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

For more info visit: www.tosohbioscience.com 105

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

106 Call customer service: 866-527-3587, technical service: 800-366-4875, option #3

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

For more info visit: www.tosohbioscience.com 107

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

100

50

0

100

50

0

Dete

ctor

resp

onse

(mAU

)

100

50

0

Retention time (minutes)

Fab peaks Fc peaks

1

20

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

108 Call customer service: 866-527-3587, technical service: 800-366-4875, option #3

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

Dete

ctor

resp

onse

(AU)

Dete

ctor

resp

onse

(AU)

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

8

Dete

ctor

resp

onse

(RFU

)

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

For more info visit: www.tosohbioscience.com 109

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

0

Dete

ctor

resp

onse

(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

50

0

25

75

Dete

ctor

resp

onse

(AU)

Dete

ctor

resp

onse

(AU)

% Buffer B

% Buffer B

100

50

0

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

ctor

resp

onse

(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

110 Call customer service: 866-527-3587, technical service: 800-366-4875, option #3

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

ctor

resp

onse

(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

Dete

ctor

resp

onse

(mAU

)

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

For more info visit: www.tosohbioscience.com 111

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

12

34

5 1

2

3 4

5

A. Analytical Column B. Preparative Column

0

Dete

ctor

resp

onse

(AU)

Dete

ctor

resp

onse

(AU)

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

ctor

resp

onse

(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)

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