Lu_et_al-2011-Journal_of_Separation_Science

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JunyuLuFangguiYeAizhuZhangZongWeiYanPengShulinZhao

KeyLaboratoryfortheChemistryandMolecularEngineeringofMedicinalResources(MinistryofEducationofChina),CollegeofChemistryandChemical

EngineeringofGuangxiNormalUniversity,Guilin,P.R.ChinaReceivedFebruary7,2011RevisedMarch18,2011AcceptedMarch18,2011

ResearchArticle

Preparationandcharacterizationofsilicamonolithmodifiedwithbovineserum

albumin-goldnanoparticlesconjugatesanditsuseaschiralstationaryphasesforcapillaryelectrochromatography

Thispaperdescribesthedevelopmentofsilicamonolithmodifiedwithbovineserumalbumin–goldnanoparticles(BSA-GNPs)conjugatesaschiralstationaryphasesforcapillaryelectrochromatography(CEC).Thebaremonolithicsilicacolumnwaspreparedbyasol–gelprocessandhasbeenmodifiedchemicallywith3-mercaptopropyl-trimethoxysilanetoprovidethiolgroups,followedbyimmobilizationofgoldnanoparticlesviatheformationofanAu-SbondandmodificationwithBSAasthechiralselectorviathenitrogenlonepairofelectrons.Ithasbeendemonstratedthatthemonolithicchiralstationaryphasescanbeusedfortheenantioseparationofanumberofphenylthio-carbamylaminoacids(PTC-D/L-AAs)byCEC.Tenpairsoftestedaminoacidsenantio-mersweresuccessfullyresolvedwithin18minunderoptimizedconditions,andtheresolutionvalueswereintherangeof1.486–2.083.WithPTC-D/L-tryptophanusedastheprobesolute,theinfluencesofappliedvoltage,organicmodifierandbufferpHinmobilephaseonapparentretentionfactor,enantioselectivityandresolutionfactorwerealsoinvestigated.

Keywords:BSA/Chiralstationaryphases/Enantioseparation/Goldnano-particles/SilicamonolithsDOI10.1002/jssc.201100102

1Introduction

Enantiomerseparationisanareaofincreasingimportanceinchemicalsystemsandthepharmaceuticalindustry[1,2].Capillaryelectrochromatography(CEC)hasbeenprovedtobeoneofthemostpopulartechniquesforenantiomerseparation[3–8],whichcombinestheadvantagesofhighselectivityofHPLCwithhighefficiencyofcapillaryelectrophoresis.TheCECmethodsforenantiomersepara-tioncurrentlyhavebeendevelopedusingawiderangeofchiralstationaryphases(CSPs)basedonthevariouschiralselectorssuchasproteins[9,10],polysaccharides[11,12],cyclodextrinsanditsderivatives[13,14],macrocyclic

Correspondence:Dr.FangguiYe,KeyLaboratoryfortheChemistryandMolecularEngineeringofMedicinalResources(MinistryofEducationofChina),CollegeofChemistryandChemicalEngineeringofGuangxiNormalUniversity,Guilin1004,P.R.China

E-mail:fangguiye@163.comFax:186-773-5832294

Abbreviations:AAs,aminoacids;CSPs,chiralstationaryphases;GNPs,goldnanoparticles;MPTMS,3-mercaptopropyltrimethoxysilane;PITC,phenyl-isothiocyanate;PTC,phenylthiocarbamyl

antibiotic[15],chiralcrownethers[16],neutraldonor–

acceptor(Pirkle-type)selectors[17],chiralion-exchangers[18]andligand-exchangetypeselectors[19].Amongthemanytypesofchiralselectorsreported,proteinsarenaturalhigh-molecular-weightpolymers,whicharecommonlyusedasthepromisingchiralselectorsforenantiomerseparation.

Uptothepresent,themostwidelyusedproteinsarebovineserumalbumin(BSA),humanserumalbumin,ovomucoidanda1-acidglycoprotein.Inthereportedenan-tioseparationswithBSAasCSPs,differentapproachesincludingsol–gelencapsulation[10,20],physicaladsorption[21]andcovalentbinding[22]methodshavebeenutilized.Forexample,Katoetal.[10]describedtheproteinencap-sulationofBSAandovomucoidforthepreparationofchiralmonolithicstationaryphasebysol–gelmethodsforCEC.Yanetal.[22]preparedaninorganic–organichybridcapil-larymonolithiccolumnwithaminopropylgroupsastheanchoringgroupstocovalentlybindingBSAasachiralselectorforCEC.Itwasnoticedthatharshreactioncondi-tionsoftenleadtothedegradationandinactivationoftheBSA[23].Thus,maintainingthenaturalconformationofBSAisessentialtochiralseparation,andthemethodsfortheimmobilizationofBSAmustbemild.Toachievethis,thereactivegroupsthroughwhichtheBSAwasbondedtomatrixaretheamino,carboxylandsulfurgroups,etc.but

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J.Luetal.theaminogroupismostlyused,suchastheglutaricdialdehyde,1,1-carbonyldiimidazole,epoxide,N,N-disucci-nimidylcarbonateands-triazinemethods.Unfortunately,mostofaboveprocesseshaveeithercomplexorrigidreac-tionconditions.

Goldnanoparticles(GNPs)havebeenusedtomodifythechromatographicmediatoenhancetheinteractionsbetweentheanalytesandstationaryphasesduetotheirpossessingthelargesurfaceareaandcompatiblewithbiomolecules,especiallyforconjugatedwithproteins.OneofitsmostimportantpropertiesisthatGNPscanbecova-lentlyattachedtosurfaces,whichbearamino,thiolorcyanofunctionalitiesduetothestrongaffinityofgoldforthesemoieties[24].Connollyetal.[25]preparedhigh-capacityGNPsfunctionalizedpolymermonoliths.GNPshavebeenimmobilizedonamethacrylatemonolithviagraftedpoly-merchainsterminatedwithaminegroups,resultinginahomogenousanddensesurfacecoverage.Recently,researchgroupofSvec[26,27]hasdevelopedanewporouspolymermonolithmodifiedwithGNPs.Thesecolumnsarepreparedfromapoly(glycidylmethacrylate-co-ethylenedimethacrylate)monoliththroughreactionofitsepoxidemoietieswithcysteaminetoaffordamonolithrichinsurfacethiolgroups;then,GNPsbecomeattachedtothesurfaceoftheporesofthemonolithviatheformationofanAu-Sbond.AstudyofemployingBSA-GNPsconjugatesasthestationaryphaseontheinnersurfaceofthemicro-channelswasperformedbyLinandhisco-workers[28]toconstructanopen-tubularCECmicrodeviceforseparatingephedrineandnorephedrineisomers.Thecouplingreactioninthismethodismild,reliableandreproducible.SinceincorporationofGNPsintothestationaryphaseenhancedseparationefficiency,itisexpectedtouseGNPsassupportstoimmobilizechiralselectorsonthemonolithiccolumnforenantioseparation.

Inthiswork,wefocusedonthedevelopmentofBSA-GNPsconjugatesmodifiedsilica(BSA-GNPs-Silica)mono-lithsasCSPsforCEC.Attheroomtemperature,amildandreliableimmobilizationprocedurewasutilizedbypreder-ivatizationofthemonolithicsilicamatrixwith3-mercapto-propyltrimethoxysilane(MPTMS)toprovidethiolgroups,whichcanbeusedforthecovalentattachmentofGNPs,andthenmodificationwithBSAasthechiralselector.ThechiralrecognitionabilityofthepreparedCSPswasinvestigatedbyseparatinganumberofaminoacids(AAs)derivatizedwithphenylisothiocyanate(PITC)(phenylthiocarbamyl(PTC)-D/L-AAs).

2Materialsandmethods

2.1Reagentsandmaterials

TetramethoxysilaneandMPTMSwerepurchasedfromAlfaAesar(WardHill,MA,USA).D/L-AAsforchiralseparation,PITCandchloroauricacidwerepurchasedfromSigma(St.Louis,MO,USA).BSA,trisodiumcitrate,sodiumdihydrogen-&2011WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim

J.Sep.Sci.2011,34,2329–2336

phosphate,disodiumhydrogenphosphateandHPLC-grademethanol(MeOH)wereobtainedfromShanghaiChemicalReagentsCorporation(Shanghai,China).Allotherreagentswereofanalyticalgrade.UltrapurewaterusedforthepreparationofsolutionswasproducedbyaMilli-Qwatersystem(Millipore,Bedford,MA,USA).AAsolutionswerepreparedwith0.1MHCl.Fused-silicacapillaryof75mmidand375mmodwasobtainedfromHebeiYongnianOpticalFiberFactory(Hebei,China).

2.2Instrumentation

AhomemadeCEsetupwithUVdetectorusedinthisworkwassimilartothatdescribedinliterature[29].Scanningelectronmicroscopy(SEM)andenergydispersiveX-rayspectrometry(EDS)werecarriedoutonFEIQuanta200FEGSEM(Philips,TheNetherlands).Transmissionelec-tronmicroscopy(TEM)observationwasconductedonaFEITecnai-12instrument(Philips).

2.3PreparationofBSA-GNPs-SilicamonolithsTheGNPsweresynthesizedbasedonthesodiumcitratesynthesismethodasreportedpreviously[24].Thebaremonolithicsilicacapillarycolumnwaspreparedaccordingtotheproceduresdescribedinthepreviousliterature[30].Asolutionof10%v/vMPTMSintoluenewaspassedthroughtheactivatedbaremonolithicsilicacapillarycolumnandmadetoreactat1101Cinthedryingovenfor1h;thisstepwasrepeatedfourtimes.Sequentially,thecolumnwasflushedwithtoluene,MeOHanddeionizedwaterinorder.Theresultingthiol-modifiedsilicamonolithsarereferredtoas‘‘SH-Silica’’.Then,asynthesizedGNPssolutionwaspumpedthroughtheSH-Silicamonolithsbythemanualsyringeuntiltheentirelengthofcolumnturnred,thepinksolutionleavingcapillarywasobserved,andrinsedwithwatertoremovetheunreactedGNPs.Finally,theGNPs-modifiedmonolithiccolumn(GNPs-Silica)wasfilledwith8mg/mLBSAina25-mMphosphatebuffer(pH7.5),sealedwithtwoGCseptaandleftovernightatroomtemperature;thisstepwasrepeatedfourtimes.Afterrinsingwithphosphatebuffer,theBSA-GNPs-Silicamonolithswereobtained.Figure1showstheschematicreactionpathwaysforthepreparationprocessofBSA-GNPs-Silicamonoliths.

2.4DerivatizationofAAswithPITC

SolutionofAAs(5mM,50mL)wastransferredintoa1.0-mLpolyethylenecentrifugetubeandisopropylalcohol(150mL),PITC(25mL)andsodiumcarbonate(0.1M,150mL)wereadded.Thesolutionwasvortexedatroomtemperaturefor30min,andthentheexcessofPITCandtheaccompanyingcomponentswereextractedwithhexane(twotimesby

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300mL).Organiclayerwasthrownoff,whilelowerwaterlayerwasgatheredintoadryEppendorfvialavoidingingressofhexanetraces,centrifuged(4000rpmfor5min)andanalyzedbyCEC.

0.45-mmmembrane.Mobilephasesolutionmustbedegassedinanultrasonicbathfor15minbeforeuse.Priortoexperiments,thecolumnwasconditionedwithmobilephaseforabout30min.Thedetectionwavelengthwassetat2nm.InCEC,theapparentretentionfactor(kÃ)wascalculatedusingtheequationküðtRÀt0Þ=t0

ð1Þ

wheretRistheretentiontimeofasoluteandt0isdeterminedbythefirstturbulenceofbaseline.Theenantio-selectivityfactor(a)wasdefinedasthefollowingequation:a¼t2=t1

ð2Þ

wheret1andt2aretheretentiontimesofthefirstandsecondelutedenantiomers.

2.5Electrochromatography

Stocksolutionsofthephosphatebufferwerepreparedbymixingdisodiumhydrogenphosphatewithsodiumdihy-drogenphosphatesolutiontoadjustthepHofbuffertothedesiredvalue.ThemobilephasewaspreparedbymixingthedesiredamountofphosphatebuffersolutionandMeOH.ThepHvaluereportedinthispaperisthatoftheaqueousportion.Alltheelectrolytesolutionswerefilteredthrough

3Resultsanddiscussion

surface of monolithsurface of monolithOSiOH(CH3O)3Si(CH2)3SHOSiOHOat 110 ºC in tolueneOO(CH2)3SHSiOSiOO3.1CharacterizationoftheBSA-GNPs-Silicamono-lithsThesizedistributionandshapecharacteristicsofthesynthesizedGNPsweremeasuredbytransmissionelectronmicroscopy.Figure2AdemonstratesthatthepreparedGNPsconsistedofsphericalparticlesandarewelldispersedandhaveanaveragesizeofabout16nm.ThepreparedGNPsweredilutedtwotimeswithdeionizedwaterforUV–visibleabsorptionmeasurements.AsshowninFig.2B,thedispersedGNPshaveaweakplasmonresonanceabsorptionmaximumintherangeof490–0nm(lmax5521.5nm),whichconfirmstheirnanometerscalesizefurther.

BSA-GNPs-Silicamonolithwascutintocross-sectionalslicesforfield-emissionscanningelectronmicroscopyimagingtoexaminetherelativesurfacecoverageasshowninFig.3.Cross-sectionalsegmentsoftraditionalsilicamonolithswererequiredforsputteringwithgoldpriortoSEMimagingbecausethenonconductingsilicainvariablyleadstouncontrollablechargingeffectsandpoortopo-graphicalresolution.However,inthiswork,goldsputteringwasnotperformedsinceGNPshavebeenimmobilizedonthesurfaceofsilicamonoliths[25].Itcanbeseenfrom

SiOSiOO(CH2)3SHsurface of monolithOO(CH2)3SSiOSiOO(GNPs)at room temperatureSiOSiOO(CH2)3Ssurface of monolithOO(CH2)3SSiOSiOSiOOOSiNH2NH2(BSA)at room temperature(CH2)3SNH2OFigure1.SchematicreactionpathwaysofthepreparedBSA-GNPs-Silicamonoliths.

AB

1.00.8Absorbance (AU) 0.60.40.20.0300400500600700wavelength (nm)Figure2.(A)TEMphotographofGNPsand(B)absorptionspectraofGNPsinaqueoussolutions.

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Figure3.SEMphotographsofBSA-GNPs-Silicamonolithsatmagnificationsof(A)3000Âand(B)20000Â.

Fig.3Athatthemonolithiccolumnhasthemorphologyofacontinuoussilicaskeletonandlargethrough-pores.Themacroporescanprovidefastflow.Besides,themonolithicbedwastightlybondedtotheinnerwallofthecapillary.Figure3BconfirmsthatGNPsremainattachedtothesurfaceofsilicamonolithsandthedensityofcoverageontheBSA-GNPs-Silicamonolithisexcellent.EDSanalysisindicatesthattheBSA-GNPs-Silicamonolithsmodifiedwith16nmGNPscontain18.49wt%Au.

ToevaluateBSA-GNPs-Silicacolumnperformance,thedependenceofcolumnefficiencyonthelinearvelocityofthemobilephasewasinvestigatedoncolumnusingthefirstelutedenantiomerandbyincreasingtheappliedvoltagefrom7to26kV(andhencethelinearvelocityofmobilephasefrom0.35to1.3mm/s).FromFig.4,itcanbeseenthatarelativelyflatVanDeemtercurvewasobtainedwhenthelinearvelocityislargerthan0.8mm/s.Itisbelievedtoberelatedtothrough-poresinthecontinuoussilicastructure,whichoffershighpermeability.Theminimumplateheightsofabout105mmwereachieved,whichwascorrespondingtothecolumnefficienciesof9500plates/m.

145140135130

PTC-D-TryptophanH(µm)125120115110105100

0.20.40.60.81.01.21.4u (mm/s)

Figure4.Plotoftheoreticplateheight(H)versusthelinearvelocityofPTC-D-Tryptophan.Experimentalconditions:mobilephase,60%(v/v)MeOHin15mMphosphatebufferatpH7.5;detectionwavelength,2nm;appliedvoltage,7–26kV;BSA-GNPs-Silicacolumn:38cmÂ75mmid(28cmeffectivelength).

3.2ChiralseparationofPTC-D=L-AAs

Toevaluatetheenantioseparationcapabilityofthesechiralmonolithiccolumnsunderaqueousmobilephasescontain-ing60%MeOHandphosphatebufferatpH7.5,tenpairsofenantiomersweretestedincludingPTC-D/L-tryptophan(PTC-D/L-Trp);PTC-D/L-tyrosine(PTC-D/L-Try);PTC-D/L-leucine(PTC-D/L-Leu);PTC-D/L-serine(PTC-D/L-Ser);PTC-D/L-phenylalanine(PTC-D/L-Phe);PTC-D/L-b-phenyl-alanine(PTC-D/L-b-Phe);PTC-D/L-asparticacid(PTC-D/L-Asp);PTC-D/L-alanine(PTC-D/L-Ala);PTC-D/L-threonine(PTC-D/L-Thr);PTC-D/L-arginine(PTC-D/L-Arg).TypicalelectrochromatogramsarepresentedinFig.5.Itcanbeseenthattenpairsofenantiomerswerecompletelybaselineresolvedwiththeseaqueousmobilephases.SeparationfactorandefficiencyforthefirstelutedenantiomerofPTC-D/L-AAswerebothsatisfactory,yetthesecondeluted

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peakrevealedalowplatenumberandastrongtailing.ThedifferentpeakshapesforthetwoenantiomerscanbeascribedtothefactthetwoenantiomersbindtototallydifferentregionsonBSA.PTC-L-AAsbindmorestronglytoBSAthanPTC-D-AAs[31,32].ElectrochromatographicdatafortheseparationofthetenpairsoftestPTC-D/L-AAsarelistedinTable1.WealsocomparedtheperformanceoftheBSA-GNPs-SilicacolumnwithGNPs-SilicaandSH-Silica1BSAmonoliths(sameBSAsolutionthroughSH-Silicamonolith).Fig.6showsthechiralseparationofPTC-D/L-TrponBSA-GNPs-Silica,GNPs-SilicaandSH-Silica1BSAcolumns.ItwasfoundthatPTC-D/L-Trpwereco-elutedwithGNPs-SilicaandSH-Silica1BSAcolumns,whereasbaselineresolutionwasobservedonBSA-GNPs-Silicamonolith.ThecomparisonresultsindicatethattheBSA-GNPs-Silicamonolithsdonothavenonspecificproteininteractionswiththeremainingsilanolgroups.Theseobtained

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2.52.01.51.00.50.0

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Figure5.EnantioseparationonBSA-GNPs-Silicamonolith.Experimentalconditions:mobilephase,65%v/vMeOHin15mMphosphatebufferatpH7.5;applied,22kV;otherconditionsarethesameasinFig.4.Solutes:(A)PTC-D/L-Trp;(B)PTC-D/L-Asp;(C)PTC-D/L-Tyr;(D)PTC-D/L-Ser;(E)PTC-D/L-Ala;(F)PTC-D/L-Leu;(G)PTC-D/L-b-Phe;(H)PTC-D/L-Phe;(I)PTC-D/L-Arg;(J)PTC-D/L-Thr.

resultsindicatedthattheBSA-GNPs-SilicamonolithasCSPsforCECwassuccessfulinthiswork.3.2.1Effectoftheappliedvoltage

SincethepIofBSAis4.7[33],theBSA-GNPs-SilicamonolithsareattributedtothecathodicEOFaswellasdissociationoftheresidualsilanolgroupsoverthe

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investigatedpHrange.ItisknowntheEOFvelocityismainlyincreasedwithincreasingappliedvoltage.TheeffectofappliedvoltageonresolutionwasinvestigatedusingPTC-D/L-Trpasthestandardmodelanalytes.ItcanbeseenfromFig.7thattheretentiontimeofPTC-D/L-Trpdecreasedwithincreasingappliedvoltagefrom18to24kV,andtheJouleheating-associatedproblemdidnotseemtobeacauseofconcern.Althoughanincreaseinappliedvoltagewill

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J.Luetal.Table1.EnantioseparationoftestPTC-D/L-AAsbyCEConthe

BSA-GNPs-SilicamonolithicCSPs

Samples

t1(min)t2(min)a(t2/t1)N1N2Rs(plates/m)(plates/m)

PTC-D/L-Arg9.75812.1741.2488337712.083PTC-D/L-Try10.06211.5731.1501112658171.653PTC-D/L-Leu9.61811.1131.155931749901.536PTC-D/L-Ser9.82811.9871.220847943681.979PTC-D/L-Phe9.93711.4581.1531369749251.620PTC-D/L-b-Phe10.35313.61.342912151301.486PTC-D/L-Trp9.82011.1241.1331471470111.618PTC-D/L-Asp9.72811.9191.225786147692.050PTC-D/L-Ala9.77812.0871.236866340531.867PTC-D/L-Thr9.795

12.599

1.286

7191

3680

1.942

Experimentalconditions:mobilephase,65%(v/v)MeOHin15mMphosphatebufferatpH7.5;appliedvoltage,22kV;detectionwavelength,2nm;BSA-GNPs-Silicacolumn;38cmÂ75mmid(28cmeffectivelength).

8

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(DL

ecnabro4

s(B) SH-Silica + BSAbADL

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Figure6.EnantioseparationofPTC-D/L-Trpon(A)BSA-GNPs-Silica,(B)SH-Silica1BSAand(C)GNPs-Silicamonoliths.AllotherexperimentalconditionsarethesameasinFig.5.

allowlesstimeforinteractionsbetweentheanalytesandthechiralselector,theresultofenantioseparationbyCECdidnotshowpoorselectivityandresolution.Forinstance,whilelowerappliedvoltage(18kV)wasused,theresolutionandenantioselectivityfactorswere2.688and1.363,respectively.Whiletheappliedvoltagewassetat24kV,theresolutionandenantioselectivityfactorswere2.052and1.302,respec-tively.

3.2.2EffectoftheMeOHcontent

Generally,thecontentoforganicmodifierinthemobilephasehasaninfluenceontheenantioseparationofanalytes.Inthiswork,PTC-D/L-Trpwereappliedasmodelanalytestostudytheinfluenceoforganicmodifier-MeOHcontentinmobilephasesonkÃ,Rsandavalues.AsshowninFig.8,whilethecontentofMeOHwasvariedfrom55to70%,thekÃvaluesofPTC-D/L-Trpenantiomersslightlydecreased,

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L

m(622kV ecnabro4D

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sb20kV

A2

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L018kV0

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1520

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Time (min)

Figure7.EffectofappliedvoltageontheseparationPTC-D/L-Trp.AllotherexperimentalconditionsarethesameasinFig.5.

2.5k*1k*22.0aRs1.51.00.50.055606570MeOH content (%)

Figure8.EffectofMeOHcontentinmobilephaseonthechiralresolution(Rs),enantioselectivity(a)andapparentretention

factors(kÃ1andkÃ

2)ofPTC-D/L-Trp.AllotherexperimentalconditionsarethesameasinFig.5.

whichindicatedhydrophobicinteractionbetweentheanalytesandCSPs.However,theenantioselectivityisalmostkeptconstant.ItwasalsofoundthatRsvaluesincreasedwithincreasingcontentofMeOHinmobilephases.ButwhenthecontentofMeOHwas70%,thefirstelutedenantiomerandtheresidualPITCcouldnotachievebaselineseparation.Therefore,65%MeOHinmobilephaseswasusedforfurtherexperiments.ItwasalsoobservedwhencontentofMeOHwasincreasedupto70%,theBSAimmobilizedontheAusurfacewasstillstable.Thisisbecauseimmobilizationofproteinsontosolidsupportsoftenincreasestheirstability.Proteinsinsolutionareusuallyinadisorderedorientationandareapttosufferfromconformationalchangeathighcontentoforganic

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2.0

k*1k*2aRs1.5

1.0

0.5

0.0

6.06.57.07.58.08.59.0pH

Figure9.EffectofbufferpHinmobilephaseonthechiralresolution(Rs),enantioselectivity(a)andretentionfactor(kÃ1andkÃ

2)ofPTC-D/L-Trp.AllotherexperimentalconditionsarethesameasinFig.5.

modifier,temperatureandpH,whichrendersthefunctionalsitesinaccessibletointeractingmolecules[34].Conforma-tionalchangeandunfavorableorientationuponimmobili-zationwouldbegreatlyreducedbyimmobilizingproteinsonGNPssurfaceviachemisorptionofNH2-groupsonAu[35].

3.2.3EffectofbufferpHinmobilephase

TheeffectofbufferpHinmobilephaseonchiralseparationofPTC-D/L-Trpwasinvestigated.FivedifferentpHs(6.0,7.0,7.5,8.0and9.0)wereusedforenantiomerseparationandtheresultisshowninFig.9.ItcanbeseenthatthekÃvaluesofPTC-D/L-TrpenantiomersslightlydecreasedwithincreasingpHvalues,whichindicatetheinteractionsbetweentheanalytesandBSAweakenedathigherpH.ItwasalsofoundthatRsandavaluesslightlyincreasedwithanincreaseinbufferpHvaluesfrom6.0to9.0.Thistendencywasalsoreportedbyotherinvestigators[36].BSAiscomposedofthreeglobulardomainsandthethreedomainsareheldtogetherbyionicandelectrostaticinteractionsatneutralpH[31].Therefore,alkalineconditionmayleadtodenaturationoftheBSAconformationanditisalsonotpreferableforsilica-basedmonolithiccolumn.AlthoughthebestresolutionofPTC-D/L-TrpenantiomerswasobservedusingbufferpHat9.0inmobilephase,pH7.5wouldbeappropriateinthiswork.

3.3Repeatabilityandstability

ToassesstheBSA-GNPs-Silicamonoliths,thestabilityandrepeatabilitywerealsoinvestigatedinthisworkbyusingthePTC-D/L-Trpasthetestanalyte.Theexperimentalresultsshowedtherelativestandarddeviations(RSDs)fortheEOF,

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kÃandRsvaluesofPTC-D/L-Trpwerelessthan1.6,2.1and2.5%(n55),respectively.Thecolumn-to-columnrepeat-abilitieswerealsoexaminedtoevaluatethepreparedCSPs,theRSDsofEOF,kÃandRsvaluesofPTC-D/L-Trpwerelessthan6.2,8.5and5.6%(n53),respectively.Moreover,theenantioselectivitywasretainedforhundredsofoperationsoverthecourseof2wkwithoutobviousdeclineofseparationperformanceandcolumncollapse.TheseresultsindicatedthatthepreparedBSA-GNPs-Silicamonolithshadgoodstabilityandrepeatability.

4Concludingremarks

AnewtypeofBSA-GNPs-SilicamonolithsasCSPsforCECwasdevelopedbysol–gelmethod.TheCSPswereemployedfortheenantioselectiveseparationofPTC-D/L-AAsbyCEC.Undertheoptimizedconditions,tenpairsofPTC-D/L-AAsweresuccessfullyresolvedbyCECusingtheaqueousmobilephase.Theeffectsoftheappliedvoltage,organicmodifierandpHinmobilephaseontheapparentretentionfactor,resolutionandenantioselectivityfactorsofPTC-D/L-Trpwerealsoinvestigated.TheobtainedresultsshowedthatthemonolithicCSPscouldwellmaintainBSAconformationandhavegoodabilitytodistinguishenantiomers.

ThefinancialsupportfromtheNationalNaturalScienceFoundationofChina(No.21065002),NationalBasicResearchProgramofChina(No.2010CB933902)andGuangxiNaturalScienceFoundationofChina2010GXNSFF013001aregrate-fullyacknowledged.

Theauthorshavedeclarednoconflictofinterest.

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