Recent advances in microemulsion electrokinetic chromatography

Review

Alex Marsh1, 2

Brian Clark2

Margo Broderick3

Joe Power3

Sheila Donegan3

Kevin Altria1

1GlaxoSmithKline R&D, NewFrontiers Science Park South,Harlow, Essex, UK

2School of Pharmacy,Bradford University,Bradford, UK

3Department of Chemical & LifeSciences,Waterford Institute of Technology,Waterford, Ireland

Recent advances in microemulsion electrokineticchromatography

Microemulsion electrokinetic chromatography (MEEKC) is an electrodriven separationtechnique. Separations are typically achieved using oil-in-water microemulsions,which are composed of nanometre-sized droplets of oil suspended in aqueous buffer.The oil droplets are coated in surfactant molecules and the system is stabilised by theaddition of a short-chain alcohol cosurfactant. The novel use of water-in-oil micro-emulsions for MEEKC separations has also been investigated recently. This reportsummarises the different microemulsion types and compositions used to-date andtheir applications with a focus on recent papers (2002–2004). The effects of key oper-ating variables (pH, surfactant, cosurfactant, oil phase, buffer, additives, temperature,organic modifier) and methodology techniques are described.

Keywords: Microemulsion electrokinetic chromatography / Review DOI 10.1002/elps.200406112

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 39702 Comparison with other capillary

electrophoretic modes . . . . . . . . . . . . . . . . . 39713 Method development options and

approaches in MEEKC . . . . . . . . . . . . . . . . . 39723.1 Cosurfactant type and concentration . . . . . 39723.2 Surfactant type and concentration. . . . . . . . 39723.3 Addition of organic solvents . . . . . . . . . . . . . 39733.4 pH of microemulsion . . . . . . . . . . . . . . . . . . . 39733.5 Buffer type and concentration . . . . . . . . . . . 39743.6 Buffer additives . . . . . . . . . . . . . . . . . . . . . . . 39743.7 Sample diluent and injection time . . . . . . . . 39753.8 Temperature. . . . . . . . . . . . . . . . . . . . . . . . . . 39753.9 Oil type and concentration . . . . . . . . . . . . . . 39753.10 Water-in-oil MEEKC . . . . . . . . . . . . . . . . . . . 39753.11 Pressure-assisted MEEKC . . . . . . . . . . . . . . 39763.12 Dual opposite injection CE . . . . . . . . . . . . . . 39763.13 High-speed MEEKC . . . . . . . . . . . . . . . . . . . 39764 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 39764.1 Chiral separations . . . . . . . . . . . . . . . . . . . . . 3978

4.2 Log P determinations . . . . . . . . . . . . . . . . . . 39784.3 Quantitative analysis . . . . . . . . . . . . . . . . . . . 39794.4 Pharmaceutical analysis . . . . . . . . . . . . . . . . 39794.5 Analysis of natural products . . . . . . . . . . . . . 39794.6 Environmental analysis . . . . . . . . . . . . . . . . . 39795 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 39796 References. . . . . . . . . . . . . . . . . . . . . . . . . . . 3980

1 Introduction

Microemulsions are solutions of nanometre-sized drop-lets dispersed throughout another, immiscible, liquid.The dispersions are either oil-in-water or water-in-oil microemulsions. Microemulsions can be used toachieve separations in the capillary electrophoretictechnique of microemulsion electrokinetic capillarychromatography (MEEKC). Oil-in-water microemulsionsare typically employed for MEEKC separations, whichcontain oil droplets (e.g., heptane or octane) suspendedin an aqueous buffer. Surfactant molecules, usuallySDS, are added at a concentration greater than theirCMC, to facilitate droplet formation by lowering the sur-face tension. A small alcohol cosurfactant is also gen-erally added as this bridges the oil-water interface whichlowers surface tension further still and stabilises themicroemulsion system.

Solutes are separated in MEEKC by a combination of theelectrophoretic mechanism and by chromatographic par-titioning with the microemulsion droplets. The partitioningoccurs between the oil droplets and the continuousaqueous phase. Water-insoluble solutes favour inclusion

Correspondence: Dr. Alex Marsh, GlaxoSmithKline R&D, NewFrontiers Science Park South, Harlow, Essex, UKE-mail: [email protected]: 1 44-1279-642330

Abbreviations: ANN, artificial neural network; DDCV, dodecoxy-carbonylvaline; MAPS, 3-(N,N,-dimethylmyristylammonio) pro-panesulfonate; MEEKC, microemulsion electrokinetic chroma-tography; O/W, oil-in water; SDOSS, sodium dioctyl sulfosucci-nate

3970 Electrophoresis 2004, 25, 3970–3980

2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Electrophoresis 2004, 25, 3970–3980 Recent advances in MEEKC 3971

into the oil droplet rather than the buffer phase, whilewater-soluble analytes reside mainly in the aqueousphase. Generally high-pH buffers are used for MEEKC asthese generate a high EOF towards the cathode when thevoltage is applied across the capillary. The SDS-coateddroplets are negatively charged and attempt to migratetowards the anode, but the EOF eventually sweeps themthrough the detector to the cathode. Charged solutesmigrate under the influence of the applied voltage de-pendent upon their size and charge. Solutes interact withthe migrating droplets and the chromatographic interac-tion is related to their hydrophobicity which enables theseparation of neutral species. Indeed, MEEKC has beenused [1–8] to assess compound hydrophobicity withgood cross-correlation to other hydrophobicity measure-ment techniques. Charged solutes may also have acharge repulsion or ion-association with the droplet whichwill also affect their migration times.

This paper briefly compares MEEKC with other capillaryelectrophoretic techniques. It reviews the current methoddevelopment options and approaches that are availablefor MEEKC. An illustrative summary of recent applicationsis also presented.

2 Comparison with other capillaryelectrophoretic modes

Comparisons are often made between MEEKC andmicellar electrokinetic chromatography (MEKC) [9–15] astheir separation basis is similar. In MEKC, surfactantmolecules group to form micelles and solutes chromato-

graphically interact with these micelles which facilitatesseparation [13]. Solutes can penetrate the surface of theMEEKC droplet more easily than the more rigid MEKCmicelle [16], which allows MEEKC to be applied to a widerrange of solutes. MEEKC has often been found to providesuperior separation efficiency to MEKC, probably due toimproved mass transfer between the microemulsiondroplet and aqueous phase, mediated by the cosurfac-tant [16]. MEEKC offers a larger separation window, andthe size of this window can be controlled [16–18], andtherefore potentially offers greater separation capabilityfor water-insoluble compounds than MEKC [19]. Investi-gations have been performed [14] comparing MEEKC tosolvent-modified MEKC, which used an electrolyte con-taining the same buffer, cosurfactant, and amount of SDSas the microemulsion. The two electrolytes were found tobe very similar with respect to separation selectivity andefficiency.

Studies have also been performed which compareMEEKC to other electrophoretic modes [18, 20–22]. Free-solution (FSCE) and nonaqueous CE (NACE), MEKC, andMEEKC separations of a range of methylquinolines havebeen compared [20], while the separation of nicotine andrelated impurities [21] was compared using MEEKC,NACE, and FSCE, with MEEKC offering the better selec-tivity. Figure 1 shows the separation of nicotine and arange of related substances by a MEEKC method. Theseparation of isomers of dienoic acids was compared bycyclodextrin-modified CE, MEEKC, and CEC [22].MEEKC was found [22] to give a superior separation tothe cyclodextrin method, but full resolution of all the iso-mers was only given by CEC.

Figure 1. Separation of nicotineand related impurities byMEEKC. Test-mix dissolved inelectrolyte; 50 mm ID630 cmstandard fused-silica capillary;sample injection, 10 mbar for 5 s;10 kV separation; 260 nm UVdetection; 407C; electrolyte,89% 10 mM tetraborate atpH 9.15 1 0.8% octane 1 3.3%SDS 1 6.6% butanol. Reprintedfrom [21], with permission.


CE

and

CE

C

3972 A. Marsh et al. Electrophoresis 2004, 25, 3970–3980

3 Method development options andapproaches in MEEKC

The MEEKC microemulsion buffer is composed of manyingredients and can also be supplemented with additionalreagents. Variation of these components can affect theseparation selectivity and quality which provides con-siderable method development options for MEEKC whenoptimising complex or difficult separations. The commonvariables encountered in MEEKC and their reportedeffects on the separation are described here. To ensure athorough approach to method development, an experi-mental design can be employed for a full assessmentof the separation-influential factors, as demonstrated re-cently for MEEKC [23]. Using a test-mixture containinganionic, cationic, and neutral drugs, a series of experi-ments were performed where the effect of variation ofsurfactant, cosurfactant and borate buffer concentra-tions, Brij 35 and propan-2-ol addition and temperaturewere measured by the change to the separation window,analyte migration, and plate height. Multiple linearregression models were used to analyse the results andSDS concentration and propan-2-ol addition were foundto have the largest effect on separation selectivity. It hasbeen shown that it is possible to predict MEEKC migra-tion times using an artificial neural network (ANN) [24],demonstrated on a set of 53 benzene derivatives andheterocyclic compounds. The ANN was used to generatea quantitative structure property relationship model be-tween the molecular structural parameters of the benzenederivatives and their observed MEEKC migration indices.Given the structural parameters of unknown molecules,the model was found to predict their migration indiceswith minimum and maximum error of 1.18 and 7.25%,respectively

3.1 Cosurfactant type and concentration

The cosurfactant is the most influential of the microemul-sion constituents on separation selectivity [16, 25–27].Butan-1-ol is the most commonly used cosurfactant inMEEKC. Migration times alter with increased co-surfac-tant concentration [28] because the solution viscosity andEOF rate change and the microemulsion droplet increa-ses in size, reducing its ability to oppose the EOF [29].Increasing the cosurfactant concentration can alterselectivity if the separation contains a mixture of neutraland ionic solutes [28]. However, for mixtures of similarcompounds, it has been found that migration times arealtered but selectivity remains consistent [30]. Increasingthe cosurfactant concentration has been found toimprove the separation [31], and to also increase migra-tion times and peak resolution [14, 18, 32]. A variety of

different alcohol molecules have been employed as thecosurfactant in MEEKC; each of a homologous series ofalcohols from propan-1-ol to hexan-1-ol markedly chan-ged the separation selectivity [33]. A mixture of eightanalytes found in green tea were separated using ninemicroemulsion compositions, each with a differentcosurfactant, resulting in four different separation selec-tivities for the analytes [27]. Branched chain alcohols suchas butan-2-ol do not enable microemulsion formationbecause they cannot bridge the oil-water interface effec-tively [28]. It has been suggested [25] that the cosurfac-tant, because it can partition into the oil droplet, canmodify the chromatographic properties of the micro-emulsion oil phase and therefore may have quite an influ-ence upon its properties.

3.2 Surfactant type and concentration

MEEKC separations are significantly affected by thechoice of microemulsion surfactant, which affects dropletcharge and size, level and direction of EOF, and thedegree of ion-pairing with solutes. SDS, an anionic sur-factant, is usually used in MEEKC at concentrations of3.3% w/w (118 mM). Increasing the SDS concentrationaffects solute migration times depending on analytecharge, and also by reducing the EOF level [9, 18, 23, 28,34]. If the solute ion-pairs with the droplet, increasingsurfactant concentration increases the droplet chargedensity and increases analyte migration and peak resolu-tion [18, 31, 32]. High surfactant concentration has alsobeen shown to produce more stable and reproduciblesystems [35]. Selectivity can be greatly altered by repla-cing SDS partly or fully with other surfactants [36]. Theuse of various anionic bile salts in place of SDS has beenshown to offer a different selectivity [37], specifically thebile salt sodium cholate was reported to offer a betterselectivity for lipophilic solutes [31]. For log P determina-tions by MEEKC, it has been reported [2] that using bio-surfactants, e.g., phosphatidylcholine gave better logPOW estimations than SDS microemulsion for a series ofsynthetic steroids. Selecting a different salt of the surfac-tant, for example LiDS instead of SDS, has been shown[28] to increase EOF and reduce operating current so thathigher voltage a faster separation can be achieved with-out generation of excessive current.

Cationic surfactants [28, 38] have been used to eliminateion-pair reactions which occur between cations (such asbasic drugs) and SDS-coated droplets. Use of a cationicsurfactant reverses the EOF direction and requires theuse of negative polarity voltage. Neutral surfactants canbe added to the microemulsion without increasing theoperating current but cannot separate neutral solutes.



They have been used to separate insoluble sun-tan lotionadditives [39], a range of methylquinolines [20], and toprovide optimum conditions for a MEEKC-dual oppositeinjection separation [40]. Mixed surfactant systems,where two different types of surfactant are used to pro-vide the optimum separation selectivity, have beenreported [6, 26, 41]. A mixed surfactant microemulsionsystem containing 0.75% w/w Brij 35 and 2.25% w/wSDS was used to separate UV filters in sunscreen lotions[41]. Mixed systems of SDS and sodium dioctyl sulfo-succinate (SDOSS), 3-(N,N-dimethylmyristylammonium)propanesulfonate; (MAPS), Tween 21 or Brij 35 gave dif-ferent separation selectivity for a mixture of neutrals [26];the four separations are shown in Fig. 2.

3.3 Addition of organic solvents

In MEKC, water-insoluble solutes partition strongly intothe micelles and are highly retained and poorly resolved.This problem can be overcome by adding organic solvent,typically acetonitrile, methanol or isopropanol, at levels ofup to 30%, to the buffer. This approach can also be takenwith MEEKC, but the amount of solvent which can beadded before the microemulsion is disrupted varies. Forinstance propan-2-ol has been used at concentrations

greater than 50% [28] but methanol caused microemul-sion demixing above 8% v/v. The addition of organic sol-vents can alter the degree of ionisation of solutes, whichaffects their electrophoretic mobility [25]. The addition ofMeOH (up to 8%) and MeCN (up to 12%) change theelectrolyte viscosity and slow the EOF, increasing themigration time [28]. This effect is greater upon insolublecompounds, and benefits their separation [6]. Propan-2-olacts as a second cosurfactant and can change separationselectivity and increase migration time [12, 23, 28, 41]. Theaddition of 30% propanol was used to achieve resolutionfor two closely migrating priority endocrine disruptingcompounds by increasing the migration time window [35].The addition of 5% MeOH, MeCN, isopropyl alcohol (IPA)or THF was found to improve the separation of isoquino-line alkaloids from herbal medicine by increasing migra-tion times and peak resolution [31].

3.4 pH of microemulsion

pH is a major factor for electrophoretic separations be-cause it affects both the EOF and the ionisation of thesolutes. A number of reports have investigated the effectof pH on MEEKC separations [10, 28, 33]. Typically a pH

Figure 2. Separation of seven neutral solutes analysed by MEEKC using four different surfactants.Test-mix of FA (formic acid) 1, benzamide; 2, nicotinic acid; 7, p-chlorobenzamide; 8, prednisone; 13,ethyl-3-nitrobenzoate; 14, b-methasone; 15, nicotinic acid butyl ester dissolved in 50/50 MeOH/H2Oat 0.2 mg?mL21. Separation conditions: sample injection, 2 s at 150 mbar; 20 kV; 48.5 cm650 mm IDfused-silica capillary; electrolyte, 0.8% w/w octane, 3.3% w/w surfactant, 6.6% w/w 1-butanol,89.3% w/w 10 mM borate in water pH 9.2; surfactant: (A) SDS, (B) SDS+SDOSS (50/50 w/w), (C) SDS1 Tween 21 (50/50 w/w), (D) SDS 1 MAPS (50/50 w/w); Reprinted from [26], with permission.



of 7–9 is used in MEEKC, giving a strong EOF and ensuringthat most ionisable compounds are ionised. Migrationtimes have been found to decrease with increases in elec-trolyte pH [31, 36]. Extremes of pH have been used for ionsuppression during hydrophobicity analysis where thesolute needs to be in its uncharged state [3, 4]. Very high pH(pH 12) buffers have been used [3] for basic compoundsand very low pH (pH 1.2–1.4) for acidic solutes. At low pH[3] there is no EOF, so a negative voltage needs to beapplied to attract the droplets toward the detector. In con-trast to a typical MEEKC separation performed at high pH,this results in the most retainedcompounds eluting first [28,42]. The separation of priority endocrine disrupting com-pounds in wastewater was performed by MEEKC at pH 2[35]. A low pH MEEKC buffer has also been employed toseparate a range of parabens from parahydroxybenzoicacid [42], a range of insoluble vitamins [43], a range ofpharmaceuticals [44], and green tea catechins [27]. Theseparation of a parabens (parahydroxybenzoate pre-servatives) test-mixture at both low and high pH is shown inFig. 3. Altering the pH changed the ionisation of the para-bens and provided a different selectivity.

3.5 Buffer type and concentration

Typically, low-ionic-strength (5–10 mM) borate or phos-phate buffers are used as the microemulsion aqueousphase, giving a swift EOF while generating low currents.

Increasing the buffer concentration of the electrolyte hasbeen shown to improve peak resolution [20, 45]. Using alow concentration of borate buffer in the microemulsiongave a faster separation because of the faster EOF at lowionic strengths, but the precision of subsequent injectionswas lower due to electrolysis effects [28]. The use of abuffer containing both positive and negative charges,such as Tris, can reduce the amount of current producedduring separation, enabling higher voltages to be appliedto give faster separations [46].

3.6 Buffer additives

Reagents can be added to the electrolyte to improveseparations. Urea is added in MEKC to aid the analysis ofinsoluble compounds. When added to MEEKC buffer, themigration time window was observed to expand, similarto the addition of organic solvents, which allows the re-solution of hydrophobic compounds [28]. Cyclodextrinscan be added to the buffer which offers additional solutesolubilisation and partitioning possibilities. The additionof 25 mM b-cyclodextrin to the microemulsion was foundto alter the separation selectivity, reducing the migrationtimes of insoluble compounds [28], while the addition of5 mM sulphated b-cyclodextrin enabled the resolution ofnine xanthones [15]. The incorporation of the ion-pairingreagent sodium octanesulphonate was found to increasethe migration times of solutes [28].

Figure 3. Separation of a paraben test-mixture using low (pH 2.1) and high (pH 9.5) pH MEEKC. Test-mix of Ac, 4-hydroxybenzoic acid; MP, methyl paraben; EP, ethyl paraben; PP, Propyl paraben; BP,butyl paraben; ISS, internal standard dissolved in electrolyte at 5 mg?mL21. Separation conditions:sample injection, 3 s at 120 mbar; 11 kV; 33 cm650 mm ID fused-silica capillary; 407C, 200 nm UV;electrolyte, 3.3% w/w SDS, 6.6% w/w 1-butanol, 0.8% w/w octane, 89.3% w/w aqueous buffer;buffer: (A) 50 mM phosphate buffer, pH 2.1, (B) 10 mM borate buffer, pH 9.2. Reprinted from [42], withpermission.



3.7 Sample diluent and injection time

It is regarded [6, 9, 12, 19, 31, 33, 47] as best practice todissolve the sample in the microemulsion electrolyte,which has a good solubilising power, for a good MEEKCseparation. Alternative sample diluents can disrupt themicroemulsion buffer inside the capillary, causing a poorquality separation. If the sample is not directly solubilisedin the required microemulsion then it can be dissolved inan alternative solvent and then diluted with microemul-sion prior to injection. The technique of “stacking”, whereimproved separation efficiencies and increased detectorresponse are obtained using a sample diluent of lesserionic strength than the microemulsion has been shown tobe effective when used in MEEKC [28, 48]. Sample injec-tion time has been reported to have an important effect onthe separation efficiency of lipophilic compounds [12, 19],with longer injection times resulting in poor peak shapeand resolution.

3.8 Temperature

The temperature of a MEEKC separation affects the hy-drophobicity of analytes and hence their partitioning withthe droplets. The electrophoretic mobility of an ion increa-ses by 2% for each 7C, and the selectivity of mixtures ofdifferent solutes can alter because the temperature affectsneutral and charged species disproportionately [25, 10].Increasing the temperature has been found to reducemigration times [6, 10, 18, 31, 36] because the EOF increa-ses due to the lower buffer viscosity.

3.9 Oil type and concentration

Generally, octane is used as the microemulsion oil. Al-though hexane, heptane and octane have been shown togive similar selectivity and migration times [26, 53],

octane has been reported to give more repeatablemicroemulsions and superior peak resolution, efficiency,and precision [12, 35]. A range of other oils have beenreported [12, 28, 36, 38, 49–51] including pentan-1-ol,hexan-1-ol, octan-1-ol, cyclohexane, chloroform, meth-ylene chloride, amyl alcohol, and butyl chloride. Addi-tionally the use of chiral oils [52] and low interfacialtension oils such as ethyl acetate [53, 54] have been used.Variation of the oil concentration within the range thatallows stable microemulsion formation has been reported[14, 18, 32] not to significantly change the separation.

3.10 Water-in-oil MEEKC

Water-in-oil microemulsions (W/O) have interesting po-tential for the separation of water insoluble compounds,and their use has been demonstrated successfully [55].Recently, the factors affecting separation by water-in-oilMEEKC have been investigated [56] and a range of neutraland acidic analytes separated (Fig. 4). The W/O micro-emulsion reported by Broderick and co-workers [56] con-sisted of 10% w/w SDS, 78% butanol, 2% octane, and10% 0.07 mM sodium acetate aqueous buffer. The advan-tage of W/O MEEKC is its ability to solubilise very water-insoluble analytes due to the oil continuous phase. Withaqueous electrolytes, sample extraction and preparationsteps are normally needed to remove the oil-soluble exci-pient materials from cream and ointments to preventinterference and precipitation. For O/W MEEKC samplepreparation, these steps are eliminated [55]. In W/OMEEKC neutral solutes do not separate in order of theirhydrophobicity, which offers a unique selectivity com-pared to O/W MEEKC, especially for the analysis of highlywater-insoluble neutral solutes. W/O MEEKC generates alow separation current, so high buffer concentrations areneeded in W/O MEEKC to generate sufficient operatingcurrent for stable and efficient resolutions to be achieved.

Figure 4. Separation of test-mixture using W/O MEEKC. Test-mix of thiourea, caffeine, napthalene,4-hydroxyacetophenone, and sorbic acid dissolved in electrolyte at 1 mg?mL21. Separation condi-tions: sample injection, 1 s at 150 mbar; electrolyte, 20% w/w SDS, 48% w/w butanol, 32% w/w0.07 M sodium acetate in water; 230 kV; 24.5 cm650 mm ID fused-silica capillary; 257C; 200 nm UVdetection. Reprinted from [56], with permission.



3.11 Pressure-assisted MEEKC

Throughout the duration of a MEEKC separation it ispossible to apply air pressure as well as voltage to thecapillary. This technique works by gently forcing the cap-illary contents towards the detector and is useful to speedup separations which may be slow due to the presence oflate-migrating very hydrophobic solutes. The use ofpressure assistance of 5 mbar during a 20 kV MEEKCseparation [57] reduced the migration time of dodeca-noacetophenone from 40 min to 25 min. Pressure assis-tance of up to 10 mbar was found not to affect theseparation, but further increases resulted in narrowing ofthe separation window [57]. Pressure-assisted MEEKChas been applied to log P determinations [57, 58] toreduce analysis time.

3.12 Dual opposite injection CE

Dual opposite injection is a sampling technique [40] wherecations and anions are injected and migrate from oppositeends of the capillary toward the detector. The detectionwindow can be situated in the middle of the capillary,which eliminates the analysis time between cationic andanionic compounds, offering the possibility of greater re-solutionwithout increasing run times. A MEEKC electrolytecontaining 10 mM zinc cations was used to suppress theEOF. A neutral surfactant (Brij 35) was used to achievechromatographic partitioning of solutes as they migratedalong the capillary towards the detector.

3.13 High-speed MEEKC

MEEKC analysis times are typically in the order of10 min. High-ionic-strength buffers are used for MEEKCseparation, which limits the voltage that can be appliedand in turn limiting the speed of analysis that couldpotentially be achieved. To form a stable microemul-sion, high concentrations of surfactant are required, butit has been reported [46] that by using a low surfacetension oil, the amount of surfactant can be reduced.By using Tris buffer, high temperature, high voltage, and“short end” capillary injection, analysis times werereduced to 1 min.

4 Applications

The number of publications involving MEEKC hasgrown considerably recently, due to method develop-ment and optimization of the technique and furtherdevelopment of new methodologies. Table 1 contains arange of applications reported in the literature over thepast two years (2003–2004) and the composition of themicroemulsion used. A brief overview of each applica-tion area (chiral separations, log P determinations,pharmaceuticals, natural products, and environmentalanalysis) is given. Honore Hansen [59] has published areview of recent applications from previous years(1996–2002).

Table 1. Range of quantitative results from MEEKC methods

Analysis Sample (label claim) Experimental results

Content of ephedrine andpseudoephedrine in tablets [63]

EphedrinePseudoephedrine

0.83 mg per tablet0.35 mg per tablet

Biphenyl nitrate purity ofindustrial samples [6]

Compound 1Compound 2Compound 4

10091.2% purity99.2

Sudafed expectorant content [9] Guaiphesin (20 mg?mL21) 20.95 mg?mL21

Pseudoephedrine (6 mg?mL21) 5.86 mg?mL21

Methyl paraben (0.1% w/v) 0.096% w/vPropyl paraben (0.01% w/v) 0.01% w/v

Troglitazone tablet assay [19] 200 mg per tablet 199.4 mg per tablet

Caffeine content mg?g21 of greentea samples [27]

Green Chinese teaGreen Indian teaOrganic green Indian tea

16.218.36 mg?g21

21.72

Eusolex UV filter content mg?g21 insunscreens [41]

Sun spraySun milkSun oil

3116 mg?g21

13



Table 2. Range of recent MEEKC buffer compositions and applications (2003–2004)

Application Microemulsion composition Ref.

Determination of nine preservatives inpharmaceutical and cosmetic products

3.3% w/w SDS, 0.81% w/w octane, 6.6% w/w butan-1-ol,89.3% w/w 50 mM phosphoric acid

[10]

Analysis of b-methasone and derivatives 1.44% w/w SDS, 0.81% w/w octane, 6.61% w/w butan-1-ol,91.14% 20 mM sodium phosphate, pH 7.5

[2]

1.9% w/w isopropylmyristate, 2.0% w/w SC/SDC, 3.5% w/w PC,0.81% w/w octane, 7.5% w/w butan-1-ol, 85.1% 20 mM sodiumphosphate, pH 7.5

Levetiracetam from otherantiepileptic drugs

1.8% w/w SDS, 0.48% w/w n-octane, 3.96% w/w butan-1-ol,93.76% w/w 10 mM Borate buffer, pH 9.2.

[33]

Separation of methylquinolines 1.66 g SDS/Brij35, 0.41 g n-alkane, 3.31 g butan-1-ol,44.6 g borate buffer, pH 9.4

[20]

Separation of isoflavonoids 3–3.5% w/w SDS, 0.2–1% w/w octane, 6–8.6% w/w butan-1-ol,10 mM sodium tetraborate, pH 9.5

[32]

Separation of isomers of dienoic acids 3.31% w/w SDS, 0.81% w/w octane, 6.61% w/w butan-1-ol,89.27% w/w 10 mM sodium tetraborate

[22]

Nicotine-related alkaloids 3.3% w/w SDS, 0.8% w/w octane, 6.6% w/w butan-1-ol,89.29% w/w 10 mM, sodium tetraborate, pH 9.15

[21]

UV filters in suncreen lotions 2.25 g SDS/0.75 g Brij35, 0.8 g n-alkane, 6.6 g1-butanol,17.5 g 2-propanol, 72.1 g 10 mM borate buffer, pH 9.2

[41]

Separation of cations and anions bynonionic microemulsion

0.6% w/w Brij35, 0.5% w/w ethyl acetate, 1.2% w/w butan-1-ol,50 mM ACES buffer, pH 6.5

[40]

Separation of neutral and acidiccompounds and a range of aromaticacids by W/O MEEKC

10% w/w SDS, 2% w/w octane, 78% w/w butan-1-ol,10% w/w 70 mM, ammonium acetate

[56]

Analysis of catechins in green teaproducts

2.31–3.32% w/v SDS, 1.36% w/v n-heptane,7.38–9.72% w/v cosurfactant, 86.3%-94.12% w/v50 mM phosphate buffer, pH 2.5.

[27]

Priority endocrine disruptingcompounds in wastewater

200 mM SDS, 80 mM octane, 900 mM butan-1-ol,20% v/v propanol, 25 mM phosphate buffer, pH 2

[35]

Analysis of catechin and galocatechinin extracts of cistus species

2.31% w/v SDS, 1.36% w/v heptane, 9.72% w/v butan-1-ol,86.61% 50 mM sodium phosphate buffer, pH 2.5

[64]

Withanolides in plant extracts 100 mM SDS, 70 mM octane, 800 mM butan-1-ol,10 mM phosphate buffer, pH 7

[34]

Separation of pharmacologically activexanthones from Securidaca inappendiculata

120 mM SDS, 80 mM heptane, 10% v/v n-butanol, 50 mM

borate buffer, pH 9.5, 5 mM sulphated b-cyclodextrin[15]

Analysis of 14 chiral compounds 1.0% w/v DDCV, 0.5% v/v ethyl acetate,1.2% v/v butan-1-ol, ACES 50 mM pH 7

[53]

Analysis of chiral compounds bychiral cyclodextrin-modified MEEKC

1.0% w/v DDCV, 0.5% v/v ethyl acetate, 1.2% v/v butan-1-ol,ACES 50 mM pH 711.0% s-b-CD, phosphate 50 mM pH7.011.0%, HS-b-CD

[60]

0.6% w/v SDS, 0.5% v/v ethyl acetate, 1.2% v/v butan-1-ol,Tris, 100 mm, pH 8.011.0% s-b-CD, phosphate 100 mM,pH 811.0% HS-b-CD

Log P screening of weakly basic, weaklyacidic, and neutral pharmaceuticals

50 mM SDS, 82 mM n-heptane, 0.87 M butan-1-ol,50 mM borate-phosphate buffer, pH 10

[57]



Table 2. Continued

Application Microemulsion composition Ref

Multiplexed MEEKC determination of logP values for neutral and basic compounds

3.3% w/v SDS, 0.8% w/v n-heptane, 6.6% w/v butan-1-ol,92% 68 mM 3-(cyclohexylamino)-1-propanesulfonic acid pH 10.3

[1]

Log P of carbonate esters and small organicmolecules

1.80% w/w SDS, 0.82% w/w n-heptane, 6.49% w/wbutan-1-ol, 0.1 M borate-0.05 M phosphate buffer, pH 7.4

[4]

1.44–2.88% w/w SDS, 0.82% w/w n-heptane,6.49% w/w butan-1-ol, 0.05 M acetate buffer, pH 4.75

2.16% w/w SDS, 0.82% w/w n-heptane,6.49% w/w butan-1-ol, 0.05 M HCl, pH 1.4

Separation of immunosuppressive drugs 1.44% w/w SDS, 0.81% w/w octane, 6.61% w/w butan-1-ol,91.14% 20 mM sodium phosphate, pH 7.5

[5]

2.0% w/w SDC, 3.5% w/w PC, 1.9% w/w IPM, 0.81% w/w octane,7.5% w/w butan-1-ol, 85.1% 20 mM sodium phosphate, pH 7.5

2.0% w/w SC, 3.5% w/w PC, 1.9% w/w IPM, 0.81% w/w octane,7.5% w/w butan-1-ol, 85.1% 20 mM sodium phosphate, pH 7.5

Six biphenyl nitrile compounds and threerelated substances

100 mM SDS, 80 mM SC, 0.81% v/v heptane, 7.5% v/v butan-1-ol,10% v/v acetonitrile, 10 mM borate

[6]

Log P estimation of neutral and weaklyacidic by MEEKC dynamically coatedcapillary columns

1.4% w/v SDS, 1.2% v/v n-heptane, 8% v/v butan-1-ol, 85% w/w50 mM sodium phosphate, pH 3

[61]

Analysis of ephedrine and pseudoephedrine 23.3 mM SDS, 16.4 mM n-heptane,180.85 mM butan-1-ol, 8% acetonitrile, 20 mM borate

[63]

Amino acid derivatives using LIF detection 2.12% w/w SDS, 0.52% w/w heptane,4.21% w/w butanol, 84 mM borate, pH 8.4

[45]

MEEKC using ANNs 1.44% w/w SDS, 0.82% w/w n-heptane, 6.49% w/w butan-1-ol,0.1 M borate-0.05 M phosphate buffer, pH 7

[24]

Separation of neutral cationic and anionicanalytes; performance evaluated bymultiple linear regression models

2–3.5% w/w SDS, 0–2.5% w/w Brij35, 5–9% w/w butan-1-ol,0–20% w/w propan-2-ol, 0–50 mM borate buffer, pH 9.2

[23]

Abbreviations: SC, sodium cholate; SDC, sodium deoxycholate; PC, phosphatidylcholine; ACES, aminoethanesulfonicacid; s-b-CD, sulphated b-cyclodextrin; HS-b-CD, highly sulphated b-cyclodextrin; IPM, isopropyl myristate

4.1 Chiral separations

Capillary electrophoresis is an important technique forenantiomer separation, and successful analyses havebeen carried out by MEEKC. To achieve chiral resolutionof a range of basic drugs, a chirally selective surfactantdodecoxycarbonylvaline (DDCV) at a concentration of1% w/v in combination with low interfacial tension oils(methyl acetate, ethyl acetate, methyl propionate,methyl formate) has been used [53, 54]. CombiningDDCV with ethyl acetate as the oil was found to have thegreatest enantioselectivity with subtle changes in selec-tivity resulting in changing the oil type. A chiral oil, (2R,3R)-di-n-butyl tartrate at a concentration of 0.5% w/w,has been successful for racemic ephedrine separation

[52]. Chiral resolution has been achieved of racemiclevetiracetam by cyclodextrin-modified microemulsions[60].

4.2 Log P determinations

The oil-water partitioning process by which solutes areseparated in MEEKC has enabled its use [1–8, 57, 61] forcompound hydrophobicity assessment with goodcross-correlation to other hydrophobicity measurementtechniques. The MEEKC method of octanol-water parti-tion coefficient determination has recently been demon-strated for a series of synthetic steroids [2], small organicmolecules [4], immunosuppressive drugs [5], biphenyl



nitrile compounds used in the synthesis of liquid crystals[6], pesticides [7], and for weakly acidic and neutralcompounds using a dynamic capillary coating [61]. Theuse of MEEKC for log P determinations has also beendemonstrated with a 96-capillary array instrument forthe high-throughput screening of compound hydropho-bicity [1], and by pressure-assisted MEEKC [57]. Basedon the same partitioning principle used to determine logPOW of compounds, the interaction between drug mole-cules and vehicle systems has been characterised usingMEEKC [8].

4.3 Quantitative analysis

The capability to perform quantitative determinations isa very important factor if an analytical method is to beused routinely. MEEKC methods have been used toperform quantitative determinations including UV filtersin sunscreen lotions [41], preservatives in pharmaceu-tical and cosmetic products [10], catechins in green tea[27], pharmaceuticals [9, 19, 51, 62] and biphenyl nitrilecompounds [6]. Using MEEKC coupled with laser-induced fluorescence detection offers more sensitivityfor quantitative methods of determining amino acidderivatives [45] and ephedrine and pseudoephedrine[63]. Table 1 shows some quantitative results obtainedfrom various MEEKC methods.

4.4 Pharmaceutical analysis

O/W MEEKC has been used for many pharmaceuticalapplications over the past two years. Huang et al. [10] hasemployed this technique in the separation of nine pre-servatives in various pharmaceutical and cosmetic prod-ucts and compared results obtained by both MEEKC andMEKC methods. Both provided a successful separationbut the MEKC analysis took 9 min compared to the 16 minMEEKC separation, due to the higher concentration ofSDS in the electrolyte. Lucangioli et al. [2] also comparedmethods with different pseudostationary phases formicroemulsion and MEKC of b-methasone and deriva-tives, and found that using biosurfactants to enhancebiopartitioning of the drugs gave a better model to esti-mate the hydrophobicity of the b-methasone series.MEEKC was found to be given superior selectivities whencompared to use of 60 mM teteraborate buffer containingb-cyclodextrin for the separation of four geometrical iso-mers of decadienoic acid [22]. MEEKC also gave a betterseparation selectivity over other capillary electrophoretictechniques in the separation of nicotine and nicotinerelated alkaloids [21].

Polar pharmaceutical compounds have been analysed inbasic microemulsion medium [32] and the separationfound to be sensitive to surfactant and cosurfactant con-centration. The method was successful for a variety ofpharmaceutical separations including a mixture of 13diuretics and 5 benzodiazepines. Levetiracetam has beenseparated [33] from 5 other anti-epileptic drugs withwhich it can be co-administered and the separationselectivity was found to be dependent on electrolytecomposition, pH and cosurfactant type. Laser-inducedfluorescence detection has been employed with MEEKCto improve sensitivity and quantify ephedrine and pseu-doephedrine [63] and amino acids [45] which had beenderivatised with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazolto make them fluoresce.

4.5 Analysis of natural products

MEEKC is an effective technique with a high separationpower for complex natural products. This has beendemonstrated in the determination of catechin and gallo-catechin in lyophilized extracts obtained from Cistusspecies [64], the separation of catechins, caffeine, andtheophylline [27]. Manipulation of surfactant, cosurfac-tant, and oil were found to offer selectivity changes in theanalysis of complex natural samples [27].

4.6 Environmental analysis

Analysis was carried out for determination of six priorityendocrine disrupting compounds in environmental sam-ples in industrial and domestic wastewater treatmenteffluents and sludges. An optimized method separatedthe six compounds in 15 min using a microemulsion of25 mM phosphate buffer pH 2, 80 mM octane, 900 mM

butanol, 200 mM sodium dodecyl sulphate, and 20%propanol, which suppressed the EOF allowing rapidanalysis of alkyl phenols [35].

5 Conclusions

The number of publications relating to MEEKC continuesto rise as the diversity of method development optionsexpands with new possibilities of generating uniqueselectivities. W/O MEEKC has been reported which isespecially useful for analysis of highly water insolublesolutes. Chiral separations have been achieved usingMEEKC through a variety of different enantioselectivemechanisms. The number of applications has continuedto expand with a particular emphasis on log P determi-nations and analysis of pharmaceuticals.

Received June 15, 2004



6 References

[1] Wong, K. S., Kenseth, J., Strasburg, R., J. Pharm. Sci 2004,93, 916–931.

[2] Lucangioli, S. E., Carducci, C. N., Scioscia, S. L., Carlucci,A., Bregni, C., Kenndler, E., Electrophoresis 2003, 24, 984–991.

[3] Gluck, S. J., Benko, M. H., Hallberg, R. K., Steele, K. P.,J. Chromatogr. A 1996, 744, 141–146.

[4] Ostergaard, J., Hansen, S. H., Larsen, C., Schou, C., Hee-gaard, N. H. H., Electrophoresis 2003, 24, 1038–1046.

[5] Lucangioli, S. E., Carducci, C. N., Scioscia, S. L., Carlucci,A., Kenndler, E., Tripodi, V. P., J. Pharm. Biomed. Anal. 2003,33, 871–878.

[6] Gong, S., Bo, T., Huang, L., Li, K. A., Liu, H., Electrophoresis2004, 25, 1058–1064.

[7] Klotz, W. L., Schure, M. R., Foley, J. P., J. Chromatogr. A2001, 930, 145–154.

[8] Reinhard, N., Mrestani, Y., Biopharm. Drug Dispos. 2001, 22,265–271.

[9] Miola, M. F., Snowden, M. J., Altria, K. D., J. Pharm. Biomed.Anal. 1998, 18, 785–797.

[10] Huang, H., Lai, Y. C., Chiu, C. W., Yeh, J. M., J. Chromatogr.A 2003, 993, 153–164.

[11] Nishi, H., J. Chromatogr. A 1997, 780, 243–264.[12] Sanchez, J. M., Salvado, V., J. Chromatogr. A 2002, 980,

241–247.[13] Ishihama, Y., Okada, Y., J. Chromatogr. 1992, 608, 23–29.[14] Hansen, S. H., Gabel-Jensen, C., Pedersen-Bjergaard, S.,

J. Sep. Sci. 2001, 24, 643–650.[15] Bo, T., Yang, X., Li, K. A., Xu, L., Liu, H., J. Sep. Sci. 2003, 26,

133–136.[16] Hansen, S. H., Gabel-Jensen, C., Tawfik Mohamed El-

Sherbiny, D., Trends Anal. Chem. 2001, 20, 614–619.[17] Pyell, U., J. Chromatogr. A 2004, 1037, 479–490.[18] Cherkaoui, S., Veuthey, J. L., J. Sep. Sci. 2002, 25, 1073–

1078.[19] Altria, K. D., J. Chromatogr. A 1999, 844, 371–386.[20] Schoftner, R., Buchberger, W., J. Sep. Sci. 2003, 26, 1247–

1252.[21] Marsh, A., Altria, K. D., Clark, B. J., Electrophoresis 2004,

259, 1270–1278.[22] Cigic, I. K., Gucek, M., Zupancic-Klali, L., Pihlar, B., J. Sep.

Sci. 2003, 26, 1253–1258.[23] Harang, V., Eriksson, J., Sänger-van-Griend, C. E., Jacobs-

son, S. P., Westerlund, D., Electrophoresis 2004, 25, 80–93.[24] Fatemi, M. H., J. Chromatogr. A 2003, 1002, 221–229.[25] Klampfl, C. W., Electrophoresis 2003, 24, 1537–1543.[26] Gabel-Jensen, C., Hansen, S. H., Pedersen-Bjergaard, S.,

Electrophoresis 2001, 22, 1330–1336.[27] Pompiono, R., Gotti, R., Luppi, B., Cavrini, V., Electropho-

resis 2003, 24, 1658–1667.[28] Altria, K. D., Clark, B. J., Mahuzier, P. E., Chromatographia

2000, 52, 758–768.[29] Hoffman, H., Phys. Chem 1996, 100, 1109–1117.[30] Szucs, R., Van Hove, E., Sandra, P., J. High Resolut. Chro-

matogr. 1996, 19, 189–192.[31] Lo, Y., Bo, T., Li, M., Gong, S., Li, K., Lui, H., J. Liq. Chro-

matogr. Rel. Technol. 2003, 26, 1719–1730.

[32] Siren, H., Kartlunen, A., J. Chromatogr. B 2003, 783, 113–124.

[33] Ivanova, M., Piunti, A., Marziali, E., Komarova, N., Raggi, M.A., Kenndler, E., Electrophoresis 2003, 24, 992–998.

[34] Cherkaoui, S., Veuthey, J. L., Cahours, X., Electrophoresis2002, 23, 336–342.

[35] Fogarty, B., Dempsey, E., Regan, F., J. Chromatogr. A 2003,1014, 129–139.

[36] Pedersen-Bjergaard, S., Gabel-Jensen, C., Hansen, S. H.,J. Chromatogr. A 2000, 897, 375–381.

[37] Miksik, I., Deyl, Z., J. Chromatogr. A 1998, 807, 111–119.[38] Boso, R. L., Bellini, M. S., Miksik, I., Deyl, Z., J. Chromatogr.

A 1995, 709, 11–19.[39] Klampfl, C. W., Leither, T., Hilder, E. F., Electrophoresis 2002,

23, 2424–2429.[40] Zhou, M. X., Foley, J. P., Electrophoresis 2004, 25, 653–663.[41] Klampfl, C. W., Leitner, T., J. Sep. Sci. 2003, 26, 1259–1262.[42] Mahuzier, P. E., Altria, K. D., Clark, B. J., J. Chromatogr. A

2001, 924, 465–470.[43] Pedersen-Bjergaard, S., Naess, O., Moestue, S., J. Chro-

matogr. A 2000, 876, 201–211.[44] Pedersen-Bjergaard, S., Chromatographia 2000, 52, 593–

598.[45] Jianping, X., Jiyou, Z., Huanxiang, L., Jiaqin, L., Jianniao, T.,

Xingguo, C., Zhide, H., Biomed. Chromatogr. 2004, in press.[46] Mahuzier, P. E., Altria, K. D., Clark, B. J., Bryant, S. M.,

Electrophoresis 2001, 22, 3819–3823.[47] Altria, K. D., J. Chromatogr. A 1999, 844, 371–386.[48] Quirino, J. P., Terabe, S., Otsuka, K., Vincent, B., J. Chro-

matogr. A 1999, 838, 3–10.[49] Fu, X., Lu, J., Zhu, A., J. Chromatogr. A 1996, 735, 353–356.[50] Miksik, I., Gabriel, J., Deyl, Z., J. Chromatogr. A 1997, 772,

297–303.[51] Vomostova, L., Miksik, I., Deyl, Z., J. Chromatogr. B 1996,

681, 107–113.[52] Aiken, J. H., Huie, C. W., Chromatographia 1993, 35, 448–

450.[53] Mertzman, M. D., Foley, J. P., Electrophoresis 2004, 25, 723–

732.[54] Pascoe, R., Foley, J., Analyst 2002, 127, 710–714.[55] Fung-Kee-Fung, C. A., Post, S., J. Liq. Chromatogr. Rel.

Technol. 2001, 24, 1133–1151.[56] Altria, K. D., Broderick, M. F., Donegan, S., Power, J., Elec-

trophoresis 2004, 25, 645–652.[57] Jia, Z., Mei, L., Lin, F., Huang, S., Killion, R. B., J. Chroma-

togr. A 2003, 1007, 203–208.[58] Poole, S. K., Durham, D., Kibbey, C., J Chromatogr. B 2000,

745, 117–126.[59] Hansen, S. H., Electrophoresis 2003, 24, 3900–3907.[60] Mertzman, M. D., Foley, J. P., Electrophoresis 2004, 25,

1188–1200.[61] Poole, S. K., Patel, S., Dehring, K., Workman, H., Dong, J.,

J. Chromatogr. B 2003, 793, 265–274.[62] Mahuzier, P. E., Clark, B. J., Crumpton, A. J., Altria, K. D.,

J. Sep. Sci. 2001, 24, 784–788.[63] Zhang, J., Xie, J., Liu, J., Tian, J., Chen, X., Hu, Z., Electro-

phoresis 2004, 25, 74–79.[64] Pomponio, R., Gotti, R., Santagati, A., Cavrini, V., J. Chro-

matogr. A 2003, 990, 215–223.


Recent advances in microemulsion electrokinetic chromatography

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