Imidazolium-based functional monomers for the imprinting of the anti-inflammatory drug naproxen: Comparison of acrylic and sol-gel approaches

ContentslistsavailableatScienceDirect

Journal

of

Chromatography

A

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / c h r o m a

Imidazolium-based

functional

monomers

for

the

imprinting

of

the

anti-inflammatory

drug

naproxen:

Comparison

of

acrylic

and

sol–gel

approaches

Porkodi

Kadhirvel

_,

_Manuel

_Azenha

_,

_Sudhirkumar

_Shinde

_,

_Eric

_Schillinger

_,

Paula

Gomes

_,

_Börje

_Sellergren

_,

_A.

_Fernando

_Silva

a_{CIQ-UP,DepartamentodeQuímicaeBioquímica,FaculdadedeCiências,UniversidadedoPorto,Portugal} b_{INFU,FacultyofChemistry,TechnicalUniversityofDortmund,Otto-Hahn-Str.6,D-44221Dortmund,Germany}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received4July2013

Receivedinrevisedform3September2013 Accepted4September2013

Available online 9 September 2013 Keywords: Molecularimprinting Cationicmonomer Sol–gel Methacrylicpolymer Naproxen Frontalanalysis

a

b

s

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Imidazolium-basedmonomerswere,forthefirsttime,employedinacomprehensiveinvestigationof

themolecularimprintingprocessofnaproxeninbothacrylicandsol–geltridimensionalnetworks.To

thisend,molecularlyimprintedpolymer(MIP)andxerogel(MIX)werebothoptimizedforperformance,

bytestingdifferentporogen,templatespeciationandcomponentratios.Thedevelopedimprintswere

characterizedfortheirporeproperties(nitrogenadsorptionanalysis),siteheterogeneity,binding

prop-ertiesandotherperformanceparameterssuchastheimprintingfactor,selectivity(HPLCcolumntests),

columnefficiencyandmasstransferkinetics(frontalanalysisstudy).MIPexhibitedmesoporosity(Dp

29nm),whereasMIXdidnot,whichwasreflectedinboththelowernumberofaccessibleimprinted

sites(4.9␮mol/gversus3.7␮mol/g)andtheslowerbinding/dissociationinMIX.Thenaproxen/ibuprofen

selectivityratiowasestimatedas6.2fortheMIXand2.5fortheMIP.Giventhehighimportanceof

capac-ityandfastmasstransferintypicalapplicationsofimprintedmaterials,andthesatisfactoryselectivityof

MIP,itcanbeconcludedthattheacrylicapproachwasgloballythemostadvantageous.Still,the

remark-ablyhighselectivityofMIXanditsreasonablecapacitydemonstratethatfutureworkdevotedtofurther

optimizationofbothformatsisworthwhile.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Molecularimprintingisaversatiletechniqueforpreparing syn-theticmaterialswith tailoredmolecularrecognitionproperties; assuch,ispresentlyattractingwidespreadinterest,especiallyin chromatography,(bio)chemicalsensing,drugdeliveryand cataly-sis[1].Thepreparationofmolecularlyimprintedpolymers(MIP) typicallyinvolvesthree steps:first,amonomer-template[M–T] complexis formedviaself-assembly;next,theM–Tcomplexis polymerizedwithanexcessofcross-linkertoformarigidpolymer; finally,thetemplateisremoved,leavingbehindbindingcavities whicharecomplementaryinshape andfunctionalgrouptothe template[2].ThevastmajorityofMIPisbasedontheuseoforganic acrylate-typepolymers:astandardprocedureusingamethacrylate monomer,withanearlyoptimalratiotothetemplate molecule andcrosslinker,isdescribedinnumerousworks[2–8].Thebroad applicabilityofmethacrylicacid(MAA)asafunctionalmonomer

∗ Correspondingauthors.Tel.:+351220402628;fax:+351220402659. E-mailaddresses:porkodikathirvel@yahoo.com(P.Kadhirvel), mazenha@fc.up.pt(M.Azenha).

inMIP productionisrelated tothefactthatthecarboxylicacid groupserveswellasahydrogenbondandprotondonoraswellas ahydrogenbondacceptor[9].Nevertheless,acommonsevere con-straintofthistypeofimprintingistheneedforanorganicsolventin whichallspeciesaresoluble.Thisfurtherprecludesuseoftemplate moleculesthatareonlysolubleinaqueousmedia,whichimplies obviouslimitationsinthedevelopmentofMIPformany environ-mental and biologicalapplications.Although somepromisehas beenshownforaqueous-basedrebindingprocedures,suchresults arerareand considerableprogressis requiredtoovercomethis limitation[10,11].

Sol–gel imprinting is one possible alternative for achieving molecular recognitionof hydrophilic targets, given the ease of preparationofsol–gelsandtheircompatibilitywithpolar environ-ments[12].Sol–gelimprintingisstraightforwardandprovidesan efficientwayforpreparinghybridmatricesthroughincorporation oforganiccomponentsintoinorganicpolymersundermildthermal conditions,whilecontrollingMIPthickness,porosityandsurface area.However,todate,amorphoussol–gelmolecularlyimprinted xerogels(MIX) havenotdemonstrated thesamedegreeof suc-cessasMIPforanalyticalapplications,namelyintheformofbulk materials.

0021-9673/$–seefrontmatter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.09.015

lycoldimethacrylate (EDMA), whereas PTMOS was used as the functionalmonomerforthesol–gelapproach.Theauthorsfound thattheacrylicsystemexhibitedhighpropranololuptake,butthis wasaccompaniedbyahighdegreeofnon-specificbindingin aque-ousbuffer.Incontrast,thesol–gelsystemdisplayedloweruptake, but remarkably lower nonspecific binding. The uptake kinetics oftheacrylicpolymer wassignificantly slowerthan thatofthe sol–gelpolymer.The imprintingof thesol–gelfilm with enan-tiomericallypure(S)-propranololresultedinitspronouncedchiral recognitionoverthe(R)-enantiomer,whilenoinformationrelated withtheenantiomeric recognition in acrylicpolymer was pro-vided.

The above unique published examples of research dealing withcomparison of acrylic and sol–gel approaches to produce molecularly imprinted materials suggest that the latter might successfully challengethedominanceof theformer. In viewof this,adeeperstudyofacrylicversussol–gelmolecular imprint-ingapproaches isjustified.Hence,we hereinreportthefirst of aseriesofstudiesconcerningthethoroughstudyofsol–geland acrylicimprintingapproachesforselectedtemplates, representa-tiveofimportantchemicalfamilies.Acomprehensive,systematic and detailed study of the molecular imprinting of naproxen (NAP),aprofen-likenonsteroidalanti-inflamatorydrug(NSAID), isdescribed,embracingpre-polymerizationissues(suchas choos-ingacommonfunctionalgroup),microstructure,thermalstability, selectivityandaffinityproperties,aswellasmasstransferkinetics.

2. Materialsandmethods 2.1. Chemicals

2.1.1. Sol–gelapproach

Ureidopropyltrimethoxysilane (UPTMOS), the template (+)-(S)-2-(6-methoxynaphthalen-2-yl) propanoic acid (S-NAP) and trifluoroacetic acid (TFA) were obtained from Sigma–Aldrich, Germany.Thesodiumsaltofnaproxen(Na-NAP)waspreparedby addingaqueousNaOHtoS-NAPinequimolaramounts,followedby solventremoval. 2-(2-pyridylethyl trimethoxysilane) (PETMOS), 3-iodopropyltrimethoxysilaneand N-(3-triethoxysilylpropyl)-4,5-dihydroimidazolewerefromABCR,Germany,andusedasreceived. Formic acid, tetramethoxysilane (TMOS), methyltrimethoxy silane (MTMOS), chlorotrimethoxysilane and 1,1,1,3,3,3-hexamethylsilanedisilazanewerepurchasedfromFluka,Germany. Tetrahydrofuran(THF),acetonitrileandmethanol(MeOH)ofHPLC gradewereobtainedfromVWRInternational.Allotherreagents usedwereofanalyticalgrade.MilliporewaterofMilli-Qquality (Millipore,Italy)wasused.

N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole(2.74g,10mmol) and (3-iodopropyl)trimethoxysilane (2.97g,10mmol) were dis-solvedin10mLofdryacetonitrile.Themixturewasrefluxedfor 14h under nitrogen. Excess acetonitrilewas distilled off using rotaryevaporatorandtheresultingproductwasisolatedasa light-brownliquid.Product’scharacterizationby1_HNMR,13_CNMRand

LC–MS,allowedtoobtaindata(seebelow)inperfectagreement withboththeexpectedstructureandpreviouslypublishedNMR dataforthesamecompound[15].

1_{HNMR(400MHz,CDCl}

3):ı0.38{m,4H CH2Si (OCH2CH3)3

and(CH3)3SiCH2 )},ı 0.96(t,9H SiOCH2CH3), ı1.53{m, 4H,

CH2CH2Si(OCH2CH3)3 and (CH3O)3 SiCH2CH2 }, ı 3.3 {s, 9H (CH3O)3Si }, ı 3.4 {m,4H NCH2CH2N and NCH2CH2N }, ı 3.5 {q, 6H, Si OCH2CH3)3, ı 3.8 {t, 4H, NCH2CH2 and

CH2CH2 N },ı8.8{s,1H, NCHN ).13CNMRData(inCDCl3) ı 6.1 and 7.3 [ CH2Si(OCH2CH3)3 and (CH3O)3SiCH2 ],18.4

( SiOCH2 CH3), 20.9 and 21.2 [ CH2CH2Si(OCH2CH3)3

and (CH3O)3SiCH2CH2 ],48.4 and 48.5 ( NCH2CH2N and

NCH2CH2N), 50.2 and 50.3 ( NCH2CH2 and CH2CH2N),

50.9 [(CH3O)3Si ], 58.7 [ Si(OCH2 CH3)3,157.8( NCHN ).MS

(ESI+MeOH)m/z:calculated437.38andfound437.38. 2.3. UV–visspectrophotometricanalysisofpre-polymerization solutions

A series of template–monomer solutions was prepared by adding different amounts of IL-SG (3) or IL-A (4) (Fig. 2) to a fixedconcentrationof0.5mmol/LS-NAPinmethanolor chloro-form,respectively.The concentrationofthecationicmonomers wasvariedfrom0to4.5mmol/L.Thechangein theabsorbance ofthesolutionswasscannedbyUV–visspectrophotometryinthe 200–1800nmrange,againsttherelevantcationicmonomer solu-tions(i.e.,lackingS-NAP)asreferences.

2.4. Synthesisofxerogelsandpolymers

Aseriesofsol–gelimprintedandnon-imprintedxerogels(MIX orNIX,respectively)waspreparedusingPETMOS(1),UPTMOS(2), orIL-SG(3)functionalmonomers(Fig.2).InallMIX,thetemplate tofunctionalmonomer,functionalmonomer tocrosslinker,and silanetowatercontentratiosweremaintainedas1:2,1:6.6,and 1:2,respectively.ThesynthesisofIL-SGderivedMIXisdescribed below(Section2.4.1),whereasdetailedproceduresforthe synthe-sisofotherxerogelsprepared(compositionsasgiveninTableS1) areprovidedasSupportingInformation.

Imprintedand non-imprinted methacrylic polymers(MIP or NIP,respectively)werealsopreparedusingtheionicliquid-based organicfunctionalmonomerIL-A(4),asdescribedinSection2.4.3.

Fig.1.Schemeofthesynthesisofthesol–gelcationic(IL-SG)precursor.

Fig.2.(1–4)StructureoffunctionalmonomerstestedfortheimprintingofS-NAP;(5–6):structuresofS-NAPanditsstructuralanalogueIBU. Theformulationofpre-polymerizationmixturesforimprinted

andnon-imprintedmaterialsissummarizedinTable1. 2.4.1. SynthesisofxerogelsusingthecationicIL-SGfunctional monomer

IL-SG(423mg,0.75mmol),Na-NAP(94.5mg,0.375mmol)and 735␮l(5mmol)ofTMOSweremixedtogetherina50mLcentrifuge tube,andthemixturedissolvedinMeOH(11.6mL),followedby water(202␮L)addition(seeTable1).

Thecentrifugetubewasthensealedwithparafilmand punc-turedwithsmallholes,allowingforslowevaporationofsolvent. Themixturestirredusingamagneticstirrerwithpellet,untilthe gelwasformed.Itwasthendriedinopenairatmosphere. 2.4.2. Endcappingofsurfacesilanolgroupsinxerogels

HalfoftheavailableamountofdryMIX/NIXxerogelswas end-cappedbyalkylationofunreactedfree OHgroups,whereasthe otherhalfwasleftasnon-endcappedtoascertaintheinfluence

Table1

Prepolymerizationsolutioncompositionusedtopreparethesol–gel(MIX/NIX)andmethacrylicpolymers(MIP/NIP).

Polymer/Xerogel Template Functionalmonomer Crosslinker Porogen Catalystorinitiator

MIX Na-NAP(0.37mmol) IL-SG(0.75mmol) TMOS(5mmol) MeOH(11.6mL) Water(102␮L)

NIX – IL-SG(0.75mmol) TMOS(5mmol) MeOH(11.6mL) Water(102␮L)

MIP S-NAP(0.37mmol) IL-A(0.75mmol) EDMA(7.5mmol) CHCl3:MeOH(2mL:0.8mL) ABDV

andthepolymerslightlycrushed. 2.4.4. Templateremoval

Apreliminarytemplateextractionfromlightlycrushed xero-gels,eitherendcapped(EC)ornon-endcapped(NEC),andpolymers, was done on a Soxhlet apparatus, using 10% formic acid in methanol.The option for coarseparticles at this stageallowed for higheryields of material.Washing wascontinueduntil the extractedliquid phase showedundetectablelevelsof S-NAPby reverse-phaseHPLCwithUVdetectionat230nm.Thematerials werethencrushedinamortarandsievedtoselecttwofractions ofparticleswithsizesranging25–45and45–75␮m,respectively. Thelarger45–75␮mparticleswereusedinsolidphaseextraction (SPE)cartridges,whereasthesmaller25–45␮moneswereusedfor packingHPLCcolumns.Finalremovaloftemplatewasthen accom-plishedbyextensivepercolationofthepackedparticleswith10% formicacidinmethanol.

2.5. Materialcharacterization

Surfacemicrographsofthepreparedmaterialswereacquired usinga Hitachi H-S4500FEG Microscopein secondary electron mode, with an acceleration voltage of 1kV. The samples were depositedonholderswithacarbonfoilwithoutgoldsputtering.

Thermogravimetricanalysiswascarried outusinga TGAQ50 (TAinstruments,Eschborn,Germany).Eachsample(10–15mg)was placedinaplatinumpan,whichwassuspendedinasensitive bal-ancetogetherwiththereferencepan.Thesamplewasthenheated inafurnaceataheatingrateof20◦C/min,underN2atmosphere.

Thesurfaceareaandtheporeparametersweredeterminedona QuantachromeAutosorb6B(QuantachromeCorporation,Boynton Beach, FL) automatic adsorption instrument. Prior to measure-ments,100–150mgofthesampleswereheatedat40–60◦Cunder highvacuum(10–5Pa)foratleast12h.Thespecificsurfaceareas (S)wereevaluatedusingtheBETmethod,thespecificporevolumes (Vp)followingtheGurvitchmethodandtheaverageporediameter

(Dp)usingtheBJHtheoryappliedtothedesorptionbranchofthe

isotherm.

2.6. Solidphaseextraction

SPEcartridgeswerepackedwith200mgoftheS-NAPimprinted (45–75␮m)andthecorrespondingnon-imprintedxerogels.The cartridgeswereconditionedwith5mLofwater,andasample con-taining3ppmS-NAPinMeOH(1mL)waspercolatedataconstant flowrateof0.5mL/min,inaVisiprep(Supelco,Bellefonte,USA) SPEstationmanifold.Aftersampleloading,1mLofwaterwasused asthewashingsolution.Thecartridgeswerethereaftersubjected

2.7. Chromatographicevaluation

Thesmaller-sizedparticlesofthepreparedmaterialswere typ-ically slurry-packed into stainless steel columns (50×4.6mm), usingMeOH/H2O80:20(v/v)aspushingsolvent,forevaluationof

theirchromatographicperformance.Puremethanolwith0.05or 0.1%aceticacidwasusedasamobilephaseforxerogelsand poly-mers,respectively. Theflowratewasfixedas0.5mL/minifnot otherwisementioned.Aliquots(20␮L)of10ppmsolutionsof S-NAPand30ppmofibuprofen(IBU)inmethanolwereinjected,and elutionmonitoredat230nm.

The retention factor (k), the separation factor (˛) and the imprintingfactor(IF)werecalculatedusingthefollowingformulae k=(t−t0)/t0;˛=knap/kibuandIF=kmip/knip,wheretisthe

reten-tiontimeofthetemplateS-NAPorofitsanalogueIBU,andt0isthe

retentiontimeofthevoidmarker.

Thecolumnefficiencyhasbeendemonstratedintermsof num-beroftheoreticalplates(N),whichwascalculatedaccordingtoEq. (2).

N=5.545



h



(2) whereNisthenumberoftheoreticalplates,tRistheretentiontime

andWhisthepeakwidthathalfheight(intimeunits).

Thebindingproperties ofthematerialsweredeterminedby classicalstaircasefrontalanalysis(foraproperunderstandingofthe technique,readingofreferences[17,18]issuggested).Forthis pur-pose,stocksolutionsofS-NAPorIBUwerepreparedin0.05%acetic acidinMeOHattwodifferentconcentrations:0.05and0.5mmol/L. Thesesolutionswereusedasmobilephasecomponentsatdifferent percentages,mixingwithpuremobilephase(0.05%aceticacidin MeOH),andthusallowinggradientelution.Allexperimentswere carriedoutatroomtemperatureand0.5mL/minflowrate,with detectionat230nm,followingtheorderofincreasing concentra-tionwithoutwashingcyclesinbetweenthestaircases.Attheend oftheexperiments,thecolumnwasflushedfor1hat1mL/min using5%formicacidinMeOH.Foraseriesofnsuccessivesteps,the sampleconcentrationinthestationaryphaseinstepn(qn),at

equi-libriumwithconcentrationCninthemobilephase,wascalculated

usingtheintegralmassbalanceequation(Eq.(3)). qn+1= qn+(Cn+1−_[VCn)FV[teq−(t0−tea)−tep]

c−Fv(t0−tea) (3)

whereFvistheflowrate,t0isthemeasuredvoidtimeofthe

col-umn,teaand tep aretheextra-columntimes(frominjector and

pump,respectively)determinedbyreplacingthecolumnwitha zerodeadvolumeconnector.teawasdeterminedbyinjectingfrom

thesampler,andtepbyrunningastepgradientwithsubsequent

determinationofbreakthroughtimes.Vcisthegeometricalvolume

ofthecolumntube. 2.8. Isothermfitting

Non-linearfittingoftheoreticalisothermstoexperimentaldata wasperformedusingOrigin5.0.Theadsorptionisotherm mod-elscommonlyevaluatedforimprintedmaterialsareLangmuir(Eq. (4)),Freundlich(Eq.(5))andLangmuir–Freundlich,L–F(Eq.(6)). q= q∗KC

1+KC (4)

q=aCm ₍₅₎

q= q∗(KC)m

1+(KC)m (6)

whereqistheconcentrationinthestationaryphaseat equilib-riumwithconcentrationCinthemobilephase.TheLangmuirmodel assumesthatoneclassofsiteispresentonthesurface,with sat-urationcapacity q* and dissociationconstant K.The Freundlich isotherm,ontheotherhand,assumessiteswithaGaussian dis-tribution of binding strengths.Here thewidth of the Gaussian distributiondescribesthe degreeof heterogeneity, throughthe indexm.

ThedifferencebetweentheL–FmodelandtheFreundlichone isevidentathighsorbateconcentrations,forwhichtheL–Fmodel isabletorepresentthesaturationbehaviour.Atlowsorbate con-centrations,theL–FequationreducestotheclassicalFreundlich equation.Ontheotherhand,asmapproachesunity,indicativeof acompletelyhomogeneoussorbentsurface(i.e.,energetic equiva-lenceofallbindingsites),theL–Fequationreducestotheclassical Langmuirequation.Thus,thehybridizedL–Fisothermisableto modeladsorptionofsolutesathighandlowconcentrationsonto bothhomogeneousandheterogeneoussorbents.

The estimates of the modelled parameters are given at the asymptotic95%confidenceinterval.Foreachmodelandeachsetof experimentaldata,theFisherparameterwascalculatedaccording tothefollowingequation(Eq.(7)).

Fcalc=

m−l



m−1



(7) whereqexp,iaretheexperimentalvaluesofthesolid-phase

concen-trationoftheadsorbateforagivensystem,qexpisthemeanvalue

ofthedataset,qexp,i,foragivensystem,qt,iistheestimateofthe

solid-phaseconcentrationoftheadsorbatebyagivenmodel,listhe numberofadjustedparametersinthemodel,andmisthenumber ofexperimentaldata-pointsforagivensystem[19].

Anadditionalmodel,theHill-coefficient,wasconsidered.This modelassumesthatthetemplatemoleculesbindtothesorbent inacooperativemanner[20].Thesorbentisassumedtobindton ligandssimultaneously,nbeingdeterminedfromtheslopeofthe followinglinearrelation(Eq.(8)):

log



(1−q)



=n.log_C−log_K (8)

wherestandsfortheratioq/q*. 3. Resultsanddiscussion

3.1. Thecommonfunctionalmonomerissue

Thefundamentalconditiontocarryoutameaningful compara-tivestudyofsol–gelandacrylicapproachestowardthepreparation

of MIM is to ensure that functional monomers used in both approaches are as similar as possible, hence establishing basi-callythesameinteractionswiththetemplatemolecule.However, tothebestofourknowledge,thisisthefirstreportwheresuch monomersimilaritywasaprimaryconcern.Thesearchfora suit-ablefunctionalmonomercommontobothapproachesbeganwith thepyridinecore,onthebasisofpreviousworkswheremethacrylic imprintsforS-NAPwerepreparedusingvinylpyridineasfunctional monomer[21–24].

Inviewoftheabove,acommerciallyavailablesiloxane func-tionalmonomerbearingthepyridinering,PETMOS(1),wastested forthesol–gelprocess.Expectedly,PETMOSshouldbecapableof a strongacid-base interactionwiththeacidic end ofthe NAP’s carboxylicgroup,whilepossiblybenefitingfromthe solvatopho-biceffectofthewater/MeOHmixture,contributingtotheoverall stabilization of the M–T complex. Evaluation of the xerogels’ performance by SPEallowedconcluding thatall those deriving fromPETMOShadanegligibleMIXtoNIXdifferenceinsorption behaviourinthecourseofloading,washingandallelutingsteps performed(seeFig.S1foratypicalresult).Therefore,no imprint-ingbehaviourwasobserved,indicatingtheessentiallynon-specific natureofthebindingsitesinthesexerogels.Inviewofthis,UPTMOS (2)wasregardedasapossiblybetteralternative,expectedtoform astableM–Tcomplexthroughmultiplehydrogenbonding interac-tionsbetweenthetemplate’scarboxylandthemonomer’sterminal ureagroup( HN CO NH2).However,unsatisfactoryresultswere

also obtained in this case (data not shown), suggesting that a strongerM–Tinteraction,suchasionicpairing,wouldbeneeded. Consequently,itwashypothesizedthattheimidazoliummoiety fromIL-SGwouldinteractmorestronglywiththeS-NAP’s carboxyl-ate,allowingtoimprovetheselectivitypropertiesoftheimprinted xerogel.Likewise,thesameshouldapplyiftheanalogouscationic functionalmonomer,IL-A(4),wasusedinthepreparationofMIP troughtheacrylicapproach.

Asapreliminaryverificationoftheaboveexpectations,UV–vis studies were undertaken to evaluate the interactions between IL-SG (3)orIL-A (4)and S-NAP(detailedspectral dataare sup-pliedasSupportingInformation).Hence,additionofthefunctional monomerstoafixedconcentrationofS-NAPresultedinthe obser-vation that an increasingconcentration of either IL-SG or IL-A provokedabathochromicshiftoftheabsorbancemaximaofS-NAP, intherange270–280nm,andanoveralldecreaseinbandintensity. ThiscorroboratestheformationofM–TcomplexesbetweenS-NAP andthemonomers,similarlytopreviousobservationsonstrong interactionsoccurringbetween4-vinylpyridine(4-VP)andS-NAP astemplatemolecule[24].Still,whilethiseffectcouldbeobserved whenusingthesiloxanemonomer(IL-SG)inmethanol,a combina-tionofmethanolwithamuchlesspolarsolvent,suchaschloroform, wasnecessarytoobtainasimilareffectwiththeacryliccounterpart, IL-A.Theseresultswereconsistentwiththoseobtainedfollowing severaltrialsofMIPsynthesiscomprisingdifferentporogens,from polarlikemethanoltoweaklypolarsuchaschloroform,with vary-ingcrosslinkers,componentratios,andtemplatespeciation(used aseithertheneutral oranionicform). Thesetrialsshowedthat animprintingeffectwasonlyobtainedwhenusingweaklypolar solventsassociatedtotheneutralformofthetemplate.Sol–gel pre-liminarytests,onthecontrary,showedthatbetterresultsemerged fromassociatingapolarporogenwiththeanionicformofthe tem-plate.Consequently,differentporogenshadtobeuseddepending ontheformat(polymerversusxerogel),hencesacrificingporogen uniformityforthesakeofoptimalimprintingeffects.

Inaddition,theUV–visspectraallowedtoroughlyestimatethe optimumratioofthefunctionalmonomertotemplateasbeing4for theacrylicand2forthesol–gelsystem,sincebeyondthoseratios therewasnegligiblechangeintheintensityandwavelengthshift ofabsorbancemaxima.

Fig.3.SPEresultofMIX/NIX-IL-NEC(nonendcapped)andMIX/NIX-IL-EC (end-capped)xerogels.SPEsteps:load:3ppmS-NAPinmethanol–1mL,WI:water– 1mL,EI:MeOH–1mL,EII:5%formicacidinmethanol–1mL.

Imprinted and non-imprinted xerogels, and polymers, have beensynthesizedfrommixturesreflectingthepresented prelimi-naryoutcomes.AquickSPEevaluationofthenon-endcapped(NEC) andend-capped(EC)sol–gelmaterials(MIX-ILand NIX-IL)was conductedtoascertaintheusefulnessoftheend-capping proce-dure,asshowninFig.3.Concerningnon-endcappedxerogels,it wasclearlydemonstratedthatMIX-IL-NEChadmuchhigher affin-ity,witharetentionofca.100%for3ppmS-NAPload(inMeOH), andcouldbeeasilyandcompletelyelutedwith5%formicacidin MeOH;inturn,about85%ofS-NAPwasrecoveredwhileloading inNIX-IL-NEC,andtheremainingwaselutedwithwaterwashing andelutionsteps.Thisconstitutedapromisingresultconcerning theimprintingofS-NAPinMIXpreparedusingIL-SG.

End-cappedxerogelsshoweddifferentretentionbehaviourin SPEascompared totheirnon-endcappedcounterparts;namely, bothMIX-IL-ECandNIX-IL-ECretainedca.100%ofloadedS-NAP(in MeOH)andsimilarrecoveryinallsubsequentsteps.Thisbehaviour may be explained by the non-specific hydrophobic interaction exertedby the insertion of CH3 groups inherent to the

end-cappingprocess,resultinginthemaskingofthelessnumerousor lessaccessibleimprintedsites.Hence,itwasdecidedtokeepthe non-end-cappedxerogelsfortheremainingofthestudy,despite thepotentialproblemsarisingfromthepresenceofresidualsilanol groupsinthenetwork,suchasnon-selectivebindingandnetwork additionalshrinkageduetoageing(additionalcondensationwithin thegel).

3.2. Comparativeporestructureandthermalstability

Imprintedpolymersandxerogels,preparedusingcationic func-tionalmonomersIL-AandIL-SG,respectively,werecharacterized byTGA,BETandSEM.TheBETspecificsurfacearea(S),specificpore volume(Vp)andaverageporediameter(Dp)werecalculatedfrom

thenitrogenadsorptionisothermsand%masslosswasobserved fromTGA,asdescribedintheexperimentalpart.Thevalues con-cerningtheporepropertiesarecollectedinTable2.Itisobserved thatthemethacrylicpolymersexhibitedlargeBETsurfacearea(ca. 320m2_{/g)whilefor thesol–gelmaterialsit wasnegligible.The}

whereasMIXpresentsadensestructuresuggestingmicroporosity. No or little difference was found between imprinted and non-imprintedmaterials,eitherpolymersorxerogels,concerning surfaceareasorporevolumeanddiameter.Thisindicatesthatthe presenceofthetemplate moleculedidnotinfluencetheporous networkoftheresultingmaterials.

The mesoporous nature of methacrylic polymers with pore size (Dp) of 29nm is expected to provide easier access tothe

inner imprinted cavities, ascompared tomicroporous xerogels (Dp<2nm)wherethediffusionisexpectedlyhindered.Formost

applicationsinliquidmedia,permanentporosityandalargesurface areaofaccessiblemesoandmacroporesarepreferred[27].

TheTGAdiagramsofimprintedandnon-imprintedmethacrylic andsol–gelmaterialsmaybefoundasSupportingInformation(Fig. S5),anddemonstratethatthetwotypesofmaterialdisplaydistinct thermal decompositionprofiles. Hence,ca.100% decomposition andweightlossbeganataround250◦C forbothimprintedand non-imprintedpolymers.Inturn,forMIXandNIXxerogels,slow degradationproceedsfrom100to400◦C,followedbyasudden32% masslossataround400◦C.Hence,thepresenceoftheorganic moi-eties(-propyldehydroimidazolium)inthehybridxerogelcauseda slowdegradationstartingatarelativelylowtemperature.Although theinitialmasslossinxerogelsstartingaround100◦Cisprobably duetothereleaseofunreactedspeciesornon-removedtemplate, it appearsfrom theTGAresultsthat themethacrylic systemis thermallymore stableup toaround250◦C. Unfortunately,TGA datagivesinformationaboutmasslossfromthematerials,butnot abouteventualimprintingsitedisruption,whichshouldoccur ear-lierthanmassloss.Itwouldbehelpfultocompareglasstransition temperaturesforboth materials,which wasactuallyattempted bydifferentialscanningcalorimetry(DSC)analysis.But,asoften occursinthistypeofDSCanalysis[28],theglasstransitionpeaks weretoo weaktobedistinguished frombaseline noise. Future evaluationofthermaldisruptionofimprintedsitesmayeventually passbyexposingtheimprintedmaterialstosuccessivelyincreasing temperatures,followedbyassessmentofthebindingperformance (affinity,selectivity)afterexposureateachtemperaturelevel. 3.3. Comparativechromatographicevaluationofimprinted materials

3.3.1. Selectivityandimprintingperformance

Methacrylicandsol–gelmaterialswerepackedinsmallcolumns andassessedbyliquidchromatographyfortheirabilitytoseparate thetemplateS-NAPfromitsstructuralanalogueIBU(Fig.2).IBU waschosenasareferenceinsteadofR-NAP,afterconcludingthat noneofthematerialswasabletoseparatetheenantiomersdespite themanychromatographicconditionstested(datanotshown).

Fig.4.Chromatographicprofileforthemethacrylic(A)andsol–gel(B)imprintedandnon-imprintedmaterials.HPLCconditions:columnsize:50×4.6mm;UVdetection: 230nm,flow:0.5mL/min;mobilephase:MeOHwith0.1%or0.05%aceticacid,respectively.Injectedsolutions(20␮L):10ppmS-NAPand30ppmofIBUinmethanol.Notice thesuperposedelutionprofilesofS-NAPandIBUwiththeNIXmaterial.

Inordertocalculatetheseparationfactor(˛)andimprinting factor(IF),methanolicsolutionsofS-NAPandIBUwereinjected intothepackedcolumns,yieldingthechromatogramsshownin Fig.4.Retentiontimeswerenotedfromthechromatographicpeaks, andthecalculatedvaluesof˛andIFofthematerialsobtainedare presentedinTable3.

ItisevidentfromFig.4Athattheimprintedpolymershowed higher retention for thetemplate S-NAPthan for its structural analogueIBU(˛=2.5),provingtheimprintingbehaviour.The non-imprintedpolymer(NIP)alsodiscriminatedthetemplateandits analogue,buttoa markedlylesserextent(˛=1.1),possiblydue tonon-specificinteractionsrelatedtothemesoporousnatureand functionalizationofthematerial[27].

Ontheotherhand,imprintedandnon-imprintedxerogels per-formedmoredistinctly.Fig.4Bclearlyshowsthattheimprinted xerogelhadamuchhigheraffinityforS-NAPthanforitsstructural analogueIBU (˛=6.23), which demonstrates thehighlyspecific recognitionoftheimprintedtemplate.Inturn,thenon-imprinted xerogelwasunabletodiscriminatethetemplateanditsanalogue (˛=1).

TheefficiencyofthepreparedHPLCcolumns,whichinpractice relatestothewidthof thechromatographicpeak observedata certainretentiontime,indirectlyprovidesinformationaboutthe masstransferkineticsbetweenthesupportsand thetemplates, andmayalsoreflectthedegreeofheterogeneityofthesorption sites.Theoreticalnumberofplatesof6and125wereestimated accordingtoEq.(2)forMIXandMIP,respectively,demonstrating themuchhighercolumnefficiencyobtainedforMIP.

In summary, the imprinted xerogel showed markedly bet-ter selectivitythan itspolymer counterpart, but withvery low columnefficiency.Inturn,themethacrylicMIP presentedmuch

better column efficiency associated to reasonable selectivity. The dissimilar efficiencies seem to indicate that the quality of templateimprintingismuchhigherinthesol–gelapproach,which isunfortunatelyeclipsedbyitsmicroporousnature.In contrast, methacrylicmaterialspresentbettermasstransferkineticsand/or lessheterogeneousbindingsitedistribution,eventuallyassociated totheirmesoporousnature.

3.3.2. Sorptionisothermanalysis

Togetdeeperinsightintothemethacrylicandsol–gelmaterials bindingpropertiessuchas,capacity,affinityconstant, heterogene-ityindexandmasstransferkinetics,frontalanalysiswasconducted. Such analysis allowed to estimatethe amount of template, or itsanalogue,adsorbed(q)ontotheimprintedandnon-imprinted stationaryphasesinequilibriumwithagivenmobilephase con-centration,C,usingEq.(3).

Datapoints(q,C)obtained(Fig.5)werethenfittedtothe differ-entisothermmodels,suchasLangmuir(Eq.(4)),Freundlich(Eq.(5)) andhybridLangmuir–Freundlich(Eq.(6))bynon-linearregression analysis.Sincesomeof theisotherms appearedtofollow a sig-moidalpattern,theHillcoefficientmodelwasalsocheckedagainst theexperimentaldata-setbyusingthelinearizationEq.(8).A sig-moidalshapemayberelatedtocooperativebindingphenomena wherebindingtothesorbentbecomesfacilitated(positively coop-erativebinding)orhampered(negativelycooperativebinding)by previouslyadsorbedligands.

Thecompletefitteddata-setispresentedasSupporting Infor-mation.Table4comprisesthemostrelevantdata,namelythose obtainedfromthemodelthatfittedbesttotheexperimentaldata for eachmaterial and sorbate.The MIP-associateddata-setwas theonlyonefittingtheHillcoefficientmodel,asshowninFig.S6,

Table3

Chromatographicpropertiesofmethacrylicandsol–gelmaterialtowardsS-NAPandIBU.

Materials Retentionfactor,kNAP Retentionfactor,kIBU Selectivity␣elekNAP/kIBU Imprintingfactor=kMIP/kNIP

IBU S-NAP

MIX 32.40 5.20 6.2 3.9 24

NIX 1.35 1.35 1.0

MIP 7.03 2.86 2.5 3.6 7.7

n− m

inpre-polymerizationmixtures,resultinginimprintedsitesforthe (template)ncomplexeswhoseabundanceintherebindingsolution

willincreasewithincreasingconcentration[29];(ii)inducedfit, whichreferstoa processwhere theinitialinteractionbetween thematerialandtheligandisrelativelyweak,butwhichrapidly inducesconformationalchangesin thestructureofthematerial thatstrengthenbinding;inducedfithastypicallybeenrelatedto highlyswellingsystemsforproteinimprinting[30]andto cova-lentlyimprintedpolymers[31].Suchmechanismisthusunlikely affectingthepresentS-NAPimprintedpolymer.Acapable elucida-tionofthisquestionisbeyondthescopeofthisstudy,anddepends onfuturefocused work.Inanycase, whatever mechanismwas activehere,seemsalsotoaccommodateIBU rebinding,another interestingfeaturedeservingfurtherstudy,whichisinlinewith thelowerselectivityfoundintheframeofMIP/MIXcomparison.

Fig.5.EquilibriumbindingisothermsforS-NAPandIBUasdeterminedfrom stair-casefrontalanalysisofthemethacrylic(MIPandNIP)andsol–gel(MIXandNIX) materials.q:sorbateconcentrationinthetestedmaterial;C:concentrationofthe sorbatepresentinthemobilephase.

capacities(q*)oftheimprintedandnon-imprintedpolymerswere estimatedforthetemplate,S-NAP:ca.4.9and2.8␮mol/g, respec-tively.ForitsanalogueIBU,therespectivevalueswereca.2.2and 3.6␮mol/g.Theimprintedmethacrylicpolymerexhibitedthusa bettercapacityforthetemplateS-NAP,roughlytwotimeshigher thanthatforthestructuralanalogueIBU.Alsosignificantlyhigher capacitytowardsS-NAPwasobservedtheMIPrelativelytoNIP, reinforcingtheconclusionthatasatisfactorilyimprintedpolymer wasproduced.

Some considerations about site heterogeneity can also be drawn.InthecaseofMIP,sitehomogeneityisanassumptionof themodelwhichiscorroboratedbythegoodnessoffitobserved. InthecaseofNIP,aheterogeneityparameterwascalculatedfrom theL–Ffitting,thematerialexhibitinghomogeneousbindingsites asindicatedby“m”valuecloseto1.Thesymmetricpeakobtained inchromatogram(Fig.4A)forS-NAPwiththeMIP/NIPmaterials agreeswiththesedata.

In what regards sol–gel materials, different adsorption behaviour was observed, as it was found that the Freundlich model allowed for the best fit of xerogel-associated data-sets. The estimatedsaturation capacity, q*, for MIXshowed a much higher value for S-NAP, 3.7␮mol/g, than for IBU,0.60␮mol/g. Furthermore, a much higher affinity was found for S-NAP (K=250Lmmol−1)overIBU(K=111Lmmol−1).Bothq*andK val-uesforMIXweremuchhigherthanthoseforNIX,confirmingthe excellentimprintingeffectonMIX.Approximatevaluesofm=0.5

Fig.6.Frontalchromatograms(partialview)obtainedforthesol–gel(MIX)and methacrylic(MIP)imprintedmaterials,allowingtheobservationofamuchfaster stabilizationofthedetectorsignalfortheMIPaftereachchangeintheconcentration ofS-NAPinthemobilephase.

indicatedsitedistributionheterogeneity.Thisisinagreementwith thepronouncedpeakbroadeningandtailing(Fig.4B)obtainedin theMIX-packedHPLCcolumn.Still,peakbroadeningmayalsobe partiallyexplainedbythepoormasstransferpropertiesassociated withthemicroporousstructureofMIX(Table2).Takentogether, frontalanalysisresultsallowtostatethatMIP exhibitedhigher capacityandmorehomogeneousbindingsitesthatMIX.

Finally, another interesting observation was obtained from frontalchromatograms,whichagreedwellwithBET,SEMandHPLC analysisalreadydescribed.AsclearlyseeninFig.6,MIPreached equilibriummuch fasterthanMIX,irrespectiveofflowrateand concentration.Forexample,forthe5–10␮Mconcentrationstep, the equilibration time required by MIP was 7.7min, while for MIX16.80minwereneeded.Again,thismaybeexplainedbythe absenceofmesoporosityinMIX,whichinfacthindersaneasyflow, resultinginlongerequilibrationtime.

4. Conclusion

Themainobjectiveofthisworkwastocomparethetwo dif-ferentsyntheticapproachesandgetimportantinsightsintermsof materialporeproperties,performanceparameterssuchas imprint-ingfactorandselectivity,andbindingpropertiesincludingbinding capacityandaffinity,heterogeneityindex,columnefficiencyand masstransferkinetics.Stemmingfromhighlysimilarcationic func-tionalmonomerstargetingthecarboxylicgroupinthetemplate, MIPand MIXmaterialswereoptimizedfor performance, impli-catingthatdifferentporogen,templatespeciationandcomponent ratioshadtobeusedforthetwoapproaches.Suchfunctional group-orientedstudyofcoursedoesnotmeanthat betterimprints in aparticularformatcouldnotbeobtainedwithotherfunctional groups,asevidencedforthemethacrylicimprintproducedwith 4-VP,whichexhibitedasuperiorselectivity(enantioseparation)as comparedtothosereportedhere.Itisthereforeessentialtokeep relativityinmindwhenfollowingthenextconsiderations,which concerncomparisonofthetwoimprintingprocessesstudied.

Pore structure plays an important role in the performance of imprints, and a very important differencebetween the two networks in this regard emerged. In fact, MIP exhibited meso-porosity and MIXdidnot, what constituteda serious handicap forMIXperformance,asreflectedintermsofdiminished acces-sibleimprintedsites(capacity)andofslowerbinding/dissociation kinetics.Globallywecanstatethatthesol–gelapproachallowed forhigherselectivityandimprintingfactor,whilethemethacrylic polymerizationapproachwassuperiorinterms ofcapacity and mass transferkineticsof theproduced imprinted material.The higherselectivityfoundforMIXmaybepartiallyattributableto the solventmemory effect (rebinding tested in methanol), but otherfactorsmaybeinvolved,suchasnothavingusedexactlythe samefunctionalgroupandtemplatespeciationinbothapproaches. Besideshighselectivity,agoodcapacityandfastkineticsarehighly desirablefeaturesintheapplicationofimprintedmaterialssuch asanimprintedstationaryphase inchromatography.Since MIP presentedaquitesatisfactoryselectivity,allconsidered,itseems reasonabletoconcludethat,withintheconstraintsinherenttothe developmentofthiscomparativework,theacrylicapproachwas themostsuccessful.

It is ourconviction that future developments in the sol–gel procedure towards mesoporosity, for example by using

macromoleculartemplating,mayaccomplishsignificant improve-ment in MIX performance. On the other hand, a deeper understanding of factors underlying the apparent cooperative binding mechanisms active for both S-NAP and IBU with MIP mayhelpdevisingmodifiedproceduresresultinginanimproved selectivity.One final word regarding use of imidazolium-based cationicfunctionalmonomers:thisvirtuallyunexplored function-ality,inthecontextofimprintingprocesses,appearstoberather interestingandpossiblyadvantageousfortheimprintingofother templatescapableofassuminganionicorhighlyelectron-donating forms.

Acknowledgements

The authors thank Fundac¸ão para a Ciência e Tecnologia (FCT),Lisbon,Portugal,andtoFEDERforfinancialsupport (PEst-C/QUI/UI0081/2011). P.K. thanks FCT also for the Ph.D. Grant SFRH/BD/44715/2008.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.chroma.2013. 09.015.

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