A silica monolithic column prepared by the sol-gel process for enantiomeric separation by

Jingwu KangDorothee WistubaVolker SchurigInstitute of Organic Chemistry, University of Tübingen,Tübingen,Germany A silica monolithic column prepared by the sol-gel process for enantiomeric separation by capillary electrochromatographyA method for the preparation of a silica monolithic capillary electrochromatography (CEC)column for the separation of enantiomers has been developed.The porous silica monolith was fabricated inside a fused-silica capillary column by using the sol-gel pro-cess.After gelation for24h,hydrothermal treatment at1007C for24h was performed to prevent the sol-gel matrix from cracking.The prepared monolith was then coated with Chirasil-b-Dex which represents a chiral polymer prepared by grafting permethyl-b-cyclodextrin to polymethylsiloxane with an octamethylene spacer.Immobilization of Chirasil-b-Dex was performed by heat treatment at1207C for48h to give a nonextract-able coating.The column performance was evaluated by using racemic hexobarbital as a model compound.The efficiency of9.26104theoretical plates/m for the first eluted enantiomer of hexobarbital was obtained at an optimal flow rate of the mobile phase.The effect of mobile phase composition on enantiomeric separation of hexo-barbital was also investigated.The column proved to be stable for more than one hun-dreds of runs during a two-months period.The enantiomers of several neutral and negatively charged chiral compounds were baseline separated on this column. Keywords:Capillary electrochromatography/Enantiomeric separation/Monolithic column/Sol-gel process EL48401IntroductionRecently,separation of enantiomers by capillary electro-chromatography(CEC)has received considerable atten-tion.Depending on the types of column design,three main approaches have been used for CEC enantiomeric separation:open tubular columns,packed columns and monolithic columns[1,2].Among these three approaches, the use of monolithic columns appears to be the most promising one because of the fritless column technology. The stationary phase of the monolithic column consists of a single piece that is chemically bonded on the inner wall of the capillary and therefore no supporting frit is required. Additionally,a monolithic column has an advantage of a high mass transfer and a low pressure drop.To date,several papers dealing with the preparation of monolithic CEC columns for enantiomeric separation have been published:(i)organic polymer-based mono-lithic columns[3–13];(ii)silica-based monolithic columns prepared by the sol-gel process[14–16]or by sintering the silica particles at high temperature[17].The organic polymer based method is characterized by simplicity, since the columns can be fabricated in one step by copolymerization of monomer and chiral selector.Silica-based monolithic columns have the advantage that the established silica modification methods used for prepar-ing HPLC chiral stationary phases can be directly trans-ferred to CEC.So far,only few papers concerning the preparation of monolithic column for CEC enantiomeric separation by the sol-gel technique have been published [14–16].Zare et al.[14]prepared a particle-loaded mono-lithic column for the enantiomeric separation of amino acids.Chen and Hobo[15]prepared a monolithic column by chemically modifying the silica sol-gel matrix with a ligand-exchange-type chiral stationary phase.Wang et al.[16]reported a method for the preparation of cyclo-dextrin based open tubular columns by the sol-gel tech-nique.Here,we describe a new method for the preparation of a cyclodextrin-based CEC monolithic column using the sol-gel process.The hydrothermal treatment was per-formed to prevent the monolith from cracking during the drying process.The prepared monolith was then modified with the chiral stationary phase Chirasil-b-Dex(Fig.1). The enantioselective column performance was evaluated and several neutral and negatively charged chiral com-pounds were successfully separated into enantiomers.Correspondence:Prof.Volker Schurig,Institute of OrganicChemistry,University of Tübingen,Auf der Morgenstelle18,D-72076Tübingen,GermanyE-mail:volker.schurig@uni-tuebingen.deFax:149-7071-295-538Abbreviations:SEM,scanning electron micrograph;TMOS,tet-ramethoxysilane1116Electrophoresis2002,23,1116–1120ªWILEY-VCH Verlag GmbH,69451Weinheim,20020173-0835/02/07-08–04–1116$17.50+.50/0C E a n d C E CFigure2.SEM of a sol-gel silica monolithic bed coated with Chirasil-b-Dex.Magnification,16506.different kind of stationary phases including chiral station-ary phases.However,the cracking of the wet gel during the drying process represents a drawback of this column technique[19,20].In order to alleviate this problem,a supercritical fluid drying technique was used in the litera-ture to prepare a cracking-free sol-gel-entrapped CEC column[20].In our hands,we found that the hydrothermal treatment was a very effective strategy to minimize the monolithic bed from extensive shrinking and to prevent it from cracking.This is probable due to the fact that hydro-thermal treatment promotes the further cross-linking of the residual silanol groups of the silica framework[21]. Figure2shows a scanning electronic micrograph(SEM) of a monolith prepared in our experiment.As can be seen,the morphology of the sol-gel matrix is reminiscent to a porous matrix formed by aggregated spherical silica particles.Chirasil-b-Dex was statically coated on the sur-face of silica matrix.Heat treatment at1207C was per-formed to result in a nonextractable coating of the immo-bilized chiral stationary phase.3.2Column performanceAs expected,the EOF velocity of the silica monolithic col-umn increased linearly with increasing the field strength in the range from0.1to0.6kV per cm.After modifying the silica monolith with the chiral stationary phase,the coat-ing of Chirasil-b-Dex shielded some of the silanol groups on the surface of silica,therefore a reduced EOF was obtained.However,the reduced EOF remained strong enough to drive the mobile phase through the capillary at an acceptable velocity when neutral analytes were investigated.For negatively charged analytes,a reversedFigure 4.Separation of enantiomers on the Chirasil-Dex monolithic column.Conditions:fused silica capillary column,25cm (effective length )650m m ID;UV de-tection was performed at 210nm.For mephobarbital,hexobarbital and benzoin:mobile phase,MES-Tris buf-fer (pH 6)/methanol (90/10v/v);applied field strength,0.4kV/cm;samples injection at 3kV for 4s;carprofen:mobile phase,MES-Tris buffer (pH 6)/methanol (60/40v/v);applied field strength,–0.4kV/cm;sample injection at –3kV for 4s.seen that the resolution (R s )is increased with a decrease of the flow rate,however,the analysis time is also in-creased.Therefore,a field strength of 0.4kV/cm (corre-sponding to a flow rate of 0.37mm/s)was selected for the further investigations.Several enantiomers including neutral and negatively charged chiral compounds were separated on this monolithic column.The chromato-grams for the baseline-separated enantiomers are shown in Fig.4.3.3Effect of mobile phase compositionon the separation of enantiomersThe effect of the methanol concentration in the mobile phase on the enantioselectivity was also investigated.As can be seen in Fig.5,the resolution factor R s decreased with raising the methanol concentration.Concurrently,an increase of the methanol concentration decreased the EOF velocity and prolonged the analysis time.Therefore,a low methanol concentration was selected.The effect of the buffer concentration on the separation of hexobar-bital enantiomers was also investigated at an MES con-centration range in the mobile phase from 20to 100m M .It was observed that the retention factor k increased slightly,however,the efficiency and resolution decreasedTable1.Reproducibility of elution time and resolution(R s) of hexobarbital enantiomersRun-to-run(n=5)Day-to-day(n=4)t1(min)t2(min)R s t1(min)t2(min)R s16.417.5 1.9216.1017.10 2.0216.317.3 2.0215.8416.96 1.9616.217.2 2.0713.7214.57 1.9916.117.1 2.0514.8015.77 2.1816.117.1 2.06RSD(%)0.880.9 2.327.207.35 4.81 Conditions:fused-silica capillary column,25cm(effective length)650m m ID;mobile phase,MES-Tris buffer(pH6)/ methanol(90/10v/v);applied field strength,0.4kV/cm; sample injection at3kV for4s.t1and t2are the elution times corresponding to the first and second eluted peaks of hexobarbital enantiomers.lithic bed as well as the Chirasil-b-Dex coating were proved to be stable.The reproducibility was evaluated via run-to-run(5runs)and day-to-day(4days).The results are summarized in Table1.A good run-to-run reproduci-bility was obtained on the same column.A relatively high deviation for day-to-day reproducibility could be due to the influence of the fluctuations of the ambient tempera-ture,since the used instrument was not thermostated.It should be noted that the day-to-day reproducibility was measured at random during two weeks.The column-to-column reproducibility was also evaluated,however,a rather poor result was obtained.Obviously,the reproduci-bility of the pore structure of the sol-gel matrix is poor between column-to-column,therefore,the thickness of the chiral polymer coating is difficult to control although the columns were coated with the same coating solution and thermally treated under the identical conditions.In conclusion,a method for the preparation of a porous sol-gel monolithic column used for the separation of enantiomers by CEC was developed.Hydrothermal treat-ment at1007C for24h was performed to prevent the silica monolith from cracking.After coating of the porous monolithic bed with Chirasil-b-Dex,the residual EOF remains strong enough to perform the separation at a reasonable time.The enantiomers of several racemic compounds were baseline-separated.The enantioselec-tive columns proved to be stable and showed a good run-to-run reproducibility.However,column-to-column reproducibility is rather poor.Further work towards the improvement of column-to-column reproducibility and enhancement of EOF is forthcoming.The authors thank Ms.Elke Nadler for SEM measure-ments and the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support. 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