Gel Filtration Chromatography- Definition, Principle, Types ...

Principle of Gel Filtration Chromatography · The stationary phase used is a porous polymer matrix whose pores are completely filled with the ... Skiptocontent Menu Home»Instrumentation»GelFiltrationChromatography-Definition,Principle,Types,Parts,Steps,Uses Subscribeustoreceivelatestnotes. EmailAddress* Biomoleculesarepurifiedusingdifferenttechniquesthatseparatethemaccordingtothedifferencesintheirspecificpropertiessuchassize,hydrophobicity,biorecognition,charge,etc. Gelfiltrationisatechniqueinwhichtheseparationofcomponentsisbasedonthedifferenceinmolecularweightorsize. Itisthesimplestandmildestofallthechromatographytechniquesandseparatesmoleculesonthebasisofdifferencesinsize. TableofContents PrincipleofGelFiltrationChromatographyTypesofGelFiltrationChromatographyStepsinGelFiltrationChromatographyApplicationsofGelFiltrationChromatographyAdvantagesofGelFiltrationChromatographyLimitationsofGelFiltrationChromatographyReferences PrincipleofGelFiltrationChromatography ImageSource:MBLLifeScience. Toperformaseparation,thegelfiltrationmediumispackedintoacolumntoformapackedbed.Themediumisaporousmatrixintheformofsphericalparticlesthathavebeenchosenfortheirchemicalandphysicalstability,andinertness(lackofreactivityandadsorptiveproperties).Thepackedbedisequilibratedwithabufferwhichfillstheporesofthematrixandthespaceinbetweentheparticles.Theliquidinsidetheporesissometimesreferredtoasthestationaryphaseandthisliquidisinequilibriumwiththeliquidoutsidetheparticles,referredtoasthemobilephase. Thestationaryphaseusedisaporouspolymermatrixwhoseporesarecompletelyfilledwiththesolventtobeusedasthemobilephase.Themoleculesinthesamplearepumpedthroughspecializedcolumnscontainingsuchmicroporouspackingmaterial(gel).Thebasisoftheseparationisthatmoleculesaboveacertainsizearetotallyexcludedfromthepores,whilesmallermoleculesaccesstheinterioroftheporespartlyorwholly.Theflowofthemobilephasehencewillcauselargermoleculestopassthroughthecolumnunhindered,withoutpenetratingthegelmatrix,whereassmallermoleculeswillberetardedaccordingtotheirpenetrationofthegel. TypesofGelFiltrationChromatography GroupSeparations Thecomponentsofasampleareseparatedintotwomajorgroupsaccordingtothesizerange.Agroupseparationcanbeusedtoremovehighorlowmolecularweightcontaminants(suchasphenolredfromculturefluids)ortodesaltandexchangebuffers. High-resolutionfractionationofbiomolecules Thecomponentsofasampleareseparatedaccordingtodifferencesintheirmolecularsize.High-resolutionfractionationcanbeusedtoisolateoneormorecomponents,toseparatemonomersfromaggregates,todeterminethemolecularweightortoperformamolecularweightdistributionanalysis. StepsinGelFiltrationChromatography Sphericalparticlesofgelfiltrationmediumarepackedintoacolumn.Thesampleisappliedtothecolumn.Buffer(mobilephase)andsamplemovethroughthecolumn.Moleculesdiffuseinandoutoftheporesofthematrix(alsodescribedasthepartitioningofthesamplebetweenthemobilephaseandthestationaryphase).Smallermoleculesmovefurtherintothematrixandsostaylongeronthecolumn.Asbufferpassescontinuouslythroughthecolumn,moleculesthatarelargerthantheporesofthematrixareunabletodiffuseintotheporesandpassthroughthecolumn.Smallermoleculesdiffuseintotheporesandaredelayedintheirpassagedownthecolumn.Separationoccursatdifferentintervalswhicharefollowedbydetectionofcomponents. ApplicationsofGelFiltrationChromatography Gelfiltrationplaysakeyroleinthepurificationofenzymes,polysaccharides,nucleicacids,proteins,andotherbiologicalmacromolecules.Gelfiltrationcanalsobeusedtofacilitatetherefoldingofdenaturedproteinsbycarefulcontrolofchangingbufferconditions.Itisusedinproteinfractionationexperiments.Gelfiltrationtechniqueisalsousedinmolecularweightdetermination.Separationofsugar,proteins,peptides,rubbers,andothersonthebasisoftheirsize.Canbeusedtodeterminethequaternarystructureofpurifiedproteins. AdvantagesofGelFiltrationChromatography GelfiltrationisarobusttechniquethatiswellsuitedtohandlingbiomoleculesthataresensitivetochangesinpH,theconcentrationofmetalionsorco-factorsandharshenvironmentalconditions.Asignificantadvantageofgelfiltrationisthatconditionscanbevariedtosuitthetypeofsampleortherequirementsforfurtherpurification,analysisorstoragewithoutalteringtheseparation.Separationscanbeperformedinthepresenceofessentialionsorcofactors,detergents,urea,guanidinehydrochloride,athighorlowionicstrength,at37°Corinthecoldroomaccordingtotherequirementsoftheexperiment.Unlikeionexchangeoraffinitychromatography,moleculesdonotbindtothechromatographymediumsobuffercompositiondoesnotdirectlyaffectresolution(thedegreeofseparationbetweenpeaks).Shortanalysistime.Welldefinedseparation.Narrowbandsandgoodsensitivity.Thereisnosampleloss.Thesmallamountofmobilephaserequired.Theflowratecanbeset. ReadAlso:Bacillusanthracis-AnOverviewBordetellapertussis-AnOverviewLactobacillusacidophilus-AnOverview14TypesofChromatography(Definition,Principle,Steps,Uses)Pseudomonasaeruginosa-AnOverview LimitationsofGelFiltrationChromatography Thelimitednumberofpeaksthatcanberesolvedwithintheshorttimescaleoftherun.Filtrationsmustbeperformedbeforeusingtheinstrumenttopreventdustandotherparticulatesfromruiningthecolumnsandinterferingwiththedetectors.Themolecularmassesofmostofthechainswillbetooclosefortheseparationtoshowanythingmorethanbroadpeaks. References http://kirschner.med.harvard.edu/files/protocols/GE_gelfiltration.pdfWilson,K.,Walker,J.(2018).PrinciplesandTechniquesofBiochemistryandMolecularBiology(8eds.).CambridgeUniversityPress:NewYork.https://www.slideshare.net/asabuwangwa/gel-permeation-chromatography-gpchttp://www.materials-talks.com/blog/2016/08/30/an-introduction-to-gel-permeation-chromatography-in-30-minutes/https://chromatography.conferenceseries.com/events-list/applications-of-chromatographyhttp://library.umac.mo/ebooks/b28050630.pdfhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/ Subscribeustoreceivelatestnotes. EmailAddress* LeaveaCommentCancelreplyCommentName Email Savemyname,email,andwebsiteinthisbrowserforthenexttimeIcomment. Δ ThissiteusesAkismettoreducespam.Learnhowyourcommentdataisprocessed. 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