TSKgel Hydrophobic Interaction Chromatography Columns
Transcript of TSKgel Hydrophobic Interaction Chromatography Columns
Hydrophobic Interaction Tips:
• TSKgelHydrophobicInteractionChromatography(HIC)columnsareofferedinglassandstainlesssteel.Stainlesssteel(SS)orPyrexfritsareembeddedinthebodyofthecolumnendfittingsofmetalandglasscolumns,respectively.ThenominalfritsizeforSScolumnsisengravedintheendfittings;Pyrexfritsintheglasscolumnshavea10µmnominalporesize.
• Halidesaltscorrodestainlesssteeltubing,fitting,andfrits.DonotstoreSScolumnsinamobilephasecontainingNaCland,wherepossible,useanothersaltintheoperatingbuffer.Chlorotrifluorethyleneandtetrafluorethylenearethematerialsintheglasscolumnfittingsthatcomeintocontactwiththemobilephaseandsample.
• Goodlaboratoryproceduresdemandthattheanalyticalcolumnbeprotectedbyaguardcolumn.TSKgelguardgelkits,containingcolumnhardwareandgelpacking,areavailabletopackyourownguardcolumn.GuardcartridgesandpackedguardcolumnsarealsoavailableforusewithTSKgelHICcolumns.
• AguardcolumnisnotavailablefortheTSKgelButyl-NPRcolumn.Besuretouseanin-linefilterwith0.5µmporestoavoidfrequentpluggingofthe1.0µmporesintheNPRcolumnfrit.Wealsorecommendapre-injectormembranefiltertopreventparticlesgeneratedbypumpsealwearfromreachingthecolumn.
• AllTSKgelHICcolumnscanberoutinelyoperatedfrompH2-12.
• TheTSKgelEther-5PWandPhenyl-5PWHICcolumnsarephysicallyandchemicallystableinwater-solubleorganicsolventsofconcentrationratiosunder50%,suchasmethanol,ethanol,andacetonitrile(DMF,DMSO,orchloroform
<30%).Changethesolventgraduallybyreducingtheflowrate(preferablywithagradient)becauserapidchange maycausedegradationofcolumnefficiency.Note:Reduceyoursaltconcentrationtopreventtheprecipitationofthe saltonthecolumn.Alsochaotropicagents(urea,SDS,etc.)willreducetheadsorptionofthebiomolecule;therefore, uselowlevelsofchaotropicagents(<2mol/L).
• TheTSKgelButyl-NPRcolumnsarecompatiblewithwater-solubleorganicsolventsofconcentrationrangeunder20%.
• Theadditionoforganicsolventsorchaotropicagentsinthefinalbuffercanimproveseparation.However,relativeelutionpositionscanchange,soaddchaotropicagentandorganicsolventinsmallquantities.
• Periodicinjectionsofonecolumnvolumeof0.2mol/LNaOHremovestronglyretainedcontaminantsfromthetopofthecolumnbyhydrolysisordissolution.Additionally,aceticacid(20-40%)canbeusedtoregeneratethecolumn.Note:Acidcanprecipitateprotein.
• Iftheinletfritofthecolumnbecomesplugged,rinsethecolumnwithwaterwhenoperatingthecolumninreverseflowdirection.Whenallelsefails,andatyourownrisk,removethecolumnendfittingatthetopofthecolumnandsonicateitin6mol/Lnitricacid.Theendfittingandfritareonepiece.Becarefulnottodisturbthepackingandrinsethefittingwellaftercleaning.
• TheshippingsolventforallTSKgelHICcolumnsisdistilledwater.
• TSKgelHICcolumnsaresuppliedwithan Inspection Datasheet,whichincludesaQCchromatogramandtestdata,andan OCSSheetsummarizingtherecommendedoperatingconditionsforoptimumcolumnperformance.
• AseparateTSKgelColumnInstructionManualthatreviewsgeneralguidelinesforcolumninstallationandcare,aswellastroubleshootingtipsforcommonlyencounteredproblems,canbedownloadedfromtheTosohBioscienceLLCwebsite(www.tosohbioscience.com).
TSKgel Hydrophobic Interaction Chromatography Columns
TSKgelButyl-NPR
TSKgelEther-5PW
TSKgelPhenyl-5PW
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About
HydrophobicInteractionChromatography(HIC)providesanadditionalchromatographicdimension,therecognitionofproteinsurfacehydrophobicity,tomakeitapowerfultechniqueforanalyticalandpreparativeseparationofbiomolecules.InHIC,aweaklynon-polarstationaryphaseisusedwithanaqueousmobilephasecontainingahighconcentrationofachaotropicsalt.Thetechniqueismainlyappliedtotheseparationofproteins,whichareelutedbygraduallyreducingthesaltconcentration.
ProteinsareretainedinHICbyinteractionwithalkylorarylfunctionalgroupsonthepackingmaterial.Thedensityofthesefunctionalgroupsislowandproteinmoleculesareadsorbedononlyoneorafewsites.Thebindingofproteinstoahydrophobicmatrixisaffectedbyanumberoffactorsincluding(1)thetypeofligand,(2)theliganddensityonthesolidsupport,(3)thebackbonematerialofthematrix,(4)thehydrophobicnatureoftheprotein,and(5)thetypeofsaltused.
Sorptiontakesplaceathighsaltconcentration,anddesorptionisaccomplishedbydecreasingthesaltconcentrationorbyaddingalowpercentageoforganicsolvent.HIChastheadvantagethatthemobilephaseconditions(primarilyaqueous)donotusuallydisrupthigher-orderproteinstructures.ThefeaturesandbenefitsofHICaredetailedinTable1below.
Table 1: Features and benefits of Hydrophobic Interaction Chromatography
Features Benefits
Choiceofthreehydrophobicligands(ether,phenyl,orbutyl)
Addedflexibilityduringmethoddevelopment
RigidpolymericbaseresinWidepHrange(2-12)ofthebaseenablingrobustcleaningoptions
SimilarchemistrytoTOYOPEARLresins
Seamlessscalabilityfromanalyticaltopreparativescale
TSKgelPhenyl-5PWcolumnsofferedinPEEKhardware
Eliminatesundesirableinteractionswithcolumnhardware
TSKgelEther-5PWandPhenyl-5PWcolumnsavailablein2mmIDformat
LC/MSapplications
Figure 1: Hydrophobic Interaction Chromatography
TSKgel Hydrophobic Interaction Chromatography Columns
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TSKgel Hydrophobic Interaction Chromatography Columns
TSKgel Hydrophobic Interaction Chromatography Columns
TSKgelHICcolumnsarepolymethacrylate-basedwithachoiceofthreeligands(butyl,ether,andphenyl)withvariedhydrophobicitiesfromlowtohigh,respectively(seeTable2).TheHICpackingmaterialsarebasedonthepolymericTSKgelG5000PWresinwhichisthenderivatizedwitholigoethylene-glycol(Ether-5PW)orphenyl(Phenyl-5PW)groups.ThebasematerialusedtoprepareTSKgel Butyl-NPRcolunmsisofthesamechemicalcompositionastheTSKgelG5000PWbasematerialusedtoprepareTSKgelPhenyl-5PWandEther-5PWcolumns.ThedifferencebetweenthetwopackingsisthattheTSKgelG5000PWpackingisporous,whereasthebasematerialoftheTSKgelButyl-NPRcolumnconsistsofspherical2.5µmnonporousparticles.Nonporousresins(NPR)aretypicallyusedforhighspeedanalyticalapplications.
TheTSKgelHICcolumnsarecompatiblewithwater-solubleorganicsolventsatconcentrationsbelow50%(20%forTSKgelButyl-NPR).SeeFigure2forthestructureoftheTSKgelHICresins.Table3listswellknownapplicationsforeachtypeofTSKgelHICcolumn.
Table 2: Comparison of TSKgel HIC columns
TSKgel Column Hydrophobicity Benefits
Phenyl-5PW Mosthydrophobic
Requiresmodestsaltconcentrationtoretainproteins.Mostpopularcolumnapplicableforthewidestrangeofhydrophobicities.
Ether-5PW Lesshydrophobic
Excellentchoiceforhydrophobicproteinssuchasmembraneproteinsormonoclonalantibodies.
Butyl-NPR Leasthydrophobic
Excellentchoiceforhighspeedapplications;usuallyhighrecoveryduetoabsenceofpores.
Table 3: Column selection for TSKgel HIC columns
Sample MM Range (Da) TSKgel Column
Peptides <1.0×104 Butyl-NPR
Mediumtolargeproteins >1.0×104
Phenyl-5PWEther-5PWButyl-NPR
DNA,RNA&PCRproducts >5.0×105 Phenyl-5PW
Butyl-NPR
Oligonucleotides >1.0×104 Phenyl-5PWButyl-NPR
5000PW (CH2CH2O)n HTSKgel Ether-5PW
NPR CH2CH2-CH2-CH3TSKgel Butyl-NPR
TSKgel Phenyl-5PW 5000PW O
O
O
O
Figure 2: Structure of TSKgel HIC resins
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About: TSKgel Butyl-NPR Hydrophobic Interaction Chromatography Columns
The2.5µmnonporousmethacrylatepackingmaterialoftheTSKgelButyl-NPRcolumnsisbondedwithbutylgroups.Intermsofhydrophobicity,theTSKgelButyl-NPRcolumnsaretheleasthydrophobicoftheHICcolumnofferingsandrequireahighersaltconcentrationforbinding.Theyarethebestchoiceforhighspeedseparationswithexcellentrecovery,evenformorehydrophobicsamples.Asinothermodesofliquidchromatography,smallerparticlesprovidehigherefficiency.Bypackingthe2.5µmnonporousresinparticlesintoshortercolumns,typicalanalysistimesarereducedtolessthan10minutes.Sincethebindingkineticsoccuronlyonthebead’ssurface,nonporousresinsaremoreefficientthanporousparticlesofthesamesize.Porediffusionisoftentheratelimitingstepintheoverallmasstransportoflargebiomoleculesthroughaporouscolumn.Eliminatingtheporesprovideshigherresolutionathigherflowrates.AnotherbenefitofNPRresinsisexcellentmassrecovery,allowingquantitationdowntonanogramlevels.Becausethesurfaceareaofnonporousparticlesismuchlower,analystsneedtobeawarethatsamplemassandvolumeneedtobeadjustedtomaintainoptimumcolumnefficiency.
Attributes and Applications
ProductattributesoftheTSKgelButyl-NPRcolumnsareshowninTable4.Theultra-efficient2.5µmnonporousresinmakesTSKgelButyl-NPRcolumnsthepreferredchoicefortime-criticalQCanalysisandsamplelimitedapplications.
Table 4: Product attributes
Attribute Value
Poresize(mean) nonporous
Particlesize(mean) 2.5µm
pHstability 2.0-12.0
Functionalgroup butyl
Antibody Fragments
Figure3showstheseparationofFabandFcfragmentsofanantibodyonaTSKgelButyl-NPRcolumn.TheappearanceofadditionalFcfragmentsisduetotheoxidationofmethionineresiduesby0.10%t-butylhydroperoxide(tBHP).ThenumbersabovetheFcpeakscorrespondtothenumber ofoxidizedresiduesineachfragment.
Mobile phase:
Gradient:Flow rate:Temperature:
Buffer A: 2mol/L (NH4)2SO4, 20mmol/L Tris, pH 7Buffer B: 20mmol/L Tris, pH 7 10%B (Omin), 100%B (34min)1mL/min30°C
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50
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Retention time (minutes)
Fab peaks Fc peaks
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C - Incubated with 0.1% tBHP
B - Incubated with 0.01% tBHP
A - Incubated with 0% tBHP (control)
10 12 14 16
10 12 14 16
10 12 14 16
TSKgel Butyl-NPR, 4.6mm ID x 3.5cm
Figure 3: Separation of Fab and Fc fragments
Column: TSKgel Butyl-NPR, 2.5 µm, 4.6 mm ID × 3.5 cmMobile phase: A: 2 mol/L (NH4)2SO4, 20 mmol/L Tris, pH 7.0 B: 20 mmol/L Tris, pH 7.0Gradient: 0 min (10%B) 34 min (100%B) Flow rate: 1 mL/min Temperature: 30 °C
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TSKgel Hydrophobic Interaction Chromatography Columns
Proteins
TheuseofaTSKgelButyl-NPRcolumnasanalternativetothesizeexclusionseparationofamonoclonalantibodyanditshighmolarmassaggregatesisshowninFigure4below.BecauseofthehighefficiencyofthenonporousparticlesintheTSKgelButyl-NPRcolumn,onlylowsampleamountsareneededforaggregateanalysis.
Phosphoglucose Isomerase
Almostidenticalseparationswereobtainedatsampleloadsfrom25µgupto100µgintheseparationofacrudesampleofphosphoglucoseisomeraseusingaTSKgelButyl-NPRcolumnasshowninFigure5.
Column: TSKgel Butyl-NPR, 4.6mm ID x 3.5cm
Sample: crude sample of phosphoglucose isomeraseLoads: A. 25µg; B. 100µg
Mobile phase: 10min linear gradient of (NH4)2SO4 from 1.8mol/Lto 0mol/L in 0.1mol/L phosphate buffer, pH 7.0
Flow Rate: 1.0mL/min
Temperature: 25°CDetection: UV@280nm
A. B.
Retention time (minutes)
0 5 10 0 5 10
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Figure 5: Effect of sample load on the separation of phosphoglucose isomerase
Column: TSKgel Butyl-NPR, 2.5 µm, 4.6 mm ID × 3.5 cmMobile phase: 10 min linear gradient of (NH4)2SO4 from 1.8 mol/L to 0 mol/L in 0.1 mol/L phosphate buffer, pH 7.0 Flow rate: 1.0 mL/min Detection: UV @ 280 nmTemperature: 25 °C Samples: crude sample of phosphoglucose isomeraseSample loads: A: 25 µg B: 100 µg
Column: TSKgel Butyl-NPR, 2.5µm, 4.6mm ID x 3.5cmMobile phase: A: 3mol/L NaCl B: H2OGradient: 0-100% B in 10minFlow rate: 1.0mL/minDetection: flourescence Ex: 280nm, Em: 348nmInjection vol.: 5µgSample: lgG1
1-9,640
2-10,530 3-11,117 4-11,7305-12,630
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(RFU
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5×105
1×106
1.5×106
2×106
2.5×106
3×106
3.5×106
4×106
4.5×106
9 10
Retention time (minutes)
11 12 13
Figure 4: Analysis of monoclonal antibody and aggregates using a TSKgel Butyl-NPR column
Column: TSKgel Butyl-NPR, 2.5 µm, 4.6 mm ID × 3.5 cmMobile phase: A: 3 mol/L NaCl B: H2OGradient: 0-100%B in 10 minFlow rate: 1.0 mL/minDetection: flourescence Ex: 280 nm, Em: 348 nmInjection vol. : 5 µgSample: lgG1
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About: TSKgel Ether-5PW Hydrophobic Interaction Chromatography Columns
OfthethreeTSKgelHICcolumns,theTSKgelEther-5PWcolumnshaveintermediatehydrophobicity.TSKgel Ether-5PWcolumnsarestableineitheracidorcausticcleaningregimensandprovideexcellentaccesstolargermoleculeswithlowdiffusioncoefficients.
Attributes and Applications
Table5liststheproductattributesoftheTSKgelEther-5PWcolumns.TheTSKgelEther-5PWcolumnsareanexcellentchoiceforseparatinghydrophobicmolecules,includingmembraneproteinsormonoclonalantibodiessuchasIgG orIgM.
Table 5: Product attributes
Attribute Value
Poresize(mean) 100nm
Particlesize(mean) 10µm
pHstability 2.0-12.0
Functionalgroup ether
Antibiotics
ATSKgelEther-5PWcolumnwasusedtodeterminetherelativepurityoftheantibioticcomponentsC-1027andC-1027-AGasshowninFigure6.AntibioticC-1027iscomposedofaproteinconsistingofmanyhydrophobicandhydroxyaminoacidswithanon-proteinchromophore.AntibioticC-1027-AGiscomposedofthehydrophobicandhydroxyaminoacidswithoutthechromophore.
Monoclonal Antibodies
Monoclonalantibodies(mAbs)playapartinmanyresearch,diagnostic,andtherapeuticapplications.Monoclonalantibodiesaregenerallythemosthydrophobicproteinsinascitesfluidandcellculturesupernatant.Figure7showstypicalresultsfromthescreeningoftwomAbsusingaTSKgelEther-5PWcolumn.
TSKgelEther-5PWcolumnshavebeenusedsuccessfullyforpurifyingmembrane-bindproteinssuchasimmunoglobulins.Figure8demonstratesthisfora50mLinjectionofmouseIgG1kmonoclonalantibodyonaTSKgelEther-5PW,8mmID×7.5cmglasscolumn.
TSKgel Ether-5PW, 7.5mm ID x 7.5cm
Sample: C-1027 C-1027-AGconcentration: 1mg/mL
Injection vol.: 20µL
Mobile phase: linear gradient from 1.5mol/L to 0mol/L (NH4)2SO4
in 0.1mol/L phosphate buffer, pH 7.0Flow Rate: 0.8mL/minDetection: UV@220nm
Retention time (minutes)0 10 205 15 25
0.06
0.04
0.02
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(AU)
Figure 6: Purification of anti-tumor antibiotic
Column: TSKgel Ether-5PW, 10 µm, 7.5 mm ID × 7.5 cm Mobile phase: linear gradient from 1.5 mol/L to 0 mol/L (NH4)2SO4 in 0.1 mol/L phosphate buffer, pH 7.0 Flow rate: 0.8 mL/min Detection: UV @ 220 nm Injection vol.: 20 µL Sample: C-1027 C-1027-AG concentration: 1 g/L
Column: TSKgel Ether-5PW, 8.0mm ID x 7.5cm, glass
Sample: A. 20µL unequilibrated mouse IgG2bκ ascitesB. 20µL unequilibrated mouse IgM
Mobile phase: linear gradient from Buffer A to B as shown Buffer A: 0.05mol/L sodium phosphate, pH 7.0, 2.0mol/Lammonium sulfate, 1.0mol/L glycineBuffer B: 0.05mol/L sodium phosphate, pH 7.0, 1.0mol/L glycine
Flow Rate: 1.0mL/minDetection: UV@280nm
A. B.
Retention time (minutes)0 10 0 1020 30 40 50 20 30 40
100
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25
75
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% Buffer B
% Buffer B
100
50
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25
75
mAb
mAb
κ ascites
Figure 7: Screening of mouse monoclonal antibodies
Column: TSKgel Ether-5PW, 10 µm, 8.0 mm ID x 7.5 cm, glass Mobile phase: linear gradient from A to B as shown A: 0.05 mol/L sodium phosphate, pH 7.0, 2.0 mol/L ammonium sulfate, 1.0 mol/L glycine B: 0.05 mol/L sodium phosphate, pH 7.0, 1.0 mol/L glycine Flow rate: 10 mL/min Detection: UV @ 280 nm Sample: A: 20 µL unequilibrated mouse IgG2b k ascites B: 20 µL unequilibrated mouse IgM k ascites
Retention time (minutes)
Column: TSKgel Ether-5PW, 8.0mm x 7.5cm glassMobile phase: 67.5min isocratic load and wash with 1mol/L (NH4)2SO4 in 1mol/L glycine, 0.5mol/L phosphate buffer pH 7.0, followed by a 37.5 min linear gradient from 1.0mol/L to 0mol/L (NH4)2SO4 in 1.0mol/L glycine, 0.05mol/L phosphate, pH 7.0Flow rate: 1.0mL/minDetection: UV@280nm, 3.0 AUFSInjection vol.: 50mLSample: 25mL raw cell culture supernatant, - 200mg total protein, - 15mg total antibody diluted to 50mL with initial elution buffer
0 50 110
MouselgG, Kappa
Dete
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(AU)
Figure 8: Monoclonal antibody purification
Column: TSKgel Ether-5PW, 10 µm, 8.0 mm ID × 7.5 cm, glass Mobile phase: 67.5 min isocratic load and wash with 1 mol/L (NH4)2SO4 in 1 mol/L glycine, 0.5 mol/L phosphate buffer, pH 7.0, followed by a 37.5 min linear gradient from 1.0 mol/L to 0 mol/L (NH4)2SO4 in 1.0 mol/L glycine, 0.05 mol/L phosphate, pH 7.0 Flow rate: 1.0 mL/min Detection: UV @ 280 nm, 3.0 AUFS Injection vol.: 50 mL Sample: 25 mL raw cell culture supernatant - 200 mg total protein - 15 mg total antibody diluted to 50 mL with initial elution buffer
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TSKgel Hydrophobic Interaction Chromatography Columns
About: TSKgel Phenyl-5PW Hydrophobic Interaction Chromatography Columns
TSKgelPhenyl-5PWcolumnswerethefirstcommerciallyavailable,polymer-basedcolumnsforhighperformanceHIC.Thesecolumnshavebeeninstrumentaltotheincreaseinpopularityofthistechniqueforanalytical,preparative,andprocessscaleseparationsofbiopolymers.ThehighporosityofTSKgelPhenyl-5PWpackingsallowsverylargeproteinstoentertheinternalporestructure,therebymaintaininghighcapacityforsuchcompounds.
Attributes and Applications
ProductattributesoftheTSKgelPhenyl-5PWcolumnsareshowninTable6.ThemosthydrophobicamongthethreeTSKgelHICcolumns,TSKgelPhenyl-5PWcolumnsareanexcellentchoicetoscreenfortheselectivity,retention,andrecoveryofmostbiomolecules.
Table 6: Product attributes
Attribute Value
Poresize(mean) 100nm
Particlesize(mean) 10µm,13µm,and20µm
pHstability 2.0-12.0
Functionalgroup phenyl
RNA
Figure9illustratestheseparationof16Sand23SribosomalRNAonaTSKgelPhenyl-5PWcolumn.TheapproximatemolarmassesoftheseRNAsare5.6×105and1.1×106Da,respectively.
Calcium-binding Proteins
Calcium-bindingproteinsareinvolvedinsignaltransductionprocesses.Oneoftheseproteins,myristoylatedfrequenin,hasamyristoylgroupthatprotrudesinthepresenceofcalcium.ThischaracteristiccanbeexploitedusingHICtopurifytheprotein,asshowninFigure10.AstepgradientisemployedonaTSKgelPhenyl-5PWglasscolumntopurifymyristoylatedfrequeninfromcrudeE. coli lysate.
Sample:
Mobile phase:
Flow Rate:Detection:
TSKgel Phenyl-5PW, 7.5mm ID x 7.5cm
16S and 23S rRNA from E. coli, 0.05mg in 0.1mL
60min linear gradient from 2mol/L to 0mol/L (NH4)2SO4 in 0.1mol/L phosphate buffer, pH 7.0 0.5mL/minUV@280nm
Retention time (minutes)0 30 60
16S rRNA
23S rRNA
Dete
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(AU)
Figure 9: Analysis of Ribosomal RNA
Column: TSKgel Phenyl-5PW, 10 µm, 7.5 mm ID × 7.5 cm Mobile phase: 60 min linear gradient from 2 mol/L to 0 mol/L (NH4)2SO4 in 0.1 mol/L phosphate buffer, pH 7.0Flow rate: 0.5 mL/minDetection: UV @ 280 nmSample: 16S and 23S rRNA from E. coli, 0.05 mg in 0.1 mL
Retention time (minutes)
Column: TSKgel Phenyl-5PW, 5mm ID x 5cmMobile phase: equilibration in 50mmol/L HEPES, 100mmol/L KCI, 1mmol/L DTT, 1mmol/L MgCl2 (pH 7.5) step gradient at 12.5 min to 50mmol/L HEPES, 1mmol/L DTT, 1mmol/L MgCl2, 2mmol/L EGTA (pH 7.5)Flow rate: 1mL/minDetection: UV@280nmTemperature: ambientSample: E. coli lysate containing myristoylated frequenin, 100µL
0 52.5
4,600
4,800
5,000
5,200
5,400
5,600
7.5 10 201510.5 17.5 22.5
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Figure 10: Purification of myristoylated frequenin
Column: TSKgel Phenyl-5PW, 10 µm, 5 mm ID × 5 cm, glass Mobile phase: equilibration in 50 mmol/L HEPES, 100 mmol/L KCI, 1 mmol/L DTT, 1 mmol/L MgCl2, 1 mmol/L CaCl2, pH 7.5 step gradient at 12.5 min to 50 mmol/L HEPES, 1 mmol/L DTT, 1 mmol/L MgCl2, 2 mmol/L EGTA, pH 7.5Flow rate: 1 mL/minDetection: UV @ 280 nmTemperature: ambientSample: E. coli lysate containing myristoylated frequenin, 100 µL
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Proteins: Scale up to Preparative Separations
Figure11comparestheresolutionofstandardproteinsonanalyticalandpreparativeTSKgelPhenyl-5PWcolumns.Differentflowratescompensatedforthechangeinparticlesizeandcolumndimensions.Highresolutionwasobtainedonbothcolumns.
Figure12comparesresolutionfora200mginjectionofcrudelipoxidaseona21.5mmID×15cmTSKgelPhenyl-5PWcolumnwiththatofa1ginjectionona55mmID×20cmcolumn.Asshown,theincreaseinparticlesizefrom13µmto20µmdidnotinfluencechromatographicresolution,keepinginmindthatthesampleloadwasonlyscaledupfive-fold.
Column: TSKgel Phenyl-5PW, A.) 7.5mm ID x 7.5cm B.) 21.5mm ID x 15cm Mobile phase: 60min linear gradient from 1.8mol/L to 0mol/L (NH4)2SO4 in 0.1mol/L phosphate buffer, pH 7.0 Flow rate: 0.5mL/min (7.5mm ID) or 4mL/min (21.5mm ID) Detection: UV@280nm Sample: 1. myoglobin 2. ribonuclease 3. lysozyme 4. a-chymotrypsinogen 5. a-chymotrypsin
Retention time (minutes)30 60 0 30 60
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5 1
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3 4
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A. Analytical Column B. Preparative Column
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Figure 11: Scale up to preparative separations
Columns: A: TSKgel Phenyl-5PW, 10 µm, 7.5 mm ID × 7. 5cm B: TSKgel Phenyl-5PW, 13 µm, 21.5 mm ID × 15 cm Mobile phase: 60 min linear gradient from 1.8 mol/L to 0 mol/L (NH4)2SO4 in 0.1 mol/L phosphate buffer, pH 7.0 Flow rate: 0.5 mL/min (7.5 mm ID) or 4 mL/min (21.5 mm ID) Detection: UV @ 280 nm Sample: 1. myoglobin 2. ribonuclease 3. lysozyme 4. a-chymotrypsinogen 5. a-chymotrypsin
Retention time (minutes)
0 60
A. 200 mg B. 1 g
120 0 60 120
Column: TSKgel Phenyl-5PW, 21.5mm ID x 15cm and 55mm ID x 20cmMobile phase: 120min linear gradient from 1.5mol/L to 0mol/L (NH4)2SO4 in 0.1mol/L phosphate buffer, pH 7.0Flow rate: 4mL/min (21.5mm) and 40mL/min (55mm)Detection: UV@280nmSample: crude lipoxidase, 200mg (21.5mm ID) and 1g (55mm ID)
Dete
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(AU)
Dete
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resp
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(AU)
Figure 12: Purify grams of protein
Columns: A: TSKgel Phenyl-5PW, 13 µm, 21.5 mm ID × 15 cm B: TSKgel Phenyl-5PW, 20 µm, 55 mm ID × 20 cm Mobile phase: 120 min linear gradient from 1.5 mol/L to 0 mol/L(NH4)2SO4 in 0.1 mol/L phosphate buffer, pH 7.0 Flow rate: 4 mL/min (21.5 mm ID) and 40 mL/min (55 mm ID) Detection: UV @ 280 nm Sample: crude lipoxidase, 200 mg (21.5 mm ID) and 1 g (55 mm ID)
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TSKgel Hydrophobic Interaction Chromatography Columns
Ordering Information
Part # Description Matrix Housing ID (mm) Length (cm) 2013 Price
14947 TSKgelButyl-NPR,2.5µm,nonporous polymer StainlessSteel 4.6 3.5 $995
42168 TSKgelButyl-NPR,2.5µm,nonporous polymer StainlessSteel 4.6 10 $1,242
14013 TSKgelEther-5PWGlass,10µm,100nm polymer Glass 5 5 $1,014
14014 TSKgelEther-5PWGlass,10µm,100nm polymer Glass 8 7.5 $1,306
18760 TSKgelEther-5PW,10µm,100nm polymer StainlessSteel 2 7.5 $985
08641 TSKgelEther-5PW,10µm,100nm polymer StainlessSteel 7.5 7.5 $1,306
14025TSKgelGlassGuardgelKitfor5mmIDand8mmIDTSKgelEther-5PWglasscolumns,20µm
polymer Glass $636
42156 TSKgelGuardCartridgefor2mmIDTSKgelEther-5PWcolumn,3pk,10µm polymer StainlessSteel 2 1 $343
19308 TSKgelGuardCartridgeHolderfor2mmIDcartridges StainlessSteel 2 1 $341
08643 TSKgelGuardgelKitfor7.5mmIDTSKgelEther-5PWcolumn,20µm polymer StainlessSteel $596
20023 TSKgelBioAssistPhenyl,10µm,100nm polymer PEEK 7.8 5 $1,306
13063 TSKgelPhenyl-5PWGlass,10µm,100nm polymer Glass 5 5 $1,014
08804 TSKgelPhenyl-5PWGlass,10µm,100nm polymer Glass 8 7.5 $1,306
18759 TSKgelPhenyl-5PW,10µm,100nm polymer StainlessSteel 2 7.5 $985
07573 TSKgelPhenyl-5PW,10µm,100nm polymer StainlessSteel 7.5 7.5 $1,306
07656 TSKgelPhenyl-5PW,13µm,100nm polymer StainlessSteel 21.5 15 $4,439
07938 TSKgelPhenyl-5PW,20µm,100nm polymer StainlessSteel 55 20 $14,495
42155 TSKgelGuardCartridgefor2mmIDTSKgelPhenyl-5PWcolumn,3pk,10µm polymer StainlessSteel 2 1 $350
19308 TSKgelGuardCartridgeHolderfor2mmIDcartridges StainlessSteel 2 1 $341
07652 TSKgelGuardgelKitfor7.5mmIDTSKgelPhenyl-5PWcolumn,20µm polymer StainlessSteel $596
16095 TSKgelGuardgelKitfor21.5mmIDTSKgelPhenyl-5PWcolumn,20µm polymer StainlessSteel $918
07936 TSKgelGuardColumnfor55mmIDTSKgelPhenyl-5PWcolumn,20µm polymer StainlessSteel 45 5 $4,098
T413113LCAT03