TSKgel Hydrophobic Interaction Chromatography Columns
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Hydrophobic Interaction Tips: • TSKgel Hydrophobic Interaction Chromatography (HIC) columns are offered in glass and stainless steel. Stainless steel (SS) or Pyrex frits are embedded in the body of the column end fittings of metal and glass columns, respectively. The nominal frit size for SS columns is engraved in the end fittings; Pyrex frits in the glass columns have a 10 µm nominal pore size. • Halide salts corrode stainless steel tubing, fitting, and frits. Do not store SS columns in a mobile phase containing NaCl and, where possible, use another salt in the operating buffer. Chlorotrifluorethylene and tetrafluorethylene are the materials in the glass column fittings that come into contact with the mobile phase and sample. • Good laboratory procedures demand that the analytical column be protected by a guard column. TSKgel guardgel kits, containing column hardware and gel packing, are available to pack your own guard column. Guard cartridges and packed guard columns are also available for use with TSKgel HIC columns. • A guard column is not available for the TSKgel Butyl-NPR column. Be sure to use an in-line filter with 0.5 µm pores to avoid frequent plugging of the 1.0 µm pores in the NPR column frit. We also recommend a pre-injector membrane filter to prevent particles generated by pump seal wear from reaching the column. • All TSKgel HIC columns can be routinely operated from pH 2-12. • The TSKgel Ether-5PW and Phenyl-5PW HIC columns are physically and chemically stable in water-soluble organic solvents of concentration ratios under 50%, such as methanol, ethanol, and acetonitrile (DMF, DMSO, or chloroform <30%). Change the solvent gradually by reducing the flow rate (preferably with a gradient) because rapid change may cause degradation of column efficiency. Note: Reduce your salt concentration to prevent the precipitation of the salt on the column. Also chaotropic agents (urea, SDS, etc.) will reduce the adsorption of the biomolecule; therefore, use low levels of chaotropic agents (<2 mol/L). • The TSKgel Butyl-NPR columns are compatible with water-soluble organic solvents of concentration range under 20%. • The addition of organic solvents or chaotropic agents in the final buffer can improve separation. However, relative elution positions can change, so add chaotropic agent and organic solvent in small quantities. • Periodic injections of one column volume of 0.2 mol/L NaOH remove strongly retained contaminants from the top of the column by hydrolysis or dissolution. Additionally, acetic acid (20-40%) can be used to regenerate the column. Note: Acid can precipitate protein. • If the inlet frit of the column becomes plugged, rinse the column with water when operating the column in reverse flow direction. When all else fails, and at your own risk, remove the column endfitting at the top of the column and sonicate it in 6 mol/L nitric acid. The end fitting and frit are one piece. Be careful not to disturb the packing and rinse the fitting well after cleaning. • The shipping solvent for all TSKgel HIC columns is distilled water. • TSKgel HIC columns are supplied with an Inspection Data sheet, which includes a QC chromatogram and test data, and an OCS Sheet summarizing the recommended operating conditions for optimum column performance. • A separate TSKgel Column Instruction Manual that reviews general guidelines for column installation and care, as well as troubleshooting tips for commonly encountered problems, can be downloaded from the Tosoh Bioscience LLC website (www.tosohbioscience.com). TSKgel Hydrophobic Interaction Chromatography Columns TSKgel Butyl-NPR TSKgel Ether-5PW TSKgel Phenyl-5PW
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
reksteen
Text Box
January, 2013 Message for Sigma-Aldrich/Supelco customers: Sigma-Aldrich Corporation is a non-exclusive distributor of TSKgel columns and TOYOPEARL bulk resins (< 1 L) in North, South and Central America, Europe, Africa and the Middle East. For the availability of TSKgel columns in Asia and Australia, please consult the website of Tosoh Corporation. Note: On the Sigma-Aldrich website and in the Supelco catalog TSKgel part numbers are preceded by the number 8, for example: 814947 for TSKgel Butyl-NPR, 3.5 cm x 4.6 mm ID, 2.5 µm
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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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B - Incubated with 0.01% tBHP
A - Incubated with 0% tBHP (control)
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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)
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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
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2-10,530 3-11,117 4-11,7305-12,630
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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
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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
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% Buffer B
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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
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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
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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
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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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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
ctor
resp
onse
(AU)
Dete
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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)
112 Call customer service: 866-527-3587, technical service: 800-366-4875, option #3
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
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