Experiments with Organic Modifiers in Reversed-Flow MEKCJoeri Vercammen, Katleen Verleysen, Pat Sandra*

Department of Organic Chemistry, University of Ghent, Krijgslaan 281 S4, B-9000 Ghent, Belgium

Ms received: July 9, 1998; accepted: September 21, 1998

Key Words:Reversed-flow MEKC; separation according to polarity; organic modifiers

SummaryReversed-flow MEKC, i.e. MEKC at low pHs to suppress the electro-osmotic flow, was evaluated for the separation of some homologousseries. At low pHs compared to MEKC, the elution order reversesand normal phase type migration is obtained. Moreover, the elutionwindow is nearly infinite. Efficiencies are very high and reproducibil-ities in absolute migration times are acceptable (%RSDa 5). Theinfluence of a series of alcohol modifiers was investigated.

1 Introduction

Micellar electrokinetic chromatography (MEKC), introduced byTerabeet al. [1], is a very efficient electrodriven separation tech-nique. In MEKC the solutes are separated based on differencesin partitioning coefficients of the solutes between the polarmobile phase and the slowly moving micellar phase. Separationoccurs according to hydrophobicity.

A drawback of MEKC lies in the pseudostationary character ofthe micellar phase resulting in an elution window. The elutionrange is limited by the migration times of a solute that is notretained by the micellar phase (t0) and of a solute, which is per-manently retained (tMC). Between this time span all neutralsolutes elute, separated or not.

The past years, different research groups have reportedapproaches to extend the elution range. An overview was givenby Muijselaar et al. [2]. These solutions offer a reasonableenlargement of the elution window for most applications, butincrease the complexity of the MEKC system. Another possibil-ity concerns the reduction of the electroosmotic flow (EOF) sothat the micelles migrate to the anodic side. Otsuka and Terabe[3] already reported possible advantages of MEKC at low pH butthey emphasized poor reproducibility. Janiniet al. [4] used capil-laries coated with polyacrylamide to suppress the EOF and creat-ing a reversal of the micellar migration direction. They calledthis mode of MEKC reversed-flow MEKC (RF-MEKC).

In this contribution we report our experiments with MEKC atlow pH values. RF-MEKC is interesting because it provides“normal phase” type migration, leading to separations accordingto polarity. Several solutes were selected to illustrate the prin-ciple. Similarly to MEKC [5] we also studied the influence ofmodifiers belonging to the homologues series of the alcohols onthe partitioning mechanism.

2 Materials and Methods

Experiments were performed on a Hewlett-Packard3DCE systemor on a Waters Quanta 4000 CE system. The capillary dimen-sions were 50lm internal diameter with a total length of 50 cm(effective length 42.5 cm). The samples were dissolved in thebuffer solutions and injected hydrodynamically for 1 s. Before arun was started the capillary was rinsed for 2 min with the buffersolution by applying pressure. Detection was with UV absorb-

ance at 214 nm. The applied voltage was –25 kV. The tempera-ture corresponded with the room temperature. For the repeatabil-ity studies, the temperature of the HP3DCE system was set at218C. 25 mM phosphoric acid at pHL 2 was used as back-ground electrolyte. Different concentrations of sodium dodecyl-sulfate (SDS) and organic modifiers were applied. All chemicalsand standards were from Fluka, Merck or Sigma-Aldrich. Themodel compounds are listed inTable 1.

3 Results and Discussion

In MEKC at basic pH values, the electroosmotic flow dominatesthe net migration and solutes are detected at the cathodic side inorder of hydrophobic character. An MEKC experiment atpH 9.35 with 35 mM SDS confirmed the elution order in MEKCgiven in Table 1.

At pH values in the acidic range the electroosmotic mobilitydecreases and at a certain point, the electroosmotic flow willhave the same value as the electrophoretic mobility of themicelles resulting in a steady state situation. For a phosphate buf-fer with 35 mM SDS we measured a steady state at a pH of 6.3.Migration to the cathodic and anodic side was not observedwithin 2 h for the solutes of Table 1. Beyond this point the netmobility of the SDS micelles will be towards the anode. Highlyhydrophobic solutes should in this case elute first. The elutionorder of the compounds is thus reversed compared to MEKCunder normal conditions. An iterative estimation oftMC, using themethod proposed by Bushy and Jorgenson [6, 7], gave a migra-tion time of 2.63 min.

This is illustrated inFigure 1 showing the RF-MEKC analysis ofthe same solutes at a pH of 2.05 with 35 mM SDS. Initial experi-ments with SDS concentrations in the range 15 to 45 mM with10 mM intervals indicated that 35 mM was the best concentra-tion in terms of efficiency and migration times. Under these con-

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Table 1. Peak numbers, names and elution order in MEKC and RF-MEKC for the model compounds.

No. Name Elution orderin MEKC

Elution orderin RF-MEKC

1 n-Butyl-4-hydroxybenzoate 8 12 n-Propyl-4-hydroxybenzoate 7 23 n-Butyrophenone 6 34 Ethyl-4-hydroxy benzoate 5 45 Propiophenone 4 56 Methyl-4-hydroxybenzoate 3 67 Acetophenone 2 78 Benzoic acid 1 8

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ditions the currentwas63 lA. As predicted,a completereversalof theelutionorderis observed.

Theefficiency at half heightis very high, e.g.375,000platesforpeak5, andmigrationtimesareshort.

As far as the repeatabilityis concerned,%RSDvaluesfor theabsolutemigration times for the five first eluting soluteswaslower than0.5andfor thethreelastelutingsolutesaround4.

In a subsequentseriesof experiments,severalalcohols,i.e. shortand long chainalcohols,wereaddedto the buffer solution.Theresultsfor theadditionof severalalcoholsin differentconcentra-tions are given in Table 2. Becausethe alcoholsn-propanol,2-propanol, and n-butanol are characterizedby a considerablehydrophobicity, it was impossible to preparesolutions higherthan25%for n-propanoland2-propanoland5%for n-butanol.

In comparisonwith the datawithout modifier, all the migrationtimesshowa clearincrease.VanHoveet al. [5] pointedout thatin MEKC shortpolar alcoholscouldbeconsidered,asmodifiersof the aqueousphasewhile the more hydrophobic,long chainalcoholsaremodifiersof themicellar phase.In theseMEKC ex-perimentsit was noticed that addition of a short chain alcohollike methanolled to a reductionof theEOF– anobservationelu-cidatedby theinfluencethesealcoholshaveon thephysicochem-

ical properties,like the dielectric constantand the viscosity, ofthe buffer. The long chain alcohols,however, mostly causedadecreasein theelectrophoreticmobility of themicelles.This wasexplainedby assuminganincorporationof thesetypeof alcoholsinto themicelles[8, 9].

To understandthe increasein the migration times of the com-poundsin RF-MEKC (seeTable2) it wasthereforenecessarytomeasurethe residualEOFwhendifferenttypesof alcoholswereaddedto the buffer solution.An indication of the extentof theEOFis obtainedby changingthepolarity of theappliedelectricalfield in comparisonto RF-MEKC andinjectingformamidein thepertinentalcohol-buffer combination.The EOF valuesaregivenin Table 3.

Figure 1. Electropherogramof the test mixture. Conditions: 25mMphosphoricacid (pH 2.05),35mM SDS,voltage –25kV, current63 lA.Peaks:seeTable1.

Figure 2. A) Electropherogramof the test mixture for 5% methanoladdition. Conditions:25mM phosphoricacid (pH 2.05), 35mM SDS,5% MeOH,voltage:–25kV, current:61 lA. PeaksseeTable1. B) Elec-tropherogramof thetestmixturefor 5% 2-propanoladdition.Conditions:25mM phosphoricacid (pH 2.05),35mM SDS,5% iPrOH, voltage: –25kV, current:60 lA. PeaksseeTable1.

Table2. Migration timeswhenalcoholsin differentconcentrationsareaddedto thebuffer solution.

MeOH EtOH PrOH 2-PrOH BuOH0% 5% 25% 50% 5% 25% 50% 5% 25% 5% 25% 5%

1 2.74 2.97 5.52 18.58 3.32 6.64 28.05 3.17 13.59 3.10 16.12 3.862 2.96 3.24 6.73 18.78 3.68 8.24 28.19 3.53 16.83 3.47 21.48 4.513 3.10 3.43 7.67 21.13 3.98 9.39 – 3.83 18.45 3.76 24.50 4.984 3.52 3.89 8.72 21.19 4.54 10.80 – 4.41 22.01 4.30 31.26 6.145 3.71 4.15 9.89 23.64 4.98 12.29 – 4.82 24.17 4.72 36.15 6.786 4.81 5.28 11.66 26.13 6.38 14.59 – 6.19 29.13 6.05 48.73 9.927 4.90 5.46 12.72 27.96 6.81 15.30 – 6.59 32.37 6.46 59.21 10.598 5.60 5.95 12.8 28.48 7.11 16.41 – 6.67 29.13 6.85 48.73 9.92

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Measurementof theEOFdefinitely madea cleardistinctionpos-sible betweenthe usedalcohols.For the long chainalcohols(n-propanol,2-propanol,and n-butanol),the reductionof the EOFcould alsobe explainedby the influenceof the modifier on thedielectric constant and the viscosity of the medium. Thisdecreasein the EOF is, however, not reflectedin an expectedincreaseof the migration times of the test compoundsin RF-MEKC (seeTable2). Sincein RF-MEKC the micellar phaseisthe mobile phasethis reductioncould be causedby aggregationof the added alcohol with the SDSmicelles, as observedinMEKC, leadingto a higheraggregationnumberandthusa lowernetcharge[5].

The influenceof theshortpolaralcoholson theEOFwastotallydifferent in RF-MEKC comparedto MEKC: the EOF increasedin RF-MEKC,causingtheincreasedretentiontimes.In our opin-ion, this is thefirst time this phenomenonhasbeenobservedandan explanationcould not yet be given.Figure 2 showsthe elec-tropherogramsof thetestmixtureat 5% methanoland5% 2-pro-panol.

4 Conclusion

The main featuresof RF-MEKC comparedto MEKC are thereversedelution order resultingin normal phasetype migration

and the nearly infinite elution window. Short chain alcoholsasmodifier directly influence the residual electroosmotic flowwhile long chainalcoholsmainly aggregatewith themicelles.

AcknowledgmentJV and KV thank the Flemish Institute for the Promotionof Scientificand TechnologicalResearchin the Industry (IWT), Flanders,Belgiumfor studygrants.

References[1] S. Terabe,K. Otsuka,K. Ichikawa, A. Tsuchiya,T. Ando, Anal.

Chem.1984,56, 113.

[2] P.G.H.M. Muijselaar, H.A. Claessens,C.A. Cramers,J. Chroma-togr. A 1995, 696, 273.

[3] K. Otsuka,S.Terabe,J. MicrocolumnSep.1989,1, 150.

[4] G.M. Janini, G.M. Muschik, H.J. Issaq,J. Chromatogr. B 1996,683, 29.

[5] E. VanHove,R. Szucs,P. Sandra,J. High Resol.Chromatogr. 1996,19, 674.

[6] M.M. Bushey, J.W. Jorgenson,Anal.Chem.1989, 61, 491.

[7] M.M. Bushey, J.W. Jorgenson,J. MicrocolumnSep.1989, 1, 125.

[8] A. Malliaris, J. Lang, J. Sturm,R. Zana,J. Phys.Chem.1987, 91,1475.

[9] P. Lianos,J. Lang,C. Strazielle,R. Zana,J. Phys.Chem.1982, 86,1019.

Table 3. Migration time of formamidein buffer systemscontainingdif-ferentalcohols.

none 5%MeOH5% EtOH5%PrOH5%2-PrOH5%BuOHt0(min.) 13.06 8.08 6.94 20.01 19.60 27.79