreased retention and hydropho-bic selectivity compared to other bilayer aggregates.

3.3.3 Hydrogen bond acidity

Despite the negative value of the b coefficient, or hydro-gen bond acidity, its large magnitude indicates integralinvolvement in the partitioning process. For each EKCsystem, the overall negative value of b results from thefact that the hydrogen bond acidity of water is muchgreater than that of the surfactant aggregates (aA . aB).In fact, most if not all of hydrogen bond acidity that canbe attributed to the micelles and/or vesicles is the directresult of water penetration into the aggregate structure.

Therefore, due to the high degree of water penetrationassociated with micelles due as a result of the single-tailed monomers and electrostatic polar head grouprepulsion, aB increases, causing an overall decrease inthe negative value of the b coefficient.

Alternatively, all vesicle systems showed decreasedhydrogen bond acidity characteristics compared tomicelles, with the CTAB/SOS vesicle showing the largestdecrease in the b coefficient. While all other investigatedvesicle systems showed a decrease in hydrogen bondacidity compared to SDS-MEKC, differentiation betweenindividual aggregates is problematic as a result of the sta-tistically similarity of each coefficient. As in the case ofcohesiveness, the decrease in water penetration betweenthe micelles and vesicles resulted in the reduction ofpseudostationary phase hydrogen bond acidity charac-teristics. The b coefficient for the AOT vesicles is theresult of the decrease in hydrogen bond donating (HBD)character of the bulk aqueous phase through introductionof methanol as well as the degree of water penetrationaffecting the pseudostationary phases HBD characteris-tics. Once again, the POPC/PS vesicles differed from theother bilayer aggregates by showing the greatest b com-pared to SDS micelles. Similar to the v coefficient, theincreased water penetration into this vesicle comparedto the others investigated resulted in the smaller negativevalue of b.

3.3.4 Hydrogen bond basicity

Although the hydrogen bond basicity coefficient, a, issmaller in magnitude compared to other coefficients, thestatistically significant degree of alteration encounteredfor certain vesicle systems warrants further discussion.For the SDS micelles as well as AOT and POPC/PS vesi-cles, the a coefficient values are statistically equivalent aswell as very close to zero. Conversely, the CTAB/SOS andOTAB/SDS systems exhibited a significant increase intheir hydrogen bond accepting capability. This was notthe result of the mobile phase or surfactant monomers di-rectly, but was in fact due to the surfactant monomercounterions. In both systems, CTAB/SOS and OTAB/SDS, bromide counterions are present, and they aregood hydrogen bond acceptors. This phenomenon hasalso been observed when LSER analysis was used tostudy DTAB micelles in EKC [38].

3.3.5 Dipolarity and molar refractivity

The dipolarity (s) and excess molar refractivity (r) coeffi-cients were small in magnitude for all EKC systems, thusindicating a small influence on solute partitioning. All



values were deemed statistically equivalent when factor-ing in the standard error for this parameter. The only sta-tistically significant increase in molar refractivity coeffi-cient, through t-test verification, (relative to SDS micelles)was observed with the AOT (0.68) and POPC/PS (0.70)vesicles. This behavior can be attributed to the polarhead groups of the surfactant monomers, which all con-tain carbamate moieties. The carbon-oxygen doublebond possesses a high degree of polarizability, i.e., theability of solute p or n electrons to interact with thesepseudostationary phases, thus producing the inflated rcoefficient.

3.3.6 System constant

The system constant, C, is related to the phase ratio of theaggregates, which is dependent on aggregate size, sur-factant concentration and critical aggregation concentra-tion. The system constant was rather consistent in magni-tude for the SDS micelles, CTAB/SOS, and AOT vesicles,if the standard error was taken into account. This is indi-cative of the relatively large phase ratio values for theseaggregates compared to the OTAB/SDS and POPC/PSvesicles. The smaller phase ratios for the OTAB/SDS andPOPC/PS systems resulted in lower values of C (21.70for OTAB/SDS and 22.04 for POPC/PS).

The LSER coefficients in Table 8 can be used to generateequations that predict solute retention with each surfac-tant aggregate (micelle, vesicle, or liposome), providedthat LSER solute descriptors are available for the solutein question. The equations are as follows:

SDS, log k = 21.36 + 2.76V2 2 0.45p*2 2

2 0.13SaH2 2 1.99SbH

2 1 0.06R2 (12)

CTAB/SOS, log k = 21.58 1 3.37V2 2 0.56p*2 1

1 0.75SaH2 2 3.66SbH

2 1 0.48R2 (13)

OTAB/SDS, log k = 21.70 1 3.01V2 2 0.50p*2 1

1 0.65SaH2 2 2.98SbH

2 1 0.54R2 (14)

AOT, log k = 21.62 1 2.85V2 2 0.43p*2 2

2 0.13SaH2 2 3.02SbH

2 1 0.68R2 (15)

POPC/PS, log k = 22.04 1 2.68V2 2 0.54p*2 1

1 0.02SaH2 2 2.90SbH

2 1 0.70R2 (16)

Analysis shows that the v and b coefficients have the lar-gest influence on retention in EKC with the employedaggregates. The especially large and positive v coefficientfor the CTAB/SOS, AOT, and OTAB/SDS systems wouldindicate that large solutes (molar volume) would preferthese vesicles to SDS micelles. Indeed, this was evidentwith the increased retention factors observed for com-pounds such as chlorobenzene, bromobenzene, butyl-

benzene, biphenyl, and naphthalene (data not shown). Incontrast, the large negative b coefficient would indicatethat solutes with hydrogen bond accepting (HBA) capabil-ities would prefer partitioning into the SDS micelles morethan the vesicles. This is the result of the increased waterpenetration associated with the micelle structure. Indeed,the retention factors for HBA solutes decreased for allvesicle pseudostationary phases (data not shown).Finally, compounds with HBD capabilities exhibited thesmallest reduction in retention in going from MEKC toVEKC, especially for the VEKC buffers that incorporatedCTAB/SOS and OTAB/SDS vesicles. In fact, the retentionfactors of m- and p-cresol actually increased slightlywhen CTAB/SOS vesicles were substituted for SDSmicelles (data not shown). This is consistent with LSERanalysis in which the CTAB/SOS and OTAB/SDS vesiclesshowed a significant increase in the a coefficient (hydro-gen bond basicity of the solvent) compared to all othersystems. However, it is important to realize that the overallretention factor of a solute is indeed a function of all LSERparameters discussed.

4 Concluding remarks

Though the electrophoretic and chromatographic perfor-mance of the four vesicles employed as pseudostationaryphases in EKC varied considerably according to the pa-rameter being evaluated, all vesicles provided a largerelution range than SDS micelles. The larger elution rangewas due to the high electrophoretic mobilities of the vesi-cles (high charge density) relative to their size, as well asthe significant decrease in EOF resulting from free cation-ic surfactant monomers in solution (CTAB/SOS, OTAB/SDS) or the effect of 10% methanol (AOT). An increase inretention time was observed for all vesicle systems, con-sequence of the increased elution ranges. However, someinteresting trends were perceived when examining reten-tion as a function of solute hydrogen bonding capability.Nonhydrogen bonding and hydrogen bond-acceptingsolutes demonstrated greater affinity for the vesiclescompared to micelles as a result of lessened water pene-tration for the bilayer aggregates. Compared to all EKCpseudostationary phases studied, the CTAB/SOS andOTAB/SDS vesicles showed the greatest hydrophobicand shape selectivity. This is believed to be the result ofthe lower degree of water penetration for these systemsdue to their tightly packed structure, resulting in a morehydrophobic environment for solute partitioning.

Through LSER analysis, it was determined that the cohe-siveness and hydrogen bond acidity of the PSP have thegreatest effect on solute retention and selectivity in EKC.The bilayer structure of the vesicles, especially those



composed of oppositely charged single-tailed surfac-tants, offers a less cohesive more hydrophobic environ-ment for solute partitioning compared to SDS micelles.This is the result of the reduced degree of water partition-ing in the aggregate structure, which resulted in theobserved overall enhanced retention and selectivity withVEKC. It was also demonstrated that aggregate composi-tion, the bulk aqueous phase, as well as the counterionscould influence the solute/pseudostationary phase parti-tioning process. Overall, it was shown that differencesexist in the partitioning process between VEKC andMEKC, and these differences could be characterizedthrough evaluation of electrophoretic and chromato-graphic parameters as well as LSER analysis.

Received December 15, 2002; in revised form August 4, 2003

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