Review

Manuel MolinaManuel Silva

Department of AnalyticalChemistry,Edificio Marie Curie (Anexo),Campus de Rabanales,University of Córdoba,Córdoba, Spain

Micellar electrokinetic chromatography:Current developments and future

This review highlights recent methodological and instrumental advances in micellarelectrokinetic chromatography (MEKC). Enhancements in sensitivity and selectivityof the technique through the use of on-line preconcentration approaches (stackingand sweeping) and nonconventional pseudostationary phases, namely nonionic andzwitterionic surfactants, mixed micelles and polymers, are discussed in detail. Laser-induced fluorescence and mass spectrometry, as alternatives to UV-absorptiondetection, have been covered to evaluate their advantages and limitations whenapplied to analysis in an MEKC format. Some thoughts on future directions in thisarea such as in-capillary reactions, coated capillaries and MEKC on microchips arealso presented.

Keywords: Coated capillaries / In-capillary reactions / Laser-induced fluorescence / Massspectrometry / Micellar electrokinetic chromatography / Microchips / Pseudostationary phases /Review / Stacking / Sweeping EL 5121

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 39072 On-line preconcentration methods . . . . . . . . 39082.1 Field-amplified sample stacking . . . . . . . . . . 39082.2 Sweeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39092.3 High-salt sample stacking . . . . . . . . . . . . . . . 39103 Nonconventional pseudostationary phases . 39113.1 Nonionic and zwitterionic surfactants . . . . . . 39113.2 Mixed micelles . . . . . . . . . . . . . . . . . . . . . . . . 39113.3 Polymers as pseudostationary phases . . . . . 39124 Detection techniques . . . . . . . . . . . . . . . . . . . 39134.1 Laser-induced fluorescence . . . . . . . . . . . . . 39134.2 Mass spectrometry . . . . . . . . . . . . . . . . . . . . 39145 Future directions . . . . . . . . . . . . . . . . . . . . . . 39155.1 In-capillary reactions . . . . . . . . . . . . . . . . . . . 39155.2 Coated capillaries . . . . . . . . . . . . . . . . . . . . . 39165.3 MEKC on microchips . . . . . . . . . . . . . . . . . . . 39166 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 39187 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 3918

1 Introduction

In recent years, CE has emerged as a versatile andpowerful technique for the separation and determinationof numerous substances in many fields [1–3]. Thisremarkable versatility coupled with a mechanism of ana-lyte resolution that is complementary to more traditionalseparation techniques, such as LC, has led to a wide-spread application of this technology in many labora-tories [4]. Among the various modes of CE, CZE andMEKC [5, 6] are the more prevalent ones. Charged ana-lytes are easily separated by CZE on the basis of differ-ences in mobility under an applied electric field, whereasneutral analytes, which cannot be separated by CZE, arereadily separated by MEKC through differences in ana-lyte affinities between the micellar pseudostationary andthe surrounding aqueous phases. Since its introductionin 1984, significant advances in theory and novel appli-cations have been documented, and nowadays, MEKCnot only allows the separation of neutral species, butalso provides added selectivity in the separation of ionicsubstances that are sometimes difficult to separate byCZE [7, 8].

MEKC can be advantageous over LC in terms of simplic-ity, resolution, and economy; however, it suffers from lowconcentration sensitivity as a consequence of the limitedsample volume and short path length for absorbance-based detection. Though off-line preconcentration meth-ods, including liquid-liquid and solid-phase extraction,have provided significant improvement in LODs [9, 10],

Correspondence: Dr. Manuel Silva, Department of AnalyticalChemistry, Edificio Marie Curie (Anexo), Campus of Rabanales,University of Córdoba, E-14071 Córdoba, SpainE-mail: qa1sirom@uco.esFax: +34-957-218614

Abbreviations: DTAF, dichlorotriazinylamino fluorescein; FASS,field-amplified sample stacking; MIPs, molecular imprinted poly-mers; MRB, micellar running buffer; OPA, o-phthalaldehyde;PAHs, polycyclic aromatic hydrocarbons; RMMs, reversemigrating micelles

Electrophoresis 2002, 23, 3907–3921 3907

2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/02/22-2312–3907 $17.50�.50/0

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3908 M. Molina and M. Silva Electrophoresis 2002, 23, 3907–3921

the huge interest to increase sensitivity in MEKC has ledmany research groups to focus on the development ofon-line preconcentration techniques based on the stack-ing effect. Therefore, the integration of this process intothe earlier steps of the MEKC method has lately emergedas an important research field, constituting a powerfulalternative to enhance the analytical properties of con-ventional MEKC methods.

The employment of additives such as organic modifiersand CD in conventional MEKC separations (using SDSor CTAB under normal or reversed polarity conditions,respectively) is a well-established means to handle theselectivity and widen the migration window and conse-quently, the number of analytes that can be determinedin a single analysis. Nevertheless, these additives canfail to separate certain multicomponent mixtures andtherefore other alternatives are required. In this context,the employment of nonconventional pseudostationaryphases such as nonionic and zwitterionic surfactants,mixed micelles and polymers as pseudostationaryphases, are beginning to show their potentials and areexpected to offer a challenging alternative to modifiedMEKC.

Among the detection techniques successfully applied tocapillary electrophoresis, only some approaches havebeen readily coupled to MEKC so far. Though this factcannot be attributed to one single reason, an intrinsicdifficulty arises from the presence of the micelle and thepartition in this phase of the analyte, whose detectionproperties can be significantly altered in the micellarenvironment. Electrochemical detection, for example, isstrongly hampered as the species can be consideredcomplexed with the micelle and consequently their be-havior may constrain the application of this technique.This handicap can hardly be overcome when the detec-tion implies the interaction of the analyte molecules withan active surface or a chemical reaction (chemilumines-cence) and thus other alternatives are preferred. In addi-tion, electrochemical, fluorimetric, and conductimetricdetectors can provide better sensitivity but are not univer-sal nor easily affordable compared to the more popularUV-detector. Mass spectrometry (MS), although universaland mass sensitive, is expensive, and further studies areneeded on coupling MS to MEKC. Despite recent devel-opments in UV detection to enhance sensitivity in MEKC,only LIF and MS are covered in this review due to theirwidespread use in CE and expected progression inMEKC in the near future. Other exciting challenges usingMEKC are also presented in this review, such as in-capil-lary reactions, covalently modified capillaries and micro-chip technology due to their interest and huge possibili-ties.

2 On-line preconcentration methods

The so-called stacking of analytes comprises severalmethodologies introduced in an effort to overcome thepoor sensitivity achieved in most CE determinations,inherent from the limited optical path length (using UVdetection) and the low sample volume injected (nL) intothe capillary. This technique is an interesting alternativeto increase the amount of sample loaded into the capillarywithout loss of CE efficiency through the optimization ofthe injection conditions and the composition of the sam-ple and the BGE, so that the analyte bands are com-pressed within the capillary just before the separation.Two physical phenomena have been used to preconcen-trate analytes, one of them involving the manipulation oftheir electrophoretic velocity (normal stacking), whereasthe second one implies the dynamic partitioning of ana-lytes into a moving pseudostationary phase (sweeping).Several CE approaches have been developed over thelast decade to perform the straightforward on-columnsample stacking of charged analytes using transientITP steps; in MEKC, however, the analytes are very oftenneutral species and thus charged micelles are required toprovide the effective mobility necessary for the stacking.The most important features of the modalities employedto perform the on-line preconcentration step in MEKC areoutlined below.

2.1 Field-amplified sample stacking

Liu et al. [11] introduced the first stacking approach forMEKC. The sample is prepared in a low-conductivitymicellar solution and the analytes, partitioned in themicelle, migrate rapidly to the boundary zone betweenthe sample and the high-conductivityBGE due to the lowerionic strength in the sample zone. As soon as the micellesreach the boundary, they are slowed down resulting in anarrow migrating zone. The stacking process, in either nor-mal or reversed-polarity mode (Fig. 1), allows a significantimprovement in sensitivity. In the first one, the whole sam-ple remains in the capillary and subsequently, the separa-tion zone is reduced and potential interfering peaks mayarise. This mode is suitable for slightly hydrophobic com-pounds, as its performance is not so much dependent onthe analyte capacity factor [12]. Oppositely, using reversedpolarity the sample is partially backed out of the capillary(sample matrix removal) and much higher preconcentra-tion factors are obtained for very hydrophobic com-pounds; however, the lesser hydrophobic ones can go outfrom the capillary during the stacking process. As a conse-quence, much work has been devoted to the latter, withreported preconcentration factors ranging from 30 to 300with respect to nonstacking conditions [13–15].

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Figure 1. Migration of micelles and neutral analyte molecules during normal and reversed electrode polarity stackingmodes. Capacity factors: ka � kb � kc. Adapted from [12] and [16].

High molecular mass surfactants were proposed as run-ning buffer constituents for enhancing the stacking pro-cess but this alternative did not appreciably improve theenrichment factors reported under normal and reversed-polarity conditions [12, 16, 17]. A preinjection plug of highconductivity and viscosity was utilized by Zhang andThormann [18], achieving a 400-fold sensitivity enhance-ment for some opioids and LODs as low as 0.1 ng/mL(UV detection); however, this approach requires a carefuladjustment of the electrophoretic (electric field strengthand mobility of the solutes) and operational (injectionsequence) conditions.

Field-enhanced sample injection (FESI), developed byTerabe et al. [15, 17, 19], is based on the injection of awater plug just before the sample so that the electric fieldis enhanced in this zone during the electrokinetic injectionand higher amount of analytes can be stacked in theboundary between the water plug and the separation solu-tion. This procedure is analogous to the reverse mode offield-amplified sample stacking (FASS), since the injectionis carried out under reversed polarity to expel the waterplug while the micelles with the analytes are injected intothe capillary, and consequently, similar enrichment factorsare attained (ca. 20–200-fold preconcentration factors).

Reverse migrating micelles (RMMs) have also been uti-lized for the successful stacking of neutral compoundsfollowed by their separation in a low-pH micellar back-ground solution [15, 19–22]. In this approach, the samplesare prepared in a low-conductivity matrix and injectedvia pressure into the capillary. The stacking process andthe separation are performed under reversed polarity insuch a way that the negatively charged micelles migratetowards the anodic tip of the capillary as the EOF is verylow in acidic pHs. Owing to the high capacity factorsof hydrophobic analytes, the micelles incorporate themalongside their movement through the sample zone andstack in the boundary with the running buffer. RMMshave been used combined with FESI to achieve 200-foldenhancement [15], and stacking with RMMs and UVdetection with a z-shaped detection cell allowed to obtainmore than a 500-fold increase in peak height [22].

2.2 Sweeping

Earlier studies on RMMs resulted in the breakthrough of anew phenomenon for trace enrichment in MEKC [20–22].This new on-line enrichment technique (sweeping), firstdescribed for hydrophobic natural compounds [23], has

3910 M. Molina and M. Silva Electrophoresis 2002, 23, 3907–3921

been systematized as a general approach to improveLODs in MEKC analyses: 1000-fold enhancement factorshave been reported for neutral analytes strongly inter-acting with the micelle [24, 25]. Oppositely to the above-mentioned FASS, now the sample conductivity mustresemble that of the micellar running buffer (MRB) solu-tion, so that the electric field is homogeneous along thecapillary. Sweeping requires low EOFs and thus is oftenconstrained to separations performed in acidic solutions.On the other hand, the injection procedure is fairly simplesince the samples are injected via pressure into the capil-lary in a nonmicellar buffering solution, followed by theapplication of the electric field for the preconcentrationwith a MRB solution placed at the inlet. Without polarityswitching in most cases, the separation is performedin reversed-polarity mode and the micelles migrate to-wards the detector “sweeping” the neutral analytes along(Fig. 2A).

The effectiveness of sweeping is closely related to theanalyte affinity for the micellar phase, in such a way thatthe length of the analyte band after the sweeping processis proportional to the injected one and inversely relatedto 1�k, where k is its retention factor. It is obvious thatpolar compounds with low capacity factors are not satis-factorily “swept” and thus sweeping needs to be com-bined with normal stacking to yield more than 100-foldincreased detector responses for slightly hydrophobiccompounds [26]. In the case of aromatic amines (withvery high capacity factors for SDS micelles), sweepingand cation-selective exhaustive injection have allowed toreach a millionfold sensitivity increase with direct UVdetection [27, 28]. Despite the amazing preconcentrationfactors reached with this technique (see Fig. 2B), somelimitations must be considered. The need for a very lowEOF implies a high irreproducibility when it is obtainedwith acidic pH solutions. Cationic surfactants have beenutilized at near-neutral pHs as an alternative [29, 30], butonly ca. 20–100-fold enhancement factors have beenachieved, very low compared to 1500–5000-fold undersuppressed EOF conditions. Moreover, most MEKCreported separations have been developed under basicconditions, and consequently sweeping can not bestraightforwardly applied. In summary, this on-line pre-concentration method can only be considered optimumfor compounds with very high capacity factors that canbe separated under acidic conditions.

2.3 High-salt sample stacking

Landers et al. [31] demonstrated the usefulness of aninnovative strategy to improve sensitivity in MEKC intro-ducing a sample with higher conductivity than the MRB.There is some controversy whether this stacking mode

Figure 2. (A) Progress of micelles and neutral analytesduring sweeping process. Capacity factor: ka � kb � kc.Adapted from [12]. (B) Almost a millionfold concentrationof dilute cations by cation-selective exhaustive injection/sweep. Conditions: BGE, 1 mM triethanolamine/15%acetonitrile/100 mM phosphoric acid; MRB, 100 mM

SDS/1 mM triethanolamine/15% acetonitrile/50 mM phos-phoric acid; high-conductivity buffer, 100 mM phosphoricacid; sample solution, (1) laudanosine and (2) 1-naphthyl-amine in water; sample concentration, (a) 2450 ppm,(b) 240 ppt; injection scheme, (a) 0.6 mm of the samplesolution, (b) 30 cm of high-conductivity buffer and then3 mm of water followed by 23 kV electrokinetic injectionof the sample solution for 1000 s; sweeping and MEKCvoltage, �23 kV with the MRB at both ends of the capil-lary. Reprinted from [18], with permission.

may be considered just a particular case of sweeping ornot [32, 33]; however, recent results suggest that itsmechanism is not completely explained so far throughnormal stacking and sweeping [33]. Anyway, this ap-proach is interesting enough to be described in a sepa-rate section since it allows the direct application of stack-

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ing to samples with a high ionic strength [34, 35]. As it wasearly proposed, the simple addition of sodium chloride(or any other salt) to the sample matrix causes the stack-ing of the charged micelles at the sample-BGE boundaryin the normal polarity mode; the plug of the stackedmicelles trap the neutral analytes coming from the samplezone with the EOF, achieving a reasonable improvementin sensitivity for several corticosteroids [31] and pesti-cides [34, 35]. As in sweeping and normal stacking, theefficiency of the preconcentration step depends on theaffinity of the analytes to the micelles. This alternativehas demonstrated to be suitable for the direct analysis ofsamples because of its robustness towards other sampleconstituents.

3 Nonconventional pseudostationaryphases

3.1 Nonionic and zwitterionic surfactants

Despite the obvious structural differences of the mono-mers of nonionic and zwitterionic surfactants, their mi-celles exhibit comparable properties and are used forsimilar purposes, involving mainly the optimization of theselectivity (including the separation of stereoisomers) andthe dynamic coating of the capillary. Nonionic surfactantswere proposed for the first time as pseudostationaryphase to enhance the selectivity in CE by Swedberg [42],who reported an apparent MEKC mechanism to explainthe analyte behavior in these media. Despite neutralanalytes should not be separated by nonionic micelles,the MEKC mechanism is clear when these surfactantsbecame charged in situ through complexation with borate[43–50]. In this field, it is worthy to mention the contribu-tions of El-Rassi et al. [51–59] based on the use of alkyl-glycoside or alkyl-N-methylglucamide surfactants forthe separation of dansyl amino acids and herbicides,among others. The separation of neutral analytes byMEKC in zwitterionic surfactant-based media (RewotericAM CAS U, Zwittergen 4–14 and polyalkylammonium pro-panesulfonate analogues) bears no difficulty since thesechemicals can own net charge depending on the condi-tions. Since their introduction to modify the selectivity ofelectrokinetic separations [42–46, 60–63], the employ-ment of both nonionic and zwitterionic surfactants hasbecome a common practice for the analysis of proteinsand peptides [51–59, 64–68], stereoisomers [64, 67, 68]and other closely related compounds [69–74].

These surfactants have also been used for the dynamicalcoating of the capillary simply washing it with the micellarsolution just before or during the MEKC separation [73–

77]. As a result, the reproducibility of the EOF of thecoated capillaries is improved and sorption of analytesonto the inner surface of the capillary is hindered. Thesesurfactants yield very efficient separation of proteins andbiogenic compounds which tend to adsorb on the wall ofa bare silica capillary, and consequently they are receivingmuch attention in the field of biochemical analysis.

3.2 Mixed micelles

The application of mixed micellar systems (MMS) forMEKC has been the focus of a considerable interest dur-ing the last decade because of their demonstrated utilityfor the analysis of highly related compounds. MMS havebeen used to solve selectivity problems arising in com-plex mixtures submitted to MEKC in one surfactant-based solutions [77–93]. All the possible combinations ofdifferent types of surfactants have been almost applied,but the most popular mixed aggregates are constitutedby one neutral (nonionic, very often Brij-35 or Tween 20)and one anionic surfactant (SDS in most cases) [77–85].Combinations of bile salts and other (charged or un-charged) surfactants allow the successful handling of theselectivity in chiral separations [79–81, 89–92]. In a sense,these systems could be compared to CD-MEKC but onlyone partition equilibrium of the analyte into the mixedpseudostationary phase takes place rather than twoseparated and competing processes. MMS are usuallyapplied for the analysis of very hydrophobic compounds,which would coelute with the common micelles, such assubstituted aromatic compounds [79, 80, 82, 84–86],steroids [79, 89, 91, 93] and a wide variety of mixtureswith a high degree of complexity and structural similarityamong their components. The results shown throughoutthe last years remark the excellent properties of mixedmicelles in combination with MEKC to resolve a greatnumber of analytical problems.

The study of thermodynamical aspects of mixed micellesand their interactions with analytes has been also thefocus of several papers taking into account their rele-vance upon the selectivity optimization [80–85, 91–101].Among them, several approaches can be remarked:(i) the Plackett-Burman statistical [94] and central com-posite [95] designs were used to optimize the high num-ber of parameters affecting the MMS-MEKC separation;(ii) the solvation parameter model [98, 99] aimed to studythe influence of the MMS composition on the capacity ofthe micelle for intermolecular interactions; and (iii) theso-called infinite elution range, in which the electroosmo-tic and micellar electrophoretic mobilities are empiricallyadjusted so that a nearly stationary micellar phase isobtained [101].

3912 M. Molina and M. Silva Electrophoresis 2002, 23, 3907–3921

3.3 Polymers as pseudostationary phases

Although conventional micelles are very useful as pseu-dostationary phases, significant limitations associatedwith their use for MEKC can seriously constraint thedevelopment of new methods: (i) from the basis of MEKCseparations, the limited migration time for unchargedanalytes and the difficulty to separate very hydrophobiccompounds (they almost coelute with the micelle) areinherent. (ii) The dynamic equilibrium which leads to theformation of the micelle is strongly dependent on the tem-perature, buffer composition and concentration of themonomer, and thus the micelle can be unstable underthe desired separation conditions, resulting in irreproduci-bility. (iii) Unless the surfactant is specifically synthesizedto possess chiral activity, the separation of stereoisomersis hampered by the micelle, which may interact with thechiral selector (cyclodextrins, for example) and minimizeits interaction with the analyte. (iv) Finally, conventionalsurfactants may be troublesome for MS detection, dueto large signals in the low-molecular-mass region, interfer-ing with most analytes. These shortcomings and the sub-sequent need for more versatile micellar structures havemade polymers the focus of intensive research since theirintroduction in this field [102–105].

A distinction between micelle polymers and the so-calledpolymer(ic) (or high-molecular-mass) surfactants may beestablished. Micelle polymers are synthesized in a micel-lar form and therefore keep the micellar structure unlessthey are solvated in organic media, whereas polymericsurfactants includes other amphiphilic polymers whosesolutions show surface active properties due to theformation of macromolecular aggregates, but have beensynthesized in a conventional way (not a micellar form).Both types of polymers and dendrimers (a different kindof highly branched polymers obtained by the successiveapplication of the polymerization reaction from a core)have been successfully applied to electrokinetic separa-tions and extensive work has been made to improve theirperformance. Figure 3 shows the schematic structuresof the above-mentioned polymers, used as pseudo-stationary phases in MEKC, compared to conventionalmicelles. Despite several reports have reviewed thisdevelopment [106–109], it may be interesting to considerthe most relevant contributions to this subject.

Palmer and co-workers reported the first successful useof a true micelle polymer of sodium-1-undecylenate forMEKC separations with buffers modified up to 50%methanol [105]. This procedure [105, 110] has demon-strated to be very effective to obtain a huge amount ofhomologues, such as SDS polymeric analogues (po-lymerization of sodium 10-undecenylsulfate) [111–117]and chiral micelle polymers (sodium N-undecylenyl-L-

valinate and analogues) [118–127]. The last stage in thisseries involves taking sodium N-undecylenyl dipeptidesas monomers to perform the polymerization; this innova-tion has allowed to obtain several types of chiral atoms inone polymer molecule, which makes possible a muchbetter enantioselectivity than that obtained with poly(so-dium N-undecylenyl-L-valinate) [123, 128–133].

Earlier works on acrylate copolymers for MEKC separa-tions focused on butyl acrylate-butyl methacrylate-methacrylic acid copolymer for the separation of veryhydrophobic compounds [134, 135]. The authors re-marked the significantly different selectivity of the poly-mers for substituted naphthalene compounds. It wasdemonstrated that changes in the surface charge as aconsequence of the presence of carboxylate groupson this polymer strongly affected the partition of theanalytes, decreasing their retention factors. The effect oforganic modifiers and other buffer constituents on acry-late polymer-based electrokinetic separations has beeninvestigated showing that much higher amounts of modi-fiers can be used than with conventional surfactantsMEKC without affecting the micelle [136–140]. Anotherimportant advantage of using high-molecular-mass sur-factants is the compatibility with MS detection but thistopic will be discussed in Section 4.

Dendrimers are highly branched polymers synthesizedfrom a core applying a multistep repetitive synthesis, inwhich globular macromolecules are obtained [141]. Thebranching structure of higher generation dendritic mole-cules results in a topology similar to that of the micellesand consequently, dendrimers have emerged as prom-ising alternatives to conventional micelles due to theirmolecular mass specificity, uniform size and unique prop-erties. Among the so-called starbust dendrimers, alsoknown as diaminobutane-based dendrimers, the mostcommon are those with 0.5–3.5 generations, furnishing4–32 nitrile terminal groups that can be functionalizedyielding molecules with different properties [141–144].The alkylation of the carboxylate terminal groups to yieldthe ester derivatives has allowed the separation of veryhydrophobic compounds such as phenols, alkyl para-bens, polycyclic aromatic hydrocarbons (PAHs) andnaphthalene derivatives [144–147]; furthermore, thesedendrimers can be used in highly modified running buf-fers (up to 90% methanol has been reported [145]) consti-tuting suitable pseudostationary phases to bridge aque-ous MEKC and nonaqueous CE. Recent works on den-drimers focus on the employment of more polar terminalgroups, such as sulfonic and carboxilyc acid, to resembleSDS micelles [148] and improve separations of proteinsand aromatic acids [149, 150]. These works have demon-strated that selectivity can be handled through the pres-

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Figure 3. Structures of the SDS micelles and nonconventional pseudostationary polymer phases used in MEKC.

ence of functional groups and thus specific dendrimersmay be developed depending on the needed applica-tion. The use of dendrimers as pseudostationary phaseshas only begun to be investigated and the comingyears should observe exciting developments in this fieldand significant improvements in pseudostationary phasetechnology.

In summary, the most salient features on the use of poly-mers in MEKC are the following: (i) they provide verystable micelles with virtually zero CMC, allowing veryhigh amounts of organic modifier in the micellar solutionat any polymer concentration. (ii) Polymers can affordunique (chiral) selectivity with respect to conventionalmicelles and can be synthesized with any electrophoreticmobility, as self-association requirement is obviated. Thepolymer technology may also offer a convincing alterna-tive to CEC due to the cheap, simple use of EKC methods

with respect to common CEC methods that involve theanalysis of the sample with packed capillaries instead ofopen-tubular ones.

4 Detection techniques

4.1 Laser-induced fluorescence

LIF detection is a highly suitable choice to improve sensi-tivity in MEKC determinations, as conventional micelles arevery often transparent to the laser beam, and furthermore,the analyte molecule inside the micelle can exhibit higherfluorescent yields [151, 152]. This detection technique isoptimal for natively fluorescent molecules; however, mostanalytes are not fluorescent and require a previous derivati-zation step with an appropriate label. An additional short-coming arises from the difficulty to excite fluorescent ana-lytes or derivatives with available laser sources.

3914 M. Molina and M. Silva Electrophoresis 2002, 23, 3907–3921

The number of papers reporting LIF with native fluores-cent is scanty [153–160] despite LODs as low as 1 ng/mLcan be readily achieved. PAHs [153, 154] and biogeniccompounds [155–159] are the most relevant analytes thatcan be detected with native fluorescence using 248 and325 nm excitation wavelengths, respectively, provided byKr/F and He/Cd laser sources. The determination of verylow concentrations of these chemicals in biological sam-ples such as body fluids [155, 157, 158], microdialyzedsamples [160] and animal tissues [157] has demonstratedthe feasibility of LIF detection for (bio)chemical analysiswhen attainable. On the contrary, as stated above mostanalytes do not show native fluorescence and therefore in-direct approaches or derivatization reactions are required.

To our knowledge, there is only one paper reporting theuse of indirect LIF for MEKC [161], probably due to thebasis of this detection mode, which implies the modifica-tion of the background fluorescent signal by the analyte.Due to the fact that the analytes are usually uncharged inMEKC, the signal depends exclusively on their quenchingefficiency, and the charge displacement phenomenon,which is the basis of indirect CZE determinations ofcharged compounds, do not contribute to it. Even ifcharged analytes were separated by MEKC, the highconcentrations of ionic surfactants employed would mini-mize charge displacement by the analytes and indirectLIF is not recommended.

Many derivatization reactions have been developed tolabel a variety of analytes with appropriate fluorescenttags for LIF. Among them, amino acids have been a fruit-ful object of study [78, 162–166], whereas sporadic workshave appeared on the determination of other compoundssuch as amines, peptides, pesticides and fatty acids [73,167–173]. Many of the reported methods have used fluo-rescein analogues (FITC, dichlorotriazinyllamino fluores-cein (DTAF), etc.) to yield fluorescent derivatives with anabsorption maximum at 488 nm, suitable to be detectedwith an inexpensive ion argon laser [73, 78, 169, 172–174]; o-phthalaldehyde (OPA)-based derivatives fit theexcitation properties of an He/Cd laser and their use isalso widely reported [166, 167, 175]. Though the above-mentioned reagents and others (continuouslyunder devel-opment) may offer a valid alternative to obtain fluorescentproducts, the performance of LIF detection is limited bythe efficiency of the labeling chemistry and the availabilityof high-purity probes.

4.2 Mass spectrometry

MS constitutes the most promising alternative to unequi-vocally identify and quantify analytes in CE due to thestraightforward on-line hyphenation of the capillary and

the spectrometer through an ESI interface. However, theemployment of high surfactant concentrations (typical inMEKC) is incompatible with MS operation and therefore,only a few papers have been published on this topic. Theinstability of the electrospray, contamination of the MSdetector, and loss in sensitivity are the more relevant pro-blems associated with the presence of surfactants in theperformance of MEKC-ESI-MS. To overcome these nega-tive effects, the more useful choices involve preventing themicelles from entering the mass spectrometer, namely, theheart-cut technique [176], the partial filling (PF) approach[35, 177–180] and the use of RMMs [35, 181].

The first mentioned attempt uses a double capillary sys-tem to pick selected plugs from the first capillary andcollect them in the second one, which sequentially intro-duced the analytes electrokinetically into the MS in amicellar free buffer. The PF technique, in which only apart of the capillary is filled with a solution containing themicelles, has been fairly widely applied because of itssimplicity and usefulness for MEKC-ESI-MS analyses; inaddition, it offers long-term operational stability as thenonvolatile additives can not interfere with the electro-spray process. RMMs prevents passage of the surfactantto the MS by adjusting the EOF at a value below the mo-bility of the micelles. This has been accomplished either inbare fused-silica capillaries keeping the pH of the electro-lyte solution lower than �5 [181] or by using coated capil-laries with a controlled, pH-independent EOF [35]. RMMshave not been so popular as the PF technique so far dueto the intrinsic difficulty to maintain a good reproducibilityof the separations in uncoated capillaries at low pHs, butthis alternative should receive more interest in the nextfuture as coated capillaries are becoming commerciallyavailable. In addition, the effective separation length issignificantly longer with RMMs than with PF and thereis no need to stop the separation process to avoid themicelles reaching the electrospray.

A significantly different line of research in MEKC-ESI-MSassumes that the micelles will reach the ESI duringthe separation, and consequently, the selection of thepseudostationary phase plays a critical role. High-molec-ular-mass surfactants have been used as alternative toconventional ones to avoid high background signals[182–184], as they are weakly ionized in normal MS con-ditions and therefore contribute to the noise only at veryhigh masses (without interfering the detection of commonanalytes). Contrasting to polymeric surfactants, fluorosur-factants have been successfully used for MEKC-MS dueto their negligible ionization [185, 186]. The introductionof MALDI-MS to MEKC have been the focus of severalworks [187–189], although further studies are required toevaluate its potential toward the standard ESI interface.

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As MEKC-MS is demonstrating its value for confirmatoryand quantitative analysis, other well-established waysto enhance MEKC features (i.e., stacking) are combinedto it [35, 190] for bringing together MEKC-MS and dailylaboratory practice.

5 Future directions

5.1 In-capillary reactions

The technique known as in-capillary electrophoresis re-action has demonstrated to be a powerful alternative fornano-scale derivatization of the analytes: multiple nano-liter aliquots of sample can be readily reacted inside thecapillary with virtually no consumption of the sample. Pre-or post capillary derivatization steps are replaced byautomated electrophoretic procedures, since the separa-tion capillary tube is also utilized as a microreactionchamber to accomplish the derivatization of the analytes,and consequently, faster and more robust analyticalmethods can be developed. This approach, first pro-posed by Taga et al. [191] for determinations of aminoacids with OPA, follows the natural progression of in-cap-illary reaction techniques to include nonenzymatic (deri-vatization) reactions over the recent years [192–202],taking into account the good results achieved by electro-

phoretically mediated microanalysis in the determinationof enzymatic activities and enzyme and substrate con-centrations [203–205].

Several alternatives have been reported using differencesin the electrophoretic mobilities to merge distinct zones ofanalyte and labeling reagent under an electric field; themost popular of them (Fig. 4) are known as at-inlet, zone-passing and throughout in-capillary derivatization (ICD).The at-inlet strategy and the zone-passing techniqueinvolve the introduction of separate plugs of sample andreagent before the application of the voltage [191–199].The first one implies the stand-by of the system while thereaction is carried out after the mixing of the zones and isused when a specific time is required to complete thereaction before the separation. The second can be per-formed when the kinetics of the reaction allow a goodderivatization yield in the short period of contact betweenthe sample and the reagent zones. The ICD approach[200–202] is quite different as the whole capillary is filledwith a running buffer containing the derivatization re-agent. The reaction takes place as the analytes are separ-ating in the buffer, and consequently this procedureresembles indirect detection methods and offers similarcharacteristics.

Though these methodologies were initially developedfor CZE, they have been successfully applied to MEKC[166, 173, 192, 201, 206–208]. It is worthy to note that

Figure 4. Strategies for in-capillary derivatization and thesubsequent separation of the labelled analytes by MEKC.Adapted from [192].

3916 M. Molina and M. Silva Electrophoresis 2002, 23, 3907–3921

the presence of the micelles may play a key role not onlyin the separation but also in the features of the in-capillaryreaction, because of its inherent properties and the parti-tion of the analyte in the pseudostationary phase. Thus,Wu et al. [208] have reported the on-column thermaldecomposition of ten N-methylcarbamates catalyzed byCTAB micelles, after their separation in this micellarmedium, followed by the derivatization of the releasedN-methylamine with OPA and fluorescence detection[208]. On the other hand, the micelle can strongly inhibitthe derivatization reaction and therefore its presencemust be prevented during this step. This is easily accom-plished injecting a surfactant-free background electrolytebetween the sample zone and the micellar solution sothat the reaction takes place in a nonmicellar medium, aspresented in Fig. 5, which shows the in-capillary deriva-tization of amino acids using dichlorotriazinylamino fluo-rescein (DTAF) and their subsequent separation by SDSmicelles and LIF detection [173].

In summary, the flexibility of the capillary to be convertedinto a microreactor is expected to promote increasedinterest on this technique for (bio)chemical analysis in thenext years, due to the high degree of automation in thesample handling and the minute sample volume con-sumed, which can be considered their major advantages.

Figure 5. Scheme for in-capillary derivatization of aminoacids with DTAF to avoid the negative effect of micelles onthe labeling reaction. Adapted from [173].

5.2 Coated capillaries

The employment of coated capillaries has been fairlydemonstrated to be a useful way to reduce or suppressthe EOF and the interactions of certain analytes withthe capillary inner wall. These capillaries can be utilizedto obtain RMMs or improve separations in MEKC duemainly to an increased migration window [35, 65, 209–212]. Commercially available coated capillaries havebeen sporadically used in MEKC [213–215] becausemost research groups prefer to use home-made poly-meric coated ones [35, 216–222]. Polyacrylamide-basedpolymers (either linear or cross-linked) are usually pre-pared to yield uncharged, hydrophobic coatings [65,216–221, 223] that are very often suitable for the separa-tion of positively charged analytes such as proteins andpolyamines preventing their sorption on the surface ofthe bare fused-silica capillary. A different research trendhas focused on the same polymerization reaction, butusing charged monomers to obtain capillaries with a con-trolled, pH-independent EOF [35, 221, 222], which areextremely flexible and effective to perform more repro-ducible separations and allow the application of RMMsunder a wide pH range [35]. The adjustment of the kindand ratio of the monomers allows to obtain any neededEOF for a particular application and consequently theuse of these specifically designed capillaries may consti-tute an important field of research in a close future. In thissense, the development of molecular imprinted polymers(MIPs) to coat the capillaries would markedly increasethe potential of this technique by combining the selectiv-ity of the micelle with that provided by the MIP coating.

5.3 MEKC on microchips

Microfluidic devices for CE are becoming a fascinatingresearch field because of the good characteristics of thedeveloped analytical miniaturized instruments. Followingthe first glass chip specifically designed for CE [224],several papers have been devoted to the application ofthese microchips for MEKC [225–233]. The earlier workabout MEKC on a microchip achieved ca. 4000 theoreti-cal plates in the separation of a test mixture of coumarins[225], contrasting to the recently reported value of 106

attained for dichlorofluorescein in a 25 cm long spiral-shaped separation channel fabricated on a glass micro-chip with a footprint of only 5 cm�5 cm [226]. The out-standing development in microfluidic devices has affectednot only fundamental aspects of the separation, but alsoallow to perform operational procedures that can not beeasily carried out with conventional CE equipment. In thissense, a remarkable contribution reported separationsusing a solvent gradient [227]; this alternative should

Electrophoresis 2002, 23, 3907–3921 Trends in MEKC 3917

widen the applicability of electrically driven separations tovery complex mixtures in a next future. Two-dimensionalseparation, a fundamental technique in proteomics andgenomics, have also been assessed to separate trypticpeptides with a peak capacity of about 1000 within10 min [228]. Recent results have shown the applicabilityof glass microchips to the chiral separation of aminoacids [229] and amphetamine and related compounds[231] labeled with FITC and 4-fluoro-7-nitrobenzofura-zane, respectively, by CD-MEKC-LIF, pointing out a trendin biochemical and pharmacological analysis.

The detection can be considered a challenging aspect ofmicrochip-based MEKC separations, because as statedin the previous section, only optical detectors can bestraightforwardly applied to MEKC. In addition, UV-ab-sorption detection for microchip separations is furtherconstrained owing to the limited optical pathway, andtherefore, LIF has constituted the most useful choice[225–232]. In addition to the inherent high sensitivity ofthis technique, it can be improved by using on-line pre-concentration steps (sweeping) to yield enhancementfactors of ca. 450 for Rhodamine B and related com-pounds [232]. Nonfluorescent analytes, however, lack ofan adequate fluorophore and can not be detected bydirect LIF, unless they are labeled with suitable tags, and

therefore indirect LIF detection has been introduced as analternative albeit less sensitive. The analysis of a mixtureof explosive compounds has been carried out by thismeans using a near-IR diode laser operating at 750 nmas excitation source; a dye (Cy7) was employed as visu-alizing agent, reaching LODs below the �g�mL�1 level[161]. A different choice uses lasers as sources for UV-visible detection on account of their collimation power,which allows for easy focusing of the beam in the micro-chip detection window. Laser-emitting diodes can beused for this purpose, as demonstrated in the determina-tion of transition metal ions complexed by 4-(2-pyridyl-azo)resorcinol recently reported by Lu and Collins [233].

Polymer-fabricated microchips have been lately appliedto MEKC and could replace glass ones due to their easierfabrication and cost-effectiveness. The respective fabri-cation processes for glass and polyester microchipsthrough photolithographic and wet-chemical etching pro-cedures are depicted in Fig. 6 [230, 234]. Imprinting meth-ods can be utilized to replicate the pattern and obtaincheap, plastic, disposable chips for a limited number ofultrafast analyses. Moreover, these chips could be veryeasily modified to obtain specific selectivities or uniqueproperties such as (bio)catalytic activity. The use of poly-mer-fabricated chips for MEKC is particularly interesting

Figure 6. Photolithographic process for (A) masking glass chips and (B) polyester chip fabrication. Reprinted from [230]and [234], with permission.

3918 M. Molina and M. Silva Electrophoresis 2002, 23, 3907–3921

because the low surface tension of the micellar runningbuffer minimizes bubble formation caused by a deficientwetting of the chip channels, as reported in CZE.

6 Conclusions

The current status of MEKC provides to analyticalchemists many tools to afford the determination of a greatvariety of substances in many real samples. MEKC is tobecome a robust and widely accepted method, whoseaccomplishment in routine laboratories is continuouslygrowing although further improvements are still needed.Basic analytical properties, such as sensitivity and selec-tivity, can be enhanced with additional studies on thestacking process and the use of new unconventionalpseudostationary phases, where micelle polymers, poly-mer surfactants and dendrimers have a great potential.While much research has gone into detector developmentin the last decade, achievements in sensitivity (LIF detec-tion) and selectivity (MS detection) require more investi-gations to develop effective fluorescent probes, mainlyfor near-infrared LIF, and to get a practical hyphenationbetween MS and MEKC. Finally, MEKC on microchips isan emerging, new technology that promises to lead thenext revolution in chemical analysis.

The authors gratefully acknowledge financial support fromSpain’s Dirección General de Investigación del Ministeriode Ciencia y Tecnología for the realization of this work aspart of Project BQU2000–0905.

Received April 18, 2002

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