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avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /aca

eview

ecent strategies to improve resolution in capillarylectrophoresis—A review

nne Varennea, Stephanie Descroixb,∗

Laboratoire Environnement et Chimie Analytique, UMR 7121 CNRS-ESPCI, 10 rue Vauquelin 75005 Paris, FranceLaboratoire d’Electrochimie et Chimie Analytique, UMR 7575 CNRS-ENSCP-Paris6, 11, rue Pierre et Marie Curie,5231 Paris Cedex 05, France

r t i c l e i n f o

rticle history:

eceived 5 May 2008

eceived in revised form

6 August 2008

ccepted 27 August 2008

ublished on line 3 September 2008

eywords:

esolution

a b s t r a c t

Besides the classical approaches used in capillary electrophoresis (CE) to improve separa-

tion, new strategies have been more recently introduced. This review presents some targeted

strategies that have been recently developed in order to enhance the resolution of elec-

trokinetic separations. Both novel electrolytes (non-aqueous and isoelectric buffers) and

additives (ionic liquids) are presented in terms of performances and mechanisms involved

to increase separation resolution. The advantages of innovative methodologies employed

in electrokinetic miniaturization are also presented, that allow resolution enhancement.

Finally, an insight into the complementarity of a specific hyphenated detection, i.e. mass

spectrometry (MS), is performed, as an original strategy to help for better characterization

of complex mixtures separation.

apillary electrophoresis

on-aqueous solvents

soelectric buffers

onic liquids

ass spectrometry hyphenation

© 2008 Elsevier B.V. All rights reserved.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10lytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12ic liquids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14at about resolution? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

iniaturization

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2. Resolution enhancement with non-conventional electro

2.1. Non-aqueous and hydro-organic electrolytes. . . . .2.2. Isoelectric buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3. A new type of additives for resolution improvement: ion4. Microdevices dedicated to electrokinetic separation: wh

5. Hyphenation of CE with mass spectrometry: a good complem6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +33 140 79 46 44; fax: +33 140 79 47 76.E-mail address: stephanie.descroix@espci.fr (S. Descroix).

003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2008.08.039

entarity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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10 a n a l y t i c a c h i m i c a

1. Introduction

Capillary electrophoresis (CE) is a very attractive separationmethod because of its low sample and electrolyte consump-tion, short analysis time, high efficiency, ease of operationand automation compared to classical gel electrophoresis. CEis also a versatile separation method as it can be appliedto a wide variety of analytes thanks to the different modesthat can be used. Numerous recent reviews illustrate the dif-ferent applications of CE analysis [1–12]. Nevertheless thecomplexity of sample matrices leads to huge needs for highresolution. In this context, main classical strategies can beapplied, that induce modification of peak efficiency, selec-tivity and thus resolution. The main parameters affectingCE resolution are capillary dimension and nature, separationelectrolyte composition (pH, ionic strength, salt nature, addi-tives), applied electric field and capillary temperature. The aimof this review is not to discuss about these classical parametersthat can be modified to get better resolution, but to more par-ticularly highlight some original recent strategies, that havebeen developed in the last few years and have demonstratedtheir ability to improve baseline resolution for electrokineticseparations. The importance of some non-conventional elec-trolytes, i.e. non-aqueous electrolytes and quasi-isoelectricbuffers, will be particularly demonstrated. New types of addi-tives are also being employed for resolution enhancement,and ionic liquids will be presented as efficient candidates inCE. In the domain of miniaturized electrophoretic systems,resolution is a main task, and some recent strategies willbe highlighted that allow baseline resolution. Last, anotherstrategy does not concern resolution during separation, butcharacterization improvement through the coupling of CEwith very specific detection methods, as CE–UV often fails forsufficient resolution. Mass spectrometry is the most adequatedetection method for such purpose.

2. Resolution enhancement withnon-conventional electrolytes

2.1. Non-aqueous and hydro-organic electrolytes

Since the 1970s [13–17], and more particularly the last 15years, organic solvents have been used for electrokineticseparations [18–22] to improve resolution, either pure or inmixture with other solvents. The range of usable solventsthat present different physico-chemical properties is verywide and leads to a high versatility. Nearly all separationmodes can employ organic solvents, such as capillary zoneelectrophoresis (CZE), micellar electrokinetic chromatogra-phy (MEKC), microemulsion electrokinetic chromatography(MEEKC) and capillary isoelectric focusing (CIEF). The mostclassical solvents used in CE are reported in Table 1. A fewrecent studies illustrate the possibility to improve the sep-aration in non-aqueous electrolytes compared to aqueous

ones. Chiu et al. reported a simple method to separate andcharacterize five nicotine-related alkaloids by non-aqueouscapillary electrophoresis (NACE) with UV and MS detection[23]. The electrolyte pH, nature and the solvent mixture have

a 6 2 8 ( 2 0 0 8 ) 9–23

been optimized. The NACE method has been coupled to elec-trospray ionization (ESI)–MS detector. This method providesbetter performance than other aqueous methods reported.And very high resolution combined with high detection sen-sitivity and an abundance of structural information can beachieved for alkaloids analyses by NACE–MS. Hansen andSheribah have also compared different electrokinetic meth-ods to determine impurities in bromazepam, namely aqueousCZE, MEKC, MEEKC with NACE [24]. NACE provided bet-ter results and was selected particularly because the lowsolubility of benzodiazepine in water was thus overcome.Aqueous and non-aqueous electrokinetic methods have beendeveloped to separate and quantify phenolic and bispheno-lic compounds in environmental samples [25]. Both methodswere suitable for the analysis of the compounds of interest,nevertheless the resolution was shown solvent-dependent,and baseline separation of the analytes and their interferingcompounds was demonstrated with NACE method. Althoughthe use of hydro-organic electrolytes cannot be considered asa recent strategy, their use during CIEF separation can be con-sidered as quite new. The possibility to improve resolutionby using hydro-organic electrolytes compared to pure aque-ous ones has thus been described in an original CIEF method[26]. Glycerol, that is well known to help protein stabilizationand solubilization of hydrophobic protein, has been tested asan alternative to commercial gels. A 30:70 (v/v) glycerol–watermedium appeared as a good compromise for performing CIEFin a bare fused-silica capillary without imposing too high aviscosity. The feasibility of this new system has been demon-strated by the successful separation of a standard mixture ofnine model proteins according to their pI, and better resolu-tion was achieved with this system than with conventionalaqueous CIEF system. Finally many recent examples illustratethe potential of non-aqueous solvents for efficient separationof a wide variety of analytes [27–36].

The main phenomena involved when using organic sol-vents, to improve resolution of electrokinetic separation haveto be discussed. By using these media, the charge of ana-lytes can be modified leading to a change of electrophoreticmobility and eventually to a resolution enhancement. In thecase of acido-basic compounds, the presence of organic sol-vents indeed leads to a pKa shift. Many papers have studiedthe influence of organic solvents on acido-basic equilibriumand have shown that pKa values can be dramatically modifiedwhen replacing water by non-aqueous solvents. According tothe analytes acido-basic properties, different behaviours areexpected when the solvent is varied. The pKa shift is mainlydue the stabilization of the molecule by the solvent comparedto water and is given by the transfer activity coefficient [37].More recently, Porras and Kenndler have written a very inter-esting review that presents a critical discussion on the useof organic solvents for electrokinetic separation. By studyingthe pKa values of phenol in water [38,39], acetonitrile (MeCN)[38,39] and methanol (MeOH) [40], they have shown that thereis no significant change in selectivity from water to MeOH,whereas there is a change in selectivity when replacing water

by MeCN. In parallel, the acido-basic properties of cationsacids (AH+/A) have also been studied and no general trendcould be observed as the selectivity was increased in MeCNand dimethylformamide (DMF) whereas it was decreased in

a n a l y t i c a c h i m i c a a c t a 6 2 8 ( 2 0 0 8 ) 9–23 11

Table 1 – Physicochemical properties of solvents used in CE [37]

Solvent Density (d,g cm−3)

Boiling point(Tb, ◦C)

Dipole moment(�, D)

Polarizability(˛, 10−30 m3)

Relativepermittivity (ε)

Viscosity (�,10−3 Pa s)

pKauto � cut off(nm)

Water 1 100 1.83 1.45 78.3 0.89 14.0 190MeOH 0.79 65 2.87 3.26 32.7 0.54 16.6 205EtOH 0.80 82 1.66 5.13 24.55 1.09 18.8 210i-PrOH 0.8 82 1.66 6.98 19.41 2.08 20.7MeCN 0.77 82 3.44 4.41 38.95 0.34 19.6 190Acetone 0.78 56 2.69 6.41 20.7 0.30 – 330THF 0.89 66 7.93 7.58 0.46 16 – 230NMF 1.01 180 3.86 6.05 128.4 1.65 10.7 245DMF 0.94 153 3.86 7.90 36.71 0.8 29.4 268FA 1.13 210 3.37 4.23 14 3.30 16.8 245Glycerol 1.26 290 2.67 8.14 42.5 945 210

nitrile

MtMarTsww

amcApti

FfsEW

MeOH, methanol; EtOH, ethanol; i-PrOH, isopropanol; MeCN, acetoformamide; FA, formamide.

eOH [40,41]. Recently Delmar Cantu et al. have determinedhe pKa of secondary and tertiary amines in water, MeOH andeCN [42]. They have observed an inversion in mobility order

ccording to the solvent of the background electrolytes and theesolution was improved when using acetonitrile electrolyte.he tertiary amines presented higher pKa values in MeCN thanecondary amines. An opposite trend was obtained in MeOH,hich can explain the mobility inversion observed in NACEith these solvents.

Another phenomenon that can modify the analyte chargend consequently the resolution is ion-pair formation. Asost of non-aqueous solvents present lower permittivity

onstant than water, ion-pair formation can be favoured.

nd ion-pairing can act as a supplementary discriminatingarameter to improve resolution of electrokinetic separa-ion. Ion-pairing effect on Ecstasy and derivatives separationn a MeCN–MeOH mixture has been clearly demonstrated

ig. 1 – Influence of the background electrolyte nature on the NAormate/1 M formic acid in MeCN–MeOH (80:20, v/v); (B) BGE 25 milica capillary 50 �m 40 cm. Applied voltage: +20 kV. Temperaturach amphetamines at 10 �g mL−1 in MeOH. Reprinted from [43]iley-VCH Verlag.

; THF, tetrahydrofuran; NMF, N-methylformamide; DMF, dimethyl-

[43]. The results have shown that in non-aqueous electrolyteselectivity and resolution can be improved as a function ofelectrolyte nature (Fig. 1). Experimental mobility values havebeen compared to theoretical mobilities to discriminate ion-pairing from ionic strength effect. Ion-pairing between acetatecounter-ion and amphetamines has been demonstrated andion-pair formation constants ranging from 6.2 to 11.8 L mol−1

have been obtained. Last year, a similar approach has beenadopted to evaluate hydrogen-bond effect and ion-pair asso-ciation on the separation of neutral calyx[4]pyrroles by NACEin MeCN electrolytes [44]. This paper has shown that thehydrogen-bond effect is not the only phenomenon involvedand that ion-pair association also occurred and influenced the

selectivity and therefore the resolution.

Considering the possibility to modify the separation selec-tivity and resolution by using non-aqueous solvents, solvationis a phenomenon that can be involved and play a major

CE amphetamines separation. (A) BGE 25 mM ammoniumM acetate ammonium/1 M acetic acid (80:20, v/v). Baree 25 ◦C, hydrodynamic injection. UV detection at 200 nm.. Copyright 2008, with permission from

a c t

12 a n a l y t i c a c h i m i c a

role. Solvation phenomenon is governed by ion-solvent andsolvent–solvent interactions. Gareil’s group has studied theinfluence of MeOH percentage in the background electrolyte(BGE) on the separation of four inorganic anions (I−, Br−, SCN−,NO3

−) [45]. In the case of these anions, the conjugated acidsremained strong whatever the MeOH percentage. Then sol-vent effect on resolution could only be due to ion-pairingand/or modification of electrophoretic friction coefficient. Bydetermining interaction constants, ion-pair formation wasdemonstrated of minor importance to explain selectivitymodification observed as a function of methanol content.In order to go deeper into the understanding of solvationinfluence, both experimental and theoretical studies havebeen performed. Whereas the theoretical model calculatedby the density functional theory (DFT) allowed explainingthe mobility selectivity in water, the approach was less effi-cient for predictions in methanol BGE. Solvation phenomenain non aqueous solvents were exploited to separate �-helicaloligopeptides [46]. This paper showed that higher separationresolution for polypeptides composed of 14–31 amino-acidsresidues was generally achieved in non-aqueous electrolytescompared to aqueous ones. The better results obtained inNACE can be due to a modification of the peptide secondarystructure according to the solvent nature of the BGE. Parallelmeasurements performed by circular dichroism have shownthat helical structure of polypeptides is favoured in methanolbuffers. This is certainly due to solvation effect and canexplain the selectivity modification observed when replacingwater by other solvents. Nevertheless, this assumption cannotexplain the similar trend observed for smaller oligopeptides.And in this case, pKa shift can also be involved in the selectivitymodification observed. Another paper illustrates the possibil-ity to improve the separation resolution by using non-aqueoussolvents for the separation of the positional isomers of a sul-fated monosaccharide, the positional isomer referring to thesulfated group [47]. While no efficient strategy was obtainedfor baseline resolution in aqueous BGE (BGE nature, pH, ionicstrength, borate complexation), this separation was achievedat alkaline pH with a BGE containing MeOH–EtOH at differ-ent proportions, the resolution being dependent of the solventcomposition (Table 2). This study illustrates the usefulness of

NACE to separate analytes with theoretical identical charge-to-mass ratios without any additives except the presence ofnon-aqueous solvent. Nevertheless, it is to notice that noquantitative experiments were performed to evaluate the own

Table 2 – Influence of the nature and composition of thesolvent on the resolution of positional isomers ofmonosulfated fucose

Solvent (%, v/v) Resolution

MeOH No effective separationMeOH–water (30:20) No effective separationMeOH–ACN (80:20) No effective separationMeOH–EtOH (80:20) No effective separationMeOH–EtOH (60:40) R1/2 = 0.5, R2/3 = 1.0MeOH–EtOH (50:50) R1/2 = 1.1, R2/3 = 1.7MeOH–EtOH–i-PrOH (40:40:20) R1/2 = 1.3, R2/3 = 2

Reprinted from [47]. Copyright 2008, with permission from Elsevier.

a 6 2 8 ( 2 0 0 8 ) 9–23

contribution of solvation and ion-pairing on the separationresolution.

Solvation phenomenon is also primordial to increase thesolubility of numerous analytes and additives. Some recentpapers illustrate this aspect. As described by Lämmerhoferin a review on chiral separation by capillary electromigra-tion techniques in non-aqueous media [48], non-aqueoussolvents present the advantage to solubilize numerous ana-lytes that are poorly hydrosoluble. Moreover, various chiralselectors present low water solubility and the use of non-aqueous solvents allows to reach the optimal concentrationleading to better resolution. Huang et al. described the enan-tiomeric separation of poorly hydrosoluble organophosphoruspesticides with pure non-aqueous electrolyte and with a mix-ture of aqueous and organic solvents [49]. An electrolytecontaining a mixture of MeOH–MeCN–water in the pres-ence of sodium cholate and �-cyclodextrine (CD) allowedachieving the enantioseparation. In this hydro-organic BGE,non-aqueous solvents were used to solubilize the analyteswhereas water was used to increase the CD solubility inorganic solvents. Furthermore, amide-type solvents are oftenused in NACE with CDs because the solubility of theseselectors can be increased. A paper has shown that �-CDcan be dissolved at concentration 40 times higher in N-methylformamide (NMF) than in water [50]. Crommen andco-workers have also emphasized the use of methanol to dis-solve amino �-CD derivatives they tested for enantiomericdrugs separation [51]. The influence of CD nature and con-centration and the BGE concentration on the enantiomericresolution have been evaluated by a multivariate approach. Amaximum resolution has been obtained for all the analytes innon-aqueous electrolyte containing 20 mM of six monodeoxy-6-mono(3-hydroxy)propylamino �-CD and 40 mM ammoniumacetate in MeOH. In the case of chiral separation, the improve-ment of resolution can also be due to a shift in bindingconstants in the presence of organic solvents. Binding con-stant is indeed solvent dependent and solvation of selector,solute and selector–solute associates plays a major role. Somereviews illustrate very well the advantages to use organic sol-vents for enantiomeric separation in particular with the goalto improve resolution [48]. As indicated previously, glycerolwas demonstrated as an ideal solvent suitable to improveresolution for protein separation by CIEF, compared to classi-cal aqueous CIEF [26]. Furthermore, the study of hydrophobicproteins, a mixture of bacteriorhodopsin isoforms, has beenperformed with either the aqueous or the glycerol–watersystem. Whereas clogging occurred with the aqueous CIEF,due to protein precipitation, bacteriorhodopsin isoforms weresuccessfully baseline separated with the glycerol–water CIEF.This new method appeared to be a promising alternative toconventional aqueous gel CIEF for hydrophobic protein char-acterization under native conditions.

2.2. Isoelectric buffers

Zone electrophoresis using isoelectric buffers (IEBs) is an old

concept since the pioneers have published their studies atthe end of the eighties. These papers were dealing with slabelectrophoresis [52], free flow electrophoresis [53,54] and cap-illary electrophoresis [55]. Amongst others, Hjerten et al. have

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bsnbs(gTssttiitsicCrcrppmtmmsipauti2luawq

a n a l y t i c a c h i m i c a

escribed the separation of proteins by CZE in isoelectricysine solution. A few years latter, Righetti et al. have evaluatedour acidic amphoteric buffers namely cysteic acid, iminodi-cetic acid, aspartic acid and glutamic acid [56–59]. Righetti’sroup that is one of the major actors in this field gave a moreundamental contribution with papers dealing with funda-

ental properties of IEBs for CZE and gave guidelines to selectroperly IEBs [60,61]. The main advantage of using IEBs for CZExperiments is their low conductivity, which allows the use ofigh electric field without observing significant Joule heatingffect. Thus very short analysis times can thus be expected.lthough the use of amino acids in particular as IEB has showneveral advantages, more theoretical studies have brought these of low conductivity background electrolytes into question

62,63]. In this context, more recent papers have confirmed theajor advantages that can be expected by employing IEBs to

mprove electrokinetic separation performed either in the CZEr the CIEF mode.

The last few years, different types of IEB or quasi-isoelectricuffers (QIEBs) have been used in the field of separationciences and have demonstrated their advantages [64,65]. Aew compound class of synthetic IEB has been introducedy Rodemann et al. [66]. This compound is designed as amall molecule with one fully or prevailingly dissociated groupsulfonic–carboxylic) and two partly protonated basic aminoroups attached onto a hydrophilic UV-transparent backbone.his new class of IEB has been successfully used for highensitivity indirect detection of a mixture of eleven anionseparated by CZE. Indeed the use of an isoelectric buffer bringshe advantage of buffered electrolytes without the concomi-ant introduction of co-ions that would be detrimental tondirect detection. Similar performances as with conventionalndirect photometric detection have been obtained and no sys-em peak due to additional co-ions has been observed. As theynthesis of new and suitable IEB is expensive and labour-ntensive other IEB preparations have been considered. Thearrier ampholytes (CA) that design the pH gradient duringIEF, can be easily fractionated to obtain narrow pH cuts of car-ier ampholytes that can be used as QIEB. This fractionationan be done by different methods. Busnel et al. obtained nar-ow pH cuts by preparative gel (sephadex G-75) [67]. In a recentaper, Antionoli et al. prepared ultra narrow pH cuts from 2-H unit wide carrier ampholytes, and presented two differentethods to obtain these QIEB for subsequent CZE separa-

ions [68]. The first procedure to sub fractionate the ampholineixture is carried out by a preparative IEF in multicompart-ent electrolyzers (MCE) with immobiline membranes. The

econd approach used a Rotofor® unit to prepare the quasi-soelectric buffers, that allowed to drastically decrease the IEBreparation time. In this case, the time required to fraction-te a CAs mixture is about 2 h and leads to 20 fractions. Priorsing these IEB as BGE to perform electrokinetic separation,his CAs fractionation has to be validated by the character-zation of the IEB obtained. Busnel et al. have characterized5 fractions they have obtained by focusing CAs on granu-ated gels, they have shown that if the narrow pH cuts are

sed at 0.8% (w/v), 20 fractions of the 25 prepared presentppropriate buffering capacity to provide satisfying separationhile maintaining low conductivity [67]. Furthermore theseuasi-isoelectric buffers composed of CAs have also been char-

6 2 8 ( 2 0 0 8 ) 9–23 13

acterized in terms of loading capacity [69]. Indeed due to theirisoelectric character, these IEB are expected to present a lowloading capacity. Even so the experiments have shown thatthe IEB tested present rather high loading capacity of thesame order of magnitude as classical buffers and far higherthan another classical IEB (an electrolyte of 10 mM histidine).Righetti’s group has characterized the different fractions theygot by MCE and Rotofor®, by measuring their pH and con-ductivity [68]. These results suggested that narrow pH cutsof carrier ampholytes should be an interesting alternativeto more conventional electrolytes to perform electrophoreticseparations.

Busnel et al. used these QIEB to perform protein sepa-ration by CZE. Fast separation (less than 2 min) as well ashigh efficiency (500 000 pL m−1) has been achieved for the sep-aration of a protein test mixture under very high electricfield (superior to 1000 V cm−1) [70]. It was also very importantto notice that compared to classical BGEs, namely sodium-phosphate and sodium-N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonate) (HEPES) the resolution was improved byusing narrow pH cuts of carrier ampholytes as BGE. In the caseof phosphate and HEPES buffers, very high ionic strength wasnecessary to achieve the protein test mixture separation thatimplied the application of lower electric fields to avoid Jouleheating effect. Righetti’s group has also used this type of IEBto perform protein separation by CZE [68]. They have shownthat the use of narrow pH cuts of carrier ampholytes as BGEallows to improve drastically the separation of a mixture of 10pI marker proteins at pH 7.9 (Fig. 2). This separation has beenperformed by CZE using an uncoated capillary. At this pH thesilanols of the silica wall should be ionized and the proteinsshould be strongly adsorbed on the silica wall. In this case,a unique property of these buffers to coat the capillary wallhas been highlighted. When using classical phosphate buffer,the resolution is indeed dramatically affected by the proteinadsorption whereas using IEB prevents the protein adsorptionleading to higher resolution. Interaction between analytes andampholytes could also been involved and explain the resolu-tion improvement achieved when using QIEB.

Recent papers of Peltre’s group have also illustrated thehigh potential of these quasi-isoelectric buffers of carrierampholytes to perform efficient separation by CZE. The ver-satility of these QIEB has been demonstrated by using themto enhance sensitivity by stacking process [71] as well as bytransient isotachophoresis [72]. These IEB have also proventheir high capability to separate complex samples, in particu-lar tryptic digests. High resolution were achieved even for largeprotein digest as the one of human transferrin (77 080 Da) [73].

These narrow pH cuts of carrier ampholytes have alsoproven their ability to improve the resolution of separationperformed by capillary isoelectric focusing. It is already wellknown that it is possible to increase resolution in a givenzone of the pH gradient by adding an amphoteric compoundto the initial ampholytes mixture [74,75]. Recently, Poitevinet al. have used narrow pH cuts of carrier ampholytes toimprove protein separation by CIEF [76]. The objective was

that the ampholytes of the narrow pH cut could interposethemselves between the initial ampholytes leading to a flat-tening of the pH gradient around the pH of the narrow pH cutadded. The separations of pI markers obtained in presence

14 a n a l y t i c a c h i m i c a a c t

Fig. 2 – Comparison of 10 pI marker proteins separation inclassical electrolyte (A) or quasi-isoelectric buffer (B). (A)Phosphate buffer at 100 mM pH 7.9. (B) Ampholine cuthaving a nominal pI of 8.29 at an ampholine concentrationof 1.4%; Capillary 50 �m × 33.5 cm. Running voltage: 25 kV,temperature: 25 ◦C, hydrodynamic injection (30 mbar/3 s),

UV detection at 214 nm. Sample at 0.2 mg mL−1. Reprintedfrom [68]. Copyright 2008, with permission from Elsevier.

or absence of the narrow pH cut in the ampholytes mixturehave been compared. When adding a narrow cut of pH 4.9, theresolution between adjacent pI markers respectively of 4.75and 4.85 was more than doubled due to the flattening of thepH gradient. Similar trend has been observed for the separa-tion of milk proteins. When adding narrow pH cut up to 3%(v/v), �-lactoglobulin A and B (pI 5.25 and 5.35) were sepa-rated illustrating the gain in resolution. Nevertheless higherconcentration cannot be used as it leads to a loss in sensi-tivity when using CE–UV. Advantage has also been taken ofthese narrow pH cuts to improve resolution of CIEF separa-tion by using them as anolyte and catholyte [77]. This workillustrates that narrow pH cuts can be considered as versa-tile buffers to improve CIEF experiments. The separation ofpI markers by CIEF is indeed dependent on the fraction ofnarrow pH cut used as anolyte or catholyte. These narrowpH cuts have been compared to more classical anolyte andcatholyte using a CIEF setup consisting in a bare silica capil-lary and 70/30 water glycerol separation medium. The resultshave shown that the resolution between neutral and acidicproteins is doubled when using narrow pH cuts. Further exper-iments have been performed to investigate the phenomena

involved. And it has been highlighted that in this case, EOFplays a crucial role when using bare silica capillary and that itis decreased drastically when using narrow pH cuts as anolyteand catholyte.

a 6 2 8 ( 2 0 0 8 ) 9–23

3. A new type of additives for resolutionimprovement: ionic liquids

A great interest is being drawn towards ionic liquids (ILs)as alternatives for conventional molecular solvents used inorganic synthesis and catalytic reactions. They supplementthe family of “green solvents” including water and supercriti-cal fluids. Amongst these, room temperature ionic liquids aredefined as materials containing only ionic species and havinga melting point below 100 ◦C. They exhibit many interestingproperties such as negligible vapour pressure, low meltingpoint, large liquid range, unique solvation ability and over-all, the versatility of their physico-chemical properties makesthem really attractive.

During these last years, a great attention has been paid tothe relevance of these new media for CE, that allow in somecases to enhance resolution, peak efficiency and peak symme-try. Due to their high viscosity and conductivity, ILs are mainlyused as additives in background electrolytes (BGE) and manyefforts have been directed towards the understanding of theseparation mechanisms involved in IL-containing BGE. Firstof all, ILs present high ability to dissolve compounds due totheir unique properties. Secondly, they have a high tendencyto be adsorbed onto the silanol groups of the capillary wall,which provides dynamic coatings [78,79]; furthermore, theycan change the conductivity and viscosity of the BGE [80].These characteristics allow modifying/inversing the electroos-motic velocity that may help for resolution enhancement.Thirdly, they can give rise to new interaction systems, espe-cially the ion-pair or ion-dipole formation, but also otherinteractions such as Van der Waals interactions or hydro-gen binding, according to the nature of the cation. As ILs areemployed diluted in a separation buffer, ILs cations and anionscan be dissociated under an electric field, each one provid-ing a selective interaction [81]. A priori, the cation of the ILhas a more relevant effect on CE resolution as (1) it can inter-act with the analyte but also with the capillary wall, and (2)competitive interactions can occur between analyte and theIL cation adsorbed onto the capillary wall or the free IL cationpresent in the BGE. However, the IL anion can also have aconsiderable effect on CE resolution that may be explainedby its interaction with the IL cation or the analyte. Somereviews detail the most important applications in various elec-trophoretic modes, i.e., CZE either in aqueous or non-aqueousmedia (NACE), electrokinetic chromatography (EKC) and capil-lary electrochromatography CEC [81–84]. We will focus on thelatest results obtained in aqueous and non-aqueous CE, witha special interest in the role of IL for resolution enhancement,i.e. either as interacting additive or electroosmotic flow (EOF)modifier.

In aqueous CE separations, IL can serve as EOF modifierfor either covalent or dynamic coating. The objectives aredual: to prevent adsorption of both small and large cationicmolecules, and to diminish or even reverse the EOF veloc-ity classically obtained in bare-fused silica capillaries. These

two phenomena can lead to resolution enhancement for CEseparations. The first attempt to reduce wall adsorption by ILcoating was done by Qin and co-workers. The coating resultedin anodic EOF and showed an enhancement in separation

a c t a

rlimtMtfdptDaaccptccaa[mwvdImp1e

m[acigctttsmccpt8Tgeoaortwa

electrophoretic mobilities, while at higher concentration, the

a n a l y t i c a c h i m i c a

esolution of both small molecules (sildenafil and its metabo-ites [85], metal ions and ammonium ions [86,87], inorganicons and alkyl phosphonic acids and esters [88]) and large

olecules [89]. The IL-coating showed no sign of any substan-ial deterioration for at least 96 h, and was compatible withS detection, contrary to dynamic modification with ILs. On

he other hand, dynamic coating is the easiest way to per-orm wall modification. The adsorbed IL cations produce aiminution in EOF value and even a change in direction; thearameters affecting this modification are the IL concentra-ion in the BGE and the alkyl chain length of the IL cation.ynamic coating with imidazolium-based ILs to prevent walldsorption is very easy to perform, as IL is introduced as andditive in the BGE, and has been widely employed for effi-ient baseline separation of various compounds: polyphenolicompounds in grape seed extracts [79], monohalogenatedhenols [90], carboxylates [91], nicotinic acid and its struc-ural isomers [92], anthraquinones [93], benzoic acid andhlorophenoxy acid herbicids [94], basic proteins (lysozyme,ytochrome c, trypsinogen and a-chymotrypsinogen) [95],mino acids (tyrosine, phenylalanine and tryptophan) andcidic drugs (fenoprofen, ibuprofen, ketoprofen and naproxen)96]. In all these studies, the authors suggest that the

echanism of separation is mainly due to the associationith the IL cation (mainly 1-ethyl-3-methyl-imidazolium)

ia electrostatic, hydrophobic, hydrogen-bonding, or ion-ipole/ion-induced interactions, with the additional effect of

L cation adsorption on the capillary wall leading to EOFodification and thus resolution modification. One work [97]

resents the baseline separation of four anthraquinones using-butyl-3-methylimidazolium-based IL as the main runninglectrolyte solutions, with �-CD as modifier.

Ionic liquids provide an interesting alternative to organicodifiers in MEKC for the separation of hydrophobic mixtures

98]. They present several advantages over organic solventss modifiers, including better water solubility and electri-al conductivity. They have been used either as a modifiern an MEKC medium or as a micellar component. Mwon-ela et al. [99] introduced for the first time ionic liquids inlassical MEKC for the baseline separation of two achiral mix-ures and one chiral mixture. 1-Butyl-3-methylimidazoliumetrafluoroborate (BMIM-BF4) was used as modifier to improvehe separation of lignans found in seeds by sodium dodecylulfate (SDS) MEKC, and was shown to provide a comple-entary method for the separation of members of this

lass of compounds [100]. It was speculated that the BMIM+

ation modifies the SDS micelles changing the micellar soluteartitioning. Qi et al. [101] developed the separation ofhree model analytes (kaempherol-3-O-glucoside, 7-hydroxy--methoxycoumarin and 8-hydroxycoumarin-7-O-glucoside).he addition of IL to the conventional BGE not only providedood resolution but also prevented Joule heating effect at highlectric field strengths. Mwogela et al. [102] compared the usef ILs and conventional molecular organic solvents (MOSs) toqueous BGE containing molecular micelles for the separationf chiral analyte mixtures. They showed that chiral and achiral

esolution and selectivity were dependent on the concentra-ion and type of modifier used. However, smaller IL volumesere needed, as compared to MOSs, in order to achieve equiv-lent chiral resolution and selectivity. Baseline separation was

6 2 8 ( 2 0 0 8 ) 9–23 15

obtained with BMIM-BF4 as additive in a classical microemul-sion electrokinetic chromatography for the analysis of activeflavones [103]. BMIM-BF4 could partially change the characterof the microemulsion droplets, which improved the separationaccordingly. Novel pseudo-phases based on ILs were studied[104] and proposed for electrokinetic chromatography. Theyare mainly imidazolium based ILs, if the alkyl tail of the ILcation is long enough. Long chain (C12 and C14) alkylimida-zolium IL were used as pseudo-stationnary phases for thebaseline separation of isomers of methylresorcinol and theseparation of some hydrophobic benzene derivatives [105].Two amino acid-derived (leucinol and N-methylpyrrolidinol)chiral ionic liquids were used as pseudostationnary phase inMEKC [106] and provided different chiral resolution depend-ing on the IL nature. The authors suggested that the rigid ringsystem of the IL surfactant undecenoxycarbonyl-l-pyrolidinolbromide apparently allowed a three-point interaction with (±)�-bromophenyl acetic acid, which is in correlation with theenhanced chiral selectivity for rigid molecules when employ-ing surfactants derived from l-proline (a rigid amino acid)[107]. These studies indicated that in this case the chiral res-olution was greatly enhanced not only due to electrostaticinteractions, but to hydrogen bonding as well. Interestinglythe monomers of the IL provided better chiral resolution thanthe corresponding polymers.

In NACE, which utilizes pure organic solvents or theirmixtures, the BGE is less polar than aqueous electrolytes.NACE separations are less affected by the EOF than aque-ous CZE. Seiman et al. showed that, when employing IL asadditives in the non-aqueous BGE, no EOF inversion seemedto occur, but the EOF generally diminished when IL concen-tration increased, except for formamide [108]. This exceptionwas explained by the fact that formamide should not be ableto destroy the ion-pairing between the cationic and anionicparts of IL. It is to note that when separation was performedin hydro-organic media (5% MeCN in water), an inversionof EOF occurred [94] and a baseline resolution was obtainedfor the separation of 2,5-dichlorobenzoic acid and 3,5-dichlorobenzoic acid. In general, the solute–IL interaction thusseems to be the main mechanism responsible for enhancedresolution in NACE. Vaher et al. were the first to use ILs inNACE, either with an aprotic solvent (MeCN) or a protic solvent(MeOH), high resolutions were achieved for dyes [109], car-boxylic acid and phenols [110] and polyphenols [79], while noseparation could be observed without the alkylimidazolium-based IL. As an explanation, the authors suggested differentdegrees of heteroconjugation between uncharged analytesand the anion of the IL (allowing the analyte to acquire somenet charge) in MeCN, whereas no such association was possi-ble when the same IL was added to the methanol because thehydrogen bonding prevented the aforementioned association.N,N′-alkylmethylimidazolium cations have been successfullyseparated in NACE when one N,N′-dialkylimidazolium-basedIL was used as an electrolyte additive to the organic solventseparation medium [111]. At low IL concentration in MeCN,the ion pairing may occur, that rapidly changed the effective

additives successfully facilitated the separation of IL cationsin these conditions. A deeper insight was performed into thephysico-chemical interactions coming into play between a

a c t a 6 2 8 ( 2 0 0 8 ) 9–23

Fig. 3 – Enantioseparation of carprofen in the presence ofTM-�-CD and chiral ILs. Fused silica capillary, 50 �m i.d.,35 cm (effective length, 26.5 cm). Electrolyte: 2.63 mM aceticacid, 5.0 mM sodium acetate buffer, pH 5.0 containing (a)30 mM TM-�-CD, (b) 30 mM TM-�-CD + 10 mM EtCholNTf2,(c) 30 mM TM-�-CD + 10 mM PhCholNTf2, (d) 30 mMTM-�-CD + 10 mM LiNTf2 in (90:10, v/v) H2O/MeOH mixture.Applied voltage: 25 kV, temperature: 25 ◦C, UV absorbanceat 230 nm, Hydrodynamic injection (30 mbar, 3 s). EOF:electroosmotic flow. Reprinted from [113]. Copyright 2008,

16 a n a l y t i c a c h i m i c a

series of arylpropionic acids (“profens”), almost fully disso-ciated, and a water-insoluble imidazolium-based IL in NACE(MeCN–MeOH) [112]. As explained for aqueous separationelectrolytes, ILs may have a double state, either free in the BGEor adsorbed on the capillary wall. Competitive ion-pair inter-actions between anionic analytes and IL cation, either free insolution or adsorbed onto the capillary wall, were proposed,that could explain resolution enhancement. For the lowest ILconcentrations, a chromatographic-like interaction betweenthe analyte and the IL cations adsorbed onto the capillarywall seems to occur, leading to increased migration times.Conversely, for the highest IL concentrations, the decrease inelectrophoretic mobility suggested that an ion-pair interac-tion between the anionic analyte and the free IL cations inthe BGE might become prevailing after the capillary wall hadbeen fully coated with the IL cation. Francois et al. [113] furtherperformed the evaluation of two chiral ILs (ethyl- and phenyl-choline of bis(trifluoromethylsulfonyl)imide, ETCholNTf2, andPhCholNTf2, respectively) by CE. No direct eniantioselectivitywas observed for these two chiral IL cations with respect to aseries of arylpropionic acids, selected as model compounds, invarious non-aqueous BGE conditions. BGEs containing both achiral IL cation and a classical chiral selector (di- or trimethyl-�-cyclodextrin) in water and water–methanol mixtures weresubsequently investigated to look for a compromise betweenthe selective formation of inclusion complexes, favoured inaqueous electrolyte, and ion-pairs, favoured in non-aqueousmedia. In most cases, an increase in resolution was observedupon adding one of the chiral IL, but this variation was mostoften due to a decrease in EOF, resulting from the increase insalt concentration and a possible wall adsorption. However,simultaneous increase in effective selectivity and resolutionwas observed in some cases, which suggests a synergisticeffect of the two selectors (Fig. 3). Apparent inclusion constantfor both chiral IL cations and the used CDs were evaluated,demonstrating an influence of the CD nature on the competi-tion between the analyte and the IL cation with respect to CDcomplexations [114].

A room temperature IL (RTIL)-mediated non-hydrolyticsol–gel (NHSG) protocol was explored for the fabrication of newmolecularly imprinted silica-based hybrid monoliths for effi-cient baseline resolution chiral separation of a basic templatezolmitriptan by CEC [115].

Very recently, ILs were employed in microchip elec-trophoresis, mainly as EOF modifier [116,117]. Zeng et al. [118]used 1-ethyl-3-methylimidazolium tetrafluoroborate aqueoussolution for the baseline chiral separation of dipeptides inglass microchannel. Compared to boric acid BGE, it exhibiteda broader separation window, with a unique EOF generationmechanism probably due to a possible hydration of the ILcation in water. These RTILs aqueous solutions seem for theauthors a revolution in the analytical methods of chiral orconformational analysis for biomolecules.

4. Microdevices dedicated to electrokinetic

separation: what about resolution?

As previously mentioned, during the last decade, the num-ber a papers dealing with miniaturized systems has been

with permission from Elsevier.

tremendously increasing, due to the numerous advantagesof microdevices. One of the major benefits of miniaturiza-tion, which is intrinsic of the microdevice dimensions, isthe decrease of sample, electrolyte and reagent consump-tion compared to classical dimensions. The decrease ofanalysis time also represents a major advantage as highthroughput analysis can be considered. The opportunity toperform different analytical operations on one chip also con-tributes to the development of miniaturization. In this context,the field of separation sciences has been widely investi-gated on microfluidic devices. The potential of electrokineticand chromatographic separation has been widely demon-strated in miniaturized format by separation according todifferent modes: zone electrophoresis [119–121], electrochro-matography [122–124], free flow electrophoresis [125,126], gelelectrophoresis [127,128], isoelectric focusing [129,130], isota-chophoresis [131]. And reviews dealing with the applicationsof microdevices for analytical issues have been published[132–135]. Many papers published in this field have highlightedthe possibility to increase resolution by using microdevices.

The microchip microfabrication permits to adapt and tooptimize the chip design according to the requirements toachieve the best separation. Different materials have beenused to fabricate microdevices: glass, silicon and polymersubstrates [136]. The first materials that have been usedare glass and silicon due to the techniques developed bythe microelectronics industry [137–141]. However the fabri-cation process is time-consuming and requires specializedfacilities and the materials are also expensive and frag-

ile. To overcome these drawbacks, different polymers havebeen investigated. The fabrication of polymers microdevicesis based on mass replication technologies (hot embossing,injection molding) or on methods for rapid prototyping (cast-

a c t a 6 2 8 ( 2 0 0 8 ) 9–23 17

imap

ttrdotcpzotacottwtbptfronroctdtbuaic(dWbimD(mDnhpDepota

Fig. 4 – separation of SDS-denaturated proteins usingcolloidal array of 160-nm silica particles in microfluidc chip.(1) trypsin inhibitor 20.1 kDa (2) ovalbumin 45 kDa (3) BSA66 kDa (4) phosphorylase B 97 kDa (5) �-galactosidase

the sum of the squares of the resolution in the two systems,

a n a l y t i c a c h i m i c a

ng, laser micromachining) [142–144]. The most widely usedaterials are polymethylmethacrylate (PMMA), polycarbon-

te (PC), polystyrene (PS), polyethyleneterephthalate (PET) orolydimethylsiloxane (PDMS).

Due to the wide variety of materials and of microfabrica-ion technologies, the chip design can be optimized accordingo the separation requirements. In this context a paper hasecently described the development of a novel microdeviceedicated to electrokinetic separation [145]. It presents anptimized combined injection system/expansion chamberhat allows injecting a flatter sample bands with increasedoncentration intensity into the separation channel. For thaturpose, this chip consists in a conventional cross-injectionone and an expansion chamber that is located at the inletf the separation channel. Numerical simulation has shownhat an expansion chamber with an expansion ratio of 2.5 andn expansion length of 500 �m delivers a sample plug with aorrect shape and orientation that should lead to a better res-lution. But most of the papers that demonstrate the potentialo tune the chip design to enhance the resolution as a func-ion of the considered electrokinetic separation mostly dealith DNA molecule separation. A microfabricated entropic

rap array has been described to separate large DNA moleculesy electrophoresis in the absence of sieving matrix [146]. Thisaper emphasizes the possibility to improve resolution byuning the chip design. Entropic traps are regular and micro-abricated structures that act as molecular sieve. In this case,esolution and band broadening depends on specific factorsf the entropic trap mechanisms. For example, the diffusion isot considered as a main source of dispersion because the bar-iers virtually block DNA diffusion across the shallow regionf the barrier. And dispersion is mainly due to the statisti-al variation of the entropic trapping life time. Resolution canhen be enhanced by increasing the number of traps of theevice. This work has also demonstrated the influence of elec-ric field on resolution because DNA molecules are not relaxedefore they are trapped. Cox and co-workers also reported these of entropic effect [147]; in this case, the entropic focusingllowed the entrance of DNA into the array in a thin band tomprove resolution. Advantage was then taken of microfabri-ation possibilities to design a hexagonal array of micropillars2 �m wide). The devices were made of quartz using stan-ard photolithography and reaction ion etching techniques.ithin the micropillar array DNA molecules were separated

y pulsed electrophoresis and based on the DNA elongationn such a device. And the migration speed depended on DNA

olecules length. Thanks to the device design, T4 and �

NA have been successfully separated in a very short time10 s) with high resolution. Baba and co-workers also reported

ore recently the use of pillars in microdevice to separateNA molecules [148]. In this work the authors described aanofabrication technique for constructing nanopillars withigh aspect ratio inside a quartz microchannel. Nanopillarsroduce a molecular sieving effect that makes possible theNA separation in absence of DNA sieving matrix. Differ-nt electropherograms are presented at different detection

oints, the resolution achieved at the third detection point isf 1.82. This paper illustrates the flexibility of the chip designhat can be adjusted to reach the better separation. Viovynd co-workers also presented an original work on long DNA

116 kDa; (2) electric field: 30.9 V cm−1. l = 4 mm, Reprintedfrom [150]. Copyright 2008, with permission from ACS.

electrophoresis in self-assembled matrixes of magnetic beadscolumns [149]. These columns were obtained by injecting anemulsion of monodisperse beads, then a magnetic field wasapplied and the beads self-organized into a quasi hexagonalarray in a PDMS microdevice. Separation of � phage, its dimerand bacteriophage T4 DNA was achieved within 150 s with aresolution greater than 2. This paper also compared theoreti-cal prediction and experimental results. They have shown thatresolution increased with the beads concentration or equiv-alently with increasing post size. More recently Harrison’sgroup reported the use of ordered colloidal array assembledinside microchannels to separate biomolecules [150]. Tak-ing advantage of microfabrication process, they developed amicrofluidic colloidal self-assembly technique to obtain effec-tive lattices of various pore sizes. These devices were used toseparate DNA fragments as well as SDS-protein complexes asa function of their size (Fig. 4). The results have shown that inthe case of DNA molecule separation, higher resolving powercan be achieved using smaller particles to reduce pore size.Concerning protein separation, the resolving power has beenestimated according to the following equation:

R = �M

Rs(1)

where �M represents the mass difference and Rs the reso-lution between two peaks. The R value they obtained wascomparable to the one achieved with microsystem using gelor polymer matrixes.

In parallel, there has been a great effort to transfer bidi-mensional separation to microchip format. In the case of twoorthogonal dimensions, it is indeed expected to achieve apeak capacity that is the product of the peak capacity of eachdimension [151]. And as the resolution is the square root of

bidimensional separations can be performed to improve res-olution. In this context, microdevices emerge as a promisingtool to perform 2D separations. Indeed minimal dead volumes

a c t

18 a n a l y t i c a c h i m i c a

can be expected due to the possibility to simplify intercon-nected channels. Different groups have been involved in thisfield especially Ramsey’s group [152,153]. They have describedthe coupling of open channel electrochromatography (OCEC)with channel zone electrophoresis (CZE) in glass microchip.The first dimension was performed in a spiral separationchannel of 25 cm and was connected to a 1.2 cm straight chan-nel for CZE separation. The authors have investigated theinfluence of sampling on the separation. And it has beendemonstrated that a higher sampling rate can improve overallresolving power. A tryptic digest of �-casein was separated onthe 2D microdevice [152]. But due to the correlation betweenOCEC and CZE, the entire 2D surface was not accessible. Thiscorrelation was observed by a diagonal location of the spots.This group has also described a 2D chip combining MEKC andCZE [153]. This paper shows the results obtained with 2D chipseparation of cytochrome c tryptic digest. The peptides werefirst separated by MEKC and every 3 s the effluent from the firstdimension was then sampled to the second dimension (CZE).The authors estimated a peak capacity ranging from 500 to1000 peaks. However this very interesting 2D chip was onlyused for peptides separation and no complex proteins mix-ture was analysed. More recently some papers have focused on2D-chip that mimic the classical 2D gel electrophoresis usedespecially for proteomics [154,155]. Griebel et al. reported thedevelopment of miniaturized 2D capillary GE device [155]. Thefirst dimension of this 2D integrated chip consisted in IEF usingimmobilized pH gradient (IPG). The second dimension (CGE)was performed in 300 microchannels. Both experimental andsimulation studies were presented. Das et al. also illustratedthe possibility to perform 2D separation in microchip [154].Their paper presents protein separation achieved in a plas-tic chip by IEF and with multichannel PAGE electrophoresis.In this case 29 parallel channels orthogonal to IEF channelwere used to perform the second dimension. The original-ity of this 2D chip is related to the presence of microfluidicpseudovalves to isolate each dimension. These pseudovalveshave been created by in-situ gel polymerization. First the pro-teins were mixed with IEF medium and introduced into theIEF channel. After IEF step, SDS was electrokinetically injectedinto the device and due to the formation of SDS-complexes,they were transferred into the second dimension. The feasi-bility of this procedure has been successfully demonstratedby the 2D separation of mixture of four fluorescently labelledproteins.

5. Hyphenation of CE with massspectrometry: a good complementarity?

The hyphenation of capillary electrophoresis with massspectrometry (MS) (CE–MS) is nowadays accepted as a mul-tidimensional analytical approach complementary and/orcompetitive to classical MS-hyphenated separation tech-niques. CE–MS combines the advantage of both techniques sothat quantitative and migration time information, in combi-

nation with molecular masses and/or fragmentation patternscan be obtained in one analysis. Due to the good comple-mentarity of both methods, their hyphenation allows a goodcompounds identification and quantification in case of over-

a 6 2 8 ( 2 0 0 8 ) 9–23

lapping peaks or even if the compounds are not separatedat all, and to help for better sensitivity. The combination ofthe rapid developments of the very powerful separation tech-nique, selective mass detection capabilities and very mildionization modes has allowed this methodology to emergeas an essential bioanalytical tool in the fields of life sciences,pharmaceutical, “omic”, environmental and forensic sciences.MS is thus nowadays coupled to CE not only for its highsensitivity but for its complementarity to CE, providing highresolution and identification power, being sometimes con-sidered as a second dimension for analyte resolution. Themain applications concern the separation of complex mix-tures of a large number of small molecules or biomolecules.We will present herein only the CE–MS separations that fullydemonstrate the interesting complementarity of both meth-ods, compared to CE–UV.

Besides optimization of the separation parameters for anefficient coupling capability, such as capillary nature, BGEpH, nature and concentration, BGE additives including typeand concentration of a chiral selector if applicable, etc., thespray chamber and transfer capillary parameters have to beoptimized as well (sheath liquid composition, nebulising anddrying gas flow rates, and temperature and spray needle posi-tion) [156,157]. Whereas the CE–MS interface generally leadsto a slight decrease in the final resolution compared to the CEseparation, the separation integrity could be preserved whileemploying the same counter-ion in separation electrolyte andsheath liquid in the case of antihistamines [158]. The greatcomplementarity of CE and MS was particularly demonstratedfor the characterization of acid hydrolysis products of (3-methacryloxypropyl)trimethoxysilane [159], and isomeric aciddegradation products of organophosphorus chemical warfareagents [160–162] (Fig. 5): while resolution was not entirelysatisfactory with CE–UV for isomeric alkyl alkylphosphonicacid, MS detection allowed to successfully characterize andquantify these products; on the other hand, CE separationwas mandatory for the identification of isomeric alkylphos-phonic acids, which led to the same fragment ion and couldnot be differentiated by MS–MS. CE–MS has recently beendeveloped as a powerful tool for metabolome analysis, as itrequires a high level of resolution, due to the huge amount ofcompounds to identify. The comprehensive, quantitative andresolutive separation of charged metabolites by CE–ESI-MSwas performed applying three different separation proto-cols [163] and current investigation in this domain is tryingto develop a single protocol [164,165]. In the more generalcontext of complex mixtures of both anionic and cationiccompounds, two effective strategies for single run baselineseparation of cationic, anionic and polyanionic compoundshas been proposed by CE–ESI-MS, and can be applied to allkinds of complex mixtures in a reasonable time (most likely inless than 30 min) and with satisfactory resolution [166]. NACEhas also been investigated for coupling with MS, as organicsolvents may favour ionization-desorption before MS detec-tion. A separation and characterization of alkaloids in severalmatrixes including chewing gums, beverages and tobaccos

was successfully performed employing non-aqueous CE–MS,that provided better separation resolution when compared toother aqueous CE–MS reports [23]. The baseline separationof chiral and achiral drugs in non-aqueous BGE, in the pres-

a n a l y t i c a c h i m i c a a c t a 6 2 8 ( 2 0 0 8 ) 9–23 19

Fig. 5 – CE–MS–MS analysis of a standard mixture of alkyl alkylphosphonic acids and alkylphosphonic acids. Bare fusedsilica capillary, 50 �m i.d. × 85 cm. Background electrolyte, 15 mM CH3COONH4 adjusted to pH 8.8 with NH3. Temperature:25 ◦C, applied voltage: +20 kV, hydrodynamic injection: 10 s, 5 kPa. Sheath liquid (75:25:2, v/v/v) MeOH/H20/NH3. Analyteconcentration, 50 �g mL−1, each in deionized water. Identification: (1) EEPA; (2) MPrPA; (3) PrMPA; (4) MEPA; (5) EMPA; (6) IPA;(7) PrPA; (8) EPA; (9) PhPA; (10) MPA. (A) CE-MS base peak electropherogram (a) and extracted ion electropherogram (m/z 95).(B) MS–MS spectra corresponding to each numbered peak obtained in CE–MS. Excitation amplitude 0.90 Vp–p; isolationw issi

ee[

baragorspi

idth, 2 Th. Reprinted from [160]. Copyright 2008, with perm

nce of cyclodextrines that migrate till the separation capillaryntrance, seems an interesting tool for drug impurity profiling167].

Concerning the separation and characterization ofiomolecules, special interest for CE–MS separations isppearing due to the great complexity of the samples. Theesolution capability of on-line CE–MS was demonstrateds a powerful tool for profiling bacterial lipopolysaccharidelycoform and isoform families [168,169] and for the analysisf complex mixtures of nucleic acid constituents [170]. High

esolution proteome/peptidome analysis of blody fluids wasuccessfully performed by CE–MS [171]. Finally, CE–MS hasroven to be as a powerful tool for interactome analysis: the

nteraction of proteins with polysaccharides has been recently

on from Elsevier.

studied with the hyphenation of frontal analysis CE (FACE)with ESI-MS, as frontal mode offered a better sensitivity thanzone mode [172]. The intact protein/polysaccharide complexbeing detected in non-denaturing sheath liquid conditions,the full resolution between the free and complexed pro-tein was achieved by frontal CE–MS, leading to interactionparameters determination, and more interestingly to themolecular mass of the complex, thus identifying the specificprotein ligand. This FACE-ESI-MS strategy opens the wayto ligand-fishing experiments performed on heterogeneous

carbohydrate mixtures and subsequent characterization ofspecifically bound carbohydrates.

Coupling MEKC with ESI-MS is very attractive and advan-tageous to achieve high resolution separation; however, the

a c t

r

20 a n a l y t i c a c h i m i c a

use of surfactant forming micelles is not compatible with ESI-MS, due to the non-volatility and high surface tension of theunpolymerized micelles. The simultaneous chiral separationand determination of ephedrine alkaloids [173,174] by MEKC-ESI-MS was performed using polymeric surfactant allowing forhigher resolution with the MS detector.

Capillary electrochromatography (CEC) is another hybridCE method where the plug like flow profile of the EOF leadsin CEC to distinctly higher efficiencies than the parabolicflow profiles of pressure driven systems in liquid chromatog-raphy (LC). The coupling of CEC has largely followed thegroundwork developed in coupling CE and MS [175,176]. Thebaseline separation of a mixture of triazines by CEC–MS usinga low-flow interface was performed successfully thanks tothe good specificity of the MS detection [177]. The on-linecoupling of packed capillary electrochromatography with ESI-MS and coordination ion spray–MS (CIS–MS) has been shownas a promising method for enantiomer analysis, providedthat for complex samples, the structural information of anMS detector is indispensable [178]. A high-efficiency peptideanalysis on monolithic multimode pressure-assisted capil-lary electrochromatography/capillary electrophoresis coupledto ESI-MS was applied to relatively complex peptide mixtures[179].

CIEF is a CE mode dedicated to separate amphoteric com-pounds, like proteins, according to their isoelectric point (pI).Analogous to 2D polyacrylamide gel electrophoresis (PAGE),CIEF–MS should be a promising alternative for fast proteomeanalysis, providing information on pI and Mr, with highprecision and accuracy, high resolving capabilities and theenhanced structural information, omitting also drawbacks ofconventional slab–gel electrophoresis, such as restrictions insensitivity and throughput as well as tedious staining pro-cedures [180]. Current research is intending to simplify theCIEF protocol for a better coupling, as for now off-line cou-pling [181] was mainly performed, since the sheath liquidnature was modified during separation [182] or a vial contain-ing the catholyte was added at the capillary outlet during thefocusing step [183,184], before connecting to MS during themobilization step. The CIEF–MS coupling with sheath liquidmodification proved the enhancement of the CE resolutionby the separation of co-migrating analytes via the MS on thebasis of their mass, and thus the viability for examination ofbiomarkers [185]. Direct on-line coupling was shown suitablefor complex peptide [186] and protein [187] mixtures, whiledemonstrating an influence of ampholytes concentration onresolution. This CIEF–MS methodology provides a consider-able dynamic range concerning the protein concentration[188], due to the concentrating capabilities of CIEF and thehigh sensitivity of MS, which helps to increase the numberof proteins identified.

Considerable efforts for the implementation of microfluidicsystems for small-scale separations are currently investigated.However, their low volume of sample injected requires sen-sitive detectors, leading to hyphenation to MS, usually viaESI. This methodology was successfully applied for the high

throughput top-down analysis basic proteins [189]. The highthroughput analysis of small molecules and peptides, withhigh efficiency and resolution, and the feasibility of proteinanalysis was demonstrated in this format [190].

a 6 2 8 ( 2 0 0 8 ) 9–23

6. Conclusion

CE is now considered as a competitive method in the fieldof separation sciences. Nevertheless, real sample analysis ofcomplex matrix as well as the “omics” developments requirehigh resolutive separation. Besides the classical strategiesthat can be applied, this review described a non-exhaustivepresentation of recent original strategies for improving elec-trokinetic separation resolutions. It has been shown that newtypes of electrolytes can improve electrokinetic separationwhile ionic liquids used as additive in the BGE can lead tobaseline separation of a wide range of analytes. No doubtthat technological developments in the fields of detectionand microfabrication have also allowed to enhance resolu-tion for CE separations. MS can indeed be considered as asecond dimension, that greatly helps for enhancing analytescharacterization for CE separations, whereas miniaturizationassociated to microfabrication progress opens the imagina-tion to improve CE separation. Some other potential originalstrategies are emerging in the few recent years, such as the useof nanoparticles for CE separations that is getting attractiveand very promising. They are employed either as dispersedadditive in the BGE or as a coating agent for the capillaryor microfluidic channel walls, and can allow the separa-tion of small molecules and biomolecules such as proteinsand DNA. It thus seems that future developments relatedto resolution enhancement will be accompanied by the useof novel compounds/materials or geometries for CE separa-tions.

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