TOSOH BIOSCIENCE Phenyl-5PW User manual

HPLC
HICColumn
TSKgelPhenyl-5PW,Ether-5PW
INSTRUCTION
MANUAL

■Useonlyinwellventilatedareas.
 Incaseofinsufficientventilation,flammableandtoxicsolventscancausefire,
 explosion,orpoisoning.
■Donotspillsolvents.
 Spillageandleakagecancausefire,electricshock,poisoning,injury,andcorrosion.
 Whencleaningupaspill,wearappropriateprotectivegear.
■Weareyeprotectionandprotectivegloves.
 Organicsolventsandacidsshouldnotcomeindirectcontactwiththeskin.
■Handlepackagewithcare.
Inappropriatehandlingmaycauserupturingandsplattering.
■Onlyusethisproductasintended.
 Thisproductisforseparationandpurification,donotuseforanyotherpurpose.
■Confirmcompoundsaresafe.
 Checkthatobtainedcompoundsandsolutionsafterseparationandpurificationare
 safe.
■Properdisposal.
 Disposeofinaccordancewithlocallawsandregulations.
■Keepthismanualforfuturereference.
 
Tohelpprotectyourpropertyfrompotentialdamageandensurepersonal
safety,pleasereadthismanualthoroughlybeforeusingtheproduct.
Alertstheusertothepotentialforseriousinjuryordeath.
AIertstheusertothepotentialfordamagetohardwareorbodilyharm.
Takeproperprecautionswhenusingflammablesolvents.Thereisthepotential
forfire,explosion,orpoisoning.
■Keepawayfromfire.
SafetyPrecautions

［NotationalConventions］
Notation Explanation
WARNING
!
WARNING
!
CAUTION
NOTE
!
CAUTION
!

TableofContents
1.Introduction …………………………………………………………… 1
2.Unpacking …………………………………………………………… 1
3.Installation …………………………………………………………… 1
4.ColumnStorage……………………………………………………… 3
5.SampleandEluentPreparation ……………………………………… 3
6.FlowRates…………………………………………………………… 4
7.Temperature…………………………………………………………… 5
8.GuardColumn………………………………………………………… 5
9.ColumnEfficiency …………………………………………………… 6
10.Troubleshooting ……………………………………………………… 7
11.QualitySpecificationsandWarranty………………………………… 7

−1−
1.Introduction
TSK-GELHICcolummnsareprepackedcolumnsfortheseparationofsample
componentsaccordingtosurfacehydrophobicity.Theseparationmechanismis
similartothatofreversedphase.
Thesecolumnsweredesignedforanalyticalandpreparativeseparationofproteins.
Wehavebothsteelcolumnandglasscolumn.
Especiallyglasscolumnisbiocompatibletypewhichconsistsofahighpreciseglass
tubewithlasticendfittings.
ThisInstructionManualcontainscrucialinformationonhowtocareforandusethese
columnsinthepropermanner,soastomakemosteffectiveuseoftheirhigh
performancecapabikities.
2.Unpacking
Checkthatnothingisthematterwiththeappearanceofpackageorthecolumn.
Fig.1 Appearanceofthepackage
Thencheckthatthefollowingdocumentsareattachedtothecolumn:
1copyInsructionManual
1copyInspectionData
3.Installation
3-1ConnectionParts
Theconnectionsofsteelcolumnareoftheswagelocktypeandthatofglasscolumn
are1/4-28UNFflangejoints.
3-2FlowDirection
UsethecolumninthedirectionshownbythearrowonthenameplateinFig.2&3.

Operatingthecolumnwiththeflowinthereversediractionforalongtimewillcause
degradationofcolumnperformance.
Fig.2 SteelColumnParts
Fig.3 GlassColumnParts
3-3PreventionofBubbles
Becarefulnottoadmitanybubblesintothecolumnduringitsinstallationorremoval
fromtheequipment.Alwaysremoveallbubblesfromallpipingsbeforeinstallingthe
column.
Admittingbubblesintothecolumnwillcausedegradationofitsperformancethrough
theoccurrenceofchanneling,etc.
3-4PreventionofPulsatoryFlow
Thistypeofcolumniseasilyaffectedbypulsatoryflowofthesolvent.
Preferably,apumpwithnofluctuationshouldbeused.
Ifapumpwithpulsationmustbeused,connectapulsedamper(accumulator)tothe
outletsideofthepumpinordertocompensateforthepulsation.
Thedampermustbehighlyresistanttocorrosion.
−２−
NamePlate
Outlet
TSKgel
TYPE
C.No.
FLOW
Inlet
Endfitting
EndPlug
Nut
Outlet
Nut
Inlet
EndPlug Endfitting
Flow
C-NO
TSKgel

4.ColumnStorage
Storethecolumnsatconstanttemperatureintherangeof4℃ to30℃.
Dontstorethesecolumnsbelow4℃ nottofreezethesolventinthecolumn.
Avoidexposingthecolumnstodirectsunlight,andstorethecolumnsinaplacesafe
fromcorrosivegases.
Ifthecolumnisbeinguseddaily,youneednotremovethecolumnfromthe
equipmentandthebuffermaybeleftinthecolumnovernight,aslongasthebuffer
salthasgoodsolubility.Whenthecolumnsaretobestoredforafewdays,removethe
columnfromtheinstrumentand seal bothendswiththeend plugs. For long-term
storage,thecolumnmustbeprotectedfromgrowthofmicroorganisms.
Thus,aqueousbuffersshouldberelacedwithdistilledanddeionizedwater.
5.SampleandEluentPreparation
5-1ReplacementofSolvents
HICtypecolumnsarefilledwithdistilledanddeionizedwaterforshipment.
Replacethissolventwithasuitablesolvent,usingone-halfoftherecommendedflow
rateshowninTable1.
Sincefrequentsolventreplacementacceleratesdegradationofcolumnefficiency,use
thesamesolventasfaraspossible.
5-2pH
ThesecolumnsareusableovertherelativelywidepHrangeof2.0 -12.0atroom
temperature.
5-3Filtration
UseonlyHPLCgradesolventsthathavebeenfilteredthroughan0.5μmfilter.
Filterallbufferedsolutionbeforeusingthem.This reduces the problemofplugged
filtersandpreservescolumnlife.
Vacuumfiltrationorsonificationmaybeusedtoremovedissolvedgasseswhichcould
affectyoursolventdeliverysystem.
5-4SaltConcentrationRange
Saltconcentrationinthesolventisnotlimiteduptonearsaturation.
−３−

5-5OrganicSolventConcentrationRange
Theconcentrationoftheorganicsolventcanrisenupto50％.
Butbecarefultoavoidsaltprecipitationwhenaddingsaltstoaqueoussolution
containingorganicsolvents.
5-6DenaturingAgents
Denaturingagentssuchasurea,guanidine-HClanddetergentscanbeused.
5-7Sample
Thesampleshouldbedissolvedinthe starting eluent and filteredthroughamicro-
porefilter(ofeg.0.5μmporesize)toprotectthecolumnfromaccumulationof
inmpurties.
6.FlowRates
Factorssuchasresolution,analyticaltime,andcolumnlifeshouldbecarefully
consideredinselectingflowrates.Ahigherflowrateresultsinashorteranalyhtical
time.Conversely lowerflowreteresultsinimprovedcolumnefficiencyandtendsto
extendcolumnlife.
Standardflowratesforphosphateorotheraqueousbuffersarerecommended
between0.4-0.8mL／min.Theflowratesandpressuredropsforthesecolumnsare
suggestedbelow:
−４−
Cat.
No.
07573
07656
13063
08804
14018
08641
08642
14013
14014
14015
Types
〈SteelColumn〉
phenyl-5PW
phenyl-5PW
〈GlassColumn〉
phenyl-5PWGlass
phenyl-5PWGlass
phenyl-5PWGlass
〈SteelColumn〉
Ether-5PW
Ether-5PW
〈GlassColumn〉
Ether-5PWGlass
Ether-5PWGlass
Ether-5PWGlass
7.5×7.5
21.5×15.0
5.0×5.0
8.0×7.5
20.0×15.0
7.5×7.5
21.5×15.0
5.0×5.0
8.0×7.5
20.0×15.0
1.2
8.0
1.0
1.2
8.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
0.5−1.0
4.0−6.0
0.5−0.8
0.5−1.0
4.0−6.0
0.5−1.0
4.0−6.0
0.5−0.8
0.5−1.0
4.0−6.0
1.2
8.0
1.0
1.2
8.0
Column
Dimensions
(mmID×cm)
Max.
FlowRate
(mL／min)
Recomended
FlowRate
(mL／min)
Max.Pres-
sureDrop
(MPa)
Table1 Recommendedflowrates

Caution:
Ontheglasscolumnsapplication,the pressure at thetopofthecolumnshould be
lessthan30MPatopreventthedamageoftheglasscolumnandtheleakageofthe
gelattheendfitting.
7.Temperature
TheoptimaloperatingtemperatureforHICtypecolumnsisbetween10-30℃.
Thesecolumnsshouldnotbeusedaboveroomtemperatureforlongtime.
Below10℃,usealowerratetoprotectthecolumns.
Forlongtermstoragethecolumnsshouldbestoredatroomtemperature.
8.GuardColumn
Whenimpuritiesthattendtobeadsorbedbythepackingmaterialpresentinthe
sample,theyareadsorbedontheinletsideofthecolumnandaccunulategradually,
causingreductionofcolumnefficiency.
Insuchcasetheoriginalcolumnefficiencycanbemaintainedbyconnectingaguard
columnbeforethe columnandreplacingitifefficiencybecomesdegradedbythe
adsorptionofmaterialtotheguardcolumn.
Formazimuminsurance against suchproblems,aguardcolumn shouldbe used as
muchaspossible.
WepreparedtheGuardcolumnkitastheguardcolumnsforTSKgelPhenyl-5PW
andEther-5PW.
PleaserefertoINSTRUCTIONMANUALforTSKguardgelforHPLC.
−５−
Cat.No.
07652
08643
08808
14025
14469
14470
Types AppliedColumns
〈TSKguardgelkit〉
TSKguardgelPhenyl-5PWkit
TSKguardgelEther-5PWkit
TSKguardgelPhenyl-5PWkitGlass
TSKguardgelEther-5PWkitGlass
〈TSKguardcolumn〉
TSKguardcolumnPhenyl-5PWGlass
TSKguardcolumnEther-5PWGlass
Phenyl-5PW(7.5×7.5)
Ether-5PW(7.5×7.5)
Phenyl-5PWGlass(8.0×7.5)
Ether-5PWGlass(8.0×7.5)
Phenyl-5PWGlass(20×15)
Ether-5PWGlass(20×15)
Table2 GuardColumns

9.ColumnEfficiency
Thenumberoftheoreticalplatesofacolumn(N),theasymmetryfactor(As)andtheir
chromatographicconditionsareshownintheInspectionData.
9-1MethodofCalculatingtheNumberofTheoreticalPlates
Fig.4 MethodofCalculatingtheNumberofTheoreticalPlates
Thenumberoftheoreticalplatesofacolumn(N)iscalculatedbythehalfpeakwidth
methodshowninFig.4andthefollowingequation.
N＝5.54(Ve／W1／2)2
Ve :Elutionvolume
W1／2 :Halfwidthvalueofpeak
h :Peakheight
9-2MethodofCalculatingtheAsymmetryFactor
Fig.5 MethodofCalculatingtheAsymmetryFactor
Theasymmetryfactorofcolumn(As)iscalculatedbythe1／10hmethod.
As＝b／a
9-3DeadVolume
Ifthedeadvolume of theequipmentortheinjectionvolumeofasamplesolution
becomestoolarge,thenumberoftheoreticalplatesmaydecrease.
−６−
Ve
h
W
Injection
1/2 1/2h
Ve
h
ab
Injection
1/10h

10.Troubleshooting
WhenusingTSKgelcolumns,someproblemscanbeavoidedbyfollowingthese
instructions.Buttheproblems(suchasthoseduetocolumnnlife,adsorptive
materials,productionofairbubbles,driedgel,orfrozensolvent)cannotbecorrected
oncetheyoccur,socareshouldbetakeninhandlingthesecolumns.
10-1CloggingoftheEndFitting
Incasethepressure-dropincreasesortheflowratedecreases,theendfittingshould
becleanedbyreversingtheflowthroughthecolumn.
Iftheclog cannotberemoved,prepareanewendfittingandreplacetheoldend
fittingwithanewone,beingverycarefulnottoloosenanyofthepackedgel
underneath.
10-2NeverElutedorElutedLate
Iftheelutiontimeisextremelylong,itmightbecausedbyadsorptiononthecolumn
packing.
Mostproblemscanbeavoidedbywashingthecolumn.
Ifthecolumnhasnotrecoveredafterwashing,itwillnotrecoveredanymore.
Inthiscaseyouhavetoexchangethecolumnwithanewone.
11.QualitySpecificationsandWarranty
11-1InspectionData
Theresultsof each inspectionare described inthe Inspection Dataenclosed in the
columnpackage.IntheInspectionData,Nisexpressedasthatpercolumn.
TheconditionsusedindeterminingtheInspectionDataareasfollows:
＊ Eluent:DistilledWater
＊ Sample:0.5％ Acetone
−７−

＊ FlowRate＆ SampleVolume
＊ Detector:UV(280nm)
11-2QualitySpecifications
11-3Warranty
Immediatelyafterreceipt,checktheappearanceofthecolumnandtestits
performanceaccordingtoSection9&11.
Iftheguaranteedspecifications(aboveTable)cannotbeobtained,contactyour
TOSOHrepresentativewihintwoweeks.
Notethatcolumnlifetimeisnotguaranteed.
−８−
Types
SteelColumn
SteelColumn
GlassColumn
GlassColumn
GlassColumn
7.5×7.5
21.5×15.0
5.0×5.0
8.0×7.5
20.0×15.0
1.0
6.0
0.8
1.0
6.0
20
100
20
20
100
ColumnDimensions
(mmID×cmL)
FlowRates
(mL／min)
SampleVolume
(μL)
Cat.
No.
07573
07656
08641
08642
13063
08804
14018
14013
14014
14015
Types
〈SteelColumn〉
TSKgelPhenyl-5PW
TSKgelPhenyl-5PW
TSKgelEther-5PW
TSKgelEther-5PW
〈GlassColumn〉
TSKgelPhenyl-5PWGlass
TSKgelPhenyl-5PWGlass
TSKgelPhenyl-5PWGlass
TSKgelEther-5PWGlass
TSKgelEther-5PWGlass
TSKgelEther-5PWGlass
7.5×7.5
21.5×15.0
7.5×7.5
21.5×15.0
5.0×5.0
8.0×7.5
20.0×15.0
5.0×5.0
8.0×7.5
21.5×15.0
≧1,000
≧3,000
≧1,000
≧3,000
≧600
≧1,000
≧3,000
≧600
≧1,000
≧3,000
1.0−2.0
0.8−1.6
1.0−2.0
0.8−1.6
1.0−2.0
1.0−2.0
0.8−1.6
1.0−2.0
1.0−2.0
1.0−2.0
Column
Dimensions
(mmID×cmL)
No .ofTheo-
reticalPlates
(N／Column)
Asymmetry
Factor
(As)

HLC, TSK-GEL, TSKgel, TSKgel SuperMultipore, BioAssist, Enantio, PStQuick, Enviropak,
TOYOPEARL, ToyoScreen, TOYOPEARL GigaCap, TOYOPEARL MegaCap, TOYOPEARLPAK
and TOYOPAK are registered trademarks of TOSOH CORPORATION in Japan and other countries.
This manual may not be reprinted or copied in whole or in part without written consent of TOSOH
CORPORATION. The contents of the manual are subject to change without notice.
Printed in Japan
TOSOH CORPORATION
BIOSCIENCE DIVISION
Shiba-Koen First Bldg.
3-8-2 Shiba, Minato-ku, Tokyo 105-8623, Japan
Phone: [phone] Fax: [phone]
Web site: http://www.separations.asia.tosohbioscience.com/
HPLC database: www2.tosoh.co.jp/hlc/hlcdb.nsf/StartE?OpenForm
TOSOH BIOSCIENCE GmbH
Zettachring 6, 70567 Stuttgart, Germany
Phone: [phone] Fax: [phone]
E-mail: [email protected]
Web site: http://www.tosohbioscience.com/
TOSOH BIOSCIENCE
SHANGHAI CO., LTD.
Room 301, Plaza B, No.1289 Yi Shan Road,
Xu Hui District, Shanghai 200233, China
Phone: [phone] Fax: [phone]
E-mail: [email protected]
Web site: http://www.separations.asia.tosohbioscience.com/
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63 Market Street #10-03 Singapore 048942
Phone: [phone] Fax: [phone]
E-mail: [email protected]
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TOSOH BIOSCIENCE LLC
3604 Horizon Drive Suite 100,
King of Prussia, PA 19406, USA
Phone: [phone] Fax: [phone]
E-mail: [email protected]
Web site: http://www.tosohbioscience.com/
Packed Columns for Aqueous
Size Exclusion Chromatography
TSKgel SuperMultiporePW-N
TSKgel SuperMultiporePW-M
TSKgel SuperMultiporePW-H
TSKgel SuperOligoPW
INSTRUCTION
MANUAL
Rev. CO073003