Gel Filtration Chromatography - Bio-Rad

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Gel filtration chromatography can be used to separate compounds such as small molecules, proteins, protein complexes, polysaccharides, and nucleic acids when in ... GelFiltrationChromatography ENrich™SizeExclusion ChromatographyColumns BuyENrichColumns Needhigh-resolutionseparationsathighflowrates?UseENrichsizeexclusionchromatographycolumns.   Bio-Spin®andMicro Bio-Spin™SizeExclusionColumns BuyBio-SpinColumns Usetheconvenient,ready-to-useBio-SpinandMicroBio-Spinsizeexclusioncolumnsforrapid,effective,andeconomicalcleanupofDNA,RNA,andproteinsamples.   Econo-Pac®10DGDesalting PrepackedGravityFlowColumns BuyEconoPacColumns UseEcono-Pac10DGprepackedgravityflowgelfiltrationchromatographycolumnsforeasyandfastdesalting.   GelFiltration ChromatographyMedia BuyGelFiltrationResin Selectgelfiltrationmediainavarietyofbeadandmeshsizesfordesalting,bufferexchange,andsizefractionationinaqueousororganicsolutions.   GelFiltrationChromatographyApplications Gelfiltrationchromatography,atypeofsizeexclusionchromatography,canbeusedtoeitherfractionatemoleculesandcomplexesinasampleintofractionswithaparticularsizerange,toremoveallmoleculeslargerthanaparticularsizefromthesample,oracombinationofbothoperations.Gelfiltrationchromatographycanbeusedtoseparatecompoundssuchassmallmolecules,proteins,proteincomplexes,polysaccharides,andnucleicacidswheninaqueoussolution.Whenanorganicsolventisusedasthemobilephase,theprocessisinsteadreferredtoasgelpermeationchromatography. Gelfiltrationchromatographycanalsobeusedfor: Fractionationofmoleculesandcomplexeswithinapredeterminedsizerange Sizeanalysisanddetermination Removaloflargeproteinsandcomplexes Bufferexchange Desalting Removalofsmallmoleculessuchasnucleotides,primers,dyes,andcontaminants Assessmentofsamplepurity Separationofboundfromunboundradioisotopes Gelfiltrationchromatographymediaforalloftheaboveusesareavailableinprepackedgravityflowcolumns,spincolumns,low-pressureandmedium-pressurechromatographycolumns,andbottledresins. GelFiltrationChromatographyMechanism Inagelfiltrationchromatographycolumn,thestationaryphaseiscomposedofaporousmatrix,andthemobilephaseisthebufferthatflowsinbetweenthematrixbeads.Thebeadshaveadefinedporesizerange,knownasthefractionationrange.Moleculesandcomplexesthataretoolargetoentertheporesstayinthemobilephaseandmovethroughthecolumnwiththeflowofthebuffer.Smallermoleculesandcomplexesthatareabletomoveintotheporesenterthestationaryphaseandmovethroughthegelfiltrationcolumnbyalongerpaththroughporesofthebeads. Anymoleculeorcomplexthatisabovethefractionationrangeforaparticulargelfiltrationchromatographycolumnwillmovethroughthecolumnfasterthananymoleculethatcanenterthestationaryphase.Therefore,anyconstituentinthesamplethatisabovethefractionationrangewillelutefirst(inthevoidvolume)beforeanythingthatisinthefractionationrange.Theminimumsizethatwillremaininthemobilephaseandnotenterthestationaryphaseisknownastheexclusionlimit.Bio-Radoffersgelfiltrationchromatographymediaandcolumnswithexclusionlimitsrangingoverthreeordersofmagnitude,from100daltonsto100,000daltons(100kDa). Moleculesandcomplexesthatcanenterthestationaryphasewillbefractionatedaccordingtotheirsizes.Smallermoleculeswillmigratedeepintotheporesandwillberetardedmorethanlargermoleculesthatdonotsoeasilyenterthepores,andarethuselutedfromthecolumnmorequickly.Thisdifferenceinporemigrationleadstofractionationofcomponentsbysizewiththelargestelutingfirst. Ingelfiltrationchromatographycolumnsdesignedfordesalting,bufferexchange,andtheremovalofsmallmoleculessuchasnucleotides,thesaltsandsmallcompoundsreadilyenterthepores,areretarded,andmigratemoreslowlythroughthecolumnthanthelargerproteinsornucleicacids.Therefore,thecomponentsofinterestinthesampleareelutedinadvanceofsalts,nucleotides,etc.DNAcleanupkitsusingthismechanismoftencontaingelfiltrationspincolumns. Resolution,heredefinedasthesharpnessoftheboundariesbetweensizefractions,isdeterminedbybeadsizeandanumberofotherfactors.Smallerbeadsizegenerallyyieldshigherresolutioninagelfiltrationchromatographycolumn.Compactmoleculesdiffusethroughthestationaryphasefasterthanlinearmolecules.Sizeexclusion,fractionationrange,andelutionrateareaffectedbybuffercomposition,ionicstrength,andpH.Forthefractionationofcomplexmixturesofproteins,elutiontimesandsizeexclusionlimitsmayneedtobedeterminedempirically. GelFiltrationChromatographyMedia Animportantcriterionforgelfiltrationchromatographymediaisthatmediaisinertandthatnothinginthesampleoranybufferbindstothemedia.Anotherconsiderationisthetypeofgelfiltrationcolumnbeingusedandwhetheritisusedinapressurizedchromatographysystemorgravityfloworspincolumns.Ifapressurizedchromatographysystemisbeingused,boththecolumnandthemediamustbeabletotoleratethepressureandflowratesused. Commonlyusedmediaforgelfiltrationchromatographyarebasedonagaroseorpolyacrylamidebeads,dextroseforgravityorlow-pressuresystems,andpolymericresinsformedium-pressuresystems.Thechoiceofmediadependsonthepropertiesofthecomponentstobeseparatedandotherexperimentalfactors.Thefollowingaregeneralconsiderationswhendeterminingthechoiceofgelfiltrationchromatographymedia: Fractionationrange Sizeexclusionlimit Operatingpressure Flowrate Sampleviscosity pHrange Autoclavability Toleranceforwater-miscibleorganicsolvents;somesamplesmaybemoresolubleinawater-organicmix Tolerancefordetergents,chaotropicagents,formamide,etc. Operatingtemperature Thetypesofsamples,choiceofmedia,andthechromatographysystemsetupwilldeterminewhichparametersarethemostimportantforagivenpurificationapplication. ChromatographySystems,ColumnsandMedia ChromatographySystems NGC™Medium-PressureChromatographySystems BioLogic™Low-PressureChromatographySystems Profinia™AffinityChromatographyProteinPurificationSystem   ChromatographyColumns Medium-PressureChromatographyColumns GravityandSpinChromatographyColumns Low-PressureChromatographyColumnsandCartridges   ChromatographyMedia SizeExclusionChromatographyMedia ChromatographyMediaSamplerPack ChromatographyStandards   RelatedTopics ChromatographySystems,HighPerformanceLiquidChromatography,BiotechnologyEquipment

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