Recent developments in capillary electrokinetic chromatography with replaceable charged pseudostationary phases or additives

Review

Iván Peric1

Ernst Kenndler2

1Faculty of Chemical Sciences,University of Concepción, Chile

2Institute for Analytical Chemistry,University of Vienna, Austria

Recent developments in capillary electrokineticchromatography with replaceable chargedpseudostationary phases or additives

Although electrochromatography in packed beds or monolithic columns has gainedenormous interest, techniques based on charged pseudostationary phases likemicelles are of high practical importance in electrically driven separation science.However, nonmicellar alternatives, e.g., using charged soluble polymers or smalleradditives are still attractive, as they allow high concentrations of organic solvents,and their application is not limited by the critical micellar concentration. This reviewdiscusses the developments in the field of electrokinetic chromatography with theseadditives in the last three years, covering ionic polymeric pseudostationary phases,dendrimers and so-called micelle polymers, but also small molecules which implementseparation selectivity due to their specific interaction with the analytes.

Keywords: Capillary electrophoresis / Electrokinetic chromatography / Polymers / Pseudosta-tionary phases / Replaceable additive / Review DOI 10.1002/elps.200305573

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 29242 Polymeric pseudostationary phases . . . . . . 29252.1 Anionic polymers . . . . . . . . . . . . . . . . . . . . . . 29262.1.1 Acrylate copolymers . . . . . . . . . . . . . . . . . . . 29262.1.2 Molecularly imprinted polymers . . . . . . . . . . 29272.1.3 Siloxane polymers . . . . . . . . . . . . . . . . . . . . . 29272.2 Cationic polymers . . . . . . . . . . . . . . . . . . . . . 29282.3 Amphiphilic block copolymers . . . . . . . . . . . 29293 Dendrimers . . . . . . . . . . . . . . . . . . . . . . . . . . 29294 Micelle polymers . . . . . . . . . . . . . . . . . . . . . . 29294.1 Achiral separations . . . . . . . . . . . . . . . . . . . . 29294.2 Chiral separations . . . . . . . . . . . . . . . . . . . . . 29304.3 Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29335 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . 29336 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 2933

1 Introduction

Electrokinetic chromatography (EKC) combines theselectivity principle of chromatography that is based onpartitioning between two nonmiscible phases, with theelectrokinetic transport of the analytes in the chromato-graphic system, caused by an electric field applied acrossthe separation distance. Noncharged analytes are trans-ported by the electroosmotic flow (EOF), a consequenceof the existence of a double layer that is formed between

Correspondence: Prof. Iván Peric, Department of Analytical andInorganic Chemistry, Faculty of Chemical Sciences, University ofConcepción, P. O. Box 160-C, Concepción, ChileE-mail: [email protected]: +56-41-245-974

Abbreviations: AGENT, allyl glycidyl ether N-methyl taurinesiloxane; AGESS, allyl glycidyl ether sulfite siloxane; AMPS, 2-acrylamido-2-methyl-1-propanesulfonic acid; APDSS, alloxypropane diol sulfate siloxane; BNA, (�)-1,1’-bi-naphthyl-2,2’-diamine; BNP, (�)-1,1’-binaphthyl-2,2’-diylhydrogen phosphate;BOH, (�)-2,2’-dihydroxy-1,1’-binaphthyl; DAGENT, dodecaneallyl glycidyl ether N-methyl taurine siloxane; DGSS, diethyleneglycol sulfate siloxane; DHCHAt, dihydrocholesteryl acry-late; Elvacite 2669, poly(methyl methacrylate-ethacrylate-methacrylic acid); HP-�-CD, hydroxypropyl-�-cyclodextrin; LAt,lauryl acrylate; LMAm, lauryl methacrylamide; LMAt, laurylmethacrylate; LSER, linear solvation energy relationship;OAGENT, octane allyl glycidyl ether N-methyl taurine siloxane;OMAt n-octyl methacrylate; OTMA, octyltrimethylammonium;PAGENT, pentene allyl glycidyl ether N-methyl taurine siloxane;PAH, polynuclear aromatic hydrocarbon; PCB, polychlorinatedbiphenyl; Polybrene, poly(N,N,N’,N’-tetramethyl-N-trimethyle-nehexamethylenediammonium); poly-D-SUV, poly(sodium-N-undecenoyl-D-valinate); poly-L-SUAL, poly(sodium-N-undeca-noyl-L-alanyl leucinate); poly-L-SUI, poly(sodium undecanoyl-L-isoleucinate); poly-L-SULV, polysodium undecanoyl-L-leucylvali-nate; poly-L-SUSL, poly(sodium-N-undecanoyl-L-seryl leuci-nate); poly-L-SUTL, poly(sodium-N-undecanoyl-L-threonyl leuci-nate), poly-L-SUVL, poly(sodium-N-undecanoyl-L-valyl leuci-nate); poly-SAU, poly(sodium 11-acrylamidoundecanoate);poly-SUA, poly(sodium 10-undecylenate); poly-SULL, poly(so-dium-N-undecanoyl leucyl leucinate); poly-SUS poly(sodium 10-undecenylsulfate; PSP, pseudostationary phase; SAGENT, sterylallyl glycidyl ether N-methyl taurine siloxane; SAm, stearyl acryl-amide; SMAt, stearyl methacrylate; tOAm, t-octyl acrylamide

2924 Electrophoresis 2003, 24, 2924–2934

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

Electrophoresis 2003, 24, 2924–2934 EKC with pseudostationary phases 2925

the liquid mobile phase and the solid surface. This doublelayer is formed mainly at the solid surface of the packedbed particles in case of electrochromatography, and atthe capillary wall in open-tubular chromatography (andcapillary zone electrophoresis as well). The mobile phasein electrochromatography is therefore driven by the EOF,and so are the neutral analytes dissolved therein.

Although many groups have developed a number of elec-trochromatographic systems in the last years, in dailypractice one is still encountered by some restrictions forthese systems. One restriction certainly lays in the practi-cal skills needed for packing the chromatographic parti-cles into the column, accompanied by the preparation ofappropriate frits to keep them inside the column. Suchlimitations led to the development of chromatographicalternatives, mainly to the fast evolution of monolithic col-umns, which is – beside theoretical reasons – related tothe more favorable kinetics of mass transport in the lattercompared to the former systems.

Another alternative to these EKC systems with a fixedstationary phase is the application of soluble and re-placeable pseudostationary phases (PSPs), with the greatadvantage that after each run a new chromatographiccolumn can be made simply by rinsing and refilling theseparation tube with a fresh background electrolyte(BGE). With such a set up, using charged micelles ormicroemulsions as PSPs, hundreds or even thousands ofapplications have been described in the literature. Theyare not the topic of the present review, which dealsmainly, in contrary, with nonmicelling additives likecharged polymers or even small molecules, dissolved inthe BGE, and interacting with the analytes. It should bementioned that this contribution also does not deal withcharged cyclodextrins as additives, which are consideredto form defined complexes with the analytes and dobehave therefore somewhat different than a phase. It isobvious that there is no sharp border between the partic-ular classes of additives – some soluble polymers canform micelles, monomeric additives form micelles abovethe critical micellar concentration, the CMC, etc. Conse-quently the classification principle in the present reviewmight be somewhat arbitrary and an overlap cannot beavoided. According to the nature of the additive, partition-ing can be based on hydrophobicity, hydrogen bonding,ionic attraction, etc. Recently, the use of additives otherthan the micelle-forming ones, which can be regarded aspseudophases in the widest sense as well, have beenreviewed [1–5].

For the development of a rational approach to plan theexperimental work, which is not only based on trial anderror, an understanding of the underlying interactionmechanisms of the PSP with the analytes is beneficial. It

is therefore the goal of this contribution to give an over-view in the field of replaceable charged PSPs for EKC,which have been published since 2000. It is meant as thecontinuation of a previous article to the same topic [1].

2 Polymeric pseudostationary phases

Polymers have shown some significant advantages withdifferent separation selectivity compared to conventionalsurfactant micelles. In addition to the desired selectivitythese polymeric systems are stable (in contrast tomicelles) under high organic solvent concentrations. Inrecent years, some reviews that include this topic havebeen published [1–4]. This section concentrates on somerelevant applications of synthetic ionic polymers as PSPs.Related papers are summarized in Table 1.

Table 1. Polymeric pseudophases

PSP Analytes Ref.

Anionic polymers(i) Acrylate copolymers

Poly(AMPS-LMAm),different feed ratios

Alkyl phenyl ketones [6]

Poly(AMPS-LAt),poly(AMPS-LMAm),poly(AMPS-LMAt),poly(AMPS-OMAt),poly(AMPS-Sam),poly(AMPS-SMAt),different ratios

Alkyl phenyl ketones,benzene derivatives

[7]

Poly(AMPS-SAm) �sweeping, � swee-ping and organic-modified separation

Alkyl phenyl ketones,quinine, proges-terone

[8]

Poly(AMPS-DHCHAt),poly(AMPS-tOAm),poly(AMPS-LAt),poly(AMPS-SAm)

Alkyl phenyl ketones,benzene derivatives,15 PAHs

[9]

Poly(AMPS-DHCHAt) PAHs (12 out of 15)within 138s

[10]

Elvacite 2669 � SDS Alkyl- and halo-sub-stituted benzenes,PAHs

[11]

(ii) Siloxane polymers

DGSS Alkyl phenyl ketones,substituted benzeneand naphthalenecompounds, PAHs

[13]

OAGENT, DAGENT,SAGENT

Alkyl phenyl ketones,substituted benzeneand naphthalenecompounds, PAHs

[14, 17]


CE

and

CE

C

2926 I. Peric and E. Kenndler Electrophoresis 2003, 24, 2924–2934

Table 1. Continued

PSP Analytes Ref.

AGESS, AGENT,APDSS, PAGENT

Alkyl phenyl ketones,substituted benzeneand naphthalenecompounds

[16]

Cationic polymersIonenes

3.6-Ionene (Poly-brene] � OTMA

Monofunctionalaromatic compounds

[19]

3.6-Ionene (Polybrene],2.4-ionene, 6.9-io-nene, 2.10-ionene,3.X-ionene

Phenols [20]

3.X-Ionene, 2.5-ionene,2.10-ionene

Alkyl phenyl ketones,phenols

[21]

2.1 Anionic polymers

2.1.1 Acrylate copolymers

Shi et al. [6–10] have shown high-speed separations ofhydrophobic solutes by EKC, comparable to that of high-speed capillary electrochromatography (CEC). The poly-meric PSPs provided efficient separations in the presenceof high concentration of organic solvents, some of themwith low electrical conductivity. These polymers weresynthesized by free-radical copolymerization of 2-acryl-amido-2-methyl-1-propanesulfonic acid (AMPS) with avariety of acrylate and acrylamide derivatives as comono-mers (Fig. 1). Linear solvation energy relationship (LSER)analysis have shown significant differences in the selec-tivity of the AMPS copolymers and sodium dodecyl sul-fate (SDS) micelles [4], whereby mainly separations of

Figure 1. Chemical structures of copolymers poly(AMPS-OMAt): nc = 7, x = CH3, Y = O; poly(AMPS-LMAt): nc = 11,x = CH3, Y = O; poly(AMPS-SMAt): nc = 17, x = CH3, Y = O;poly(AMPS-LAt): nc = 11, x = H, Y = O; poly(AMPS-LMAm): nc = 11, x = CH3, Y = NH; poly(AMPS-SAm):nc = 17, x = H, Y = NH. Reprinted from [7], with permis-sion.

neutral model compounds have been performed. Copoly-mers of AMPS and methacrylates with different pendantchain lengths were studied and a negligible difference inseparation selectivity was observed among them. Never-theless, it was shown that the spacer bonding chemistrycontributes to significant chemical selectivity differen-ces as poly(AMPS-lauryl methacrylate (LMAt)) towardspoly(AMPS-lauryl methacrylamide (LMAm)). Poly(AMPS-alkyl methacrylate) have shown a weaker hydrogen-bonding ability and less cohesiveness than poly(AMPS-alkyl methacrylamide).

Poly(AMPS-stearyl acrylamide (SAm)) has shown a nota-ble sweeping ability. Signal increase of more than 1000times for quinine, heptanophenone, and progesteroneusing sweeping in reversed-flow EKC at low pH wasobserved. Moreover, the cationic hydrophobic solute qui-nine could be concentrated up to 10 000 times, due to itsrelatively high retention factor. It was separated fromother hydrophobic solutes by using a buffer with a rela-tively high concentration of organic modifier. Detection of12.5 ppb of quinine with a signal-to-noise ratio of 15 wasachieved. Reproducible migration times and peak heightsof hydrophobic solutes were observed [8].

Copolymerization of AMPS with dihydrocholesteryl acry-late (DHCHAt) and t-octyl acrylamide (tOAm) was carriedout and characterized; the product (Fig. 2) was used asnew polymeric PSP. Efficiency with theoretical plates upto 870 000/m was achieved. LSER analysis of 20 soluteswas accomplished to study the retention mechanism.Fifteen polycylic aromatic hydrocarbons (PAHs) were al-most all resolved in the presence of 30% v/v acetonitrile.Nine benzene derivatives were separated by poly(AMPS-DHCHAt) showing a significantly different selectivity fromthat of SDS. PAHs and n-dodecanophenone show a dif-ferent retention mechanism by comparing poly(AMPS-DHCHAt) and poly(AMPS-lauryl acrylate). Only minordifference in selectivity for small aromatic compoundswas observed. On the other hand, a significant selectivitydifference for selected small aromatic compounds onpoly(AMPS-tOAm) and poly(AMPS-SAm) was found [9].A high-speed capillary-based EKC of PAHs using poly(so-dium AMPS-DHCHAt) as the PSP resulted in the separa-tion of 12 out of 15 PAHs within 138 s. The RSDs of migra-tion times were lower than 1.5% [10].

The mixed system of poly(methyl methacrylate-ethacry-late-methacrylic acid) (Elvacite 2669; Fig. 3) and SDSmicelles as PSP has been investigated. In this systemSDS has a CMC of 2 mM, and the fluorescence datashows that its microenvironment is similar to SDS alone.Moreover, by using LSER analysis the solvation proper-ties of the individual and the mixed system were com-pared. The mixed pseudophase is a weaker hydrogen



Figure 2. Chemical structuresof poly(AMPS-DHCHAt) (left)and poly(AMPS-tOAm) (right).Reprinted from [9], with permis-sion.

Figure 3. Structure of the triblock copolymer Elvacite2669.

bond donor, possesses stronger polarization propertiesand different functional group selectivities compared toSDS micelles. The functional group selectivities of non-hydrogen bonding and hydrogen bonding solutes weredetermined providing a further support for the LSER find-ings [11].

2.1.2 Molecularly imprinted polymers

In order to study molecular recognition, a novel approachthat includes the use of molecularly imprinted polymers(MIPs) as a free-moving PSP in a fritless capillary hasbeen reported [12]. Monodisperse microspheres (100–200 nm diameter) with (�)-ephedrine as the templatemolecule were obtained by polymerization of methacrylicacid and 1,1,1-tris(hydroxymethyl)propanetrimethacry-late as cross-linker. A partial filling of the capillary with0.1% w/v suspension in an aqueous 10 mM phosphatebuffer, pH 2.5, with 40% acetonitrile was used to separateracemic ephedrine. Under these conditions ephedrineenantiomers are cationic, whereas the MIPs are almostuncharged. As the EOF is low, the polymer particles arekept out from the detection window to avoid light scatter-ing. It is expected that this would be the initial step in fur-ther developments in this new type of chiral selectors.

2.1.3 Siloxane polymers

Many conventional chromatographic stationary phasesare based on silica or siloxane chemistry, so that siloxanemonomers are commercially available over a full rangeof molecular weights and structures. These chemistries

have been used by Peterson and Palmer [13–17] todevelop and characterize novel siloxane polymers as pol-ymeric PSPs for EKC. The very low aqueous solubilityof siloxane-based polymers has been overcome, butsome degradation in performance has been observed;thus, solutions must be freshly prepared daily [13]. Hy-droxyl groups of poly[dimethylsiloxane-co-[3-[2-(2-hy-droxyethoxy)ethoxy]propyl]methylsiloxane] were convertedto sulfate groups [13] obtaining the corresponding water-soluble diethylene glycol sulfate siloxane (DGSS); addi-tion to borate buffer at 1% w/v enabled the separation ofmodel hydrophobic compounds. A narrower migrationwindow resulted due to the lower electrophoretic mobilityof DGSS; thus only 10 of the 15 PAHs assayed were sepa-rated. Alternatively, hydroxyl groups in a polymethylhy-drosiloxane (PMHS) were converted into the amino-func-tional sulfonate. Then, alkyl chains were attached toincrease the lipophilic character, obtaining three differentsiloxanes as a family of PSPs, i.e., octane allyl glycidylether N-methyl taurine siloxane (OAGENT), dodecaneallyl glycidyl ether N-methyl taurine siloxane (DAGENT)and steryl allyl glycidyl ether N-methyl taurine siloxane(SAGENT) series (Fig. 4), where the amount of alkyl chainvaried from 10 up to 30%. Further allyl glycidyl ether sul-fite siloxane (AGESS); allyl glycidyl ether N-methyl taurinesiloxane (AGENT), alloxy propane diol sulfate siloxane(APDSS), and pentene allyl glycidyl ether N-methyl taurinesiloxane (PAGENT) were obtained by modification ofPMHS [16]. The methylene selectivity can be less orgreater than SDS by varying the alkyl chain and its con-centration. According to this variation a complex behaviorof the electrophoretic migration was found. Efficiencyand chemical selectivity were studied as usual, whereDAGENT with 15–20% of alkyl substitution provided thebest overall performance and a significantly differentselectively compared with SDS [14]. Nevertheless, withmost of these novel PSPs a narrower migration windowpersists, and only a partial resolution of 14 PAHs in ace-tonitrile-modified buffers was achieved [17]. Selectivityappeared to be caused by the size of the ring structureof the analyte. Selectivity differences were investigated



Figure 4. Structure of anionic siloxane surfactants. (A)OAGENT (C8), (B) DAGENT (C12), (C) SAGENT (C18). Re-printed from [14], with permission.

by LSER studies. Results reveal that siloxanes are gener-ally more cohesive, less polar, more able for interactionwith n- and �-electrons, and capable in accepting hydro-gen bonds more readily than SDS micelles [15].

Recently, a novel anionic siloxane polymer with a shorterlinker arm between the siloxane backbone and the sulfo-nate head group has been synthesized [18]. In this case,PMHS was modified with AGESS without a tertiary aminestructure found in previous siloxane-based PSPs. Theresults indicate that the structure and chemistry of thislinker arm has appreciable effects on the chemical selec-tivity of the PSPs. LSER studies show that these polymersare not as nonpolar as those previously studied. The fur-ther development of the siloxane polymers will be of inter-est due to the range of chemistries that could be devel-oped based on these backbones.

2.2 Cationic polymers

Charged polymers that have hydrophobic domains,called ionenes, can be successfully used for the separa-tion of neutral compounds in EKC, similarly to micellarsystems. Recently, a comparison of the retention pro-perties of additives like poly(N,N,N’,N’-tetramethyl-N-trimethylene hexamethylenediammonium) (Polybrene),monomeric octyltrimethyl ammonium (OTMA) and micel-lar OTMA applied as positively charged PSPs for EKC ofneutral analytes was made [19]. In all systems a previousdynamic coating of the capillary wall with Polybrene was

carried out, in order to establish an EOF directed towardsthe anode and a counter-migration of the additive. Alladditives possess a quaternary ammonium as functionalgroup. The BGE was 20 mM acetate buffer at pH 5.2.Based on the measurement of the mobility of 15 analytes(monofunctional aromatic compounds with different func-tional groups), their capacity or retention factors, ki, weredetermined in all systems. Low correlation of the ki-valuesbetween the particular systems was observed, indicatingtheir different selectivity at least for individual pairs of ana-lytes. Based on the log ki-values, LSER analysis wasapplied to elucidate which of the main types of chemicalinteraction are responsible for retention. As a result, cavityformation and n- or �-electron interactions were found tobe significant for the micellar OTMA system, which is inagreement with findings described in the literature forother ionic micellar systems. The polymeric system aswell as the monomeric OTMA system show a significantretention parameter indicating n- and �-electron interac-tions.

Synthesized 2,10-ionene and 6,9-ionene have shown abetter separation selectivity than commercially availablepoly(diallyl dimethyl ammonium) (PDADMA) and 3,6-ionene (Polybrene), for the determination of phenols byCZE as well as MEKC when compared as modifiers ofEOF [20]. Fast (subminute) separation of phenols as mod-el compounds by injecting the samples from the short endof the capillary was attained. In the CZE mode, phenolswith concentrations of approximately 0.1 ppm at detec-tion wavelength 254 nm were determined with a linearrange between 0.05–3 ppm.

The influence of the structure and concentration of the2,5-ionene, 2,10-ionene and aromatic 3.X-ionene basedPSPs in the BGE on migration and separation of neutralanalytes was examined [21]. These systems are suitablefor the separation of uncharged analytes with hydropho-bic or aromatic groups. Upon applications of the cationicionenes always a reversal of the EOF resulted due to thedynamic coating of the capillary wall.

2.3 Amphiphilic block copolymers

Micelle-like polymer aggregates are a class of self-orga-nized assemblies and provide functional fine particles,which recently have been used as PSPs [22]. Here, anamphiphilic block copolymer 1 (Fig. 5) that consists

Figure 5. Block copolymer 1.



Figure 6. Electropherogram of phenols. Samples: 1, phe-nol; 2, m-cresol; 3, o-cresol; 4, p-cresol; 5, 2-naphthol; 6,1-naphthol. Running solution: (a) 1.0 mg mL�1 1-AG,7.0 mM SDS, 20 mM phosphate buffer (pH 7); (b) same as(a) without 1-AG; (c) same as (a) without SDS. Reprintedfrom [22], with permission.

of poly[(N-acetylimino)ethylene] (hydrophilic part) andpoly[(N-pentanoylimino)ethylene] (hydrophobic part) gen-erates aggregates that provide a hydrophobic pool inaqueous media. In order to confer charges to these neu-tral aggregates, SDS was added to the BGE below itsCMC. Phenols were used as test analytes and their elec-trophoretic mobilities were near to zero in the absence ofeither the aggregate (1-AG) or SDS. When 1-AG and SDSare added together to the BGE the electrophoretic mobil-ity of phenols increased with increasing SDS concentra-tion (Fig. 6).

The self-assembly and micellization behavior of amphi-philic triblock copolymers containing polyoxyethylene,polyoxypropylene and polyoxybutylene blocks in aque-ous solution have been summarized [23]. This studyshows that semiquantitative analysis and prediction ofmicellar parameters and phase behavior are possible. Ithas been shown that the hydrophobic blocks form micel-lar cores. The coassociation behavior of a mixture of twodifferent block copolymers and the effect of block lengthon phase separation have been discussed. Conse-quently, this deeper understanding provides an incentiveto explore new applications on these self-assembled sys-tems.

3 Dendrimers

The use of a relatively new class of synthetic organicmacrocompounds called dendrimers as PSPs, whichare characterized by interesting properties like multiva-lency, encapsulation, molecular size, and molecular

entanglement is particularly interesting [24]. A survey ofdifferent separations performed utilizing dendrimers aswell as of several future plausible uses of various classesof dendrimers has been reviewed [1, 3, 25]. Generally,addition of dendrimers of different generations to theBGE provided higher performance with respect to classi-cal MEKC separations, which has been attributed partlyto a higher homogeneity of the dendrimer phase andpartly to a wider migration time window, connected tothe reduced electroosmotic mobility under the givenexperimental conditions and higher hydrophobic charac-ter of dendrimers. Nevertheless, relatively few applica-tions of dendrimers as potential EKC pseudophasehave been described, possibly because only few ofthem are commercially available. Recently, capillaryelectrophoresis has been used to separate and charac-terize poly(amidoamine) dendrimers with an ethylenedia-mine core [26] as well as to measure the electrophoreticmobility of carboxylic acid-terminated dendrimers focus-ed on its dependence on pH and ionic strength [27].From the results a critical condition was concluded fornonspecific counterion binding, which is analogues tothe well-known Manning condition for counterion con-densation on polyelectrolytes.

4 Micelle polymers

4.1 Achiral separations

Micelle polymers are high-molecular-weight surfactantsthat are usually synthesized by polymerizing a surfactant.The great variety of so-called micelle polymers can beseen in Table 2. These types of polymers have receivedparticular attention as PSPs in EKC, like those based on10-undecylenic acid. In this context, the effects of threedifferent free-radical polymerization initiators on the be-havior as PSPs of poly(sodium 10-undecylenate) (poly-SUA) and poly(sodium 10-undecenylsulfate) (poly-SUS)have been reported [28]. No significant differences inseparation efficiencies were noted with the two micellepolymers in spite of the difference in molecular weight.Consequently, it is claimed that polydispersity will not bea limiting factor in the performance of polymeric PSPs.

On the other hand, the separation of pyrethrin esters hassystematically modified including poly-SUS and acetoni-trile as constituents of the BGEs. Shorter analysis timecompared to the SDS-mediated separation and HPLCwas obtained [29]. Also, baseline resolution of nine mod-erately to highly hydrophobic polychlorinated biphenyl(PCB) congeners by EKC using poly-SUS in the absenceof cyclodextrins was obtained. Elution order for each PCBcongener depends on the degree of chlorination andhydrophobic character [30].



Table 2. Micelle polymers

PSP Analytes Ref.

Achiral separationsPoly-SUA Alkyl phenyl ketones [28]Poly-SUS Pyrethrin esters [29]

PCBs [30]Poly-SAU Alkyl phenyl ketones,

substituted benzeneand naphthalenecompounds

[31, 32]

Oligo SUA Cold medicineingredients

[33, 34]

Oligo SUS PAHs

Chiral separations

(i) Monopeptide surfactants

Poly-L-SUI BNP and BOH [38]Poly-L-SUV BNP and BOH [38]

BOH [39]Poly-D-SUV� HP-�-CD

PCBs [40]

(ii) Dipeptide surfactantsPoly-L-SUAL BNP and BOH,

benzodiazepinederivatives

[41]

Poly-L-SUVL BNP and BOHPoly-L-SUSL Benzodiazepine

derivativesPoly-L-SULV 75 Racemic compounds

including binaphthyl,paveroline, benzo-diazepinones, andcumarinic derivatives

[42]

Poly-L-SUTL, poly-LL-SULL, poly-LD-SULL

BNA, BOH, and BNP [43]

All posible combinat-ions of the L-formof alanine, valine,and leucine with theachiral glycine

BOH, BNP, lorazepam,temazepam, andpropanolol

[46]

Polymerization ofN-acryloyl-L-valineand N-acryloyl-L-ala-nine derivatives

N-3,5-Dinitrobenzoylphenylalanine iso-propyl esters

[46]

Synthesis, characterization and use of high-molecular-weight poly(sodium 11-acrylamidoundecanoate) (poly-SAU) (Fig. 7) as PSPs in EKC to separate uncharged com-pounds were described [31, 32]. This polymer has a highstability in the presence of organic modifiers and showedsignificantly different solute migration behaviors from

Figure 7. Structure of poly-SAU. Reprinted from [32],with permisson.

conventional micelles giving high separation efficiencies(� 200 000 theoretical plates/m). It is highly charged andhas a densely packed chain structure. To evaluate andcharacterize the chemical interactions that influence theretention behavior LSER was used. The remarkable fea-ture is that the system has a slightly higher capacity forelectron-pair interactions as well as a higher capacity fordipole-dipole and dipole-induced dipole interactions thanthe aqueous phase. Due to its improved selectivity, thepoly-SAU micellar system would become a new choicefor the adjustment of resolution (see Table 3).

Synthesis of oligomers derived from SUS and SUA mono-mers has been described. Degree of polymerization wasestimated to be from 3 to 9. Performance of each oligo-mer was briefly investigated in comparison with that inSDS-MEKC for the separation of components in coldmedicine [33] and hydrophobic compounds or PAHs,which were used as test analytes [34].

4.2 Chiral separations

As it is not the goal of the present contribution to give adetailed review on enantioseparations by EKC readersare referred to other reviews [35–37] for detailed discus-sion of fundamentals of chiral separations, or of applica-tions of chiral micelle polymers and combination ofpolymerized chiral surfactants and cyclodextrins. Thechiral discrimination of racemic mixtures of (�)-1,1’-binaphthyl-2,2’-diylhydrogen phosphate (BNP) and (�)-2,2’-dihydroxy-1,1’-binaphthyl (BOH) by poly(sodium-N-undecenoyl-D-valinate) (poly-L-SUV) and poly(sodiumundecanoyl-L-isoleucinate) (poly-L-SUI) in EKC separationhas been investigated by NMR and fluorescence spec-troscopy [38]. In the hydrophobic micellar pockets of thepolymeric chiral surfactants (PCSs), BNP enantiomers arelocalized and complexes with 1:1 stoichiometry areformed. A stronger affinity of the R-enantiomer than ofthe S-enantiomer of each solute to PCS was found.

The binding constants of PCS and BOH were determinedin [38] from a Benesi-Hildebrand treatment of the fluores-cence data. In consideration of the intermolecular inter-



Table 3. System constants for different EKC separation systems

SDS poly-SUS poly-SAU

Coef. S.E. S.Coef. Coef. S.E. S.Coef. Coef. S.E. S.Coef.

c �1.65 0.11 �1.86 0.09 �2.28 0.11m 2.74 0.11 0.78 2.11 0.09 0.79 1.64 0.11 0.75r 0.27 0.08 0.13 0.26 0.06 0.18 0.18 0.08 0.15s �0.37 0.07 �0.13 �0.16 0.06 �0.08 0.45 0.08 0.27a �0.23a) 0.13 �0.06 �0.27 0.11 �0.09 �0.15a) 0.13 �0.06b �1.82 0.16 �0.40 �1.05 0.13 �0.30 �1.18 0.17 �0.41

Coef., partial regression coefficient; S.E., standard error in the estimate; S. Coef., standard partialregression coefficienta) Value is not statistically significant at the 95% confidence level.Reproduced from [32], with permission

actions observed by 1H NMR data, a model which ratio-nalizes the chiral discrimination of the enantiomers ofBNP was proposed. On the other hand, the feasibility ofusing poly-L-SUV in EKC-MS has been demonstrated[39]. At optimum ESI-MS conditions, enantioseparationof BOH was successfully attained.

A dual chiral system consisting of poly-D-SUV and hydro-xypropyl-�-cyclodextrin (HP-�-CD) to achieve the simul-taneous enantioseparation of five PCBs was developed[40]. In this context, the use of a conventional chiralmicelle or a PCS as the single chiral selector is difficultdue to the strongest hydrophobic interactions betweenthe chiral PCB and the monomeric or polymeric surfac-tant. An improved chiral resolution for the PCBs wasachieved by adding HP-�-CD to the BGE containingpoly-D-SUV. Separations needed a relatively long analysistime (�50 min) (Fig. 8).

In order to investigate the effect of an extra heteroatomat the polar head group of the micelle polymer on theenantiomeric separation of binaphthyl derivatives andbenzodiazepines, four specific dipeptide-terminatedmicelle polymers, poly(sodium-N-undecanoyl-L-alanylleucinate) (poly-L-SUAL), poly(sodium-N-undecanoyl-L-valyl leucinate) (poly-L-SUVL), poly(sodium-N-undeca-noyl-L-seryl leucinate) (poly-L-SUSL), and poly(sodium-N-undecanoyl-L-threonyl leucinate (poly-L-SUTL) (Fig. 9),were synthesized [41]. In case of temazepam, oxaze-pam, BOH, and BNP the three chiral centers in poly-L-SUTL provided improved resolution over that of twochiral-centered dipeptide-terminated micelle polymer(Fig. 10). It has been stated that the presence of the extraheteroatom on the polar head group of poly-L-SUTL hasa predominant influence on the chiral recognition mech-anisms.

Figure 8. Electropherograms showing comparison ofSDS to poly-D-SUV for the simultaneous enantiosepara-tion of five PCBs. Reprinted from [40], with permission.

Moreover, enantioseparation of 75 racemic compoundswith polysodium undecanoyl-L-leucylvalinate (poly-L-SULV) as chiral PSPs at neutral and basic pH was investi-gated [42]. As a result, a total of 58 out of 75 racemic com-pounds were resolved under optimum concentration ofpoly-L-SULV. Even though anionic chiral analytes are diffi-



Figure 9. Chemical structures of binaphthyl and benzo-diazepine derivatives. Reprinted from [41], with permis-sion.

Figure 10. Comparison of various micelle polymers forthe enantiomeric separation of (�)-binaphthol. (A) Poly-L-SUAL; (B) poly-L-SUVL; (C) poly-L-SUSL; (D) poly-L-SUTL. EKC conditions: (A)–(D) 100 mM Tris, 10 mM borate(pH 10); applied voltage, 30 kV; current, �48 �A; detec-tion, 254 nm; temperature, 25�C; pressure, 5 s�10 mbar;sample stock, 1–2 mg/mL 50% methanol/water mixture.Reprinted from [41], with permission.

cult to resolve by using poly-L-SULV, it was found that thesuccess rate for chiral resolution of cationic as well asneutral racemates was high. In this context, the authorsdiscussed aspects regarding electrostatic, steric, hydro-phobic, and hydrogen-bonding interactions.

In order to determine the site of chiral recognition, thetwo diasteromeric forms of sodium N-undecanoyl leucylleucinate (SULL) were used in order to determine thedepth of penetration of chiral analytes into the micellarcore of polymeric and monomeric surfactants [43].Three binaphthyl derivatives, (�)-1,1’-bi-naphthyl-2,2’-diamine (BNA), BOH and BNP, were assayed. The resultssuggest that interaction of BNP with both the C- andN-terminal amino acid of poly-SULL occurs, while mono-SULL mainly interacts with the C-terminal amino acid.Accordingly, a deeper penetration of BNP enantiomersinto the micellar core of the poly-SULL than that of themono-SULL takes place. By varying the temperature achange in the depth of penetration of BNP into the micel-lar core of the poly-SULL results. Nevertheless, inde-pendently of the temperatures assayed, the enantiomersof BNA and BOH always interact preferentially with theN-terminal amino acid of poly-SULL and mono-SULLsurfactants.

A cationic chiral polymer derived from 3-(N-10-unde-cenoyl-L-valyl)aminopropyltrimethylammonium bromideby UV-irradiation was obtained [44]. With this polymer,the chiral separation of enantiomers of amino acid deri-vatives was less than that of micelles of ordinary sur-factant 3-(N-undecanoyl-L-valyl)aminopropyltrimathyl-ammonium bromide. It was concluded that the cessa-tion of kinetic association-dissociation of micelles bypolymerization was not able to improve the chiralseparation. The performance of 18 monomeric and pol-ymeric amino acid-based surfactants in chiral sepa-rations was compared [45]. It was concluded that thepolymeric surfactants under consideration are usuallybetter chiral selectors for neutral as well as cationicenantiomers.

Finally, newly synthesized linear polymers containingL-amino acid moieties have been applied to chiral separa-tions by EKC [46]. Here, chiral linear polymers wereobtained by thermal polymerization of N-acryloyl-L-valineand N-acryloyl-L-alanine derivatives and subsequent hy-drolysis. The hydrophobic solute retention was quiteweak but a racemic mixture of 3,5-dinitrobenzoylatedamino isopropyl esters could be enantiomerically sepa-rated with the polymer derived from N-acryloyl-L-valineesters and with the copolymer from N-acryloyl-L-valineMe ester and N-acryloyl-L-valine N-methylamide at pH7.0. These separations are unsuccessful at pH 9.0 inBGEs containing anionic linear polymers. This pH de-pendence was discussed from the viewpoint of the micro-scopic hydrophobicity of the polymers, as estimated fromthe fluorescence of pyrene adsorbed onto the polymers inwater.



4.3 Others

The use of liposomes – small membrane-enclosed vesi-cles formed of either natural or synthetic lipids – in EKChas been reported [47–49]. They can be made with well-defined compositions, and their sizes can be adjusted ona wide scale as well. In order to investigate phospholipidliposomes as carriers in EKC, the effect of the composi-tion of the liposomes and the choice of phospholipids onthe separation of model corticosteroides were studied(Fig. 11). The retention factors of uncharged solutes inliposome EKC have shown a direct proportionality to their

Figure 11. EKC separation of six corticosteroids withliposomes as carriers. Running conditions: 58.5 cmuncoated fused-silica capillary (50 cm to the detector);UV detection at 245 nm; 20 kV; injection at 50 mbar for5 s; temperature, 25 or 42�C. Buffers: (A) 3 mM 1-palmi-toyl-2-oleyl-sn-glycero-3-phosphocholine (POPC/phos-phatidyl serine 80/20 mol%) in 50 mM CHES, pH 9(25�C); (B) 4.5 mM POPC/cardiolipin (CL) 80/20 mol%in 50 mM CHES, pH 9 (25�C); 3 mM 1,2-dipalmitoyl–sn-glycero-3-phosphocholine/CL (DPPC/CL 80/20 mol%) in50 mM CHES, pH 9 (43�C). Compounds: 1, 1-dehydro-aldosterone; 2, cortisone; 3, cortisol; 4, 21-deoxycortisol;5, 11-deoxycortisol; 6, dexamethasone. Reprinted from[47], with permission.

liposome – water partition coefficients. To unravel thecontributions from various types of interactions for solutepartitioning into the liposomes, LSER models were devel-oped. The hydrogen bond-acceptor strength as well asthe size of solutes have been shown to be the main fac-tors that determine partitioning into lipid bilayers [49]. Ithas been concluded that liposome EKC does not provideany particular advantage as an analytical technique.However, it has been considered as powerful tool for thestudy of drug-membrane interaction [48].

5 Conclusions

Several methods have been developed which use theEOF as vehicle for the transport of the mobile phase inchromatographic systems. Mostly used in practice aresystems with charged micelles as replaceable PSPs, butpacked and, moreover, monolithic beds for introducingnew column technologies expanded in the last years.However, as an alternative replaceable charged additivesto the BGE like polymers or nonmicelling small moleculesare still of interest, as they do not show some of therestrictions of the other methods: they do not needadvanced column technologies, have no need for frits,no CMC limits the use of organic solvents, etc. A success-ful separation with these PSPs depends mainly on thechoice of the charged soluble additive, and on operationconditions resulting in an acceptable migration window.However, for complex separations the relatively smallmigration window may make these PSPs less useful inpractice, especially in those cases, when the value of theelectrophoretic mobility of the PSP is much smaller thanthat of the mobility of the counterdirected EOF.

Financial support for the stay of I. P. at the Institute forAnalytical Chemistry, University of Vienna, Austria, fromChile’s Ministerio de Educación (Project MECESUP UCO9905), Fondo Nacional de Ciencia y Tecnología (ProjectFONDEF No DO1I1115) and Dirección de Investigaciónde la Universidad de Concepción (Project DIUC DI201.021.014-1.0) is gratefully acknowledged.

Received April 17, 2003

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Recent developments in capillary electrokinetic chromatography with replaceable charged pseudostationary phases or additives