Gel permeation chromatography - Wikipedia

Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC), that separates analytes on the basis of size, typically in organic ... Gelpermeationchromatography FromWikipedia,thefreeencyclopedia Jumptonavigation Jumptosearch Gelpermeationchromatography(GPC)isatypeofsize-exclusionchromatography(SEC),thatseparatesanalytesonthebasisofsize,typicallyinorganicsolvents.Thetechniqueisoftenusedfortheanalysisofpolymers.Asatechnique,SECwasfirstdevelopedin1955byLatheandRuthven.[1]ThetermgelpermeationchromatographycanbetracedbacktoJ.C.MooreoftheDowChemicalCompanywhoinvestigatedthetechniquein1964.TheproprietarycolumntechnologywaslicensedtoWatersCorporation,whosubsequentlycommercializedthistechnologyin1964.[2]GPCsystemsandconsumablesarenowalsoavailablefromanumberofmanufacturers.Itisoftennecessarytoseparatepolymers,bothtoanalyzethemaswellastopurifythedesiredproduct. Whencharacterizingpolymers,itisimportanttoconsiderthedispersity(Đ)aswellthemolecularweight.Polymerscanbecharacterizedbyavarietyofdefinitionsformolecularweightincludingthenumberaveragemolecularweight(Mn),theweightaveragemolecularweight(Mw)(seemolarmassdistribution),thesizeaveragemolecularweight(Mz),ortheviscositymolecularweight(Mv).GPCallowsforthedeterminationofĐaswellasMvand,basedonotherdata,theMn,Mw,andMzcanbedetermined. Contents 1Howitworks 2Application 3Materialandmethods 3.1Instrumentation 3.2Gel 3.3Column 3.4Eluent 3.5Pump 3.6Detector 4Dataanalysis 5Advantages 6Disadvantages 7Orthogonalmethods 8References Howitworks[edit] GPCseparatesbasedonthesizeorhydrodynamicvolume(radiusofgyration)oftheanalytes.Thisdiffersfromotherseparationtechniqueswhichdependuponchemicalorphysicalinteractionstoseparateanalytes.[3]Separationoccursviatheuseofporousbeadspackedinacolumn(seestationaryphase(chemistry)).SchematicofporevsanalytesizeThesmalleranalytescanentertheporesmoreeasilyandthereforespendmoretimeinthesepores,increasingtheirretentiontime.Thesesmallermoleculesspendmoretimeinthecolumnandthereforewillelutelast.Conversely,largeranalytesspendlittleifanytimeintheporesandareelutedquickly.Allcolumnshavearangeofmolecularweightsthatcanbeseparated.RangeofmolecularweightsthatcanbeseparatedforeachpackingmaterialIfananalyteistoolarge,itwillnotberetained;conversely,iftheanalyteistoosmall,itmayberetainedcompletely.Analytesthatarenotretainedareelutedwiththefreevolumeoutsideoftheparticles(Vo),whileanalytesthatarecompletelyretainedareelutedwithvolumeofsolventheldinthepores(Vi).Thetotalvolumecanbeconsideredbythefollowingequation,whereVgisthevolumeofthepolymergelandVtisthetotalvolume:[3] V t = V g + V i + V o {\displaystyleVt=Vg+Vi+Vo} Ascanbeinferred,thereisalimitedrangeofmolecularweightsthatcanbeseparatedbyeachcolumn,andthereforethesizeoftheporesforthepackingshouldbechosenaccordingtotherangeofmolecularweightofanalytestobeseparated.Forpolymerseparationstheporesizesshouldbeontheorderofthepolymersbeinganalyzed.IfasamplehasabroadmolecularweightrangeitmaybenecessarytouseseveralGPCcolumnsintandemtoresolvethesamplefully. Application[edit] GPCisoftenusedtodeterminetherelativemolecularweightofpolymersamplesaswellasthedistributionofmolecularweights.WhatGPCtrulymeasuresisthemolecularvolumeandshapefunctionasdefinedbytheintrinsicviscosity.Ifcomparablestandardsareused,thisrelativedatacanbeusedtodeterminemolecularweightswithin±5%accuracy.Polystyrenestandardswithdispersitiesoflessthan1.2aretypicallyusedtocalibratetheGPC.[4]Unfortunately,polystyrenetendstobeaverylinearpolymerandthereforeasastandarditisonlyusefultocompareittootherpolymersthatareknowntobelinearandofrelativelythesamesize. Materialandmethods[edit] Instrumentation[edit] AtypicalGPCinstrumentincluding:A.Autosampler,B.Column,C.Pump,D.RIdetector,E.UV-visdetector Insideofanautosamplerforrunningseveralsampleswithoutuserinteraction,e.g.overnight Gelpermeationchromatographyisconductedalmostexclusivelyinchromatographycolumns.Theexperimentaldesignisnotmuchdifferentfromothertechniquesofliquidchromatography.Samplesaredissolvedinanappropriatesolvent,inthecaseofGPCthesetendtobeorganicsolventsandafterfilteringthesolutionitisinjectedontoacolumn.Theseparationofmulti-componentmixturetakesplaceinthecolumn.Theconstantsupplyoffresheluenttothecolumnisaccomplishedbytheuseofapump.Sincemostanalytesarenotvisibletothenakedeyeadetectorisneeded.Oftenmultipledetectorsareusedtogainadditionalinformationaboutthepolymersample.Theavailabilityofadetectormakesthefractionationconvenientandaccurate. Gel[edit] GelsareusedasstationaryphaseforGPC.Theporesizeofagelmustbecarefullycontrolledinordertobeabletoapplythegeltoagivenseparation.Otherdesirablepropertiesofthegelformingagentaretheabsenceofionizinggroupsand,inagivensolvent,lowaffinityforthesubstancestobeseparated.CommercialgelslikePLgel&Styragel(cross-linkedpolystyrene-divinylbenzene),[5][6]LH-20(hydroxypropylatedSephadex),[7]Bio-Gel(cross-linkedpolyacrylamide),HW-20&HW-40(hydroxylatedmethacrylicpolymer),[8]agarosegelandareoftenusedbasedondifferentseparationrequirements.[9] Column[edit] ThecolumnusedforGPCisfilledwithamicroporouspackingmaterial.Thecolumnisfilledwiththegel. Eluent[edit] Theeluent(mobilephase)shouldbeagoodsolventforthepolymer,shouldpermithighdetectorresponsefromthepolymerandshouldwetthepackingsurface.ThemostcommoneluentsforpolymersthatdissolveatroomtemperatureGPCaretetrahydrofuran(THF),o-dichlorobenzeneandtrichlorobenzeneat130–150 °Cforcrystallinepolyalkynesandm-cresolando-chlorophenolat90 °Cforcrystallinecondensationpolymerssuchaspolyamidesandpolyesters. Pump[edit] TherearetwotypesofpumpsavailableforuniformdeliveryofrelativelysmallliquidvolumesforGPC:pistonorperistalticpumps. Detector[edit] InGPC,theconcentrationbyweightofpolymerintheelutingsolventmaybemonitoredcontinuouslywithadetector.Therearemanydetectortypesavailableandtheycanbedividedintotwomaincategories.ThefirstisconcentrationsensitivedetectorswhichincludesUVabsorption,differentialrefractometer(DRI)orrefractiveindex(RI)detectors,infrared(IR)absorptionanddensitydetectors.Thesecondcategoryismolecularweightsensitivedetectors,whichincludelowanglelightscatteringdetectors(LALLS)andmultianglelightscattering(MALLS).[10]Theresultingchromatogramisthereforeaweightdistributionofthepolymerasafunctionofretentionvolume.GPCchromatogram;Vo=noretention,Vt=completeretention,AandB=partialretentionThemostsensitivedetectoristhedifferentialUVphotometerandthemostcommondetectoristhedifferentialrefractometer(DRI).Whencharacterizingcopolymer,itisnecessarytohavetwodetectorsinseries.[4]Foraccuratedeterminationsofcopolymercompositionatleasttwoofthosedetectorsshouldbeconcentrationdetectors.[10]ThedeterminationofmostcopolymercompositionsisdoneusingUVandRIdetectors,althoughothercombinationscanbeused.[11] Dataanalysis[edit] Gelpermeationchromatography(GPC)hasbecomethemostwidelyusedtechniqueforanalyzingpolymersamplesinordertodeterminetheirmolecularweightsandweightdistributions.ExamplesofGPCchromatogramsofpolystyrenesampleswiththeirmolecularweightsanddispersitiesareshownontheleft. GPCSeparationofAnionicallySynthesizedPolystyrene;Mn=3,000g/mol,Đ=1.32 GPCSeparationofFree-RadicalSynthesizedPolystyrene;Mn=24,000g/mol,Đ=4.96 Standardizationofasize-exclusioncolumnBenoitandco-workers[citationneeded]proposedthatthehydrodynamicvolume,Vη,whichisproportionaltotheproductof[η]andM,where[η]istheintrinsicviscosityofthepolymerintheSECeluent,maybeusedastheuniversalcalibrationparameter.IftheMark–Houwink–SakuradaconstantsKandαareknown(seeMark–Houwinkequation),aplotoflog[η]Mversuselutionvolume(orelutiontime)foraparticularsolvent,columnandinstrumentprovidesauniversalcalibrationcurvewhichcanbeusedforanypolymerinthatsolvent.Bydeterminingtheretentionvolumes(ortimes)ofmonodispersepolymerstandards(e.g.solutionsofmonodispersedpolystyreneinTHF),acalibrationcurvecanbeobtainedbyplottingthelogarithmofthemolecularweightversustheretentiontimeorvolume.Oncethecalibrationcurveisobtained,thegelpermeationchromatogramofanyotherpolymercanbeobtainedinthesamesolventandthemolecularweights(usuallyMnandMw)andthecompletemolecularweightdistributionforthepolymercanbedetermined.Atypicalcalibrationcurveisshowntotherightandthemolecularweightfromanunknownsamplecanbeobtainedfromthecalibrationcurve. Advantages[edit] Asaseparationtechnique,GPChasmanyadvantages.Firstofall,ithasawell-definedseparationtimeduetothefactthatthereisafinalelutionvolumeforallunretainedanalytes.Additionally,GPCcanprovidenarrowbands,althoughthisaspectofGPCismoredifficultforpolymersamplesthathavebroadrangesofmolecularweightspresent.Finally,sincetheanalytesdonotinteractchemicallyorphysicallywiththecolumn,thereisalowerchanceforanalytelosstooccur.[3]Forinvestigatingthepropertiesofpolymersamplesinparticular,GPCcanbeveryadvantageous.GPCprovidesamoreconvenientmethodofdeterminingthemolecularweightsofpolymers.Infactmostsamplescanbethoroughlyanalyzedinanhourorless.[12]Othermethodsusedinthepastwerefractionalextractionandfractionalprecipitation.Astheseprocesseswerequitelabor-intensivemolecularweightsandmassdistributionstypicallywerenotanalyzed.[13]Therefore,GPChasallowedforthequickandrelativelyeasyestimationofmolecularweightsanddistributionforpolymersamples Disadvantages[edit] TherearedisadvantagestoGPC,however.First,thereisalimitednumberofpeaksthatcanberesolvedwithintheshorttimescaleoftheGPCrun.Also,asatechniqueGPCrequiresaroundatleasta10%differenceinmolecularweightforareasonableresolutionofpeakstooccur.[3]Inregardstopolymers,themolecularmassesofmostofthechainswillbetooclosefortheGPCseparationtoshowanythingmorethanbroadpeaks.AnotherdisadvantageofGPCforpolymersisthatfiltrationsmustbeperformedbeforeusingtheinstrumenttopreventdustandotherparticulatesfromruiningthecolumnsandinterferingwiththedetectors.Althoughusefulforprotectingtheinstrument,thereisthepossibilityofthepre-filtrationofthesampleremovinghighermolecularweightsamplebeforeitcanbeloadedonthecolumn.Anotherpossibilitytoovercometheseissuesistheseparationbyfield-flowfractionation(FFF). Orthogonalmethods[edit] Field-flowfractionation(FFF)canbeconsideredasanalternativetoGPC,especiallywhenparticlesorhighmolarmasspolymerscausecloggingofthecolumn,sheardegradationisanissueoragglomerationtakesplacebutcannotbemadevisible.FFFisseparationinanopenflowchannelwithouthavingastaticphaseinvolvedsonointeractionsoccur.Withonefield-flowfractionationversion,thermalfield-flowfractionation,separationofpolymershavingthesamesizebutdifferentchemicalcompositionsispossible.[14] References[edit] ^Lathe,G.H.;Ruthven,C.R.J.TheSeparationofSubstanceand'1956,62,665–674.PMID 13249976 ^Moore,J.C.Gelpermeationchromatography.I.Anewmethodformolecularweightdistributionofhighpolymers.J.Polym.Sci.,1964,2,835-843.[1][deadlink]doi:10.1002/pol.1964.100020220 ^abcdSkoog,D.A.PrinciplesofInstrumentalAnalysis,6thed.;ThompsonBrooks/Cole:Belmont,California,2006,Chapter28. ^abSandler,S.R.;Karo,W.;Bonesteel,J.;Pearce,E.M.PolymerSynthesisandCharacterization:ALaboratoryManual;AcademicPress:SanDiego,1998. ^AgilentTechnologies."AGILENTORGANICGPC/SECCOLUMNS"(PDF).Retrieved2019-12-06. ^WatersCorporation."STYRAGELCOLUMNCAREANDUSEMANUAL"(PDF).Retrieved2019-12-06. ^GEHealthcare."SephadexLH-20".Retrieved2019-12-06. ^TOSOHBIOSCIENCE."TOYOPEARLHW-40".Retrieved2019-12-06. ^Helmut,D.GelChromatography,GelFiltration,GelPermeation,MolecularSieves:ALaboratoryHandbook;Springer-Verlag,1969. ^abTrathnigg,B.DeterminationofMWDandChemicalCompositionofPolymersbyChromatographicTechniques.Prog.Polym.Sci.1995,20,615-650.[2]doi:10.1016/0079-6700(95)00005-Z ^Pasch,H.HyphenatedTechniquesinLiquidChromatographyofPolymers.Adv.Polym.Sci.2000,150,1-66.[3]doi:10.1007/3-540-48764-6 ^Cowie,J.M.G.;Arrighi,V.Polymers:ChemistryandPhysicsofModernMaterials,3rded.CRCPress,2008. ^OdianG.PrinciplesofPolymerization,3rded.;WileyIntersciencePublication,1991. ^ThermalField-FlowFractionation:Ultra-BroadPolymerSeparation|http://www.chemeurope.com/en/products/77045/thermal-field-flow-fractionation-ultra-broad-polymer-separation.htmlArchived2013-10-19attheWaybackMachine Retrievedfrom"https://en.wikipedia.org/w/index.php?title=Gel_permeation_chromatography&oldid=1090993889" Categories:BiochemicalseparationprocessesChromatographyPolymersPolyolefinsHiddencategories:AllarticleswithdeadexternallinksArticleswithdeadexternallinksfromFebruary2019WebarchivetemplatewaybacklinksAllarticleswithunsourcedstatementsArticleswithunsourcedstatementsfromSeptember2019 Navigationmenu Personaltools NotloggedinTalkContributionsCreateaccountLogin Namespaces ArticleTalk English Views ReadEditViewhistory More Search Navigation MainpageContentsCurrenteventsRandomarticleAboutWikipediaContactusDonate Contribute HelpLearntoeditCommunityportalRecentchangesUploadfile Tools WhatlinkshereRelatedchangesUploadfileSpecialpagesPermanentlinkPageinformationCitethispageWikidataitem Print/export DownloadasPDFPrintableversion Inotherprojects WikimediaCommons Languages العربيةBosanskiČeštinaDeutschEestiفارسیHrvatskiItalianoNederlands日本語SlovenčinaSvenskaУкраїнська Editlinks