LC Columns
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THE ESSENTIAL CHROMATOGRAPHY & SPECTROSCOPY CATALOG LC & LC/MS
For more information
LC AND LC/MS
Your Essential Resource for Columns & Supplies
LC and LC/MS CoLuMnS
LC and LC/MS Columns
ThelargestportfolioofFastLCcolumns,
andabroadfamilyofphasesacrossallparticlesizes
forexceptionalflexibilityandscalability
Whetheryouareperformingconventionalorultra-fastchromatography,separatingbiomolecules,
oranalyzingcomplexbasiccompounds,youcantrustAgilentfortheindustry’shighest-performing
columnsthatdeliverthefast,reproducibleresultsyouneed–allengineeredwithAgilent’sunparalleled
qualityandreliability.
• Poroshell 120 columns –highefficiencyandhighresolutionwithupto50%lesspressurethan
sub-2µmcolumns.
• ZoRBaX Rapid Resolution High definition (RRHd) columns –1.8µmcolumnsfeatureimproved
packingprocessestoachievestabilityupto1200barforusewiththeAgilent1290InfinityLCand
otherUHPLCinstrumentsandareavailableinmorethan14phases,plusHILIC.
• ZoRBaX Eclipse Plus columns –C18andC8columnsdeliversuperiorpeakshape,whilethe
phenyl-hexylbondedphaseandC18bondedphaseforPAHseparationsexpandselectivityoptionsfor
moreapplications.AllEclipsePlusphasesareavailableinFastLC/UHPLCRRHDandRRHTcolumns,
1.8µm.Forscalability,theEclipsePlusC18phaseisverysimilartothePoroshell120EC-C18phase.
• InadditiontoPoroshell120andRRHDcolumns,ZoRBaX Rapid Resolution High Throughput
(RRHT) columns areathirdFastLCoptionwithover1401.8µmcolumnschoices.RRHTcolumns
areavailablein2.1,3.0and4.6mmids,allwith600barstability.
Andremember,whenyouchooseAgilentZORBAXLCcolumns,yougetmorethanjustadependable
product.Youalsogetover40yearsofexpertise–alongwithunmatchedtechnicalsupport–fromthe
world’slargestchromatographysupplier.Ontheweb,byphoneorinperson,Agilenthelpsyousolve
theproblemsthatcanslowyoudownandgetinthewayofyourresults.
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CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
TableofContents
LC and LC/MS Columns for Small Molecule Separations
HPLC Column Selection.................................................... 206
Preparative HPLC Columns.............................................. 311
Fast Columns for Reversed-Phase HPLC/uHPLC.........227
Agilent Prep LC Columns .................................................311
Poroshell 120 ......................................................................228
ZORBAX PrepHT ................................................................314
ZORBAX Rapid Resolution High Definition 1.8 µm......233
Pursuit and Pursuit XRs Prep ..........................................319
ZORBAX Rapid Resolution High Throughput 1.8 µm......239
Polaris Prep Columns........................................................321
Agilent UHPLC Guards......................................................246
Load & Lock ........................................................................322
Columns for Reversed-Phase analytical HPLC .............247
Columns for Other HPLC Techniques.............................323
ZORBAX Eclipse Plus........................................................248
ZORBAX HILIC Plus...........................................................324
ZORBAX Eclipse PAH........................................................254
Normal-Phase Columns....................................................326
ZORBAX Eclipse XDB........................................................256
ZORBAX Ion-Exchange Columns – SAX and SCX.........333
ZORBAX 80Å StableBond.................................................264
Hi-Plex Columns for Carbohydrate Analysis .................335
ZORBAX Rx .........................................................................272
appendices......................................................................... 343
ZORBAX 80Å Extend-C18.................................................274
Quick Guide to USP Designations
for HPLC Columns..............................................................343
ZORBAX Bonus-RP............................................................278
ZORBAX Original Reversed-Phase Columns.................283
Kits for Analytical HPLC ...................................................284
Pursuit..................................................................................287
oligo Solutions .................................................................. 347
StratoSpheres DNA Cartridges .......................................347
TOP, TOP-DNA and TOP-RNA Cartridges ......................348
Polaris ..................................................................................298
TC-C18(2) and HC-C18(2) .................................................304
PLRP-S .................................................................................306
www.agiLEnT.CoM/CHEM/LC
205
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
HPLC Column Selection
Tousethecolumnselectionguidediagrambelow,simplyfollowthepathforyouranalyteandmobilephase.Atthefarright,followyourfinalcolumn
selectiontothepagesindicated.
Hexane Soluble
Organic Solvent
Soluble
MeOH and MeOH:
H2O or ACN and
ACN: H2O Soluble
Normal-Phase Mode with Bare Silica
Pages 326-328
Normal-Phase Mode
with Bonded Phase
Pages 326-328
Reversed-Phase Mode
with Bonded Phase
Page 248
Gel Permeation (Small Molecule)
Page 488
Reversed-Phase Mode
with Bonded Phase
Page 248
Reversed-Phase Mode with
Bonded Phase Using Ionization Control
Page 247
Reversed-Phase Mode with
Bonded Phase Using Ion-Pair Agent
Page 247
Reversed-Phase Mode
with Bare Silica (HILIC)
Page 324
Ion-Exchange Mode
Page 333
Gel Permeation Chromatography
Page 488
Gel Filtration Chromatography
Page 431
Ion-Exchange Mode
with Wide-Pore Material
Page 397
Reversed-Phase Mode
with Wide-Pore Material
Page 523
Size Exclusion Chromatography
Page 523
THF Soluble
Molecular Weight
< 3000
Non-Ionic
Water Soluble
Ionic
Sample
BioMoLECuLES
Organic Solvent
Soluble
Molecular Weight
> 3000
Water Soluble
Informationaboutbiocolumnswithmolecularweight>3000canbe
foundintheAgilentBioHPLCColumnsection,turntopage350.
Adaptedwithpermissionfrom"PracticalHPLCMethodologyandApplications,"BrianA.Bidlingmeyer,JohnWiley&Sons,Inc.,NewYork,p.109
206
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Quick guide to agilent Reversed-Phase Bonded Phases
ZoRBaX RP-HPLC Columns
Recommended uses and applications
Page no.
Poroshell120
ü Superficiallyporousparticlesforhighefficiencyatlowpressure
ü Sub-2µmefficiencywitha2.7µmparticle
ü Endcappedandnon-endcappedC18andC8phases,andavarietyofotherphases,
forselectivityoptimization
ü Compatiblewith400barand600barLC's
228
EclipsePlus
AvailableinRRHD(1200bar)
andRRHT(600bar)configurations,1.8µm
ü Excellentfirstchoiceformethoddevelopment
ü LonglifefrompH2-9forreliableseparationsofbasic,acidicandneutralcompounds
ü Superiorpeakshapewithbasiccompounds
ü Highresolutionandefficiencywith1.8,3.5and5µmcolumns
ü RigorousQA/QCtestingforgreaterlong-termreproducibility
248
EclipseXDB
AvailableinRRHD(1200bar)
andRRHT(600bar)configurations,1.8µm
ü Fourselectivitychoicesforflexiblemethoddevelopment
ü HighperformanceoverawidepHrange(2-9)
ü Goodpeakshapeforacids,basesandneutrals
ü LonglifetimewitheXtraDenseBondinganddoubleendcapping
ü Fast,ultra-fast,andhighresolutionseparationsusing1.8and3.5µmcolumns
ü Choicesfromcapillarytoprep
256
StableBond(SB)
AvailableinRRHD(1200bar)
andRRHT(600bar)configurations,1.8µm
ü Basic,acidic,neutralcompounds
ü ExceptionalstabilityatlowpH(1-2)
ü Useofhightemperature(upto90°CforC18,80°CforC8,C3,Phenyl,CN,andAq)
andlowpHasanaddedselectivitytool
ü Widestselectionofbondedphasesfordifferentselectivity(C18,C8,C3,CN,Phenyl,Aq)
ü UsesmobilephasesforLC/MSwithformicacid,aceticacid,orTFA
ü UsesmobilephaseswithTFAforpeptideandproteinseparation
ü Rapidseparationsusing1.8and3.5µmcolumns
264
(Continued)
Information about biocolumns can be found in the section beginning on page 350
www.agiLEnT.CoM/CHEM/LC
207
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Quick guide to agilent Reversed-Phase Bonded Phases
ZoRBaX RP-HPLC Columns
Recommended uses and applications
Page no.
ZORBAXRx
AvailableinRRHD(1200bar)
andRRHT(600bar)configurations,1.8µm
ü Generalseparationofbasic,acidicandneutralcompoundsatlowpHwithdifferentselectivity
thanSBcolumns
ü Rx-C8isthesameasSB-C8
272
Bonus-RP
AvailableinFastLC/UHPLCRRHD(1200bar)
andRRHT(600bar)configurations,1.8µm
ü Separatingbasiccompoundsinhigheraqueousmobilephases
ü Generalseparationofbasic,neutral,acidiccompoundsatmid-rangepHorlowpH;
especiallystableatlowpH
ü Separatingpeptidesfordifferentselectivity
ü Rapidseparationsusing3.5µmcolumns
278
Extend-C18
AvailableinFastLC/UHPLCRRHD(1200bar)
andRRHT(600bar)configurations,1.8µm
ü SeparatingbasiccompoundsabovetheirpKainfreebaseform;separationofbasic,acidic,
neutralcompoundsathighpH;uptopH11.5
ü UsesammoniumhydroxideasmobilephaseadditivewithLC/MSwithsmallmolecules
orpeptides
ü Separatingathigh,mid-rangeandlowpHforselectivitychanges
ü Rapidseparationsusing3.5µmcolumns
274
original ZoRBaX Columns
Recommended uses and applications
Page no.
ZORBAX
ü Generalseparationofbasic,acidic,neutralcompoundsatlowpHwithdifferentselectivity
thanSBcolumns;highernumberofactivesilanolsthanSB
ü "Mixedmode"separationatmoreneutralpHvalues
ü AvailableinODS,C8,CNandODS"Classic"(non-endcapped)
283
TiPS & TooLS
The LC Handbook: guide to LC Columns and Method development
ThishandyguidemakesiteasytochoosetherightLCcolumn,andcontainsplentyoftipsandtrickstomake
yourjobeasierandmoreproductive(publication#5990-7595EN).
Request a copy or download a mobile copy at www.agilent.com/chem/lchandbook
208
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Quick guide to additional agilent Reversed-Phase Columns
Pursuit Family
Recommended uses and applications
Page no.
PursuitHPLC
ü Fullrangeofphases,includingC18andC8
ü Diphenylutilizesstrongdipole-dipolehydrogenbondingandpi-pimechanismsfordifferent
selectivitywitharomaticcompounds
ü PFPprovidesexcellentseparationofpolar(halogenated)analytesandpositionalisomersunder
standardreversed-phaseconditions
287
PursuitXRsandPursuitXRsUltra
ü Offerlargersurfaceareaandsmallerporesize,incomplementaryphasestoPursuitfamily
ü Ultraoffersstabilityto600bar,duetospecialhardwareandloading
287
Polaris Family
Recommended uses and applications
Page no.
C18-AandC8-A
ü C18-AandC8-Aofferalternateselectivitiesforgeneralpolarapplications
Availablein3.0,5.0,and10µm(C18-Aonly) ü Designedwithhydrogen-bond-acceptingendcapping
298
Amide-C18
Availablein3.0and5.0µm
ü Subtlealternativeselectivityduetotheabsenceofstericprotection
ü Utilizeanembeddedamide,similartoZORBAXBonus-RP
298
C18-EtherandC8-Ether
Availablein3.0and5.0µm
ü Endcappedwithanethergrouptocreateamorepolarsurfaceforselectivityvariation
298
other agilent Columns
Recommended uses and applications
Page no.
TC-C18(2)
Availablein5µm
ü Anexcellentchoiceformixturesofpolarandnon-polarcompounds,
includingstrongbasiccompounds
304
HC-C18(2)
Availablein5µm
ü High-value,highlyretentiveoption
ü Carbonloadof17%
ü Superiorpeakshapeforbasiccompounds
304
www.agiLEnT.CoM/CHEM/LC
209
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
ZORBAX Reversed-Phase HPLC Column Selection Flow Chart
For small and large molecules
Mostchromatographersusereversed-phaseHPLCasoneoftheirkeyanalysistechniques.Reversed-phaseHPLCcanbeusedtoanalyzeionicandnonionic
analytes.ThereforethisZORBAXColumnSelectionFlowChartwillfocusonreversed-phasecolumns.Tomoreeasilyselectareversed-phasecolumnfor
methoddevelopmentofsmallandlargemolecules,followtheoutlineonthesepages.
Thisflowchartprovidesinformationonchoosinganinitialcolumnformethoddevelopmentofsmallmoleculeandproteinandpeptidesamples,andincludes
decisionsonbondedphaseandcolumnconfiguration.
Small Molecules
Large Molecules
MW < 3000
MW > 3000
80-120Å
Packing Pore Size
300Å
First choice of a packing pore size is based on the size of molecules to be analyzed. Typical small molecules can diffuse easily in and out of
standard 80-120Å pore packings, but larger peptides and proteins may not. For this reason, it is recommended to use 300Å pore packings (300SB)
for isocratic or gradient separations of peptides and proteins.
Eclipse Plus C18
or Poroshell 120 EC-C18
Starting Column Bonded Phase
StableBond 300SB-C18
A C18 is recommended as the starting column bonded phase for most samples since it maximizes retention for moderately polar to non-polar
compounds. Shorter chain phases should be considered if resolution cannot be optimized with a C18 phase or if you are analyzing larger proteins,
or very hydrophobic compounds that are difficult to elute from C18 with conventional reversed-phase solvents. Start with Poroshell 120 EC-C18
or Eclipse Plus RRHT/RRHD for fast LC performance.
Small Molecules
Large Molecules
MW < 3000
MW > 3000
Standard analysis
Fast analysis
Standard analysis
Fast analysis
Eclipse Plus C18
Poroshell 120 EC-C18
ZoRBaX RRHd 300SB-C18
ZoRBaX 300SB-C18
4.6 x 150 mm, 3.5 µm
4.6 x 100 mm, 2.7 µm
4.6 x 150 mm, 5 µm
4.6 x 50 mm, 3.5 µm
959963-902
695975-902
883995-902
865973-902
Poroshell 120 EC-C18
ZoRBaX RRHd*
Eclipse Plus C18
Poroshell 300SB-C18
4.6 x 75 mm, 2.7 µm
697975-902
2.1 x 50 mm, 1.8 µm
660750-902
2.1 x 75 mm, 5 µm
959757-902
* Firstchoiceforuseonthe1290InfinityLCorotherUHPLCinstrumentswith1000+barpressurelimit.
Information about biocolumns can be found in the section beginning on page 350
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CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Column and Mobile Phase Guidelines:
Reversed-Phase
HPLCcolumnsconsistoftwoparts:thecolumnchemistryandhardware.Forthepropercolumnchemistry,
consultthecatalogsectionforeachtypeofbondedphase.Forchoosingcolumnhardwareandparticle
sizes,consultthesectiononcolumnsizesandrapidseparations,includingAgilentZORBAXRapid
ResolutionHT,SolventSaver,CapillaryandPrepHTcolumns.
Pore Size Selection
Chooseacolumnpackingwithsmallpore(60-120Å)ifthesolutemolecularweightislessthanabout3000.
Otherwise,usecolumnpackingwiththe300Åporesize.
Particle Size Selection
ZORBAXRapidResolution
HighThroughput(RRHT)Columns
ThetypicalparticlesizeforHPLCcolumnsis5µmwith3.5µmandsmaller,nowcommoninmethod
development.Ifhigh-speedanalysesorhigherresolutionanalysesarerequired,packingwith1.8µm
and2-3µmparticlescanbeused.Shortercolumnswiththeseparticlescanproducefasterhigh-resolution
separations,withthe1.8µmparticlesizeprovidingthehighestefficiencyand2.7µmsuperficiallyporous
providingsimilarresults.With1.8,2.7,3.5and5µmparticlesizestochoosefrom,startwiththesmallest
particlesizeforyourHPLCorUHPLC–400bar,600bar,or1200bar–toachievethebestresults.
Column Configuration
Choosingthebestcolumnsizeformethoddevelopmenthaschangeddramaticallyinthepastfewyears.
Smaller3.0mmidor2.1mmidcolumnsarenowusedmorethan4.6mmidtolowersolventuseand
achievecompatibilitywithMSdetectors.Andshorter50,75and100mmlongcolumnscanbeagreat
startingchoice,withlongercolumnsusedonlywhenmoreresolutionisneededorwhen3.5and5µm
particlesizesareused.
TiPS & TooLS
NeedhelpselectingtherightLCcolumnforyourmethod?
TrytheNavigator:AselectiontoolforLCcolumnsandsampleprep.
Lookforitonlineandviayourmobiledeviceathttp://navigator.chem.agilent.com
www.agiLEnT.CoM/CHEM/LC
211
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Silica, Polymers, and Bonded Phase
Base Material
ThebasematerialforanLCcolumnismostoftenhighpuritysilicamaterialwithtotallyporousparticlessuch
asthatusedinmostAgilentcolumns,includingZORBAX,Pursuit,andPolaris.However,morechoicesare
available,includingpolymermaterialwithhighpHstabilityusedinPLRP-Scolumnsandsuperficiallyporous
silicaparticlessuchasthoseusedinPoroshell120columns.ThehighpurityTypeBsilicas,includingthe
ZORBAXRx-SilusedinZORBAXEclipsePlus,andsuperficiallyporousPoroshell120,areanexcellentfirst
choiceformostmethods.TypeAsilicas,suchasZORBAXSIL,usedinOriginalZORBAXcolumns,arestill
manufacturedandusedinmanymethods.
Bonded Phase
AgoodfirstchoiceforbondedphaseisC18orC8,andtherecommendedstartingcolumnchoicesare
EclipsePlusC18orPoroshell120EC-C18.Thesetwochoicesprovideexcellentpeakshapeandcanbe
usedoverthepHrange2-9,accommodatingmosttypicalLCandLC/MSmobilephases.Ifthesample
solutesofinterestarenotadequatelyseparatedonthesecolumns,CNandPhenylcolumns–including
Phenyl,Phenyl-HexylandDiphenyl–mayoffersignificantdifferencesinselectivityfromstraight-chainalkyl
phasestoeffecttheseparation.
Ingeneral,largersolutes,suchasproteins,arebestseparatedonshort-chainreversed-phasecolumns
(C3,CN,C8)andpeptidesandsmallmoleculesareseparatedonlonger-chaincolumns(C18).However,
therearemanycaseswherethisconventionalwisdomdoesnotapply.Forexample,peptidescanalsobe
effectivelyseparatedusingshort-chaincolumns,andhydrophobicpeptidescanshowbetterrecovery
onlonger-chainphases.Therefore,itisbesttoinitiallyselectaphaseinthemiddleofthehydrophobic
spectrum(e.g.,C8),thenchangetoamorehydrophobicphaseormorehydrophilicphasedependingon
initialresultsandsolubilitypropertiesofyoursample.
Polymers
WhenacolumnisneededthatcanoperateatverylowandveryhighpH,polymericpackingsprovide
analternativetosilica-basedmaterials.Polymericparticlesaregoodforsmall-scalechromatography,
particularlyLC/MS,astheyarechemicallystableanddonotleachsolubleorparticulatespecies.
Reversed-phasesphericalpolymericpackingsusedinAgilentPLRP-Scolumns,forexample,arebasedona
styrene/divinylbenzenecopolymerwithaninherentlyhydrophobicsurface.Nobondedphaseisrequiredfor
reversed-phasechromatographywithpolymericparticles.Theserigidmacroporousparticlescanbecoated
andderivatizedtogivearangeoffunctionalities,includingweakandstrongcationandanionexchangers.
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CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
pH and Mobile Phase
Thechoiceofmobilephaseforareversed-phasesystemstartswithselectingtheorganicmodifier.
Acetonitrileisthemostcommonlyusedorganicmodifier.However,selectivitydifferencesandsample
retentionwillvarysignificantlyamongmobilephasescontainingacetonitrile,methanol,andtetrahydrofuran
(THF).Samplesolubilityislikelytodifferinsuchsolventsanddictateuseofaspecificsolventorsolvents.
UVdetectionatcertainwavelengthsisnotpossiblewithcertainmodifiers(e.g.,methanolat200nm).
BothpHandionicstrengthoftheaqueousportionofmobilephasesareimportantparametersindeveloping
ruggedmethodsthatarenotsensitivetosmallvariationsinconditions.Withioniccompounds,retentionof
typicalspeciesshowssignificantchangeswithpH.ItisveryimportanttocontrolpHinsuchreversed-phase
systemstostabilizeretentionandresolution.ApHbetween2and4generallyprovidesthemoststable
conditionsforretentionvs.smallchangesinpH,andthispHisrecommendedforstartingmethod
developmentformostsamples,includingbasiccompoundsandtypicalweakacids.
HPLC Column
Column
dimensions
Stationary
Phase
Typeof
Surface
PoreSize
Particle
Size
Length
Chemical Properties
Physical Properties
ChemicalLifetime/Selectivity
RetentionFactor
Efficiency
Sensitivity
Speed
Inner
Diameter
www.agiLEnT.CoM/CHEM/LC
213
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Working with LC/MS
WhenchoosingHPLCcolumnsforLC/MS,chromatographersoftenneedtoconsiderseveralaspectsof
theirmethodandseparation,typicallyincludingresolution,flowrate,andstationaryphasechoice.Often,
forrelativelysimpleanalytes,shorterhighresolutioncolumnsarethebestchoice.Thesecolumnsallowfor
highthroughputwhilemaintaininghighseparationefficiency.NarrowboreRapidResolutionHighDefinition
(RRHD)forseparations(>600bar)andPoroshell120columns(<600bar)offerhighresolutionevenin
shortercolumnsdimensions.Formoredifficultsamples,usersshouldseeklongercolumnlengths.
SincemanyLC/MSanalysesarerunatlowerflowrates(typicallyfromµL/minflowratesupto1mL/min),
movingtosmallerinternaldiametercolumnsisthebestchoicefortheuser.Agilent’sSolventSaver
(3.0mmid)andnarrowbore(2.1mmid)willoftenresultinlowersolventusageforthemethod,
andareexcellentoptionsforhighresolutionandhighersensitivitythanthelargeridcolumns.
Mostoften,thebestbondedphasechoiceisanendcappedC18phase.EclipsePlusC18isahigh
performanceendcappedC18phaseavailableinsub-2µmRRHDandRRHTcolumnformats.Forfast
high-throughputseparationswithLC/MS,Poroshell120EC-C18isanexcellentchoice.Poroshellhas
alargerfrit,soit’swellsuitedfordirtierLC/MSsamples,suchasbloodplasma,whichmayoftenclog
columnswithsmallerporosityfrits.
BothEclipsePlusC18andPoroshell120EC-C18phasesarestableoverawidepHrangeandare
compatiblewiththevolatilebufferssuchasaceticandformicacids.
TiPS & TooLS
LC Flow Rate Calculator app
ThisFREESmartphoneappletsyouquicklyadjustyourflowratetoaccommodateothermethodchanges.
Downloadatwww.agilent.com/chem/lcapp
214
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Transferring your method
to a high efficiency column
HighefficiencycolumnsforUHPLC/FastLCwillhelpyouincreaseyouranalyticalspeedandresolution.
Dependingontheinstrumentconfigurationyouareusing,youmayneedtomakeafewadjustments
togetthemostfromthesecolumns.
Becauseoftheirhighefficiency,verynarrowpeakselutefromhigherefficiencycolumnsquickly.
WhilemodernHPLCinstrumentationanddatasystemsareabletocapturethebenefitsoftheseparticles,
attentiontoinstrumentalconfigurationisimportanttogetthebestresults.
Stepstotransferyourmethod:
Check the specifications that came with your instrument –Yourinstrumentmayalreadybe
configuredappropriatelyforhighefficiencycolumns.Ifnot,thencontinue.
optimize the data collection rate for LC and LC/MS (at least 40 Hz detector with fast
response time for uV) –Setthedetectortothefastestsetting,thentothesecondfastestsetting
andevaluateiftheresolutionisdifferent.
use a semi-micro or micro-flow cell –Smallervolumeflowcellssuchasthesemi-micro(6mm/5µL)
ormicro(3mm/2µL)arerecommendedforbestperformance.Therearenewercartridgeflowcells
(e.g.theUltraLow-DispersionMax-LightUltraFlowCell,P/NG4212-60007)designedtooptimize
UHPLCinstrumentperformance.
TiPS & TooLS
FortheAgilent1290InfinityLC,insituationsrequiringextremelylowdeadvolumes,usetheultra-lowdispersionkit,whichincludesanultra-low
dispersionflowcelland0.08mmidcapillaries.
www.agiLEnT.CoM/CHEM/LC
215
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Minimize tubing volume in the instrument –UseRed(0.12mmid)tubinginsteadofGreen
(0.17mmid)asithasonlyhalfofthevolumethatthesamplehastotravelthrough.Thiscutsdown
extracolumnbandbroadening.Ensurethatyourconnectionsareasshortaspossible.Thekeylocations
tocheckare:
• Theautosamplerneedleseat
• TheautosamplertotheThermostattedColumnCompartment–or‘TCC’
• TheTCCtothecolumn
• Thecolumntotheflowcell,includingtheinternaldiameteroftheintegralflowcellinletcapillary
all of these specific capillaries can be ordered individually
from agilent, in the lengths you need, and for your instrument.
Turn to pages 36-39.
Scale your gradient profile and injection volume –Ifusinggradientelution,scalethegradient
profileandinjectionvolumetothenewsmallercolumntoquicklytransferthemethodandavoid
overloading.Forisocraticandgradientelution,makesurethatyouscaletheinjectionvolumetomatch
theoverallcolumnvolume.
Minimize injection sample dispersion in the column –Useaninjectionsolventwithsolventstrength
thatisequivalenttoorweakerthanthemobilephase,especiallywhenusinganisocraticmethod.Thisis
goodpracticeingeneralforanycolumn,andmoreimportantwithhighefficiencycolumns.
TiPS & TooLS
Seeavideothattakesyouthroughthesestepsat
www.agilent.com/chem/poroshell120video
Also,checkouttheLCMethodTranslatorToolat
www.agilent.com/chem/lcmethodtranslator
216
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Take care to make proper connections –AgilentrecommendsSwagelokfittingswithfrontandback
ferrules,whichgivebestsealingperformancethroughoutourLCsystem(usethisontheinstrument
connections,i.e.valves,heaters,etc).Polyketonefittingsarehighlyrecommendedforupto600bar.Use
thisfitting(P/N5042-8957)oncolumnconnectionswithPoroshell120.ForRRHDcolumns,useAgilent's
removable1200barfitting(P/N5067-4733).
optimize your flow rate –ForPoroshell120,ifyou’reusinga2.1mmid,thesuggestedstartingflow
rateis0.42mL/min;for3.0mmidPoroshell120columns,wesuggeststartingat0.85mL/min,andfor
4.6mmid,wesuggeststartingat1.5-2mL/min.
1200barremovablefitting(SV),5067-4733
Fast analysis of cefepime
and related impurities
1.819
0
5.190
2.152
0.988
3
5
6
3
4
5
6
1
2
3
4
8.741
8
9
min
7
8
9
min
8
9
min
2.0mL/min
300bar
4.428
2
7
ImpurityE
Cefepime
ImpurityF
ImpurityA
ImpurityB
1.5mL/min
225bar
3.439
1.421
1
4
5.825
0.696
0.816
0
2
2.687
mAU
20
15
10
5
0
1
1.202
0
mAU
20
15
10
5
0
1mL/min
155bar
4
3
1.148
DAD,254nm
1
0.473
0.544
0.659
Detector:
Agilent1200InfinitySeries
RapidResolutionLCSystem
5
2
mAU
20
15
10
5
0
0.954
Instrument:
Poroshell 120 EC-C18
697975-902
4.6 x 75 mm, 2.7 µm
0.419
0.510
Column:
1.
2.
3.
4.
5.
5
6
7
VHP FiTTingS
Agilent’s1200barremovablefitting(for1/16inodcapillaries)consistsofastainlesssteelscrew,aninternalstainlesssteel
ferruleandafrontferruleinPEEK.Thefittingcanbeusedthroughouttheflowpath,butbecauseitcanbere-usedwithout
losingtightness,itisespeciallysuitablefortheconnectionbetweentheheatexchangerandthecolumn.Thisnewand
improvedfittingreplacesthestandardstainlesssteelSwagelokfittingwhichwasnotremovable.TheVeryHighPressure
(VHP)fittingisavailableinthreesizes–short(P/N5067-4733),long(P/N5067-4738)andextralong(P/N5067-4739).
Theshortfittingistheonethatismostcommonlyused,andwillbeappropriate90%ofthetime.Insomecases,ifusing
columnswithlongernuts,alongerfittingwillbeneeded.
www.agiLEnT.CoM/CHEM/LC
217
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
agilent LC Columns overview: Small Molecules
Start with Poroshell 120 for Fast LC performance on any HPLC – phases align with ZoRBaX family.
up to 50% less pressure than sub-2 µm; a total lab productivity enhancer
1.7 µm solid core; 0.5 µm porous outer layer for a 2.7 µm particle, id's: 4.6 mm, 3.0 mm, 2.1 mm, Lengths: 30-150 mm.
New phases coming soon! Check www.agilent.com/chem/poroshell120
Poroshell 120
Compatible with HPLC and UHPLC instruments. Suitable for analysis of acids, bases, and neutrals. Also great for peptide mapping.
Poroshell 120 is for any lab looking for increased analytical speed and resolution with less backpressure.
Poroshell 120 EC-C18** (uSP L1),
EC-C8** (uSP L1), Phenyl-Hexyl (uSP L11)
Carbon Load: Phenyl-Hexyl - 8%
Poroshell 120 EC-Cn (uSP L10)
ZoRBaX Eclipse Plus**
ZoRBaX StableBond
ZoRBaX Eclipse XdB
RRHd: 1.8 µm, stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 30-250 mm
IDs: 4.6 mm, 3.0 mm, 2.1 mm, 1.0 mm; Prep
RRHd: 1.8 µm, stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 20-250 mm
IDs: 4.6 mm , 3.0 mm, 2.1 mm, 1.0 mm;
Prep, Capillary (C18)
RRHd: 1.8 µm, stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 15-250 mm
IDs: 4.6 mm, 3.0 mm, 2.1 mm, 1.0 mm;
Capillary and Prep
Poroshell 120 SB-C18 (uSP L1), SB-C8
Carbon Load: SB-C18 - 7.5%, SB-C8 - 4.5%
**Best Phase for Method development
ZoRBaX Family
CH3
O
C18 (USP L1),
C8 (USP L7),
Phenyl-Hexyl (USP L11),
PaH (USP L1)
O
CH3
O
Si
CH3
CH3
CH3
O
Si
CH3
CH3
O
Si
CH3
SB-C18 (USP L1),
SB-C8 (USP L7),
SB-C3 (USP L56),
SB-Phenyl (USP L11),
SB-Cn (USP L10),
SB-aq
O
Si
C18 (USP L1),
C8 (USP L7),
Phenyl (USP L11),
Cn (USP L10)
R1
R
OH
R
O
Si
CH3
CH3
O
Si
O
Si
O
Si
OH
CH3
CH3
CH3
R
O
O
CH3
CH3
CH3
CH3
CH3
Si
CH3
Si
R1
Si
CH3
High performance and excellent peak shape
with acids, bases and neutrals.
High performance with acids, bases, and
neutrals with superior lifetime at low pH.
Good peak shape for basic, acidic, and
neutral compounds with high performance
over a wide pH range (pH 2-9). eXtra Dense
Bonding and double endcapping help give
this column a long lifetime.
Sample applications
Environmental: EPA Method 1694,
Illicit and prescribed drugs in wastewater
Food Safety: Quinolone antibiotics
Pharmaceutical: Chloramphenicol,
Simvastatin, Chrysophenol (TCM),
amphetamine, ranitidine
Sample applications
Chemical/industrial: Triton
Environmental: Organic acids,
pesticides in drinking water
Food Safety: Anthocyanine,
parabenes, melamine
Pharmaceutical: Analgesics,
anesthetics, traditional Chinese medicine
Sample applications
Environmental: Herbicides/pesticides,
steroids in water
Food Safety: Food colors,
aromatic flavorings, mycotoxins,
epoxyphenolic-based can coatings
Pharmaceutical: Goldenseal and related
alkaloids, antidepressants, triamcinolone
R
double Endcapped
(except PAH, which
is not endcapped)
Temp limit: 60 °C
Pore size: 95Å
Surface area:
160 m2/g
Particle sizes:
1.8, 3.5, 5 µm
pH: 2.0-9.0 for C18,
C8; 2.0-8.0 for PAH,
Phenyl-Hexyl
Carbon load:
C18: 9%; C8: 7%;
Phenyl-Hexyl: 9% ;
PAH: 14%
Best all around – exceptional peak shape,
efficiency, resolution, and lifetime
non-Endcapped
Temp limit: 80 °C
(90 °C for SB-C18)
Pore size: 80Å
Surface area:
180 m2/g
Particle sizes:
1.8, 3.5, 5, 7 µm
pH: 1.0-8.0
(0.8-8.0 for SB-C18)
Carbon Load:
C18: 10%; C8: 5.5%;
C3: 4%; Phenyl: 5.5%;
CN: 4%,
Aq: Proprietary
Best for low pH mobile phases –
great for method development
Lengths: 30-250 mm
IDs: 2.0 mm, 3.0 mm, 4.6 mm; Prep
Pursuit/
Pursuit XRs
C18 (USP L1), C8 (USP L7),
diphenyl (USP L11), PFP (USP L43),
PaH (USP L1), Si (USP L3)
Pursuit XRs offers higher loadability
and Pursuit XRs Ultra is loaded
for higher pressure stability.
double Endcapped
Temp limit: 60 °C
Pore size: 80Å
Surface area:
180 m2/g
Particle sizes:
1.8, 3.5, 5, 7 µm
pH: 2.0-9.0
(2.0-8.0 for CN)
Carbon load:
C18: 10%; C8: 7.6%;
Phenyl: 7.2%;
CN: 4.3%
High performance
over a wide pH range
Endcapped
(except Pursuit XRs Si)
Pore Size: 200Å
(Pursuit),
100Å (Pursuit XRs)
Surface area: 200 m2/g
(Pursuit); 440 m2/g
(Pursuit XRs)
Particle Sizes:
3, 5, 10 µm
Reliable Selectivity alternatives
218
Si
CH3
R1
R
CH3
O
CH3
R
Si
CH3
pH: 2.0-9.0
Carbon Load:
Pursuit C18: 12.9%;
Pursuit C8: 7.4%;
Pursuit Diphenyl: 7.3%;
PFP: 6.3%; XRs
C18: 22%; XRs Ultra
C18: 23.3%; XRs Ultra
C8: 15%; XRs Ultra
Diphenyl: 14.6%
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
O
HO
O
N
HO
OH
N
O
O
O
N
Endcapped: EC-C18, EC-C8, Phenyl-Hexyl, Bonus-RP (triple), EC-Cn
non-endcapped: SB-C18, SB-C8 and SB-aq
Temp Limit: 60 °C (EC-C18, EC-C8, Phenyl-Hexyl, Bonus-RP); 80 °C (SB-C8, SB-Aq); 90 °C (SB-C18)
Pore Size: 120Å ; Surface Area: 130 m2/g; pH: 2.0-8.0 (EC-C18, EC-C8, Phenyl-Hexyl); 1.0-8.0 (SBC18, SB-C8, SB-Aq); 2.0-9.0 (Bonus-RP); Carbon Load: 8% (EC-C18); 7% (EC-C8)
Poroshell 120 Bonus-RP (uSP L60)
Carbon Load - 7.5%
Poroshell 120 SB-aQ
Carbon Load: Proprietary
PoLaR Compounds
ZoRBaX Extend-C18
ZoRBaX Bonus-RP
SB-aQ
Polaris
RRHd: 1.8 µm, stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 20-250 mm
IDs: 4.6 mm, 3.0 mm, 2.1 mm, 1.0 mm
RRHd: 1.8 µm, stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 30-250 mm
IDs: 4.6 mm, 3.0 mm, 2.1 mm, 1.0 mm; Prep
RRHd: 1.8 µm stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 20 - 250 mm
IDs: 4.6 mm, 3.0 mm, 2.1 mm; Prep
Lengths: 30-250 mm,
(available in
3 µm and 5 µm particles)
IDs: 2.0 mm, 3.0 mm, 4.6 mm; Prep
ZoRBaX SB-aq
C18-a (USP L1), C8-a (USP L7),
C18-Ether (USP L1), C8-Ether (USP L7),
amide-C18 (USP L60), nH2 (USP L8),
Si-a (USP L3)
R1
O
C18 (USP L1)
C18
O
Si
Bonus-RP (USP-L60)
Si
PG
R
R1
CH3
Si
CH3
CH3
O
Si
R1
C18
O
Si
PG
R
PG
R
Silica Support
R1
CH3
Si
CH3
CH3
R1
High efficiency and long life at high pH –
up to pH 11.5. Improve retention,
resolution and peak shape of basic
compounds. High sensitivity for LC/MS
separations of peptides. Unique
bidendate bonding and double
endcapping provides high pH stability.
Polar-embedded to improve peak shapes;
for basic compounds at low and mid pH.
Proprietary phase ideal for polar
compounds and high aqueous conditions.
Hydrogen-bond accepting and ether group
endcapping provide alternate selectivities.
Sample applications
Environmental: EPA 8330 (explosives)
Food Safety: Aflatoxins, mycotoxins
Pharmaceutical: Antihistamines,
xanthines
Sample applications
Environmental: Triazine pesticides
Food Safety: Hydroxymethylfurfural
Pharmaceutical: Antifungal medications,
anorectics, ulcer medications
Sample applications
Environmental: Pesticides in drinking water
Food Safety: Pesticides in food
Pharmaceutical: Water-soluble vitamins
Sample applications
Environmental: Triazine pesticides
Food Safety: Hydroxymethylfurfural
Pharmaceutical: Antifungal medications,
anorectics, ulcer medications
double-Endcapped
Temp limit: 60 °C
Pore Size: 80Å
Surface area:
180 m2/g
Particle sizes:
1.8, 3.5, 5 µm
pH: 2.0-11.5
Carbon load: 12.5%
O
Si
R1
Triple-Endcapped
Temp limit: 60 °C
Pore size: 80Å
Surface area:
180 m2/g
Particle sizes:
1.8, 3.5, 5 µm
a good option for
separations at high pH
pH: 2.0-9.0
Carbon load: 9.5%
alternative selectivity to alkyl,
phenyl, cyano phases
See ZORBAX StableBond for specification
and structure.
Endcapped
Pore size: 180Å
Surface Area:
200 m2/g
Particle Sizes:
3, 5, 10 µm
pH: 2.0-9.0
Exceptional lifetime at low pH –
no endcapping
Carbon load: Polaris
C18-A: 13.8%;
Polaris C8-A: 7.4%;
Polaris C18-Ether:
12.1%; Polaris
C8-Ether: 7.1%
More options
for Polar Compounds
Looking for a HiLiC column?
HILIC Plus is a HILIC column based on Eclipse Plus silica for excellent peak shapes
information about biocolumns
can be found in the section
beginning on page 350
Poroshell 120 HiLiC: 2.7 µm, stable to 600 bar
non-bonded silica
Pore size: 95Å (120Å, Poroshell 120)
Surface Area: 160 m2/g(130 m2/g for Poroshell 120)
Particle Sizes: 1.8, 2.7, 3.5 µm
pH: 0-8.0
RRHd: 1.8 µm,
stable to 1200 bar
Lengths: 50, 100, 150 mm
IDs: 4.6 mm (3.5 µm only), 3.0 mm, 2.1 mm
High sensitivity for LC/MS applications and recommended for EPA 1694.
www.agiLEnT.CoM/CHEM/LC
219
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Method Development from pH 1-12
Start method development at low pH (pH 2-3)
Withsomanycolumnchoicesavailable,howdoyouknowwheretostartyourmethoddevelopment?Therecommendedstartingpointformethod
developmentisusingabufferedlowpHmobilephase–aroundpH2-3.UsingalowpHmobilephasemostoftenresultsinthebestpeakshapeforbasic
compoundsonsilica-basedcolumns.AtlowpH,thesilanolsonthesilicaarefullyprotonatedsopositivelychargedbasiccompoundsdonotinteractstrongly.
Theresultisgoodpeakshape.Manyacidiccompoundsarenon-charged,maximizingtheirretentionatlowpH.Theseobservationsarekeyadvantagesto
methoddevelopmentatlowpH.
Forstandardanalyticalwork,startmethoddevelopmentwithacetonitrileasthemobilephaseorganicmodifierand20-50mMphosphatebuffer(pH2-3)
astheaqueouscomponentfornon-LC/MSapplications.TheseconditionsprovidegoodpHcontrol,necessaryforthemostreproducibleanalysesofionizable
compounds.ForLC/MSapplicationsformicacidorTFAaregoodmobilephaseadditivesforlowpH.
Optimize solvents and bonded phases at low pH
Theinitialmethoddevelopmentstepsmayleadveryquicklytoasatisfactoryseparation.Butifmoreoptimizationisneeded,acetonitrilecanbereplaced
withmethanolortetrahydrofuranandtheseparationre-optimized.Thisstepmayleadtoasatisfactorysolution,butifstillmoreselectivityoptimization
isneeded,thecolumnbondedphasecanbechanged.
AtlowpHtherearemanybondedphasechoicesavailableforoptimization.TheseincludetheEclipsePlusphasesaswellastheEclipseXDBfamilywith
C18,C8,PhenylandCN.AlternatechoicesincludefivedifferentStableBondbondedphases:SB-C18,SB-C8,SB-Phenyl,SB-CN,andSB-C3.Forpolar
analytes,tryBonus-RP,SB-AqorthePolarisfamily,includingC18-A,C8-A,C18-EtherandAmide-C18phases.
ItmaybenecessaryatlowpHtoimprovetheretentionofacidiccompounds.Forthesesituations,lowerthepHevenfurther,downtopH1-2,
anduseStableBondcolumns.ThesecolumnsprovidethegreateststabilityatverylowpHandprovidemanyselectivityoptionsforachievingthe
highestresolutionseparations.
TiPS & TooLS
LC Method Translator
Usethisonlinetooltoquicklyfactorinchangestocolumnlength,diameter,flowrate,
andmore–andtocalculatemethodadjustments.Thisisparticularlyusefulfor
gradientmethods.
Todownload,gotowww.agilent.com/chem/lcmethodtranslator
220
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Choose Agilent ZORBAX Eclipse Plus or Poroshell 120
for method development at mid pH (pH 4-9)
TherearesomesamplesthatmaynotberesolvedatlowpHormayhavebettersolubilityandstabilityatmidpH.TheEclipsePlusC18and
Poroshell120EC-C18columnscanbeusedatthemidpHrangeformethoddevelopment.TheEclipsePluscolumnisstabletopH9soitisequally
reliableatmidpH.Thesedoubleendcappedcolumnshavetwokeyadvantages–goodpeakshapeatlowandmidpH,aswellassufficientbondedphase
densitytoprotectthecolumnfromsilicadegradationfrompH6-9.
AtmidpH,basiccompounds(e.g.,amines)maystillhaveapositivechargeandthesilanolsonthesilicasurfacemayhaveanegativecharge.
ThereforecoveringasmanysilanolsaspossibleleadstothebestpeakshapeatmidpH.ThismakestheEclipsePlusC18thebeststartingchoicefor
acolumnatmidpH.PhosphatebufferisusuallythefirstchoiceformobilephasemodifieratpH7becauseitsbufferrangeispH6.1-8.1.Asecondchoice
formidpHisacetatebuffersinceitbuffersfrompH3.8-5.8anditsvolatilitymakesitagoodchoiceforLC/MScompatibility.
Choose Agilent ZORBAX Extend-C18 columns for method development at high pH (pH 9-12)
AtlowormidpH,someseparationsofbasiccompoundsmaystillnothaveenoughretentionorthedesiredselectivity.Forthesesamples,highpH
separationsmaybeappropriate.Untilrecently,highpHseparationsonsilica-basedcolumnswereavoidedbecauseofshortcolumnlifetimes,dueto
dissolutionoftheunderlyingsilicagel.SpecialbondedphasessuchastheZORBAXExtend-C18,canprotectthesilicafromdissolution,sothata
reasonablecolumnlifetimecanbeachievedandtheselectivityadvantagesofhighpHcanbeexplored.
ThemobilephasebufferchoicesathighpHwiththeExtend-C18columnareorganicbuffersliketriethylamineandammoniumhydroxide.Thesebuffersare
bestusedwithmethanolastheorganicmodifiertoextendthecolumnlifetimeathighpH.Thisisanothergoodoptiontoconsiderwhenworkingwithhigh
pHandPLRP-Scolumns,whicharemadefromapolymericmaterial.
EaSy, RELiaBLE PH TESTing
AgilentoffersafulllineofpHmetersandelectrodes.Designedforchromatographers,
thesepHmetersofferintuitiveuserdesignandexceptionalruggednessforyourlab.Learnmoreat
www.agilent.com/chem/agilentpH
www.agiLEnT.CoM/CHEM/LC
221
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Method development guidelines from Low to High pH
START
SAMPLE
Poorly retained compounds
Low pH 3.0
Initial separation
STEP 1
ü ZORBAXEclipsePlusC18,C8,or
Poroshell120EC-C18
ü pH2.5(2-3)20-50mMbuffer
ü T=30°C(ambientto60°C)
ü Adjust%ACNfor0.5<k<20
STEP 1a
STEP 2
ZoRBaX Eclipse Plus – 1 column for pH 2-9
Increaseordecrease%oforganicmodifier
by5%(v/v)
ü ZORBAXSB-C18orSB-C8
ü pH1-320-50mMbuffer
ü T=30°C(ambientto90°C)
ü Adjust%ACNfor0.5<k<20
Band spacing problems
Band spacing problems
STEP 1b
STEP 3
ü Changeorganicmodifier(MeOHorTHF)
ü Adjust%organicfor0.5<k<20
ü Varycolumntemperature,upto60°Cfor
Eclipseupto80-90°CforStablebond
ü RestartatSTEP2
STEP 1c
Increaseordecrease%of
organicmodifierby5%(v/v)
Band
spacing
problems
STEP 1d
STEP 4
Increaseordecrease%oforganicmodifier
by5%(v/v)
ü Changebonded-phasefunctionalitytoEclipse
PlusPhenyl-Hexyl,Bonus-RP,EclipseXDB-CN,
SB-CN,SB-Aq
ü RestartatSTEP1
STEP 1e
Band spacing problems
ü Changeorganicmodifier(ACNorTHF)
ü Adjustfor0.5<k<20
STEP 5
Mid pH 7.0
BASICCOMPOUNDS(Ion-Pairing)
ü UseMeOHorganicmodifier,10mMhexane
sulfonicacid,25-50mMpH3buffer
ü Adjust%MeOHfor0.5<k<20
Band spacing problems
Band spacing problems
pH 6.0
OR
OR
Acids
Band spacing problems
pH 4.0
Bases
Poorly retained compounds
ü ZORBAXEclipsePlusC18,C8or
Poroshell120EC-C18
ü pH7(6-9),20-50mMbuffer
ü T=30°C(ambientto40°C)
ü Adjust%MeOHfor0.5<k<20
Poorly retained acidic compounds
Band spacing problems
STEP 5a
STEP 6
ACIDICCOMPOUNDS(Ion-Pairing)
ü UseMeOHorganicmodifier,10mM
tetrabutylammoniumphosphate,25-50mM
pH7buffer
ü Adjust%MeOHfor0.5<k<20
Increaseordecrease%oforganicmodifier
by5%(v/v)
Band spacing problems
STEP 7
pH 8.0
ü Changeorganicmodifier(ACNorTHF)
ü Adjustfor0.5<k<20
ü RestartatSTEP6
Band spacing problems
STEP 8
ü ChangebondedphasefunctionalitytoZORBAX
EclipsePlusPhenyl-HexylorBonus-RP
ü RestartatSTEP5
Band spacing problems
Band spacing problems
STEP 5c
Increaseordecrease%of
organicmodifierby5%(v/v)
Band
spacing
problems
STEP 5b
Varytemperaturewithinrecommended
rangeforbondedphase
Band spacing problems
STEP 5d
ü Changeorganicmodifier(ACNorTHF)
ü Adjustfor0.5<k<20
STEP 9
High pH 9.0
ü ZORBAXExtend-C18
ü pH10.5(9-11.5);5-20mMammoniaorTEA,
or10-50mMorganicbuffer,orboratebuffer
ü T=25°C(ambientto40°C)
ü Adjust%MeOHfor0.5<k<20
Band spacing problems
STEP 10
pH 10.0
ü Changeorganicmodifier(ACNorTHF)
ü Adjustfor0.5<k<20
ü Varytemperaturewithinrecommendedrange
forbondedphase
pH 11.0
Band spacing problems
Try different HPLC mode
Contact agilent Technologies
contact_us@agilent.com
pH 12.0
222
Band spacing problems
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
guard Columns
TheValueofGuardColumns
Guardcolumnscanhelpextendthelifeofyouranalyticalcolumn.Choosingtouseguardcolumnscanhelp
reduceoperatingexpenses,byreducingthefrequencyofanalyticalcolumnreplacement.
Theguardcolumnpreventsdamagecausedbyparticulatematterandstronglyadsorbedmaterial.
Tomaintainanadequatecapacityforsampleimpurities,chooseaguardcolumnwithaninternaldiameter
similartothecolumninternaldiameter.Ideally,thepackingoftheguardcolumnshouldbethesameasthe
analyticalcolumnsothatthechromatographyoftheanalyticalcolumnisnotaltered.
Guardcolumnscontributetotheseparation,soyoushouldincludeaguardcolumnin-lineduring
methoddevelopment.
AgilentUHPLCguardsprovideprotectionforhigh-efficiencyPoroshell120andZORBAXRRHDand
RRHTcolumns,withoutreducingperformance.Partnumbersforallguardcolumnsareincorporated
intothedifferentproductfamilytables.
Judgingwhentoreplaceaguardcolumncanbedifficult.Asaroughguide,ifplatenumber,pressureor
resolutionchangebymorethan10%,theguardcolumnprobablyneedsreplacing.Youwillneedtomake
ajudgmentcallonhowoftentoreplaceyourguardcolumnsbasedonyourapplicationtype.Itisalways
preferabletochangetheguardcolumnsoonerratherthanlater.
UHPLCGuard,1200bar,821725-903
www.agiLEnT.CoM/CHEM/LC
223
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Cartridge Selection guide
icon*
Type of Cartridge
Features
Benefits
AgilentHPLCCartridge
Canreversecolletsintheendfittingtoadd
guardcartridges
Inexpensive
Extendscolumnlifetime
Permitsrapidcolumnchanges
Canuse2,3,4and4.6mmcartridges
Cartridgeshaveauniquefilterandsieve
ateachend
Helpspreventblockage
Highefficiency,standalone,
low-dead-volumecartridge
Sealsupto5000psi(340bar)or3000psi
withaPEEKfitting
Polymericcartridgedesigned
forleak-tightsealsagainstmetalsurfaces
Nogasketsrequired
Moresolvent-resistantthanPEEK
Reusablefittings
Adaptforconnectionsto1/16inLCfittings
ZORBAXGuardCartridge:
Standalonesystem
ZORBAXRapidResolutionandRapid
ResolutionHTCartridgeColumns:
3.5µmand1.8µmpackings,
Standalonesystem
ForhighthroughputLC/MS,LC/MS/MS
andcombinatorialseparations
PackedwithEclipseXDBforpHusefrom2-9
PackedwithStableBondforlowpHuse
Forallanalytetypes
Lowbleed
Soldindividuallyorasthree-packs
ZORBAXSemi-PreparativeGuard
HPLCHardwareKit:
Standalonesystem
ZORBAXandAgilentPrepPreparative
CartridgeColumnandGuardHPLCSystem:
Standaloneandintegralhardwareoptions
PolymericAnalyticalColumn
andGuardCartridge
Easy,low-dead-volumeassembly
Sealsupto2000psi(135bar,13.5MPa)
Tubing(polyphenylenesulfone)designed
forleak-tightsealsagainstmetalsurfaces
Nogasketsrequired
Reusablefittings
Adaptforconnectionsto1/16inLCfittings
Easy,low-dead-volumeassembly
Extendscolumnlifetime
Reusablefittings
Permitsrapidcolumnchanges
Hardwareoptionsforintegralandexternalguards
Canusewith21.2and30mmidcolumns
Highefficiency
Inexpensive
Lowdeadvolume
Rapidcartridgechanges
Reusableholder
Extendscolumnlifetime
ChromSepColumnHardware:
Completesystemsandreplacementcartridges
Easy,no-dead-volumeassembly
Economicalformat
MetaGuardColumnHardware:
Completesystemsandreplacementcartridges
Easy,no-dead-volumeassembly
Notoolsrequired
Modularflexibility
Economicalformat
Notoolsrequired
Modularflexibility
AgilentFastGuardsforUHPLC
Requiresnospecialhardware–
connectsrighttotheanalyticalcolumn
AvailableinmatchingphasesforPoroshell120,
RRHDandRRHTcolumns
*Lookfortheseiconstohelpyouselecttheproperguardcartridgesandcolumns.
224
Extendscolumnlifetime
withoutimpactingperformance
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Cartridge/guard Cartridge Systems Compatibility guide*
icon
id
(mm)
Column Type
guard Cartridge Holder
Phases
Cartridgecolumncartridgeholder
5021-1845
Guardcartridge(internalsystem)cartridgeholder
5021-1845
2.0
3.0
4.0
4.6
LiChrospher
Nucleosil
Purospher
Superspher
ZORBAX
Standardfitting
Columnguardcartridge(standalone)
cartridgeholder
820999-901
2.1
3.0
4.6
ZORBAX
RapidResolutioncartridgeholder
820555-901
Noguardcartridgeholder
4.6
ZORBAX
Semi-preparativecolumn
Semi-prepguardcartridge(standalone)
cartridgeholder
840140-901
9.4
ZORBAX
(Continued)
www.agiLEnT.CoM/CHEM/LC
225
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Cartridge/guard Cartridge Systems Compatibility guide*
icon
nEw!
id
(mm)
Column Type
guard Cartridge Holder
PrepHT
Guardcartridge
820444-901
21.2
ZORBAX
AgilentPrep
Analytical
Guardcartridgeholder(PL1310-0016)andPLRP-S
guardcartridges,2/pk(PL1612-1801)
3.0
PLRP-S
Singlereplacementcolumn
Noguardcartridgeholder
1.0
2.0
4.6
Pursuit
PursuitXRs
Polarisphases
FastGuardsforUHPLC:
Singlereplacementguardcolumn
Noguardcartridgeholder
2.1
3.0
4.6
Poroshell120:
EC-C18
EC-C8
SB-C18
Phenyl-Hexyl
Sub-2µm:
EclipsePlusC18
EclipseXDB-C18
SB-C18
SB-C8
*StandaloneguardcartridgesfitallcartridgeandstandardfittingcolumnsavailablefromAgilent.Allcolumnswithouticonsarestandardfittingcolumns.
226
Phases
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Fast Columns
for Reversed-Phase HPLC/UHPLC
Thepastdecadehasseenasteadyincreaseintheefficiencyandspeedofchromatography,startingwith
smallerparticlesizes,thatenablehigherresolution,andcontinuingwithnewtechnologicaladvancesin
particledesign–superficiallyporousparticles–thatenablethesesameresolutionenhancementswith
lowerbackpressure.
Designedespeciallyforhigh-productivityanalysis(FastLC),AgilentZORBAXandPoroshellcolumnsare
thebestfirstchoiceforanyanalysis,becausetheygiveyou:
• Theproductivityyouneedtostayaheadofyourcompetition:technologicaladvanceslikesub-2µm
particlesandsuperficiallyporousPoroshell120columnsdeliverincreasedspeedandresolution.
• Flexibilityandmethodscalabilityfromlabtolabandaroundtheworld–forsmallmoleculeand
biomoleculeanalyses.
• Unbeatablechromatographicperformance:ZORBAXsilica–thebasesilicausedforallZORBAX
andPoroshell120columns–isultra-pure,verystrong,andhighlyuniformforultimatereliability.
• Thebroadestrangeofphasesandcolumnconfigurationstosuityourspecificapplicationneeds.
Recommendations for Fast LC Columns
your Lab Situation
agilent Recommends
Rationale
You’reusingbothUHPLC(1000+bar)
andHPLCinstruments
(e.g.Agilent1290InfinityLC
and1260InfinityLC–600bar)
1.Poroshell120
2.ZORBAXRRHD1.8µm
Poroshell120isaneasycolumntouseonbothinstrument
types.ZORBAXRRHDwillhelpyouoptimizethecapabilities
ofthe1290InfinityLCforUHPLC.
Only400-600barHPLCs–
Agilent1200s,Agilent1100s(400bar)
aswellasthe1220InfinityLC
or1260InfinityLC
(600bar)
1.Poroshell120
WithPoroshell120,youcanenhancetheperformanceofolder
400-barinstruments,andalsogetevenbetterperformancefrom
2.ZORBAXEclipsePlus3.5µmand5µm
newer600barUHPLCinstruments.Forestablishedmethodsthat
youcan’ttransfer,theZORBAXEclipsePluscolumnwillprovide
exceptionalpeakshapeandperformance.
AmixofUHPLCinstruments
1.ZORBAXRRHD1.8µm
(Agilent1290InfinityLC,
2.Poroshell120
other1000+barinstruments)
andsomeHPLCinstruments(e.g.1200LC)
ZORBAXRRHDcandeliveroptimumperformanceonallthese
instruments.Poroshell120canbeusedonthe600barinstruments
tooptimizetheirperformance.
TiPS & TooLS
AgilentCrossLaboffersarangeofPEEKcapillariesandtubing.Usedincombinationwiththerightfittings,
theyprovideaninertsurfacefortheFastLCofsensitivebiomolecules. Turntopage130.
www.agiLEnT.CoM/CHEM/LC
227
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Poroshell 120
Solidcore,1.7µmdiameter
Porousshell,0.5µmthick
• Highefficiencyandhighresolution,withupto50%lessbackpressurethansub-2µmcolumns
• 2µmfrit,forruggedperformancewithdirtysamples
• Compatiblewith400barand600barLCs,aswellasUHPLCinstruments
• AnexpandingfamilyofbondedphasestoalignwiththeZORBAXFamily,forreliablescalability
• Excellentselectivityandpeakshapes
• Designedforexceptionalreproducibility
AgilentPoroshell120columnsarea2.7µmparticlewitha1.7µmsolidcoreand0.5µmporousouterlayer.
Thissmallparticlesizeprovideshighefficiency,similartosub-2µmcolumns,butwith40-50%lesspressure.
Thesehighefficiency,highresolutioncolumnscanbeusedonanytypeofLC.Theporousouterlayerand
solidcorelimitdiffusiondistanceandimproveseparationspeedwhilethenarrowparticlesizedistribution
improvesefficiencyandresolution.Thecolumnscansupporthighpressureandmultiplecolumnscanbe
usedforthehighestresolutionandefficiencypossible.ThesameprinciplesareusedinPoroshell300
columns,idealforfast,highresolutionseparationsofbiomolecules.
Column Specifications
Bonded Phase
Pore Size Temp Limits
EC-C18
120Å
60°C
2.0-8.0
Double
10%
130m2/g
EC-C8
120Å
60°C
2.0-8.0
Double
5%
130m2/g
Phenyl-Hexyl
120Å
60°C
2.0-8.0
Double
9%
130m2/g
SB-C18
120Å
90°C
1.0-8.0
No
8%
130m2/g
SB-C8
120Å
80°C
1.0-8.0
No
5.5%
130m2/g
SB-Aq
120Å
80°C
1.0-8.0
No
Proprietary
130m2/g
Bonus-RP
120Å
60°C
2.0-9.0
Triple
9.5%
130m2/g
EC-CN
120Å
60°C
2.0-8.0
Double
3.5%
130m2/g
HILIC
120Å
60°C
0.0-8.0
No
N/A
130m2/g
Specificationsrepresenttypicalvaluesonly
TiPS & TooLS
WatchthePoroshell120MethodTransferVideotolearnhoweasyitistotransferexisting
methodstoPoroshell120atwww.agilent.com/chem/poroshell120video
228
pH Range Endcapped Carbon Load Surface area
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Poroshell 120
(Maximum pressure: 600 bar)
Hardware
Particle
Size EC-C18
(µm) uSP L1
EC-C8
uSP L7
Phenyl-Hexyl SB-C18
uSP L11
uSP L1
SB-C8
uSP L7
Bonus-RP
uSP L60
description
Size (mm)
Analytical
4.6x150
2.7
693975-902 693975-906 693975-912
Analytical
4.6x100
2.7
695975-902 695975-906 695975-912
685975-902 685975-906 685975-914 695968-901
Analytical
4.6x75
2.7
697975-902 697975-906
687975-902
Analytical
4.6x50
2.7
699975-902 699975-906 699975-912
689975-902 689975-906 689975-914 699968-901
Analytical
4.6x30
2.7
691975-902 691975-906
681975-902
UHPLCGuard, 4.6x5
600bar,3/pk
2.7
820750-911 820750-913 820750-914
820750-912
SolventSaver
3.0x150
2.7
693975-302 693975-306 693975-312
683975-302 683975-306 683975-314 693968-301
SolventSaver
3.0x100
2.7
695975-302 695975-306 695975-312
685975-302 685975-306 685975-314 695968-301
SolventSaver
3.0x75
2.7
697975-302 697975-306
687975-302
SolventSaver
3.0x50
2.7
699975-302 699975-306 699975-312
689975-302 689975-306 689975-314 699968-301
SolventSaver
3.0x30
2.7
691975-302 691975-306
681975-302
UHPLCGuard, 3.0x5
600bar,3/pk
2.7
823750-911 823750-913 823750-914
823750-912
NarrowBore
2.1x150
2.7
693775-902 693775-906 693775-912
683775-902 683775-906 683775-914 693768-901
NarrowBore
2.1x100
2.7
695775-902 695775-906 695775-912
685775-902 685775-906 685775-914 695768-901
NarrowBore
2.1x75
2.7
697775-902 697775-906
687775-902
NarrowBore
2.1x50
2.7
699775-902 699775-906 699775-912
689775-902 689775-906 689775-914 699768-901
NarrowBore
2.1x30
UHPLCGuard, 2.1x5
600bar,3/pk
SB-aq
683975-902 683975-906 683975-914 693968-901
2.7
691775-902 691775-906
681775-902
2.7
821725-911 821725-913 821725-914
821725-912
Poroshell120Columns
www.agiLEnT.CoM/CHEM/LC
229
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Environmental phenols on Poroshell 120
Column a:
Poroshell 120 EC-C18
695975-902
4.6 x 100 mm, 2.7 µm
Column B:
Eclipse Plus C18
959964-902
4.6 x 100 mm, 1.8 µm
1.
2.
3.
4.
5.
A
1
Gradient:
A:Water0.1%formicacid
B:Acetonitrile0.1%formicacid
2mL/min
Initial:8%B
10min:30%B
Hydroquinone
Resorcinol
Catechol
4-Nitrophenol
p-cresol
6.
7.
8.
9.
10.
o-cresol
2-Nitrophenol
2,3Dimethylphenol
2,5Dimethylphenol
1-Naphthol
2
3
Detector:
Injection:
P = 332 bar
275nm,2mmflowcell
9
4
5
10µL
Agilent1200SL40°C
0
2
4
10
8
6
8
10
Time (min)
Nopulsedamper
Nomixer3µLheater
7
6
B
1
2
3
P = 510 bar
9
4
5
7
6
10
8
Poroshell120providessub-2µmlikeefficiencyatlowerpressure.
0
2
4
6
8
10
Time (min)
EP_Poro120
RRLCcheckoutsample(P/N5188-6529)spiked
w/50µL2mg/mLthioureainwater/acetonitrile(65:35)
2.223
9.753
6.273
4.132
2.816
n = 27295, Press = 386 bar
2.5
5
13.041
Sample:
10
8.218
DADSig=254,4nm
Ref=360,100nm
7.5
5.281
Detector:
B
3.494
InjectionVolume: 4µL
5
2.705
26°C
2.5
1.756
Temperature:
n = 25053, Press = 182 bar
2.418
0.58mL/min
1.148
60%Acetonitrile:40%water
FlowRate:
0.646
MobilePhase:
A
1.518
2.016
Eclipse Plus C18
959964-302
3.0 x 100 mm, 1.8 µm
0.865
Column B:
1.275
Poroshell 120 EC-C18
695975-302
3.0 x 100 mm, 2.7 µm
0.649
Column a:
0.826
uHPLC efficiency at HPLC pressures
7.5
10
12.5
Forthissampleofneutralalkylphenones,thePoroshell120columndelivered>90%oftheefficiencyattainedbythe1.8µmcolumn.Alsonotethatthepressureonthe
Poroshell120columnisabout50%ofthepressureonthe1.8µmcolumn.
230
min
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
HPLC separation of 12 phenols performed
in just 5 minutes – and under 400 bar –
using an agilent Poroshell 120 EC-C18 column
Column:
Poroshell 120 EC-C18
699975-902
4.6 x 50 mm, 2.7 µm
MobilePhase:
SolventA:Waterwith0.1%formicacid
SolventB:Acetonitrile
Gradient:
5%Bin0.8min
60%Bin6.8min
1200SLcontrolledtemperature
at25°C2mmflowcell
Detector:
DAD,270nm
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
2.5 mL/min, 274 bar
Importantly,theflowratewaskeptto2.5mL/min,
reducingtheamountofmobilephaseconsumedper
analysistoabout15mL.
AgilentPoroshell120giveshighefficiency,high
resolutionseparationsquicklyatHPLCpressures.
5
0
Hydroquinone
Resourcinol
Catechol
Phenol
4-Nitrophenol
p-cresol
o-cresol
2-Nitrophenol
3,4dimethylphenol
2,3dimethylphenol
2,5dimethylphenol
1-napthol
min
12 phenols analyzed using a longer (4.6 x 100 mm)
agilent Poroshell 120 EC-C18 column
Column:
Poroshell 120 EC-C18
695975-902
4.6 x 100 mm, 2.7 µm
MobilePhase:
SolventA:Waterwith0.1%formicacid
SolventB:Acetonitrile
Gradient:
5%Bin2min
60%Bin17min
1200RRLCSLcontrolledtemperature
at25°C2mmflowcell
Detector:
DAD,270nm
Byreducingtheflowrateto2.0mL/min,the
pressurewaskepttolessthan400barimproving
theseparationofalate-elutingpeakpair
(highlighted)withonlyaminorincreaseinanalysis
time.ThisseparationcanbeachievedusingHPLCor,
ifahigherflowrateisdesired,aUHPLC.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
2.0 mL/min, 394 bar
mAU
100
80
60
Hydroquinone
Resourcinol
Catechol
Phenol
4-Nitrophenol
p-cresol
o-cresol
2-Nitrophenol
3,4dimethylphenol
2,3dimethylphenol
2,5dimethylphenol
1-napthol
40
20
0
2
4
6
8
www.agiLEnT.CoM/CHEM/LC
10
12
min
231
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Poroshell 120 EC-C18
for fast uHPLC separations
Column:
Poroshell 120 EC-C18
695975-302
3.0 x 100 mm, 2.7 µm
MobilePhase:
65%A:0.2%Formicacid
35%B:Methanol
Isocratic
FlowRate:
Varies
Temperature:
26°C
Detector:
Sig=220,4nm,Ref=Off
1.
2.
3.
4.
5.
6.
mAU
300
Saccharin
Caffeine
P-hydroxybenzoicacid
Aspartame
Dehydroaceticacid
Benzoicacid
Flow Rate: 0.5 mL/min,
P: 300 bar
200
100
0
2
Thisexampleshowsafastseparationusingamobile
phasethatgenerateshigherpressures.Inthetop
chromatogram,a3.0mmidcolumnwasused,
withaflowrateof0.5mL/minandapressure
below400bar–makingthisatypicalLCseparation.
Althoughthetopseparationwasfast(justunder
6minutes),themiddleandbottomchromatograms
showthatyoucanreduceruntimestounder3minutes
byincreasingtheflowrate.Thesefasteranalyseswill
takeyourpressureto400-560bar;looktotheAgilent
1200InfinitySeriesflexibleupgradeoptionstohelp
youtakeadvantageofUHPLCcapabilities.
6
Flow Rate: 0.75 mL/min,
P: 433 bar
200
100
0
2
4
mAU
Flow Rate: 1.0 mL/min,
P: 559 bar
200
100
0
Moreviscoussolventslikemethanolcanbeused
atHPLCorUHPLCpressures.
TiPS & TooLS
ForafulllistingofourLCcapillaryportfolio,turntopages16-46.
232
4
mAU
300
2
4 min
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
ZoRBaX Rapid Resolution High definition
(RRHd) 1.8 µm
• Highpressure(1200bar)columnsforoptimumresultswiththe1290InfinityLCorother
UHPLCinstruments
• 1.8µmparticlesdelivermaximumresolutionforthemostdefinedseparations
• Availablein12ZORBAXphases,includingEclipsePlusC18forsuperiorpeakshape,
ZORBAXStableBondC18forlowpHstability,Bonus-RP,EclipsePAH,EclipsePlusPhenyl-Hexyl
andExtend-C18
• AlsoavailableinHILICPlus
• Achievethesameselectivityon3.5and5µmZORBAXcolumnswiththesamebondedphasefor
compatibilitywithanyLC
ZORBAXRapidResolutionHighDefinition(RRHD)columnsareanexpansionoftheZORBAX1.8µmparticle
columnline.ThenewRRHDcolumnsuseimprovedpackingprocessestoachievestabilityupto1200bar
forusewiththeAgilent1290InfinityLCorotherUHPLCinstruments.RRHD1.8µmcolumnsareavailable
in50,100and150mmlengthsforfastorhighresolution–trulyhighdefinition–separationsofyourmost
complexsamples.
ZoRBaX RRHd Column Specifications
Bonded Phase
Pore Size Surface area pH Range Endcapped Temp Limit
ZORBAXEclipsePlusC18
95Å
160m2/g
2.0-9.0
Double
60°C
ZORBAXEclipsePlusC8
95Å
160m2/g
2.0-9.0
Double
60°C
ZORBAXEclipsePlusPhenyl-Hexyl
95Å
160m2/g
2.0-9.0
Double
60°C
ZORBAXEclipseXDB-C18
80Å
180m2/g
2.0-9.0
Double
60°C
ZORBAXExtend-C18
80Å
180m2/g
2.0-11.5**
Double
60°C
ZORBAXBonusRP
80Å
180m2/g
2.0-9.0
Triple
60°C
ZORBAXStableBondSB-C18
80Å
180m2/g
1.0-8.0*
No
80°C
ZORBAXStableBondSB-C8
80Å
180m2/g
1.0-8.0*
No
80°C
ZORBAXStableBondSB-Phenyl
80Å
180m2/g
1.0-8.0*
No
80°C
ZORBAXStableBondSB-CN
80Å
180m2/g
1.0-8.0*
No
80°C
ZORBAXStableBondSB-Aq
80Å
180m2/g
1.0-8.0*
No
80°C
ZORBAXEclipsePAH
95Å
160m2/g
2.0-8.0
No
60°C
ZORBAXHILICPlus
95Å
160m2/g
0.0-8.0
No
60°C
ZORBAXStableBond300SB-C8
300Å
45m2/g
1.0-8.0*
No
80°C
ZORBAXStableBond300SB-C18
300Å
45m2/g
1.0-8.0*
No
80°C
ZORBAXStableBond300SB-C3
300Å
45m2/g
1.0-8.0*
No
80°C
ZORBAX300-Diphenyl
300Å
45m2/g
1.0-8.0*
No
80°C
*StableBondcolumnsaredesignedforoptimaluseatlowpH.AtpH>6,highestcolumnstabilityforallsilicabasedcolumns
isobtainedbyoperatingattemperatures<40°Candusinglowerbufferconcentrations–10-20mMororganicbuffers.
300SB-C18maybeusedupto90°C.ForpH6-8,selecttheEclipsePlusC18column.
**Temperaturelimitsare60°CuptopH8,40°CfrompH8-11.5.
www.agiLEnT.CoM/CHEM/LC
ZORBAXRapidResolutionHighDefinition(RRHD)
1.8µmColumns
233
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Separation of licorice root on RRHd columns
Column a:
ZoRBaX RRHd SB-C18
857700-902
2.1 x 50 mm, 1.8 µm
Column B:
858700-902
2.1 x 100 mm, 1.8 µm
Column C:
859700-902
2.1 x 150 mm, 1.8 µm
MobilePhase:
10-100%B/30min
A:0.1%Formicacid(fa)
B:Acetonitrilewith0.1%fa
FlowRate:
F=0.4mL/min
Gradient:
30minutegradientoneachlength
Temperature:
Ambient
Detector:
280nmUV
Instrument:
1290InfinityLC
50 mm
A
7 peaks
Rs: 0
mAU
200
150
100
50
0
2
0
2
4
6
8
10
12
4
14
16
18
min
100 mm
B
8 peaks
Rs: 1.37
mAU
200
150
100
50
0
2
0
2
4
6
8
10
12
4
14
16
18
min
150 mm
C
9 peaks
Rs: 2.40
mAU
200
150
100
50
0
2
0
234
2
4
6
8
10
12
4
14
6
16
18
min
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Sub 1 minute separations with RRHd columns
Column:
ZoRBaX RRHd SB-C18
857700-902
2.1 x 50 mm, 1.8 µm
Gradient:
H2O(0.05%trifluoroaceticacid)/10-40%ACN/1min
InjectionVolume: 0.5µLx100ppmeach
Temperature:
60°C
Detector:
UV,275nm
DataRate:
160Hz
3
mAU
500
6compoundbiocidemixin0.5min
400
F=2.0mL/min
P=975bar
300
1.
2.
3.
4.
5.
6.
1
2
200
0.5 min
6
100
5
4
0
0
.05
.1
.15
.2
.25
.3
.35
.4
.45
2-methyl-4-isothiazolin-3-one
5-chloro-2-methyl-4-isothiazolin-3-one
Carbendazim
Benzisothiiazol-3(2H)-one
2-phenoxyethanol
Methylparaben
min
RRHDSub1m
new levels of sensitivity and resolution
Column a:
ZoRBaX RRHd Eclipse Plus C18
959758-302
3.0 x 100 mm, 1.8 µm
Column B:
×107
2
1
ZoRBaX RRHd Eclipse Plus C18
959757-302
3.0 x 50 mm, 1.8 µm
IonSource:
360°C,12L/min.50psi,
3500V.
Temperature:
Ambient,notemperaturecontrol
(approx24°C)
MobilePhase:
A:0.2%Formicacidinwater
B:ACN
Detector:
Agilent1290InfinityLC
with6410MS/MS
Sample:
20µL(10µLfor50mmcolumn)
of1µg/mLstandard
A
1.5
Average PW1/2 = 0.025
nc = 43
1
0.5
0
0.1
× 107
1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.873
1.9
2
CountsversusAcquisitiontime(min)
B
1
Average PW1/2 = 0.0092
nc = 39
0.8
0.6
0.4
0.2
1.006
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
CountsversusAcquisitiontime(min)
BytransferringyourmethodtoanAgilentRRHDcolumn,youcanenhanceresolutionfordifficultanalyses–allowingyouto
savetimebyusingshortercolumnswithoutcompromisingperformance.
TheRRHDcolumnsavesanalyticaltimewithoutsacrificingperformance.
www.agiLEnT.CoM/CHEM/LC
Compounds(inelutionorder)withidentifyingmass:
1. Acetaminophen,m/z109
2. Caffeine,m/z194
3. 2-acetamidophenol,m/z109
4. Acetanilide,m/z135
5. Acetylsalicylicacid,m/z120
6. Phenacetin,m/z179
7. Salicylicacid,m/z120
8. Sulindac,m/z356
9. Piroxicam,m/z332
10. Tolmetin,m/z257
11. Ketoprofen,m/z254
12. Diflunisal,m/z332
13. Diclofenac,m/z235
14. Celecoxib,m/z351
15. Ibuprofen,m/z160
235
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Selectivity comparison: C18 columns
Column a:
ZoRBaX RRHd Eclipse Plus C18
959758-902
2.1 x 100 mm, 1.8 µm
Column B:
ZoRBaX RRHd Eclipse XdB-C18
981758-902
2.1 x 100 mm, 1.8 µm
Column C:
Column d:
MobilePhase:
ZoRBaX RRHd Extend-C18
758700-902
2.1 x 100 mm, 1.8 µm
ZoRBaX RRHd SB-C18
858700-902
2.1 x 100 mm, 1.8 µm
A:0.1%HCOOHinH2O(30%)
B:0.1%HCOOHinCH3CN(70%)
1.
2.
3.
4.
×10 2
1
0.9
0.8
Anadamine(AEA),m/z348
Palmitoylethanolamide(PEA),m/zPEA
2-arachinoylglycerol(2-AG),m/z379*
Oleoylethanolamide(OEA),m/z326
*Afifthpeakwithamassof379wasalsodetected.
Thisimpurityisbelievedtobe1,3arachidonolyglycerol,
arearrangementof2-AG.
OEA
0.7
0.6
PEA
0.5
a
0.4
AEA
0.3
0.2
2-AG
0.1
*
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
Counts(%)vs.AcquisitionTime(min)
×10 2
1
2.6
2.8
3
3.2
3.4
2.6
2.8
3
3.2
3.4
2.6
2.8
3
3.2
3.4
2.8
3
3.2
3.4
OEA
0.9
0.8
PEA
0.7
FlowRate:
1mL/min,isocratic
Temperature:
30°C
0.5
Sample:
1µL
0.3
MS2Scan:
290-390,ESIpositivemode,
scantime:500,fragmentor:
135V;dryinggas:12L/min,325°C;
nebulizerpressure:35psig;
capillaryvoltage:3000
0.6
B
0.4
AEA
0.2
2-AG
0.1
*
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
Counts(%)vs.AcquisitionTime(min)
×10 2
1
2.4
OEA
0.9
0.8
0.7
0.6
Selectivitydifferencesareduetosubtle,yetimportant
variations,suchasbondingtype,endcapping,ortheamount
andtypeofsilanolsonthesilica.Otherfactorsthatinfluence
selectivityincludemobilephasecomposition,temperature,
andpH.(Notethatthesefactorsareidenticalinthe
followingexample.)
HerewecomparedtheselectivityoffourAgilent
ZORBAXRRHDC18columnsusinganendocannabinoid
analysismethod.
PEA
0.5
C
0.4
AEA
0.3
0.2
2-AG
0.1
*
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
Counts(%)vs.AcquisitionTime(min)
×10 2
1
OEA
0.9
0.8
0.7
0.6
0.5
0.4
PEA
d
AEA
0.3
0.2
0.1
2-AG
*
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Counts(%)vs.AcquisitionTime(min)
TiPS & TooLS
Forfulldetails,seeAgilentpublication5990-7166EN,www.agilent.com/chem/library
236
2.2
2.4
2.6
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Selectivity comparison:
Phenyl and other columns
Column a:
ZoRBaX RRHd Eclipse Plus C18
959758-902
2.1 x 100 mm, 1.8 µm
Column B:
ZoRBaX RRHd Eclipse Plus Phenyl-Hexyl
959758-912
×10
2.1 x 100 mm, 1.8 µm
Cyanidin,m/z286
Peonidin,m/z300
2
Column C:
ZoRBaX RRHd SB-aq
858700-914
2.1 x 100 mm, 1.8 µm
Column d:
ZoRBaX RRHd SB-Phenyl
858700-912
2.1 x 100 mm, 1.8 µm
MobilePhase:
A:5%HCOOHinH2O
B:CH3CN
FlowRate:
0.65mL/min
Gradient:
10-50%Bin15min
Temperature:
30°C
MS2Scan:
ESI+,200-1000
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Delphinidin,m/z302
a
Malvidin,m/z330
1
×10 2
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
×10 2
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
3
4
5
6
7
8
9
10
11
12
13
14
15
2
3
4
5
6
7
8
9
10
11
12
13
14
15
2
3
4
5
6
7
8
9
10
11
12
13
14
15
2
3
4
5
6
7
8
9
10
11
12
13
14
15
C
1
×10 2
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
2
B
1
ExtractedionchromatogramsfromLC/MSscandataof
blueberryanthocyanins.
Petunidin,m/z316
d
1
Counts(%)versusAcquisitiontime(min)
TiPS & TooLS
Forfulldetails,seeAgilentpublication5990-8470EN,www.agilent.com/chem/library
www.agiLEnT.CoM/CHEM/LC
237
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Rapid Resolution High definition (RRHd) Columns for High Pressure use (Maximum Pressure: 1200 bar)
Hardware description
Size (mm)
Particle Size
(µm)
Eclipse
Plus C18
uSP L1
Eclipse
Plus C8
uSP L7
Eclipse Plus
Phenyl-Hexyl
uSP L11
Eclipse
PaH
uSP L1
SolventSaverRRHD,1200bar
3.0x150
1.8
959759-302 959759-306
SolventSaverRRHD,1200bar
3.0x100
1.8
959758-302 959758-306 959758-312
959758-318
SolventSaverRRHD,1200bar
3.0x50
1.8
959757-302 959757-306 959757-312
959757-318
UHPLCGuard,1200bar,3/pk
3.0x5
1.8
823750-901
NarrowBoreRRHD,1200bar
2.1x150
1.8
959759-902 959759-906 959759-912
959763-918
NarrowBoreRRHD,1200bar
2.1x100
1.8
959758-902 959758-906 959758-912
959764-918
959741-918
NarrowBoreRRHD,1200bar
2.1x50
1.8
959757-902 959757-906 959757-912
UHPLCGuard,1200bar,3/pk
2.1x5
1.8
821725-901
Rapid Resolution High definition (RRHd) Columns for High Pressure use (Maximum Pressure: 1200 bar)
Hardware description
Size (mm)
Particle Size SB-C18
(µm)
uSP L1
SB-C8
uSP L7
SB-Cn
uSP L10
SB-Phenyl
uSP L11
SB-aq
SolventSaverRRHD,1200bar
3.0x150
1.8
859700-302 859700-306
SolventSaverRRHD,1200bar
3.0x100
1.8
858700-302 858700-306 858700-305 858700-905 858700-314
SolventSaverRRHD,1200bar
3.0x50
1.8
857700-302 857700-306 857700-305 857700-312 857700-314
UHPLCGuard,1200bar,3/pk
3.0x5
1.8
823750-902 823750-904
NarrowBoreRRHD,1200bar
2.1x150
1.8
859700-902 859700-906 859700-905 859700-912 859700-914
NarrowBoreRRHD,1200bar
2.1x100
1.8
858700-902 858700-906 858700-905 858700-912 858700-914
NarrowBoreRRHD,1200bar
2.1x50
1.8
857700-902 857700-906 857700-905 857700-912 857700-914
UHPLCGuard,1200bar,3/pk
2.1x5
1.8
821725-902 821725-904
Rapid Resolution High definition (RRHd) Columns for High Pressure use (Maximum Pressure: 1200 bar)
Hardware description
Size (mm)
Extend-C18
Particle Size (µm) uSP L1
Bonus-RP
uSP L60
HiLiC Plus
SolventSaverRRHD,1200bar
3.0x150
1.8
759700-302
981759-302
SolventSaverRRHD,1200bar
3.0x100
1.8
758700-302
981758-302
959758-301
SolventSaverRRHD,1200bar
3.0x50
1.8
757700-302
981757-302
959757-301
UHPLCGuard,1200bar,3/pk
3.0x5
1.8
823750-903
NarrowBoreRRHD,1200bar
2.1x150
1.8
759700-902
981759-902 859768-901 959759-901
NarrowBoreRRHD,1200bar
2.1x100
1.8
758700-902
981758-902 858768-901 959758-901
757700-902
981757-902 857768-901 959757-901
NarrowBoreRRHD,1200bar
2.1x50
1.8
UHPLCGuard,1200bar,3/pk
2.1x5
1.8
ZORBAXRRHDcolumnsarealsoavailablein300Åconfigurationsforbiomolecules.Turntopage364.
ZORBAXRRHD300-HILICwillbeavailablein2013.
238
Eclipse
XdB-C18
uSP L1
821725-903
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
ZoRBaX Rapid Resolution High Throughput
(RRHT) 1.8 µm
• Highpressure(600bar)columnsforultrahighspeedormaximumresolutionanalyses
withRapidResolutionHTcolumnspackedwithtotallyporous,1.8µmpackings
• Carefullyengineeredparticlesdelivermaximumresolutionat25%lesspressure
thanothersub-2µmmaterials
• Reduceanalysistimebyupto95%
• DevelopHPLCmethodsmorequickly
• Securelytransferconventionalmethodswithover140RRHTcolumnchoices
• Analyzecomplexsamplesonshortercolumnsfasterandmaximizepeakcapacity
• Matchingselectivityin3.5,5and7µmparticlesizesforcompletemethodscalability
• Short(50mmlongandless)columncanbeusedonsomeconventionalLCs
AgilentZORBAXRapidResolutionHT(1.8µm)columnsuseatotallyporous,1.8µmparticletoprovide
maximumresolutioninfast,ultra-fastandhighresolutionanalyses.Youcanreduceanalysistimebyupto
95%incomparisonto250mmlengthcolumns.Withmorethan140RRHTcolumnchoices,includingthe
highperformanceZORBAXEclipsePlusandmanyotherZORBAXcolumnchoices(EclipseXDB,StableBond,
Extend,Bonus-RP),methodscanbedevelopedquicklyorsecurelytransferredtoasmallerparticlesize
columnwithnolossinresolution.Thesmallparticlesizeprovidesdoubletheefficiencyofa3.5µmcolumn
inthesamecolumnlength,providingthehighestefficiencyandresolutionpossible.Thispermitstheanalysis
ofcomplexsamplesonshortercolumnswiththehighestresolutionandpeakcapacity.The1.8µmRapid
ResolutionHTcolumnstakehigh-speed,high-resolutionHPLCtoanewlevel.
ZORBAXRapidResolutionHighThroughput
(RRHT)1.8µmColumns
The600barcolumnscanbeusedwiththeAgilent1260InfinityLCSystemuptothishighpressurelimit.
Inaddition,theshortercolumnscanbeusedonmanyotherLC's,includingtheAgilent1200Rapid
ResolutionLCSystem.
www.agiLEnT.CoM/CHEM/LC
239
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Rapid Resolution HT (RRHT) provides double
the efficiency of Rapid Resolution columns
Column a:
ZoRBaX Rapid Resolution SB-C18
835975-902
4.6 x 50 mm, 3.5 µm
Column B:
ZoRBaX RRHT SB-C18
827975-902
4.6 x 50 mm, 1.8 µm
MobilePhase:
25%Water,75%MeOH
FlowRate:
1.5mL/min
Temperature:
Ambient
Detector:
Plates (n)
Plates (n)
1.
3476
1.
6560
2.
4585
2.
8958
3.
5673
3.
11508
4.
6180
4.
12266
A
B
2
2
3
UV,254nm
4
3
1.
2.
3.
4.
1
ThisfigureshowsthatRapidResolutionHTcolumnscanprovide
doubletheefficiencyofa3.5µmcolumninthesamecolumn
length.Thishighefficiencycanbeusedforveryhigh-resolution,
highthroughputanalyses.
0
Uracil
Phenol
4-CI-Nitrobenzene
Toluene
4
1
0.0
1
0.5
Time (min)
Time (min)
1.0
LCRR002
increase peak capacity with RRHT columns
Column a:
Eclipse RRHT XdB-C8
928700-906
2.1 x 100 mm, 1.8 µm
Column B:
Eclipse XdB-C18
961753-902
2.1 x 100 mm, 3.5 µm
MobilePhase:
A:H2O
B:ACN
Peakcapacity:
A:461
B:343
FlowRate:
0.5mL/min
Gradient:
0.0min50%B
20.0min100%B
Temperature:
40°C
Detector:
UV,254nm
Sample:
Alkylphenones
A
mAU
120
100
80
60
40
20
0
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
1
11
0
2
4
6
8
10
12
Time (min)
14
16
18
B
mAU
100
1
80
60
11
40
20
0
0
2
4
6
8
10
12
Time (min)
14
16
18
LCRR004
240
Uracil
C3-Alkylphenone
C4-Alkylphenone
C5-Alkylphenone
C6-Alkylphenone
C7-Alkylphenone
C8-Alkylphenone
C9-Alkylphenone
C10-Alkylphenone
C12-Alkylphenone
C14-Alkylphenone
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Reduce analysis time dramatically with Rapid
Resolution HT columns
Column a:
Eclipse XdB-C18
990967-902
4.6 x 250 mm, 5 µm
Column B:
Eclipse XdB-C18
963967-902
4.6 x 150 mm, 3.5 µm
A
1
2
B
Column C:
Eclipse XdB-C18
966967-902
4.6 x 75 mm, 3.5 µm
Column d:
ZoRBaX Eclipse XdB-C18
935967-902
4.6 x 50 mm, 3.5 µm
1
1
5
MobilePhase:
Eclipse RRHT XdB-C18
925975-902
4.6 x 50 mm, 1.8 µm
73%MeOH:27%20mM
PhosphateBuffer,pH7.0
FlowRate:
1mL/min
Temperature:
Ambient
Detector:
UV,254nm
D
3
1
2 3 4
1
E
5
5
2
6
4
3 4
0
6
5
1.
2.
3.
4.
5.
6.
7.
7
7
6 7
2
4
7
6
7
6
4
3
2
Column E:
5
2
C
4
3
6
Uracil
Naproxen
Mefanamicacid
Butylparaben
Propranolol
Naphthalene
Dipropylphthalate
8
Time (min)
10
12
LCRR003
ThisfigureshowsthedramaticreductioninanalysistimemadepossiblebyusingRapidResolutionHTcolumns.ChromatogramAshowsaseparationthattakes11.5minuteson
a25cm,5µmcolumn.RapidResolution(3.5µm)columns,showninchromatogramBandC,reduceanalysistimesubstantially,butwithaslightcompromiseinresolution.
TheRapidResolutionHTcolumnreducesanalysistimeto2.2minutes,an80%reduction,whilestillmaintainingbaselineresolution.
Long lifetime of RRHT columns at elevated
temperatures
Column:
ZoRBaX RRHT SB-C18
827700-902
2.1 x 50 mm, 1.8 µm
MobilePhase:
A:60%H2O
B:40%ACN
FlowRate:
1mL/min
Temperature:
80°C
Detector:
UV,254nm
Sample:
QCtestmix
Plates
12000
10000
8000
6000
4000
0
1
499
718
853
988
1153
1305
1506
1641
1776
1911
2046
2181
2319
2508
2643
2778
2913
3085
3220
3468
3603
3738
3887
4054
4189
4324
4537
4756
4892
5033
5201
2000
Injection Number
www.agiLEnT.CoM/CHEM/LC
LCRR005
241
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Comparison of efficiencies – Rapid Resolution High definition (RRHd)/RRHT
(1.8 µm) and Rapid Resolution (3.5 µm) columns
Column Length (mm)
Poroshell 120
Resolving
Power
n (3.5 µm)*
Resolving
Power
n (1.8 µm)
High Resolution
150
32,000
21,000
32,500
100
21,000
14,000
24,000
75
16,000
10,500
17,000**
50
11,000
7,000
12,000
30
5,500
4,200
6,000
20
—
—
3,500
—
2,100
2,500
ultra Fast
15
Resolutiona N
1/2
*5µmHPLCcolumnsofthesamelengthhave40%fewerplates(N-value);4.6mmid
**Availableasacustomcolumn
Dataisbasedon4.6mmidcolumns
TiPS & TooLS
TheLCRackfromAgilentcanhelpyoureducecapillarylengthsandminimizeextra-columnvolume.
Italsoprotectsyourinstrumentandenablesyoutoswitchoutmodulesasneeded.
AgilentrackforLCsystems,5001-3726
242
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Rapid Resolution HT Columns for High Pressure use (Maximum Pressure: 600 bar, 9000 psi)
Hardware description
Size
(mm)
Particle Eclipse
Size Plus C18
(µm) uSP L1
Eclipse
Plus C8
uSP L7
Eclipse Plus Eclipse
Phenyl-Hexyl PaH
uSP L11
uSP L1
Eclipse
XdB-C18
uSP L1
Eclipse
XdB-C8
uSP L7
Extend-C18
uSP L1
RapidResolution
HT,600bar
4.6x150
1.8
959994-902
RapidResolution
HT,600bar
4.6x100
1.8
959964-902 959964-906 959964-912
RapidResolution
HT,600bar
4.6x75
1.8
959951-902
RapidResolution
HT,600bar
4.6x50
1.8
959941-902 959941-906 959941-912
959941-918 927975-902 927975-906 727975-902
RapidResolution
HT,600bar
4.6x30
1.8
959931-902 959931-906 959931-912
959931-918 924975-902 924975-906 724975-902
RapidResolution
HT,600bar
4.6x20
1.8
UHPLCGuard,
600bar,3/pk
4.6x5
1.8
820750-901
820750-903
SolventSaverHT, 3.0x100
600bar
1.8
959964-302 959964-306 959964-312
928975-302
SolventSaverHT, 3.0x50
600bar
1.8
959941-302 959941-306 959941-312
927975-302 927975-306 727975-302
SolventSaverHT, 3.0x30
600bar
1.8
924975-302 924975-306 724975-302
SolventSaverHT, 3.0x20
600bar
1.8
926975-302 926975-306 726975-302
UHPLCGuard,
1200bar,3/pk
3.0x5
1.8
823750-901
NarrowBore
RRHT,600bar
2.1x150
1.8
959794-902
NarrowBore
RRHT,600bar
2.1x100
1.8
959764-902 959764-906 959764-912
959764-918 928700-902 928700-906 728700-902
NarrowBore
RRHT,600bar
2.1x50
1.8
959741-902 959741-906 959741-912
959741-918 927700-902 927700-906 727700-902
NarrowBore
RRHT,600bar
2.1x30
1.8
959731-902 959731-906 959731-912
924700-902 924700-906 724700-902
NarrowBore
RRHT,600bar
2.1x20
1.8
UHPLCGuard,
1200bar,3/pk
2.1x5
1.8
959964-918 928975-902
728975-902
926975-902 926975-906 726975-902
728975-302
823750-903
926700-902 926700-906 726700-902
821725-901
www.agiLEnT.CoM/CHEM/LC
821725-903
243
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Rapid Resolution HT Columns for High Pressure use (Maximum Pressure: 600 bar, 9000 psi)
Hardware description
244
Size
(mm)
Particle
Size SB-C18
(µm) uSP L1
SB-C8
uSP L7
SB-Phenyl
uSP L11
SB-Cn
uSP L10
SB-aq
Rx-SiL
uSP L3
Bonus-RP
uSP L60
RapidResolution
HT,600bar
4.6x150
1.8
829975-902 829975-906 829975-912 829975-905 829975-914
RapidResolution
HT,600bar
4.6x100
1.8
828975-902 828975-906 828975-912 828975-905 828975-914 828975-901 828668-901
RapidResolution
HT,600bar
4.6x75
1.8
RapidResolution
HT,600bar
4.6x50
1.8
827975-902 827975-906 827975-912 827975-905 827975-914 827975-901 827668-901
RapidResolution
HT,600bar
4.6x30
1.8
824975-902 824975-906 824975-912 824975-905 824975-914
RapidResolution
HT,600bar
4.6x20
1.8
826975-902 826975-906
UHPLCGuard,
600bar,3/pk
4.6x5
1.8
820750-902 820750-904
SolventSaverHT, 3.0x150
600bar
1.8
829975-302 829975-306 829975-312 829975-305
SolventSaverHT, 3.0x100
600bar
1.8
828975-302 828975-306 828975-312 828975-305 828975-314 828975-301 828668-301
SolventSaverHT, 3.0x50
600bar
1.8
827975-302 827975-306 827975-312 827975-305 827975-314 827975-301 827668-301
SolventSaverHT, 3.0x30
600bar
1.8
824975-302 824975-306
SolventSaverHT, 3.0x20
600bar
1.8
826975-302 826975-306
UHPLCGuard,
1200bar,3/pk
3.0x5
1.8
823750-902 823750-904
NarrowBore
RRHT,600bar
2.1x150
1.8
820700-902 820700-906 820700-912 820700-905
NarrowBore
RRHT,600bar
2.1x100
1.8
828700-902 828700-906 828700-912 828700-905 828700-914 828700-901 828768-901
NarrowBore
RRHT,600bar
2.1x50
1.8
827700-902 827700-906 827700-912 827700-905 827700-914 827700-901 827768-901
NarrowBore
RRHT,600bar
2.1x30
1.8
824700-902 824700-906 824700-912 824700-905 824700-914
NarrowBore
RRHT,600bar
2.1x20
1.8
826700-902 826700-906
UHPLCGuard,
1200bar,3/pk
2.1x5
1.8
821725-902 821725-904
830975-906
830668-901
824975-305
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
Rapid Resolution HT Columns and Cartridges (Maximum Pressure: 400 bar, 6000 psi)
Particle
Size
Size (mm)
(µm)
Eclipse
XdB-C18
uSP L1
RapidResolutionHT,400bar
4.6x50
1.8
922975-902 922975-906 922975-902 822975-906 722975-902
RapidResolutionHT,3/pk,400bar
4.6x50
1.8
922975-932
922975-932
NarrowBoreRRHT,400bar
2.1x50
1.8
922700-902
922700-902
NarrowBoreRRHT,3/pk,400bar
2.1x50
1.8
922700-932
922700-932
Hardware description
Eclipse
XdB-C8
uSP L7
SB-C18
uSP L1
SB-C8
uSP L7
Extend-C18
uSP L1
Rapid Resolution HT Cartridges (require hardware kit 820555-901)
RapidResolutionHTCartridge
4.6x50
1.8
925975-902
825975-902
RapidResolutionHTCartridge,3/pk
4.6x50
1.8
925975-932
825975-932
RapidResolutionHTCartridge
2.1x50
1.8
925700-902
825700-902
RapidResolutionHTCartridge,3/pk
2.1x50
1.8
925700-932
825700-932
RapidResolutionHTCartridge
4.6x30
1.8
923975-902
823975-902
RapidResolutionHTCartridge,3/pk
4.6x30
1.8
923975-932
823975-932
RapidResolutionHTCartridge
2.1x30
1.8
923700-902
823700-902
RapidResolutionHTCartridge,3/pk
2.1x30
1.8
923700-932
823700-932
RapidResolutionHTCartridge
4.6x15
1.8
921975-902
821975-902
RapidResolutionHTCartridge,3/pk
4.6x15
1.8
921975-932
821975-932
RapidResolutionHTCartridge
2.1x15
1.8
921700-902
821700-902
RapidResolutionHTCartridge,3/pk
2.1x15
1.8
921700-932
821700-932
820555-901
820555-901
HardwareKitforRRandRRHTCartridges
www.agiLEnT.CoM/CHEM/LC
245
CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS
agilent Fast guards for uHPLC
UHPLCGuard,1200bar,821725-903
• HighperformanceguardcolumnsforFastLCcolumns
• Twoformats–oneforPoroshell120columns,stableto600bar,RRHDcolumns,1.8µm
(stableto1200bar),andRRHTcolumns,1.8µm(stableto600bar)
AgilentUHPLCGuardsarehighperformanceguardsdesignedbyAgilentforitsFastLCcolumnsfamilies.
AgilentUHPLCGuardsuseeasy-to-installhardwarethatfitsdirectlyontheendofthecolumn;noextra
hardwareisneeded.Theyaresoldinpackagesofthree.
AgilentUHPLCGuardsextendthelifetimeofanalyticalcolumnswithoutdiminishingperformance.
Fast guards for uHPLC
ZoRBaX RRHd columns, 1.8 µm (1200 bar), and ZoRBaX RRHT columns, 1.8 µm (600 bar)
Hardware
Eclipse
Plus C18
uSP L1
description
Size
(mm)
Particle Size
(µm)
Eclipse XdB-C18
uSP L1
SB-C18
uSP L1
SB-C8
uSP L7
UHPLCGuard,1200bar,3/pk
2.1x5
1.8
821725-901 821725-903
821725-902 821725-904
UHPLCGuard,1200bar,3/pk
3.0x5
1.8
823750-901 823750-903
823750-902 823750-904
UHPLCGuard,600bar,3/pk
4.6x5
1.8
820750-901 820750-903
820750-902 820750-904
description
Size
(mm)
Particle Size
(µm)
EC-C18
uSP L1
SB-C18
uSP L1
UHPLCGuard,600bar,3/pk
2.1x5
2.7
821725-911 821725-913
821725-912 821725-914
UHPLCGuard,600bar,3/pk
3.0x5
2.7
823750-911 823750-913
823750-912 823750-914
UHPLCGuard,600bar,3/pk
4.6x5
2.7
820750-911 820750-913
820750-912 820750-914
Poroshell 120 columns, 2.7 µm (600 bar)
Hardware
TiPS & TooLS
LearnaboutFastGuardsforUHPLC–aneasywaytoextendthelifeofyouranalytical
FastLCcolumnwithoutlosingperformance.www.agilent.com/chem/fastguardsvideo
246
EC-C8
uSP L7
Phenyl-Hexyl
uSP L11
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Other Columns for Reversed-Phase
Analytical HPLC
Achieve excellent peak shape and resolution every time –
leveraging the industry’s broadest selection
of reversed-phase columns
Whether you are using Fast LC or working with more conventional HPLC applications, Agilent’s LC family
offers you a range of phases and selectivities to help you perfect your separation.
The ZORBAX Family of phases scales readily to Fast LC columns in the Rapid Resolution High Throughput
(RRHT) and Rapid Resolution High Definition (RRHD) families and Poroshell 120 columns, see previous
section, page 227.
In this section, we’ll provide overviews of other key analytical columns from Agilent:
ZORBAX Rapid Resolution, 3.5 µm, configurations are an ideal choice for initial method development,
providing increased sample throughput for any application when compared to 5 µm columns.
ZORBAX Solvent Saver 3.0 mm id column configurations provide 60% mobile phase reduction over
4.6 mm id columns.
ZORBAX Eclipse Plus HPLC columns are designed to reliably produce superior peak shapes for basic
compounds, and are available across all ZORBAX column configurations.
More than 13 additional ZORBAX phases including StableBond, Eclipse PAH, Eclipse XDB,
ZORBAX Rx, Extend-C18, Bonus-Rx and Original ZORBAX columns – in total, more than
1400 configurations for reliable scalability and method transfer.
ZORBAX Method Development kits contain three columns for the price of two! Each as a different
bonded phase for optimizing selectivity.
ZORBAX Method Validation kits – choose as many columns as you need (or as few) to make method
validation easier and less expensive.
Pursuit, Pursuit XRs and Pursuit XRs Ultra columns provide alternate selectivities to
the ZORBAX family.
Polaris Columns provide polar-modified phases for routine polar applications.
Other Columns for Reversed-Phase Analytical HPLC.
www.AgiLEnt.COM/CHEM/LC
247
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse Plus
• The ideal column for method development – excellent results for a wide range of compounds
• High level of performance – peak shape, efficiency, resolution, and lifetime – with all sample types:
acids, bases and neutrals
• Superior reproducibility with more rigorous QA/QC testing
• Improved, patented silica manufacturing with start-to-finish product control
• Available in 1.8, 3.5, and 5 µm particle sizes for all analytical, high resolution, and fast LC analyses
Agilent ZORBAX Eclipse Plus columns provide the ultimate in performance for silica-based columns.
Peak shape is excellent for the most challenging basic compounds, improving efficiency and resolution with
these sample types. These results are achieved by improvements in the silica manufacturing and bonding
technology, which is completely controlled by Agilent.
ZORBAX Eclipse Plus Columns
Because of their high level of performance, Eclipse Plus columns are the ideal first choice for method
development of all samples. If you need to achieve fast method development and superior productivity, then
choose a column with high-resolution 1.8 µm particles. For standard methods, conventional 5 µm and Rapid
Resolution 3.5 µm columns are your best choice. With all particle sizes, easy method transfer is possible.
With more rigorous QA and QC testing, column lot-to-lot reproducibility is also improved, resulting in
long-term reliable results for all analyses.
Column Specifications
tiPS & tOOLS
Bonded Phase
Pore
Size
Surface
Area
temp.
Limits
ZORBAX Eclipse Plus C18
95Å
160 m2/g
60 °C
2.0-9.0
Double
9%
ZORBAX Eclipse Plus C8
95Å
160 m2/g
60 °C
2.0-9.0
Double
7%
2
pH Range* Endcapped
Carbon
Load
ZORBAX Eclipse PAH
95Å
160 m /g
60 °C
2.0-8.0
No
14%
ZORBAX Eclipse Plus
Phenyl-Hexyl
95Å
160 m2/g
60 °C
2.0-8.0
Double
9%
Specifications represent typical values only.
*Column lifetime will be reduced significantly at pH >7 and temperature >40 °C. At pH 6-9, highest column stability for
all silica based columns is obtained by operating at temperatures <40 °C and using lower buffer concentrations in range
of 0.01-0.02 M, especially with phosphate and carbonate buffers.
The EC-C18, EC-C8 and Phenyl-Hexyl
phases on Poroshell 120 are very
similar to Eclipse Plus C18,
Eclipse Plus C8 and Eclipse Plus
Phenyl-Hexyl phases.
Turn to page 228.
248
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse Plus: Best peak shape
in the industry without tailing
Column:
1
Eclipse Plus C18
959996-902
4.6 x 100 mm, 5 µm
Mobile Phase:
A: 60% Water
B: 40% Acetonitrile
Flow Rate:
1.0 mL/min
Temperature:
Ambient
Detector:
UV, 254 nm
Sample:
Pyridine, Phenol
1. Pyridine
2. Phenol
2
Eclipse Plus C18
Ace 5 C18
SunFire C18
XBridge C18
Discovery HS C18
Luna C18 (2)
Gemini C18
XTerra MS C18
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time (min)
LCEC001
Peak shape and efficiency are better
with ZORBAX Eclipse Plus
Column A:
XBridge C18, 4.6 x 150 mm, 5 µm
Column B:
Eclipse Plus C18
959993-902
4.6 x 150 mm, 5 µm
Mobile Phase:
A: 0.1% Formic acid
B: 0.1% Formic acid in ACN
Flow Rate:
1.0 mL/min
Gradient:
0.0 min 10% B
15 min 30% B
Temperature:
40 °C
A
1
N = 14000
Tf = 1.25
0
Detector:
UV, 254 nm
Sample:
Sulfonamides
2
B
1
2
5
4
4
6
Time (min)
6
8
10
N = 23000
Tf = 1.01
2
0
N = 41000
Tf = 1.13
3
2
4
1.
2.
3.
4.
5.
6.
Sulfanilamide
Sulfadiazine
Sulfathiazole
Sulfamerazine
Sulfmethazine
Sulfamethoxazole
12
N = 60000
Tf = 1.00
3
4
6
Time (min)
www.AgiLEnt.COM/CHEM/LC
6
5
8
10
12
LCEC003
249
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Eliminate tailing and maximize resolution
with Eclipse Plus Columns
Column A:
A
Eclipse Plus C18, 4.6 x 50 mm
Column B:
Competitive C18, 4.6 x 50 mm
Mobile Phase:
65% ACN:35% 25 mM phosphate buffer (pH 7.4)
Eclipse Plus C18, 4.6 x 50 mm
1
Tf = 1.20
1. Nortriptyline
2. Dipropylthalate
Superior peak shape and better selectivity with Eclipse Plus means more resolution,
easier quantitation and better results in your separations.
B
Competitive C18, 4.6 x 50 mm
1
Tf = 4.86
2
2
0
0.5
1
1.5
min
2
Eclipse Plus C18 vs. C8
Column A:
Eclipse Plus C18
4.6 x 50 mm, 5 µm
Column B:
Eclipse Plus C8
4.6 x 50 mm, 5 µm
Mobile Phase:
Water: acetonitrile (30:70)
Flow Rate:
2.0 mL/min
Temperature:
30 °C
0
Detector:
UV, 230 nm
B
Sample:
Lip balm extract in ACN
(melted at 100 °C, cooled and 0.45 µm filtered)
Less retention can save significant time – the C8 is a good choice here.
1. Oxybenzone
2. Internal Std.
3. Octylmethoxycinnamate
A
N: 8800
Pw : 0.014 min.
TF: 1.20
N: 11600
Pw : 0.047 min.
TF: 1.06
1
N: 5000
Pw : 0.019 min.
TF: 1.09
0
2
N: 6600
Pw : 0.037 min.
TF: 0.99
1
N: 11800
Pw : 0.093 min.
TF: 1.02
3
N: 6300
Pw : 0.061 min.
TF: 0.98
2
4
min
4
min
2.2 min.
3
Rapid analysis of an analgesic tablet,
selectivity differences at pH 2 and pH 7
Column:
Gradient:
Eclipse Plus C8
959946-906
4.6 x 50 mm, 5 µm
1.
2.
3.
4.
pH 2.7
3
1
10-60% B/3 min
pH 2.7: A: 0.1% Formic acid B: 0.1% fa in ACN
pH 7.0: A: 20 mM Na phosphate B: ACN
2
0
Sample:
Acetaminophen
Caffeine
Acetylsalicylic acid
Unknown
0.5
1
generic Excedrin tablet
1.5
4
2
2.5
3
3.5
min
pH 7.0
1
3
Both Eclipse Plus C18 and C8 can be used over a wide pH range to optimize
selectivity or analysis time.
250
2 min.
2
4
0
0.5
1
1.5
2
2.5
3
3.5
min
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Eclipse Plus C8 is less retentive than Eclipse Plus C18
Column A:
Eclipse Plus C8
959996-906
4.6 x 100 mm, 5 µm
Column B:
Eclipse Plus C18
959996-902
4.6 x 100 mm, 5 µm
Mobile Phase:
80% Methanol 8 mM (total) K2HPO4 pH 7
Flow Rate:
1.0 mL/min
Detector:
UV, 215 nm
Sample:
Amitriptyline 0.05 µg/µL (0.5 µL injection)
A
3.789
0
1
2
3
Tf=1.04
4
5
B
6
7
8
9
min
7
8
9
min
5.777
Tf=1.00
0
1
2
3
4
5
6
A C8 column is typically selected because it will retain less than a C18 column,
reducing analysis time.
The Eclipse Plus C8 column shows the same behavior with excellent peak shape
on difficult basic compounds.
fast and ultra-fast analysis of basic compounds
on Eclipse Plus
Column A:
Column B:
Eclipse Plus C18
959941-902
4.6 x 50 mm, 1.8 µm
A
O
Eclipse Plus C18
959993-902
4.6 x 150 mm, 5 µm
Mobile Phase:
A: 50% 8 mM K2HPO4, pH 7
B: 50% ACN
Flow Rate:
1.0 mL/min
Temperature:
Ambient
Detector:
UV, 254 nm
Sample:
Berberine, 0.4 mg/mL, 2 µL
N+
O
Cl-
1. Berberine
OCH3
Tf = 1.00
OCH3
0
0.5
1
1.5
2
2.5
min
B
Tf = 1.06
0
0.5
1
1.5
2
2.5
min
EP103
ZORBAX Eclipse Plus Columns
www.AgiLEnt.COM/CHEM/LC
251
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse Plus
Hardware
Description
Size (mm)
Analytical
4.6 x 250
Particle Size
(µm)
5
Eclipse
Plus C18
USP L1
Eclipse
Plus C8
USP L7
Eclipse Plus
Phenyl-Hexyl
USP L11
959990-902 959990-906 959990-912
Eclipse
PAH
USP L1
959990-918
Analytical
4.6 x 150
5
959993-902 959993-906 959993-912
959993-918
Analytical
4.6 x 100
5
959996-902 959996-906 959996-912
959996-918
Analytical
4.6 x 50
5
959946-902 959946-906
Rapid Resolution
4.6 x 150
3.5
959963-902 959963-906 959963-912
959963-918
Rapid Resolution
4.6 x 100
3.5
959961-902 959961-906 959961-912
959961-918
Rapid Resolution
4.6 x 75
3.5
959933-902 959933-906 959933-912
Rapid Resolution
4.6 x 50
3.5
959943-902 959943-906 959943-912
Rapid Resolution
4.6 x 30
3.5
959936-902 959936-906 959936-912
Rapid Resolution HT, 600 bar
4.6 x 100
1.8
959964-902 959964-906 959964-912
Rapid Resolution HT, 600 bar
4.6 x 75
1.8
959951-902
Rapid Resolution HT, 600 bar
4.6 x 50
1.8
959941-902 959941-906 959941-912
959941-918
Rapid Resolution HT, 600 bar
4.6 x 30
1.8
959931-902 959931-906 959931-912
959931-918
UHPLC Guard, 600 bar, 3/pk
4.6 x 5
1.8
820750-901
Solvent Saver
3.0 x 250
Solvent Saver
3.0 x 150
5
Solvent Saver Plus
3.0 x 150
3.5
959963-302 959963-306 959963-312
Solvent Saver Plus
3.0 x 100
3.5
959961-302 959961-306 959961-312
Solvent Saver RRHD, 1200 bar
3.0 x 150
1.8
959759-302 959759-306
Solvent Saver RRHD, 1200 bar
3.0 x 100
1.8
959758-302 959758-306
Solvent Saver RRHD, 1200 bar
3.0 x 50
1.8
959757-302 959757-306
Solvent Saver HT, 600 bar
3.0 x 100
1.8
959964-302 959964-306 959964-312
Solvent Saver HT, 600 bar
3.0 x 50
1.8
959941-302 959941-306 959941-312
5
959943-918
959964-918
959990-318
959993-302 959993-306
(Continued)
Agilent HILIC Plus uses the same manufacturing processes as the Eclipse Plus family.
See information about ZORBAX HILIC Plus on page 324.
252
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse Plus
Hardware
Description
Size (mm)
UHPLC Guard, 1200 bar, 3/pk
3.0 x 5
Particle Size
(µm)
1.8
Eclipse
Plus C18
USP L1
Eclipse
Plus C8
USP L7
Eclipse Plus
Phenyl-Hexyl
USP L11
Eclipse
PAH
USP L1
823750-901
Narrow Bore
2.1 x 250
5
Narrow Bore
2.1 x 150
5
959701-902 959701-906 959701-912
959790-918
Narrow Bore
2.1 x 50
5
959746-902 959746-906
Narrow Bore RR
2.1 x 150
3.5
959763-902 959763-906 959763-912
Narrow Bore RR
2.1 x 100
3.5
959793-902 959793-906 959793-912
Narrow Bore RR
2.1 x 50
3.5
959743-902 959743-906 959743-912
Narrow Bore RR
2.1 x 30
3.5
959733-902 959733-906 959733-912
Narrow Bore RRHD, 1200 bar
2.1 x 150
1.8
959759-902 959759-906
Narrow Bore RRHD, 1200 bar
2.1 x 100
1.8
959758-902 959758-906
Narrow Bore RRHD, 1200 bar
2.1 x 50
1.8
959757-902 959757-906
Narrow Bore RRHT, 600 bar
2.1 x 100
1.8
959764-902 959764-906 959764-912
959764-918
Narrow Bore RRHT, 600 bar
2.1 x 50
1.8
959741-902 959741-906 959741-912
959741-918
959701-918
959793-918
Narrow Bore RRHT, 600 bar
2.1 x 30
1.8
959731-902 959731-906 959731-912
UHPLC Guard, 1200 bar, 3/pk
2.1 x 5
1.8
821725-901
Guard Cartridges, 4/pk
4.6 x 12.5
5
820950-936 820950-937 820950-938
820950-939
Guard Cartridges, 4/pk
2.1 x 12.5
5
821125-936 821125-937 821125-938
821125-939
820999-901 820999-901 820999-901
820999-901
Guard Hardware Kit
www.AgiLEnt.COM/CHEM/LC
253
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse PAH
• High resolution separation of 16 PAHs in EPA Method 610
• Extensive range of particle sizes (1.8, 3.5 and 5 µm) and sizes for fast and high resolution separations
• Each batch of material is specifically tested with PAHs for maximum reproducibility under expected
ZORBAX Eclipse PAH Columns
operating conditions
• Excellent performance using the high quality, improved silica of Eclipse Plus columns
• Good for applications requiring "shape selectivity" or the separation of geometric isomers
Agilent ZORBAX Eclipse PAH columns are recommended for the separation of polycyclic aromatic
hydrocarbons. PAHs are considered priority pollutants and the analysis of these potentially carcinogenic
compounds in water, soil and food is of major importance. Eclipse PAH columns separate all 16 PAHS in
EPA method 610 quickly and with high resolution.
Column Specifications
Bonded Phase
Pore
Size
Surface
Area
temp.
Limits
Carbon
pH Range Endcapped Load
ZORBAX Eclipse PAH
95Å
160 m2/g
60 °C
2.0-8.0
No
14%
Specifications represent typical values only.
High resolution and fast analysis
on RRHt Eclipse PAH column
Column:
Eclipse PAH
959941-918
4.6 x 50 mm, 1.8 µm
Mobile Phase:
A: Water; B: Acetonitrile
Gradient:
Time (Min)
0.00
3.5
5.2
5.5
6.5
%B
40
100
100
40
40
mAU
2000
1750
1500
1250
Rs = 2.2 vs. 2.0 for
4.6 x 50 mm,
3.5 µm
Rs = 2.2
1000
750
Flow Rate:
2.0 mL/min
500
Temperature:
25 °C
250
Detector:
DAD 220, 4 nm No Ref. DAD Stop Time = 6.0 min
Stop Time = 7.0
0
1
2
3
Time (min)
4
5
LCEPlusPAH
254
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse PAH
Hardware Description
Analytical
Particle Size
Size (mm)
(µm)
Eclipse
PAH
USP L1
4.6 x 250
959990-918
5
Analytical
4.6 x 150
5
959993-918
Analytical
4.6 x 100
5
959996-918
Rapid Resolution
4.6 x 150
3.5
959963-918
Rapid Resolution
4.6 x 100
3.5
959961-918
Rapid Resolution
4.6 x 50
3.5
959943-918
Rapid Resolution HT, 600 bar
4.6 x 100
1.8
959964-918
Rapid Resolution HT, 600 bar
4.6 x 50
1.8
959941-918
Rapid Resolution HT, 600 bar
4.6 x 30
1.8
959931-918
Solvent Saver
3.0 x 250
5
959990-318
Narrow Bore
2.1 x 250
5
959790-918
Narrow Bore
2.1 x 150
5
959701-918
Narrow Bore RR
2.1 x 100
3.5
959793-918
Narrow Bore RRHT, 600 bar
2.1 x 100
1.8
959764-918
Narrow Bore RRHT, 600 bar
2.1 x 50
1.8
959741-918
Guard Cartridges, 4/pk
4.6 x 12.5
5
820950-939
Guard Cartridges, 4/pk
2.1 x 12.5
5
821125-939
Guard Hardware Kit
820999-901
www.AgiLEnt.COM/CHEM/LC
255
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse XDB
CH3
O
Si
CH3
CH3
O
Si
CH3
CH3
CH3
O
Si
CH3
CH3
O
Si
CH3
CH3
CH3
O
Si
CH3
eXtra Densely Bonded and Double Endcapped
Eclipse XDB Bonded Phase
• Four selectivity choices for method development optimization
• Good peak shape for basic, acidic and neutral compounds
• High performance over a wide pH range – pH 2-9
• Particle sizes from 1.8 to 7 µm
• Long lifetime with eXtra Dense Bonding and double endcapping
Agilent ZORBAX Eclipse XDB columns – C18, C8, Phenyl and CN – provide four bonded phase choices for
method development optimization. These columns provide good peak shape over a wide pH range (2-9)
for additional method development flexibility with one family of columns. Eclipse XDB columns can be used
for method development at low pH (2-3) and the same column can be used for method development in the
mid pH (6-8) region. In the mid pH region residual silanols are more active and tailing interactions are more
likely. To overcome these interactions, Eclipse XDB columns are eXtra Densely Bonded and double
endcapped through a proprietary process to cover as many active silanols as possible. The result is superior
peak shape of basic compounds from pH 2-9. Eclipse XDB columns are available in 1.8, 3.5, 5 and 7 µm
particle sizes for high speed, high resolution, analytical and prep scale separations.
Column Specifications
Bonded Phase
Pore
temp.
pH
Carbon
Size Surface Area Limits Range* Endcapped Load
ZORBAX Eclipse XDB-C18
80Å
180 m2/g
2
60 °C
2.0-9.0
Double
10%
ZORBAX Eclipse XDB-C8
80Å
180 m /g
60 °C
2.0-9.0
Double
7.6%
ZORBAX Eclipse XDB-Phenyl
80Å
180 m2/g
60 °C
2.0-9.0
Double
7.2%
60 °C
2.0-8.0
Double
4.3%
ZORBAX Eclipse XDB-CN
80Å
2
180 m /g
Specifications represent typical values only
*Eclipse XDB columns are designed for operation over a wide pH range.At pH 6-9, highest columns stability for all silica
based columns is achieved by operating at temperatures <40 °C and using low buffer concentrations in the range
of 0.01-0.02 M.
tiPS & tOOLS
Poroshell 120 EC-CN is very similar to ZORBAX XDB-CN. Page 228
256
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
good peak shape over a wide pH range
with ZORBAX Eclipse XDB
Column:
Mobile Phase:
Eclipse XDB-C8
993967-906
4.6 x 150 mm, 5 µm
Sample:
A
A: pH 3.0 75% 25 mM phosphate buffer 25% ACN
B: pH 7.0 90% 20 mM phosphate 10% ACN
Flow Rate:
1.5 mL/min
Temperature:
40 °C
B
2
A:
1. Maleate
2. Doxylamine
3. Chlorpheniramine
4. Triprolidine
5. Diphenhydramine
B:
1. Procainamide
2. N-acetylprocainamide
3. N-propionylprocainamide
2
3
1
3
4
5
1
ZORBAX Eclipse XDB columns provide good peak shape over a wide pH
range and are an excellent choice for method development from pH 2-9.
0
2.5
5.0
Time (min)
7.5
0
2.5
5.0
Time (min)
7.5
LCEC004
Column stability testing at pH 3 and 60 °C
ZORBAX SB-C8
883975-906
4.6 x 150 mm, 5 µm
Column:
Eclipse XDB-C8
993967-906
4.6 x 150 mm, 5 µm
Mobile Phase:
Purge Conditions:
70% 50 mM NaAc-HCl, pH 3.0
30% ACN
Retention Test Conditions:
65% Methanol
35% Water
Flow Rate:
1.0 mL/min
Temperature:
60 °C
Sample:
Tricyclic antidepressants
7.0
6.5
K of Amitriptyline
Column:
6.0
ZORBAX SB-C8
5.5
ZORBAX Eclipse XDB-C8
Competitive C8
5.0
0
2
4
6
8
Column Volumes of Mobile Phase (X1000)
10
12
LCEC005
Eclipse XDB columns are stable over a wide pH range. At low pH an Eclipse endcapped column is extremely stable and shows equivalent stability to a non-endcapped column,
SB-C8, at pH 3. The columns were purged with a pH 3 mobile phase at 60 °C. Then they were tested with a strongly basic compound to determine if the endcapping or bonded
phase had been hydrolyzed from the silica surface. The Eclipse XDB column was very stable, as shown by the consistency of the retention of amitriptyline over the 12,000 column
volumes of the test. Another endcapped column shows less stability under these same conditions.
www.AgiLEnt.COM/CHEM/LC
257
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Column stability testing at pH 7.0
Column A:
Competitive C8
SiL-type
After 1826 column volumes
Column B:
Eclipse XDB-C8
993967-906
4.6 x 150 mm, 5 µm
Sol-type
After 1843 column volumes
Mobile Phase:
60% ACN
40% 250 mM Phosphate Buffer, pH 7.0
Flow Rate:
1.5 mL/min
Temperature:
60 °C
Sample:
Tricyclic antidepressants
A
B
1
5
1.
2.
3.
4.
5.
5
1
2, 3
4
2
Uracil
Nortriptyline
Doxepin
Amitriptyline
Trimipramine
3
4
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (min)
0
1
2
3
4
5
Time (min)
6
7
8
LCEC006
Double endcapping, dense bonding and the durable Rx-Sil particles (sol-type) combine to provide long lifetime at pH 7 when compared to single endcapped sil-gel columns
used here. The conditions used for this test – high temperature (60 °C) and high salt concentration (250 mM), accelerate the dissolution of silica, causing premature failure of
the sil-gel type column.
Selectivity changes for basic compounds
with Eclipse XDB and StableBond
Column A:
Eclipse XDB-C8
966967-906
4.6 x 75 mm, 3.5 µm
Column B:
ZORBAX Rx/SB-C8
866953-906
4.6 x 75 mm, 3.5 µm
Mobile Phase:
70% 25 mM NaH2PO4, pH 3.0
30% Methanol
Flow Rate:
1.0 mL/min
Temperature:
35 °C
A
B
2
2
3
3
1
1
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (min)
1. Barbital
2. Sulfamethoxazole
3. Caffeine
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (min)
LCEC008
Eclipse XDB and StableBond columns are based on the same silica but have different bonding and endcapping. Therefore, they can have very different selectivity for the same
sample under the same conditions, as this example shows.
258
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Optimize separations with Eclipse XDB
selectivity options
Column A:
Column B:
Column C:
Mobile Phase:
A
1
Eclipse XDB-Phenyl
963967-912
4.6 x 150 mm, 3.5 µm
2
3
0
B
Eclipse XDB-C8
963967-906
4.6 x 150 mm, 3.5 µm
1.0 mL/min
35 °C
Sample:
Sunscreens
4
6
8
10
12
14
16
10
12
14
16
1
3
0
C
15% H2O:85% MeOH
Flow Rate:
2
2
Eclipse XDB-C18
963967-902
4.6 x 150 mm, 3.5 µm
Temperature:
1. Oxybenzone
2. Padimate-O
3. Ethylhexylsalicylate
2
4
6
8
1
2
3
0
2
4
6
8
10
Time (min)
12
14
16
LCEC007
This separation of sunscreens on all three Eclipse XDB bonded phases – C18, C8 and Phenyl – shows that different bonded phases can be used to optimize a separation. While all
three bonded phases provide an adequate separation, the Eclipse XDB-Phenyl provides a different peak elution order and a much shorter overall analysis time. All three bonded
phases also provide excellent peak shape with no mobile phase additives.
Selectivity for urea pesticides
Column A:
Column B:
Eclipse XDB-C18
993967-902
4.6 x 150 mm, 5 µm
Eclipse XDB-Cn
993967-905
4.6 x 150 mm, 5 µm
A
2
3
5
6
0
10
B
Column C:
A. 60:40 MeOH:Water
B. 60:40 MeOH:Water
C. 77:23 MeOH:Water
Flow Rate:
1.0 mL/min
34
1
0
2
C
2
1
25 °C
Sample:
Urea pesticides
0
30
Time (min)
2
5
6
4
6
Time (min)
34
Temperature:
20
40
50
2
Eclipse XDB-C18
993967-902
4.6 x 150 mm, 5 µm
Mobile Phase:
4
1
8
10
8
10
1.
2.
3.
4.
5.
6.
Fenuron
Monuron
MonoLinuron
Diuron
Linuron
Pencycuron
5
6
4
6
Time (min)
LCEC010
The Eclipse XDB-CN column reduces retention time and provides good selectivity for Urea pesticides when compared to an Eclipse XDB-C18 column.
www.AgiLEnt.COM/CHEM/LC
259
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse XDB
Hardware Description
Eclipse
XDB-C18
Size (mm) Particle Size (µm) USP L1
Eclipse
XDB-C8
USP L7
9.4 x 250
990967-206
Eclipse
XDB-Phenyl
USP L11
Eclipse
XDB-Cn
USP L10
Standard Columns (no special hardware required)
Semi-Preparative
5
990967-202
Analytical
4.6 x 250
5
990967-902
990967-906
990967-912
990967-905
Analytical
4.6 x 150
5
993967-902
993967-906
993967-912
993967-905
Analytical
4.6 x 50
5
946975-902
946975-906
Rapid Resolution
4.6 x 150
3.5
963967-902
963967-906
963967-912
963967-905
966967-905
Rapid Resolution
4.6 x 100
3.5
961967-902
961967-906
Rapid Resolution
4.6 x 75
3.5
966967-902
966967-906
966967-912
961967-905
Rapid Resolution
4.6 x 50
3.5
935967-902
935967-906
935967-912
Rapid Resolution
4.6 x 30
3.5
934967-902
934967-906
Rapid Resolution
4.6 x 20
3.5
932967-902
932967-906
UHPLC Guard, 1200 bar, 3/pk
4.6 x 5
1.8
820750-903
Rapid Resolution HT, 600 bar
4.6 x 100
1.8
928975-902
928975-906
Rapid Resolution HT, 600 bar
4.6 x 50
1.8
927975-902
927975-906
Rapid Resolution HT, 600 bar
4.6 x 30
1.8
924975-902
924975-906
Rapid Resolution HT, 600 bar
4.6 x 20
1.8
926975-902
926975-906
Solvent Saver
3.0 x 250
5
990967-302
990967-306
990967-312
990967-305
Solvent Saver
3.0 x 150
5
993967-302
993967-306
993967-312
993967-305
Solvent Saver Plus
3.0 x 150
3.5
963954-302
963954-306
963954-312
963954-305
Solvent Saver Plus
3.0 x 100
3.5
961967-302
961967-306
961967-312
Solvent Saver Plus
3.0 x 75
3.5
966954-302
Solvent Saver RRHD, 1200 bar
3.0 x 150
1.8
981759-302
Solvent Saver RRHD, 1200 bar
3.0 x 100
1.8
981758-302
Solvent Saver RRHD, 1200 bar
3.0 x 50
1.8
981757-302
Solvent Saver HT, 600 bar
3.0 x 100
1.8
928975-302
928975-306
Solvent Saver HT, 600 bar
3.0 x 50
1.8
927975-302
927975-306
Solvent Saver HT, 600 bar
3.0 x 30
1.8
924975-302
924975-306
926975-306
Solvent Saver HT, 600 bar
3.0 x 20
1.8
926975-302
UHPLC Guard, 1200 bar, 3/pk
3.0 x 5
1.8
823750-903
Narrow Bore
2.1 x 150
5
993700-902
993700-906
993700-912
993700-905
Narrow Bore
2.1 x 50
5
960967-902
960967-906
960967-912
960967-905
Narrow Bore RR
2.1 x 150
3.5
930990-902
930990-906
Unless indicated, column pressure limit is 400 bar.
*These columns are packed with Eclipse XDB-C18, 5 µm.
(Continued)
260
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse XDB
Eclipse
XDB-C18
Size (mm) Particle Size (µm) USP L1
Eclipse
XDB-C8
USP L7
Narrow Bore RR
2.1 x 100
3.5
961753-902
961753-906
Narrow Bore RR
2.1 x 75
3.5
966735-902
Narrow Bore RR
2.1 x 50
3.5
971700-902
971700-906
Narrow Bore RR
2.1 x 30
3.5
974700-902
974700-906
Narrow Bore RR
2.1 x 20
3.5
972700-902
972700-906
Narrow Bore RRHD, 1200 bar
2.1 x 150
1.8
981759-902
Narrow Bore RRHD, 1200 bar
2.1 x 100
1.8
981758-902
Narrow Bore RRHD, 1200 bar
2.1 x 50
1.8
981757-902
Narrow Bore RRHT, 600 bar
2.1 x 100
1.8
928700-902
928700-906
Narrow Bore RRHT, 600 bar
2.1 x 50
1.8
927700-902
927700-906
Narrow Bore RRHT, 600 bar
2.1 x 30
1.8
924700-902
924700-906
Narrow Bore RRHT, 600 bar
2.1 x 20
1.8
926700-902
926700-906
UHPLC Guard, 1200 bar, 3/pk
2.1 x 5
1.8
821725-903
MicroBore RR
1.0 x 150
3.5
963600-902
Hardware Description
Eclipse
XDB-Phenyl
USP L11
Eclipse
XDB-Cn
USP L10
Standard Columns (no special hardware required)
961753-905
963600-906
MicroBore RR
1.0 x 50
3.5
965600-902
965600-906
MicroBore RR
1.0 x 30
3.5
961600-902
961600-906
5185-5921
MicroBore Guard, 3/pk
1.0 x 17
5
5185-5921
Guard Cartridge
9.4 x 15
5
820675-112* 820675-112* 820675-112*
820675-112*
Guard Cartridges, 4/pk
4.6 x 12.5
5
820950-925
820950-926
820950-927
820950-935
Guard Cartridges, 4/pk
2.1 x 12.5
5
821125-926
821125-926
821125-926
821125-935
Guard Hardware Kit
840140-901
840140-901
840140-901
840140-901
Guard Hardware Kit
820999-901
820999-901
820999-901
820999-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 250
7
977250-102
977250-106
PrepHT Cartridge
21.2 x 150
7
977150-102
977150-106
PrepHT Cartridge
21.2 x 150
5
970150-902
970150-906
PrepHT Cartridge
21.2 x 100
5
970100-902
970100-906
PrepHT Cartridge
21.2 x 50
5
970050-902
970050-906
PrepHT Guard Cartridge
17.0 x 7.5
5
820212-925
820212-926
Guard Cartridge Hardware
820444-901
820444-901
PrepHT Endfittings, 2/pk
820400-901
820400-901
Unless indicated, column pressure limit is 400 bar.
*These columns are packed with Eclipse XDB-C18, 5 µm.
www.AgiLEnt.COM/CHEM/LC
261
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse XDB
Hardware Description
Eclipse
XDB-C18
Size (mm) Particle Size (µm) USP L1
Eclipse
XDB-C8
USP L7
Agilent Cartridge Columns (require hardware kit 5021-1845)
Analytical
4.6 x 250
5
7995118-585 7995108-585
Analytical
4.6 x 150
5
7995118-595 7995108-595
Rapid Resolution
4.6 x 75
3.5
7995118-344 7995108-344
Solvent Saver Plus
3.0 x 75
3.5
Guard Cartridges, 10/pk
4.0 x 4
5
Cartridge Holder
7995230-344
7995118-504 7995118-504
5021-1845
5021-1845
922975-906
Standard Columns (no special hardware required)
Rapid Resolution HT, 400 bar
4.6 x 50
1.8
922975-902
Rapid Resolution HT, 3/pk, 400 bar
4.6 x 50
1.8
922975-932
Narrow Bore RRHT, 400 bar
2.1 x 50
1.8
922700-902
Narrow Bore RRHT, 3/pk, 400 bar
2.1 x 50
1.8
922700-932
4.6 x 30
3.5
933975-902
933975-906
Rapid Resolution Cartridge, 3/pk
4.6 x 30
3.5
933975-932
933975-936
Rapid Resolution Cartridge
4.6 x 15
3.5
931975-902
931975-906
Rapid Resolution Cartridge, 3/pk
4.6 x 15
3.5
931975-932
931975-936
Rapid Resolution Cartridge
2.1 x 30
3.5
973700-902
973700-906
Rapid Resolution Cartridge, 3/pk
2.1 x 30
3.5
973700-932
973700-936
Rapid Resolution Cartridge
2.1 x 15
3.5
975700-902
975700-906
Rapid Resolution Cartridge, 3/pk
2.1 x 15
3.5
975700-932
975700-936
Rapid Resolution HT Cartridge, 400 bar
4.6 x 50
1.8
925975-902
Rapid Resolution HT Cartridge, 3/pk, 400 bar
4.6 x 50
1.8
925975-932
Rapid Resolution HT Cartridge, 400 bar
4.6 x 30
1.8
923975-902
Rapid Resolution HT Cartridge, 3/pk, 400 bar
4.6 x 30
1.8
923975-932
Rapid Resolution Ht Cartridges (require hardware kit 820555-901)
Rapid Resolution Cartridge
(Continued)
262
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Eclipse XDB
Eclipse
XDB-C18
Size (mm) Particle Size (µm) USP L1
Hardware Description
Eclipse
XDB-C8
USP L7
Rapid Resolution Ht Cartridges (require hardware kit 820555-901)
Rapid Resolution HT Cartridge, 400 bar
4.6 x 15
1.8
921975-902
Rapid Resolution HT Cartridge, 3/pk, 400 bar
4.6 x 15
1.8
921975-932
Rapid Resolution HT Cartridge, 400 bar
2.1 x 50
1.8
925700-902
Rapid Resolution HT Cartridge, 3/pk, 400 bar
2.1 x 50
1.8
925700-932
Rapid Resolution HT Cartridge, 400 bar
2.1 x 30
1.8
923700-902
Rapid Resolution HT Cartridge, 3/pk, 400 bar
2.1 x 30
1.8
923700-932
Rapid Resolution HT Cartridge, 400 bar
2.1 x 15
1.8
921700-902
Rapid Resolution HT Cartridge, 3/pk, 400 bar
2.1 x 15
1.8
Hardware Kit for RR and RRHT Cartridges
921700-932
820555-901
Capillary glass-lined Columns
Capillary
0.5 x 250
5
5064-8286
Capillary
0.5 x 150
5
5064-8287
Capillary RR
0.5 x 150
3.5
5064-8288
Capillary RR
0.5 x 35
3.5
5064-8298
Capillary
0.3 x 250
5
5064-8269
Capillary
0.3 x 150
5
5064-8291
Capillary RR
0.3 x 150
3.5
5064-8271
Capillary
0.5 x 35
5
5064-8296
Capillary
0.3 x 35
5
5064-8297
www.AgiLEnt.COM/CHEM/LC
263
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX 80Å StableBond
R
O
Si
R1
R
and SB-Aq
OH
• High purity (Type B) silica for good peak shape
R
O
Si
R1
R
OH
R
O
• Longest column lifetime and best reproducibility for low pH separations – down to pH 1
• Patented stable column chemistry allows use at high temperature and low pH without degradation
• Six different bonded phases provide broad selectivity – SB-C18, SB-C8, SB-CN, SB-Phenyl, SB-C3,
Si
Agilent ZORBAX StableBond columns use patented, unique, nonfunctional silanes with bulky diisobutyl
(SB-C18) or diisopropyl (SB-C8, SB-C3, SB-Phenyl, SB-CN, and SB-Aq) side chain groups that sterically
protect the key siloxane bond to the silica surface from hydrolytic attack at low pH. StableBond packing
materials are not endcapped in order to provide exceptional stability and to maximize lifetime and
reproducibility under acidic mobile phase conditions. The high purity, low acidity silica provides excellent
peak shape with acidic, basic and neutral compounds making StableBond columns an excellent choice for
low pH method development. ZORBAX StableBond columns are compatible with all common mobile
phases, including very high aqueous mobile phases.
R1
Column Specifications
R
Sterically Protected StableBond Bonded Phase
Bonded Phase
Pore
Size
Surface
Area
temp.
Carbon
Limits* pH Range* Endcapped Load
ZORBAX SB-C18
80Å
180 m2/g
90 °C
0.8-8.0
No
10%
ZORBAX SB-C8
80Å
180 m2/g
80 °C
1.0-8.0
No
5.5%
ZORBAX SB-C3
80Å
180 m2/g
80 °C
1.0-8.0
No
4%
ZORBAX SB-Phenyl
80Å
180 m2/g
80 °C
1.0-8.0
No
5.5%
ZORBAX SB-CN
80Å
180 m2/g
80 °C
1.0-8.0
No
4%
ZORBAX SB-Aq
80Å
180 m2/g
80 °C
1.0-8.0
No
proprietary
Specifications represent typical values only
*StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-based columns
is obtained by operating at temperatures <40 °C and using lower buffer concentrations in the range of 0.01-0.02 M. At
mid-range pH, Eclipse Plus, Eclipse XDB and Bonus-RP are recommended.
tiPS & tOOLS
ZORBAX StableBond SB-C18, SB-C8 and SB-Aq phases are also available on Poroshell 120.
Turn to page 228
264
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
StableBond SB-C18 shows excellent stability
at low pH and high temperature (pH 0.8, 90 °C)
ZORBAX SB-C18
883975-902
4.6 x 150 mm, 5 µm
Column:
ZORBAX Rx-C18
883967-902
4.6 x 150 mm, 5 µm
Mobile Phase:
50% Methanol/50% Water
with 1.0% TFA
Test Solute: Toluene
Temperature:
90 °C
As an indicator of column breakdown, retention time
of toluene was measured after purging the column
with mobile phase. Only the StableBond SB-C18 is
unchanged after three working months of use under
these very low pH (0.8) and high temperature (90 °C)
conditions. ZORBAX Rx-C18 also provides a stable
matrix, and can be used as an alternative selectivity
to StableBond SB-C18.
StableBond SB-C18
(Diisobutyl-C18)
Relative Retention
Column:
1.0
ZORBAX Rx-C18
(Dimethyl-C18)
0.8
Competitor A-C18
Competitor B-C18
0.6
Competitor C-C18
0.4
Competitor D-C18
0.2
Competitor E-C18
0.0
0
5,000
10,000
15,000
20,000
25,000
30,000
Column Volumes of Mobile Phase Purge
LCSB001
Shorter chain ZORBAX SB-Cn is also stable
at low pH (pH 2.0, 50 °C)
ZORBAX SB-Cn
883975-905
4.6 x 150 mm, 5 µm
Mobile Phase:
0.1% TFA, pH 2:ACN
Flow Rate:
1 mL/min
Gradient:
0-100% ACN
Temperature:
50 °C
Sample:
1-phenylheptane @ 50% AC/50% water
with 0.1% TFA
SB-CN
100
80
% k Remaining
Column:
60
40
Si-(Me)2PrCN
20
ZORBAX StableBond SB-CN and other short chain StableBond bonded
phases are also exceptionally stable at low pH. Conventional dimethyl
CN and similar bonded phases lack this stability.
0
0
1,000
2,000
3,000
4,000
5,000
6,000
Column Volumes
LCSB002
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265
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
SB-Cn optimizes retention and resolution
Column A:
Column B:
ZORBAX SB-C18
866953-902
4.6 x 75 mm, 3.5 µm
A
B
2
ZORBAX SB-Cn
866953-905
4.6 x 75 mm, 3.5 µm
5
1
4
2
3
4
5
Mobile Phase:
30% ACN
70% 25mM NaH2PO4, pH 2.5
Flow Rate:
1.0 mL/min
Temperature:
35 °C
1
0
5
10
15
Time (min)
20
25
0
1.
2.
3.
4.
5.
Estriol
Daidzen
Quercetin
Genistein
Diethylstilbestrol
3
2
4
6
8 10
Time (min)
12
LCSB003
The SB-CN column is used here to reduce analysis time by 50%. The retention of the most hydrophobic analyte is cut in half. At the same time, retention of the more polar, early
eluting peaks increases slightly.
five different bonded phases provide selectivity options
Column A:
ZORBAX SB-C18
883975-902
4.6 x 150 mm, 5 µm
A
3
Sample:
1
5
4
2
Column B:
ZORBAX SB-C8
883975-906
4.6 x 150 mm, 5 µm
Column C:
ZORBAX SB-C3
883975-909
4.6 x 150 mm, 5 µm
Column D:
ZORBAX SB-Phenyl
883975-912
4.6 x 150 mm, 5 µm
Column E:
ZORBAX SB-Cn
883975-905
4.6 x 150 mm, 5 µm
Mobile Phase:
0-100% B in 18.8 min
A: 50 mM NaH2PO4,
pH 2.5 in 95% H2O / 5% ACN
B: 50 mM NaH2PO4,
pH 2.5 in 47% H2O / 53% ACN
Flow Rate:
1.0 mL/min
Temperature:
26 °C
Detector:
254 nm
SB-C3 is just one of the five different StableBond selectivity choices. In this example,
optimum resolution is obtained with SB-C3. All are based on the same high purity Rx-SIL.
Selectivity changes are therefore dependent only on the bonded phases, making method
development more reliable.
3
B
1
5
4
2
3
C
1
5
4
2
3
D
1
5
4
2
E
13
5
4
2
0
5
10
15
Time (min)
LCSB004
266
1. Procaine
2. Lidocaine
3. d-Cinchonine
4. Butacaine
5. Tetracaine
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX 80Å StableBond
Hardware Description
Size (mm)
Particle
Size (µm)
SB-C18
USP L1
SB-C8
USP L7
SB-Cn
USP L10
SB-C3
USP L56
SB-Phenyl
USP L11
SB-Aq
Standard Columns (no special hardware required)
Semi-Preparative
9.4 x 250
5
880975-202 880967-201 880975-205 880975-209 880975-212
Semi-Preparative
9.4 x 150
5
883975-202
Semi-Preparative
9.4 x 100
5
884975-202
Semi-Preparative
9.4 x 50
5
846975-202
Analytical
4.6 x 250
5
880975-902 880975-906 880975-905 880975-909 880975-912 880975-914
Analytical
4.6 x 150
5
883975-902 883975-906 883975-905 883975-909 883975-912 883975-914
Analytical
4.6 x 50
5
846975-902 846975-906
Rapid Resolution
4.6 x 250
3.5
884950-567
Rapid Resolution
4.6 x 150
3.5
863953-902 863953-906 863953-905
846975-914
863953-912 863953-914
Rapid Resolution
4.6 x 100
3.5
861953-902 861953-906 861953-905
861953-912 861953-914
Rapid Resolution
4.6 x 75
3.5
866953-902 866953-906 866953-905
866953-912 866953-914
835975-912 835975-914
Rapid Resolution
4.6 x 50
3.5
835975-902 835975-906 835975-905
Rapid Resolution
4.6 x 30
3.5
834975-902 834975-906
Rapid Resolution
4.6 x 20
3.5
832975-902 832975-906
Rapid Resolution HT, 600 bar
4.6 x 150
1.8
829975-902 829975-906 829975-905
829975-912 829975-914
Rapid Resolution HT, 600 bar
4.6 x 100
1.8
828975-902 828975-906 828975-905
828975-912 828975-914
Rapid Resolution HT, 600 bar
4.6 x 75
1.8
830975-906
Rapid Resolution HT, 600 bar
4.6 x 50
1.8
827975-902 827975-906 827975-905
827975-912 827975-914
824975-912 824975-914
Rapid Resolution HT, 600 bar
4.6 x 30
1.8
824975-902 824975-906 824975-905
Rapid Resolution HT, 600 bar
4.6 x 20
1.8
826975-902 826975-906
UHPLC Guard, 600 bar, 3/pk
4.6 x 5
1.8
820750-902 820750-904
Solvent Saver
3.0 x 250
5
880975-302 880975-306 880975-305 880975-309 880975-312 880975-314
Solvent Saver
3.0 x 150
5
883975-302 883975-306 883975-305 883975-309 883975-312 883975-314
Solvent Saver Plus
3.0 x 150
3.5
863954-302 863954-306 863954-305
Solvent Saver Plus
3.0 x 100
3.5
861954-302 861954-306 861954-305 861954-309 861954-312 861954-314
863954-312 863954-314
Solvent Saver Plus
3.0 x 75
3.5
866953-302
Unless indicated, column pressure limit is 400 bar.
(Continued)
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267
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX 80Å StableBond
Hardware Description
Size (mm)
Particle
Size (µm)
SB-C18
USP L1
SB-C8
USP L7
SB-Cn
USP L10
SB-C3
USP L56
SB-Phenyl
USP L11
SB-Aq
Standard Columns (no special hardware required)
Solvent Saver RRHD,
1200 bar
3.0 x 150
1.8
859700-302 859700-306
Solvent Saver RRHD,
1200 bar
3.0 x 100
1.8
858700-302 858700-306 858700-305
858700-312
Solvent Saver RRHD,
1200 bar
3.0 x 50
1.8
857700-302 857700-306 857700-305
857700-312
Solvent Saver HT, 600 bar
3.0 x 150
1.8
829975-302 829975-306 829975-305
829975-312
Solvent Saver HT, 600 bar
3.0 x 100
1.8
828975-302 828975-306 828975-305 828975-309 828975-312 828975-314
Solvent Saver HT, 600 bar
3.0 x 50
1.8
827975-302 827975-306 827975-305
Solvent Saver HT, 600 bar
3.0 x 30
1.8
824975-302 824975-306 824975-305
Solvent Saver HT, 600 bar
3.0 x 20
1.8
826975-302 826975-306
1.8
823750-902 823750-904
UHPLC Guard, 1200 bar, 3/pk 3.0 x 5
Narrow Bore
2.1 x 150
5
Narrow Bore
2.1 x 50
5
Narrow Bore RR
2.1 x 150
3.5
827975-312 827975-314
883700-922 883700-906 883700-905 883700-909 883700-912
860975-902 860975-906 860975-905 860975-909 860975-912 860975-914
830990-902 830990-906
830990-914
Narrow Bore RR
2.1 x 100
3.5
861753-902 861753-906 861753-905
Narrow Bore RR
2.1 x 75
3.5
866735-902
861753-912 861753-914
Narrow Bore RR
2.1 x 50
3.5
871700-902 871700-906
Narrow Bore RR
2.1 x 30
3.5
874700-902 874700-906
Narrow Bore RR
2.1 x 20
3.5
872700-902 872700-906
Narrow Bore RRHD, 1200 bar 2.1 x 150
1.8
859700-902 859700-906 859700-905
859700-912
Narrow Bore RRHD, 1200 bar 2.1 x 100
1.8
858700-902 858700-906 858700-905
858700-912
Narrow Bore RRHD, 1200 bar 2.1 x 50
1.8
857700-902 857700-906 857700-905
857700-912
871700-914
Unless indicated, column pressure limit is 400 bar.
(Continued)
268
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX 80Å StableBond
Hardware Description
Size (mm)
Particle
Size (µm)
SB-C18
USP L1
SB-C8
USP L7
SB-Cn
USP L10
SB-C3
USP L56
SB-Phenyl
USP L11
SB-Aq
Standard Columns (no special hardware required)
Narrow Bore RRHT, 600 bar
2.1 x 150
1.8
820700-902 820700-906 820700-905
820700-912
Narrow Bore RRHT, 600 bar
2.1 x 100
1.8
828700-902 828700-906 828700-905
828700-912 828700-914
Narrow Bore RRHT, 600 bar
2.1 x 50
1.8
827700-902 827700-906 827700-905
827700-912 827700-914
Narrow Bore RRHT, 600 bar
2.1 x 30
1.8
824700-902 824700-906 824700-905
824700-912 824700-914
Narrow Bore RRHT, 600 bar
2.1 x 20
1.8
826700-902 826700-906
UHPLC Guard, 1200 bar, 3/pk 2.1 x 5
1.8
821725-902 821725-904
MicroBore RR
1.0 x 150
3.5
863600-902 863600-906 863600-905
MicroBore RR
1.0 x 50
3.5
865600-902 865600-906
MicroBore RR
1.0 x 30
3.5
861600-902 861600-906
MicroBore Guard, 3/pk
1.0 x 17
5
5185-5920 5185-5920
Guard Cartridge, 2/pk
9.4 x 15
7
820675-115 820675-115 820675-124 820675-124 820675-115
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-920 820950-915 820950-916 820950-922 820950-917 820950-933
Guard Cartridge, 4/pk
2.1 x 12.5
5
821125-915 821125-915 821125-924 821125-924 821125-915 821125-933
Guard Hardware Kit
9.4 x 15
0
840140-901 840140-901 840140-901 840140-901 840140-901
Guard Hardware Kit
820999-901 820999-901 820999-901 820999-901 820999-901 820999-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 250
7
877250-102 877250-106 877250-105
PrepHT Cartridge
21.2 x 150
7
877150-102 877150-106
877150-114
PrepHT Cartridge
21.2 x 150
5
870150-902 870150-906
870150-914
PrepHT Cartridge
21.2 x 100
5
870100-902 870100-906
870100-914
PrepHT Cartridge
21.2 x 50
5
870050-902 870050-906
870050-914
PrepHT Guard Cartridge, 2/pk 17.0 x 7.5
5
820212-920 820212-915 820212-915
877250-112 877250-114
820212-915 820212-933
Guard Cartridge Hardware
820444-901 820444-901 820444-901 820444-901 820444-901 820444-901
PrepHT Endfittings, 2/pk
820400-901 820400-901 820400-901 820400-901 820400-901 820400-901
Unless indicated, column pressure limit is 400 bar.
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269
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX 80Å StableBond
Hardware Description
SB-C18
Size (mm) Particle Size (µm) USP L1
SB-C8
USP L7
SB-Phenyl
USP L11
Agilent Cartridge Columns (require hardware kit 5021-1845)
Analytical
4.6 x 250
5
7995218-585 7995208-585
Analytical
4.6 x 150
5
7995218-595 7995208-595
Rapid Resolution
4.6 x 75
3.5
7995218-344 7995208-344
Guard Cartridges, 10/pk
4.0 x 4
5
Cartridge Holder
7995118-504 7995118-504
5021-1845
5021-1845
822975-902
822975-906
Standard Columns (no special hardware required)
Rapid Resolution HT
4.6 x 50
1.8
Rapid Resolution HT, 3/pk
4.6 x 50
1.8
822975-932
Narrow Bore RRHT
2.1 x 50
1.8
822700-902
Narrow Bore RRHT, 3/pk
2.1 x 50
1.8
822700-932
Rapid Resolution Cartridge
4.6 x 30
3.5
833975-902
833975-906
Rapid Resolution Cartridge, 3/pk
4.6 x 30
3.5
833975-932
833975-936
Rapid Resolution Cartridge
4.6 x 15
3.5
831975-902
831975-906
Rapid Resolution Cartridge, 3/pk
4.6 x 15
3.5
831975-932
831975-936
Rapid Resolution Cartridge
2.1 x 30
3.5
873700-902
873700-906
Rapid Resolution Cartridge, 3/pk
2.1 x 30
3.5
873700-932
873700-936
Rapid Resolution Cartridge
2.1 x 15
3.5
875700-902
875700-906
Rapid Resolution Cartridge, 3/pk
2.1 x 15
3.5
875700-932
875700-936
Rapid Resolution HT Cartridge
4.6 x 50
1.8
825975-902
Rapid Resolution HT Cartridge, 3/pk
4.6 x 50
1.8
825975-932
Rapid Resolution HT Cartridge
4.6 x 30
1.8
823975-902
Rapid Resolution HT Cartridge, 3/pk
4.6 x 30
1.8
823975-932
Rapid Resolution HT Cartridge
4.6 x 15
1.8
821975-902
Rapid Resolution HT Cartridge, 3/pk
4.6 x 15
1.8
821975-932
Rapid Resolution HT Cartridge
2.1 x 50
1.8
825700-902
Rapid Resolution HT Cartridge, 3/pk
2.1 x 50
1.8
825700-932
Rapid Resolution HT Cartridge
2.1 x 30
1.8
823700-902
Rapid Resolution HT Cartridge, 3/pk
2.1 x 30
1.8
823700-932
Rapid Resolution HT Cartridge
2.1 x 15
1.8
821700-902
Rapid Resolution HT Cartridge, 3/pk
2.1 x 15
1.8
821700-932
Rapid Resolution Cartridges (require hardware kit 820555-901)
Rapid Resolution Ht Cartridges (require hardware kit 820555-901)
Hardware Kit for RR and RRHT Cartridges
270
820555-901
833975-912
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX 80Å StableBond
Description
Size (mm)
SB-C18
Particle Size (µm) USP L1
Capillary glass-lined Columns
Capillary
0.5 x 250
5
5064-8258
Capillary
0.5 x 150
5
5064-8256
Capillary
0.5 x 35
5
5064-8254
Capillary RR
0.5 x 150
3.5
5064-8262
Capillary RR
0.5 x 35
3.5
5064-8260
Capillary
0.3 x 250
5
5064-8257
Capillary
0.3 x 150
5
5064-8255
Capillary
0.3 x 35
5
5064-8253
Capillary RR
0.3 x 150
3.5
5064-8261
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271
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Rx
• Recommended for alternate selectivity at low pH relative to Eclipse Plus C18, Eclipse XDB-C18 and
StableBond SB-C18; for higher temperature applications, StableBond is recommended
• Higher carbon load than SB-C18 columns (12% vs. 10%)
• High stability and good peak shape for low pH applications (up to pH 8)
• Manufactured using dimethyloctadecylsilane and non-endcapped
• ZORBAX Rx-C8 is the same product as SB-C8
Column Specifications
Bonded Phase
Pore
Size
Surface
Area
temp.
Limits
pH
Range*
Endcapped
Carbon
Load
ZORBAX Rx-C18
80Å
180 m2/g
60 °C
2.0-8.0
No
12%
ZORBAX Rx-C8
80Å
180 m2/g
80 °C
1.0-8.0
No
5.5%
Specifications represent typical values only
*At pH 6-9 highest column stability for all silica based columns is obtained by operating at temperatures <40 °C and using
lower buffer concentrations in the range of 0.01-0.02 M.
Analysis of diazepam on Rx-C18
Column:
ZORBAX Rx-C18
880967-302
3.0 x 250 mm, 5 µm
Mobile Phase:
35% H2O:65% MeOH
Flow Rate:
0.5 mL/min
1
2
1. Ethylparaben
2. Diazepam
An Rx-C18 column is used for this USP analysis of diazepam and the internal standard
ethylparaben. The Solvent Saver 3.0 mm id Rx-C18 column reduces solvent usage by 60%
over what would be used if the analysis was done on a 4.6 x 250 mm column.
0.0
2.5
Time (min)
5.0
7.5
LCRX001
272
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Rx
Hardware Description
Size (mm)
Particle Size Rx-C18
(µm)
USP L1
Rx-C8
USP L7*
Semi-Preparative
9.4 x 250
5
880967-202 880967-201
Analytical
4.6 x 250
5
880967-902 880967-901
Analytical
4.6 x 150
5
883967-902 883967-901
Rapid Resolution
4.6 x 150
3.5
863967-902
Rapid Resolution
4.6 x 100
3.5
861967-902
Rapid Resolution
4.6 x 75
3.5
866967-902
Solvent Saver
3.0 x 250
5
880967-302
Solvent Saver
3.0 x 150
5
883967-302
Solvent Saver Plus
3.0 x 150
3.5
863967-302
Solvent Saver Plus
3.0 x 100
3.5
861967-302
Narrow Bore
2.1 x 150
5
883700-902
Narrow Bore RR
2.1 x 100
3.5
861767-902
Guard Cartridge, 2/pk
9.4 x 15
7
820675-115 820675-115
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-914 820950-913
Guard Cartridge, 4/pk
2.1 x 12.5
5
821125-915 821125-915
Guard Hardware Kit
9.4 x 15
840140-901 840140-901
Guard Hardware Kit
820999-901 820999-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 250
7
877967-102 877250-106
PrepHT Cartridge
21.2 x 150
7
877150-106
PrepHT Cartridge
21.2 x 150
5
870150-906
PrepHT Cartridge
21.2 x 100
5
870100-906
PrepHT Cartridge
21.2 x 50
PrepHT Guard Cartridge, 2/pk
5
870050-906
5
820212-914 820212-915
Guard Cartridge Hardware
820444-901 820444-901
PrepHT Endfittings, 2/pk
820400-901 820400-901
*Rx-C8 is the same product as SB-C8. For other sizes and configurations, see the ZORBAX StableBond section, page 264.
www.AgiLEnt.COM/CHEM/LC
273
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX 80Å Extend-C18
C18
O
Si
O
Si
C18
Silica Support
• High efficiency and long life at high pH – up to pH 11.5
• Unique bidentate bonding and double endcapping provides high pH stability
• More efficiency and better peak shape than polymer-based columns
• Improve retention, resolution and peak shape of basic compounds
• High sensitivity for LC/MS separations of peptides
The Agilent ZORBAX Extend-C18 column uses a novel bidentate C18-C18 bonding technology to
make it possible to develop high-resolution separations at high pH with a silica-based column.
At high pH, non-charged basic compounds will not interact with the underlying silica. The result is
high efficiency separations with superior peak shape and improved resolution. High pH separations
are also the best choice for compounds that are more stable or more soluble in high pH solutions.
Some of the mobile phase buffer options for high pH include triethylamine, pyrrolidine, glycine, borate
and ammonium hydroxide. Ammonium hydroxide at pH 10.5 is an excellent mobile phase modifier for
the LC/MS of peptides and small molecules with improved sensitivity compared with TFA containing
mobile phase at low pH. The Extend-C18 column is stable from pH 2-11.5 with good peak shape for
all types of compounds. Extend-C18 columns also provide an additional selectivity choice at low pH.
Column Specifications
tiPS & tOOLS
Always use Agilent Certified Lamps
for Best LC Performance
Bonded Phase
Pore
Size
Surface
Area
ZORBAX Extend-C18
80Å
180 m2/g
temp.
Carbon
Limits* pH Range** Endcapped Load
60 °C
2.0-11.5
Double
12.5%
Specifications represent typical values only.
Agilent detector lamps are built to the
tightest specifications and quality
standards. They are designed to increase
light intensity and decrease noise, which
improves chromatographic results.
Agilent rigorously tests its lamps for
lowest lamp-to-lamp variability.
Trust Agilent lamps for robust,
long-lasting performance and lower
cost of ownership. To learn more, visit
www.agilent.com/chem/lamps
274
*Temperature limits are 60 °C up to pH 8, 40 °C from pH 8-11.5.
**Above pH 6 highest column stability for all silica based columns is obtained by reducing the operating temperature to
40 °C or below and using lower buffer concentrations (0.01-0.02 M) or organic buffers.
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Basic antihistamines on Extend-C18 at high pH
Column:
ZORBAX Extend-C18
773450-902
4.6 x 150 mm, 5 µm
pH 7
1.
2.
3.
4.
5.
6.
7.
7
4
2, 3
1
Mobile Phase:
pH 7:
30% 20 mM Na2HPO4 70% MeOH
pH 11:
30% 20 mM TEA 70% MeOH
Flow Rate:
1.0 mL/min
Temperature:
Ambient
Detector:
254 nm
5
6
0
Maleate
Scopolamine
Pseudoephedrine
Doxylamine
Chlorpheniramine
Triprolidine
Diphenhydramine
5
Time (min)
pH 11
7
4
Sample:
Antihistamines
3
5
1
Pseudoephedrine and scopolamine are difficult to retain at low and mid pH. Pseudoephedrine is
often analyzed by ion exchange methods. The Extend-C18 column retains these compounds in a
noncharged form at high pH and improves resolution.
2
6
5
0
10
Time (min)
LC80001
Long life at high pH with Extend-C18
ZORBAX Extend-C18
773450-902
4.6 x 150 mm, 5 µm
Mobile Phase:
20% Methanol
80% 0.1 M Carbonate buffer, pH 10.0
Flow Rate:
1.0 mL/min
Temperature:
Ambient
30
ZORBAX Extend-C18
At high pH, columns will fail due to silica dissolution. The example here shows
extended lifetime of ZORBAX Extend-C18 at high pH in comparison to competitor
W. This was measured by the amount of dissolved silica.
Amount of Dissolved Silica (mg)
Column:
Competitor w
20
10
0
0
3
5
9
12
15
Volume Eluent (L)
LC80002
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275
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Extend-C18 provides good peak shape at low pH
Column:
ZORBAX Extend-C18
773450-902
4.6 x 150 mm, 5 µm
Mobile Phase:
80% 25 mM NaH2PO4, pH 3.0
20% Methanol
Flow Rate:
1.0 mL/min
Temperature:
35 °C
Detector:
UV, 254 nm
Sample:
Basic compounds
1
1.
2.
3.
4.
3
Theobromine
Dimethylxanthine
Theophylline
Caffeine
4
2
These basic compounds are separated on the Extend-C18 at low pH with
excellent peak shape. The Extend-C18 column can be used at high and low pH.
0
1
2
3
4
5
6
Time (min)
7
LC80003
ZORBAX 80Å Extend-C18
Hardware Description
Size (mm)
Particle Size
(µm)
Extend-C18
USP L1
Standard Columns (no special hardware required)
Analytical
4.6 x 250
5
770450-902
Analytical
4.6 x 150
5
773450-902
Analytical
4.6 x 50
5
746450-902
Rapid Resolution
4.6 x 150
3.5
763953-902
Rapid Resolution
4.6 x 100
3.5
764953-902
Rapid Resolution
4.6 x 75
3.5
766953-902
Rapid Resolution
4.6 x 50
3.5
735953-902
Rapid Resolution HT, 600 bar
4.6 x 100
1.8
728975-902
Rapid Resolution HT, 600 bar
4.6 x 50
1.8
727975-902
Rapid Resolution HT, 600 bar
4.6 x 30
1.8
724975-902
Rapid Resolution HT, 600 bar
4.6 x 20
1.8
726975-902
Solvent Saver
3.0 x 250
5
770450-302
Solvent Saver
3.0 x 150
5
773450-302
Solvent Saver Plus
3.0 x 150
3.5
763954-302
Solvent Saver Plus
3.0 x 100
3.5
764953-302
Solvent Saver Plus
3.0 x 50
3.5
735954-302
Unless indicated, column pressure limit is 400 bar.
(Continued)
276
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX 80Å Extend-C18
Hardware Description
Size (mm)
Particle Size
(µm)
Extend-C18
USP L1
Standard Columns (no special hardware required)
Solvent Saver RRHD, 1200 bar
3.0 x 100
1.8
758700-302
Solvent Saver RRHD, 1200 bar
3.0 x 50
1.8
757700-302
Solvent Saver HT, 600 bar
3.0 x 100
1.8
728975-302
Solvent Saver HT, 600 bar
3.0 x 50
1.8
727975-302
Solvent Saver HT, 600 bar
3.0 x 30
1.8
724975-302
Solvent Saver HT, 600 bar
3.0 x 20
1.8
726975-302
Narrow Bore
2.1 x 150
5
773700-902
Narrow Bore
2.1 x 50
5
760450-902
Narrow Bore RR
2.1 x 100
3.5
761753-902
Narrow Bore RR
2.1 x 50
3.5
735700-902
Narrow Bore RRHD, 1200 bar
2.1 x 150
1.8
759700-902
Narrow Bore RRHD, 1200 bar
2.1 x 100
1.8
758700-902
Narrow Bore RRHD, 1200 bar
2.1 x 50
1.8
757700-902
Narrow Bore RRHT, 600 bar
2.1 x 100
1.8
728700-902
Narrow Bore RRHT, 600 bar
2.1 x 50
1.8
727700-902
Narrow Bore RRHT, 600 bar
2.1 x 30
1.8
724700-902
Narrow Bore RRHT, 600 bar
2.1 x 20
1.8
726700-902
MicroBore RR
1.0 x 150
3.5
763600-902
MicroBore RR
1.0 x 50
3.5
765600-902
MicroBore RR
1.0 x 30
3.5
761600-902
MicroBore Guard, 3/pk
1.0 x 17
5
5185-5923
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-930
Guard Cartridge, 4/pk
2.1 x 12.5
5
821125-930
Guard Hardware Kit
820999-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 150
5
770150-902
PrepHT
21.2 x 100
5
770100-902
PrepHT
21.2 x 50
5
770050-902
17.0 x 7.5
5
820212-930
PrepHT Endfittings, 2/pk
PrepHT Guard Cartridge, 2/pk
Guard Cartridge Hardware
820400-901
820444-901
Unless indicated, column pressure limit is 400 bar.
www.AgiLEnt.COM/CHEM/LC
277
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Bonus-RP
R1
O
Si
PG
R
R1
CH3
Si
CH3
CH3
R1
O
Si
PG
R
R1
CH3
Si
• Excellent peak shape for challenging basic compounds at low and mid pH
• Unique reversed-phase selectivity
• Novel bonding technology with embedded polar group and steric protection
• Usable in 100% aqueous mobile phases
The Agilent ZORBAX Bonus-RP column has a polar amide group embedded in a long alkyl chain.
This novel bonding reduces interactions between basic compounds and the silica support, improving
peak shape for the most difficult basic compounds. Peak shape and column lifetime are further improved
by triple endcapping. In addition, diisopropyl side groups provide steric protection against acid hydrolysis
for good lifetime at low pH. The Bonus-RP column provides an alternate selectivity to C18 and C8 alkyl
bonded phases.
CH3
CH3
Column Specifications
R1
O
Si
PG
R1
Unique, Polar Alkyl Bonus-RP Bonded Phase
Bonded Phase
Pore
Size
Surface
Area
temp.
Limits*
ZORBAX Bonus-RP
80Å
180 m2/g
60 °C
R
Specifications represent typical values only.
*Temperature limits are 60 °C up to pH 8, 40 °C from pH 8-9.
tiPS & tOOLS
ZORBAX Bonus-RP is also available on Poroshell 120.
Turn page 228
278
pH Range Endcapped
2.0-9.0
Triple
Carbon
Load
9.5%
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
improved peak shape of basic compounds
using Bonus-RP
Column:
Alkyl-C8
4.6 x 150 mm, 5 µm
Mobile Phase:
75% 25 mM NH4OAc, pH 5.5
25% ACN
Flow Rate:
1.5 mL/min
Temperature:
40 °C
Detector:
254 nm
1
2
TF
1. 1.8
2. 1.7
3. 2.1
0
Column:
ZORBAX Bonus-RP
883668-901
4.6 x 150 mm, 5 µm
Mobile Phase:
80% 25 mM NH4OAc, pH 5.5
20% ACN
Flow Rate:
1.5 mL/min
Temperature:
40 °C
Detector:
254 nm
3
1
2
3
4 5 6
Time (min)
7
1. Doxylamine
2. Chlorpheniramine
3. Triprolidine
8
9
10
LCBR001
3
TF
1. 1.1
2. 1.1
3. 1.1
0
1
1. Doxylamine
2. Chlorpheniramine
3. Triprolidine
2
1
2
3
4
Time (min)
5
6
LCBR002
Bonus-RP eliminates peak tailing of these basic compounds in comparison to a typical alkyl C8 bonded phase. In the mid-pH region, residual silanols can interact more strongly
with basic compounds to cause peak tailing. The polar group in the Bonus-RP bonded phase eliminates peak tailing of these basic compounds by reducing interactions with
residual silanols.
ZORBAX Bonus-RP is stable at low and mid pH
ZORBAX Bonus-RP
883668-901
4.6 x 150 mm, 5 µm
Mobile Phase:
60% 25 mM
Phosphate Buffer,
pH 7.0:40% ACN
Flow Rate:
1.5 mL/min
Temperature:
23 °C
0.0018
Toluene Plate Height, cm
Column:
Competitive alkyl/amide
Bonus-RP
0.0016
0.0014
0.0012
0.0010
Triple endcapping of Bonus-RP enhances stability at pH 7. Each 10,000
column volume is equivalent to approximately one working month.
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
Column Volume of Purge
LCBR003
www.AgiLEnt.COM/CHEM/LC
279
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Dimethyl-C18/amide, Bonus-RP
ZORBAX Bonus-RP
883668-901
4.6 x 150 mm, 5 µm
Mobile Phase:
Aging:
50% MeOH
50% 0.1% TFA
Test:
80% MeOH
20% H2O
Flow Rate:
1.0 mL/min
Temperature:
Aging:
60 °C
Test:
23 °C
0.0055
Dimethyl-C18/amide
Bonus-RP
0.0050
Toluene Plate Height, cm
Column:
0.0045
0.0040
0.0035
0.0030
0.0025
0.0020
0
10,000
20,000
30,000
40,000
Column Volume of Purge
LCBR004
Sterically protecting side groups provide good low pH stability and longer column lifetime than similar polar alkyl bonded phases.
ZORBAX Bonus-RP provides unique selectivity
Column A:
Column B:
ZORBAX Bonus-RP
883668-901
4.6 x 150 mm, 5 µm
Eclipse XDB-C8
993967-906
4.6 x 150 mm, 5 µm
1.
2.
3.
4.
A
1
mAu
15
Cephalexin
Cephaclor
Cephuroxime
Cephoxitin
10
3
Mobile Phase:
75% 25 mM Na Citrate, pH 6
25% MeOH
5
2
4
0
Flow Rate:
1.0 mL/min
Temperature:
Ambient
Detector:
254 nm
0
2
4
6
8
10
Time (min)
B
1
mAu
Sample:
3 µL
Cephalosporins
15
3
10
4
5
Peak elution order can change dramatically when using Bonus-RP.
In this example, the elution order of the first three peaks changes.
2
0
0
2
4
6
Time (min)
280
8
10
LCBR005
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Bonus-RP
Hardware Description
Size (mm)
Particle Size Bonus-RP
(µm)
USP L60
Standard Columns (no special hardware required)
Analytical
4.6 x 250
5
880668-901
Analytical
4.6 x 150
5
883668-901
Rapid Resolution
4.6 x 250
3.5
884950-577
Rapid Resolution
4.6 x 150
3.5
863668-901
Rapid Resolution
4.6 x 100
3.5
864668-901
Rapid Resolution
4.6 x 75
3.5
866668-901
Rapid Resolution
4.6 x 50
3.5
835668-901
Rapid Resolution HT, 600 bar
4.6 x 100
1.8
828668-901
Rapid Resolution HT, 600 bar
4.6 x 75
1.8
830668-901
Rapid Resolution HT, 600 bar
4.6 x 50
1.8
827668-901
Solvent Saver
3.0 x 250
5
880668-301
Solvent Saver
3.0 x 150
5
883668-301
Solvent Saver Plus
3.0 x 150
3.5
863668-301
Solvent Saver Plus
3.0 x 100
3.5
864668-301
Solvent Saver HT, 600 bar
3.0 x 100
1.8
828668-301
Solvent Saver HT, 600 bar
3.0 x 50
1.8
827668-301
Rapid Resolution HD, 1200 bar
2.1 x 150
1.8
859768-901
Rapid Resolution HD, 1200 bar
2.1 x 100
1.8
858768-901
Rapid Resolution HD, 1200 bar
2.1 x 50
1.8
857768-901
Narrow Bore
2.1 x 150
5
883725-901
Narrow Bore
2.1 x 50
5
861971-901
Unless indicated, column pressure limit is 400 bar.
(Continued)
ZORBAX Bonus-RP bonding is also available on Poroshell 120
columns. Turn to page 228.
www.AgiLEnt.COM/CHEM/LC
281
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Bonus-RP
Hardware Description
Size (mm)
Particle Size Bonus-RP
(µm)
USP L60
Standard Columns (no special hardware required)
Narrow Bore RR
2.1 x 150
3.5
863700-901
Narrow Bore RR
2.1 x 100
3.5
861768-901
Narrow Bore RR
2.1 x 50
3.5
861700-901
Narrow Bore RRHT, 600 bar
2.1 x 100
1.8
828768-901
Narrow Bore RRHT, 600 bar
2.1 x 50
1.8
827768-901
MicroBore RR
1.0 x 150
3.5
863608-901
MicroBore RR
1.0 x 50
3.5
865608-901
MicroBore RR
1.0 x 30
3.5
861608-901
MicroBore Guard, 3/pk
1.0 x 17
5
5185-5922
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-928
Guard Cartridge, 4/pk
2.1 x 12.5
5
821125-928
Guard Hardware Kit
820999-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 250
7
878250-101
PrepHT Cartridge
21.2 x 150
7
878150-101
PrepHT Cartridge
21.2 x 150
5
868150-901
PrepHT Cartridge
21.2 x 100
5
868100-901
PrepHT Cartridge
21.2 x 50
5
PrepHT Endfittings, 2/pk
PrepHT Guard Cartridge, 2/pk
Guard Cartridge Hardware
Unless indicated, column pressure limit is 400 bar.
tiPS & tOOLS
Watch LC troubleshooting videos featuring Agilent chromatographic experts at
www.agilent.com/chem/lctroubleshooting
282
868050-901
820400-901
17.0 x 7.5
5
820212-928
820444-901
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Original Reversed-Phase Columns
Agilent Original ZORBAX columns are made with Type A silica and are useful for many applications of acidic or neutral compounds. These columns have a
higher activity level and are therefore useful for separating isomers (e.g. cis-trans, geometric) or other compounds where silanol activity enhances selectivity.
These columns are used in many established methods.
Column Specifications
Bonded Phase
Pore Size Surface Area temp Limit pH Range Endcapped Carbon Load
ZORBAX C18
70Å
300 m2/g
60 °C
2.0 - 8.0
Yes/No
20%
ZORBAX C8
70Å
300 m2/g
60 °C
2.0 - 8.0
Yes
12%
ZORBAX-Phenyl
70Å
300 m2/g
60 °C
2.0 - 8.0
Yes
12%
ZORBAX CN
70Å
300 m2/g
60 °C
2.0 - 8.0
N/A
7%
ZORBAX-TMS
70Å
300 m2/g
60 °C
2.0 - 7.0
N/A
4%
ZORBAX Original Reversed-Phase Columns
Hardware Description
Size (mm)
Particle ODS (C18)
Size (µm) USP L1
C8
USP L7
Phenyl
USP L11
Cn
USP L10
tMS
USP L13
Standard Columns (no special hardware required)
Semi-Preparative
9.4 x 250
5
880952-202 880952-206
Analytical (Endcapped)
4.6 x 250
5
880952-702 880952-706 880952-712 884950-507 880952-710
Analytical (Non-endcapped)
4.6 x 250
5
884950-543
Analytical
4.6 x 150
5
883952-702 883952-706 883952-712 884950-526 883952-710
Solvent Saver
3.0 x 250
5
880952-302
Solvent Saver
3.0 x 150
5
883952-302
guard Columns (hardware required)
Guard Cartridge, 2/pk
9.4 x 15
7
820675-115 820675-115 820675-115 820675-124
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-902 820950-906 820950-912 820950-905 820950-924
Guard Hardware Kit
840140-901 840140-901 840140-901 840140-901 840140-901
Guard Hardware Kit
820999-901 820999-901 820999-901 820999-901 820999-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
PrepHT Endfittings, 2/pk
21.2 x 250
7
877952-102 877952-106
877952-105
820400-901 820400-901
820400-901
www.AgiLEnt.COM/CHEM/LC
283
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Kits for Analytical HPLC
ZORBAX Method Development Kits
ZORBAX Method Development Kits
Agilent offers a series of kits that allow for fast method development at an attractive price. Each kit contains
3 columns. Six new kits have been added and are recommended for use with the new Agilent Automated
Method Development LC. Several of these kits contain Rapid Resolution HT (1.8 µm) columns in a variety
of bonded phases for easy method optimization and several kits contain Rapid Resolution (3.5 µm) columns
in the same variety of bonded phases. These kits contain some of the Eclipse Plus family of columns for
excellent peak shape and optimum performance with a wide variety of compounds.
ZORBAX Method Development Kits
Recommended for use with the Agilent Automated Method Development LC System
284
Description
Part no.
Rapid Resolution HT (RRHT) Selectivity Method Development Kit, 2.1 mm id
Includes 2.1 x 50 mm, 1.8 µm, 600 bar columns: one each Eclipse Plus C18, Eclipse Plus Phenyl-Hexyl and Bonus-RP
5190-1431
Rapid Resolution HT (RRHT) pH Method Development Kit, 2.1 mm id
Includes 2.1 x 50 mm, 1.8 µm, 600 bar columns: one each Eclipse Plus C18, SB-C18 and Extend-C18
5190-1432
Rapid Resolution HT (RRHT) Selectivity Method Development Kit, 4.6 mm id
Includes 4.6 x 50 mm, 1.8 µm, 600 bar columns: one each Eclipse Plus C18, Eclipse Plus Phenyl-Hexyl and Bonus-RP
5190-1433
Rapid Resolution HT (RRHT) pH Method Development Kit, 4.6 mm id
Includes 4.6 x 50 mm, 1.8 µm, 600 bar columns: one each Eclipse Plus C18, SB-C18 and Extend-C18
5190-1434
Rapid Resolution Selectivity Method Development Kit, 4.6 mm id
Includes 4.6 x 100 mm, 3.5 µm columns: one each Eclipse Plus C18, Eclipse Plus Phenyl-Hexyl and Bonus-RP
5190-1435
Rapid Resolution pH Method Development Kit, 4.6 mm id
Includes 4.6 x 100 mm, 3.5 µm columns: one each Eclipse Plus C18, SB-C18 and Extend-C18
5190-1436
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Method Development Kits
Description
Part no.
StableBond Method Development Kit
Includes 4.6 x 150 mm, 5 µm columns; one each: SB-C18, SB-CN and SB-Phenyl phases
5183-4624
Fast StableBond Method Development Kit
Includes 4.6 x 75 mm, 3.5 µm columns; one each: SB-C18, SB-CN and SB-Phenyl phases
5183-4625
Eclipse XDB Method Development Kit
Includes 4.6 x 150 mm, 5 µm columns; one each: XDB-C18, XDB-C8, XDB-Phenyl phases
5183-4626
Fast Eclipse XDB Method Development Kit
Includes 4.6 x 75 mm, 3.5 µm columns; one each: XDB-C18, XDB-C8 and XDB-Phenyl phases
5183-4627
pH Method Development Kit
Includes 4.6 x 150 mm, 5 µm columns; one each: SB-C18, XDB-C18 and Extend-C18 phases
5185-5807
Fast pH Method Development Kit
Includes 4.6 x 75 mm, 3.5 µm columns; one each: SB-C18, XDB-C18 and Extend-C18 phases
5185-5808
Aqueous Method Development Kit
Includes 4.6 x 150 mm, 5 µm columns; one each: SB-Aq, Bonus RP and SB-C18
5185-5809
Fast Aqueous Method Development Kit
Includes 4.6 x 75 mm, 3.5 µm columns; one each: SB-Aq, Bonus RP and SB-C18
5185-5810
ZORBAX Cartridge Column Starter Kits
Hardware
Description
Part no.
ZORBAX C18 Kit
Includes one 4.6 x 150 mm, 5 µm Eclipse XDB-C18 column; one 4.6 x 150 mm, 5 µm StableBond C18 column; cartridge holder;
mounting tool; replacement filter (2/pk); and open-end wrench
5183-2021
ZORBAX C8 Kit
Includes one 4.6 x 150 mm, 5 µm Eclipse XDB-C8 column; one 4.6 x 150 mm, 5 µm StableBond C8 column; cartridge holder;
mounting tool; replacement filter (2/pk); and open-end wrench
5183-2022
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285
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
ZORBAX Method Validation Kits
ZORBAX Method Validation Kits are supplied to customers who need the same HPLC column type (bonded
phase, particle size, configuration) but from different manufacturing lots. To request columns from different
lots, contact Agilent Technologies or your local Agilent Authorized Distributor using the following procedure:
• Request Validation Kits (columns from different lots) by using Part Number 899999-888
• Indicate the Part Number of the current column you are using
• Indicate the Lot Number of the current column you are using
• Indicate the number of additional columns needed from different lots
(example: you have a current column and may need two additional lots)
• Please fax your request to (302) 993-5354 (United States and Canada) or email to
cag_sales-na@agilent.com. You will receive a quote from your Customer Service Representative
within 1-2 business days. Delivery of your method validation kit is usually 3 weeks or less from
the time your order is placed, depending on lot availability.
Custom HPLC Column Ordering
Columns not listed can be easily ordered using the following procedure:
• Request a Special Products Quotation (SPQ) using Part Number 899999-999
• Indicate column dimensions (example: 4.6 x 50 mm); bonded phase type (example: StableBond C3);
particle size (example: 5 µm); and pore size (example: 80Å)
• Please fax your request to (302) 993-5354 (United States and Canada) or email to
cag_sales-na@agilent.com. You will receive a quote from your Customer Service Representative
within 1-2 business days. Delivery of your custom column is usually 3 weeks or less from
the time your order is placed, depending on lot availability.
Custom columns are priced with a minimal surcharge over the price of stocked columns.
286
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Pursuit HPLC Columns
Beginning in drug discovery and drug metabolism, Pursuit columns are ideal for analyzing lead compounds
and biological samples. The column's performance is due to the unique combination of advanced bonding
chemistry and ultra-high purity silica. These factors combine to provide rapid separations with excellent first
time resolution and symmetrical peaks for polar compounds, whether at pH 1.5 or 10. Additionally, the need
for ion-pairing agents such as TFA is often eliminated, thus maximizing the performance of single and
parallel multi-channel LC/MS systems.
Culminating in QC, Pursuit is ideal for implementing dependable trouble-free analysis of raw materials and
approved drugs. Rigorous control and validation of each step in the manufacturing process ensures column
reproducibility. With Pursuit, your laboratory can spend its energy on producing results.
Special columns, such as Pursuit PFP (for very polar compounds) and Pursuit PAH (environmental), give you
the extra selectivities you need for your most challenging applications.
Pursuit HPLC Columns
Pursuit
For LC/MS and high throughput applications, the Pursuit column is built on the larger 200Å pore size silica.
High ligand density delivers up to 40% faster separations without sacrificing resolution. This is accomplished
by optimizing mass transfer with the larger pore size.
Pursuit XRs
Pursuit XRs columns are for performance in analytical R&D, QC and preparative applications. Combining
high ligand density with a 100Å pore size, high surface area silica, Pursuit XRs columns are designed to
increase productivity, as they offer maximum loadability, excellent stability and easy scalability while
maintaining superior resolution.
Pursuit XRs Ultra
For the ultimate in speed and good resolution on any instrument, we designed the Pursuit XRs Ultra around
an optimized 2.8 µm particle and an advanced packing procedure. Now you can decrease your run time
while maintaining resolution. Lower backpressure allows high flow rates to be used, and the 2.8 µm
particles of ultra-pure silica delivers 10-15% higher efficiency than 3 µm columns.
www.AgiLEnt.COM/CHEM/LC
287
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Column Specifications
Pore Size
Surface Area
pH Range
Endcapped
Carbon
Load
Pore Volume
200Å
200 m2/g
1.5-10
Yes
12.9%
11 mL/g
3.5 µmol/m2
Pursuit C8
200Å
200 m2/g
1.5-10
Yes
7.4%
11 mL/g
3.8 µmol/m2
Pursuit Diphenyl
200Å
200 m2/g
1.5-8.0
Yes
7.3%
11 mL/g
2.8 µmol/m2
Pursuit PFP
200Å
200 m2/g
1.5-10
Yes
6.3%
11 mL/g
3.4 µmol/m2
Pursuit PAH
200Å
200 m2/g
1.5-10
Yes
Pursuit XRs C18
100Å
440 m2/g
1.5-10
Yes
22%
11 mL/g
2.9 µmol/m2
Pursuit XRs C8
100Å
440 m2/g
1.5-10
Yes
15%
11 mL/g
3.7 µmol/m2
100Å
440 m2/g
1.5-8.0
Yes
14.6%
11 mL/g
2.6 µmol/m2
100Å
440 m2/g
1.5-10
Yes
100Å
440 m2/g
1.5-10
Yes
23.2%
11 mL/g
3.2 µmol/m2
Pursuit XRs Ultra C8
100Å
440 m2/g
1.5-10
Yes
15%
11 mL/g
3.7 µmol/m2
Pursuit XRs Ultra Diphenyl
100Å
440 m2/g
1.5-8.0
Yes
14.6%
11 mL/g
2.6 µmol/m2
Bonded Phase
Pursuit C18
Pursuit XRs Diphenyl
Pursuit XRs Si
Pursuit XRs Ultra C18
Specifications represent typical values only
tiPS & tOOLS
Request custom LC columns online at www.agilent.com/chem/customlccol
288
Ligand Coverage
11 mL/g
11 mL/g
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
tricyclic antidepressants and benzodiazepines
Column:
Pursuit XRs C18
A6000150X046
4.6 x 150 mm, 5 µm
Mobile Phase:
A: Water+0.1% HCOOH
B: MeCN+0.1% HCOOH
Gradient:
30-40% B in 15 min, hold at 40% B for 15 min
Flow Rate:
1.0 mL/min
Temperature:
Ambient
Detector:
UV, 254 nm
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
24
2
1
5
3
mAU
4
67
8
9
10
11
13
12
14
7-Aminoclonazepam
7-Aminoflunitrazepam
Nordoxepin
Doxepin
Desipramine
Imipramine
Nortriptyline
Amitriptyline
Trimipramine
Clomipramine
Nordiazepam
Clonazepam
Flunitrazepam
Diazepam
0
0
min
24
VLC0001
Mechanical stability of Pursuit XRs
Pursuit XRs C18
A6000050X020
2.0 x 50 mm, 5 µm
Sample:
DMSO mix
Mobile Phase:
A: MeOH:water, 10:90 + 0.1% HCOOH
B: MeOH:water, 90:10 + 0.1% HCOOH
1. 4-Methoxybenzenesulfonamide
2. Methyl 3-aminothiophene-2-carboxylate
3. Trimipramine
injection 1
1
2
3
400
Gradient:
0-100% B in 3 min, back to 0% B in 0.5 min, hold at
0% B for 3.5 min
mAU
Column:
Flow Rate:
0.4 mL/min
0
Temperature:
Ambient
Detector:
UV, 254 nm
0
min
injection 5000
6
1
2
3
mAU
400
0
0
min
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6
VLC0002
289
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Antifungals
Column:
Pursuit XRs Ultra Diphenyl
A7521050X020
2.0 x 50 mm, 2.8 µm
Mobile Phase:
Water+0.1% HCOOH:MeCN+0.1% HCOOH, 80:20
Flow Rate:
0.4 mL/min
2
1.
2.
3.
4.
240,000
Temperature:
Ambient
µV
1
Detector:
UV, 254 nm
0
4
3
0
min
4-Aminobenzoic acid
Sorbic acid
Benzoic acid
Salicylic acid
6
VLC0003
Liquid chromatography phase test mixture
(LPtM) on Pursuit C8
Column:
Pursuit C8
A3031050X020
2.0 x 50 mm, 3 µm
Mobile Phase:
A: 0.05% HCOOH in water
B: 0.05% HCOOH in MeCN
Flow Rate:
0.6 mL/min
Detector:
UV, 220 nm
4
2
1.
2.
3.
4.
3
Aspartame
Cortisone
Reserpine
Dioctyl phthalate
1
2
min
5
VLC0004
Adrenocorticosteroids on Pursuit PfP and C18
Mobile Phase:
MeCN:water, 22.5:77.5
Flow Rate:
1.5 mL/min
Pursuit PfP
300
Ambient
Detector:
UV, 240 nm
3
4
mAU
Temperature:
1 2
5
7
6
9
8
10
0
0
min
25
Pursuit C18
300
mAU
1
3
2
4
10
5
6 7
8
9
0
0
min
25
VLC0005
290
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Triamcinolone
Prednisolone
Cortisone
Methylprednisolone
Corticosterone
Beclomethasone
Prednisolone acetate
Triamcinolone acetonide
Cortisone acetate
Fluocinolone acetonide
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Pursuit HPLC Columns
Semi-Prep Scale
Pursuit C18
USP L1
Pursuit C8
USP L7
Size (mm)
Particle Size (µm)
Pursuit Diphenyl Pursuit PfP
10.0 x 250
10
A3002250X100 A3032250X100
10.0 x 150
5
A3000150X100
A3050150X100
10.0 x 250
5
A3000250X100 A3030250X100
A3050250X100
Pursuit PAH
USP L1
Pursuit HPLC Columns
Analytical Scale
Size (mm)
Particle Size (µm)
Pursuit C18
USP L1
Pursuit C8
USP L7
Pursuit Diphenyl Pursuit PfP
4.6 x 250
10
A3002250X046 A3032250X046
4.6 x 150
10
A3002150X046 A3032150X046
4.6 x 100
10
A3002100X046 A3032100X046
4.6 x 250
5
A3000250X046 A3030250X046 A3040250X046
Pursuit PAH
USP L1
A3050250X046 A7000250X046
4.6 x 150
5
A3000150X046 A3030150X046 A3040150X046
A3050150X046 A7000150X046
4.6 x 100
5
A3000100X046 A3030100X046 A3040100X046
A3050100X046
4.6 x 50
5
A3000050X046 A3030150X046 A3040050X046
A3050050X046
4.6 x 250
3
A3001250X046 A3031250X046 A3041250X046
A3051250X046
4.6 x 150
3
A3001150X046 A3031150X046 A3041150X046
A3051150X046
4.6 x 100
3
A3001100X046 A3031100X046 A3041100X046
A3051100X046 A7001100X046
4.6 x 50
3
A3001050X046
A3051050X046
4.6 x 30
3
A3001030X046
4.0 x 250
5
A3000250X040
4.0 x 125
5
A3000125X040
3.9 x 300
10
A3002300X039
3.9 x 300
5
A3000300X039
A3041050X046
3.9 x 150
5
A3000150X039
3.0 x 250
5
A3000250X030
A3040250X030
3.0 x 150
5
A3000150X030
A3040150X030
3.0 x 100
5
A3000100X030
A3050150X030
A3050100X030
3.0 x 250
3
A3001250X030
3.0 x 150
3
A3001150X030
A3041150X030
A3051150X030
3.0 x 100
3
A3001100X030
A3041100X030
A3051100X030 A7001100X030
(Continued)
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291
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Pursuit HPLC Columns
Analytical Scale
Size (mm)
Particle Size (µm)
Pursuit C18
USP L1
Pursuit C8
USP L7
Pursuit Diphenyl Pursuit PfP
3.0 x 50
3
A3001050X030
2.0 x 250
5
A3000250X020
A3041050X030
2.0 x 150
5
A3000150X020 A3030150X020 A3040150X020
2.0 x 100
5
A3000100X020 A3030100X020 A3040100X020
A3050100X020
2.0 x 50
5
A3000050X020 A3030050X020 A3040050X020
A3050050X020
2.0 x 30
5
A3000030X020
A3050030X020
2.0 x 20
5
A3000020X020
2.0 x 250
3
A3001250X020
A3040030X020
Pursuit PAH
USP L1
A3051050X030
A3050020X020
A3041250X020
2.0 x 200
3
A3041200X020
2.0 x 150
3
A3001150X020 A3031150X020 A3041150X020
A3051150X020
2.0 x 100
3
A3001100X020 A3031100X020 A3041100X020
A3051100X020 A7001100X020
2.0 x 50
3
A3001050X020 A3031050X020 A3041050X020
A3051050X020
2.0 x 30
3
A3001030X020 A3031030X020 A3041030X020
A3051030X020
2.0 x 20
3
A3001020X020
A3051020X020
Size (mm)
Particle Size (µm)
Pursuit C18
USP L1
50.0 x 250
10
A3002250X500 A3032250X500
21.2 x 250
10
A3002250X212 A3032250X212
A3041020X020
Pursuit HPLC Columns
Prep Scale
292
Pursuit C8
USP L7
Pursuit Diphenyl Pursuit PfP
21.2 x 150
10
A3002150X212
21.2 x 250
5
A3000250X212
A3050250X212
21.2 x 150
5
A3000150X212
A3050150X212
21.2 x 100
5
A3040100X212
Pursuit PAH
USP L1
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Pursuit ChromSep Complete Cartridge Systems
Hardware
Size (mm)
Particle Size (µm)
Pursuit C18
USP L1
Pursuit C8
USP L7
Pursuit PAH
USP L1
A3000250C046
A3030250C046
A7000250C046
4.6 x 250
5
4.6 x 250
3
4.6 x 150
5
A3000150C046
A3030150C046
4.6 x 100
5
A3000100C046
A3030100C046
4.6 x 150
3
A3001150C046
A3031150C046
A7001150C046
4.6 x 100
3
A3001100C046
A3031100C046
A7001100C046
4.6 x 50
3
A3001050C046
3.0 x 250
5
A3000250C030
3.0 x 150
5
A3000150C030
3.0 x 100
5
A3000100C030
3.0 x 150
3
A3001150C030
3.0 x 100
3
A3001100C030
2.0 x 250
5
A3000250C020
2.0 x 150
5
A3000150C020
2.0 x 100
5
A3000100C020
2.0 x 150
3
A3001150C020
2.0 x 100
3
A3001100C020
2.0 x 50
3
A3001050C020
A3031250C046
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A7000150C046
A7000100C030
A3030150C020
293
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Pursuit ChromSep Replacement Cartridges
Hardware
Size (mm)
Particle Size (µm)
4.6 x 250
5
4.6 x 150
5
4.6 x 150
3
4.6 x 100
3
Unit
Pursuit C18
USP L1
Pursuit C8
USP L7
A7000250R046
3/pk
A7000250T046
A3000150R046
3/pk
A3000150T046
3/pk
A3030150R046
A3030150T046
A7000150T046
A7001150R046
A3031150T046
A7001150T046
A7001100R046
3
5
3.0 x 100
3
3.0 x 100
3
2.0 x 50
3
A3001050T046
A3000150R030
3/pk
A3000150T030
A3000100R030
A7000100R030
3/pk
A3000100T030
A7000100T030
5
3.0 x 150
A7001100T046
A3001050R046
3/pk
3.0 x 150
A7000150R046
A3031150R046
3/pk
4.6 x 50
Pursuit PAH
USP L1
A3001150R030
3/pk
A3001150T030
A3001100R030
3/pk
A7001100R030
A3001100T030
A7001100T030
A3031050R020
3/pk
A3031050T020
Metaguard Columns, 3/pk
Hardware
294
iD (mm)
Particle Size (µm)
4.6
10
A3002MG
Pursuit C18 Pursuit C8 Pursuit DP Pursuit PfP
2.0
10
A3002MG2
4.6
5
A3000MG
A3030MG
2.0
5
A3000MG2
A3030MG2 A3040MG2 A3050MG2
1.0
5
A3000MG1
4.6
3
A3001MG
A3031MG
2.0
3
A3001MG2
A3031MG2 A3041MG2 A3051MG2
1.0
3
A3040MG
A3050MG
A3040MG1
A3041MG
A3041MG1
A3051MG
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Pursuit XRs HPLC Columns
Semi-Prep Scale
Pursuit
XRs C8
USP L7
Pursuit
XRs C18
USP L1
Size (mm)
Particle Size
(µm)
10.0 x 250
10
A6002250X100
10.0 x 250
5
A6000250X100
10.0 x 150
5
A6000150X100
10.0 x 50
5
A6000050X100
10.0 x 150
3
Pursuit
XRs Diphenyl
USP L11
Pursuit
XRs Si*
USP L3
A6004250X100
A6020250X100
A6021150X100
*Pursuit XRs Si is a normal phase column.
Pursuit XRs HPLC Columns
Analytical Scale
Size (mm)
Particle Size
(µm)
4.6 x 250
10
Pursuit
XRs C18
USP L1
Pursuit
XRs C8
USP L7
Pursuit
XRs Diphenyl
USP L11
A6002250X046
Pursuit
XRs Si*
USP L3
A6004250X046
4.6 x 50
10
A6002050X046S
4.6 x 250
5
A6000250X046
A6010250X046 A6020250X046
4.6 x 150
5
A6000150X046
A6010150X046 A6020150X046
4.6 x 100
5
A6000100X046
A6010100X046 A6020100X046 A6006100X046
4.6 x 50
5
A6000050X046
A6020050X046 A6006050X046
4.6 x 250
3
A6001250X046
A6021250X046
4.6 x 150
3
A6001150X046
A6010150X046 A6021150X046
4.6 x 100
3
A6001100X046
A6011100X046 A6021100X046 A6005100X046
4.6 x 50
3
A6001050X046
A6011050X046 A6021050X046 A6005050X046
4.6 x 30
3
A6001030X046
4.0 x 250
5
A6000250X040
A6010250X040
4.0 x 150
5
A6000150X040
A6010150X040
3.0 x 250
5
A6000250X030
A6010250X030 A6020250X030
3.0 x 150
5
A6000150X030
A6010150X030 A6020150X030
3.0 x 100
5
A6000100X030
A6010100X030 A6020100X030
3.0 x 150
3
A6001150X030
A6011150X030 A6021150X030
3.0 x 100
3
A6001100X030
A6011100X030 A6021100X030
3.0 x 50
3
A6001050X030
A6011050X030 A6021050X030
3.0 x 30
3
A6001030X030
A6021030X046
*Pursuit XRs Si is a normal phase column.
(Continued)
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295
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Pursuit XRs HPLC Columns
Analytical Scale
Size (mm)
Particle Size
(µm)
Pursuit
XRs C18
USP L1
Pursuit
XRs C8
USP L7
Pursuit
XRs Diphenyl
USP L11
Pursuit
XRs Si*
USP L3
2.1 x 100
5
2.0 x 250
5
A6000250X020
A6020250X020
2.0 x 150
5
A6000150X020
A6010150X020 A6020150X020
2.0 x 100
5
A6000100X020
A6010100X020
2.0 x 50
5
A6000050X020
A6010050X020 A6020050X020
2.0 x 30
5
A6000030X020
2.0 x 250
3
A6001250X020
A6021250X020
2.0 x 150
3
A6001150X020
A6011150X020 A6021150X020
2.0 x 100
3
A6001100X020
A6011100X020 A6021100X020
A6001050X020
A6011050X020 A6021050X020 A6005050X020
2.0 x 50
3
2.0 x 30
3
A6006100X021
A6021030X020
2.0 x 20
3
1.0 x 150
3
A6001020X020
A6001150X010
1.0 x 100
3
A6001100X010
A6021100X010
*Pursuit XRs Si is a normal phase column.
Pursuit XRs HPLC Columns
Prep Scale
Size (mm)
Particle Size
(µm)
50.0 x 250
10
Pursuit
XRs C8
USP L7
A6002250X500
30.0 x 250
5
A6000250X300
30.0 x 150
5
A6000150X300
30.0 x 100
5
A6000100X300
30.0 x 50
5
A6000050X300
21.2 x 250
10
A6002250X212 A6012250X212
21.2 x 250
5
A6000250X212
21.2 x 150
5
A6000150X212
21.2 x 100
5
A6000100X212
21.2 x 50
5
A6000050X212
21.2 x 30
5
A6000030X212
*Pursuit XRs Si is a normal phase column.
296
Pursuit
XRs C18
USP L1
Pursuit
XRs Diphenyl
USP L11
Pursuit
XRs Si*
USP L3
A6002250X500 A6004250X500
A6004250X300
A6020150X300
A6004250X212
A6020250X212
A6020100X212
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Metaguard Columns, 3/pk
Hardware
Pursuit XRs
Pursuit XRs C18 Pursuit XRs Si Pursuit XRs C8 Diphenyl
iD (mm)
Particle Size (µm)
4.6
10
A6002MG
4.6
5
A6000MG
3.0
5
2.0
5
A6000MG2
A6010MG2
A6020MG2
4.6
3
A6001MG
A6011MG
A6021MG
3.0
3
2.0
3
Pursuit PAH
A6004MG
A6010MG
A6020MG
A7000MG3
A7001MG3
A6001MG2
A6011MG2
A6021MG2
A6001MG2
Pursuit XRs Ultra HPLC Columns
Size (mm)
Particle Size
(µm)
Pursuit XRs
Ultra C18
Pursuit XRs
Ultra C8
3.0 x 150
2.8
A7501150X030 A7511150X030
3.0 x 100
2.8
A7501100X030
Pursuit XRs
Ultra Diphenyl
2.0 x 150
2.8
A7501150X020
2.0 x 100
2.8
A7501100X020 A7511100X020 A7521100X020
2.0 x 50
2.8
A7501050X020 A7511050X020 A7521050X020
2.0 x 30
2.8
A7501030X020 A7511030X020 A7521030X020
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297
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Polaris HPLC Columns
In areas like drug discovery where target compounds are increasingly polar, it is critical to have a
reversed-phase column that performs well under aqueous conditions. Retention is critical, but cannot
come with troublesome secondary interactions. Likewise, phase collapse and shifting retention times
need to be avoided. The answer is our Polaris line of polar-modified columns.
From the collapse-resistant pore structure of our base silica, to the "wettability" engineered into the
bonded phases, Polaris columns have been designed for high aqueous conditions. The combination of
high phase density bonding, ultra pure silica, and silanol shielding leads to excellent peak shape among
polar-modified columns.
As a family, Polaris offers a variety of polar modifications in both C18 and C8 chemistries.
Polaris C18-A
Polaris HPLC Columns
Polaris C18-A is the best starting place for separations where the benefits of polar-modified columns
are desired. The polar modifications of C18-A help it avoid poor peak shape and retention issues in low
organic conditions.
Polaris C8-A
Polaris C8-A offers an alternative selectivity to standard C8 phases and has a lower hydrophobicity than
Polaris C18-A, making it ideal for polar samples, or faster overall analysis times.
Polaris C18-Ether
Polaris C18-Ether offers an alternative selectivity to Polaris C18-A and standard C18 phases, and typically
delivers increased retention of polar compounds away from the void volume.
Polaris C8-Ether
Polaris C8-Ether offers an alternative selectivity to Polaris C8-A with particular utility for hydrogen
bonding compounds.
Column Specifications
Pore Size
Surface
Area
Carbon
Load
Endcapped
Pore
Volume
Ligand Coverage
Polaris C18-A
180Å
200 m2/g
13.8%
Yes
1.1 cm3/g
3.9 µmol/m2
Polaris C8-A
180Å
200 m2/g
7.4%
Yes
1.1 cm3/g
4.8 µmol/m2
Polaris C18-Ether
180Å
200 m2/g
12.1%
Yes
1.1 cm3/g
3.3 µmol/m2
Polaris C8-Ether
180Å
200 m2/g
7.1%
Yes
1.1 cm3/g
4.5 µmol/m2
Polaris Amide C18
180Å
200 m2/g
15%
Yes
1.1 cm3/g
4.4 µmol/m2
Polaris NH2
180Å
200 m2/g
5.5%
Amide
1.1 cm3/g
3.8 µmol/m2
180Å
200 m2/g
N/A
1.1 cm3/g
N/A
Bonded Phase
Polaris Si-A
Specifications represent typical values only.
298
N/A
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Selectivity test mix for Polaris columns
Mobile Phase:
MeCN:water 70:30
Flow Rate:
1.0 mL/min
Temperature:
Ambient
Detector:
UV, 254 nm
Polaris C8-A
5+8
6
mAU
400
12 3
4
7
0
0
14
min
Polaris C18-A
8
400
5
mAU
1
6
4
2 3
1.
2.
3.
4.
5.
6.
7.
8.
Uracil
Caffeine
Phenol
Toluene
Butylbenzene
o-Terphenyl
Amylbenzene
Triphenylene
1.
2.
3.
4.
Aspartame
Cortisone
Reserpine
Dioctyl phthalate
7
0
0
14
min
VLC0007
LC/MS performance test mix for Polaris C8-A
Column:
Polaris C8-A
A2011030X030
3.0 x 30 mm, 3 µm
Mobile Phase:
A: Water+0.05% HCOOH
B: MeCN+0.05% HCOOH
3
4
5-90% B in 3 min and hold for 4 min
Flow Rate:
0.6 mL/min
Temperature:
Ambient
Detector:
UV, 220 nm
400
1
mAU
Gradient:
2
0
0
min
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8
VLC0008
299
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Polaris HPLC Columns
Polaris
C18-A
Polaris
C8-A
Polaris
C18-Ether
Polaris
C8-Ether
Polaris
Amide C18
Polaris
nH2*
Polaris
Si-A*
Size (mm)
Particle
Size (µm)
50.0 x 250
10
A2002250X500
30.0 x 100
5
A2000100X300
30.0 x 3.0
3
21.2 x 250
10
A2002250X212
A2008250X212
A2004250X212
21.2 x 250
5
A2000250X212 A2010250X212 A2020250X212 A2030250X212 A2006250X212
A2013250X212 A2003250X212
A2004250X500
A2007030X030
21.2 x 150
5
A2000150X212
21.2 x 100
5
A2000100X212
21.2 x 50
5
10.0 x 250
10
10.0 x 250
5
A2003150X046
A2003050X212
A2008250X100
A2000250X100
A2020250X100 A2030250X100 A2006250X100
A2013250X100
10.0 x 50
3
4.6 x 250
10
A2002250X046
A2021050X100
4.6 x 250
5
A2000250X046 A2010250X046 A2020250X046 A2030250X046 A2006250X046
4.6 x 200
5
A2000200X046
4.6 x 150
5
A2000150X046 A2010150X046 A2020150X046 A2030150X046 A2006150X046
A2013150X046 A2003150X046
4.6 x 100
5
A2000100X046 A2010100X046
A2006100X046
A2013100X046 A2003100X046
4.6 x 50
5
A2000050X046
A2006050X046
A2013050X046 A2003050X046
4.6 x 30
5
A2000030X046
4.6 x 250
3
A2001250X046
A2021250X046 A2031250X046 A2007250X046
A2014250X046 A2005250X046
4.6 x 150
3
A2001150X046 A2011150X046
A2007150X046
A2014150X046 A2005150X046
4.6 x 100
3
A2001100X046 A2011100X046
A2007100X046
A2014100X046 A2005100X046
4.6 x 75
3
A2001075X046 A2011075X046
A2003250X046
A2020050X046
A2013250X046
*Normal phase columns.
(Continued)
300
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Polaris HPLC Columns
Size (mm)
Particle
Size (µm)
Polaris
C18-A
Polaris
C8-A
Polaris
C18-Ether
Polaris
C8-Ether
4.6 x 50
3
A2001050X046
4.6 x 30
3
A2001030X046
4.0 x 250
5
A2000250X040 A2010250X040 A2020250X040 A2030250X040
Polaris
Amide C18
A2021050X046 A2031050X046 A2007050X046
Polaris
nH2*
Polaris
Si-A*
A2014050X046 A2005050X046
A2013250X040 A2003250X040
4.0 x 150
5
A2000150X040 A2010150X040 A2020150X040 A2030150X040
A2013150X040 A2003150X040
4.0 x 125
5
A2000125X040 A2010125X040 A2020125X040 A2030125X040
A2013125X040 A2003125X040
3.0 x 250
5
A2000250X030 A2010250X030 A2020250X030 A2030250X030 A2006250X030
A2013250X030 A2005250X046
3.0 x 150
5
A2000150X030 A2010150X030 A2020150X030 A2030150X030 A2006150X030
A2013150X030 A2003150X030
3.0 x 100
5
A2000100X030 A2010100X030 A2020100X030 A2030100X030 A2006100X030
A2013100X030 A2003100X030
3.0 x 50
5
A2000050X030
3.0 x 250
3
A2001250X030
3.0 x 200
3
A2001200X030
3.0 x 150
3
A2001150X030
A2003050X030
A2021150X030
A2007250X030
A2014250X030 A2003250X030
A2007150X030
A2014150X030 A2005150X030
3.0 x 100
3
A2001100X030
A2007100X030
A2014100X030 A2005100X030
3.0 x 50
3
A2001050X030
A2021050X030 A2031050X030 A2007050X030
A2014050X030 A2005050X030
3.0 x 30
3
A2001030X030 A2011030X030
2.0 x 250
5
A2000250X020
A2020250X020 A2030250X020 A2006250X020
A2013250X020 A2003250X020
2.0 x 150
5
A2000150X020 A2010150X020 A2020150X020 A2030150X020 A2006150X020
A2013150X020 A2003150X020
2.0 x 100
5
A2000100X020
A2006100X020
A2013100X020 A2003100X020
2.0 x 50
5
A2000050X020 A2010050X020 A2020050X020 A2030050X020 A2006050X020
A2013050X020 A2003050X020
2.0 x 30
5
A2000030X020
2.0 x 20
5
A2000020X020
A2006030X020
A2013030X020 A2003030X020
A2013020X020 A2003020X020
2.0 x 250
3
A2001250X020 A2011250X020 A2021250X020 A2031250X020 A2007250X020
A2014250X020 A2005250X020
2.0 x 150
3
A2001150X020 A2011150X020 A2021150X020 A2031150X020 A2007150X020
A2014150X020 A2005150X020
2.0 x 100
3
A2001100X020
A2014100X020 A2005100X020
A2021100X020 A2031100X020 A2007100X020
2.0 x 75
3
A2021075X020
2.0 x 50
3
A2001050X020 A2011050X020 A2021050X020 A2031050X020 A2007050X020
2.0 x 30
3
A2001030X020
2.0 x 20
3
A2001020X020
A2021050X020
A2007030X020
A2014050X020 A2005050X020
A2014030X020 A2005030X020
A2014020X020 A2005020X020
*Normal phase columns.
www.AgiLEnt.COM/CHEM/LC
301
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Polaris ChromSep Complete Cartridge Systems
Hardware
Size (mm)
Polaris
C18-A
Particle Size (µm)
4.6 x 250
5
A2000250C046
4.6 x 150
5
A2000150C046
4.6 x 100
5
A2000100C046
4.6 x 250
3
A2001250C046
4.6 x 150
3
A2001150C046
3.0 x 250
5
A2000250C030
3.0 x 100
5
A2000100C030
2.0 x 100
5
A2000100C020
2.0 x 150
3
A2001150C020
2.0 x 100
3
A2001100C020
2.0 x 50
3
A2001050C020
Polaris ChromSep Replacement Cartridges
Hardware
302
Size (mm)
Particle Size (µm)
4.6 x 250
5
4.6 x 150
5
4.6 x 100
5
4.6 x 150
3
4.6 x 100
3
3.0 x 150
5
3.0 x 100
5
3.0 x 100
3
2.0 x 150
3
2.0 x 50
3
Unit
Polaris
C18-A
A2000250R046
3/pk
A2000250T046
3/pk
A2000150T046
3/pk
A2000100T046
3/pk
A2001150T046
3/pk
A2001100T046
3/pk
A2000150T030
A2000150R046
A2000100R046
A2001150R046
A2001100R046
A2000150R030
A2000100R030
3/pk
A2000100T030
A2001100R030
3/pk
A2001100T030
A2001150R020
3/pk
A2001150T020
3/pk
A2001050T020
A2001050R020
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Metaguard Columns
Hardware
Dimensions
Particle
Size Polaris
(µm) C18-A
Polaris
C8-A
Polaris
C18-Ether
Polaris
C8-Ether
Polaris
Amide C18
Polaris
nH2*
A2002MG
Polaris
Si-A*
4.6
10
2.0
10
A2004MG
4.6
5
A2000MG
A2010MG
A2020MG
A2006MG
A2013MG
A2003MG
2.0
5
A2000MG2
A2010MG2
A2020MG2
A2006MG2
A2013MG2
A2003MG2
4.6
3
A2001MG
A2011MG
A2021MG
A2007MG
A2014MG
A2005MG
2.0
3
A2011MG2
A2011MG2
A2021MG2
A2007MG2
A2014MG2
A2005MG2
1.0
3
A2001MG1
A2008MG2
A2030MG
A2031MG2
A2004MG2
*Normal phase columns.
www.AgiLEnt.COM/CHEM/LC
303
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Agilent tC-C18(2) and HC-C18(2)
For cost-conscious chromatographers who need traditional LC columns and don't need the individual
testing of ZORBAX, Pursuit or Polaris columns, the Agilent TC(2)/HC(2) columns provide an alternative.
TC-C18(2)
Agilent TC-C18(2) is the ideal choice for complex natural product extract samples, traditional medicines
and environmental samples or any sample where you need to analyze mixtures of polar and non-polar
compounds, including strong basic compounds.
• Lower carbon load – 12%
• Ideal for polar compounds and gradient separations that start at low % organic or cover a wide
organic range
• Good choice for samples dissolved in water, or mostly water
• Use with most common mobile phases, including formic acid, acetic acid, trifluoroacetic acid (TFA)
and phosphate buffers with acetonitrile and methanol as the organic modifiers
• Excellent performance from pH 2-8
HC-C18(2)
Agilent HC-C18(2) is a more retentive C18 with a higher carbon load. An excellent value alternative to
other high carbon load columns, it also provides superior peak shape for basic compounds.
• Higher carbon load – 17% – provides greater retention for moderately polar
and non-polar compounds
• Ideal for non-polar compounds and separations that start at mid-level % organic
(at least greater than 10% organic)
• Good choice for industrial samples or samples dissolved in organic/mostly organic solvents
• Stable over a very wide pH range (2-9) for maximum flexibility
304
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Column Specifications
Bonded
Phase
Pore Size
Surface Area
temp.
Limits
pH Range*
Endcapped
Carbon
Load
TC-C18(2)
170Å
290 m2/g
60 °C
2.0-8.0
Yes
12%
HC-C18(2)
170Å
290 m2/g
60 °C
2.0-9.0
Yes
17%
Specifications represent typical values only.
Agilent HC-C18(2) and tC-C18(2)
Description
Size (mm)
Agilent HC-C18(2)
4.6 x 250
5
588905-902
Agilent HC-C18(2)
4.6 x 150
5
588915-902
Agilent TC-C18(2)
4.6 x 250
5
588925-902
Agilent TC-C18(2)
4.6 x 150
5
588935-902
Agilent HC-C18(2) guards, 2/pk
4.6 x 12.5
5
520518-904
Agilent TC-C18(2) guards, 2/pk
4.6 x 12.5
5
520518-905
Guard Hardware Kit
Particle Size (µm)
Part no.
820999-901
tiPS & tOOLS
Don't forget, we have special offers throughout the year.
To learn more, visit www.agilent.com/chem/specialoffers
www.AgiLEnt.COM/CHEM/LC
305
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
PLRP-S HPLC Columns
• Contain durable and resilient polymer particles that deliver reproducible results over longer lifetimes
• Thermally and chemically stable
• Comply with USP L21 designation
• Used in bioscience, chemical, clinical research, energy, environmental, food and agriculture, material
science and pharmaceutical industries
• Pore sizes (100Å-4000Å) for separations of small molecules to large complexes and polynucleotides
The PLRP-S family of columns consists of a range of pore sizes and particle sizes, all with identical chemistry
and fundamental adsorptive characteristics. The particles are inherently hydrophobic, therefore no bonded
phase, alkyl ligand is required for reversed-phase separations. This gives a highly reproducible material that
is free from silanols and heavy metal ions. Columns within the extensive product range are suitable for micro
separations, including both bottom-up and top-down proteomics, analytical separations, and preparative
purifications. In addition, process columns can be packed with bulk media.
Column Specifications
pH Range
1-14
Buffer Content
Unlimited
Organic Modifier
1-100%
Temperature Limits
200 °C
Maximum Pressure
5-8 µm: 3000 psi (210 bar)
3 µm: 4000 psi (300 bar)
PLRP-S Applications
306
Pore Size
Application
100Å
Small molecules/synthetic biomolecules
300Å
Recombinant peptides/proteins
1000Å
Large proteins
4000Å
DNA/high speed
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
HPLC of 25 bp DnA ladder
Column:
PLRP-S, 2.1 x 150 mm
Mobile Phase:
A: 0.1 M TEAA
B: 0.1 M TEAA in 50% water:50% ACN
Flow Rate:
200 µL/min
Gradient:
12.5-50% B in 150 min
HPLC
Response, UV, 260 nm
PLRP-S 100Å
PLRP-S 300Å
PLRP-S 1000Å
PLRP-S 4000Å
Time
VLCPLRPSResp
Polyethylene glycols
Column:
PLRP-S 100Å
PL1111-3500
4.6 x 150 mm, 5 µm
Mobile Phase:
A: Water
B: ACN
Gradient:
10-30% B in 12 min, held at 30% B for 3 min
Flow Rate:
1.0 mL/min
PEG 400
min
0
15
Injection Volume: 10 µL
Sample Conc:
1 mg/mL
Detector:
ELS (neb=50 °C, evap=70 °C, gas=1.6 SLM)
PEG 1080
0
min
15
VLC0048
www.AgiLEnt.COM/CHEM/LC
307
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
Exploiting chemical stability –
nH4OH concentration
2
3
1
Column:
PLRP-S 100Å
PL1512-5500
4.6 x 250 mm, 5 µm
1 mM
Mobile Phase:
A: NH4OH (various mM) in water
B: NH4OH (various mM) in ACN
10 mM
Gradient:
Linear 10-100% B in 15 min
Flow Rate:
1.0 mL/min
Detector:
ELS (neb=80 °C, evap=85 °C, gas=1.0 SLM)
1. Angiotensin II
2. Angiotensin I
3. Angiotensin III
100 mM
min
0
10
VLC0049
5
Alberta Peptide institute test mix
Column:
PLRP-S 100Å
PL1512-5500
4.6 x 250 mm, 5 µm
Mobile Phase:
A: 0.1% TFA in 99% water:1% ACN
B: 0.1% TFA in 70% water:30% ACN
Gradient:
0-100% B in 30 min
Flow Rate:
1.0 mL/min
Detector:
UV, 220 nm
4
3
1
0
2
min
1.
2.
3.
4.
5.
Ala3-Gly4 (free amino)
Gly3-Gly4 (Na-acetylated)
Ala3-Gly4 (Na-acetylated)
Val3-Gly4 (Na-acetylated)
Val3-Val4 (Na-acetylated)
30
VLC0050
308
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
2
Large fibrous proteins
Column:
PLRP-S 300Å
PL1512-3801
4.6 x 150 mm, 8 µm
Column:
PLRP-S 1000Å
PL1512-3802
4.6 x 150 mm, 8 µm
Mobile Phase:
1
1
A: 0.25% TFA in water
B: 0.25% TFA in 5% water:95% ACN
Flow Rate:
1.0 mL/min
Gradient:
20-60% B in 15 min
Detector:
UV, 220 nm
1. Collagen (120,000 MW)
2. Fibrinogen (340,000 MW)
2
PLRP-S 300Å
0
min
12
www.AgiLEnt.COM/CHEM/LC
PLRP-S 1000Å
0
min
18
VLC0051
309
COLUMnS fOR SMALL MOLECULE SEPARAtiOnS
PLRP-S HPLC Columns
Hardware
Size (mm)
Particle Size (µm)
4.6 x 250
PLRP-S
300Å
USP L21
PLRP-S
1000Å
USP L21
PLRP-S
4000Å
USP L21
8
PL1512-5800 PL1512-5801 PL1512-5802
PL1512-3800 PL1512-3801 PL1512-3802 PL1512-3803
4.6 x 150
8
4.6 x 50
8
4.6 x 250
5
PL1512-1801 PL1512-1802 PL1512-1803
PL1512-5500 PL1512-5501
4.6 x 150
5
PL1111-3500 PL1512-3501
4.6 x 50
5
PL1512-1500 PL1512-1501 PL1512-1502 PL1512-1503
4.6 x 150
3
PL1512-3300 PL1512-3301
4.6 x 50
3
PL1512-1300 PL1512-1301
2.1 x 250
8
PL1912-5801
2.1 x 150
8
PL1912-3801 PL1912-3802 PL1912-3803
2.1 x 50
8
2.1 x 250
5
PL1912-5500 PL1912-5501
2.1 x 150
5
PL1912-3500 PL1912-3501
PL1912-1801 PL1912-1802 PL1912-1803
2.1 x 50
5
PL1912-1500 PL1912-1501 PL1912-1502 PL1912-1503
2.1 x 150
3
PL1912-3300 PL1912-3301
2.1 x 50
3
PL1912-1300 PL1912-1301
PLRP-S Guard Cartridges for 5 x 3 mm, 2/pk
PL1612-1801 PL1612-1801 PL1612-1801 PL1612-1801
Guard Cartridge holder for 3.0 x 5.0 mm cartridges
PL1310-0016 PL1310-0016 PL1310-0016 PL1310-0016
*Prep columns are also available for the PLRP-S family. Turn to pages 467-471
310
PLRP-S
100Å
USP L21
CoLumns for smaLL moLeCuLe seParations
Preparative HPLC Columns
Flexible, cost-effective options for scaling and prep
Whether you are scaling up a routine analytical method, or maintaining precise separations throughout
every phase of production, Agilent can help you rise to the challenge.
• Agilent Prep LC columns are a cost-effective prep solution designed for high loadability to purify
milligram to gram quantities of product
• ZORBAX PrepHT columns are designed for rapid scale-up from the ZORBAX family of phases
• Scalable prep columns are also available for Pursuit and Polaris columns
• Bulk materials are available for all phases and can be ordered through Agilent’s Custom Ordering
Process, www.agilent.com/chem/customlc
Agilent Prep LC Columns
• High loadability for maximum sample purification
• Easy scalability from 4.6 up to 50 mm id for rapid method development
• High throughput 21.2 mm id cartridges for fast purification
• Exceptional column stability and loadability up to pH 10
Prep LC Columns
Agilent Prep LC columns are designed for high loadability to purify milligram to gram quantities of products.
Preparative sized columns are available in 21.2, 30, and 50 mm internal diameters with lengths ranging
from 50-250 mm. Columns are available in 5 and 10 µm particle sizes with very high efficiency in every
dimension. These column choices accommodate almost every preparative sample.
Agilent Prep 21.2 mm id columns are available with Agilent’s Preparative Cartridge Hardware. This reliable
cartridge hardware makes it simple to use columns with different lengths to increase sample load. Guard
columns are easily integrated onto these columns, providing superior protection of the analysis column.
Analytical size 4.6 mm id scalar columns are available for method development and optimization prior to
scaling up to larger columns. Bulk material is also available.
Agilent Prep columns are available in a C18 bonded phase suitable for purification of a wide variety of
non-polar and polar compounds. Unbonded silica columns are also available.
www.agiLent.Com/CHem/LC
311
CoLumnS foR SmALL moLECuLE SEPARATionS
Column Specifications
Bonded Phase
Surface
Area
Pore Size
Temp. Limits
2
pH Range Endcapped
Carbon Load
C18
100Å
400 m /g
60 °C*
2.0-10.0
Single
24%
Silica
100Å
400 m2/g
**
1.0-8.0
N/A
N/A
Specifications represent typical values only.
*Temperature limits are 60 °C up to pH 8, 40 °C from pH 8-10.
**Temperature limits for bare silica are determined by the pH of the mobile phase.
Agilent Prep C18
Superior loadability on Agilent Prep C18
with basic compounds
Column:
50% 0.1%TFA:50% ACN
Flow Rate:
1 mL/min
Sample:
10 µL
Doxepin/Amitriptyline
0.5-50 mg/mL
mAU
4000
Agilent Prep C18
443905-902
4.6 x 150 mm, 5 µm
Mobile Phase:
Competitor W-C18
mAU
40
35
30
25
20
15
10
5
0
α = 1.6
3500
3000
2500
2000
1500
1000
500
0
0
1
2
3
4 5 6
Time (min)
7
8
9
α = 1.57
0
1
2
3
4 5 6
Time (min)
7
8
9
LCPLC01
Agilent Prep columns show better resolution and loadability than competitor columns.
Steroids: Easy scalability using
Agilent Prep columns
Column A:
Column B:
Column C:
Column D:
Agilent Prep C18
443905-902
4.6 x 150 mm, 5 µm
Agilent Prep C18
443905-102
21.2 x 150 mm, 5 µm
Agilent Prep C18
413910-302
30.0 x 150 mm, 10 µm
1. Hydrocortisone
2. Testosterone (in MeOH @ 1mg/mL)
Mobile Phase:
55% Water:45% ACN
Flow Rate:
0.7 mL/min
14.87 mL/min
29.77 mL/min
85.37 mL/min
Temperature:
Ambient
Detector:
240 nm
Sample:
2 µL
42.4 µL
170 µL
488 µL
Agilent Prep C18
413910-502
50.0 x 150 mm, 10 µm
Agilent Prep C18 shows excellent scalability, making method transfer
simple and predictable.
A
0
1
2
B
4
2
6
8
10
12
1
2
0
2
C
4
6
8
10
12
1
2
0
2
D
4
6
8
10
12
1
2
0
312
2
4
6
Time (min)
8
10
12
LCPLC02
CoLumns for smaLL moLeCuLe seParations
agilent Prep LC Columns
Hardware Description
size (mm)
Particle size
(µm)
C18
silica
standard Columns (no special hardware required)
Scalar
4.6 x 250
10
440910-902 440910-901
Scalar
4.6 x 150
10
443910-902 443910-901
Scalar
4.6 x 100
10
449910-902
Scalar
4.6 x 250
5
440905-902 440905-901
Scalar
4.6 x 150
5
443905-902 443905-901
Scalar
4.6 x 100
5
449905-902 449905-901
Scalar
4.6 x 50
5
446905-902 446905-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)*
PrepHT
21.2 x 250
10
410910-102 410910-101
PrepHT
21.2 x 150
10
413910-102 413910-101
PrepHT
21.2 x 50
10
446910-102
PrepHT
21.2 x 150
5
443905-102 443905-101
PrepHT
21.2 x 100
5
449905-102 449905-101
PrepHT
21.2 x 50
5
PrepHT Endfittings, 2/pk
446905-102 446905-101
820400-901 820400-901
standard Columns (no special hardware required)
Prep 30
30.0 x 250
10
410910-302 410910-301
Prep 30
30.0 x 150
10
413910-302 413910-301
Prep 30
30.0 x 100
10
419910-302 419910-301
Prep 30
30.0 x 100
5
449905-302 449905-301
Prep 30
30.0 x 50
5
446905-302 446905-301
Prep 50
50.0 x 250
10
410910-502 410910-501
Prep 50
50.0 x 150
10
413910-502 413910-501
Prep 50
50.0 x 100
10
419910-502 419910-501
Prep 50
50.0 x 100
5
449905-502 449905-501
21.2 x 10
10
420212-902 420212-901
guard Columns (hardware required)
PrepHT Guard Cartridges, 2/pk
Guard Cartridge Hardware
820444-901 820444-901
PrepHT External Guard Hardware Kit
420420-901 420420-901
Bulk Packing (1 kg)
10
420910-902 420910-901
*All PrepHT cartridge columns require hardware kit P/N 820400-901. If a guard column is desired for the 21.2 mm id
columns, the PrepHT Guard Hardware Kit, P/N 820444-901, is also required. If the guard column is used on a 30 mm id
column then the external guard column hardware kit, P/N 420420-901, is required.
www.agiLent.Com/CHem/LC
313
CoLumns for smaLL moLeCuLe seParations
ZorBaX PrepHt
• Easy scale-up from analytical to preparative scale with ZORBAX phases
• Fast preparative separations, up to 2000 mg
• 5 to 7 µm particles for high efficiency and high yield
• Easy to install finger-tight connections seal up to 5000 psi/350 bar
• Use to maintain selectivity of the analytical phase in your prep separations
High purity, high recovery and high throughput can be easily achieved with Agilent ZORBAX PrepHT
columns. These are available in a variety of bonded phases – Eclipse XDB, StableBond, Bonus-RP, and
Extend-C18 – for optimized resolution and loadability under any conditions.
ZORBAX PrepHT Columns
ZORBAX PrepHT columns are packed with 5 and 7 µm particle sizes for very high resolution. The high
resolution allows high loadability, high yield, and high purity of compounds. The larger diameter columns
and mechanically stronger ZORBAX particles allow for flow rates up to 100 mL/min, thus increasing
throughput.
ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scale without losing
resolution. For complex separations on larger columns (21.2 mm id, 150 mm length and longer), Agilent has
carefully chosen the 7 µm particle size to achieve a balance between high efficiency and high loadability.
314
CoLumns for smaLL moLeCuLe seParations
High purity and high recovery with ZorBaX
PrepHt columns
Antianginal drugs
Mass-based fraction collection using ZORBAX SB-C18 column
shows high purity and high recovery of each compound
(Application Note publication number 5988-7113EN).
The separation of the three antianginal drugs was successfully
done in a single run with high recovery and >90% purity.
Separations up to 2000 mg are possible depending on the
complexity of separation.
1
2
3
DAD
1. Nifedipine
2. Nifmodipine
3. Nifsoldipine
Absorbance (mAU)
Sample:
DMSO
0
1
2
3
4
Time (min)
5
6
7
MSD
Vial 2 – 343
Vial 1 – 347
Vial 3 – 389
8000000
6000000
4000000
2000000
0
1
2
3
4
Time (min)
5
6
7
LCPHT01
amount nifedipine
[mg]
amount nifmodipine
[mg]
amount nifsoldipine
[mg]
fraction 1
18.90
0.11
0.16
Purity Nifedipine
98.6%
fraction 2
0.29
17.66
0.77
Purity Nifmodipine
94.4%
fraction 3
0.49
1.66
18.36
Purity Nifsoldipine
89.5%
recovery [mg]
19.68
19.43
19.29
recovery [%]
101.3
102.0
101.9
www.agiLent.Com/CHem/LC
315
CoLumns for smaLL moLeCuLe seParations
ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scale without
losing resolution. For complex separations for larger columns (21.2 mm id and higher, 150 mm length
and higher), Agilent has carefully chosen the 7 µm particle size to achieve a balance between high
efficiency and high loadability.
scale-up from analytical to prep ZorBaX sB-C18 columns
using the same pump
Column size
Column 1 50 x 150 mm
flow
(mL/min)
injection
(µL)
Detector Cell
Part no.
100
2200
0.3 mm quartz
Custom Column
Column 2 21.2 x 150 mm
18
400
0.3 mm quartz
877150-102
Column 3 9.4 x 150 mm
3.5
80
0.3 mm quartz
883975-202
Column 4 4.6 x 150 mm
0.85
2.0
3 mm SS
883975-902
Using the same 1100 pump, a scale-up from 4.6 mm to 50 mm id was possible without any loss of resolution. This increases
throughput by reducing the time required for redeveloping and adjusting the method.
Scale-up to PrepHT
2
3500
3
3000
Absorbance (mAU)
2500
2000
Column 1
1500
1
1. Theobromine
2. Theophylline
3. Caffeine
Column 2
1000
Column 3
500
Column 4
0
0
2.5
5.0
7.5
10.0
12.5
Time (min)
15.0
17.5
20.0
LCPHT_ScaleUp
316
CoLumns for smaLL moLeCuLe seParations
ZorBaX PrepHt 80stableBond (require hardware 820400-901)
Hardware Description
size (mm)
Particle size sB-C18
(µm)
usP L1
sB-C8
usP L7
sB-aq
sB-Cn
usP L10
sB-Phenyl
usP L11
PrepHT Cartridge
21.2 x 250
7
877250-102 877250-106 877250-114 877250-105 877250-112
PrepHT Cartridge
21.2 x 150
7
877150-102 877150-106 877150-114
PrepHT Cartridge
21.2 x 150
5
870150-902 870150-906 870150-914
PrepHT Cartridge
21.2 x 100
5
870100-902 870100-906 870100-914
PrepHT Cartridge
21.2 x 50
5
870050-902 870050-906 870050-914
PrepHT Guard Cartridge, 2/pk
17.0 x 7.5
5
820212-920 820212-915 820212-933 820212-933 820212-915
ZorBaX PrepHt 300stableBond (require hardware 820400-901)
300sB-C18
usP L1
300sB-C8
usP L7
300sB-C3
usP L56
300sB-Cn
usP L10
size (mm)
Particle size
(µm)
PrepHT Cartridge
21.2 x 250
7
897250-102 897250-106 897250-109 897250-105
PrepHT Cartridge
21.2 x 150
7
897150-102 897150-106 897150-109
PrepHT Cartridge
21.2 x 150
5
895150-902 895150-906 895150-909
PrepHT Cartridge
21.2 x 100
5
895100-902 895100-906 895100-909
PrepHT Cartridge
21.2 x 50
5
895050-902 895050-906 895050-909
PrepHT Guard Cartridge, 2/pk
17.0 x 7.5
5
Hardware Description
820212-921 820212-918 820212-924 820212-924
Guard Cartridge Hardware
Includes guard column end fitting, polymeric seal, and seal insertion tool
(seal holder and seal pusher)
820444-901 820444-901 820444-901 820444-901
PrepHT Endfittings, 2/pk
820400-901 820400-901 820400-901 820400-901
ZorBaX PrepHt original (require hardware 820400-901)
Hardware Description
PrepHT Cartridge
PrepHT Endfittings, 2/pk
size (mm)
Particle size
(µm)
21.2 x 250
7
oDs (C18)
usP L1
C8
usP L7
Cn
usP L10
nH2
usP L8
siL
usP L3
877952-102 877952-106 877952-105 877952-108 877952-101
820400-901 820400-901 820400-901 820400-901 820400-901
www.agiLent.Com/CHem/LC
317
CoLumns for smaLL moLeCuLe seParations
ZorBaX PrepHt eclipse XDB (require hardware 820400-901)
Hardware Description
size (mm)
eclipse
XDB-C18
Particle size (µm) usP L1
eclipse
XDB-C8
usP L7
PrepHT Cartridge
21.2 x 250
7
977250-102 977250-106
PrepHT Cartridge
21.2 x 150
7
977150-102 977150-106
PrepHT Cartridge
21.2 x 150
5
970150-902 970150-906
PrepHT Cartridge
21.2 x 100
5
970100-902 970100-906
PrepHT Cartridge
21.2 x 50
5
970050-902 970050-906
PrepHT Guard Cartridge, 2/pk
17.0 x 7.5
5
820212-925 820212-926
Guard Cartridge Hardware
Includes guard column end fitting, polymeric seal, and seal insertion tool (seal holder and seal pusher)
820444-901 820444-901
PrepHT Endfittings, 2/pk
820400-901 820400-901
ZorBaX PrepHt Bonus-rP and extend-C18 (require hardware 820400-901)
Hardware Description
size (mm)
Bonus-rP
Particle size (µm) usP L60
extend-C18
usP L1
PrepHT Cartridge
21.2 x 250
7
878250-101
PrepHT Cartridge
21.2 x 150
7
878150-101
PrepHT Cartridge
21.2 x 150
5
868150-901 770150-902
PrepHT Cartridge
21.2 x 100
5
868100-901 770100-902
PrepHT Cartridge
21.2 x 50
5
868050-901 770050-902
PrepHT Guard Cartridge, 2/pk
17.0 x 7.5
5
820212-928 820212-930
Guard Cartridge Hardware
Includes guard column end fitting, polymeric seal, and seal insertion tool (seal holder and seal pusher)
820444-901 820444-901
PrepHT Endfittings, 2/pk
820400-901 820400-901
ZorBaX PrepHt rx-siL (require hardware 820400-901)
Hardware Description
size (mm)
rx-siL
Particle size (µm) usP L3
rx-C18
usP L1
PrepHT Cartridge
21.2 x 250
7
877250-101
PrepHT Cartridge
21.2 x 250
7
877967-102
PrepHT Guard Cartridge, 2/pk
17.0 x 7.5
5
820212-919 820212-914
Guard Cartridge Hardware
Includes guard column end fitting, polymeric seal, and seal insertion tool (seal holder and seal pusher)
820444-901 820444-901
PrepHT Endfittings, 2/pk
820400-901 820400-901
ZorBaX PrepHt accessories
Hardware Description
318
Part no.
Guard Cartridge Hardware
820444-901
PrepHT Endfittings, 2/pk
820400-901
Replacement Seals
820385-901
CoLumns for smaLL moLeCuLe seParations
Pursuit and Pursuit Xrs Prep
• Prep-scalable columns for Pursuit and Pursuit XRs columns
• Particle sizes to 10 µm and column diameters up to 50 mm
• High surface area silica
Pursuit and Pursuit XRs Prep columns are designed for high loadability with a high surface area.
natural products – capsaicin and dihydrocapsaicin
on Pursuit Xrs C18
Column a:
Column B:
Pursuit Xrs C18
a6001150X046
4.6 x 150 mm, 3 µm
Pursuit Xrs C18
a6000150X046
4.6 x 150 mm, 5 µm
Column C:
Pursuit Xrs C18
a3002150X046
4.6 x 150 mm, 10 µm
Mobile Phase:
CH3CH:H20 - 70:30
Flow Rate:
1.0 mL/min
Temperature:
Ambient
Detector:
220 nm
Sample:
1. Capsaicin
2. Dihydrocapsaicin
230
220
210
200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
1
mAU
2
3 µm
RT [min]
0
200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
-10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
6
6.5
7
7.5
8
8.5
9
9.5
6
6.5
7
7.5
8
8.5
9
9.5
10
mAU
5 µm
RT [min]
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
10
mAU
240
220
200
Demonstrating an easy, linear scale-up of natural products from
Pursuit XRs C18 3 µm and 5 µm analytical columns to a 10 µm
preparative column.
180
160
140
10 µm
120
100
80
60
40
20
0
RT [min]
0
0.5
1
1.5
2
2.5
3
3.5
4
www.agiLent.Com/CHem/LC
4.5
5
5.5
10
319
CoLumns for smaLL moLeCuLe seParations
agilent Pursuit Prep Columns
Pursuit C18
usP L1
Pursuit C8
usP L7
size (mm)
Particle size
(µm)
10.0 x 250
5
A3000250X100 A3030250X100
10.0 x 250
10
A6002250X100 A3032250X100
21.2 x 250
10
A6002250X212
21.2 x 250
10
A6002250X212 A3032250X212
Pursuit Diphenyl
Pursuit PfP
A3040250X100
A3050250X100
agilent Pursuit Xrs Prep Columns
320
size (mm)
Particle size
(µm)
Pursuit
Xrs C18
usP L1
Pursuit
Xrs C8
usP L7
21.2 x 250
10
A6002250X212
21.2 x 250
5
A6000250X212
21.2 x 150
5
A6000150X212
A6010150X212
21.2 x 100
5
A6000100X212
A6010100X212
21.2 x 50
5
A6000050X212
30.0 x 250
10
A6002250X300
30.0 x 150
10
A6002150X300
30.0 x 250
5
A6000250X300
30.0 x 150
5
A6000150X300
30.0 x 100
5
A6000100X300
50.0 x 250
10
A6002250X500
Pursuit
Xrs Diphenyl
Pursuit
Xrs si
usP L3
A6004250X212
A6020250X212
A6020100X212
A6004250X300
A6010250X300
A6022250X500
A6004250X500
CoLumns for smaLL moLeCuLe seParations
Polaris Prep Columns
• Prep-scalable columns for Polaris phases
• 10.0 and 21.2 mm ids available, with particles up to 10 µm
scale-up from analytical to prep
Column a:
Column B:
Polaris C18-a
a2001050X046
4.6 x 50 mm, 3 µm
1
2
Polaris C18-a
a2000050X046
4.6 x 50 mm, 5 µm
Column C:
Custom column
Mobile Phase:
A: H20 + 0.1% NH40H, pH 10.0 (25 mM)
B: CH3CN
A:B 65:35
Flow Rate:
1.0 mL/min
Temperature:
40 °C
Detector:
215 nm
Sample:
1. Pindolol
2. Metoprolol
3. Propranolol
3
a: 3 µm
1
2
3
B: 5 µm
1
2
C: 10 µm
3
Polaris Prep Columns
size (mm)
Particle size
(µm)
10.0 x 250
5
Polaris
C18-a
Polaris
C18-ether
Polaris
amide C18
Polaris
si-a
A2000250X100 A2020250X100 A2006250X100
Polaris
C8-a
Polaris
C8-ether
Polaris
nH2
A2010250X100 A2030250X100 A2013250X100
21.2 x 250
5
A2000250X212 A2030250X212
A2003250X212 A2010250X212
21.2 x 250
10
A2002250X212
A2004250X212
A2013250X212
Bulk materials for prep
Agilent has bulk materials available for all phases. Most materials and quantities can be ordered through the Custom Column and Bulk Ordering process,
and can be produced in multiple kg. quantities. Quotes are able to be delivered within 48 hours. Contact your Agilent product specialist for support in placing
a custom order.
www.agiLent.Com/CHem/LC
321
CoLumns for smaLL moLeCuLe seParations
Load & Lock Preparative HPLC Column
Packing systems
Agilent offers a complete range of Load & Lock column systems for laboratory and process preparative
LC. They are designed to let you easily and quickly pack your own preparative high efficiency columns.
This is the right solution for applications ranging in scale from development (multigrams) to production
(multi-kilo) of pharmaceutical compounds, peptides, and natural products. Our Load & Lock columns
have a unique fluid/sample distribution system to maximize productivity. The system provides dynamic
axial compression (DAC) and static "locked" axial compression (SAC) and is designed for easy operation
to deliver greater convenience.
Laboratory Load & Lock Columns
Load & Lock Columns
• Mobile packing station supports three different column sizes
• Runs on compressed air with no need for a power supply
• Quick and easy packing and unpacking within minutes
Agilent's laboratory scale Load & Lock columns combine excellent packed-bed stability with enhanced flow
distribution to deliver the highest quality purification possible with maximum speed, flexibility, and ease of
operation. Three different column sizes are supported: 1 in, 2 in and 3 in id. Because the station is powered
by compressed air, it is the perfect solution for hazardous environments. The quick-release single bolt clamp
offers speedy and easy packing and unpacking within minutes.
Load & Lock Preparative HPLC Column Packing systems
Description
Load & Lock 4001 Column
water Jacket
size (mm)
No
25.0 x 500
PCG93LL500X25
Yes
25.0 x 500
PCG93LL500X25WJ
Spare parts kit
Load & Lock 4002 Column
PCG931AAKIT
No
50.0 x 500
PCG93LL500X50
Yes
50.0 x 500
PCG93LL500X50WJ
No
75.0 x 500
PCG93LL500X75
Yes
75.0 x 500
Spare parts kit
Load & Lock 4003 Column
Spare parts kit
Mobile packing station (air driven hydraulic)
322
Part no.
PCG932AAKIT
PCG93LL500X75WJ
PCG933AAKIT
PCG93LLSTAND123
CoLumns for smaLL moLeCuLe seParations
Columns for Other HPLC Techniques
Reproducible results for Normal Phase and beyond
Agilent's extended family of HPLC columns support every technique, providing you with the
Agilent quality you depend on for every application.
• ZORBAX HILIC Plus – good retention of small, polar analytes and high sensitivity for LC/MS –
in Fast LC 1.8 µm options
• ZORBAX normal phase columns – bonded and non-bonded silica packings
• ZORBAX ion-exchange columns – based on rugged ZORBAX Silica, stable from pH 2-7
• Hi-Plex columns for carbohydrate analysis – ligand-exchange columns
• Ultron ES Chiral columns – with two complimentary protein-based chiral stationary phases –
are an excellent choice for enantiomeric separations. Ideal for many pharmacological applications.
www.agiLent.Com/CHem/LC
323
CoLumns for smaLL moLeCuLe seParations
ZorBaX HiLiC Plus
• HILIC column for good retention of small, polar analytes
• Based on Eclipse Plus silica for excellent peak shape
• High sensitivity for LC/MS applications
• Recommended for EPA Method 1694
Agilent ZORBAX HILIC Plus columns are for use in hydrophilic interaction chromatography (HILIC)
applications, which are typically used for the retention and resolution of small polar compounds. HILIC Plus
columns are non-bonded silica columns based on the high performance silica used in ZORBAX Eclipse Plus
columns. This silica provides excellent peak shape, critical for many polar, basic analytes. These columns
ship prepared for use in HILIC mode – containing acetonitrile:water – in order to reduce the extensive
equilibration typically required for HILIC separations. HILIC Plus columns are available in a 3.5 µm particle
size for high resolution and in 2.1 and 4.6 mm id for compatibility with mass spectrometers or with standard
UV detectors.
Column specifications
Phase
Non-bonded silica
Specifications represent typical values only.
tiPs & tooLs
Poroshell 120 HILIC is very similar to
ZORBAX HILIC Plus.
Turn to page 228
324
Pore size
95Å
surface area
2
160 m /g
pH range
0-8.0
CoLumns for smaLL moLeCuLe seParations
separation of group 4 analytes in ePa 1694
on ZorBaX HiLiC Plus column
Column:
ZorBaX HiLiC Plus
959793-901
2.1 x 100 mm, 3.5 µm
metformin
Cimetidine
x102
Mobile Phase:
90% Acetontrile:10% Water
Flow Rate:
0.25 mL/min
Gradient:
Linear gradient to 55% acetonitrile in 7 min
Held at 55%
Temperature:
25 °C
1.00
0.95
0.90
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
albuterol
ranitidine
x102
1.00
0.95
0.90
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
Duplicate runs for column USCJP0004;
10 min equilibration between two runs
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5
EPA1694
ZorBaX HiLiC Plus
Description
size (mm)
Analytical
4.6 x 100
Particle size (µm) Part no.
3.5
959961-901
Analytical
4.6 x 50
3.5
959943-901
Narrow Bore
2.1 x 100
3.5
959793-901
Narrow Bore
2.1 x 50
3.5
959743-901
ZorBaX HiLiC Plus rrHD, stable to 1200 bar
size (mm)
Particle size (µm) Part no.
3.0 x 100
1.8
959758-301
3.0 x 50
1.8
959757-301
2.1 x 150
1.8
959759-901
2.1 x 100
1.8
959758-901
2.1 x 50
1.8
959757-901
Poroshell 120 HiLiC Plus
size (mm)
Particle size (µm)
Part no.
2.1 x 50
2.7
699775-901
2.1 x 100
2.7
695775-901
2.1 x 150
2.7
693775-901
3 x 50
2.7
699975-301
3 x 150
2.7
693975-301
4.6 x 50
2.7
699975-901
4.6 x 100
2.7
695975-901
4.6 x 150
2.7
693975-901
www.agiLent.Com/CHem/LC
325
CoLumns for smaLL moLeCuLe seParations
ZorBaX normal-Phase Columns
For normal-phase chromatography, the Agilent ZORBAX product line offers a choice of bonded and
non-bonded silica packings.
ZORBAX Rx-SIL
• Made from highly pure (> 99.995%) porous silica microspheres (pore size is the space between the
solid silica microparticles)
• Available in 1.8 and 5 µm particle sizes
• Stronger than other silica types
• Less acidic than ZORBAX SIL, lower metal content
• Low acidity and low metal content make ZORBAX Rx-SIL ideal for normal-phase separation of polar
compounds that exhibit poor peak symmetry on more acidic silica
• Useful for very hydrophilic compounds with high organic mobile phases in HILIC mode
ZORBAX Eclipse XDB-CN
• Made from highly pure Rx-SIL
• Excellent choice for normal-phase applications with basic compounds
• Equilibrates more rapidly than ZORBAX Rx-SIL and is used for many of the same
normal-phase applications
326
CoLumns for smaLL moLeCuLe seParations
ZORBAX CN
• Cyanopropyldimethylsilane monolayer bonded to ZORBAX SIL
• Equilibrates more rapidly than ZORBAX SIL, and used for many of the same normal-phase applications
• Less prone to fouling and less water sensitive than silica
Pursuit XRs Si
• 100Å silica for higher surface area and good loadability
• 14.6% carbon load
• Available in 3 µm, 5 µm and 10 µm
Polaris NH2
• 180Å silica for high surface area and loadability
• 5.5% carbon load
• Available in 3 µm, 5 µm, and 10 µm sizes
• Polar-modified with silanol shielding
• Designed for high-aqueous conditions
Polaris Si-A
• 180Å silica with highest surface area and loadability
• Available in 3 µm, 5 µm, and 10 µm
High resolution normal-phase separation of
octylphenoxy ethanol surfactant on ZorBaX Cn
Column:
ZorBaX Cn
880952-705
4.6 x 250 mm, 5 µm
Mobile Phase:
Primary: Heptane
Secondary: 2-Methoxyethanol/Isopropanol (50/50)
Flow Rate:
2 mL/min
Gradient:
2-20% Secondary in 10 min, Linear Hold at 20%
Temperature:
50 °C
Detector:
278 nm
Sample:
Octylphenoxy (polyethylene oxy)
Ethanol Surfactant (n=10)
0
5
10
15
Time (min)
20
LCNP001
Polaris ordering information can be found on pages 300-303
Pursuit ordering information can be found on pages 291-297
www.agiLent.Com/CHem/LC
327
CoLumns for smaLL moLeCuLe seParations
ZORBAX NH2
• Amino-propyl silane phase bonded to ZORBAX SIL
• Used for normal-phase and weak anion-exchange, and reversed-phase HPLC of polar compounds
• Vitamins A and D are separated in the normal-phase mode
• Carbohydrates and sugars are separated in the reversed-phase mode
Column specifications
Phase
ZORBAX Rx-SIL
tiPs & tooLs
Pursuit XRs Silica is another
choice for normal-phase
chromatography. For more
information, see pages 295-296.
328
Pore size surface area pH range endcapped Carbon Load
80Å
180 m2/g
2
0-8.0
No
ZORBAX Eclipse XDB-CN
80Å
180 m /g
2.0-8.0
Yes
ZORBAX SIL
70Å
300 m2/g
0-8.0
No
4.3%
ZORBAX CN
70Å
300 m2/g
2.0-7.0
Yes
7%
ZORBAX NH2
70Å
300 m2/g
2.0-7.0
Yes
4%
CoLumns for smaLL moLeCuLe seParations
normal-Phase Columns Based on ZorBaX rx-siL
Hardware Description
size (mm)
rx-siL**
Particle size (µm) usP L3
eclipse
XDB-Cn
usP L10
standard Columns (no special hardware required)
Semi-Prep
9.4 x 250
5
880975-201
Analytical
4.6 x 250
5
880975-901 990967-905*
Analytical
4.6 x 150
5
883975-901 993967-905*
Rapid Resolution HT, 600 bar
4.6 x 100
1.8
828975-901
Rapid Resolution HT, 600 bar
4.6 x 50
1.8
827975-902
Rapid Resolution HT, 600 bar
3.0 x 100
1.8
828975-301
Rapid Resolution HT, 600 bar
3.0 x 50
1.8
827975-301
Narrow Bore
2.1 x 150
5
Rapid Resolution HT, 600 bar
2.1 x 100
1.8
828700-901
883700-901 993700-905*
Rapid Resolution HT, 600 bar
2.1 x 50
1.8
827700-901
Guard Cartridge, 2/pk
9.4 x 15
5
820675-119
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-919 820950-935
Guard Cartridge, 4/pk
2.1 x 12.5
5
821125-919 821125-935
Guard Hardware Kit
9.4 x 15
guard Columns (hardware required)
840140-901
Guard Hardware Kit
820999-901 820999-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 250
7
PrepHT Endfittings, 2/pk
PrepHT Guard Cartridge, 2/pk
877250-101
820400-901
17.0 x 7.5
Guard Cartridge Hardware
5
820212-919
820444-901
*These columns ship containing reversed-phase solvents. Flush with isopropanol before using normal-phase solvents.
**These columns can also be used in HILIC mode.
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329
CoLumns for smaLL moLeCuLe seParations
normal-Phase Columns Based on ZorBaX original siL
Hardware Description
size (mm)
Particle size
(µm)
siL
usP L3
Cn
usP L10
nH2
usP L8
Carbohydrate
analysis*
standard Columns (no special hardware required)
Semi-Prep
9.4 x 250
5
880952-201 880952-205 880952-208
Analytical
4.6 x 250
5
880952-701 880952-705 880952-708 840300-908
Analytical
4.6 x 150
5
883952-701 883952-705 883952-708 843300-908
Narrow Bore
2.1 x 50
5
Guard Cartridge, 2/pk
9.4 x 15
5
820675-119 820675-111 820675-111
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-901 820950-905 820950-908 820950-908
Guard Cartridge, 4/pk
2.1 x 12.5
5
Guard Hardware Kit
9.4 x 15
860700-708
guard Columns (hardware required)
840140-901 840140-901 840140-901
Guard Hardware Kit
820999-901 820999-901 820999-901 820999-901
PrepHt Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 250
877952-101 877952-105 877952-108
PrepHT Endfittings, 2/pk
820400-901 820400-901 820400-901
Guard Cartridge Hardware
820444-901
*Columns ship in acetonitrile:water and are tested with a mix of sugars.
330
7
CoLumns for smaLL moLeCuLe seParations
Pursuit Xrs si, usP L3
size (mm)
Particle size (µm)
Part no.
semi-Prep scale
10.0 x 250
10
A6004250X100
analytical scale
4.6 x 250
10
A6004250X046
4.6 x 100
5
A6006100X046
4.6 x 50
5
A6006050X046
4.6 x 100
3
A6005100X046
4.6 x 50
3
A6005050X046
2.1 x 100
5
A6006100X021
2.0 x 50
3
A6005050X020
50.0 x 250
10
A6004250X500
30.0 x 250
10
A6004250X300
21.2 x 250
10
A6004250X212
Prep scale
Polaris HPLC Columns
size (mm)
Polaris
Particle size (µm) nH2
50.0 x 250
10
21.2 x 250
10
21.2 x 250
5
Polaris
si-a
A2004250X500
A2004250X212
A2013250X212
A2003250X212
21.2 x 150
5
A2003150X046
21.2 x 50
5
A2003050X212
10.0 x 250
5
A2013250X100
(Continued)
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331
CoLumns for smaLL moLeCuLe seParations
Polaris HPLC Columns
Polaris
Particle size (µm) nH2
size (mm)
4.6 x 250
10
4.6 x 250
5
A2013250X046
4.6 x 150
5
A2013150X046
Polaris
si-a
A2003250X046
A2003150X046
4.6 x 100
5
A2013100X046
A2003100X046
4.6 x 50
5
A2013050X046
A2003050X046
4.6 x 250
3
A2014250X046
A2005250X046
4.6 x 150
3
A2014150X046
A2005150X046
4.6 x 100
3
A2014100X046
A2005100X046
4.6 x 50
3
A2014050X046
A2005050X046
4.0 x 250
5
A2013250X040
A2003250X040
4.0 x 150
5
A2013150X040
A2003150X040
4.0 x 125
5
A2013125X040
A2003125X040
3.0 x 250
5
A2013250X030
A2005250X046
3.0 x 150
5
A2013150X030
A2003150X030
3.0 x 100
5
A2013100X030
A2003100X030
A2014250X030
A2003250X030
3.0 x 50
5
3.0 x 250
3
A2003050X030
3.0 x 150
3
A2014150X030
A2005150X030
3.0 x 100
3
A2014100X030
A2005100X030
3.0 x 50
3
A2014050X030
A2005050X030
2.0 x 250
5
A2013250X020
A2003250X020
2.0 x 150
5
A2013150X020
A2003150X020
2.0 x 100
5
A2013100X020
A2003100X020
2.0 x 50
5
A2013050X020
A2003050X020
2.0 x 30
5
A2013030X020
A2003030X020
2.0 x 20
5
A2013020X020
A2003020X020
2.0 x 250
3
A2014250X020
A2005250X020
2.0 x 150
3
A2014150X020
A2005150X020
2.0 x 100
3
A2014100X020
A2005100X020
2.0 x 50
3
A2014050X020
A2005050X020
2.0 x 30
3
A2014030X020
A2005030X020
2.0 x 20
3
A2014020X020
A2005020X020
metaguard Columns
Hardware
332
Dimensions
Particle size (µm)
Polaris nH2
Polaris si-a
4.6
10
A2004MG
2.0
10
A2004MG2
4.6
5
A2013MG
A2003MG
2.0
5
A2013MG2
A2003MG2
4.6
3
A2014MG
A2005MG
2.0
3
A2014MG2
A2005MG2
CoLumns for smaLL moLeCuLe seParations
Ion-Exchange Columns
ZorBaX ion-exchange Columns – saX and sCX
• ZORBAX SAX and 300SCX columns are based on rugged ZORBAX silica
• Stable from pH 2-7
• Provide high efficiency, rapid separations
• Compatible with organic mobile phase modifiers
Agilent ZORBAX Strong Ion-Exchange columns are available as both Strong Anion-Exchange (SAX) and
Strong Cation-Exchange (300SCX) columns. Each column is packed with bonded, 5 µm, spherical silica
particles for optimum efficiency.
ZORBAX SAX packing has a permanently bonded quaternary amine. A trifunctional organo-silane reagent is
used in producing this packing to maximize its stability with aqueous mobile phases. This column is ideal for
separation of water-soluble compounds such as aromatic and aliphatic carboxylic acids and sulfonic acids.
ZORBAX SCX packing has 300Å pore size silica particles chemically bonded to an aromatic sulfonic acid
group. This column is used for separations of basic, water-soluble compounds and bio-molecules.
Column specifications
Bonded Phase
Pore size surface area pH range
2
functionality
max Pressure
ZORBAX SAX
70Å
300 m /g
2.0-7.0
Quaternary amine
350 bar
ZORBAX 300SCX
300Å
50 m2/g
2.0-7.0
Sulfonic acid
350 bar
Specifications represent typical values only.
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333
CoLumns for smaLL moLeCuLe seParations
Cough/cold remedies on ZorBaX 300sCX
1
Column:
ZorBaX 300sCX
880952-704
4.6 x 250 mm, 5 µm
Mobile Phase:
100 mM NaH2PO4 (pH 6.5)
Flow Rate:
3 mL/min
Temperature:
20 °C
Detector:
210 nm
Sample:
Cold remedies
1.
2.
3.
4.
5.
2
3
0
4
Pyrilamine
Theophylline
Glyceryl Guaicolate
Caffeine
Phenylephrine
5
4
2
Time (min)
LCIE001
ZorBaX ion-exchange Columns – saX and sCX
Description
size (mm)
Particle size
(µm)
saX
Semi-preparative
9.4 x 250
5
880952-203 880952-204
Analytical
4.6 x 250
5
880952-703 880952-704
Analytical
4.6 x 250
5
Analytical
4.6 x 150
5
880952-714*
883952-703 883952-704
Analytical
4.6 x 150
5
883952-714*
Analytical
4.6 x 50
5
846952-704
Solvent Saver
3.0 x 50
5
860700-304
Narrow Bore
2.1 x 150
5
883700-704
Narrow Bore
2.1 x 150
5
883700-714*
Narrow Bore
2.1 x 50
5
Guard Hardware Kit
860700-704
820999-901 820999-901
*These columns have been modified to provide less retention, for those who desire that in their application.
334
300sCX
CoLumns for smaLL moLeCuLe seParations
Hi-Plex Columns for Carbohydrate analysis
• Agilent's recommended column for accurate, low-pressure analysis of typical carbohydrates,
providing leading-edge features for reliable quantitative and qualitative analysis
• Enable reduced column operating pressures for repeatable performance and longer column life
• Wide range of ligand counter ions and column configurations meet requirements of challenging
organic applications
• Simplified HPLC system requirements through isocratic separation capabilities; excellent
batch-to-batch reproducibility for ultimate confidence in your results
• Can be used with water or diluted acid as an eluent
• Available in 8 µm and 10 µm particle sizes in a range of choices for USP media types – L17,
L19, L34 and L58
The least complicated LC methods for detecting sugars, sugar alcohols and organic acids call for
ligand-exhange columns with a simple mobile phase. However, the wide particle size distribution
of conventional resins can lead to high backpressures and reduced productivity.
Hi-Plex columns are engineered with monodisperse sulfonated particles, creating a high-performance
media uniquely suited to stringent USP methods for analyzing carbohydrates, alcohols and organic acids.
Unlike the ZORBAX NH2 column used for carbohydrate analysis with an acetonitrile:water mobile phase,
Hi-Plex ligand-exchange columns provide more resolution for mono- and disaccharides due to the
interaction of the hydroxyl groups with the metal ion associated with the cation-exchange functionality
of the sulfonic acid group.
Column specifications
Bonded Phase
Hi-Plex Ca
temperature
range
flow rate
(mL/min)
eluent
80-90 °C
0.6
Water
Hi-Plex Ca USP L19
80-90 °C
0.3
Water
Hi-Plex Pb
70-90 °C
0.6
Water
Hi-Plex H for carbohydrates
60-70 °C
0.6
Water
Hi-Plex H for organic acids
40-60 °C
0.6
Dilute Acid
Hi-Plex Ca (Duo)
80-90 °C
0.6
Water
Hi-Plex K
80-90 °C
0.6
Water
Hi-Plex Na (Octo)
80-90 °C
0.6
Water, Sodium Hydroxide
Hi-Plex Na
80-90 °C
0.3
Water
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335
CoLumns for smaLL moLeCuLe seParations
Hi-Plex Column Selection
USP methods specify the type of HPLC media and column dimensions which should be used for
the analysis. The Hi-Plex product range has four materials that comply with USP definitions.
media type L17
Strong cation-exchange resin consisting of sulfonated, cross-linked styrene-divinylbenzene copolymer
in the hydrogen form, 7 to 11 µm in diameter – Hi-Plex H.
media type L19
Strong cation-exchange resin consisting of sulfonated, cross-linked styrene-divinylbenzene copolymer
in the calcium form, 9 µm in diameter – Hi-Plex Ca and Hi-Plex Ca (Duo).
media type L34
Strong cation-exchange resin consisting of sulfonated, cross-linked styrene-divinylbenzene copolymer
in the lead form, about 9 µm in diameter – Hi-Plex Pb.
media type L58
Strong cation-exchange resin consisting of sulfonated, cross-linked styrene-divinylbenzene copolymer
in the sodium form, 6 to 30 µm diameter – Hi-Plex Na and Hi-Plex Na (Octo).
In addition to the standard column sizes, the media is also packed in specific column dimensions
for different USP methods, including sugar alcohol analysis.
For some application areas there are several column options, and the choice of the most appropriate
Hi-Plex media will depend on sample matrix and exact carbohydrate composition.
336
CoLumns for smaLL moLeCuLe seParations
Hi-Plex Column selection
application area
recommended Column
USP Methods Specifying L17 Media
Hi-Plex H
USP Methods Specifying L19 Media
Hi-Plex Ca and Hi-Plex Ca (Duo)
USP Methods Specifying L34 Media
Hi-Plex Pb
USP Methods Specifying L58 Media
Hi-Plex Na and Hi-Plex Na (Octo)
Mono- and Disaccharides
Hi-Plex Ca
Hi-Plex Pb
Hi-Plex H
Hi-Plex Na (Octo)
Anomer Separations
Hi-Plex Ca
Organic Acids
Hi-Plex H
Alcohols
Hi-Plex Ca
Hi-Plex K
Hi-Plex H
Hi-Plex Pb
Adulteration of Food and Beverages
Hi-Plex Ca and Hi-Plex Pb
Food Additives
Hi-Plex Ca and Hi-Plex Pb
Dairy Products
Hi-Plex Ca and Hi-Plex H
Sweetened Dairy Products
Hi-Plex Pb
Confectionery
Hi-Plex Ca and Hi-Plex Pb
Fruit Juice
Hi-Plex Ca
Wine
Hi-Plex H
Wood Pulp Hydrolysates (cellulose/hemi-cellulose)
Hi-Plex Pb
Fermentation Monitoring
Hi-Plex H
Oligosaccharides
Hi-Plex Na
Samples with High Salt Content (molasses)
Hi-Plex Na (Octo)
Oligosaccharides <Dp5 with Monosaccharides
Hi-Plex Ca (Duo)
Corn Syrups
Hi-Plex Na
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337
CoLumns for smaLL moLeCuLe seParations
1
analysis of fruit juice
Column:
Hi-Plex Ca
PL1170-6810
7.7 x 300 mm, 8 µm
Mobile Phase:
Water
Flow Rate:
0.6 mL/min
Temperature:
85 °C
Detector:
RI
orange juice
2
0
3
1.
2.
3.
4.
Sucrose
Glucose
Fructose
Sorbitol
1.
2.
3.
4.
5.
Oxalic acid
Citric acid
Tartaric acid
Succinic acid
Lactic acid
20
min
2
3
1
tomato juice
0
20
min
3
apple juice
1
2
4
0
25
min
VLC0009
organic acid analysis
Column:
Mobile Phase:
Hi-Plex H
PL1170-6830
7.7 x 300 mm, 8 µm
2 3
Water with acid as specified
Flow Rate:
0.6 mL/min
Temperature:
60 °C
Detector:
ELS (neb=80 °C,
evap=90 °C,
gas=0.7 SLM), RI
1
4
0.1% tfa using eLs
0
5
15
min
2 3
1
4
5 mm H2so4 using ri
0
min
5
15
VLC0010
338
CoLumns for smaLL moLeCuLe seParations
usP methods for sugar alcohols
Column:
Hi-Plex Ca usP L19
PL1570-5810
4.0 x 250 mm, 8 µm
1
2
3
Mobile Phase:
Water
Flow Rate:
0.3 mL/min
Temperature:
60 °C
Detector:
RI
1.
2.
3.
4.
5.
6.
7.
4
5
6
7
0
25
min
VLC0011
Corn syrup, Hi-Plex
Column:
Hi-Plex na
PL1171-6140
7.7 x 300 mm, 10 µm
Mobile Phase:
Water
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
14-10
1
2
Pressure:
11 bar
Flow Rate:
0.3 mL/min
Temperature:
80 °C
Detector:
RI
3
4
9-5
0
min
Iso-erythritol
Adonitol
Arabitol
Mannitol
Xylitol
Dulcitol
Sorbitol
Dp1
Dp2
Dp3
Dp4
Dp5
Dp6
Dp7
Dp8
Dp9
Dp10
Dp11
Dp12
Dp13
Dp14
30
VLC0012
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339
CoLumns for smaLL moLeCuLe seParations
analysis of sweeteners on Hi-Plex Ca columns
Column:
Hi-Plex Ca
PL1170-6810
7.7 x 300 mm, 8 µm
Mobile Phase:
Water
Flow Rate:
0.6 mL/min
Temperature:
85 °C
Detector:
ELSD
3
12
0
7
5
4
8
6
1.
2.
3.
4.
5.
6.
7.
8.
Stachyose
Raffinose
Sucrose
Glucose
Galactose
Fructose
Mannitol
Sorbitol
1.
2.
3.
4.
Maltotriose
Lactose
Glucose
Fructose
23
min
Hi-Plex Ca columns are ideal for analyzing most sweeteners,
including glucose and fructose (monosaccharides), sucrose
(disaccharide), and mannitol and sorbitol (sugar alcohols).
analysis of carbohydates on Hi-Plex H columns
Column:
1
Hi-Plex H
PL1170-6830
7.7 x 300 mm, 8 µm
Mobile Phase:
Water
Flow Rate:
0.6 mL/min
Temperature:
70 °C
Detector:
RI
2
3
4
0
For carbohydrate analysis of samples containing high levels of
organic acids, Hi-Plex H columns deliver sharp, reproducible peaks.
Note, however, that some sugars (such as raffinose) can undergo acid
hydrolysis even when water is used as the eluent.
340
min
15
CoLumns for smaLL moLeCuLe seParations
analysis of sugars with high sodium matrix
Column:
1
Hi-Plex na (octo)
PL1170-6840
7.7 x 300 mm, 8 µm
Mobile Phase:
0.015 M NaOH
Flow Rate:
0.6 mL/min
Temperature:
85 °C
Detector:
RI
Food products containing high levels of sodium ions are
best analyzed with Hi-Plex Na (Octo) columns. This saves
time when sodium hydroxide is used as the eluent with
PAD, because it eliminates the need for the post-column
addition of sodium hydroxide.
1. Sucrose
2. Glucose
3. Arabinose
3
2
0
23
min
usP method for sorbitol
Column:
Hi-Plex Pb usP L34
PL1170-2820
7.7 x 100 mm, 8 µm
Mobile Phase:
Water
Flow Rate:
0.7 mL/min
Temperature:
50 °C
Detector:
RI
USP method for sorbitol – a sugar alcohol and
alternative sweetener – using mannitol as the internal
standard. Hi-Plex Pb columns are recommended for
alcoholic drinks that also contain glycerol, as well as
sweetened dairy-based food products.
1
1. Mannitol
2. Sorbitol
2
0
www.agiLent.Com/CHem/LC
min
23
341
CoLumns for smaLL moLeCuLe seParations
Hi-Plex Columns for Carbohydrate analysis
size (mm)
Particle
size (µm)
Crosslink
Content (%)
Counter
ion
Hi-Plex Ca USP L19
4.0 x 250
8
8
Ca2+
PL1570-5810
Hi-Plex Ca (Duo)
6.5 x 300
8
8
Ca2+
PL1F70-6850
Hi-Plex Ca
7.7 x 300
8
8
Ca2+
PL1170-6810
Hi-Plex Pb USP L34
7.7 x 100
8
8
Pb2+
PL1170-2820
PL1170-6820
Description
Part no.
Hi-Plex Pb
7.7 x 300
8
8
Pb2+
Hi-Plex K
7.7 x 300
8
8
K+
PL1170-6860
PL1F70-6830
Hi-Plex H
6.5 x 300
8
8
H+
Hi-Plex H
7.7 x 300
8
8
H+
PL1170-6830
PL1170-2823
Hi-Plex H USP L17
7.7 x 100
8
8
H+
Hi-Plex Na
7.7 x 300
10
4
Na+
PL1171-6140
Hi-Plex Na (Octo)
7.7 x 300
8
8
Na+
PL1170-6840
size (mm)
Particle
size (µm)
Crosslink
Content (%)
Counter
ion
PL1170-1810
Hi-Plex guard Columns
Description
Part no.
Hi-Plex Ca
7.7 x 50
8
8
Ca2+
Hi-Plex Ca (Duo)
7.7 x 50
8
8
Ca2+
PL1170-1850
PL1170-1820
Hi-Plex Pb
7.7 x 50
8
8
Pb2+
Hi-Plex K
7.7 x 50
8
8
K+
PL1170-1860
PL1170-1830
Hi-Plex H
7.7 x 50
8
8
H+
Hi-Plex Na
7.7 x 50
10
4
Na+
PL1171-1140
Hi-Plex Na (Octo)
7.5 x 50
8
8
Na+
PL1170-1840
Particle
size (µm)
Crosslink
Content (%)
Counter
ion
PL1670-0810
Hi-Plex guard Cartridges, 2/pk
Description
size (mm)
Hi-Plex Ca
3.0 x 5.0
8
8
Hi-Plex Ca (Duo)
3.0 x 5.0
8
8
Ca2+
PL1670-0850
PL1670-0820
Hi-Plex Pb
3.0 x 5.0
8
8
Pb2+
Hi-Plex K
3.0 x 5.0
8
8
K+
PL1670-0860
Hi-Plex H
3.0 x 5.0
8
8
H+
PL1670-0830
PL1671-0140
PL1670-0840
Hi-Plex Na
3.0 x 5.0
10
4
Na+
Hi-Plex Na (Octo)
3.0 x 5.0
8
8
Na+
Guard Cartridge holder for 3.0 x 5.0 mm cartridges
342
Part no.
Ca2+
PL1310-0016
CoLumns for smaLL moLeCuLe seParations
Quick Guide to USP Designations
for HPLC Columns
The US Pharmacopeia (USP) is a standard source for many pharmaceutical methods. The USP specifies
columns by packing materials rather than by manufacturer. The USP has updated its L1 definitions, listed
below you will see the most recent definitions and columns that apply. Rapid Resolution High Throughput
(RRHT) columns are now choices in the L1, L7, and L11 categories.
usP Designations
usP
method
usP Packing materials
Column
Particle size
(µm)
Pore size
(Å)
L1
Octadecyl silane chemically bonded to porous silica or ceramic
microparticles, 1.5 to 10 µm in diameter, or a monolithic rod
Poroshell 120 EC-C18
Poroshell 120 SB-C18
Poroshell 300SB-C18
Poroshell 300 Extend-C18
ZORBAX Eclipse Plus C18
ZORBAX Eclipse XDB-C18
ZORBAX StableBond SB-C18
ZORBAX Rx-C18
ZORBAX Extend-C18
ZORBAX ODS
ZORBAX ODS classic
Pursuit XRs C18
Pursuit C18
Pursuit C18-A
Polaris C18-Ether
SepTech ST60 C18
SepTech ST150 C18
Agilent Prep C18
2.7
2.7
5
5
1.8, 3.5, 5
1.8, 3.5, 5, 7
1.8, 3.5, 5, 7
3.5, 5
1.8, 3.5, 5, 7
3, 5, 7
5
3, 5, 10
3, 5, 10
3, 5, 10
3, 5
10
10
5, 10
120
120
300
300
95
80
80, 300
80
80, 300
70
70
100
200
180
200
60
150
100
L3
Porous silica particles, 1.5 to 10 µm in diameter, or a monolithic silica
rod
ZORBAX HILIC Plus
ZORBAX SIL
ZORBAX Rx-SIL
Pursuit XRs Si
Polaris Si-A
Agilent Prep
1.8, 3.5
5
3.5, 5, 7
3, 5, 10
5, 10
5, 10
95
70
80, 300
100
180
100
L7
Octylsilane chemically bonded to totally porous silica particles, 1.5 to 10 Poroshell 120 EC-C8
µm in diameter, or a monolithic silica rod
Poroshell 120 SB-C8
Poroshell 300SB-C8
ZORBAX Eclipse Plus C8
ZORBAX Eclipse XDB-C8
ZORBAX SB-C8
ZORBAX Rx-C8
ZORBAX C8
Pursuit XRs C8
Pursuit C8
Polaris C8-A
Polaris C8-Ether
2.7
2.7
5
1.8, 3.5, 5
1.8, 3.5, 5, 7
1.8, 3.5, 5, 7
1.8, 3.5, 5, 7
5
3, 5, 10
3, 5, 10
3, 5
3, 5
120
120
300
95
80
80, 300
80
70
100
200
180
200
(Continued)
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343
CoLumns for smaLL moLeCuLe seParations
usP Designations
usP Packing materials
Column
Particle size
(µm)
Pore size
(Å)
L8
An essentially monomolecular layer of aminopropylsilane chemically
bonded to totally porous silica gel support, 3 to 10 µm in diameter
ZORBAX NH2
Polaris NH2
5
5
70
180
L9
Irregular or spherical, totally porous silica gel having a
chemically bonded, strongly acidic cation-exchange coating,
3 to 10 µm in diameter
ZORBAX SCX
5 spherical
300
L10
Nitrile groups chemically bonded to porous silica particles,
3 to 10 µm in diameter
ZORBAX CN
ZORBAX SB-CN
ZORBAX Eclipse XDB-CN
5
3.5, 5
3.5, 5
70
80, 300
80
L11
Phenyl groups chemically bonded to porous silica particles,
1.5 to 10 µm in diameter
ZORBAX Eclipse XDB Phenyl
ZORBAX Eclipse Plus Phenyl-Hexyl
ZORBAX Phenyl
Poroshell 120 Phenyl-Hexyl
Pursuit XRs DiPhenyl
Pursuit DiPhenyl
5
1.8, 3.5, 5
3.5
2.7
3, 5, 10
3, 5, 10
70
95
80
120
100
200
L13
Trimethylsilane chemically bonded to porous silica particles,
3 to 10 µm in diameter
ZORBAX TMS
5
70
L14
Silica gel having a chemically bonded, strongly basic quaternary
ammonium anion-exchange coating, 5 to 10 µm in diameter
ZORBAX SAX
IonoSpher A
5
5
70
120
L17
Strong cation-exchange resin consisting of sulfonated cross-linked
styrene-divinylbenzene copolymer in the hydrogen form, 7 to 11 µm
in diameter
Hi-Plex H
8
N/A
L19
Strong cation-exchange resin consisting of sulfonated cross-linked
Hi-Plex Ca
styrene-divinylbenzene copolymer in the calcium form, 9 µm in diameter Hi-Plex Ca (Duo)
8
8
N/A
N/A
L20
Dihydroxypropane groups chemically bonded to porous
silica particles, 3 to 10 µm in diameter
5
N/A
usP
method
LiChrospher Diol
(Continued)
344
CoLumns for smaLL moLeCuLe seParations
usP Designations
usP
method
L21
Particle size
(µm)
Pore size
(Å)
PLRP-S
3, 5, 8, 10, 1015, 15-20, 50
100
PLRP-S
3, 5, 8, 10, 1015, 15-20, 50
300
usP Packing materials
Column
A rigid spherical styrenedivinylbenzene copolymer,
5 to 10 µm in diameter
PLRP-S
5, 8, 10, 30, 50
1000
PLRP-S
5, 8, 10, 30, 50
4000
PLgel
3, 5, 10, 20
50, 100, 500,
103, 105,
105, 106,
MIXED
L22
A cation-exchange resin made of porous polystyrene gel
with sulfonic acid groups, about 10 µm in size
Hi-Plex H
8
N/A
L25
Packing having the capacity to separate compounds with a MW range
from 1,000 to 5,000 da (as determined by the polyethylene oxide),
applied to neutral, anionic and cationic water-soluble polymers. A
polymethacrylate resin base, crosslinked with polyhydroxylated ether
(surface contained some residual carboxyl functional gruops) was
found suitable
PL aquagel-OH
5, 8
30
L33
Packing having the capacity to separate dextrons by molecular size
over a range of 4,000 to 500,000 da. It is spherical, silica-based,
and processed to provide pH stability
ZORBAX GF-250
4
150
Bio SEC-3
3
100, 150,
300
Bio SEC-5
5
100, 150,
300, 500,
1000, 2000
ProSEC
5
300
(Continued)
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345
CoLumns for smaLL moLeCuLe seParations
usP Designations
usP
method
346
usP Packing materials
Column
Particle size
(µm)
Pore size
(Å)
L34
Strong cation-exchange resin consisting of sulfonated cross-linked
styrene-divinylbenzene copolymer in the lead form, about 9 µm in
diameter
Hi-Plex Pb
8
N/A
L35
A zirconium-stabilized spherical silica packing with a hydrophilic (dioltype) molecular monolayer bonded phase having a pore size of 150Å
ZORBAX GF-250
ZORBAX GF-450
4
6
150, 300
L43
Pentafluorophenyl groups chemically bonded to silica particles by a
propyl spacer, 5 to 10 µm in diameter
Pursuit PFP
3, 5
200
L45
Beta cyclodextrin bonded to porous silica particles, 5 to 10 µm in
diameter
ChiraDex Chiral
5
100
L50
Multifunction resin with reversed-phase retention and strong anionZORBAX 300SCX
exhange functionalities. The resin consists of ethylvinylbenzene, 55%
cross-linked with divinylbenzene copolymer, 3 to 15 µm in diameter, and
a surface area of not less than 350 m2 per g. Substrate is coated with
quarternary ammonium functionalized latex particles consisting of
styrene cross-linked with divinylbenzene
5
300
L52
Weak cation-exchange resin made of porous silica with sulfopropyl
groups, 5 to 10 µm in diameter
5
120
L53
Bio SAX
Weak cation-exchange resin consisting of ethylvinylbenzene, 55%
crosslinked with divinylbenzene copolymer, 3 to 15 µm diameter.
Substrate is surface grafted with carboxylic acid and/or phosphoric acid
functionalized monomers. Capacity not less than 400 µEq/column
3, 5, 10
300
L56
Propyl silane chemically bonded to totally porous silica particles, 3 to 10 ZORBAX SB-C3
µm in diameter
3, 5
80
L57
A chiral-recognition protein, ovomucoid, chemically bonded to silica
particles, about 5 µm in diameter, with a pore size of 120Å
5
120
L58
Strong cation-exchange resin consisting of sulfonated cross-linked
Hi-Plex Na
styrene-divinylbenzene copolymer in the sodium form, about 6 to 30 µm Hi-Plex Na (Octo)
in diameter
10
8
N/A
N/A
L60
Spherical, porous silica gel, 10 µm in diameter, the surface has been
covalently modified with alkyl amide groups and endcapped
1.8, 3.5, 5
2.7
3, 5
80
120
180
IonoSpher C
Ultron ES-OVM
ZORBAX Bonus-RP
Poroshell 120 Bonus-RP
Polaris Amide-C18
CoLumns for smaLL moLeCuLe seParations
Oligo Solutions
stratospheres Dna Cartridges
• Greater yields of full length products than controlled-pore glass
• Inert support prevents side reactions and improves quality of the end product
• 1000Å pore size permits synthesis of longer oligonucleotide sequences, up to 70-mer
• Certificate of Analysis offered for every batch
StratoSpheres DNA Cartridges
StratoSpheres DNA Synthesis Cartridges make it easy to obtain high-quality synthetic DNA oligonucleotides.
The high-yielding polystyrene packing delivers more full-length product than conventional controlled-pore
glass supports. In addition, the hydrophobic nature of the polystyrene promotes coupling and minimizes
non-specific binding to maximize production efficiency. These high-throughput cartridges deliver very
economical oligonucleotide synthesis, and provide the high performance expected from macroporous
polystyrene supports. StratoSpheres DNA synthesis cartridges deliver maximum flexibility in high-throughput
environments.
stratospheres Dna Cartridges
Description
StratoSpheres DNA DMT bz dA
StratoSpheres DNA DMT bz dC
StratoSpheres DNA DMT ac dC
size
(nmol)
Part no.
40
PL3554-1602dAbz
200
PL3554-4602dAbz
40
PL3554-1602dCbz
200
PL3554-4602dCbz
40
PL3554-1602dCac
200
PL3554-4602dCac
StratoSpheres DNA DMT ibu dG
40
PL3554-1602dGibu
200
PL3554-4602dGibu
StratoSpheres DNA DMT dmf dG
40
PL3554-1602dGdmf
200
PL3554-4602dGdmf
40
PL3554-1602dT
200
PL3554-4602dT
StratoSpheres DNA DMT dT
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347
CoLumns for smaLL moLeCuLe seParations
toP, toP-Dna and toP-rna Cartridges
• Superior yield and purity come from proprietary polymeric resins and optimized buffers
• Typical yield is more than 85% and typical purity is over 90%, eliminating the need for multiple
sample-loading steps
• Agilent TOP cartridges use up to two thirds less reagent than products from other vendors
TOP, TOP-DNA and TOP-RNA Cartridges
TOP, TOP-DNA and TOP-RNA cartridges provide a high-throughput, simple, cost-effective solution for
DNA and RNA oligonucleotide purification. The TOP product range incorporates a unique 96-well plate
with removable tubes, streamlined gravity flow or vacuum procedure, and proprietary polymeric resin.
Agilent's innovative technology delivers superior yield and purity for standard oligos up to 1 µmol synthesis
scale and up to 150-mer in length. Flexibility is assured from a choice of simple gravity flow (for walk-away
and low initial setup cost) or vacuum procedure (for fast turnaround – less than 15 minutes for the entire
purification process). Up to 10 minutes drying time between each step is permissible with no effect on
purification results (drying time after the acetonitrile conditioning step should be kept to a minimum).
TOP and TOP-DNA Cartridges
• Fast throughput improves production efficiency
• Pre-HPLC "sample prep" ability maximizes utility
• Gravity (TOP) or vacuum flow (TOP-DNA) ensures flexibility
TOP-DNA is a high-throughput, simple, fast, cost-effective solution that purifies oligos up to 150-mer
in length. Its high binding capacity can purify DNA oligos from 200 nmol to 1 µmol synthesis scales.
TOP-DNA can also be used for sample preparation before HPLC purification for very high quality oligos
in large-scale analysis. The proprietary polymeric resin is compatible with direct loading of AMA
deprotected oligo solutions.
tiPs & tooLs
For more information on TOP RNA, view this Application Note on-line: High Performance RNA oligonucleotide purification
using Agilent TOP-RNA (publication # 5990-8974EN), www.agilent.com/chem/library
348
CoLumns for smaLL moLeCuLe seParations
TOP-RNA Cartridges
• A complete solution for RNA oligo purification to enhance productivity
• High throughput and automation friendly, freeing up operator time
• Less reagent use reduces operating costs
With TOP-RNA you can purify short and long RNA oligos, siRNA to 21-mer and long RNA to 60-80-mer.
The high binding capacity purifies RNA oligos up to 1 µmol. The proprietary polymeric resin and validated
protocol allow deprotection of 2'hydroxyl group without removal of the 5'trityl group.
toP, toP-Dna and toP-rna Cartridges
Description
sorbent
mass (mg)
Volume
(mL)
unit
Part no.
7573915C
TOP-RNA well plate tubes for 1 µmol scale
100
1.8
96/pk
TOP-RNA well plate tubes for 1 µmol scale
100
1.8
20 x 96/pk 7573915B
TOP-DNA well plate tubes for 1 µmol scale
150
1.8
96/pk
7572915C
TOP cartridge
500
6
30/pk
12102301
TOP cartridge
300
6
30/pk
12102300
Mega Bond Elut TOP
3g
20
20/pk
14251921
TOP-DNA well plate tubes for 1 µmol scale
150
1.8
20 x 96/pk 7572915B
TOP well plate tubes for 50 nmol scale
25
1.8
96/pk
75719025
TOP well plate tubes for 200 nmol scale
50
1.8
96/pk
75719050
TOP well plate tubes for 200 nmol scale,
high capacity
100
1.8
96/pk
7571901C
96-well plate sealing mat
50/pk
5133005
Disposable waste tray
25/pk
5133001
100/pk
75700001
TOP reusable base plate
VersPlate Base Plate
75400001
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349
CoLUMNS for BioMoLeCULe SePArATioNS
Table of Contents
LC and LC/MS Columns for Biomolecules
Biocolumn Selection Guidelines ..................................... 351
Method Development........................................................ 444
Biomolecule Separations ................................................. 353
ZORBAX Column Methods...............................................444
UHPLC/HPLC Techniques ................................................ 364
Reversed-Phase LC/MS Methods..................................446
Reversed-Phase HPLC ......................................................365
Bio Ion-Exchange Column Methods...............................447
ZORBAX 300Å StableBond ..............................................367
SEC Column Methods .......................................................449
ZORBAX 300Å Extend-C18...............................................376
High Sensitivity Capillary Column Methods..................451
Poroshell 300 ......................................................................380
Capillary and Nano Columns ........................................... 452
Poroshell 120 ......................................................................385
2-D LC/MS Analyses Using ZORBAX
Capillary and Nano LC Columns......................................456
PLRP-S .................................................................................387
MicroBore (1.0 mm id) Columns..................................... 461
Ion-Exchange Chromatography .......................................397
Purification – Prep HPLC.................................................. 464
Agilent Bio MAb HPLC Columns ....................................399
ZORBAX PrepHT ................................................................466
Agilent Bio IEX HPLC Columns .......................................402
PLRP-S for Prep to Process..............................................467
PL-SAX Strong Anion-Exchange Columns ....................406
PL-SAX and PL-SCX for Prep to Process.......................472
PL-SCX Strong Cation-Exchange Columns....................410
Peptide Purification ...........................................................475
Agilent Bio-Monolith Ion-Exchange
HPLC Columns....................................................................412
VariTide RPC Columns for Synthetic Peptides .............475
Size Exclusion Chromatography......................................416
Agilent Bio SEC-3 ..............................................................418
Agilent Bio SEC-5 ..............................................................424
ProSEC 300S .......................................................................428
ZORBAX GF-250 and GF-450
Gel Filtration Columns ......................................................431
Affinity Chromatography ..................................................434
Agilent Bio-Monolith Protein A HPLC Columns...........434
Agilent Protein Fractionation System
and Proteomics Reagents ................................................437
Multiple Affinity Removal System ..................................438
Multiple Affinity Removal System Starter Kits.............441
mRP-C18 High-Recovery Protein Columns....................442
350
ZORBAX Bio-SCX Series II...............................................458
ZORBAX Amino Acid Analysis (AAA)
Columns and Supplies ......................................................394
VariPure IPE ........................................................................476
Appendices......................................................................... 477
BioHPLC Columns Literature ...........................................477
ZORBAX 300 Citations ......................................................485
Poroshell 300 Citations.....................................................486
PLRP-S Citations ................................................................486
PL-SAX Citations................................................................487
PL-SCX Citations................................................................487
CoLUMNS for BioMoLeCULe SePArATioNS
From sample purification to analysis, Agilent's biomolecule columns and supplies are easy to integrate into
your workflow for a complete, reproducible, and high-quality solution.
In this section of the catalog you will also find advice and tips on solvent choice, mobile phase modification,
optimization, and example separations to assist you in column selection and method development.
Agilent has complete solutions for your needs. These include the Agilent 1260 Infinity Bio-inert LC system
with a metal-free sample path and the Agilent 1290 Infinity LC, designed to provide highest speed,
resolution, and ultra-sensitivity for UHPLC applications, including those utilizing Agilent wide-pore 300Å
ZORBAX StableBond columns. Biomolecules may be complex in structure, but their analysis is simplified
by using Agilent HPLC columns, systems, and supplies.
What is a biomolecule?
Biomolecules are compounds made by living organisms. They can range in size from amino acids and small
lipids to large polynucleotides such as DNA or RNA.
In this section, we deal with the separation of:
Proteins – separation based on size with size exclusion chromatography, charge with ion-exchange
chromatography, and hydrophobicity with reversed-phase chromatography.
Peptides – biocolumns for the analysis and purification of the full range of peptides, including hydrophobic,
hydrophilic, basic and acidic peptides across the full size range. Also, columns for peptide mapping by HPLC
and UHPLC.
DNA/rNA oligonucleotides – reversed-phase and ion-exchange options for DNA and RNA oligos,
and with particle pore sizes to cover the full range of oligonucleotide sizes, from small synthetic oligos to
large plasmids.
Amino acids – the ZORBAX Eclipse Amino Acid Analysis HPLC columns provide a high efficiency solution
for rapid analysis of 24 amino acids. Typical analysis times range from 14 minutes, with a 75 mm column,
to 24 minutes with a 150 mm column.
Broad-distribution polymers – analysis of lipids, polysaccharides and drug delivery compounds
using polymeric columns and standards to determine their molecular weight distribution and composition.
These compounds tend to exhibit broad MW distributions, in contrast to other biomolecules that have
narrow MW distributions or a defined molecular weight.
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351
CoLUMNS for BioMoLeCULe SePArATioNS
What is a biocolumn?
Biochromatography columns, or biocolumns, are liquid chromatography columns used for the separation
of biological compounds such as peptides and proteins, oligonucleotides and polynucleotides, and other
biomolecules and complexes. Biocolumns are specifically designed for biomolecule analysis with larger
pore sizes to accommodate the larger molecule sizes. Media is designed to minimize non-specific binding
of analytes for improved recovery. Separation mechanisms are chosen to either retain biological function
so bioactivity is not lost during analysis, or to deliberately denature for primary structure characterization.
Typically, HPLC has been used to separate biomolecules. Now, advanced techniques such as UHPLC are
becoming a popular choice because multiple separation mechanisms are needed in the characterization of
biomolecules. Therefore, Agilent offers advanced chemistries developed specifically for the separation of
biomolecules using size exclusion, reversed-phase, ion-exchange, and affinity functionalities, all of which are
covered in this section of the catalog.
352
CoLUMNS for BioMoLeCULe SePArATioNS
Protein Separations
Proteins are complex molecules that require multiple techniques to provide full characterization. They exist as three-dimensional structures and it is this
structure that confers their biological activity.
The sequence of the amino acid chains defines the primary structure of the protein. Hydrogen bonding between amino acids of the primary structure then
confers a secondary structure typically in the form of alpha helices and pleated sheets. A further series of interactions, hydrogen bonding, ionic, hydrophobic
and disulphide bridges, between regions of the secondary structure, then provides the tertiary protein structure, or three-dimensional conformation. If the
protein is composed of a number of amino acid chains, the interaction between these chains gives the quaternary structure.
When looking at methods for protein characterization, it is therefore clear from Figure 1 that techniques will be required that characterize the protein in its
native state, without disrupting the tertiary and quaternary structures. We also need techniques for assessing the primary amino acid sequence, in the fully
denatured state with the three-dimensional structure stripped away.
Primary protein structure
is a sequence of amino acids
Amino
acids
Pleated
sheet
Alpha
helix
Hydrogen bonds
Pleated
sheet
Hydrogen bonds
Ionic interactions
Hydrophobic interactions
Disulphide bridges
Alpha
helix
Secondary protein structure
occurs when the sequence of amino acids are linked by hydrogen bonds
Tertiary protein structure
occurs when certain attractions are present between alpha helices
and pleated sheets
Quaternary protein structure
is a protein consisting of more than one amino acid chain
figure 1. Schematic showing the various levels of protein structure.
The environment of the protein can influence, stabilize, or disrupt the structure of the protein. Factors to consider include pH, temperature, salt
concentrations, aqueous or organic solvent content, and for some proteins, the presence of a stabilizing small molecule or metal ion. Protein structure
can also be disrupted by the use of sulfhydryl reducing agents to break -S-S- bonds or chaotropic agent, like urea or guanidine HCI. With the complexity
of proteins and the intramolecular interactions that determine the three-dimensional structure, you can also expect that there will be intermolecular
associations between protein molecules and other molecular entities and the surfaces with which they come into contact. This can result in protein
complexes, aggregation (with possible precipitation), and deposition on surfaces, including those of the HPLC column and system. Therefore, you
should consider the handling and environment in which the protein is maintained.
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CoLUMNS for BioMoLeCULe SePArATioNS
Protein Column Selection Guide
Application
Technique
Agilent Columns
Notes
Primary structure
analysis
UHPLC/HPLC
reversed-phase
separations
ZORBAX 300SB
Poroshell 300SB
PLRP-S
Reversed-phase separations require (or cause) denaturing of the protein to obtain
detailed information about the amino acid sequence and/or amino acid modifications
(including post-translational modifications).
Aggregation
analysis
Size exclusion
separations
Bio SEC-3
Bio SEC-5
ProSEC 300S
ZORBAX GF
Aggregates in protein biopharmaceuticals are of major concern as they can induce an
immunogenic response and can influence the composition of the final formulation.
Charge variant
analysis
Ion-exchange
separations
Agilent Bio IEX
Agilent Bio MAb
PL-SAX
PL-SCX
The ratio of individual amino acids determines the net charge of the protein molecule.
The pH at which the net charge is zero is called the isoelectric point (pI). When the
solution pH is less than the pI, the protein will be positively charged (acidic), and when
the solution pH is greater than the pI, the protein is negatively charged (basic).
For ion-exchange analysis, we recommend the eluent pH be at least one pH unit
away from its pI. Protein analysis using ion-exchange columns requires buffered
mobile phase and either salt gradients or pH gradients for elution.
Higher resolution of oxidation study
17.5
15
12.5
Flow Rate:
1.0 mL/min
Gradient:
33 to 50% B, 0 to 4 min
10
Detector:
1290 Infinity LC with diode array detector
at 280 nm
7.5
Sample:
Insulin, insulin chain A and chain B,
oxidized (bovinesigma, 1 mg/mL)
oxidized insulin
chain B
insulin
oxidized insulin
chain A
5
1.648
A: 0.1% TFA
B: 0.01% TFA + 80% ACN
mAU
1.384
Mobile Phase:
ZorBAX rrHD 300SB-C18
857750-902
2.1 x 50 mm, 1.8 µm
0.148
Column:
Subspecies of oxidized
insulin chain B
2.5
0
min
It is evident that the oxidized insulin chains are resolved from insulin in under 2 minutes using the
Agilent ZORBAX RRHD 300SB-C18 2.1 x 50 mm, 1.8 µm column.
354
CoLUMNS for BioMoLeCULe SePArATioNS
intact MAb monomer and dimer separation
Column:
Bio SeC-3, 300Å
5190-2511
7.8 x 300 mm, 3 µm
Buffer:
Sodium phosphate buffer, pH 7.0, 150 mM
Isocratic:
0-100% Buffer A from 0-30 min
Flow Rate:
1.0 mL/min
Sample:
CHO-humanized MAb, 5 mg/mL – intact
Injection:
5 µL
Detector:
UV 220 nm
Temperature:
Ambient
mAU
300
Monomer
250
Dimer
200
150
100
50
0
4
6
8
10
12
14 min
VLC_MD_Mab
Separation of charge variants of human igG1
with pH gradient
Column:
Mobile Phase:
Agilent Bio MAb
5190-2411
2.1 x 250 mm, 5 µm
A: 10 mM Na2HPO4, pH 6.0
B: A + 0.5 M NaCl or just 0.5 M Na2HPO4, pH 6.0
Flow Rate:
2 mL/min
Gradient:
0.5 min hold with mobile phase A followed by a linear
gradient to 45% B in 15 min (elapsed time 15.5 min);
then 60% B at 15.6 min continued to 20 min.
Column flushed with 100% B for 15 min before
re-equilibration for the next run.
pH Gradient: A: 5 mM Na2HPO4, buffer pH 5.5
and B: 40 mM NA2HPO4 (not buffered, pH 8.9).
2% B/min at 1 mL/min for 15 min, followed by
a column wash with 90% B for 5 min.
Detector:
UV at 220 nm
Sample:
One mg each/mL in mobile phase A
Monoclonal antibodies (MAb) -human IgG1 (5 mg/mL
stock solution) derived from CHO cells
Instrument:
Before carboxypeptidase B digestion
After carboxypeptidase B digestion
mAU
50
40
30
20
10
0
10
10.5
11
11.5
12
12.5
13
13.5
14
14.5 min
Agilent 1200 SL system with diode array detector
MAb c-terminal cleavage: Human IgG1 MAb, 1 mg/mL in 25 mM Na2HPO4 buffer, pH 7.5, was incubated with approximately 25 units of the
carboxypeptidase B for 18 hours and 10 µL samples were injected.
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CoLUMNS for BioMoLeCULe SePArATioNS
Peptide Separations
Peptide Mapping
Peptide mapping is required for the characterization of proteins. It is used to confirm the identity of a protein
and to identify and quantify post-translational modifications.
The purified protein is first digested using an enzyme, such as trypsin, yielding a range of peptide fragments.
The specificity of the enzyme cleavage produces a fingerprint of peptides which is characteristic of that
protein. Identification of the peptide fragments confirms the identity of the protein, and changes in the
profile of the peptide digest can be used to identify post-translational modifications to that protein that
may have occurred during the manufacturing or purification processes.
Reversed-phase UHPLC/HPLC is the preferred technique for the analysis of peptide digests with either
MS or UV detection. LC/MS is used for the identification of the peptide fragments and determination
of sequence coverage whereas LC/UV is more commonly used for peptide map comparisons in the
monitoring/QC segments. To achieve sufficient resolution for quantification and identification, longer column
lengths or higher efficiency particles such as the sub-2 µm ZORBAX RRHD, or superficially porous Poroshell
are recommended.
Peptide digests are complex mixtures, and for complete coverage, i.e. resolution of the individual peptides,
a high efficiency/high resolution column is required. The peptide fragments can range in size and
hydrophobicity, so Agilent offers several columns for peptide mapping. There are three options: pore sizes,
particle sizes, and superficially porous and fully porous for UHPLC separations.
TiPS & TooLS
Capillary electrophoresis is an alternative technique to liquid chromatography for the separation of complex peptide mixtures. Further information
can be found in the following Case Study:
An orthogonal view of peptide mapping – analysis of bovine serum albumin digest using capillary electrophoresis and quadrupole time-of-flight
mass spectrometry (publication # 5990-7631EN)
www.agilent.com/chem/library
356
CoLUMNS for BioMoLeCULe SePArATioNS
Peptide Mapping Column Selection
Recommended column choices determined by system/column pressure maximum and peptide size/hydrophobicity.
Application
Technique
Agilent Columns
Notes
Large peptide
fragments/hydrophobic
peptide core
400 bar HPLC
Poroshell 300 SB-C18
ZORBAX 300SB-C18, 3.5 µm
Agilent 1200 Infinity LC
Small hydrophobic
peptides
600 bar UHPLC
Poroshell 300 SB-C18
Agilent 1260 Infinity LC and 1260 Infinity Bio-inert Quaternary LC
1200 bar UHPLC
ZORBAX RRHD 300SB-C18,
1.8 µm
Poroshell 300 SB-C18
Agilent 1290 Infinity LC
400 bar HPLC
Poroshell 120 EC-C18
Poroshell 120 SB-C18
Agilent 1200 Infinity LC
600 bar UHPLC
Poroshell 120 EC-C18
Poroshell 120 SB-C18
Agilent 1260 Infinity LC and 1260 Infinity Bio-inert Quaternary LC
1200 bar UHPLC
Poroshell 120 EC-C18
Poroshell 120 SB-C18
Agilent 1290 Infinity LC
If you have an Agilent 1290 Infinity LC in your lab, we recommend starting with a ZORBAX RRHD 300SB-C18 column to screen your peptide map.
increased resolution for peptide mapping
Column:
ZorBAX 300SB-C18
858750-902
2.1 x 100 mm, 1.8 µm
Mobile Phase:
A: 0.1% TFA
B: 0.01% TFA + 80% ACN
Flow Rate:
0.5 mL/min
Gradient:
2% B for 1 min, 2 to 45% B for 8.8 min, 45 to 95% B
for 0.2 min, 95% B for 2 min, 95 to 2% B for 0.2 min
Norm
350
300
250
200
150
Temperature:
50 °C
Detector:
1290 Infinity LC with diode array detector at 280 nm
Sample:
Enzymatic protein digest (MAb)
100
50
0
-50
1
2
3
4
5
6
7
8
9
min
The longer 100 mm Agilent ZORBAX RRHD 300SB-C18 column provides maximum resolution for protein digests – in this sample the total run time,
including washing and equilibration, is under fifteen minutes.
www.AGiLeNT.CoM/CHeM/LC
357
CoLUMNS for BioMoLeCULe SePArATioNS
Separation of Natural and Synthetic Peptides
Purification columns and media are required for the isolation and analysis of natural and synthetic
peptides. Purity and recovery determination of the isolated or purified peptide requires the use of high
efficiency columns. The primary technique used for the isolation and purification, and analysis, is
reversed-phase HPLC.
The fractions from a purification or isolation workflow and the final peptide product are analyzed for
purity using high efficiency columns. The peptides will vary in size, charge and hydrophobicity and so,
as with peptide mapping applications, Agilent offers a range of columns to provide optimum separations
of the full range of peptides. For small peptides, typically less than 10 amino acid residues, the smaller
pore UHPLC materials are used, but if the peptide is larger, contains more amino acid residues, or exists
in a dimeric or multimeric form, then the larger pore size 300Å columns provide better separations due to
improved mass transfer.
Natural and Synthetic Peptides
Column Selection
Recommended column choices as determined by system/column pressure maximum for the analysis of
natural and synthetic peptides.
Application
Technique
Agilent Columns
Notes
Poroshell 300 SB-C18
ZORBAX 300SB-C18, 3.5 µm
PLRP-S
Agilent 1200 Infinity LC
600 bar UHPLC
Poroshell 300 SB-C18
Agilent 1260 Infinity LC and 1260 Infinity Bio-inert Quaternary LC
1200 bar UHPLC
ZORBAX RRHD 300SB-C18, 1.8 µm
Agilent 1290 Infinity LC
400 bar HPLC
Poroshell 120 EC-C18
Poroshell 120 SB-C18
PLRP-S
Agilent 1200 Infinity LC
600 bar UHPLC
Poroshell 120 EC-C18
Poroshell 120 SB-C18
Agilent 1260 Infinity LC and 1260 Infinity Bio-inert Quaternary LC
Larger peptides
400 bar HPLC
with more than
10 amino acid residues
Peptides with typically
less than 10 amino
acid residues
Reversed-phase columns are also the first choice for purifying large numbers of individual peptides or larger
amounts of a particular peptide. High efficiency, small particle pre-packed prep columns are available for
the high efficiency purification of small amounts of peptides, and larger particle columns and bulk media for
the larger scale purifications, as shown in Table 1.
358
CoLUMNS for BioMoLeCULe SePArATioNS
Table 1. Agilent columns for small- to large-scale peptide purifications.
Amount of Peptide required
Agilent Column
mg
g
kg
ZORBAX Prep HT 300StableBond
VariTide RPC
PLRP-S
After solid phase synthesis (SPS) using a polystyrene resin such as one of the Agilent StratoSpheres
products, the peptide is cleaved from the support and the resultant mixture is separated to obtain the target
peptide. A high efficiency column is needed for the purification as the candidate peptide must be resolved
from peptides that are very similar in structure. Check www.agilent.com for further information.
Preparative scale purification of
Leuprolide by concentration overload
Column:
PLrP-S 100Å, 10 µm
PL1412-4100
Bulk Media: Load & Lock 4001 Column
PCG93LL500X25
Mobile Phase:
Isocratic separation
using 0.1% TFA
in 28% ACN:72% water
Flow Rate:
Linear velocity 360 cm/hr
Time (min)
0
40
Crude leuprolide separation of 30 mg on-column load.
% Purity (Pre-impurity 1)
% Purity (Leuprolide)
% Purity (Post-impurity)
Fraction analysis – the concentration overload purification.
www.AGiLeNT.CoM/CHeM/LC
359
CoLUMNS for BioMoLeCULe SePArATioNS
DNA and RNA Oligonucleotide Separations
There is a renewed interest in oligonucleotides (oligos) as they are used in more and more applications,
including potential therapeutics. The synthesis workflow is similar to that used for the more established
synthetic peptide production, i.e. an activated solid phase synthesis resin is used with sequential addition
of specific nucleotides to build the desired sequence.
The nucleotide building blocks are protected at the 5’ hydroxyl end with a dimethoxytrityl (DMT) group and
the cleaved target oligo will have this protected group still attached. As DMT is hydrophobic, it is a useful
handle that can be used for the first stage step. To increase the stability of the oligonucleotide, particularly to
enzyme degradation, it may be chemically modified, for example by replacing oxygen with sulfur to produce
phosphorothioates.
When using chemical synthesis to produce biomolecules, the coupling efficiency of each additional cycle
is never 100%. The sample, after cleavage from the solid phase synthesis support, will contain deletion
sequences, oligos where one or more residues are missing, and some amount of larger oligos produced by
double coupling or branching. The sample mixture is complex and high efficiency techniques are required
for analysis.
There are three UHPLC/HPLC techniques that are routinely used for oligonucleotide separations:
Trityl-on: This procedure is relatively simple to perform and separates the full-length target oligo, which still
has the DMT group attached, from the deprotected failure sequences. The analytical information obtained
is limited and this is generally considered to be a purification method.
ion-exchange separations of the trityl-off, deprotected oligos: This method uses the negative
charge on the backbone of the oligo to facilitate the separation. Resolution is good for the shorter oligos but
decreases with increasing chain length. Aqueous eluents are used but oligos are highly charged, and high
concentrations of salt are needed to achieve elution from the column.
ion-pair reversed-phase separation of the trityl-off, deprotected oligos: This technique uses
organic solvents and volatile ion-pairing agents and is suitable for LC/MS. The technique is best performed
with high efficiency particles. Conditions that fully denature the oligos and prevent association with
complimentary sequences are required. Thus, the separation is best performed at elevated temperatures.
360
CoLUMNS for BioMoLeCULe SePArATioNS
DNA and RNA Oligonucleotide Column Selection
Application
Technique
Agilent Columns
Notes
Trityl-on/trityl-off
oligonucleotides
Trityl-on
PLRP-S 50 µm media
Deprotected
oligonucleotides
Ion-pair reversed-phase PLRP-S 3 µm to
separation of the trityl-off, 50 µm
deprotected oligos
Separates due to differences in hydrophobicity. Ideal for the separation of
trityl-on from trityl-off oligos and is also used for ion-pair reversed-phase
separations of deprotected oligos.
Deprotected
oligonucleotides
Ion-exchange separations PL-SAX 1000Å
of the trityl-off,
deprotected oligos
Separates deprotected oligos under denaturing high pH conditions.
The quaternary amine functionality on the polymeric particles enables
ion-exchange separations at high pH, improving chromatography for
self-complementary sequences.
TiPS & TooLS
Further information can be found in the following publications:
Agilent PLRP-S 100Å HPLC Columns and Media (publication # 5990-8187EN)
Agilent PL-SAX 1000Å HPLC Columns and Media (publication # 5990-8200EN)
www.agilent.com/chem/library
www.AGiLeNT.CoM/CHeM/LC
361
CoLUMNS for BioMoLeCULe SePArATioNS
Amino Acid Analysis
Agilent offers several good options for separation of amino acids, including the Agilent ZORBAX Eclipse AAA
column which uses an updated protocol and is specially tested using amino acids. The ZORBAX Eclipse AAA
high efficiency column rapidly separates amino acids following an updated and improved protocol.
Total analysis from injection to injection can be achieved in as little as 14 min (9 min analysis time)
on shorter, 7.5 cm columns and 24 min (18 min analysis time) on the 15 cm column. Exceptional sensitivity
(5 to 50 pmol with diode array or fluorescence detectors) and reliability are achieved using both OPAand FMOC-derivatization chemistries in one fully automated procedure using the Agilent 1200 Infinity LC.
The newer ZORBAX Eclipse Plus C18 column is also an excellent choice for amino acid separations.
ZorBAX eclipse AAA Column Selection
Application
Diameter x Length (mm) Particle Size (µm)
Analytical routine sensitivity
4.6 x 150
5.0
Analytical routine sensitivity, high-resolution using FLD
4.6 x 150
3.5
Analytical routine sensitivity, high-throughput
4.6 x 75
3.5
Solvent Saver high sensitivity, high-resolution
3.0 x 150
3.5
For more information on the ZORBAX Eclipse Plus C18 column,
turn to page 248.
H
155.16
137.14
C6 H 9 N 3 O 2
D
133.10
115.09
C 4 H 7 NO 4
His
Asp
Chart of the Amino Acids
Histidine
R
174.20
156.19
C 6 H 14 N 4 O 2
Arg
Aspartic Acid
F
165.19
147.18
C 9 H 11 NO 2
Arginine
K
146.19
128.17
C 6 H 14 N 2 O 2
Lys
Phenylalanine
L
131.17
113.16
C 6 H 13 NO 2
Lysine
I
131.18
113.16
C 6 H 13 NO 2
Ile
Phe
A
89.09
71.08
C 3 H 7 NO2
Leu
Alanine
M
149.21
131.20
C 5 H 11 NO 2 S
Leucine
W
204.23
186.21
C 11 H 12 N 2 O 2
Trp
Ala
C
121.16
103.14
C 3 H 7 NO 2 S
Met
Pro
Gly
Cysteine
N
132.12
114.10
C4H 8 N 2O3
Methionine
P
115.13
97.12
C 5 H 9 NO 2
Cys
G
75.07
57.05
C 2 H 5 NO 2
Asn
Glycine
S
105.09
87.08
C 3 H 7 NO 3
Ser
Asparagine
V
117.15
99.13
C 5 H 11 NO 2
Val
Q
146.15
128.13
C 5 H 10 N 2 O 3
Gln
E
147.13
129.11
C 5 H 9 NO 4
Glutamine
Y
181.19
163.17
C 9 H 11 NO 3
Serine
Basic
Nonpolar (hydrophobic)
Tyr
Glu
Glutamic Acid
T
119.12
101.10
C 4 H 9 NO 3
Tyrosine
Thr
Threonine
1-Letter
Amino Acid Code
Molecular Weight
Polar, uncharged
Isoleucine
Tryptophan
Proline
Valine
Acidic
S
105.09
87.08
C 3 H 7 NO 3
Ser
MW-H2O
Chemical Structure
Molecular Formula
Serine
362
3-Letter
Amino Acid Code
Chemical Name
CoLUMNS for BioMoLeCULe SePArATioNS
Broad Distribution Biomolecules
Carbohydrates, Lipids and PEGs
Aqueous size exclusion chromatography employing columns packed with polymeric media can be extremely
useful when investigating biomolecules and their derived species with broad molecular weight distributions.
Examples include PEGylated proteins and complex polysaccharides which find use in biopharma
applications. The wide pore size distribution of polymeric SEC columns compared to silica-based material
are excellent for samples with polydispersities greater than one.
Broad Distribution Biomolecule Column Selection
Low MW polymers
and oligomers,
oligosaccharides, PEGs,
lignosulfonates
2 or 3 PL aquagel-OH columns
The PL aquagel-OH analytical
series has a pH range of 2-10,
compatible with organic solvents
(up to 50% methanol),
mechanical stability up to 140 bar
(2030 psi) and low column
operating pressures.
ü PL aquagel-OH 8 µm
ü PL aquagel-OH 20 5 µm
ü PL aquagel-OH MIXED-M 8 µm
Polydisperse biopolymers,
polysaccharides, cellulose
derivatives
2 or 3 PL aquagel-OH columns
Very high MW polymers,
hyaluronic acids,
starches, gums
PL aquagel-OH 60/50/40 15 µm in series
ü PL aquagel-OH MIXED-H 8 µm
ü PL aquagel-OH 60/50/40 8 µm
www.AGiLeNT.CoM/CHeM/LC
363
CoLUMNS for BioMoLeCULe SePArATioNS
UHPLC/HPLC Techniques
High-performance liquid chromatography, HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry
and analytical chemistry to identify, quantify and purify the individual components of the mixture. There has been an evolution toward ultra high-performance
liquid chromatography (UHPLC) which is widely accepted for high-efficiency separations of small- to medium-sized molecules, and has been used to reduce
analysis time and/or to increase resolution. The use of UHPLC has been extended to large biomolecules with the introduction of wide pore chromatographic
media in columns that can withstand pressures of 600 to 1200 bar.
On the following pages you will see the wide range of columns that Agilent offers for the HPLC and UHPLC separation of proteins and other biomolecules.
UHPLC/HPLC Techniques for Biomolecule Analysis
364
Technique
Advantages
Disadvantages
Reversed-Phase
ü Denaturing conditions
ü High resolution
ü High efficiency silica columns cannot be cleaned using aggressive
ü High capacity
solvents when performing purifications
ü Relatively simple
ü Sample concentrated on-column
ü Small particle, 1.8 µm, for UHPLC separations
ü Polymeric media for unsurpassed chemically and thermally stable
Ion-Exchange
ü Good recovery of biological activity
ü High capacity
ü Sample concentrated on-column
ü Limited MS compatibility due to presence of salts
Size Exclusion
ü Good recovery of biological activity
ü Non-interactive technique with good sample recovery
ü No sample concentration
ü Limited capacity
Affinity
ü Highly selective
ü Good recovery of biological activity
ü Sample concentrated on-column
ü Often single step isolation
ü No sample concentration
ü Limited capacity
CoLUMNS for BioMoLeCULe SePArATioNS
reversed-Phase HPLC
Confidently perform
high-resolution separations
Reversed-phase UHPLC/HPLC separates solutes based on differences in hydrophobicity, with the least
hydrophobic peak eluting first. This high-resolution technique is capable of separating peptides, proteins
and oligonucleotides that differ by only one amino acid or nucleotide residue.
Because HPLC uses organic solvents (such as acetonitrile, methanol, ethanol and propanol) it is also a
denaturing technique that disrupts a biomolecule’s three-dimensional structure. This allows you to obtain
information about a molecule’s primary structure and sequence, as well as variations in the sequence to
be identified.
Agilent offers the industry’s broadest range of wide-pore reversed-phase columns, all backed by
technical support experts and application chemists around the globe. This section features the following
column innovations:
• ZorBAX 300Å pore silica columns – an industry first for reversed-phase protein and biomolecule
separations – are available in 6 phases, along with a broad array of sizes. For fast UHPLC separations,
we also offer a 1.8 µm particle size option that withstands pressures up to 1200 bar, and can be used
with high-pressure instruments, such as Agilent’s 1290 Infinity LC.
• Agilent Poroshell columns feature the industry’s first solid core/porous shell particle. Our wide-pore
Poroshell 300 columns are ideal for fast chromatography, and are available in a variety of phases.
• Agilent PLrP-S columns contain polymer particles, and can be used to separate peptides and
proteins of various sizes and DNA/macromolecular complexes. These columns are unique in that they
are 100% organic, can withstand temperatures as high as 200 °C, and can be used under conditions
from pH 1 to pH 14.
• Choose from a range of column sizes, particle sizes (3-8 µm for analytical separations) and pore sizes
(100Å to 4000Å). Preparative columns (10-50 µm) are also available, either prepacked in columns or
as bulk material.
www.AGiLeNT.CoM/CHeM/LC
365
CoLUMNS for BioMoLeCULe SePArATioNS
reversed-Phase Column Selection
Application
Agilent Columns
Notes
Proteins and polypeptides
ZORBAX 300Å, 1.8 µm
Improved packing processes achieve stability up to 1200 bar for use with the Agilent
1290 Infinity LC. RRHD 1.8 µm columns are available in 50 and 100 mm lengths for
fast or high resolution – truly high definition – separations of the most complex
samples.
ü RRHD 300SB-C18
ü RRHD 300SB-C8
ü RRHD 300SB-C3
ü RRHD 300-Diphenyl
ü RRHD 300-HILIC
ZORBAX 300Å StableBond
ü 300SB-C18
ü 300SB-C8
ü 300SB-C3
ü 300SB-CN
Peptides and proteins up to
1,000 kDA, monoclonal antibodies
and intact proteins
ZORBAX 300Å Extend-C18
Incorporate a unique patented bidentate silane, combined with a doubleendcapping process that protects the silica from dissolution at high pH –
up to pH 11.5.
Poroshell 300
Poroshell columns use a unique particle made with a layer of porous silica on a solid
core of silica. This reduces the diffusion distance for proteins making practical, rapid
HPLC separations of peptides and proteins.
ü 300SB-C18
ü 300SB-C8
ü 300SB-C3
ü 300Extend-C18
Small hydrophilic peptides
in protein digests
Poroshell 120
The 120Å pore size is ideal for the fast high resolution analysis of small hydrophilic
peptides and peptide fragments in protein digests.
Peptides to DNA
PLRP-S
Particles are inherently hydrophobic so an alkyl ligand bonded phase is not required
for reversed-phase separations. This gives a highly reproducible material that is free
from silanols and heavy metal ions.
ü 100Å
ü 300Å
ü 1000Å
ü 4000Å
Small molecules/peptides/oligonucleotides PLRP-S 100Å
Recombinant peptides/proteins
366
Wide-pore, 300Å columns are necessary for an efficient separation of proteins and
peptides, or other large molecules, to allow these analytes to completely access the
bonded phase. C18 and C8 are ideal for complex protein and protein digest
separations. StableBond provides enhanced stability for low pH.
PLRP-S 300Å
Large proteins
PLRP-S 1000Å
DNA/high speed separation
PLRP-S 4000Å
CoLUMNS for BioMoLeCULe SePArATioNS
ZorBAX 300Å StableBond
Agilent ZORBAX 300Å StableBond columns are an ideal choice for the reproducible separations of proteins
and peptides for two key reasons. First, wide-pore, 300Å columns are necessary for an efficient separation
of proteins and peptides, or other large molecules, in order to allow these analytes to completely access the
bonded phase. Second, 300StableBond columns are unmatched in their durability at low pH, such as with
TFA-containing mobile phases typically used for protein and peptide separations. For LC/MS separations
at low pH, 300StableBond columns can also be used with formic acid and acetic acid mobile phase
modifiers. These columns are available in five different bonded phases (C18, C8, C3, CN, and Diphenyl*)
for selectivity and recovery optimization of proteins and polypeptides. To further increase sample recovery
and improve efficiency for difficult proteins, 300StableBond columns can be used up to 80 °C. 300SB-C18
and 300SB-C8 columns are an ideal choice for complex protein and protein digest separations. These
columns are also available in capillary (0.3 and 0.5 mm id) and nano (0.075 and 0.10 mm id) dimensions
for reversed-phase LC/MS separations of protein digests. Capillary and nano columns can be used for
either 1-D or 2-D proteomics separations.
*Diphenyl is available in a 1.8 µm particle size only.
Column Specifications
Bonded Phase
Pore
Size
Surface
Area
Temp
Limits*
pH
range*
endcapped
Carbon
Load
ZORBAX 300SB-C18
300Å
45 m2/g
90 °C
1.0-8.0
No
2.8%
2
ZORBAX 300SB-C8
300Å
45 m /g
80 °C
1.0-8.0
No
1.5%
ZORBAX 300SB-C3
300Å
45 m2/g
80 °C
1.0-8.0
No
1.1%
2
ZORBAX 300SB-CN
300Å
45 m /g
80 °C
1.0-8.0
No
1.2%
ZORBAX 300-Diphenyl
300Å
45 m2/g
80 °C
1.0-8.0
Yes
1.9%
Specifications represent typical values only
Sterically Protected 300StableBond Bonded Phase
*300StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-based
columns is obtained by operating at temperatures <40 °C and using low buffer concentrations in the range of 0.01-0.02 M.
At mid or high pH, 300Extend-C18 is recommended.
TiPS & TooLS
Further information can be found in the following publication:
Comparison of ZORBAX StableBond 300Å LC Columns to Optimize Selectivity for Antibody Separations
Using HPLC and LC/MS (publication # 5989-6840EN)
www.agilent.com/chem/library
www.AGiLeNT.CoM/CHeM/LC
367
CoLUMNS for BioMoLeCULe SePArATioNS
Higher resolution of intact monoclonal antibody
Column:
ZorBAX rrHD 300SB-C8
857750-906
2.1 x 50 mm, 1.8 µm
Gradient A
0.5 mL/min
optimized resolution
mAU
250
200
Mobile Phase:
A: H20:IPA (98:2) + 0.1% TFA (v/v)
B: IPA:ACN:H20 (70:20:10) + 0.1% TFA (v/v)
Flow Rate:
Between 0.5 mL/min and 1.0 mL/min
Gradient:
Multi-segmented and linear elution
Temperature:
80 °C
Detector:
Agilent 1290 Infinity LC with auto injector (ALS),
binary pump and thermostatted oven and diode array
detector (DAD), UV, 225 nm
150
Shoulder
100
resolved
50
0
0
2
4
mAU
6
8
mAU
350
100
increased
sensitivity
250
150
10
min
Gradient B
1.0 mL/min
High speed anaysis time
80
60
40
20
0
50
0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8 min
Expanded View
0
2
4
6
8
10
min
Higher resolution of oxidation study
Mobile Phase:
A: 0.1% TFA
B: 0.01% TFA + 80% ACN
Flow Rate:
1.0 mL/min
Gradient:
33 to 50% B, 0 to 4 min
Detector:
1290 Infinity LC with diode array detector at 280 nm
mAU
17.5
15
oxidized insulin
chain B
1.384
ZorBAX rrHD 300SB-C18
857750-902
2.1 x 50 mm, 1.8 µm
0.148
Column:
insulin
12.5
10
Insulin, insulin chain A and chain B, oxidized
(bovinesigma, 1 mg/mL)
oxidized insulin
chain A
5
1.648
Sample:
7.5
Subspecies of
oxidized insulin chain B
2.5
0
min
It is evident that the oxidized insulin chains are resolved from insulin in under 2 minutes using the
Agilent ZORBAX RRHD 300SB-C18 2.1 x 50 mm, 1.8 µm column.
TiPS & TooLS
Typical mobile phases for protein and peptide separations combine a very low pH with TFA (or other acids) to solubilize proteins. StableBond columns
have extremely long lifetimes under these conditions. They are available in 300Å pore size for proteins up to 100-500 kDa.
368
CoLUMNS for BioMoLeCULe SePArATioNS
improved reproducibility
of monoclonal antibodies
Column:
mA
Mobile Phase:
A: H20:IPA (98.2), 0.1% TFA
B: IPA:ACN:H20 (70:20:10), 0.1% TFA
Flow Rate:
1.0 mL/min
Temperature:
80 °C
Detector:
1290 Infinity LC with diode array detector at 225 nm
Sample:
MAb
1st injection
150th injection
400
ZorBAX rrHD 300SB-C8
857750-906
2.1 x 50 mm, 1.8 µm
300
200
100
0
0.5
mA
Gradient timescale
% Solvent B
0.00
25
3.00
35
4.00
90
5.00
25
1.5
2
2.5
3 min
bar
750
pre- and post-150
injection column
blank run overlays
700
Time (min)
1
500
500
pressure
300
Excellent column reproducibility and protein
recovery using Agilent ZORBAX 300SB-C8.
gradient wash and
re-equilibration region
250
UV
100
5
0
min
increased resolution for peptide mapping
Column:
ZorBAX 300SB-C18
858750-902
2.1 x 100 mm, 1.8 µm
Norm
350
300
Mobile Phase:
Flow Rate:
A: 0.1% TFA
B: 0.01% TFA + 80% ACN
0.5 mL/min
250
200
150
Gradient:
2% B for 1 min, 2 to 45% B for 8.8 min, 45 to 95% B
for 0.2 min, 95% B for 2 min, 95 to 2% B for 0.2 min
100
50
Temperature:
50 °C
Detector:
1290 Infinity LC with diode array detector at 280 nm
0
-50
Sample:
Enzymatic protein digest (MAb)
1
2
3
4
5
6
7
8
9
min
The longer 100 mm Agilent ZORBAX RRHD 300SB-C18 column provides maximum resolution for protein
digests – in this sample the total run time, including washing and equilibration, is under fifteen minutes.
www.AGiLeNT.CoM/CHeM/LC
369
CoLUMNS for BioMoLeCULe SePArATioNS
Peptides: effect of TfA concentration
Peptide Sequence
Column:
ZorBAX 300SB-C8
883995-906
4.6 x 150 mm, 5 µm
0.05% TfA
Mobile Phase:
A: Water and TFA
B: ACN and TFA
Flow Rate:
1.0 mL/min
Gradient:
0 min 0% B
30 min 30% B
Temperature:
40 °C
Detector:
UV 254 nm
Sample:
Peptide Standards S1-S5, decapeptides
differing slightly in hydrophobicity, 6 µL
6
10
8
12
14
16
6
8
10
12
14
16
20
8
10
12
18
14
16
Retention Time (min)
Mobile Phase:
ZorBAX 300SB-C3
883995-909
4.6 x 150 mm, 5 µm
A: 5:95
ACN:Water with 0.10% TFA (v/v%)
B: 95:5
ACN:Water with 0.085% TFA (v/v%)
Flow Rate:
1.0 mL/min
Gradient:
15-53% in 20 min, posttime 12 min
Temperature:
Ambient – 60 °C
Detector:
UV 215 nm
Sample:
Polypeptides
1
2
5
3
1.
2.
3.
4.
5.
6.
7.
8.
6
7
8
5
5
4
Leucine Enkephalin
Angiotensin II
RNase A
Insulin (BOV)
Cytochrome c
Lysozyme
Myoglobin
Carbonic anhydrase
60 °C
35 °C
2
24
4
4
0
22
LCBP021
4, 5
Ambient
24
S5
20
45 °C
4
22
S4
18
24
S5
20
Peptides/proteins: effect of elevated
temperature
Column:
22
S4
S3
S2 S1
1.0% TfA
6
18
S1 S2 S3
0.25% TfA
S5
S4
S3
S1 S2
6
8 10 12 14 16 18 20
Time (min)
0
2
4
6
8 10 12 14 16 18 20
Time (min)
LCBP026
TiPS & TooLS
The Agilent 1290 Infinity LC delivers significantly faster results and higher data quality – enabling more informed decisions in shorter time. This higher
productivity gives you competitive advantages and provides you a higher return on investment. Calculate for yourself how much you can save by
deploying the 1290 Infinity technology. The online method translator and cost savings calculator helps you to transfer your HPLC methods and calculate
your cost savings, at www.agilent.com/chem/hplc2uhplc
370
CoLUMNS for BioMoLeCULe SePArATioNS
Short-chain ZorBAX 300SB-C3 is stable at low
pH, high temperature
ZorBAX 300SB-C3
883995-909
4.6 x 150 mm, 5 µm
Mobile Phase:
Gradients 0-100% B in 80 min
A: 0.5% TFA in Water
B: 0.5% TFA in Acetonitrile
Isocratic Retention Test Conditions:
1-phenylheptane 50% A, 50% B
Flow Rate:
1.0 mL/min
Temperature:
60 °C
90
% k Phenyltheptane Remaining
Column:
100
80
ZorBAX 300 SB-C3
70
Competitor C4
60
50
40
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
Volume of Mobile Phase (mL)
LCSB005
four different 300SB bonded phases optimize
separation of large polypeptides
2, 3
A
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
1
8
7
5
4
6
Column A:
Column B:
Column C:
ZorBAX rrHD 300SB-C18
883995-902
4.6 x 150 mm, 5 µm
9
10
B
4
ZorBAX 300SB-C8
883995-906
4.6 x 150 mm, 5 µm
ZorBAX 300SB-C3
883995-909
4.6 x 150 mm, 5 µm
6, 7
2
1
5
8
3
9
10
C
2
1
3
4
RNase
Insulin
Cytochrome c
Lysozyme
Parvalbumin
CDR
Myoglobin
Carbonic Anhydrase
S-100b
S-100a
7
5
8
6
9
10
Column D:
Mobile Phase:
ZorBAX 300SB-CN
883995-905
4.6 x 150 mm, 5 µm
Linear Gradient, 25-70% B in 40 min
A: 0.1% TFA in Water
B: 0.09% TFA in 80% Acetonitrile/20% Water
D
2
1
3
4
5
7
8
6
9
10
0
5
10
15
20
Time (min)
25
30
35
40
LCSB006
Flow Rate:
1.0 mL/min
Temperature:
60 °C
Sample:
3 µg each protein
The 300SB-C18, C8, C3, and CN bonded phases all provide a different separation of this
group of polypeptides. This adds an important parameter for quickly optimizing protein
separations. The 300SB-CN column offers unique selectivity for more hydrophilic polypeptides.
www.AGiLeNT.CoM/CHeM/LC
371
CoLUMNS for BioMoLeCULe SePArATioNS
ZorBAX 300Å StableBond
Hardware Description
Size (mm)
300SB-C18
Particle Size (µm) USP L1
300SB-C8
USP L7
300SB-CN
USP L10
300SB-C3
USP L56
300-Diphenyl
USP L11
Standard Columns (no special hardware required)
Semi-Preparative
9.4 x 250
5
880995-202
880995-206 880995-205
880995-209
Analytical
4.6 x 250
5
880995-902
880995-906 880995-905
880995-909
Analytical
4.6 x 150
5
883995-902
883995-906 883995-905
883995-909
Analytical
4.6 x 50
5
860950-902
860950-906 860950-905
860950-909
Rapid Resolution
4.6 x 150
3.5
863973-902
863973-906 863973-905
863973-909
Rapid Resolution
4.6 x 100
3.5
861973-902
861973-906
Rapid Resolution
4.6 x 50
3.5
865973-902
865973-906 865973-905
865973-909
Solvent Saver Plus
3.0 x 150
3.5
863974-302
863974-306
863974-309
Solvent Saver Plus
3.0 x 100
3.5
Narrow Bore
2.1 x 250
5
881750-902
Narrow Bore
2.1 x 150
5
883750-902
Narrow Bore RR
2.1 x 150
3.5
Narrow Bore RR
2.1 x 100
3.5
861775-902
861775-906
Narrow Bore RR
2.1 x 50
3.5
865750-902
865750-906
Narrow Bore RRHD
2.1 x 100
1.8
858750-902
858750-906
858750-909 858750-944
Narrow Bore RRHD
2.1 x 50
1.8
857750-902
857750-906
857750-909 857750-944
MicroBore
1.0 x 250
5
861630-902
MicroBore RR
1.0 x 150
3.5
863630-902
863630-906
MicroBore RR
1.0 x 50
3.5
865630-902
865630-906
MicroBore Guard, 3/pk
1.0 x 17
5
5185-5920
5185-5920
Guard Cartridge, 2/pk
9.4 x 15
7
820675-124
820675-124 820675-124
820675-124
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-921
820950-918 820950-923
820950-924
Guard Cartridge, 4/pk
2.1 x 12.5
5
821125-918
821125-918 821125-924
821125-924
Guard Hardware Kit
840140-901
840140-901 840140-901
840140-901
Guard Hardware Kit
820999-901
820999-901 820999-901
820999-901
861973-306
883750-906 883750-905
883750-909
863750-906
(Continued)
372
CoLUMNS for BioMoLeCULe SePArATioNS
ZorBAX 300Å StableBond
Hardware Description
Size (mm)
300SB-C18
Particle Size (µm) USP L1
300SB-C8
USP L7
300SB-CN
USP L10
300SB-C3
USP L56
300-Diphenyl
USP L11
PrepHT Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 250
7
897250-102
897250-106 897250-105
897250-109
PrepHT Cartridge
21.2 x 150
7
897150-102
897150-106
897150-109
PrepHT Cartridge
21.2 x 150
5
895150-902
895150-906
895150-909
PrepHT Cartridge
21.2 x 100
5
895100-902
895100-906
895100-909
PrepHT Cartridge
21.2 x 50
5
895050-902
895050-906
895050-909
820400-901
820400-901 820400-901
820400-901
820212-921
820212-918 820212-924
820212-924
820444-901
820444-901 820444-901
820444-901
PrepHT Endfittings, 2/pk
PrepHT Guard Cartridge, 17.0 x 7.5
2/pk
5
Guard Cartridge Hardware
Capillary Glass-lined Columns
Capillary
0.5 x 250
5
5064-8266
Capillary
0.5 x 150
5
5064-8264
Capillary
0.5 x 35
5
5064-8294
Capillary RR
0.5 x 150
3.5
5064-8268
Capillary RR
0.5 x 35
3.5
5065-4459
Capillary
0.3 x 250
5
5064-8265
Capillary
0.3 x 150
5
5064-8263
Capillary
0.3 x 35
5
5064-8295
Capillary RR
0.3 x 150
3.5
5064-8267
5065-4460
Capillary RR
0.3 x 100
3.5
5064-8259
5065-4461
Capillary RR
0.3 x 35
3.5
5064-8270
5065-4462
Capillary RR
0.3 x 50
3.5
5064-8300
5065-4463
Nano Columns (PeeK fused silica)
Nano RR
0.1 x 150
3.5
5065-9910
Nano RR
0.075 x 150
3.5
5065-9911
Nano RR
0.075 x 50
3.5
5065-9924
5065-9923
Trap/Guard, 5/pk
0.3 x 5
5
5065-9913
5065-9914
5065-9915
5065-9915
Trap/Guard Hardware kit
www.AGiLeNT.CoM/CHeM/LC
373
CoLUMNS for BioMoLeCULe SePArATioNS
ZorBAX rrHD 300-Diphenyl
Utilizing the same unique chemistry as the Pursuit 3.5 µm and 5 µm Diphenyl columns, the unique wide pore
300Å Diphenyl phase offers additional selectivity through pi-pi interactions with aromatic amino acids in the
primary sequence. Agilent ZORBAX 1.8 µm 300Å Rapid Resolution High Definition (RRHD) columns bring
UHPLC performance to the reversed-phase separation of intact proteins and protein digests.
The diphenyl column can be used for:
• Analysis of intact and modified proteins and polypeptides including protein structural analysis
• Detection of post-translational modifications
• Impurity analysis
• Confirming protein identity
The ZORBAX RRHD 300-Diphenyl provides:
• Stability at low pH – allowing you to run your protein and peptide separations down to pH 1 using
trifluoroacetic acid (TFA), and formic acid eluents with complete confidence
• Temperature stability – you can run your separations up to 80 °C to improve efficiency and reduce
eluent viscosity, without compromising column lifetime
• UHPLC compatible – enabling higher order characterization with reduced analysis time
Column Specifications
Bonded Phase
Pore
Size
Surface
Area
Temp
Limits
ZORBAX RRHD 300-Diphenyl
300Å
45 m2/g
80 °C
Specifications represent typical values only
374
pH range endcapped
1.0-8.0
Yes
Carbon
Load
1.9%
CoLUMNS for BioMoLeCULe SePArATioNS
fast separation of reduced monoclonal antibody
Agilent ZorBAX rrHD 300-Diphenyl
858750-944
2.1 x 100 mm, 1.8 µm
Mobile Phase:
A: 0.1% TFA in water
B: 80% n-propyl alcohol,
10% ACN, 9.9% water, and 0.1% TFA
Sample:
Reduced monoclonal
antibody (IgG1) (1.0 mg/mL)
4.010
Column:
100
Heavy chain 1
Light chain
80
Heavy chain 2
3.897
0.569
120
Flow Rate:
0.5 mL/min
Gradient:
0 min-1%B, 2 min-20% B, 5 min-50% B
Temperature:
74 °C
Detector:
UV, 280
40
20
0.501
2 µL
0.454
Sample
Injection:
0.766
60
0
2
3
4
5
4.299
1
120
100
4.119
0.594
Heavy chain 2
0
0.953
20
0.479
0.499
60
40
Description
Heavy chain 1
Light chain
80
1
2
3
4
5
Dimensions Particle Size (µm) Part No.
ZORBAX RRHD 300-Diphenyl
2.1 x 50
1.8
857750-944
ZORBAX RRHD 300-Diphenyl
2.1 x 100
1.8
858750-944
www.AGiLeNT.CoM/CHeM/LC
375
CoLUMNS for BioMoLeCULe SePArATioNS
ZorBAX 300Å extend-C18
• Rugged, high and low pH separations of polypeptides and peptides from pH 2-11.5
• Different selectivity possible at high and low pH
• High efficiency and good recovery of hydrophobic peptides at high pH
• Ideal for LC/MS with ammonium-hydroxide-modified mobile phase
Agilent ZORBAX 300Å Extend-C18 is a wide-pore HPLC column for high efficiency separations of peptides
from pH 2-11.5. The unique, bidentate bonded phase provides excellent lifetime and reproducibility at high
and low pH. At high pH, retention and selectivity of peptides and polypeptides can change dramatically as
a result of changes in charge on molecules. Excellent recoveries of hydrophobic polypeptides have been
achieved at room temperature and high pH. LC/MS sensitivity of peptides and polypeptides can also be
improved at high pH using a simple ammonium-hydroxide-containing mobile phase.
Novel Bidentate C18-C18 Bonding
for Extend-C18 Bonded Phase
Column Specifications
Bonded Phase
Pore
Size
Surface
Area
ZORBAX 300Å Extend-C18
300Å
45 m2/g
Specifications represent typical values only.
*Temperature limits are 60 °C up to pH 8, 40 °C from pH 8-11.5.
TiPS & TooLS
Selecting the right column is only
part of the total solution. Don't forget
key supplies such as our wide range
of LC lamps. Turn to page 90.
376
Temp.
Carbon
Limits* pH range endcapped Load
60 °C
2.0-11.5
Double
4%
CoLUMNS for BioMoLeCULe SePArATioNS
LC/MS analysis of angiotensin on extend-C18
Gradient:
15-50% B in 15 min
Temperature:
35 °C
12.050
100
12.5
0
100
80
4.0E7
AIII
1.0E7
20
0
0
0
2.5
5.0 7.5 10
Time (min)
12.5
1468.8
1277.9
1326.3
1373.3
1179.1
433.0
40
+3
659.8
AI
8.477
5.499
AII
2.0E7
+2
60
9.621
3.0E7
m/z
1000
500
0
500
1296.6
5.0 7.5 10
Time (min)
649.0
2.5
5.0E7
TIC (200-1500 m/z)
1025.2
0
0
B
Angiotensin I
Max: 367225
High pH
40
20
0
+2
60
AI
1.0E6
2.5 µL sample (50 pmol each)
Angiotensin I, II, III
844.8
2.0E6
Sample:
659.7
720.3
767.3
AII + AIII
3.0E6
Pos. Ion ESI- Vf 70 V, Vcap 4.5 kV,
N2- 35 psi, 12 L/min., 325 °C
+3
80
13.261
4.0E6
MS Conditions:
649.5
0.2 mL/min
5.0E6
TIC (200-1500 m/z)
A
Angiotensin I
Max: 10889
Low pH
Flow Rate:
433.0
Acidic Conditions:
A: 0.1% TFA in water
B: 0.085% TFA in 80% acetonitrile (ACN)
Basic Conditions:
A: 10 mM NH4OH in water
B: 10 mM NH4OH in 80% ACN
227.1
Mobile Phase:
250.9
ZorBAX extend-C18
773700-902
2.1 x 150 mm, 5 µm
340.3
398.0
458.0
Column:
1000
+1
m/z
LC30003
Both small and large peptides demonstrate selectivity changes at high and low pH. At high pH, due to a change in charge, all three Angiotensins can be resolved. In addition,
the spectral clarity of Angiotensin I is dramatically improved at high pH with the ammonium hydroxide mobile phase. The Extend-C18 column can be used for the analysis of
small peptides at high pH as well.
Reference: B.E. Boyes. Separation and Analysis of Peptides at High pH Using RP-HPLC/ESI-MS, 4th WCBP, San Francisco, CA, Jan. 2000.
www.AGiLeNT.CoM/CHeM/LC
377
CoLUMNS for BioMoLeCULe SePArATioNS
Long life at high pH with 300extend-C18
ZorBAX extend-C18
773450-902
4.6 x 150 mm, 5 µm
Mobile Phase:
20% 20 mM NH4OH, pH 10.5
80% Methanol
Flow Rate:
1.5 mL/min
Temperature:
Aging 24 °C
Tests 40 °C
4-Chloro-1-nitrobenzene
Trimipramine
6
k Values
Column:
8
4
2
Each 10,000 column volume is approximately one working month.
0
0
10,000
20,000
30,000
40,000
50,000
Column Volumes of Mobile Phase
LC30001
Use ZorBAX extend-C18 for alternate
selectivity at high pH
Column:
ZorBAX extend-C18
773700-902
2.1 x 150 mm, 5 µm
A
3E7
2.5E7
2E7
Mobile Phase:
A: 0.1% TFA in Water
B: 0.085% TFA in 80% ACN
1.5E7
LLL-NH2
1E7
A: 20 mM NH4OH in Water
B: 20 mM NH4OH in 80% ACN
LHG
.5E7
0
Flow Rate:
0.25 mL/min
0
Gradient:
5-60% B in 20 min
B
Temperature:
25 °C
MS Conditions:
Pos. Ion ESI-Vf 70V, Vcap 4.5 kV
N2 – 35 psi, 12 L/min, 300 °C
4 µL (50 ng each peptide)
LLL
LLVF
LHL
2
4
6
8
10
12
14
16
18
20
LLL-NH2
3E7
LLL
2.5E7
LHG
2E7
LLVF
1.5E7
LHL
1E7
The Extend column can be used for high pH separations of peptides.
At high and low pH, very different selectivity can result. Just by
changing pH, a complimentary method can be developed and it is
possible to determine if all peaks are resolved. The Extend column
can be used at high and low pH, so the complimentary separation
can be investigated with one column. Better MS sensitivity for this
sample is also achieved at high pH.
378
.5E7
0
0
2
4
6
8
10
Time (min)
12
14
16
18
20
LCBP017
CoLUMNS for BioMoLeCULe SePArATioNS
ZorBAX 300Å extend-C18
Hardware Description
Size (mm)
Particle Size (µm) Part No.
Analytical
4.6 x 250
5
770995-902
Analytical
4.6 x 150
5
773995-902
Rapid Resolution
4.6 x 150
3.5
763973-902
Rapid Resolution
4.6 x 100
3.5
761973-902
Rapid Resolution
4.6 x 50
3.5
765973-902
Narrow Bore RR
2.1 x 150
3.5
763750-902
Narrow Bore RR
2.1 x 100
3.5
761775-902
Narrow Bore RR
2.1 x 50
3.5
765750-902
Guard Cartridge, 4/pk
4.6 x 12.5
5
820950-932
Guard Cartridge, 4/pk
2.1 x 12.5
5
Guard Hardware Kit
821125-932
820999-901
Capillary Glass-lined Columns
Capillary RR
0.3 x 150
3.5
5065-4464
Capillary RR
0.3 x 100
3.5
5065-4465
Capillary RR
0.3 x 75
3.5
5065-4466
Capillary RR
0.3 x 50
3.5
5065-4467
www.AGiLeNT.CoM/CHeM/LC
379
CoLUMNS for BioMoLeCULe SePArATioNS
Poroshell 300
Solid core, 4.5 µm diameter
• UHPLC separations of biomolecules with superficially porous particles
• 300Å pore provide high efficiency and recovery with proteins (up to 1,000 kDa)
and monoclonal antibodies
• Achieve long lifetime at low pH with Poroshell 300SB; at high pH with 300Extend-C18
• Optimize recovery and selectivity with four different bonded phases – 300SB-C18, 300SB-C8,
300SB-C3, and 300Extend-C18
Agilent Poroshell 300 columns are ideal for fast separations of proteins and peptides because the
superficially porous particle allows for fast flow rates to be used while maintaining sharp, efficient peaks.
Peptides and proteins are typically separated slowly to reduce the potential peak broadening of these slow
diffusing analytes. However, Poroshell columns use a superficially porous particle made with a thin layer of
porous silica, 0.25 µm thick, on a solid core of silica. This reduces the diffusion distance for proteins making
practical rapid HPLC separations of peptides and proteins up to 500-1,000 kDA possible with 400/600 bar
HPLC systems, including the Agilent 1260 Infinity Bio-inert. Poroshell columns bonded with StableBond
bonded phases provide excellent stability and selectivity choices with TFA and formic acid mobile phases.
The Poroshell 300Extend-C18 column can be used from pH 2-11 for unique separations. These columns can
be used for analytical protein separations as well as LC/MS separations.
Porous shell,
0.25 µm thick
Column Specifications
Bonded Phase
Pore Size
Temp. Limits*
pH range endcapped
Poroshell 300SB-C18, C8, C3
300Å
90 °C
1.0-8.0
No
Poroshell 300Extend-C18
300Å
40 °C above pH 8
60 °C below pH 8
2.0-11.0
Yes
Specifications represent typical values only.
Poroshell 300 Columns
380
*300StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-based
columns is obtained by operating at temperatures <40 °C and using low buffer concentrations in the range of 0.01-0.02 M.
At mid or high pH, 300Extend-C18 is recommended.
CoLUMNS for BioMoLeCULe SePArATioNS
Poroshell 300 columns separate
proteins and peptides in seconds
Column:
5
4
3
Poroshell 300SB-C18
660750-902
2.1 x 75 mm, 5 µm
Mobile Phase:
A: 0.1% TFA in H20
B: 0.07% TFA in ACN
Flow Rate:
3.0 mL/min
Gradient:
5-100% B in 1.0 min
Temperature:
70 °C, 260 bar pressure
Detector:
215 nm
Sample:
Proteins and Peptides
This separation of eight polypeptides and
proteins is completed in less than 60 seconds.
Each peak is sharp and efficient.
1
2
7
6
8
0
1.
2.
3.
4.
5.
6.
7.
8.
Angiotensin II
Neurotensin
RNase
Insulin
Lysozyme
Myoglobin
Carbonic Anhydrase
Ovalbumin
0.5
Time (min)
1.0
LCPO001
TiPS & TooLS
Further information can be found in the following publications:
Poroshell 300SB-C18 (publication # 5988-2100ENUS)
Rapid HPLC Analysis of Monoclonal Antibody IgG1 Heavy Chains Using ZORBAX Poroshell 300SB-C8 (publication # 5989-0070EN)
Use of Temperature to Increase Resolution in the Ultrafast HPLC Separation of Proteins with ZORBAX Poroshell 300SB-C8 HPLC Columns
(publication # 5989-0589EN)
Using the High-pH Stability of ZORBAX Poroshell 300Extend-C18 to Increase Signal-to-Noise in LC/MS (publication # 5989-0683EN)
www.agilent.com/chem/library
www.AGiLeNT.CoM/CHeM/LC
381
CoLUMNS for BioMoLeCULe SePArATioNS
reduce peptide map analysis time
by 90% with Poroshell 300SB
Column A:
Poroshell 300SB-C18
660750-902
2.1 x 75 mm, 5 µm
Column B:
ZorBAX 300SB-C18
883750-902
2.1 x 150 mm, 5 µm
Mobile Phase:
A: 95% H2O, 5% ACN, 0.1% TFA
B: 5% H2O, 95% ACN, 0.07% TFA
Flow Rate:
1 mL/min
0.208 mL/min
Gradient:
0-100% B = 12 min
0-100% B = 120 min
Temperature:
70 °C
Sample:
20 µL (0.22 µg/1 µL)
BSA Tryptic Digest
(15 hours, 70 pmol)
A
1
2
3
4
Time (min)
5
6
7
10
20
30
40
Time (min)
50
60
70
8
B
80
LCPO002
A single chromatographic run of a protein tryptic digest can require one hour or more to complete. With Poroshell columns, the same complex separation can be completed in
1/10th the time.
MicroBore Poroshell 300 columns
provide maximum sensitivity for LC/MS
Column:
Poroshell 300SB-C18
661750-902
1.0 x 75 mm, 5 µm
Mobile Phase:
A: Water + 0.1% Formic Acid
B: ACN + 0.1% Formic Acid
Flow Rate:
600 µL/min
Gradient:
20-100% B in 5.5 min
Temperature:
80 °C
MS Conditions:
LC/MS: Pos. Ion ESI – Vcap 6000 V
Drying Gas Flow: 12 L/min
Drying Gas Temperature: 350 °C
Nebulizer: 45 psi
Fragmentor Volatage: 140 V
Scan: 600-2500
Stepsize: 0.15 amu
Peak width: 0.06 min
Sample:
1EB
80000000
0.5
0.75
1.0
2.5
5.0
60000000
40000000
20000000
0
0.5
1.0
1.5
2.0
2.5
Time (min)
3.0
3.5
4.0
LCPO003
1 µL
With narrow bore diameters of 2.1 mm, 1.0 mm, and 0.5 mm, Poroshell columns make an ideal LC/MS partner. When the sample is very limited, the 1.0 mm or 0.5 mm id
Poroshell columns are an excellent choice for high sensitivity LC/MS analyses. Sensitive MS molecular weight determinations are possible with as little as 0.5 to 5 pmole of
protein on Poroshell columns. Poroshell columns have also been used for rapid MS identification of intact proteins, even in the presence of stabilizers and tissue culture media.
382
CoLUMNS for BioMoLeCULe SePArATioNS
Monoclonal igG1 chains:
Separation on Poroshell 300SB-C8
Column:
Mobile Phase:
Poroshell 300SB-C8
660750-906
2.1 x 75 mm, 5 µm
A: 90% water:
10% ACN + 3 mL/L of MW 300 PEG
B: 10% water:
90% ACN + 3 mL/L of MW 300 PEG
Flow Rate:
1.0 mL/min
Gradient:
0 min 25% B
10 min 40% B
10.1 min 25% B
12 min 25% B
mAU
Heavy Chains
120
Light Chains
80
igG treated with:
DTT
40
0
0
70 °C
Sample:
Monoclonal IgG1
4
6
8
min
igG treated with:
DTT
Peptide-N-Glycosidase F
80
40
0
2
4
6
8
10
min
mAU
120
igG treated with:
DTT
Peptide-N-Glycosidase F
Carboxypeptidase-B
80
Courtesy of:
Novartis Pharma,
Biotechnology, Basel
Dr. Kurt Forrer
Patrik Roethlisberger
10
Non-Glycosylated?
Glycosylated?
120
0
Temperature:
2
mAU
40
0
0
2
4
6
8
10
min
IgG_Poroshell
TiPS & TooLS
Agilent offers an extensive selection of certified chromatography sample vials
including polypropylene and deactivated and siliconized glass.
For more information see (publication # 5990-9022EN).
www.agilent.com/chem/library
www.AGiLeNT.CoM/CHeM/LC
383
CoLUMNS for BioMoLeCULe SePArATioNS
Column:
Poroshell 300SB-C8
660750-906
2.1 x 75 mm, 5 µm
Mobile Phase:
Flow Rate:
A: 0.1% TFA in H20
B: 0.1% TFA in ACN
Column Temperature 40 °C
453
B: 20 to 70% in 3 min
Detector:
UV (214 nm)
1.
2.
3.
4.
5.
6.
7.
8.
7
23 4
406
1.0 mL/min
Gradient:
5
6
500
Response (mV)
Protein elution pattern
on ZorBAX Poroshell 300SB-C8
359
8
312
265
218
Glycoprotein X, MW ~ 22 kDa
Protein I, MW ~ 4 kDa
Glucagon, MW ~ 3.5 kDa
Biosynthetic human insulin, MW ~ 6 kDa
Protein J, MW ~ 3 kDa
Protein K, MW ~ 6 kDa
Glycoprotein Y, MW ~ 45 kDa
Glycoprotein Z, MW ~ 30 kDa
171
1
124
77
30
0
21
42
63
84
105
126
Time (s)
Column Temperature 60 °C
490
168
189
210
5
444
2 3
398
Response (mV)
147
6
4
7
352
8
306
260
214
168
1
122
76
30
0
21
42
63
84
105
Time(s)
Column Temperature 75 °C
480
5
435
4
Response (mV)
147
168
189
210
6
7
3 2
390
126
345
8
300
255
210
165
1
120
75
30
0
21
42
63
84
105
126
Time (s)
147
168
189
210
Prot Elution
Poroshell 300
Hardware Description
Size
(mm)
Particle Size
(µm)
Poroshell
300SB-C3
Poroshell
300extend-C18
Narrow Bore
2.1 x 75
5
660750-902 660750-906 660750-909 670750-902
MicroBore
1.0 x 75
5
661750-902 661750-906 661750-909 671750-902
Capillary
0.5 x 75
5
5065-4468
Guard Cartridge, 4/pk
2.1 x 12.5
5
821075-920 821075-918 821075-924
1.0 x 17
5
5185-5968
Guard Hardware Kit
MicroBore Guard, 3/pk
384
Poroshell
Poroshell
300SB-C18 300SB-C8
820999-901 820999-901 820999-901
5185-5968
5185-5968
5185-5968
CoLUMNS for BioMoLeCULe SePArATioNS
Poroshell 120
• 120Å pore size for shorter chain peptide mapping
• UHPLC performance on 600 bar systems
• Up to 90% of the efficiency of sub-2 µm
• 2X the efficiency of 3.5 µm
• Up to 50% less pressure than sub-2 µm columns
Agilent Poroshell 120 columns are a 2.7 µm particle with a 1.7 µm solid core and 0.5 µm porous outer layer.
This small particle size provides high efficiency, similar to sub-2 µm columns, but with 40-50% less pressure.
These high efficiency, high resolution columns can be used on any type of LC. The porous outer layer and
solid core limit diffusion distance and improve separation speed while the narrow particle size distribution
improves efficiency and resolution. The columns can support high pressure and multiple columns can be
used for the highest resolution and efficiency possible. The smaller 120Å pore size is ideal for fast high
resolution analysis of small hydrophilic peptides in protein digests.
Column Specifications
Bonded Phase
Pore Size Temp Limits
pH range endcapped
Carbon
Load
EC-C18
120Å
60 °C
2.0-8.0
Double
10%
SB-C18
120Å
90 °C
1.0-8.0
No
8%
Specifications represent typical values only
For information on the full family of Poroshell 120 phases,
see page 228.
www.AGiLeNT.CoM/CHeM/LC
385
CoLUMNS for BioMoLeCULe SePArATioNS
Poroshell 120
386
Description
Size (mm)
Analytical
4.6 x 150
Particle Size
(µm)
2.7
eC-C18
USP L1
SB-C18
USP L1
693975-902 683975-902
Analytical
4.6 x 100
2.7
695975-902 685975-902
Solvent Saver
3.0 x 150
2.7
693975-302 683975-302
Solvent Saver
3.0 x 100
2.7
695975-302 685975-302
Narrow Bore
2.1 x 150
2.7
693775-902 683775-902
Narrow Bore
2.1 x 100
2.7
695775-902 685775-902
CoLUMNS for BioMoLeCULe SePArATioNS
PLrP-S
• Contain durable and resilient polymer particles that deliver reproducible results over longer lifetimes
• Thermally and chemically stable
• Comply with USP L21 designation
• Used in bioscience, chemical, clinical research, energy, environmental, food and agriculture, material
10 µm
A
10 µm
10 µm
B
C
science and pharmaceutical industries
• Pore sizes (100Å-4000Å) for separations of small molecules to large complexes and polynucleotides
The PLRP-S family of columns consists of a range of pore sizes and particle sizes, all with identical chemistry
and fundamental adsorptive characteristics. The particles are inherently hydrophobic, therefore no bonded
phase, alkyl ligand is required for reversed-phase separations. This gives a highly reproducible material that
is free from silanols and heavy metal ions. Columns within the extensive product range are suitable for
nano/capillary separations, including both bottom-up and top-down proteomics, analytical separations,
and preparative purifications. In addition, process columns can be packed with bulk media.
Scanning electron micrographs (SeM)
of PLrP-S 10 µm particles.
The difference in pore size is clearly seen.
A is the small pore 100Å
B the larger pore 300Å
C the gigaporous 4000Å
Column Specifications
pH Range
1-14
Buffer Content
Unlimited
Organic Modifier
1-100%
Temperature Limits
200 °C
Maximum Pressure
5-8 µm: 3000 psi (210 bar)
3 µm: 4000 psi (300 bar)
PLrP-S Applications
Pore Size
Application
100Å
Small molecules/peptides/oligonucleotides
300Å
Recombinant peptides/proteins
1000Å
Large proteins
4000Å
DNA/high speed
www.AGiLeNT.CoM/CHeM/LC
387
CoLUMNS for BioMoLeCULe SePArATioNS
HPLC of 25 bp DNA ladder
PLrP-S, 2.1 x 150 mm
Mobile Phase:
A: 0.1 M TEAA
B: 0.1 M TEAA in 50% water:50% ACN
Flow Rate:
200 µL/min
Gradient:
12.5-50% B in 150 min
PLRP-S 100Å
Response, UV, 260 nm
Column:
HPLC
PLRP-S 300Å
PLRP-S 1000Å
PLRP-S 4000Å
Time
VLCPLRPSResp
Polyethylene glycols
Column:
PLrP-S 100Å
PL1111-3500
4.6 x 150 mm, 5 µm
Mobile Phase:
A: Water
B: ACN
Gradient:
10-30% B in 12 min, held at 30% B for 3 min
Flow Rate:
1.0 mL/min
PEG 400
1 mg/mL
Detector:
ELS (neb=50 °C, evap=70 °C, gas=1.6 SLM)
15
PEG 1080
Injection Volume: 10 µL
Sample Conc:
min
0
0
min
15
VLC0048
388
CoLUMNS for BioMoLeCULe SePArATioNS
exploiting chemical stability –
TfA concentration
Column:
PLrP-S 100Å
PL1512-5500
4.6 x 250 mm, 5 µm
Mobile Phase:
A: TFA (various %) in water
B: TFA (various %) in ACN
Gradient:
Linear 12-40% B in 15 min
Flow Rate:
1.0 mL/min
Detector:
1. Angiotensin III
2. Angiotensin II
3. Angiotensin I
1
2
3
0.0025%
0.02%
0.1%
0
15
min
VLC0068
ELS (neb=75 °C, evap=85 °C, gas=1.0 SLM)
Selectivity in peptide rP-LC
Column:
PLrP-S 100Å
PL1512-5500
4.6 x 250 mm, 5 µm
Mobile Phase:
A: 0.1% TFA/1% 2-Propanol/Water
B: 0.1% TFA/1% 2-Propanol/ACN
Flow Rate:
1.0 mL/min
Gradient:
95% A (0-3 min) to 50% A (13 min)
Detector:
UV, 220 nm
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
YG
GYG
PY
YV
YY
GLY
YF
GFL
YGGFM
Oxalic acid (marker)
Benzoic acid (marker)
(see J.Chromatography 512 (1990) 315-23)
Good separation of peptide standards on Agilent PLRP-S
www.AGiLeNT.CoM/CHeM/LC
389
CoLUMNS for BioMoLeCULe SePArATioNS
exploiting chemical stability –
NH4oH concentration
Column:
PLrP-S 100Å
PL1512-5500
4.6 x 250 mm, 5 µm
Mobile Phase:
A: NH4OH (various mM) in water
B: NH4OH (various mM) in ACN
Gradient:
Linear 10-100% B in 15 min
Flow Rate:
1.0 mL/min
Detector:
ELS (neb=80 °C, evap=85 °C, gas=1.0 SLM)
1. Angiotensin II
2. Angiotensin I
3. Angiotensin III
2
3
1
1 mM
10 mM
100 mM
min
0
10
VLC0049
Alberta Peptide institute test mix
5
Column:
PLrP-S 100Å
PL1512-5500
4.6 x 250 mm, 5 µm
Mobile Phase:
A: 0.1% TFA in 99% water:1% ACN
B: 0.1% TFA in 70% water:30% ACN
Gradient:
0-100% B in 30 min
Flow Rate:
1.0 mL/min
Detector:
UV, 220 nm
1.
2.
3.
4.
5.
Ala3-Gly4 (free amino)
Gly3-Gly4 (Na-acetylated)
Ala3-Gly4 (Na-acetylated)
Val3-Gly4 (Na-acetylated)
Val3-Val4 (Na-acetylated)
4
3
1
0
2
min
30
VLC0050
390
CoLUMNS for BioMoLeCULe SePArATioNS
whey proteins in dairy samples – milk
Column:
PLrP-S 300Å
PL1512-3801
4.6 x 150 mm, 8 µm
Mobile Phase:
A: 0.1% TFA in 99% water:1% ACN
B: 0.1% TFA in 1% water:99% ACN
Gradient:
36-48% B, 0-24 min, 48-100% B, 24-30 min
100% B, 30-35 min, 100-36% B, 35-40 min
Flow Rate:
1.0 mL/min
2
1
3
1. a-Lactalbumin
2. b-Lactoglobulin (B chain)
3. b-Lactoglobulin (A chain)
Injection Volume: 10 µL
Detector:
UV, 220 nm
min
0
Temperature as a tool to enhance mass transfer
and improve resolution of oligonucleotides
in ion-pair reversed-phase HPLC
Mobile Phase:
A: 100 mM TEAA
B: 100 mM TEAA in 25% ACN
Gradient:
5% change in buffer B over 5 min
Flow Rate:
1.0 mL/min
Temperature:
35 °C, 60 °C, or 80 °C
Detector:
UV, 254 nm
1
25
mV
2
35 ºC
1. 29-mer
2. 30-mer
0
0
12.5
min
25
12
mV
PLrP-S 100Å
PL1512-1300
4.6 x 50 mm, 3 µm
VLC0074
60 ºC
0
0
25
min
1
12.5
2
mV
Column:
24
80 ºC
0
0
min
12.5
www.AGiLeNT.CoM/CHeM/LC
VLC0072
391
CoLUMNS for BioMoLeCULe SePArATioNS
Large fibrous proteins
Column:
PLrP-S 300Å
PL1512-3801
4.6 x 150 mm, 8 µm
2
Column:
PLrP-S 1000Å
PL1512-3802
4.6 x 150 mm, 8 µm
Mobile Phase:
A: 0.25% TFA in water
B: 0.25% TFA in 5% water:95% ACN
Flow Rate:
1.0 mL/min
Gradient:
20-60% B in 15 min
Detector:
UV, 220 nm
1. Collagen (120,000 MW)
2. Fibrinogen (340,000 MW)
1
2
1
PLRP-S 300Å
0
392
min
12
PLRP-S 1000Å
0
min
18
VLC0051
CoLUMNS for BioMoLeCULe SePArATioNS
PLrP-S HPLC Columns
Hardware Size (mm)
PLrP-S
Particle Size 100Å
(µm)
USP L21
PLrP-S
300Å
USP L21
PLrP-S
1000Å
USP L21
PLrP-S
4000Å
USP L21
4.6 x 250
8
PL1512-5800 PL1512-5801 PL1512-5802
4.6 x 150
8
PL1512-3800 PL1512-3801 PL1512-3802 PL1512-3803
4.6 x 50
8
4.6 x 250
5
PL1512-5500 PL1512-5501
4.6 x 150
5
PL1111-3500 PL1512-3501
4.6 x 50
5
PL1512-1500 PL1512-1501 PL1512-1502 PL1512-1503
4.6 x 150
3
PL1512-3300 PL1512-3301
4.6 x 50
3
PL1512-1300 PL1512-1301
PL1512-1801 PL1512-1802 PL1512-1803
2.1 x 250
8
PL1912-5801
2.1 x 150
8
PL1912-3801 PL1912-3802 PL1912-3803
2.1 x 50
8
PL1912-1801 PL1912-1802 PL1912-1803
2.1 x 250
5
PL1912-5500 PL1912-5501
2.1 x 150
5
PL1912-3500 PL1912-3501
2.1 x 50
5
PL1912-1500 PL1912-1501 PL1912-1502 PL1912-1503
2.1 x 150
3
PL1912-3300 PL1912-3301
2.1 x 50
3
PL1912-1300 PL1912-1301
1.0 x 50
8
1.0 x 50
5
1.0 x 10
5
1.0 x 150
3
1.0 x 50
3
PL1312-1802
PL1312-1500
PL1312-1502
PL1C12-2502
PL1312-3300
PL1312-1300
PLRP-S Guard Cartridges
for 5 x 3 mm, 2/pk
PL1612-1801 PL1612-1801 PL1612-1801 PL1612-1801
Guard Cartridge holder
for 3.0 x 5.0 mm cartridges
PL1310-0016 PL1310-0016 PL1310-0016 PL1310-0016
TiPS & TooLS
For prep columns and media
ordering information,
turn to pages 470-471.
For microbore columns
ordering information,
turn to page 463.
www.AGiLeNT.CoM/CHeM/LC
393
CoLUMNS for BioMoLeCULe SePArATioNS
Amino Acid Analysis (AAA)
Columns and Supplies
ZorBAX eclipse Amino Acid
Analysis (AAA) Columns
• High resolution and rapid analysis of 24 amino acids
• Tested for amino acid analysis
• Uses well-known OPA and FMOC precolumn derivatization chemistry
• Easily automated using a detailed online, derivatization protocol available for use with Agilent
1100/1200 autosampler
The Agilent ZORBAX Eclipse AAA high efficiency column rapidly separates amino acids following an
updated and improved protocol. Total analysis from injection-to-injection can be achieved in as little as
8 min (7 min analysis time) on a 50 mm 1.8 µm column, 14 min (9 min analysis time) on shorter, 75 mm
length columns and 24 min (18 min analysis time) on the 150 mm column length. Exceptional sensitivity
(5-50 pmol with DAD, FLD) and reliability are achieved using both OPA and FMOC derivatization
chemistries in one fully automated procedure using the Agilent 1100/1200 HPLC instrument.
ZORBAX Eclipse Plus C18 columns are another excellent
choice for Amino Acid Analysis. For more information about
ZORBAX Eclipse Plus Columns, see page 248.
ZorBAX eclipse Amino Acid Analysis (AAA) Columns
Hardware Description
TiPS & TooLS
Further information can be found in the
following publication:
High-Speed Amino Acid Analysis (AAA)
on 1.8 µm Reversed-Phase (RP) Columns
(publication # 5989-6297EN)
www.agilent.com/chem/library
394
Size (mm)
Particle Size (µm)
Part No.
Analytical routine sensitivity
4.6 x 150
5
993400-902
Analytical routine sensitivity,
high-resolution using FLD
4.6 x 150
3.5
963400-902
Analytical routine sensitivity,
high-throughput
4.6 x 75
3.5
966400-902
Solvent Saver high sensitivity,
high-resolution
3.0 x 150
3.5
961400-302
Guard Cartridges, 4/pk
4.6 x 12.5
5
820950-931
Guard Hardware Kit
820999-901
CoLUMNS for BioMoLeCULe SePArATioNS
Amino Acid Standards
Each amino acid standard contains the following amino acids:
• Glycine
• L-cysteine
• L-histidine
• L-tyrosine
• L-leucine
• L-methionine
• L-serine
• L-alanine
• L-phenylalanine
• L-glutamic acid
• L-proline
• L-isoleucine
• L-arginine
• L-threonine
• L-valine
• L-lysine
• L-aspartic acid
Amino Acid Standards, 10 x 1 mL ampoules*
Description
Part No.
1 nmol/µL
5061-3330
250 pmol/µL
5061-3331
100 pmol/µL
5061-3332
25 pmol/µL
5061-3333
10 pmol/µL
5061-3334
Amino acids supplement kit
Includes 1 g each of norvaline, sarcosine, asparagine, glutamine, tryptophan,
and 4-hydroxyproline
5062-2478
*Consider shelf-life and buy limited quantities, P/N 5062-2478 ships as 1 g vials
Amino Acid Separations reagents
Description
Part No.
OPA reagent, 10 mg/mL each in 0.4 M borate buffer o-phthalaldehyde (OPA) and
3-mercaptopropionic acid, 6 x 1 mL ampoules
5061-3335
FMOC reagent, 2.5 mg/mL in acetonitrile, 9-fluorenylmethylchloroformate, 1 mL, 10 ampoules
5061-3337
Borate buffer, 100 mL
5061-3339
DTDPA (Dithiodiproprionic) reagent, for analysis of cysteine, 5 g
5062-2479
www.AGiLeNT.CoM/CHeM/LC
395
CoLUMNS for BioMoLeCULe SePArATioNS
High resolution of 24 amino acids
using ZorBAX eclipse-AAA protocol
Column:
ZorBAX eclipse AAA
963400-902
4.6 x 150 mm, 3.5 µm
Mobile Phase:
A: 40 mM Na2HPO4, pH 7.8
B: ACN:MeOH:Water,
45:45:10 v/v
Flow Rate:
2 mL/min
Temperature:
40 °C
Detector:
Fluorescence
Sample:
24 Amino Acids
3
8
2
7
4
1
22
16 17
18
19
15
9
10
14
11
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
23
20
Asp
Glu
Asn
Ser
Gln
His
Gly
Thr
Cit
Arg
Ala
Tyr
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Cys
Val
Met
Nva
Trp
Phe
Ile
Leu
Lys
Hyp
Sar
Pro
12
24
6
21
5
0
5
10
15
20
25
Time (min)
LCPAH01
This high resolution separation of 24 amino acids is done in 18 minutes. If the Rapid Resolution 4.6 x 75 mm Eclipse AAA column is selected, these amino acids are resolved in 9 minutes.
396
CoLUMNS for BioMoLeCULe SePArATioNS
ion-exchange Chromatography
Purify proteins and other charged molecules
Ion-exchange chromatography (IEX) is a highly sensitive technique that allows you to separate ions and
polar molecules based on their charge. Like SEC, IEX can be used to separate proteins in their native state.
Applying IEX to charge variant analysis
During production and purification, antibodies can exhibit changes in charge heterogeneity as a result
of amino acid substitutions, glycosylation, phosphorylation, and other post-translational or chemical
modifications. Because these changes can impact stability and activity – or cause immunologically adverse
reactions – the analysis of charge heterogeneity in monoclonal antibody (MAb) preparations is critical to
biopharmaceuticals.
In protein analysis, charge variations at a given pH indicate a change in the primary molecular structure –
resulting in additional forms of the protein in question. These are called isoforms (or charge variants),
and can be resolved by IEX chromatography. IEX is also useful as a preparative technique.
The pages that follow describe Agilent’s family of weak and strong ion-exchangers – both anionic
and cationic.
• Agilent non-porous Bio ieX columns are designed for high-resolution, high-efficiency,
and high-recovery separations.
• Agilent Bio MAb columns are optimized for separating charge isoforms of monoclonal antibodies.
• Agilent porous ieX columns (PL-SAX and PL-SCX) are chemically stable, and are available
in two pore sizes – allowing you to separate peptides, oligonucleotides, and very large proteins.
• Bio-Monolith ieX columns are uniquely suited to separating antibodies, viruses, and DNA.
www.AGiLeNT.CoM/CHeM/LC
397
CoLUMNS for BioMoLeCULe SePArATioNS
ion-exchange Column Selection
Application
Agilent Columns
Notes
Monoclonal antibodies
Agilent Bio MAb
Thorough characterization of monoclonal antibodies includes the identification and
monitoring of acidic and basic isoforms. Agilent Bio MAb HPLC columns feature a unique
resin specifically designed for high-resolution charge-based separations of monoclonal
antibodies.
Peptides and proteins
Agilent Bio IEX
Agilent Bio Ion-Exchange columns are packed with polymeric, nonporous, ion-exchange
particles. Bio IEX columns are designed for high resolution, high recovery and highly
efficient separations.
PL-SAX
The strong anion-exchange functionality, covalently linked to a fully porous chemically
stable polymer, extends the operating pH range. In addition, the anion-exchange capacity
is independent of pH. For synthetic oligonucleotides, separations using denaturing
conditions of temperature, organic solvent, and high pH are all possible. The 5 µm media
delivers separations at high resolution with the 30 µm media used for medium pressure
liquid chromatography.
Proteins, peptides and deprotected
synthetic oligonucleotides
Globular proteins and peptides
ü 1000Å
ü 4000Å
PL-SAX 1000Å
Very large biomolecules/high speed
PL-SAX 4000Å
Small peptides to large proteins
PL-SCX
ü 1000Å
ü 4000Å
Globular proteins
398
PL-SCX 1000Å
Very large biomolecules/high speed
PL-SCX 4000Å
Antibodies (IgG, IgM), plasmid DNA,
viruses, phages and other macro
biomolecules
Bio-Monolith
ü Bio-Monolith QA
ü Bio-Monolith DEAE
ü Bio-Monolith SO3
ü Bio-Monolith Protein A
Viruses, DNA, large proteins
Bio-Monolith QA
Plasmid DNS, bacteriophages
Bio-Monolith DEAE
Proteins, antibodies
Bio-Monolith SO3
PL-SCX is a macroporous PS/DVB matrix with a very hydrophilic coating and strong
cation-exchange functionality. This process is controlled to provide the optimum density
of strong cation-exchange moieties for the analysis, separation and purification of a wide
range of biomolecules. The 5 µm media delivers separations at higher resolution with the
30 µm media used for medium pressure liquid chromatography.
Strong cation-exchange, strong and weak anion-exchange, and Protein A phases.
Bio-Monolith HPLC columns are compatible with preparative LC systems,
including Agilent 1100 and 1200 HPLC systems.
CoLUMNS for BioMoLeCULe SePArATioNS
Agilent Bio MAb HPLC Columns
• A packing support composed of a rigid, spherical, highly cross-linked polystyrene divinylbenzene
(PS/DVB) non-porous bead
• Particles grafted with a hydrophilic, polymeric layer, virtually eliminating non-specific binding of
antibody proteins
• A different process is used to layer the weak cation-exchange phase to the particle making it a
higher density than the Agilent Bio WCX column particles
• Specifically designed for the separation of charge isoforms of monoclonal antibodies
Thorough characterization of monoclonal antibodies includes the identification and monitoring of
acidic and basic isoforms. Agilent Bio MAb HPLC columns feature a unique resin specifically designed
for high-resolution, charge-based separations of monoclonal antibodies. Compatible with aqueous
solution buffers, acetonitrile/acetone/methanol and water mixtures. Commonly used buffers: phosphate,
tris, MES and acetate.
Bio MAb columns are available in 1.7, 3, 5 and 10 µm sizes, providing higher resolution with
smaller particles.
Column Specifications
Bonded Phase
Weak Cation-Exchange
(carboxylate)
iD
2.1 and 4.6 mm
Particle
Size
1.7, 3, 5
and 10 µm
operating
pH
Temperature
Stability
Limit
2-12
80 °C
flow rate
0.1-1.0 mL/min
TiPS & TooLS
Capillary electrophoresis is an
alternative technique to liquid
chromatography for the separation
of charged isoforms. Further
information can be found in the
following Technical Note:
Capillary electrophoreseis focusing
on the Agilent Capillary Electrophoresis
system (publication # 5989-9852EN)
www.agilent.com/chem/library
www.AGiLeNT.CoM/CHeM/LC
399
CoLUMNS for BioMoLeCULe SePArATioNS
Consistent ion-exchange MAb separation
Column:
mAU
Bio MAb, PeeK
5190-2411
2.1 x 250 mm, 5 µm
Buffer:
A: Sodium phosphate buffer, 20 mM
B: Buffer A + 400 mM NaCl
Gradient:
15-35% Buffer B from 0-30 min
Flow Rate:
0.65 mL/min
Sample:
CHO-humanized MAb, 1 mg/mL
Injection:
2.5 µL
Detector:
UV 220 nm
Temperature:
Ambient
120
100
80
60
40
20
0
1st run
2
6
4
8
10
12
14
16
18
min
mAU
140
120
100
80
60
40
20
0
To provide a metal free flow path, Bio MAb PEEK columns
are available.
10th run
4
2
6
8
10
12
14
16
18
min
VLC_IE_Mab
Virtually eliminate retention time variations
Column:
Bio MAb, stainless steel
5190-2413
4.6 x 250 mm, 10 µm
Mobile Phase:
A: 10 mM phosphate, pH 6.0
B: A + 1.0 M NaCl
Flow Rate:
1.0 mL/min
Gradient:
0-100% B in 42 min
Temperature:
25 °C
Detector:
UV 214 nm
2
1
3
1. Cytochrome c
2. Lysozyme
3. Ribonuclease A
Lot A
Lot B
Lot C
0
2
4
6
8
10
12
14
16
18
min
The combination of well-controlled resin production, column surface chemistry, and column packing virtually eliminates retention time variations
from column-to-column and lot-to-lot.
400
CoLUMNS for BioMoLeCULe SePArATioNS
Charge isoform analysis
of monoclonal antibodies
Absorbance (AU)
Column:
Bio MAb, PeeK
5190-2407
4.6 x 250 mm, 5 µm
Mobile Phase:
A: 10 mM Sodium Phosphate, pH 7.50
B: A + 100 mM NaCl, pH 7.50
0.40
Flow Rate:
0.8 mL/min
0.30
Gradient:
15-95% B in 60 min
Sample:
5 µL, 5 mg/mL, MAb
0.60
0.50
Acidic
isoforms
Basic
isoforms
0.20
0.10
0.00
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
High resolution separation of acidic and basic charge variants using the Agilent Bio MAb NP5 column
Agilent Bio MAb HPLC Columns
Particle Size (µm)
Bio MAb
PeeK
Pressure Limit
Bio MAb
Stainless Steel
Pressure Limit
4.6 x 250
10
5190-2415
275 bar, 4000 psi
5190-2413
275 bar, 4000 psi
4.6 x 50, Guard
10
5190-2416
275 bar, 4000 psi
5190-2405
413 bar, 6000 psi
Size (mm)
4.6 x 250
5
5190-2407
400 bar, 5800 psi
4.6 x 50, Guard
5
5190-2408
400 bar, 5800 psi
4.6 x 50
3
5190-2403
551 bar, 8000 psi
4.6 x 50
1.7
5190-2401
600 bar, 8700 psi
4.0 x 10, Guard
10
5190-2414
275 bar, 4000 psi
4.0 x 10, Guard
5
5190-2406
413 bar, 6000 psi
4.0 x 10, Guard
3
5190-2404
551 bar, 8000 psi
4.0 x 10, Guard
1.7
5190-2402
600 bar, 8700 psi
2.1 x 250
10
5190-2419
275 bar, 4000 psi
2.1 x 50, Guard
10
5190-2420
275 bar, 4000 psi
2.1 x 250
5
5190-2411
400 bar, 5800 psi
2.1 x 50, Guard
5
5190-2412
400 bar, 5800 psi
www.AGiLeNT.CoM/CHeM/LC
401
CoLUMNS for BioMoLeCULe SePArATioNS
Agilent Bio ieX HPLC Columns
• Highly cross-linked and rigid nonporous poly(styrene divinylbenzene) (PS/DVB) particles
are grafted with a hydrophilic, polymeric layer, eliminating nonspecific binding
• Uniform, densely packed ion-exchange functional groups are chemically bonded to the hydrophilic
layer (multiple ion-exchange groups per anchoring) to increase column capacity
• Particles, coating and bonding are resistant to high pressures, promoting higher resolution
and faster separations
• Multiple ion-exchange groups are captured on one anchoring to increase capacity
Agilent Bio IEX HPLC columns are packed with polymeric, nonporous, ion-exchange particles
and are designed for high resolution, high recovery and highly efficient separations of peptides,
oligonucleotides and proteins.
The Bio IEX family offers strong cation-exchange (SCX), weak cation-exchange (WCX), strong
anion-exchange (SAX) and weak anion-exchange (WAX) phases. All phases are available in
1.7, 3, 5 and 10 µm non-porous particles sizes.
Column Specifications
Bonded Phase
SCX (Strong cation-exchange) - S03H
iD
Particle Size
2.1 and 4.6 mm
1.7, 3, 5 and 10 µm
operating
pH Stability Temperature Limit
2-12
WCX (Weak cation-exchange) - C00H
SAX (Strong anion-exchange) - N(CH3)3
WAX (Weak cation-exchange) - N(C2H5)2
TiPS & TooLS
More information is a click away. We have a variety of educational primers, application notes, maintenance guides,
and literature available from Agilent for free.
To learn more, visit www.agilent.com/chem/library
402
80 °C
flow rate
0.1-1.0 mL/min
CoLUMNS for BioMoLeCULe SePArATioNS
exceptional separating power
Column:
mAU
Agilent Bio SCX, stainless steel
5190-2423
4.6 x 50 mm, 3 µm
Buffer:
10 mM Phosphate, pH 6.0
Flow Rate:
0.5 mL/min
Gradient:
0-1.0 M NaCl, 15 min
Detector:
280 nm
2
1
5
1.
2.
3.
4.
5.
4
40
3
30
Ovalbumin, 4.6 pl
Ribonuclease A, 8.7 pl
Cytochrome c, 9.6 pl
Aprotinin, 10.0 pl
Lysozyme, 11.0 pl
N > 100,000/50 mm for Lysozyme
20
The hydrophilic, polymeric layer and densely packed ion-exchange functional
groups provide extremely sharp peak shapes and high resolution of a mixture
of proteins with a broad range of isoelectric points (pI).
10
0
2
4
6
8
10
12 min
Separation of protein standards on Agilent 3 µm
ion-exchange columns by cation-exchange
chromatography
Column A:
Agilent Bio SCX, NP 3, 4.6 x 50 mm, SS
Column B:
Agilent Bio wCX, NP 3, 4.6 x 50 mm, SS
Column C:
Agilent Bio MAb, NP 3, 4.6 x 50 mm, SS
Mobile Phase:
A: 10 mM NaH2PO4.2H2O, pH 5.70
B: A + 1 M NaCl
Flow Rate:
0.5 mL/min
Gradient:
0 min - 100% A : 0% B
25 min - 0% A : 100% B
3
1
3
2
3
SCX
1
Temperature:
Ambient
Detector:
Agilent 1260 Infinity Bio-inert Quaternary LC
with diode array detector at 220 nm
Sample:
Cytochrome c, ribonuclease A, lysozyme
and protein mix
2
wCX
2
1
MAb
illustration that Bio wCX, SCX and MAb columns are capable of producing protein separations
Agilent column
Bio WCX NP, 3 µm
Bio SCX NP, 3 µm
Bio MAb NP, 3 µm
Peak number
Peak name
rT [min] Height [mAU] Area [mAU*s] Plates width [min] resolution
1
Cytochrome c
7.86
124
1833
7844
0.2089
-
2
RNase A
9.03
241
3358
10800
0.2044
3.32
3
Lysozyme
13.13
636
7274
44488
0.1466
13.73
1
RNase A
7.06
396
2616
39847
0.0832
-
2
Cytochrome c
7.66
297
2778
28920
0.1060
1.08
3
Lysozyme
10.49
763
7186
44828
0.1167
1.37
1
Cytochrome c
10.04
203
2369
21814
0.1600
-
2
RNase A
11.37
256
2690
33314
0.1467
3.11
3
Lysozyme
12.59
652
6616
56734
0.1244
5.28
www.AGiLeNT.CoM/CHeM/LC
403
CoLUMNS for BioMoLeCULe SePArATioNS
Brand B wCX-10
4.0 x 250 mm, 10 µm
Mobile Phase:
A: 20 mM sodium phosphate (pH = 6.0)
B:20 mM sodium phosphate (pH = 6.0)
containing 1.0 M sodium chloride
Flow Rate:
0.5 mL/min
Gradient:
10% B 0 min, 35% B 35 min,
10% B 36 min, 10% B 45 min
Detector:
UV, 220 nm/4 nm, Reference: Off
(data also acquired at 220, 230, 240,
and 280 nm)
Sample:
P128
mAU
350
300
250
200
150
100
50
0
22
mAU
140
26.359
26.618
Column C:
A
mAU
400
350
300
250
200
150
100
50
0
22
23
24
25
23
24
25
26
27
28
29
30
31
min
26.867
Bio MAb, PeeK
5190-2415
4.6 x 250 mm, 10 µm
B
25.860
Column B:
24.786
Bio MAb, PeeK
5190-2407
4.6 x 250 mm, 5 µm
26
27
28
29
30
31
min
26
27
28
29
30
31
min
25.210
Column A:
27.564
weak cation-exchange chromatography for P128 therapeutic protein sample
on the Agilent 1260 Bio-inert Quaternary LC system using different cation-exchange columns
C
120
23.976
100
80
Sample was desalted by ultrafiltration and extracted into 20 mM
sodium phosphate.
60
40
20
22
404
23
24
25
CoLUMNS for BioMoLeCULe SePArATioNS
Agilent Bio ieX HPLC Columns, PeeK
Particle Size (µm)
Pressure Limit
Bio SCX
Part No.
Bio wCX
Part No.
Bio SAX
Part No.
Bio wAX
Part No.
4.6 x 250
10
275 bar, 4000 psi
5190-2435
5190-2455
5190-2475
5190-2495
4.6 x 50, Guard
10
275 bar, 4000 psi
5190-2436
5190-2456
5190-2476
5190-2496
4.6 x 250
5
400 bar, 5800 psi
5190-2427
5190-2447
5190-2467
5190-2487
Size (mm)
4.6 x 50, Guard
5
400 bar, 5800 psi
5190-2428
5190-2448
5190-2468
5190-2488
2.1 x 250
10
275 bar, 4000 psi
5190-2439
5190-2459
5190-2479
5190-2499
2.1 x 50, Guard
10
275 bar, 4000 psi
5190-2440
5190-2460
5190-2480
5190-2500
2.1 x 250
5
400 bar, 5800 psi
5190-2431
5190-2451
5190-2471
5190-2491
2.1 x 50, Guard
5
400 bar, 5800 psi
5190-2432
5190-2452
5190-2472
5190-2492
Bio SCX
Part No.
Bio wCX
Part No.
Bio SAX
Part No.
Bio wAX
Part No.
Agilent Bio ieX HPLC Columns, Stainless Steel
Size (mm)
Particle Size (µm)
Pressure Limit
4.6 x 250
10
275 bar, 4000 psi
5190-2433
5190-2453
5190-2473
5190-2493
4.6 x 250
5
413 bar, 6000 psi
5190-2425
5190-2445
5190-2465
5190-2485
4.6 x 50
3
551 bar, 8000 psi
5190-2423
5190-2443
5190-2463
5190-2483
4.6 x 50
1.7
600 bar, 8700 psi
5190-2421
5190-2441
5190-2461
5190-2481
4.0 x 10, Guard
10
275 bar, 4000 psi
5190-2434
5190-2454
5190-2474
5190-2494
4.0 x 10, Guard
5
413 bar, 6000 psi
5190-2426
5190-2446
5190-2466
5190-2486
4.0 x 10, Guard
3
551 bar, 8000 psi
5190-2424
5190-2444
5190-2464
5190-2484
4.0 x 10, Guard
1.7
275 bar, 4000 psi
5190-2422
5190-2442
5190-2462
5190-2482
www.AGiLeNT.CoM/CHeM/LC
405
CoLUMNS for BioMoLeCULe SePArATioNS
PL-SAX Strong Anion-exchange Columns
• Small particles deliver excellent chromatographic performance
• Wide range of particle sizes and 2 pore sizes for flexible analysis to scale-up purification
• Exceptional stability for long column lifetime
PL-SAX -N(CH3)3+ is ideal for the anion-exchange HPLC separations of proteins, peptides and deprotected
synthetic oligonucleotides under denaturing conditions. The strong anion-exchange functionality, covalently
linked to a chemically stable fully porous polymer, extends the operating pH range. In addition, the
anion-exchange capacity is independent of pH. For synthetic oligonucleotides, separations using
denaturing conditions of temperature, organic solvent, and high pH are all possible. PL-SAX delivers
improved chromatography for self-complementary or G-rich sequences that may associate to form
aggregates or hairpin structures. The 5 µm material provides high efficiency separations of n
and n-1 sequences. A wide range of particle sizes and column geometries permits analysis scale-up
to purification. The strong anion-exchange functionality provides a material with exceptional chemical
and thermal stability, even with sodium hydroxide eluents, leading to long column lifetime.
Column Specifications
Bonded Phase
Strong Anion-Exchange
406
iD
(mm)
2.1, 4.6, 7.5,
25, 50
and 100
Particle Size
(µm)
Pore Size
5, 8, 10
and 30
1000Å
and 4000Å
operating
Temperature
pH Stability
Limit
1-14
80 °C
CoLUMNS for BioMoLeCULe SePArATioNS
Standard ion-exchange protein separation
Column:
PL-SAX 1000Å
PL1551-1502
4.6 x 50 mm, 5 µm
Mobile Phase:
A: 10 mM Tris HCl pH 8
B: A+0.35 M NaCl pH 8
Gradient:
0-100% B in 20 min
Flow Rate:
1.0 mL/min
Detector:
UV, 220 nm
1.
2.
3.
4.
Myoglobin
Bovine carbonic anhydrase
Ovalbumin
Soybean trypsin inhibitor
3
1
0
4
2
25
min
VLC0070
Analysis of choline kinase
on PL-SAX 4000Å
Column:
PL-SAX
PL1551-1803
4.6 x 50 mm, 8 µm
Mobile Phase:
A: 20 mM Tris 5% ethylene
glycol, pH 7.5
(The following are required
to retain enzyme activity)
1.0 mM Ethylene glycol
tetraacetic acid
2.0 mM ß-Mercaptoethanol
0.2 mM
Phenylmethylsulfonyl fluoride
B: A + 1 M KCI
Flow Rate:
3.0 mL/min
Detector:
UV, 280 nm
%B
100
enzyme activity
0
0
min
40
Separation courtesy of T Porter, Purdue University, USA
www.AGiLeNT.CoM/CHeM/LC
407
CoLUMNS for BioMoLeCULe SePArATioNS
Analysis of representative whey proteins
Column:
1
PL-SAX 1000Å
PL1551-1802
4.6 x 50 mm, 8 µm
Mobile Phase:
A: 0.02 M Tris HCI, pH 7
B: A + 0.5 M CH3COONa, pH 7
Flow Rate:
1.0 mL/min
Gradient:
Linear 0-50% B in 10 min
Detector:
UV, 280 nm
2
1.
2.
3.
4.
Carbonic anhydrase
a-lactalbumin
b-lactoglobulin B
b-lactoglobulin A
4
3
0
High resolution separation of a
Poly-T-oligonucleotide size standard
spiked with 10-mer, 15-mer, 30-mer
and 50-mer (main peaks)
Column:
408
min
18
2000
10
15
PL-SAX 1000Å
PL1551-1802
4.6 x 50 mm, 8 µm
Mobile Phase:
A: 7:93 v/v ACN: 0.1 M TEAA, pH 8.5
B: 7:93 v/v ACN: 0.1 M TEAA,
1 M ammonium chloride, pH 8.5
Gradient:
0-40% B in 10 min, followed by
40-70% B in 14 min and
70-100% B in 25 min
Flow Rate:
1.5 mL/min
Temperature:
60 °C
Detector:
UV, 220 nm
30
50
0
0
min
50
VLC0045
CoLUMNS for BioMoLeCULe SePArATioNS
PL-SAX Strong Anion-exchange Columns
Particle Size (µm)
Pressure Limit
PL-SAX
1000Å
PL-SAX
4000Å
1.0 x 50
5
207 bar, 3000 psi
PL1351-1502
PL1351-1503
2.1 x 50
5
207 bar, 3000 psi
PL1951-1502
PL1951-1503
4.6 x 50
5
207 bar, 3000 psi
PL1551-1502
PL1551-1503
2.1 x 50
8
207 bar, 3000 psi
PL1951-1802
PL1951-1803
2.1 x 150
8
207 bar, 3000 psi
PL1951-3802
PL1951-3803
4.6 x 50
8
207 bar, 3000 psi
PL1551-1802
PL1551-1803
4.6 x 150
8
207 bar, 3000 psi
PL1551-3802
PL1551-3803
4.6 x 250
10
207 bar, 3000 psi
PL1551-5102
PL1551-5103
4.6 x 150
10
207 bar, 3000 psi
PL1551-3102
PL1551-3103
25 x 50
10
207 bar, 3000 psi
PL1251-1102
PL1251-1103
25 x 150
10
207 bar, 3000 psi
PL1251-3102
PL1251-3103
50 x 150
10
207 bar, 3000 psi
PL1751-3102
PL1751-3103
100 x 300
10
207 bar, 3000 psi
PL1851-2102
PL1851-2103
4.6 x 250
30
207 bar, 3000 psi
PL1551-5702
PL1551-5703
4.6 x 150
30
207 bar, 3000 psi
PL1551-3702
PL1551-3703
25 x 150
30
207 bar, 3000 psi
PL1251-3702
PL1251-3703
50 x 150
30
207 bar, 3000 psi
PL1751-3702
PL1751-3703
100 x 300
30
207 bar, 3000 psi
PL1851-3102
PL1851-3103
PL-SAX
1000Å
PL-SAX
4000Å
Size (mm)
PL-SAX Strong Anion-exchange Bulk Media
Size
Particle Size (µm)
100 g
10
PL1451-4102
PL1451-4103
1 kg
10
PL1451-6102
PL1451-6103
100 g
30
PL1451-4702
PL1451-4703
1 kg
30
PL1451-6702
PL1451-6703
www.AGiLeNT.CoM/CHeM/LC
409
COLUMNS FOR BIOMOLECULE SEPARATIONS
PL-SCX Strong Cation-Exchange Columns
• Optimal design for effective separation of biomolecules
• Pore sizes allow use of a range of solute sizes
• Exceptional stability for long column lifetime
PL-SCX -SO3- is a macroporous PS/DVB matrix with a very hydrophilic coating and strong cation-exchange
functionality. This process is controlled to provide the optimum density of strong cation-exchange moieties
for the analysis, separation and purification of a wide range of biomolecules, from small peptides to large
proteins. Two pore sizes are available, 1000Å and 4000Å, to provide good mass transfer characteristics for
a range of solute sizes. The 5 µm media delivers separations at higher resolution with the 30 µm media used
for medium pressure liquid chromatography.
Column Specifications
ID
(mm)
Bonded Phase
Strong Cation-Exchange
Particle Size
(µm)
Pore Size
2.1, 4.6,
7.5, 25, 50
and 100
5, 8, 10
and 30
Standard protein separation
2
1
Column:
PL-SCX 1000Å
PL1545-1502
4.6 x 50 mm, 5 µm
Mobile Phase:
A: 20 mM KH2PO4, pH 6.0
B: A + 1 M NaCl
Gradient:
0-100% B in 20 min
Flow Rate:
1.0 mL/min
Detector:
UV, 280 nm
1000Å
and 4000Å
3
Lot B
Lot C
410
2
4
6
8
10
12
14
16
1-14
1.
2.
3.
4.
Lot A
0
Operating
Temperature
pH Stability
Limit
18
min
Myoglobin
Chymotrypsinogen A
Cytochrome c
Lysozyme
80 °C
COLUMNS FOR BIOMOLECULE SEPARATIONS
PL-SCX Strong Cation-Exchange Columns
Particle Size (µm)
Pressure Limit
PL-SCX
1000Å
PL-SCX
4000Å
1.0 x 50
5
207 bar, 3000 psi
PL1345-1502
PL1345-1503
2.1 x 50
5
207 bar, 3000 psi
PL1945-1502
PL1945-1503
4.6 x 50
5
207 bar, 3000 psi
PL1545-1502
PL1545-1503
2.1 x 50
8
207 bar, 3000 psi
PL1945-1802
PL1945-1803
2.1 x 150
8
207 bar, 3000 psi
PL1945-3802
PL1945-3803
4.6 x 50
8
207 bar, 3000 psi
PL1545-1802
PL1545-1803
4.6 x 150
8
207 bar, 3000 psi
PL1545-3802
PL1545-3803
4.6 x 150
10
207 bar, 3000 psi
PL1545-3102
PL1545-3103
4.6 x 250
10
207 bar, 3000 psi
PL1545-5102
PL1545-5103
25 x 50
10
207 bar, 3000 psi
PL1245-1103
PL1245-1103
25 x 150
10
207 bar, 3000 psi
PL1245-3103
PL1245-3103
50 x 150
10
207 bar, 3000 psi
PL1745-3103
PL1745-3103
100 x 300
10
207 bar, 3000 psi
PL1845-2103
PL1845-2103
4.6 x 150
30
207 bar, 3000 psi
PL1545-3702
PL1545-3703
4.6 x 250
30
207 bar, 3000 psi
PL1545-5703
PL1545-5703
25 x 150
30
207 bar, 3000 psi
PL1245-3702
PL1245-3703
50 x 150
30
207 bar, 3000 psi
PL1745-3703
PL1745-3703
100 x 300
30
207 bar, 3000 psi
PL1845-3102
PL1845-3103
PL-SCX
1000Å
PL-SCX
4000Å
Size (mm)
PL-SCX Strong Cation-Exchange Bulk Media
Size
Particle Size (µm)
100 g
10
PL1445-4102
PL1445-4102
1 kg
10
PL1445-6102
PL1445-6103
100 g
30
PL1445-4702
PL1445-4703
1 kg
30
PL1445-6702
PL1445-6703
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411
COLUMNS FOR BIOMOLECULE SEPARATIONS
Agilent Bio-Monolith Ion-Exchange
HPLC Columns
• Polymer-based, monolith HPLC columns designed for macro biomolecule separations
• Flow-rate independent separations; no diffusion, no pores and no void volume make transport
between mobile and stationary phase very rapid
• Monolith disk is 5.2 mm x 4.95 mm (100 µL column volume) with continuous channels, eliminating
diffusion mass transfer
• Extremely fast separations speed up method development time and decrease costs; locking in
method parameters takes significantly less time and buffer
Bio-Monolith Ion-Exchange HPLC Column
Agilent Bio-Monolith Ion-Exchange HPLC columns provide high resolution and rapid separations of
antibodies (IgG, IgM), plasmid DNA, viruses, phages and other macro biomolecules. The product family
offers strong cation-exchange, strong and weak anion-exchange and Protein A phases. Bio-Monolith
HPLC columns are compatible with HPLC and preparative LC systems, including Agilent 1100 and 1200
HPLC systems.
Agilent Bio-Monolith HPLC Column Selection Guide
Column
Description
Key Applications
Part No.
Bio-Monolith QA
The quaternary amine bonded phase (Strong AnionExchange) is fully charged over a working pH range
of 2-13, binding negatively charged biomolecules.
ü Adenovirus process monitoring and quality control
ü IgM purification monitoring and quality control
ü Monitoring DNA impurity removal
ü Monitoring endotoxin removal
ü HSA Purity
5069-3635
Bio-Monolith DEAE
The diethylaminoethyl bonded phase (Weak AnionExchange) offers increased selectivity of biomolecules
with negative charge over a working pH range of 3-9.
ü Process monitoring and quality control of
bacteriophage manufacturing and purification
ü Process monitoring and quality control of plasmid
DNA purification
5069-3636
Bio-Monolith SO3
The sulfonyl bonded phase (Strong Cation-Exchange) is
fully charged over a working pH range of 2-13, binding
positively charged biomolecules.
ü Fast and high resolution analytical separations of
large molecules such as proteins and antibodies
ü Hemoglobin A1c fast analytics
5069-3637
TIPS & TOOLS
Agilent also offers a Protein A Bio-Monolith column for affinity chromatography. For more information, see pages 434-436.
412
COLUMNS FOR BIOMOLECULE SEPARATIONS
Column Specifications
Dimensions
5.2 mm x 4.95 mm
Column volume
100 µL
Maximum pressure
150 bar (15 MPa, 2200 psi)
Temperature min/max
Working: 4-40 °C
Storage: 4-30 °C
Recommended pH
Working range: 2-13
Materials of construction
Hardware: Stainless steel
Color ring identifier
Bio-Monolith QA: Blue
Cleaning-in-place: 1-14
Packing: poly(glycidyl methacrylate-co-ethylene dimethacrylate) highly porous monolith
Bio-Monolith DEAE: Green
Bio-Monolith SO3: Red
Shelf life/expiration date
SO3, QA, DEAE: 24-36 months
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413
COLUMNS FOR BIOMOLECULE SEPARATIONS
8
UV at 220 nm
Sample:
One mg each/mL in mobile phase A.
1. RNAse from bovine pancreas (pI 9.6)
2. Cytochrome c from bovine heart (pI 10.37-10.8)
3. Lysozyme from chicken egg (pI 11.35) (0.5 mg)
4
mAU
14
Instrument:
414
Agilent 1200 SL with diode array detector
8
6
16.410
9.072
8
10
12
14
16
10.335
4
10
12.170
6
18
min
18
min
16.901
10
12
Detector:
A with NaCl gradient
12
9.271
0.5 min hold with mobile phase A followed by a linear
gradient to 45% B in 15 min (elapsed time 15.5 min);
then 60% B at 15.6 min continued to 20 min. Column
flushed with 100% B for 15 min before reequilibration for the next run.
pH Gradient: A: 5 mM Na2HPO4, buffer pH 5.5 and B:
40 mM NA2HPO4 (not buffered, pH 8.9). 2% B/min at
1 mL/min for 15 min, followed by a column wash with
90% B for 5 min.
3
6.834
Gradient:
2
6.357
2 mL/min
1
5.653
Flow Rate:
mAU
14
6.482
7.059
7.604
8.162
A: 10 mM Na2HPO4, pH 6.0
B: A + 0.5 M NaCl or just 0.5 M Na2HPO4, pH 6.0
4.483
4.968
Mobile Phase:
4.945
Agilent Bio-Monolith CM15,
5.5 x 15 mm
4.284
4.520
4.688
Column:
7.832
Baseline expansion of a separation of
protein standards
B with Na2HPO4 gradient
6
4
4
6
8
10
12
B shows a better resolution of protein contaminants.
14
16
COLUMNS FOR BIOMOLECULE SEPARATIONS
120
50
Time: 36 min
Time: 158 min
Time: 191 min
% Buffer B
45
DEAE
5069-3636
5.2 x 4.95 mm
Mobile Phase:
A: 125 mM Phosphate buffer, pH 7.0
B: 125 mM Phosphate buffer + 1 M NaCl, pH 7.0
Flow Rate:
1 mL/min
Gradient:
100% buffer A (2.5 min)
0-100% buffer B (10 min)
100% buffer A (2 min)
40
Relative Absorbance (mAU)
Column:
35
2
100
80
30
Phages and other components
25
60
20
% Gradient
Bio-Monolith DEAE column monitors phage
production during fermentation
40
15
3
1
10
20
Detector:
UV at 280 nm
5
Instrument:
High pressure gradient HPLC system,
Agilent 1200 Infinity LC
0
0
0
2
4
6
8
10
12
min
As phage proliferation progresses, the genomic DNA (gDNA) concentration increases as the host cells are being lysed. In the late stages of
fermentation, gDNA begins to degrade into fragments. These gDNA fragments cannot be easily removed by purification media, therefore it is
critical to stop the fermentation cycle prior to the degradation of the genomic DNA. The chromatogram above represents three samples taken
from the bioreactor at 36, 158 and 191 minutes. Peak 1 represents phage, media and host cells, peak 2 the intact gDNA and peak 3 the
fragmented gDNA.
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415
COLUMNS FOR BIOMOLECULE SEPARATIONS
Size Exclusion Chromatography (SEC)
Accurately determine biomolecule
aggregation, fragmentation, and chemical
ligation/modification
Size exclusion chromatography (SEC) is a technique for separating proteins, oligonucleotides, and other
complex biopolymers by size using aqueous eluents.
Applying SEC to aggregation studies
The size, type, and content of aggregates present in protein biopharmaceuticals can affect both efficacy
and formulation – or worse, induce an immunogenic response. Aggregation formations occur through a
variety of mechanisms, including disulfide bond formation and non-covalent interactions.
Because the size of protein aggregates, including dimers, is sufficiently different from the protein monomer,
you can separate the various forms using SEC. In fact, SEC with UV or light scattering is a standard
technique for quantifying protein aggregation.
Applying SEC to quantitation and molecular
weight determination
For proteins and other molecules of discreet molecular weight, SEC can be used to detect and quantitate
monomers, dimers, aggregates and fragments. SEC can also separate oligonucleotide mixtures.
For biopolymers of varying sizes, like starches and other polysaccharides, SEC can provide data on
molecular weight distribution and branching (with the proper detectors).
416
COLUMNS FOR BIOMOLECULE SEPARATIONS
As a leading manufacturer of SEC columns and instruments for over 30 years, Agilent is continually
developing new SEC products that will provide even higher resolution and quicker separations. This section
highlights Agilent’s broad family of SEC columns for protein biopolymer analysis:
• Bio SEC-3 and Bio SEC-5 columns are available in a variety of pore sizes, and are well suited for
protein analysis – especially when determining the presence of dimers and aggregates in therapeutic
biologicals. Note that 3 µm Bio SEC-3 columns provide higher resolution than our industry-standard
5 µm Bio SEC-5 columns.
• ProSec 300S columns work well with globular proteins under high salt conditions.
• ZORBAX GF-250 and GF-450 columns are best for preparative SEC of proteins, because of their
larger column size and higher flow rates.
• PL aquagel-OH columns can be used to analyze biopolymers of broad molecular weights, such as
PEGs, oligo- and polysaccharides, starches, and gums.
Size Exclusion Chromatography (SEC)
Application
Agilent Columns
Notes
Peptides, proteins
Agilent Bio SEC-3
Higher resolution and faster separations from 3 µm particles, with 100Å,
150Å, and 300Å pore sizes.
Large biomolecules and samples
with multiple molecular weight
components
Agilent Bio SEC-5
More pore size options (100Å, 150Å, 300Å, 500Å, 1000Å, and 2000Å)
to cover a wider range of analytes.
Globular proteins, antibodies
ProSEC 300S
Single column option for protein analysis in high salt conditions.
Proteins, globular proteins
ZORBAX GF-250/450
Higher flow rate capabilities and larger column size for SEC semi-prep
and prep.
Low MW polymers and oligomers,
oligosaccharides, PEGs,
lignosulfonates
2 or 3 PL aquagel-OH
The PL aquagel-OH analytical series has a pH range of 2-10,
compatibility with organic solvent (up to 50% methanol), mechanical
stability up to 140 bar (2030 psi), and low column operating pressures.
Polydisperse biopolymers,
polysaccharides, cellulose
derivatives
Very high MW polymers,
hyaluronic acids, starches, gums
ü PL aquagel-OH 8 µm
ü PL aquagel-OH 20 5 µm
ü PL aquagel-OH MIXED-M 8 µm
2 or 3 PL aquagel-OH
ü PL aquagel-OH MIXED-H 8 µm
ü PL aquagel-OH 60/50/40 8 µm
PL aquagel-OH 60/50/40 15 µm in series
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417
COLUMNS FOR BIOMOLECULE SEPARATIONS
Agilent Bio SEC-3
• Exceptional loading capacity, stability, and reproducibility for size-based biomolecule separations
• Sharper peaks, higher resolution, and better protein recovery
• Faster separations than large-particle SEC columns
• Compatibility with most aqueous buffers
• Excellent stability in high-salt and low-salt conditions
Agilent Bio SEC-3 HPLC columns are a breakthrough technology for size exclusion chromatography (SEC).
They are packed with spherical, narrowly dispersed 3 µm silica particles coated with a proprietary
hydrophilic layer. This thin polymeric layer is chemically bonded to pure, mechanically stable silica under
controlled conditions, ensuring a highly efficient size exclusion particle.
Agilent Bio SEC-3 HPLC columns are available in 100Å, 150Å and 300Å pore sizes to accommodate most
peptide and protein size exclusion separations.
Column Specifications
Pore Size
100Å
Particle Size
MW Range
pH Range
Max Pressure
3 µm
100-100,000
2-8.5
240 bar, 3500 psi
Flow Rate
0.1-1.25 mL/min (7.8 mm id)
0.1-0.4 mL/min (4.6 mm id)
150Å
3 µm
500-150,000
2-8.5
240 bar, 3500 psi
0.1-1.25 mL/min (7.8 mm id)
0.1-0.4 mL/min (4.6 mm id)
300Å
3 µm
5,000-1,250,000
2-8.5
240 bar, 3500 psi
0.1-1.25 mL/min (7.8 mm id)
0.1-0.4 mL/min (4.6 mm id)
TIPS & TOOLS
Deactivated/silanized vials have inert surfaces that will not interact with metals, biologicals or proteins, and
will not cause pH shifts. Avoid standard polypropylene vials for biological or light-sensitive compounds.
418
COLUMNS FOR BIOMOLECULE SEPARATIONS
Calibration curves – Bio SEC-3
Bio SEC-3
7.8 x 300 mm, 3 µm
Mobile Phase:
150 mM Na phosphate, pH 7.0
Flow Rate:
1.0 mL/min
Detector:
UV
1,000,000,000
100,000,000
Proteins
MWt
300Å
150Å
100Å
Thyroglobulin
670000
6.34
5.50
5.63
Gamma globulin
158000
8.03
6.24
5.74
BSA
67000
8.90
7.00
6.03
Ovalbumin
45000
9.57
7.70
6.41
Myoglobin
17000
10.12
8.50
7.10
Ribonuclease A
12700
10.40
8.80
7.46
Vitamin B-12
1350
11.90
11.40
10.20
Intact MAb monomer and dimer separation
Column:
Bio SEC-3, 300Å
5190-2511
7.8 x 300 mm, 3 µm
Buffer:
Sodium phosphate buffer, pH 7.0, 150 mM
Isocratic:
0-100% Buffer A from 0-30 min
Flow Rate:
1.0 mL/min
Sample:
CHO-humanized MAb, 5 mg/mL – intact
Injection:
5 µL
Detector:
UV 220 nm
Temperature:
Ambient
10,000,000
Molecular Weight
Column:
1,000,000
100,000
300Å
10,000
150Å
100Å
1,000
100
5
4
6
7
8
9
10
11
12
13
MWvsRet
Retention Volume (mL)
mAU
300
Monomer
250
Dimer
200
150
100
50
0
4
6
8
10
12
14 min
VLC_MD_Mab
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419
COLUMNS FOR BIOMOLECULE SEPARATIONS
Comparison of Agilent Bio SEC-3
and competitor column in the analysis
of a monoclonal antibody
Column:
Bio SEC-3, 300Å
5190-2511
7.8 x 300 mm, 3 µm
Competitor 7.8 x 300 mm
Mobile Phase:
150 mM sodium phosphate +
100 mM Na sulfate (with salt)
150 mM sodium phosphate (without salt)
UV, 220 nm
Sample:
MAb (2 mg/mL)
Monomer
30
Dimer
With salt
With salt
20
1.0 mL/min
Detector:
mAU
40
Monomer
30
Column:
Flow Rate:
mAU
40
Dimer
20
10
10
Without salt
Without salt
0
8 minutes
8 minutes
0
Agilent Bio SEC-3
0
2
4
6
Competitor column
8
10 min
0
2
4
6
8
10 min
Mono_Dimer1
The Agilent Bio SEC-3 column reveals the presence of smaller MW species missed by the competitor column.
Monoclonal Antibody Monomer and Dimer Analysis using Agilent Bio SEC-3
and a Competitor Column
Column
Resolution Ratio
Monomer:Dimer
Monomer
Efficiency
Percentage
Dimer
With salt
Agilent
2.04
7,518
0.59
With salt
Competitor
1.88
3,967
0.59
Without salt
Agilent
2.08
7,942
0.60
Without salt
Competitor
1.92
4,164
0.57
Eluent
420
COLUMNS FOR BIOMOLECULE SEPARATIONS
Pore Size Choice
The choice of media pore size will influence the resolution in SEC. As the separation is based on differences in molecular size in solution, the sample
must be able to permeate the porous structure of the particles – if the pore size is too small, the samples will be excluded from the pores and elute
in the void volume of the column, and if too large then, all will be able to fully permeate the particles and so there will be very little separation.
Pore size choice: Proteins
Column A:
Bio SEC-3, 100Å
5190-2503
4.6 x 300 mm, 3 µm
Column B:
Bio SEC-3, 150Å
5190-2508
4.6 x 300 mm, 3 µm
Column C:
Bio SEC-3, 300Å
5190-2513
4.6 x 300 mm, 3 µm
Mobile Phase:
50 mM Na2HPO4, 50 mM NaH2PO4 +
0.15 M NaCl, pH 6.8
Flow Rate:
0.35 mL/min
Detector:
UV, 220 nm
Sample:
BioRad Gel Filtration Standards Mix
mAU
2500
5
300Å
Thyroglobulin Aggregates
Thyroglobulin
IgA
g-globulin
Ovalbumin
Myoglobin
Vitamin B12
4
2
2000
6
1.
2.
3.
4.
5.
6.
7.
7
3
1
1500
150Å
1000
500
100Å
0
0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
PoreSz_Proteins
Pore size choice: Mouse IgG
Column A:
Column B:
Column C:
Bio SEC-3, 100Å
5190-2503
4.6 x 300 mm, 3 µm
Bio SEC-3, 150Å
5190-2508
4.6 x 300 mm, 3 µm
Bio SEC-3, 300Å
5190-2513
4.6 x 300 mm, 3 µm
mAU
1.
2.
3.
4.
1400
1200
1000
Dimer
Monomer
Monomer Fragment
Azide
2
4
800
1
Mobile Phase:
50 mM Na2HPO4, 50 mM NaH2PO4 +
0.15 M NaCl, pH 6.8
600
Flow Rate:
0.35 mL/min
400
Detector:
UV, 220 nm
Sample:
Mouse IgG
3
300Å
150Å
200
100Å
0
0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
PoreSz_Mouse
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421
COLUMNS FOR BIOMOLECULE SEPARATIONS
Column Length
Where the separation time is a critical parameter, shorter columns packed with the higher efficiency, 3 µm media are used. With the shorter
columns, higher flow rates are used to reduce the analysis time but without compromising the quality of the data – quantitation of monoclonal
antibody monomer and dimer.
Agilent Bio SEC-3 column length comparison,
150 mm
Agilent Bio SEC-3 column length comparison,
300 mm
Column:
Bio SEC-3, 300Å
5190-2512
7.8 x 150 mm, 3 µm
Column:
Bio SEC-3, 300Å
5190-2511
7.8 x 300 mm, 3 µm
Mobile Phase:
150 mM sodium phosphate
Mobile Phase:
Flow Rate:
1.0 mL/min (56 bar), 1.5 mL/min (75 bar)
150 mM sodium phosphate + 100 mM Na sulfate
(with salt)
150 mM sodium phosphate (without salt)
Detector:
UV, 220 nm
Flow Rate:
1.0 mL/min
Sample:
MAb (2 mg/mL)
Detector:
UV, 220 nm
Sample:
MAb (2 mg/mL)
150 mm
1.5 mL/min
1.0 mL/min
300 mm
Monomer
Monomer
Dimer
With salt
Dimer
Monomer
Dimer
4 min
Without salt
8 min
422
COLUMNS FOR BIOMOLECULE SEPARATIONS
Agilent Bio SEC-3
Particle Size (µm)
Bio SEC-3
100Å
USP L33
7.8 x 300
3
5190-2501
5190-2506
5190-2511
7.8 x 150
3
5190-2502
5190-2507
5190-2512
4.6 x 300
3
5190-2503
5190-2508
5190-2513
4.6 x 150
3
5190-2504
5190-2509
5190-2514
7.8 x 50, Guard
3
5190-2505
5190-2510
5190-2515
Size (mm)
Bio SEC-3
150Å
USP L33
Bio SEC-3
300Å
USP L33
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423
COLUMNS FOR BIOMOLECULE SEPARATIONS
Agilent Bio SEC-5
• Maximum recovery for a broad range of size-based, biomolecule separations
• Outstanding reproducibility and column lifetime
• Excellent stability, even under high-pH, high-salt, and low-salt conditions
• Compatibility with most aqueous buffers
Agilent Bio SEC-5 HPLC columns are packed with 5 µm silica particles coated with a proprietary, neutral,
hydrophilic layer for maximum efficiency and stability. Our specially designed packing also provides high
pore volume, improving both peak capacity and resolution.
Bio SEC-5 columns are available in 5 µm particles with 100Å, 150Å, 300Å, 500Å, 1000Å, and 2000Å
nominal pore sizes.
Column Specifications
Particle Size
MW Range
pH Range
Max Pressure
Flow Rate
100Å
Pore Size
5 µm
100-100,000
2-8.5
240 bar, 3500 psi
0.1-1.25 mL/min (7.8 mm id)
150Å
5 µm
500-150,000
2-8.5
240 bar, 3500 psi
0.1-1.25 mL/min (7.8 mm id)
300Å
5 µm
5,000-1,250,000
2-8.5
240 bar, 3500 psi
0.1-1.25 mL/min (7.8 mm id)
0.1-0.4 mL/min (4.6 mm id)
0.1-0.4 mL/min (4.6 mm id)
0.1-0.4 mL/min (4.6 mm id)
500Å
5 µm
15,000-5,000,000
2-8.5
240 bar, 3500 psi
0.1-1.25 mL/min (7.8 mm id)
0.1-0.4 mL/min (4.6 mm id)
1000Å
5 µm
50,000-7,500,000
2-8.5
240 bar, 3500 psi
0.1-1.25 mL/min (7.8 mm id)
0.1-0.4 mL/min (4.6 mm id)
2000Å
5 µm
>10,000,000
2-8.5
240 bar, 3500 psi
0.1-1.25 mL/min (7.8 mm id)
0.1-0.4 mL/min (4.6 mm id)
424
COLUMNS FOR BIOMOLECULE SEPARATIONS
Calibration curves – Bio SEC-5
Column:
Bio SEC-5
7.8 x 300 mm, 5 µm
Mobile Phase:
150 mM Na phosphate, pH 7.0
Flow Rate:
1.0 mL/min
Detector:
UV, 214 nm
1,000,000,000
Molecular Weight
100,000,000
10,000,000
1,000,000
1000Å
100,000
500Å
10,000
300Å
150Å
1,000
100Å
100
4
5
6
7
8
9
10
Retention Volume
11
12
13
CalCurves_SEC5
Retention Volume (mL)
Proteins
MW
1000Å 500Å 300Å 150Å 100Å
Thyroglobulin
670000
10.07
8.23
7.03
5.82
5.77
Gamma globulin
158000
10.88
9.80
8.57
6.55
5.79
BSA
67000
11.13
10.44
9.44
7.29
6.00
Ovalbumin
45000
11.28
10.83
9.89
7.90
6.40
Myoglobin
17000
11.44
11.28
10.42
8.66
7.05
Ribonuclease A
12700
11.52
11.41
10.58
8.93
7.32
Vitamin B-12
1350
12.00
12.59
11.78
11.49
10.30
Side-by-side comparison
Agilent Bio SEC-5, 150Å
TSK G2000SWxl
Column:
Bio SEC-5
5190-2521
7.8 x 300 mm, 5 µm
Mobile Phase:
150 mM Na phosphate, pH 7.0
Flow Rate:
1.0 mL/min
Detector:
UV, 214 nm
1.
2.
3.
4.
5.
6.
Thyroglobulin, 5.43 min
BSA dimer, 6.19 min
BSA monomer, 6.93 min
Ribonuclease A, 8.74 min
Poly-DL-alanine (1-5 kDa), 9.90 min
Uracil, 12.13 min
1.
2.
3.
4.
5.
6.
Thyroglobulin, 5.64 min
BSA dimer, 6.23 min
BSA monomer, 7.02 min
Ribonuclease A, 9.22 min
Poly-DL-alanine (1-5 kDa), 10.02 min
Uracil, 11.81 min
Separation of a protein mixture on an Agilent Bio SEC-5 HPLC
column and a Tosoh TSK-Gel column. Notice the sharper peaks
and better resolution on the Agilent Bio SEC-5 HPLC column.
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425
COLUMNS FOR BIOMOLECULE SEPARATIONS
Exceptional lifetime,
and lot-to-lot reproducibility
Column:
Mobile Phase:
Lifetime
Reproducibility
Bio SEC-5, 150Å
5190-2521
7.8 x 300 mm, 5 µm
AU
.80
150 mM Phosphate
Buffer, pH 7.0
.60
3. Ribonuclease A
.50
4. Uracil
1
1
1. Thyroglobulin
.70
2. BSA
4
3
2
.40
Lot 1
.30
3
2
Lot 2
.20
4
.10
The four protein mixture shows
excellent retention time
reproducibility over 300 injections
and on three columns from
different manufacturing lots.
Lot 3
.00
0
2
4
6
8
10
12 14 min
0
2
4
6
8
10
12
14 min
3rd run
After 300 injections
Comparison between Agilent Bio SEC-3 and Agilent Bio SEC-5
Analysis of monoclonal antibody
Column:
Column:
Bio SEC-3, 300Å
5190-2511
7.8 x 300 mm, 3 µm
Bio SEC-5, 300Å
5190-2526
7.8 x 300 mm, 5 µm
Mobile Phase:
150 mM Sodium Phosphate, pH 7
Flow Rate:
1 mL/min
Detector:
UV @ 220 nm
Sample:
Humanized monoclonal antibody
1. Dimer
2. Monomer
3. Monomer fragment
mAU
120
3
100
3 µm
80
1
2
60
40
5 µm
20
1
0
3
2
The 3 µm column gives better separation
0
2
4
6
8
10
12
14
16
18
min
Mono_Antibody
426
COLUMNS FOR BIOMOLECULE SEPARATIONS
Agilent Bio SEC-5
Size (mm)
7.8 x 300
Particle Size
(µm)
Bio SEC-5
100Å
USP L33
Bio SEC-5
150Å
USP L33
Bio SEC-5
300Å
USP L33
Bio SEC-5
500Å
USP L33
Bio SEC-5
1000Å
USP L33
Bio SEC-5
2000Å
USP L33
5
5190-2516
5190-2521
5190-2526
5190-2531
5190-2536
5190-2541
7.8 x 150
5
5190-2517
5190-2522
5190-2527
5190-2532
5190-2537
5190-2542
4.6 x 300
5
5190-2518
5190-2523
5190-2528
5190-2533
5190-2538
5190-2543
4.6 x 150
5
5190-2519
5190-2524
5190-2529
5190-2534
5190-2539
5190-2544
7.8 x 50, Guard
5
5190-2520
5190-2525
5190-2530
5190-2535
5190-2540
5190-2545
TIPS & TOOLS
The Agilent rack can be used to optimize your 1290 Infinity LC for ultra-low dispersion, which can enhance
performance of high-efficiency columns. Further information can be found in application note 5990-9502EN
at www.agilent.com/chem/library
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427
COLUMNS FOR BIOMOLECULE SEPARATIONS
ProSEC 300S
• Mechanically robust silica particles that do not bleed during use
• Single column with extended linear resolving range
• Column dimensions for use with multi-detector systems
The Agilent ProSEC 300S column is specifically designed as a single column solution for globular protein
analysis. The pore size selection and optimization provides an extended linear resolving range so that this
single column can be used for analysis across the full range of globular proteins.
The particles are extremely robust and do not fragment during use to leach particulates. This gives
exceptionally stable baselines making this column an ideal choice for use with light scattering detectors.
Two column dimensions, 7.5 mm id and 4.6 mm id, to suit multi-detector size exclusion chromatography
provide an option for the analysis of small masses.
ProSEC 300S Column Specifications
Bonded Phase
Pore
Size
Particle
Size
Protein MW
Range
pH
Range
ProSEC 300S
300Å
5 µm
1,500-800,000
2-7.5
Flow Rate
Max Pressure
<1.5 mL/min
(7.5 mm id)
250 bar, 3700 psi
<0.5 mL/min
(4.6 mm id)
ProSEC 300S
Dimensions
Particle Size (µm)
Part No.
4.6 x 250
5
PL1547-5501
7.5 x 300
5
PL1147-6501
4.6 x 50
5
PL1547-1501
7.5 x 50
5
PL1147-1501
Guard Columns
428
COLUMNS FOR BIOMOLECULE SEPARATIONS
Calibration of the ProSEC 300S column
with globular proteins
Mobile Phase:
50 mM KH2PO4-K2HPO4 (@ pH 6.8)
containing 0.3 M NaCl
Flow Rate:
1.0 mL/min
Detector:
UV, 280 nm
Sample:
Protein samples
6
5.5
Molecular weights of the proteins
Mw/Daltons
Protein
670,000
155,000
66,430
44,287
29,000
16,700
12,384
1,423
Thyroglobulin
g-Globulin
Bovine serum albumin
Ovalbumin
Carbonic anhydrase
Myoglobulin
Cytochrome c
Bacitracin
Log M
5
4.5
4
3.5
3
5
7
9
Retention time (min)
10
13
PSEC300Sglob
Analysis of Bovine Serum Albumin by light
scattering using ProSEC 300S columns
ProSEC 300S
PL1147-6501
7.5 x 300 mm, 5 µm
Mobile Phase:
Water + 120 mM NaCl, 2.7 mM KCI, 10 mM
NaH2PO4
Flow Rate:
1.0 mL/min
Detector:
Differential refractive index + PL-GPC 50 Dual Angle
Light Scattering Detector
Sample:
Bovine serum albumin
Monomer
Response
Column:
Trimer
Dimer
Tetramer
RI
Molecular Weights
Monomer
66,900 Daltons, 88.5%
Dimer
34,900 Daltons (2.02 x monomer
molecular weight), 9.8%
197,000 Daltons (2.94 x monomer
molecular weight), 1.2%
279,300 Daltons (5.17 x monomer
molecular weight), 0.5%
Trimer
Tetramer
Aggregation
15 Deg
90 Deg
0
Retention time (min)
14
BovineSerAlb
Overlay of differential refractive index and dual angle light scattering sample.
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429
COLUMNS FOR BIOMOLECULE SEPARATIONS
Column:
ProSEC 300S
PL1147-6501
7.5 x 300 mm, 5 µm
Mobile Phase:
0.1 M KH2PO4 containing 0.3 M NaCl, pH 8.0
Flow Rate:
1.0 mL/min
Temperature:
5 °C
Detector:
UV at 310 nm + PL-GPC 50 Dual Angle
Light Scattering Detector
Sample:
Proteins
Response (mV)
Overlay of UV and light scattering 90°
for a sample of g-globulins, illustrating monomer,
dimer, and trimer peaks
UV
LS 90°
Retention time (min)
UV_Overlay
Overlay of UV and light scattering 90° for a
sample of BSA, illustrating monomer, dimer,
trimer and aggregate peaks
430
ProSEC 300S
PL1147-6501
7.5 x 300 mm, 5 µm
Mobile Phase:
0.1 M KH2PO4 containing 0.3 M NaCl, pH 8.0
Flow Rate:
1.0 mL/min
Temperature:
5 °C
Detector:
UV at 310 nm + PL-GPC 50 Dual Angle
Light Scattering Detector
Sample:
Proteins
UV
Response (mV)
Column:
LS 90°
Retention time (min)
UV_Overlay_BSA
COLUMNS FOR BIOMOLECULE SEPARATIONS
ZORBAX GF-250 and GF-450
Gel Filtration Columns
• High efficiency and reproducibility with short analysis time
• Semi-prep and prep column dimensions
• Compatible with organic modifiers and denaturants
• Wide usable pH range (3-8)
GF-250 Gel Filtration Columns
Agilent ZORBAX GF-250 and GF-450 size exclusion (gel filtration) columns are ideal for size separations of
proteins and other biomolecules. The separation range is 4,000-900,000 for globular proteins when using
GF-250 and GF-450 columns in series. The GF-250/GF-450 size exclusion columns have a hydrophilic diol
bonded phase for high recovery of proteins (typically >90%) and a unique zirconia modification of the silica
for a pH operating range from 3-8. The GF-250 and GF-450 columns are packed with precisely sized porous
silica microspheres with narrow pore size and particle size distributions. The result is a highly efficient,
rugged and reproducible size exclusion column that can be used for both analytical and preparative
separations of proteins with flow rates of up to 3 mL/min. These columns are compatible with organic
modifiers (<25%) and denaturants in the mobile phase to reduce protein aggregation. Some common
applications include separations of protein monomers, dimers and aggregates, desalting, protein molecular
weight estimation and separations of modified proteins.
Column Specifications
Bonded Phase
Pore Size Particle Size
MW Range
Surface Area pH Range
2
Flow Rate
Max Pressure
ZORBAX GF-250
150Å
4 µm
4,000-400,000
140 m /g
3.0-8.0
<3.0 mL/min
350 bar
ZORBAX GF-450
300Å
6 µm
10,000-900,000
50 m2/g
3.0-8.0
<3.0 mL/min
350 bar
Specifications represent typical values only
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431
COLUMNS FOR BIOMOLECULE SEPARATIONS
Retention volume versus log (MW)
for the Bio-Rad standards separated
on an Agilent ZORBAX GF-250 column
Thyroglobulin (670 kDa)
ZORBAX GF-250
884973-901
9.4 x 250 mm, 4 µm
Mobile Phase:
200 mM Sodium phosphate, pH 7.0
Temperature:
Ambient
Detector:
UV, 254 nm
Gamma globulin (158 kDa)
Ovalbumin (44 kDa)
Log (MW)
Column:
Myoglobulin (17 kDa)
Vitamin B12 (1.35 kDa)
Retention volume (mL)
Separations of proteins on preparative columns
Column:
Column:
ZORBAX GF-250
884973-901
9.4 x 250 mm, 4 µm
5
ZORBAX GF-250
ZORBAX GF-450
884973-902
9.4 x 250 mm, 6 µm
Mobile Phase:
0.2 M Na2HPO4, pH 7.0
Flow Rate:
5.0 mL/min
Detector:
UV, 280 nm
Sample:
200 µL
1.
2.
3.
4.
5.
6.
6
3
4
2
1
0
5
10
15
5
ZORBAX GF-450
3 4
6
2
1
0
5
10
Retention time (min)
432
15
Thyroglobulin
Catalase
BSA
ß-Lactoglobulin B
Myoglobin
Tyr-Gly-Gly
COLUMNS FOR BIOMOLECULE SEPARATIONS
ZORBAX GF-250 (USP L33) and GF-450 (USP L35) Gel Filtration Columns
Hardware Description
GF-250, 150Å
Size (mm)
9.4 x 250
Particle Size
(µm)
Part No.
4
884973-901
GF-250, 150Å
4.6 x 250
4
884973-701
GF-450, 300Å
9.4 x 250
6
884973-902
GF-250 Diol, Guard Cartridge, 2/pk
9.4 x 15
6
820675-111
GF-250 Diol, Guard Cartridge, 4/pk
4.6 x 12.5
6
820950-911
GF-450 Diol, Guard Cartridge, 2/pk
9.4 x 15
6
820675-111
GF-250 Diol, Guard Cartridge, 4/pk
4.6 x 12.5
6
820950-911
Guard Columns (hardware required)
Prep Guard Hardware Kit
840140-901
Guard Hardware Kit
820999-901
PrepHT Columns
PrepHT GF-250, 150Å
21.2 x 250
6
877974-901
PrepHT GF-450, 300Å
21.2 x 250
6
877974-910
PrepHT GF-250, Guard Cartridge, 2/pk
17.0 x 7.5
5
820212-911
PrepHT GF-450, Guard Cartridge, 2/pk
17.0 x 7.5
5
820212-911
PrepHT Endfittings, 2/pk
Guard Cartridge Hardware
820400-901
820444-901
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433
COLUMNS FOR BIOMOLECULE SEPARATIONS
Affinity Chromatography
Affinity chromatography is a powerful technique which takes advantage of highly specific molecular
interactions, frequently between specific proteins (e.g. antigen/antibody). Agilent offers several specialty
affinity products, a monolithic Protein A column for the isolation and quantitation of IgG and a series of
Multiple Affinity Removal Systems for the elimination of high abundance proteins in biological samples.
Agilent Bio-Monolith Protein A HPLC Columns
• Designed for the analytical separation of all IgG (human and mouse), except for IgG class3
• Flow-rate independent separations; no diffusion, no pores and no void volume make transport
between mobile and stationary phase very rapid
Bio-Monolith Protein A Column, 5069-3639
• Extremely fast separations speed up method development time and decrease costs
• Locking in method parameters takes significantly less time and buffer
Agilent Bio-Monolith Protein A HPLC columns are part of the Agilent Bio-Monolith column family.
Protein A Bio-Monolith columns are compatible with HPLC and preparative LC systems, including
Agilent 1100 and 1200 HPLC systems.
TIPS & TOOLS
For information on Ion-Exchange
Bio-Monolith columns,
turn to pages 412-415.
434
COLUMNS FOR BIOMOLECULE SEPARATIONS
Column Specifications
Dimensions
5.2 mm x 4.95 mm
Column volume
100 µL
Maximum pressure
150 bar (15 MPa, 2200 psi)
Temperature min/max
Working: 4-40 °C
Recommended pH
Working range: 2-13
Materials of construction
Hardware: Stainless steel
Storage: 4-30 °C
Cleaning-in-place: 1-14
Packing: poly(glycidyl methacrylate-co-ethylene dimethacrylate) highly
porous monolith
Color ring identifier
Bio-Monolith Protein A: White
Shelf life/expiration date
Protein A: 12 months
Bio-Monolith Protein A
Column
Description
Key Applications
Part No.
Bio-Monolith Protein A
The Protein A affinity column is designed for the
analytical separation of all IgG (human and mouse),
except for IgG class3.
ü Quantitative determination of IgG (fermentation titer
calculation)
5069-3639
TIPS & TOOLS
Further information can be found in
the following application note:
Rapid Human Polyclonal IgG
Quantification Using the Agilent
Bio-Monolith Protein A HPLC
Column (publication # 5989-9733EN)
www.agilent.com/chem/library
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435
COLUMNS FOR BIOMOLECULE SEPARATIONS
Protein A
5069-3639
5.2 x 4.95 mm
Mobile Phase:
PBS buffer, pH 7.4
0.5 M acetic acid, pH 2.6
Gradient:
Stepwise gradient: 100% buffer
A-100% buffer B-100% buffer A (0.5 min each step)
Flow Rate:
1 mL/min
Detector:
A high pressure gradient HPLC system,
Agilent 1200 Infinity LC - UV at 280 nm
Sample:
Human Plasma diluted with binding buffer (PBS
buffer, pH 7.4)
Relative absorbance [mAU]
Column:
1200
120
1000
100
800
80
600
60
400
40
200
20
0
% Gradient
Rapid human polyclonal IgG quantification using
the Agilent Bio-Monolith Protein A HPLC column
0
0
0.5
1
1.5
2
2.5
3
Time [min]
The selectivity of the Bio-Monolith Protein A column for the IgG from human plasma. IgG binds to protein A, a 100% buffer B step gradient is applied, and IgG elutes at 1.5 min.
Key:
Lane 1: Whole serum prior to separation
Lane 2: IgG standard
Lane 3: Peak 1 (flow-through fraction)
Lane 4: Peak 2 (Protein A-bound fraction; i.e. IgG1 and IgG2)
SDS PAGE analysis of fractions from the separation.
436
COLUMNS FOR BIOMOLECULE SEPARATIONS
Agilent Protein Fractionation System
and Proteomics Reagents
• LC/MS analysis of biological samples
• Preparation for electrophoretic analysis
• Sample preparation for biomarker discovery
• Instrument and workflow validation
• Cost-effective immunodepletion
• Sample desalting, concentration, and fractionation
In order to more easily isolate and identify proteins in biological samples, such as serum, plasma, and
cerebro-spinal fluid (CSF), the Agilent Multiple Affinity Removal System is designed to chromatographically
eliminate interfering high-abundance proteins from biological samples. Removal of these abundant proteins
improves the subsequent LC/MS and electrophoretic analysis of the sample by effectively expanding the
dynamic range.
For sample fractionation and desalting, the Agilent mRP-C18 High-Recovery Protein column is designed to
simultaneously desalt, concentrate, and fractionate in one easy step with extremely high recovery of
samples as compared to conventional RP HPLC columns that are fully compatible with LC/MS analysis.
In addition, validated reagents for sample preparation in biomarker discovery and other proteomics
applications are also available, including a complex standard, and proteomics grade trypsin. For your
convenience, these reagents are fully compatible with Agilent LC/MS methods and require no additional
sample pretreatments.
Large volume requirements and custom column dimensions can also be addressed with our
custom configurations.
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437
COLUMNS FOR BIOMOLECULE SEPARATIONS
Multiple Affinity Removal System
The Multiple Affinity Removal System from Agilent enables the identification and characterization of
high-value, low abundant proteins and biomarkers found in serum, plasma, and other biological fluids.
The Multiple Affinity Removal System reproducibly and specifically removes up to 14 high abundant
proteins found in human biological fluids and 3 high abundant proteins found in mouse biological fluids.
The Multiple Affinity Removal System is available in a variety of LC column dimensions and in spin cartridge
format. When combined with Agilent's optimized buffers, convenient spin filters and concentrators, the
Agilent Multiple Affinity Removal System creates an automated, integrated depletion solution compatible
with most LC instruments (columns), and bench top centrifuges (spin cartridges).
Multiple Affinity Removal System
438
Samples depleted using the Multiple Affinity Removal System are ready for downstream analyses such as
2-D gel electrophoresis, LC/MS, and other analytical techniques.
COLUMNS FOR BIOMOLECULE SEPARATIONS
Multiple Affinity Removal System Selection Guide
Total Protein
Removed
Dimension
Load Capacity
Albumin, IgG, antitrypsin, IgA, transferrin,
haptoglobin, fibrinogen, alpha2macroglobulin, alpha1-acid glycoprotein,
IgM, apolipoprotein Al, apolipoprotein All,
complement C3, transthyretin
94%
Spin Cartridge
8 - 10 µL
Albumin, IgG, IgA, transferrin, haptoglobin,
antitrypsin, fibrinogen
88-92%
Product
Proteins Removed
MARS Human-14
MARS Human-7
MARS Human-6
MARS Human-6
High Capacity
MARS Human-2
MARS Human-1
MARS Mouse-3
Albumin, IgG, IgA, transferrin, haptoglobin,
antitrypsin
Albumin, IgG, IgA, transferrin, haptoglobin,
antitrypsin
Albumin, IgG
Albumin
Albumin, IgG, transferrin
85-90%
85-90%
69%
50-55%
80%
Part No.
5188-6560
4.6 x 50 mm
20 µL
5188-6557
4.6 x 100 mm
40 µL
5188-6558
10.0 x 100 mm
250 µL
5188-6559
Spin Cartridge
12 - 14 µL
5188-6408
4.6 x 50 mm
30 - 35 µL
5188-6409
4.6 x 100 mm
60 - 70 µL
5188-6410
10.0 x 100 mm
250 - 300 µL
5188-6411
Spin Cartridge
7 - 10 µL
5188-5230
4.6 x 50 mm
15 - 20 µL
5185-5984
4.6 x 100 mm
30 - 40 µL
5185-5985
Spin Cartridge
14 - 16 µL
5188-5341
4.6 x 50 mm
30 - 40 µL
5188-5332
4.6 x 100 mm
60 - 80 µL
5188-5333
10.0 x 100 mm
up to 340 µL
5188-5336
Spin Cartridge
50 µL
5188-8825
4.6 x 50 mm
100 µL
5188-8826
Spin Cartridge
65 µL
5188-5334
4.6 x 50 mm
130 µL
5188-6562
Spin Cartridge
25 - 30 µL
5188-5289
4.6 x 50 mm
37 - 50 µL
5188-5217
4.6 x 100 mm
75 - 100 µL
5188-5218
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439
COLUMNS FOR BIOMOLECULE SEPARATIONS
Illustration of high abundance proteins removed by Agilent Multiple Affinity Removal
Columns and Spin Cartridges
Transferrin
Fibrinogen
IgA
Haptoglobin
a1-Antitrypsin
Complement C3
a1-Acid Glycoprotein
a2-Macroglobulin
IgG
15%
6% Remaining for Analysis
Albumin
Apolipoprotein AI
Apolipoprotein AII
IgM
Transthyretin
TIPS & TOOLS
Learn more about Agilent’s
complete services portfolio at
www.agilent.com/chem/services
440
COLUMNS FOR BIOMOLECULE SEPARATIONS
Multiple Affinity Removal System Starter Kits
The LC Column and Spin Cartridge Reagent Starter Kits include all the required supplies to use with
Multiple Affinity Removal System. These buffers provide optimal conditions for column longevity and
sample reproducibility.
• The kits provide enough Buffer A and Buffer B for approximately 200 sample depletions using the
4.6 x 50 mm LC columns, approximately 100 sample depletions using the 4.6 x 100 mm LC columns
and 200 spin cartridge uses.
• Buffer A, the loading buffer, minimizes protein-protein interactions, allowing low abundant proteins
LC Column Reagent Starter Kit, 5185-5986
often bound to high abundant proteins to pass through the column, while the targeted high abundant
proteins bind to their associated antibodies.
• Buffer B, the elution buffer, then disrupts the antibody-protein interaction eluting the high abundant
proteins off the column.
Multiple Affinity Removal System Starter Kits
Description
Part No.
LC Column Reagent Starter Kit
Includes:
5185-5986
Buffer A, 1 L, for loading, washing, and equilibrating, qty 2
5185-5987
Buffer B, 1 L, for eluting
5185-5988
0.22 µm cellulose acetate, 25/pk, qty 2
5185-5990
Spin concentrators, 5K MWCO, 4 mL, 25/pk
5185-5991
Multiple Affinity Removal Spin Cartridge Reagent Kit
Includes:
5188-5254
Buffer A, 1 L, for loading, washing, and equilibrating
5185-5987
Buffer B, 1 L, for eluting
5185-5988
Spin filters, 0.22 µm cellulose acetate, 25/pk, qty 2
5185-5990
Spin concentrators, 5K MWCO, 4 mL, 25/pk
5185-5991
Luer-Lok adapters, 2/pk
5188-5249
Plastic syringe, 5 mL, Luer-Lok, 2/pk
5188-5250
Microtube, 1.5 mL, screw top, 100/pk, qty 6
5188-5251
Caps and plugs, 6/pk
5188-5252
PTFE needles, Luer-Lok, 10/pk
5188-5253
High concentration sample dilution buffer, 50 mL
Luer-Lok adapters, 5188-5249
Luer-Lok syringe, 5188-5250
5188-8283
Luer-Lok needles, 5188-5253
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441
COLUMNS FOR BIOMOLECULE SEPARATIONS
mRP-C18 High-Recovery Protein Columns
The mRP (macroporous reversed-phase) C18 High-Recovery Protein column is designed for high recovery,
high resolution separation, fractionation, and simultaneous desalting of complex protein samples (like
immunodepleted serum or plasma proteins).
• Greater than 95-99% protein sample recovery has been observed with immunodepleted serum
using the Agilent Multiple Affinity Removal System – LC column
• Can load up to 380 µg of total protein mass without reducing chromatographic resolution
of the proteins
• Column packed with macroporous C18-bonded ultrapure 5 µm particle silica designed to
reduce or eliminate strong adsorption of proteins
mRP-C18 High-Recovery Protein Column,
4.6 x 50 mm, 5188-5231
• Maximum operating pressure of 250 bar (4000 psi)
• Compatible with water and all common organic solvents
mRP-C18 High-Recovery Protein Columns
Description
Protein Load Capacity
Part No.
mRP-C18, 0.5 x 100 mm
10 ng - 5 µg
5188-6510
mRP-C18, 2.1 x 75 mm
8 - 85 µg
5188-6511
mRP-C18, 4.6 x 50 mm
40 - 380 µg
5188-5231
Proteomics Reagents for LC/MS Analysis
The Agilent Complex Proteomics Standard is a soluble Pfu protein extract containing over 1,500 proteins.
Together with our TPCK-treated proteomics grade trypsin this is an ideal combination for workflow validation
in LC/MS biomarker discovery and other proteomic studies.
Proteomics Reagents for LC/MS Analysis
Description
442
Part No.
Complex Proteomics Standard
400510
Proteomics Grade Trypsin
204310
COLUMNS FOR BIOMOLECULE SEPARATIONS
Protein Fractionation of Complex Samples on the mRP Column
mRP-C18, 4.6 x 50 mm
mAU
7
6
5
4
3
2
1
0
10
20
30
40
50
Time (min)
60
70
mAU
8
7
6
5
4
3
2
1
0
0
80
HeLa Membrane Prep
mAU
25
20
15
10
5
0
0
10
20
30
Time (min)
40
50
20
30
Time (min)
40
50
60
HeLa Cell Lysate (352 µg)
Highest Recovery
mAU
25
20
15
10
5
0
10
0
"Top-6" Depleted Human Serum
Lipids
10
20
30
40
Time (min)
50
60
MRPC18
Human Brain Membrane Lipid Raft Prep (500 µg)
mRP-C18 High-Recovery Protein Column, 0.5 x 100 mm, 5188-6510
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443
COLUMNS FOR BIOMOLECULE SEPARATIONS
Method Development
ZORBAX Column Methods
This ZORBAX Column Selection Strategy for Proteins and Peptides provides some critical details on method development for proteins or polypeptides.
Choose the Initial Column and Conditions for Peptides, Polypeptides, and Proteins
Peptides, Polypeptides, Proteins
MW >50 kDa
Peptides, Polypeptides, Proteins
MW <50 kDa
Initial Bonded Phase
Poroshell 300SB-C18
StableBond 300SB-C8
300SB columns are wide-pore columns with unbeatable lifetime in
TFA-containing mobile phases. This makes them an ideal first choice
for separations of peptides and proteins.
Poroshell 300SB columns use an innovative particle technology to
deliver rapid protein separations. Short anaysis times with efficient
peaks are easily obtained with Poroshell columns.
• C8 is an excellent starting bonded phase because of its moderate
• C18 is a good starting bonded phase choice with Poroshell for
hydrophobicity
• C18 and C8 are generally selected for peptides and protein
most peptides, polypeptides and proteins because the retention
is maximized
• C8 is generally selected for moderate size proteins but can be
digests but can also be used for proteins
• C3, C4 and CN are generally selected for larger, hydrophobic
polypeptides and proteins but can also be used for peptides
• PLRP-S when chemical and thermal stability are needed
used with polypeptides or very large proteins
• C3 is generally selected for antibodies or large proteins but can
be used for peptides and polypeptides
Initial Separation Conditions
444
Column:
StableBond 300SB-C8
4.6 x 150 mm, 3.5 or 5 µm
883995-906
863973-906
Mobile Phase:
A: 95% H2O: 5% ACN with 0.1% TFA
B: 5% H2O: 95% ACN with 0.085% TFA
Gradient:
0-60% B in 60 min
Temperature:
35-40 °C
Flow Rate:
1 mL/min
Column:
Poroshell 300SB-C18
2.1 x 75 mm, 5 µm
660750-902
Mobile Phase:
A: 95% H2O: 5% ACN with 0.1% TFA
B: 5% H2O: 95% ACN with 0.085% TFA
Gradient:
0-60% B in 60 min
Temperature:
35-40 °C
Flow Rate:
2 mL/min
COLUMNS FOR BIOMOLECULE SEPARATIONS
Start at Low pH with Simple Aqueous/Organic Gradient
Typically, a water/acetonitrile with 0.1% TFA gradient is used to elute all components of interest. A typical high resolution gradient on a 300Å pore
size column requires 30-50 min. A Poroshell column requires a shorter analysis time and a higher flow rate and still provides exceptional resolution.
To improve resolution, increase the gradient time, decrease column length, or increase flow rate.
Optimize Sample Solubility
For best peak shape and recovery at any pH, it is important to completely solubilize a sample. Highly acidic or neutral solvents can be used with
ZORBAX 300StableBond and Poroshell 300SB, while neutral solvents and dilute bases can be used with ZORBAX 300Extend-C18.
Solvent Choices to Solubilize Proteins and Peptides
Water/phosphate Buffer
Weakest
Dilute acid (TFA, Acetic Acid or HCI)
Neutral pH, 6-8 M guanidine-HCI or isthiocyanate
5% HOAc/6 M urea
Dilute acid + aqueous/organic solvents (ACE, MeOH, THF)
Dilute base (ammonium hydroxide)
DMSO or 0.1%-1% in DMSO
Strongest
Formamide
Increase the Temperature
Separations of proteins and peptides are influenced by temperature and higher column temperature can dramatically improve both resolution and
recovery of proteins and hydrophobic and aggregating peptides.
Poroshell 300SB - up to 80 °C
StableBond 300SB - up to 80 °C
Optimize Mobile Phase pH
Try Mid and High pH if Low pH does not work
If an optimized, low pH method does not provide an ideal separation, then mid or high pH mobile phase can be used. At high pH, selectivity is often
very different because acidic amino acids become negatively charged and some basic amino acids may lose their charge. ZORBAX 300Extend-C18
is an excellent choice for mid to high pH separation.
Column:
Mobile Phase:
ZORBAX 300Extend-C18
4.6 x 150 mm, 5 µm
773995-902
Gradient:
5-60% B in 30 min
Temperature:
25-30 °C (<60 °C)
A: 20 mM NH4OH in H2O
B: 20 mM NH4OH in 80% ACN
Flow Rate:
1 mL/min
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COLUMNS FOR BIOMOLECULE SEPARATIONS
Starting Column Choices for Analytical Separations of Peptides, Polypeptides, and Proteins
Low pH
Mid and High pH
MW <50 kDa
MW >50 kDa
MW <25 kDa
ZORBAX 300SB-C8
4.6 x 150 mm, 3.5 µm
Poroshell 300SB-C18
2.1 x 75 mm, 5 µm
ZORBAX 300Extend-C18
2.1 x 150 mm, 3.5 µm
863973-906
660750-902
763750-902
Reversed-Phase LC/MS Methods
LC/MS of proteins and peptides is used to provide information for protein characterization, to accurately identify post-translational modifications of
proteins, and to determine the molecular weight of synthetic and natural peptides. LC/MS is also used to provide protein identification in 2-D separations
for proteomics applications. Therefore, LC/MS of proteins and peptides is a critical separation area, which requires some special column and mobile phase
recommendations. In general, smaller column sizes are used for LC/MS and TFA is generally not used in mobile phase because of reduced sensitivity in the
MS with this mobile phase additive.
Initial Column Choices for LC/MS Separations of Proteins and Polypeptides
Analytical LC/MS applications – 2.1 mm id columns provide good sensitivity when sample size is not limited.
With Poroshell columns, smaller 1 mm column ids are used.
Low pH
Mid and High pH
MW <50 kDa
MW <1000 kDa
MW <25 kDa
ZORBAX 300SB-C8
2.1 x 150 mm, 3.5 µm
Poroshell 300SB-C18
1.0 x 75 mm, 5 µm
ZORBAX 300Extend-C18
2.1 x 150 mm, 3.5 µm
863750-906
661750-902
763750-902
High Sensitivity/Proteomics Applications
Capillary columns are used for high sensitivity protein and peptide applications. The 0.5 mm id columns are used for protein and protein
digest separations while the 0.3 mm id columns are most often used for protein digests. These can be analyzed at high pH with an ammonium
hydroxide mobile phase. Nano columns (0.1 and 0.075 mm id) are often used in 2-D LC/MS systems for proteomics and the initial choice is
C18 bonded phase.
446
COLUMNS FOR BIOMOLECULE SEPARATIONS
Bio Ion-Exchange Column Methods
Bio MAb Monoclonal Antibodies
Cation-Exchange (SCX, WCX)
Monoclonal Antibodies, Proteins,
Peptides, and Glycoproteins
Anion-Exchange (SAX, WAX)
Oligonucleotides, Proteins
Recommended Initial Conditions
Mobile Phase:
A: 20 mM sodium
phosphate, pH 5.5
B: Buffer A plus
400 mM NaCl
Gradient:
10-35% B in 50 min
Sample Size:
2 mg/mL; 5 µL injection
Temperature:
Ambient
Detection:
UV 220 nm
A: 20 mM sodium
phosphate, pH 5.0
for WCX or pH 6.0
for SCX
B: Buffer A plus
500 mM NaCl
Mobile Phase:
Gradient:
1-100% B in 30 min
for 50 mm columns;
60 min for 250 mm
columns
Gradient:
1-100% B in 30 min
for 50 mm columns
and 60 min for
250 mm columns
Sample Size:
2 mg/mL; 5 µL injection
Sample Size:
2 mg/mL; 5 µL injection
Temperature:
Ambient
Temperature:
Ambient
Detection:
UV 220/280 nm
Detection:
UV 220/280 nm
Mobile Phase:
A: 10 mM Tris buffer,
pH 8 for WAX or
pH 9.0 for SAX
B: Buffer A plus
400 mM NaCl
Select Flow Rate Based on Column Diameter and Particle Size
2.1 mm id Columns
Particle Size, µm
4.6 mm id Columns
Flow Rate, mL/min
Particle Size, µm
Flow Rate, mL/min
5
0.1-0.5
1.7
0.1-0.3
1.7
0.1-0.8
3
0.1-0.5
5
0.1-0.8
10
0.1-1.0
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COLUMNS FOR BIOMOLECULE SEPARATIONS
Optimize Conditions
Some separations may require a specific buffer, ionic strength, pH, and/or temperature
Ionic Strength:
Certain ionic strength is required to sustain the function of columns. Usually, a minimal concentration of 10-20 mM salt is required. However,
greater than 20 mM strength may prevent the adsorption of biomolecules onto the column. Commonly used salts are sodium and potassium
chloride and acetate. For elution, a typical salt concentration is 400-500 mM.
Note: Never use water alone for washing columns as it causes a significant increase in backpressure.
Selection of Buffers and pH:
Buffers play a key role in the optimization of separations. Phosphate buffers are typically
used for antibodies and many biomolecules. The following are also recommended: MES,
Tris, and ACES buffers. Use buffers of pH 5.0-6.5. pH can be adjusted usually by +/- 0.2
units. For some specific proteins, buffers with higher pH (>pH 6.5) may be needed.
Phosphoric acid, acetic acid, HCl and NaOH can be used to adjust pH.
Selection of Buffers and pH:
For anion-exchange, acetate and phosphate
buffers of pH 8.0-9.0 are recommended. pH can
be adjusted usually by +/- 0.2 units. For some
specific proteins, buffers with higher or lower
pH may be needed. Phosphoric acid, acetic
acid, HCl and NaOH can be used to adjust pH.
pH gradients can also be used for elution.
pH gradients can also be used for elution.
Additives
Additives
Organic Solvents:
Organic Solvents:
Acetonitrile, ethanol, methanol, and other
similar solvents can be used up to 50%.
Acetonitrile, ethanol, methanol, and other
similar solvents can be used up to 50%.
Detergents:
Detergents:
Non-ionic, anionic, and zwitterionic
detergents can be used. Cationic detergents
are not recommended.
Non-ionic, cationic, and zwitterionic
detergents can be used. Anionic detergents
are not recommended.
Temperature:
Agilent Bio MAb and IEX columns are stable up to 80 °C. However, many proteins and biomolecules are heat labile. Be sure to establish the
temperature stability of your sample before routinely using high temperature for separation.
448
COLUMNS FOR BIOMOLECULE SEPARATIONS
SEC Column Methods
Choose Initial Columns and Conditions for Size-Based Separation of Biomolecules,
Aggregation Analysis – Peptides, Polypeptides, and Proteins
Peptides, Polypeptides, Proteins
MW >0.1-1,250 kDa
Peptides, Polypeptides, Proteins
MW >0.1-10,000 kDa
Select Column Based on Molecular Weight Range and Pore Size
Agilent Bio SEC-3 (3 µm)
Agilent Bio SEC-5 (5 µm)
Pore Size
MW range, kDa
Pore Size
MW range, kDa
100Å
0.1-100
100Å
0.1-100
150Å
0.5-150
150Å
0.5-150
300Å
5-1,250
300Å
5-1,250
500Å
15-5,000
1000Å
50-7,500
2000Å
>10,000
Recommended Initial Separation Conditions
Column:
Agilent Bio SEC (3 µm and 5 µm)
Temperature:
Recommended: 10-30 °C, Maximum: 80 °C
Mobile Phase:
150 mM phosphate buffer, pH 7.0*
Flow Rate:
Gradient:
Isocratic in 30-60 min range
0.1-0.4 mL/min for 4.6 mm id columns
0.1-1.25 mL/min for 7.8 mm id columns
Sample Size:
≤ 5% of total column volume
*Other aqueous buffers with high and low salt can be used
For additional information, see application note: Defining the Optimum Parameters for Efficient Size Separations of Proteins
(publication # 5990-8895EN)
www.agilent.com/chem/library
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449
COLUMNS FOR BIOMOLECULE SEPARATIONS
After the initial chromatogram, additional changes may be needed to improve the separation, maintain protein solubility, or to decrease sample
interaction with the chromatographic media. The ionic strength of the mobile phase can be adjusted up or down in strength to attain an optimized
separation. pH can also be adjusted usually + 0.2 units. If further optimization is necessary, the upward or downward range should be expanded.
A change of temperature or addition of an organic solvent can also be used.
For protocols requiring additional salt, these buffers are typical:
100-150 mM sodium chloride in 50 mM sodium phosphate, pH 7.0
100-150 mM sodium sulfate in 50 mM sodium phosphate, pH 7.0
50-100 mM urea in 50 mM sodium phosphate, pH 7.0
Other similar salts (e.g. KCl) and guanidine hydrochloride can also be used
pH Range:
2.0-8.5
Potential organic solvent additions include:
5-10% ethanol (or other similar solvents) in 50 mM sodium phosphate, pH 7.0
5% DMSO in 50 mM sodium phosphate, pH 7.0
Temperature:
Typically, SEC separations are run at 20-30 °C. Separation of proteins and peptides may require higher temperature to improve both resolution
and recovery of proteins and hydrophobic peptides.
Maximum temperature of Bio SEC columns is 80 °C
450
COLUMNS FOR BIOMOLECULE SEPARATIONS
High Sensitivity Capillary Column Methods
Mobile Phase Considerations
Low pH
Mid and High pH
TFA is generally not used for LC/MS separations of proteins and
peptides. The first step is normally to replace TFA with 0.1 to 1%
formic acid. Acetic acid, up to 1% can also be used as an alternative
mobile phase modifier. At low pH, the best separation may still be
obtained with TFA in the mobile phase. In some cases, the TFA can be
displaced post-column with an alternative acid, such as propionic acid.
LC/MS can also be done at high pH with 10-20 mM NH4OH as a
mobile phase additive.
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451
COLUMNS FOR BIOMOLECULE SEPARATIONS
Capillary and Nano Columns
Nano Columns
• Highest sensitivity for your smallest sample sizes
• Compatible with all LC/MS interfaces
• Internal diameters of 0.5, 0.3, 0.1, and 0.075 mm
• Packings/phases for both small and large molecules (80Å and 300Å pore sizes, respectively)
• Ideal for 1-D and 2-D (proteomics) applications
Agilent ZORBAX Capillary (0.5 and 0.3 mm id) and Nano (0.1 and 0.075 mm id) columns are now available
in a wide variety of phases, pore sizes, and dimensions. These columns are ideal for very sample-limited
applications because they provide enhanced sensitivity by reducing on-column sample dilution. This high
sensitivity can be provided with exceptional reproducibility using Agilent columns and low dispersion
HPLC instruments. The fastest growing application for capillary and nano columns is 2-D LC/MS for
complex proteomics samples. Agilent provides all the columns needed for the 2-D separation – the SCX
columns for the first dimension, the reversed-phase trapping column, and the reversed-phase column for
the second dimension.
TIPS & TOOLS
Agilent offers a variety of e-Seminars and
on-site training to help you learn how to
be a more effective chromatographer.
For more information, visit
www.agilent.com/chem/education
452
COLUMNS FOR BIOMOLECULE SEPARATIONS
ZORBAX Nano columns for high
sensitivity protein digest analysis
by LC/MS
Column:
ZORBAX 300SB-C18
5065-9911
0.075 x 150 mm, 3.5 µm
Mobile Phase:
A: Water + 0.1% Formic acid,
B: ACN + 0.1% Formic acid
Flow Rate:
600 nL/min
Gradient:
2% B to 52% B in 25 min
Detector:
Positive Ion Nano Electrospray MS
Sample:
100 fm (1 µL) Digest of 8 Proteins
Intens.
1.5
1.0
0.5
2.5
5.0
7.5
10.0
12.5
15.0
Time (min)
17.5
20.0
22.5
LCSB008
A ZORBAX Nano HPLC column, 0.075 mm id, is used for high sensitivity LC/MS analysis of a protein digest sample.
High sensitivity with capillary columns
Column:
Column:
Column:
Column:
Sample:
ZORBAX SB-C18
5064-8255
0.3 x 150 mm, 5 µm
ZORBAX SB-C18
5064-8256
0.5 x 150 mm, 5 µm
ZORBAX SB-C18
863600-902
1.0 x 150 mm, 3.5 µm
ZORBAX SB-C18
883975-902
4.6 x 150 mm, 5 µm
mAU
0.3 mm
1400
1200
1000
800
600
0.5 mm
400
300
1.0 mm
200
4.6 mm
0
200 ng Biphenyl
0
2.5
5.0
7.5
10.0
Time (min)
12.5
15.0
17.5
LCCN002
Sample-limited applications require capillary column dimensions to minimize on-column sample dilution and to enhance sensitivity. The 0.3 mm capillary in this example provides
100 times more sensitivity than the standard 4.6 mm column. Agilent Nanobore (0.1 mm to 0.075 mm id) columns can provide up to 2,000 times more sensitivity for your most
limited sample applications.
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453
COLUMNS FOR BIOMOLECULE SEPARATIONS
Human serum: Low abundance protein isolation
and identification from 1-D gel band by LC/MS
Column:
Mobile Phase:
A: Water + 0.1% Formic acid
B: Acetonitrile + 0.1% Formic acid
Flow Rate:
6 µL/min
Gradient:
0 min 3% B
5 min 3% B (loading)
50 min 45% B
52 min 80% B
57 min 80% B
60 min 3% B
Sample:
Proteins Identified
ZORBAX 300SB-C18
Trap: 0.3 x 5 mm, 5 µm, 5065-9913
Analytical: 0.3 x 150 mm, 5 µm, 5064-8263
1. a-1-Antichymotrypsin
2. Antithrombin-III Precursor
3. Complement Factor B Precursor
Base Peak Chromatogram
Intens.
x107
3
2
Band from 1-D in gel digest
1
0
20
25
30
35
40
Time (min)
45
50
55
LCBP014
Sample Preparation of Human Serum:
Major serum proteins removed using Multiple Affinity Removal Column:
4.6 x 100 mm, P/N 5185-5985
Followed by 1-D gel digest
Peptide phosphorylation sites LC and
LC/MS using Capillary LC columns
5-55% B in 50 min, to
85% B from 55-57 min
Detector:
UV, 206 nm
MS Conditions:
LC/MS:
Vcap:
Drying gas flow:
Drying gas
temperature:
Nebulizer:
Capillary Exit Volt:
Accum Time:
Total Averages:
Isolation Width:
Frag Ampitude:
Sample:
454
x107
0.8
0.6
phosphopeptide
0.4
0.2
0.0
% Relative Abundance
Gradient:
5.5 µL/min
Full Scan MS
x105
2.5
Pos. Ion ESI
with LC/MSD trap
4000 V
7 L/min
250 °C
15 psi
50 V Max
300 ms
3
3 m/z
1.0 V
Beta case in digest, 100 nL (4 pmol)
688.4
2.0
1.5
1032.0
1.0
0.5
0.0
10 15 20 25 30 35 40 45 50
Time (min)
343.0 559.5
400
1275.4 1582.0
800
1600
m/z
1600
MS/MS of [M+2H] 2+ at m/z 1032
UV
x104
2.5
100
% Relative Abundance
Flow Rate:
A: Water + 0.1% Formic acid
B: Acetonitrile + 0.1% Formic acid
MS
% Relative Abundance
Mobile Phase:
ZORBAX 300SB-C18
5064-8268
0.5 x 150 mm, 3.5 µm
UV at 206 nm
Column:
80
60
40
20
0
10 15 20 25 30 35 40 45 50
Time (min)
2.0
982.5 1022.9
[MH2-H2PO4]2+
[MH2-H20]2+
1.5
1.0
747.3
0.5
1106.7
1490.8
1690.0
0.0
400
800
1600
1600
m/z
LCBP037
COLUMNS FOR BIOMOLECULE SEPARATIONS
Capillary columns for HPLC analyses
with UV and MS detection
Flow Rate:
5.5 µL/min
Detector:
UV, 206 nm
MS Conditions:
LC/MS:
Sample:
Pos. Ion ESI with
LC/MSD trap-Vcap 4000 V
Drying Gas Flow:
7 L/min
Drying Gas Temperature: 250 °C
Nebulizer: 15 psi
Capillary Exit Volt:
50 V
Max Accum Time:
300 ms
Total Averages:
3
Isolation Width:
3 m/z
Frag Ampitude:
1.0 V
100 nL
Beta Casein Digest (4 pmol)
A ZORBAX 300SB-C18 capillary column (0.3 mm id) is used for
the separation of the protein digest. Detection is by both UV and
Electrospray MS. MS detection can be used for identification of
peptide fragments.
UV
80
60
40
20
0
10
x10
5-55% B in 50 min, to 85% B from 55-57 min
A: Water + 0.1% Formic acid
B: Acetonitrile + 0.1% Formic acid
100
UV at 206 nm
Mobile Phase:
ZORBAX 300SB-C18
5064-8263
0.3 x 150 mm, 5 µm
% Relative Abundance
Column:
15
20
25
30
35
Time (min)
40
45
50
15
20
25
30
35
Time (min)
40
45
50
MS
0.8
0.6
0.4
0.2
0.0
10
LCSB007
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COLUMNS FOR BIOMOLECULE SEPARATIONS
2-D LC/MS Analyses Using ZORBAX Capillary
and Nano LC Columns
Typical Column Configuration for 2-D HPLC
1100/1200 Series
quaternary pump
1100/1200 Series
micro well plate
autosampler
Reversed-phase
enrichment column
Waste
Nanopump
Detection
(1100/1200 Series
LC/MSD Trap)
SCX Strong
cation-exchange column
Reversed-phase column 1100/1200 Series column
compartment
1100/1200 Series
quaternary pump
1100/1200 Series
micro well plate
autosampler
Reversed-phase
enrichment column
Waste
Nanopump
Detection
(1100/1200 Series
LC/MSD Trap)
SCX Strong
cation-exchange column
Reversed-phase column 1100/1200 Series column
compartment
Flow path of an Agilent customized Nanoflow Proteomics Solution system.
1. Sample loading, elution from SCX and trapping on enrichment column
2. Valve switch in column compartment, elution from enrichment column; separation on RP, and MS anaylsis
456
COLUMNS FOR BIOMOLECULE SEPARATIONS
Proteins in a complex sample by 2-D HPLC
with Nano HPLC columns
Column:
ZORBAX 300SB-C18
5065-9913
0.3 x 5 mm, 5 µm
Column:
ZORBAX 300SB-C18
5065-9911
0.075 x 150 mm, 3.5 µm
Mobile Phase:
Quaternary Pump: 3% Acetonitrile/0.1% Formic acid
Nanopump: A = Water, 0.1% Formic acid, B = ACN,
0.1% Formic acid
Flow Rate:
Quaternary Pump: 30 µL/min
Nanopump: 300 nL/min
Gradient:
Quaternary Pump: Isocratic
Nanopump:
6 min = 3% B, 120 min = 60% B, 125 min = 80% B,
130 min = 80% B, 131 min = 3% B, 140 min = 3% B
MS Conditions:
Source: Nano ESI, drying gas flow: 5 L/min, drying gas temp: 225 °C
Ion Trap: Skim: 1:35 V, cap exit offset: 115 V, octupole 1:12 V,
octupole 2:3.5 V, trap drive: 80 V. ICC: on, averages: 4, max accu
time: 150 ms; target 60.000, ion mode positive, MS/MS mode.
Sample:
Tryptic Digest of bovine serum albumin
Volume: 1 to 8 µL
Salt Step Elution: 8 mL of 10 mM-100 mM KCI (10 mM increments),
125 mM, 150 mM, 200 mM, 300 mM, 500 mM, 1 M.
Intens.
x106
1.0
30 mM KCI
0.5
x105
6
4
2
60 mM KCI
x105
6
75 mM KCI
4
2
Tryptic digest of bovine serum albumin (BSA).
The base peak chromatograms show a
selection of fractions from a 2-D HPLC
separation. Single chromatograms represent
peptides from BSA eluting at a given salt
concentration followed by enrichment and
reversed-phase chromatography.
x106
1.0
120 mM KCI
0.5
5
10
15
20
25
Time (min)
30
35
40
LCCN004
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COLUMNS FOR BIOMOLECULE SEPARATIONS
ZORBAX Bio-SCX Series II
Nano Columns
ZORBAX has Bio-SCX Series II columns designed for optimized 2-D separations of peptides and
proteins using LC/MS. This packing is based on ultra-pure 3.5 µm ZORBAX silica particles, bonded
with a bio-friendly polymer that is functionalized with sulfonic acid groups. This gives strong retention
and good peak shape in the ion-exchange step of 2-D analysis of peptides and proteins.
Column Specifications
Bonded Phase
Pore
Size
Surface
Area
pH
Range
Functionality
Max
Pressure
ZORBAX Bio-SCX Series II
300Å
90 m2/g
2.5-8.5
Sulfonic acid
350 bar
ZORBAX Bio-SCX Series II
458
Bio-SCX
Particle Size (µm) Series II
Description
Size (mm)
Capillary
0.3 x 35
3.5
5065-9912
Capillary
0.8 x 50
3.5
5065-9942
COLUMNS FOR BIOMOLECULE SEPARATIONS
ZORBAX Bio-SCX Series II provides
more retention of small peptides
Column:
ZORBAX Bio SCX Series II
5065-9912
0.3 x 35 mm, 3.5 µm
Mobile Phase:
95% 40 mM NaCl: 5% ACN,
0.3% Formic acid
Flow Rate:
5 µL/min
Detector:
230 nm
Sample:
Synthetic Dipeptides
2
1. Trp - Tyr
2. Mct - Trp
1
mAU
40
20
0
0
2
4
6
8
10
6
8
10
Time (min)
2
mAU
40
20
0
1
0
2
4
Time (min)
LCIE002
The new ZORBAX Bio-SCX Series II column retains smaller peptides more strongly than some other SCX columns. The result is increased resolution of more hydrophilic peptides
fragments and more accurate identification when these columns are used in 2-D HPLC analysis.
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COLUMNS FOR BIOMOLECULE SEPARATIONS
ZORBAX HPLC Capillary Columns (glass-lined stainless steel)
Poroshell
Particle Size (µm) 300SB-C18 300SB-C8 300SB-C8
Description
Size (mm)
Capillary
0.8 x 50
3.5
Capillary
0.5 x 250
5
5064-8266
Capillary
0.5 x 150
5
5064-8264
Capillary RR
0.5 x 150
3.5
5064-8268
Capillary
0.5 x 75
5
Bio-SCX
300Extend-C18 Series II
5065-9942
5065-4468
Capillary
0.5 x 35
5
5064-8294
Capillary RR
0.5 x 35
3.5
5065-4459
Capillary
0.3 x 250
5
5064-8265
Capillary
0.3 x 150
5
5064-8263
Capillary
0.3 x 35
5
5064-8295
Capillary
0.3 x 35
3.5
Capillary RR
0.3 x 150
3.5
5064-8267
5065-4460
5065-4464
Capillary RR
0.3 x 100
3.5
5064-8259
5065-4461
5065-4465
Capillary RR
0.3 x 75
3.5
5064-8270
5065-4462
5065-4466
Capillary RR
0.3 x 50
3.5
5064-8300
5065-4463
5065-4467
5065-4427
5065-4427 5065-4427
5065-4427
Replacement Screens, 10/pk
5065-9912
ZORBAX Nano HPLC Columns (PEEK)
ZORBAX 300SB-C18 trap/guard, 5065-9913
460
Particle Size
(µm)
300SB-C18
USP L1
0.1 x 150
3.5
5065-9910
Nano RR
0.075 x 150
3.5
5065-9911
Nano RR
0.075 x 50
3.5
5065-9924
Trap/Guard, 5/pk
0.3 x 5
Description
Size (mm)
Nano RR
Trap/Guard Hardware kit
5
300SB-C8
USP L7
5065-9923
5065-9913
5065-9914
5065-9915
5065-9915
COLUMNS FOR BIOMOLECULE SEPARATIONS
MicroBore (1.0 mm id) Columns
• High sensitivity for small sample sizes
• Compatible with LC/MS interfaces
• Wide variety of bonded phases
• Silica and polymeric particles
Agilent MicroBore (1.0 mm id) columns are a good choice when sample sizes are limited. They can improve
detection limits 5 times over 2.1 mm id columns when the same sample mass is used. This increase in
sensitivity can be critical. MicroBore columns use low flow rates (typically ~ 50 µL/min). Therefore, these
columns are ideal for use with detectors requiring low flow rates such as some mass spectrometers and
with capillary LC systems.
Optimum performance is achieved when MicroBore columns are used with UHPLC/HPLC Microbore
systems. A wide variety of bonded phases is available for use up to 400 bar including StableBond,
300SB-C18, 300SB-C8, and Poroshell columns. Polymeric reversed-phase, PLRP-S, and ion-exchange
PL-SAX and PL-SCX are also available for applications requiring exceptionally stable wide pore particles.
Guard columns are also now available with an adjustable tube stop depth to provide a perfect zero dead
volume connection every time.
Sterically Protected 300StableBond Bonded Phase
Separation of a tryptic digest on
ZORBAX MicroBore 300SB-C18
Column:
ZORBAX 300SB-C18
863630-902
1.0 x 150 mm, 3.5 µm
Mobile Phase:
Gradient: 2-60% B in 60 min
A: 0.1% TFA
B: 0.075% TFA/80% ACN
Flow Rate:
50 µL/min
Temperature:
50 °C
Detector:
UV, 215 nm
Sample:
2 µL
Tryptic Digest of rhGH
This example of a tryptic digest separated on a
MicroBore column demonstrates the high sensitivity
and resolution possible with 1.0 mm id columns.
0
5
10
15
20
25
30
35
Time (min)
40
45
50
55
LCMB001
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461
COLUMNS FOR BIOMOLECULE SEPARATIONS
Microbore HPLC for sensitive peptide analysis
Column:
PLRP-S 100Å 5 µm, 150 mm x various id
Mobile Phase:
A: 0.01 M Tris HCI, pH 8
B: A + 0.35 M NaCl, pH 8
Flow Rate:
1 mL/min
Gradient:
Linear 20% ACN, 0.1% TFA to 50% ACN, 0.1% TFA
over 15 min
Peak Identification
A. 1.0 mm id (flow rate 47 µL/min)
B. 2.1 mm id (flow rate 200 µL/min)
C. 4.6 mm id (flow rate 1 mL/min)
2
1.
2.
3.
4.
Injection Volume: 0.5 µL
Sample Conc:
0.25 mg/mL
Detector:
UV, 220 nm
Oxytocin
Angiotensin II
Angiotensin I
Insulin
4
1
3
A
B
C
Peptide separation on Agilent PLRP-S 100Å 5 µm columns
0
min
8
Peak Identification
1.0 mm
2.1 mm
4.6 mm
120
Insulin
90
160
Oxytocin
80
90
Angiotensin II
Angiotensin I
80
140
100
70
80
60
60
40
60
80
60
40
30
Peak Area
50
Peak Area
100
Peak Area
Peak Area
70
120
40
30
40
20
50
20
20
20
10
0
0
0
0
50
100
conc (ng)
150
0
50
100
conc (ng)
Standard curve data-point graphs on Agilent PLRP-S columns
462
10
150
0
0
50
100
conc (ng)
150
0
50
100
conc (ng)
150
COLUMNS FOR BIOMOLECULE SEPARATIONS
MicroBore (1.0 mm id)
Particle Size
(µm)
300SB-C18
USP L1
1.0 x 250
5
861630-902
MicroBore RR
1.0 x 150
3.5
863630-902
863630-906
MicroBore RR
1.0 x 50
3.5
865630-902
865630-906
MicroBore Guard, 3/pk
1.0 x 17
5
5185-5920
5185-5920
Particle Size
(µm)
Poroshell
300SB-C18
Poroshell
300SB-C8
Poroshell
300SB-C3
Poroshell
300Extend-C18
671750-902
Description
Size (mm)
MicroBore
300SB-C8
USP L7
Description
Size (mm)
MicroBore
1.0 x 75
5
661750-902
661750-906
661750-909
MicroBore Guard, 3/pk
1.0 x 17
5
5185-5968
5185-5968
5185-5968
PLRP-S
100Å
USP L21
PLRP-S
300Å
USP L21
PLRP-S
1000Å
USP L21
Particle Size
(µm)
PLRP-S
4000Å
USP L21
Description
Size (mm)
MicroBore
1.0 x 150
3
PL1312-3300
MicroBore
1.0 x 50
3
PL1312-1300 PL1312-1301
MicroBore
1.0 x 50
5
PL1312-1500 PL1312-1501 PL1312-1502 PL1312-1503
MicroBore
1.0 x 50
8
PL1312-1802 PL1312-1803
Description
Size (mm)
MicroBore
1.0 x 50
Particle Size
(µm)
5
PL-SCX
1000Å
PL-SCX
4000Å
PL-SCX
1000Å
PL-SCX
4000Å
PL1351-1502 PL1351-1503 PL1345-1502 PL1345-1503
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463
COLUMNS FOR BIOMOLECULE SEPARATIONS
Purification – Prep HPLC
Agilent has a comprehensive range of silica and polymeric HPLC columns and media designed for
biomolecule purification. There are high efficiency small particle prep columns optimized for the purification
of µg and mg amounts of a biopharmaceutical drug candidate and fully porous bulk media, to pack
development and process columns to purify multiple 100 g, kg and multi-kg of API.
Some columns are specifically designed to address the needs of high efficiency purification, while
other products provide easy scale-up from small particle analytical columns to full scale API production.
Table 1 shows prep column/media options and the quantity of product that can be purified.
Polymeric Prep HPLC Columns
Discovery
BioPharmaceutical Lifecycle
µg
Development
mg
g
high efficiency
Reversed-Phase
mRP-C18
ZORBAX Prep HT
300Å StableBond
VariTide RPC
PLRP-S 100Å, 300Å,
1000Å, 4000Å
PL-SAX
Ion-Exchange
PL-SCX
Size Exclusion
ZORBAX GF-250/450
Table 1: Agilent columns and media for biomolecule purification – chromatographic type, product family and purification scale.
464
Production
kg
multi-kg
high throughput
COLUMNS FOR BIOMOLECULE SEPARATIONS
Purification Column Selection
Application
Technique
Notes
Agilent Columns
Proteomics
Reversed-Phase
A specialist high recovery column for proteomics applications. It is designed for µg scale
purifications with maximum recovery.
mRP-C18
All Biomolecules
Reversed-Phase
High efficiency 300Å silica-based particles.
ZORBAX PrepHT 300SB
Synthetic Peptides Reversed-Phase
Polymeric material designed for the purification of synthetic peptides. It is a high efficiency
single-column solution for the full range of synthetic peptides, acidic, basic, hydrophobic
and hydrophilic, and covers the size range of peptides produced by both solution and solid
phase synthesis.
VariTide RPC
All Biomolecules
The premium polymeric reversed-phase family with a range of pore sizes and particle sizes
to enable high efficiency laboratory scale purification using small particle prep column,
and scale-up to high yield production purification with larger particles at the process
scale. Use PLRP-S when purification will be scaled up to produce APIs and will need
regulatory documentation.
PLRP-S
Reversed-Phase
ü 3 µm and 5 µm for high efficiency
ü 8 µm, 10 µm, 10-15 µm, 15-20 µm, 30 µm and 50 µm particles for larger scale
and low pressure purification
All Biomolecules
Ion-Exchange
A fully porous strong anion-exchanger
PL-SAX
ü 5 µm particle size for high efficiency separations
ü 8 µm, 10 µm and 30 µm particles for larger scale medium and low pressure purification
A fully porous strong cation-exchanger
PL-SCX
ü 5 µm particle size for high efficiency separations
ü 8 µm, 10 µm and 30 µm particles for larger scale medium and low pressure purification
TIPS & TOOLS
Further information can be found in
the following publication:
Biomolecule Purification
(publication # 5990-8335EN)
www.agilent.com/chem/library
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465
COLUMNS FOR BIOMOLECULE SEPARATIONS
ZORBAX PrepHT
High purity, high recovery, and high throughput can be easily achieved with Agilent ZORBAX PrepHT
columns. These are available in a variety of bonded phases – StableBond 300Å, C18, C8, C3, and CN –
for optimized resolution and loadability under any conditions.
ZORBAX PrepHT columns are packed with 5 and 7 µm particle sizes for very high resolution.
The high resolution allows high loadability, high yield, and high purity of compounds. The larger
diameter columns and mechanically stronger ZORBAX particles allow for flow rates up to 100 mL/min,
thus increasing throughput.
ZORBAX 300Å StableBond Prep HT
Cartridge Columns
ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scale without losing
resolution. For complex separations on larger columns (21.2 mm id, 150 mm length and longer), Agilent has
carefully chosen the 7 µm particle size to achieve a balance between high efficiency and high loadability.
ZORBAX 300Å StableBond
Hardware Description
Size
(mm)
Particle Size
(µm)
300SB-C18
USP L1
300SB-C8
USP L7
300SB-CN
USP L10
300SB-C3
USP L56
PrepHT Cartridge Columns (require endfittings kit 820400-901)
PrepHT Cartridge
21.2 x 250
7
897250-102
897250-106 897250-105
897250-109
PrepHT Cartridge
21.2 x 150
7
897150-102
897150-106
897150-109
PrepHT Cartridge
21.2 x 150
5
895150-902
895150-906
895150-909
PrepHT Cartridge
21.2 x 100
5
895100-902
895100-906
895100-909
PrepHT Cartridge
21.2 x 50
5
895050-902
895050-906
895050-909
820400-901
820400-901 820400-901
820400-901
17.0 x 7.5
5
820212-921
820212-918 820212-924
820212-924
820444-901
820444-901 820444-901
820444-901
PrepHT Endfittings, 2/pk
PrepHT Guard Cartridge, 2/pk
Guard Cartridge Hardware
466
COLUMNS FOR BIOMOLECULE SEPARATIONS
PLRP-S for Prep to Process
• Discovery stage to multi-kg cGMP production reduces method development time
• Chemical stability for separations, optimization, sanitation, and regeneration increases selectivity
and column lifetime
• Single batch packing of multiple columns reduces system downtime and validation costs
The PLRP-S media, rigid poly(styrene/divinylbenzene) particles, are available in a range of pore sizes for
small molecule, synthetic biomolecule and macromolecule purification. Their thermal and chemical stability
makes them ideal for purifications that require extreme conditions for sample preparation, compound
elution, and column regeneration.
Capacity and resolution are two key parameters for maximizing the throughput of a purification. With a wide
choice of pore sizes and extended range of operating conditions, PLRP-S provides more options to achieve
the optimum process. Particle sizes range from 3 µm to 50 µm for scale-up from the µg/mg discovery stage
to multi-kg cGMP production. Excellent chemical stability, up to 1 M NaOH, permits sanitation and
regeneration that increase column lifetime. PLRP-S media batch sizes of up to 600 L are available, providing
single batch packing of multiple columns.
As part of our commitment to quality and continuity of supply, all manufacturing is carried out under a fully
documented process. A Type II Drug Master File and regulatory support files are available for process
materials, and facility audits are routinely conducted.
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467
COLUMNS FOR BIOMOLECULE SEPARATIONS
PLRP-S Prep to Process Application Guide
PLRP-S Media Pore Size
Application
100Å
300Å
Synthetic biomolecules, peptides, and oligonucleotides
3
3
Recombinant biomolecules, peptides, and proteins
3
3
3
Large biomolecules, antibodies, DNA fragments
Small molecules, unstable compounds including metal sensitivity
Column Specifications
pH Range
1-14
Buffer Content
Unlimited
Organic Modifier
1-100%
Temperature Limits
200 °C
Maximum Pressure
5-8 µm: 3000 psi (210 bar)
3 µm: 4000 psi (300 bar)
468
1000Å 4000Å
3
3
COLUMNS FOR BIOMOLECULE SEPARATIONS
Purification of a 25-mer trityl-off oligonucleotide
and analytical quantitation of the fraction
using PLRP-S 100Å, 4.6 x 50 mm
PLRP-S 100Å
PL1512-1300
4.6 x 50 mm, 3 µm
Mobile Phase:
A: 100 mM Triethylammonium acetate (TEAA)
B: 100 mM TEAA in 25:75 Acetonitrile:water
Flow Rate:
Purity (CE): 90.54%
Recovery: 83.20%
mV
Column:
200
0
1 mL/min
25% B 0 min, 35% B 2 min, 45% B 22.5 min,
45% B 23 min, 25% B 23.05 min, 25% B 26 min
Temperature:
80 ºC
min
0
min
25
mV
Gradient:
0
80
0
12.5
VLC0053
Crude bradykinin prep load
Column:
PLRP-S 100Å
PL1512-5100
4.6 x 250 mm, 10 µm
Sample:
30 µL containing 1.5 mg of crude peptide
Mobile Phase:
0.1% TFA in 21% ACN:79% water
Flow Rate:
1 mL/min (360 cm/hr)
0
min
30
VLCbradykinin
HPLC analysis of the fractions collected across the
peak showed that fractions 1 to 4 contained only
the peptide of interest and that the level of the critical
impurity increased with increasing fraction number.
Using the high efficiency PLRP-S column it was
possible to obtain from the crude, 91.7% pure,
a recovery of 97% with 100% purity. For more
information, see application note 5990-7736EN.
HPLC Purity %
100
% HPLC Purity
Fraction analysis –
the concentration
overload purification
% Purity (Bradykinin)
% Purity (Impurity)
Fraction number
0
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469
COLUMNS FOR BIOMOLECULE SEPARATIONS
Prep to Process PLRP-S
PLRP-S
100Å
PLRP-S
300Å
Size (mm)
Particle Size (µm)
100 x 300
30
100 x 300
15-20
PL1812-6200
PL1812-6201
100 x 300
10-15
PL1812-6400
PL1812-6401
100 x 300
10
PL1812-6100
PL1812-6101
100 x 300
8
PL1812-6800
PL1812-6801
50 x 300
8
PL1712-6800
PL1712-6801
50 x 150
30
50 x 150
15-20
PL1712-3200
PL1712-3201
50 x 150
10-15
PL1712-3400
PL1712-3401
50 x 150
10
PL1712-3100
PL1712-3101
50 x 150
8
PL1712-3800
PL1712-3801
25 x 300
15-20
PL1212-6200
PL1212-6201
25 x 300
10-15
PL1212-6400
PL1212-6401
25 x 300
10
PL1212-6100
PL1212-6101
25 x 300
8
PL1212-6800
PL1212-6801
25 x 150
30
25 x 150
10
PL1212-3100
PL1212-3101
25 x 150
8
PL1212-3800
PL1212-3801
25 x 50
10
PLRP-S
1000Å
PLRP-S
4000Å
PL1812-3102
PL1812-3103
PL1712-3702
PL1712-3703
PL1712-3102
PL1712-3103
PL1212-3702
PL1212-3703
PL1712-3102
PL1712-3103
PL1212-1102
PL1212-1103
PL1512-5702
PL1512-5703
PL1512-5102
PL1512-5103
PL1512-3702
PL1512-3703
PL1512-3102
PL1512-3103
PLRP-S Method Development Columns
470
4.6 x 250
30
4.6 x 250
15-20
PL1512-5200
PL1512-5201
4.6 x 250
10-15
PL1512-5400
PL1512-5401
4.6 x 250
10
PL1512-5100
PL1512-5101
4.6 x 250
8
PL1512-5800
PL1512-5801
4.6 x 150
30
4.6 x 150
15-20
4.6 x 150
10-15
4.6 x 150
10
PL1512-3100
PL1512-3101
4.6 x 150
8
PL1512-3800
PL1512-3801
PL1512-3200
PL1512-3201
PL1512-3401
COLUMNS FOR BIOMOLECULE SEPARATIONS
PLRP-S Bulk Media
Particle Size (µm)
Unit
PLRP-S
100Å
PLRP-S
300Å
PLRP-S
1000Å
50
1 kg
PL1412-6K00
PL1412-6K01
PL1412-6K02
100 g
PL1412-4K00
PL1412-4K01
PL1412-4K02
30
1 kg
15-20
1 kg
PL1412-6200
PL1412-6201
PL1412-6702
PL1412-6703
PL1412-4702
PL1412-4703
100 g
PL1412-4200
PL1412-4201
1 kg
PL1412-6400
PL1412-6401
100 g
PL1412-4400
PL1412-4401
1 kg
PL1412-6100
100 g
PL1412-4100
PL1412-6101
PL1412-6102
PL1412-6103
PL1412-4101
PL1412-4102
PL1412-4103
1 kg
PL1412-6800
PL1412-6801
100 g
10-15
10
8
PLRP-S
4000Å
For larger quantities, please contact your local Agilent sales office
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471
COLUMNS FOR BIOMOLECULE SEPARATIONS
PL-SAX and PL-SCX for Prep to Process
• Ion-exchange purifications over a wider pH range extend applications
• HPLC flow rates and rapid equilibration reduce purification cycle times
• Large pore size for improved mass transfer delivers high speed, high resolution purifications
These rigid, strong ion-exchange materials are extremely hydrophilic and are designed for purification of
biomolecules. The PL-SAX and PL-SCX materials are totally polymeric and are chemically and thermally
stable over a full range of HPLC conditions. The strong ion-exchange functionalities, covalently linked to a
chemically stable polymer, facilitate ion-exchange purifications over a wider pH range. This stability can be
exploited for column sanitation and clean-up. Thermal stability also enables the use of denaturing conditions
and stabilizing/solubilizing agents for the purification of target compounds, as encountered in the
purification of synthetic oligonucleotides with self-complementary sequences.
Both the 1000Å and 4000Å wide-pore materials are mechanically stable and robust and can be operated
over a wide range of linear velocities, with fast loading of dilute solutions and wash cycles. HPLC flow rates
and rapid equilibration reduces purification cycle times.
Packing in dynamic axial compression (DAC) column hardware is straightforward and high efficiency
columns are achieved with excellent reproducibility and lifetimes. The 1000Å pore size is for high-capacity
purifications and the 4000Å gigaporous particles with improved mass transfer are intended for large
biomolecules and high-speed, high-resolution purifications.
472
COLUMNS FOR BIOMOLECULE SEPARATIONS
Column Specifications
PL-SAX
PL-SCX
Matrix
Fully polymeric
Fully polymeric
Pore Sizes
1000Å, 4000Å
1000Å, 4000Å
Particle Sizes
10 µm, 30 µm
10 µm, 30 µm
Bead Form
Rigid spherical
Rigid spherical
Functionality
Quaternary amine
Sulfonic acid
Pressure Stability
3000 psi
3000 psi
Temperature Stability
80 °C
80 °C
pH Range
1-14
1-14
Eluent Compatibility
All anion-exchange buffers
All cation-exchange buffers
Packed Bed Density
0.39 g/mL
0.39 g/mL
Column:
PL-SAX 1000Å, 8 µm
Mobile Phase:
A: 93% 0.1 M TEAA, pH 7:7% ACN
B: 93% 0.1 M TEAA, 3.24 M ammonium acetate, pH
7:7% ACN
Gradient:
0-100% B in 20 min
Flow Rate:
1.5 mL/min
Temperature:
60 °C
Detector:
UV, 290 nm
80
91-mer
%B
Purification of a large oligonucleotide
0
0
min
35
VLC0054
Preparative fractionation of a culture
filtrate containing amyloglucosidases
on Agilent PL-SAX 4000Å
Column:
PL-SAX
PL1551-1803
4.6 x 50 mm, 8 µm
Mobile Phase:
A: 0.01 M Tris HCI, pH 8
B: A + 0.5 M NaCl, pH 8
Flow Rate:
4.0 mL/min
Gradient:
Linear 0-100% B in 2 min
Detector:
UV, 280 nm
Preparative fraction
0
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min
30
473
COLUMNS FOR BIOMOLECULE SEPARATIONS
Prep to Process PL-SAX and PL-SCX
Dimensions
PL-SAX
Particle Size (µm) 1000Å
100 x 300
30
PL-SAX
4000Å
PL-SCX
1000Å
PL-SCX
4000Å
PL1851-3102 PL1851-3103 PL1845-3102 PL1845-3103
100 x 300
10
PL1851-2102 PL1851-2103 PL1845-2102 PL1845-2103
50 x 150
30
PL1751-3702 PL1751-3703 PL1745-3702 PL1745-3703
50 x 150
10
PL1751-3102 PL1751-3103 PL1745-3102 PL1745-3103
25 x 150
30
PL1251-3702 PL1251-3703 PL1245-3702 PL1245-3703
25 x 150
10
PL1251-3102 PL1251-3103 PL1245-3102 PL1245-3103
25 x 50
10
PL1251-1102 PL1251-1103 PL1245-1102 PL1245-1103
7.5 x 150
8
PL1151-3802 PL1151-3803
7.5 x 50
8
PL1151-1802 PL1151-1803 PL1145-1802 PL1145-1803
PL-SAX and PL-SCX Method Development Columns
Prep to Process PL-SAX and PL-SCX Columns
and Bulk Media
4.6 x 250
30
PL1551-5702 PL1551-5703 PL1545-5702 PL1545-5703
4.6 x 250
10
PL1551-5102 PL1551-5103 PL1545-5102 PL1545-5103
4.6 x 150
30
PL1551-3702 PL1551-3703 PL1545-3702 PL1545-3703
4.6 x 150
10
PL1551-3102 PL1551-3103 PL1545-3102 PL1545-3103
PL-SAX and PL-SCX Bulk Media
Particle Size (µm) Unit
PL-SAX
1000Å
30
1 kg
PL1451-6702 PL1451-6703 PL1445-6702 PL1445-6703
100 g
PL1451-4702 PL1451-4703 PL1445-4702 PL1445-4703
10
1 kg
PL1451-6102 PL1451-6103 PL1445-6102 PL1445-6103
100 g
PL1451-4102 PL1451-4103 PL1445-4102 PL1445-4103
For larger quantities, please contact your local Agilent sales office
474
PL-SAX
4000Å
PL-SCX
1000Å
PL-SCX
4000Å
COLUMNS FOR BIOMOLECULE SEPARATIONS
Peptide Purification
VariTide is a cost-effective solution for the production of synthetic peptides. This column lets you manage
the cost and efficiency of high-volume synthetic peptide purification, from µg to g scale. VariTide provides
a solution for peptide houses that manufacture small quantities of hundreds or thousands of peptides where
manufacturing time is the economic driving force.
VariTide RPC Columns
VariTide RPC Columns for Synthetic Peptides
• A single column to cover the full range of synthetic peptides
• Small particle size for maximum efficiency, even with 1 and 2 in prep columns
• Bulk media to pack 1 and 2 in prep columns for the purification of mg to g quantities
VariTide RPC columns and media are part of the VariPep Peptide Solution. This is the recommended option
for cost-effective separation and purification of synthetic peptides using generic methods.
VariTide RPC Columns for Synthetic Peptides
Size (mm)
Part No.
21.2 x 250
PL1E12-5A05
10.0 x 250
PL1012-5A05
4.6 x 250
PL1512-5A05
VariTide RPC Bulk Media
Description
Part No.
100 g
PL1412-4A05
1 kg
PL1412-6A05
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475
COLUMNS FOR BIOMOLECULE SEPARATIONS
Crude peptide screen
Column:
VariTide RPC
PL1512-5A05
4.6 x 250
Mobile Phase:
Acidic
A: 0.1% TFA in 95% water: 5% ACN
B: 0.1% TFA in 50% water: 50% ACN
Basic
A: 5% ACN, 95% 20 mm ammonium
carbonate pH 9.5
B: 50% ACN, 50% 20 mm ammonium
carbonate pH 9.5
Flow Rate:
1.0 mL/min (360 cm/h)
Gradient:
0-100% B in 30 min
Detector:
UV, 220 nm
Acidic
Major impurity not
separated at acidic pH
Basic
min
0
30
VLC0052
VariPure IPE
• Pre-packed for convenience
• Removal of ion-pairing agents for improved productivity
• High performance and economy for excellent efficiency
VariPure IPE is a polymer-supported quaternary-amine resin with a bicarbonate counter ion, designed for
removing acidic ion-pair reagents, such as trifluoroacetic acid (TFA), formic acid or acetic acid. VariPure IPE
is a high performance and economical acid removal material conveniently supplied as pre-packed SPE type
devices. The particle size, capacity and device geometry are matched to provide sufficient residence time to
achieve effective ion-air extraction under gravity flow. For acid labile peptides, removal of the ion-pairing
agent prevents acid degradation of the peptide during post-HPLC work-up, and increases the yield of
purified product.
VariPure IPE
Loading
Unit
Part No.
100 mg per 3 mL tube
~ 5 mL 0.1% TFA
50/pk
PL3540-D603VP
500 mg per 6 mL tube
~ 25 mL 0.1% TFA
50/pk
PL3540-C603VP
1 g per 20 mL tube
~ 50 mL 0.1% TFA
25/pk
PL3540-P603VP
25 g
476
Counter-ion
Removal Capacity
PL3549-3603VP
COLUMNS FOR BIOMOLECULE SEPARATIONS
BioHPLC Columns Literature
Title
Column/Product
Application
Publication Number Publication Type
Rapid Analysis of Adenovirus Type 5 Particles with
Bio-Monolith Anion-Exchange HPLC Columns to Support
the Development of a High-Titre Manufacturing Platform
Bio-Monolith QA
Adenovirus
5990-5524EN
Application Note
Separation of Two Sulfurated Amino Acids with other
Seventeen Amino Acids by HPLC with Pre-Column
Derivatization
Eclipse Plus-C18
Amino acid analysis
5990-5977EN
Application Note
Rapid, Accurate, Sensitive, and Reproducible HPLC
Analysis of Amino Acids
ZORBAX Eclipse AAA
Amino acid analysis
5980-1193EN
Application Note
High-Speed Amino Acid Analysis (AAA) on 1.8 µm
Reversed-Phase (RP) Columns
ZORBAX Eclipse Plus
Amino acid analysis
5989-6297EN
Application Note
Improved Amino Acid Methods Using
Agilent ZORBAX Eclipse Plus C18 Columns for a Variety
of Agilent LC Instrumentation and Separation Goals
ZORBAX Eclipse Plus
Amino acid analysis
5990-4547EN
Application Note
Rapid and Precise Determination of Cellular Amino Acid
Flux Rates using HPLC with Automated Derivatization
with Absorbance Detection
ZORBAX Eclipse Plus
Amino acid analysis
5990-3283EN
Application Note
Agilent PL-SAX 1000Å HPLC Columns and Media
PL-SAX
Analysis/Prep Oligonucleotides
5990-8200EN
Flyer
Compliance for Biopharmaceutical Laboratories
LC columns
Compliance
5990-7001EN
Primer
Macroporous Reversed-Phase C18 High-Recovery
Protein Fractionation HPLC Column
mRP-C18
Human serum,
Biomarkers
5989-2714EN
Brochure
Rapid Human Polyclonal IgG Quantification using the
Agilent Bio-Monolith Protein A HPLC Column
Bio-Monolith
IgG
5989-9733EN
Application Note
Rapid IgM Quantification in Cell Culture Production
and Purification Process Monitoring using the
Agilent Bio-Monolith QA Column
Bio-Monolith QA
IgM
5989-9674EN
Application Note
Optimization of Protein Separations on Weak
Cation-Exchange Columns – a Study of the Particle Size,
Buffer Salts and Gradients
Bio IEX
MAbs
5990-8833EN
Technical Poster
(Continued)
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477
COLUMNS FOR BIOMOLECULE SEPARATIONS
BioHPLC Columns Literature
Title
Column/Product
Application
Publication Number Publication Type
pH Gradient Elution for Improved Separation of
Monoclonal Antibody Charged Variants
Bio MAb
MAbs
5990-9629EN
Application Note
Characterization of Monoclonal Antibodies on the
Agilent 1260 Infinity Bio-inert Quaternary LC by
Size Exclusion Chromatography using the
Agilent Bio SEC Columns
Bio SEC
MAbs
5990-6414EN
Application Note
Agilent BioHPLC Columns for the Characterization of
Monoclonal Antibodies
Biocolumns
MAbs
5990-7753EN
Flyer
Fast Separation of Monoclonal Antibody and Dimer by
SEC with Agilent Bio SEC
Bio SEC
MAbs
5990-8613EN
Application Note
Choosing a ZORBAX Poroshell Phase (C3, C8, or C18) for
Fast Separation of Monoclonal Antibodies
Poroshell 300
MAbs
5989-0071EN
Application Note
Determination of the Glycosylation Status of Intact
Recombinant Human Antibodies using Time of Flight
Mass Spectrometry
Poroshell 300
MAbs
N/A
Technical Poster
High Speed and Ultra-High Speed Peptide Mapping of
Human Monoclonal IgG on Poroshell 300SB-C18, C8,
and C3
Poroshell 300
MAbs
5989-0590EN
Application Note
Rapid HPLC Analysis of Monoclonal Antibody IgG1 Heavy
Chains using ZORBAX Poroshell 300SB-C8
Poroshell 300
MAbs
5989-0070EN
Application Note
Comparison of ZORBAX StableBond 300Å LC Columns to
Optimize Selectivity for Antibody Separations Using HPLC
and LC/MS
ZORBAX 300SB
MAbs
5989-6840EN
Application Note
Ultra High Speed and High Resolution Separations of
Reduced and Intact Monoclonal Antibodies with Agilent
ZORBAX RRHD Sub-2 µm 300 Diphenyl UHPLC Column
ZORBAX RRHD 300-Diphenyl MAbs
5990-9668EN
Application Note
Reversed-Phase Optimization for Ultra Fast Profiling of
Intact and Reduced Monoclonal Antibodies using
Agilent ZORBAX Rapid Resolution High Definition
300SB-C3 Column
ZORBAX RRHD 300SB-C3
5990-9667EN
Application Note
MAbs
(Continued)
478
COLUMNS FOR BIOMOLECULE SEPARATIONS
BioHPLC Columns Literature
Title
Column/Product
Application
Publication Number Publication Type
Reversed-Phase Separation of Intact Monoclonal
Antibodies (MAb) using Agilent ZORBAX RRHD
300SB-C8
ZORBAX RRHD 300SB-C8
MAbs
5990-9016EN
Application Note
Rapid UHPLC Analysis of Reduced Monoclonal
Antibodies using an Agilent ZORBAX Rapid Resolution
High Definition (RRHD) 300SB-C8 Column
ZORBAX RRHD 300SB-C8
MAbs
5990-9631EN
Application Note
Increased UV-Sensitivity in Combination with Novel
WCX Column Separation for Better Detectability of
Charge State Variants of Biotherapeutic Proteins
Bio MAb
MAbs and other
proteins
N/A
Technical Poster
Agilent HPLC Column Selection Guide
HPLC columns
Many
5990-4435EN
Selection Guide
The LC Handbook: Guide to LC Columns
and Method Development
LC columns
Method development 5990-7595EN
Primer
Agilent PLRP-S 100Å HPLC Columns and Media
PLRP-S
Oligonucleotides
5990-8187EN
Flyer
HPLC Purification of 26-bp Serial Analysis of Gene
Expression Ditags
PLRP-S
Oligonucleotides
5990-7739EN
Application Note
Improved Column Lifetime with Thermally Stable Polymer
Columns for Oligonucleotide Ion-Pair RP HPLC
PLRP-S
Oligonucleotides
5990-7764EN
Application Note
Ion-Pair Reversed-Phase Purification of De-Protected
Oligonucleotides – Choice of Pore Size
PLRP-S
Oligonucleotides
5990-7763EN
Application Note
Use Temperature to Enhance Oligonucleotide Mass
Transfer and Improve Resolution in Ion-Pair RP HPLC
PLRP-S
Oligonucleotides
5990-7765EN
Application Note
High Resolution Separations of Oligonucleotides using
PL-SAX Strong Anion-Exchange HPLC Columns
PL-SAX
Oligonucleotides
5990-8297EN
Application Note
Fast Impurity Profiling of Synthetic Oligonucleotides
with the Agilent 1290 Infinity LC System and
Agilent 6530 Accurate-Mass QTOF LC/MS
ZORBAX Eclipse Plus C18
RRHD
Oligonucleotides
5990-5825EN
Application Note
Agilent PLRP-S Media and Load & Lock Columns –
The Future of Prep/Process Chromatography
Prep/Process
Oligonucleotides,
Peptides, Proteins
5990-8201EN
Flyer
Agilent PLRP-S 50 µm HPLC Media
PLRP-S
Oligonucleotides,
Peptides, Small
proteins
5990-8188EN
Flyer
(Continued)
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479
COLUMNS FOR BIOMOLECULE SEPARATIONS
BioHPLC Columns Literature
Title
Column/Product
Application
Publication Number Publication Type
Analysis of Peptides on a PLRP-S 100Å 10 µm with ELS
Detection and Acetonitrile-Free Eluents
PLRP-S
Peptides
5990-7760EN
Application Note
Investigation into the Alternatives to Acetonitrile for the
Analysis of Peptides
PLRP-S
Peptides
5990-7740EN
Application Note
Investigation into the Alternatives to Acetonitrile for the
Analysis of Peptides on a SepTech ST150 10-C18
SepTech
Peptides
5990-7951EN
Application Note
Investigation into the Alternatives to Acetonitrile for the
Analysis of Peptides on a VariTide RPC
VariTide RPC
Peptides
5990-8145EN
Application Note
Fast Monitoring of Bacteriophage Production
During Fermentation Using the Agilent Bio-Monolith
HPLC Column
Bio-Monolith
Phage production,
process monitoring
5990-3247EN
Application Note
Physicochemical Characterization of a Therapeutic
Protein by Peptide Mapping, SEC and IEX using the
Agilent 1260 Infinity Bio-inert Quaternary LC System
Bio MAb, Bio SEC,
ZORBAX Eclipse Plus,
Poroshell 120
Protein analysis
5990-6192EN
Application Note
Optimization of the Agilent 1100 HPLC System for
Superior Results with ZORBAX Poroshell Columns
Poroshell 300
Protein analysis
5988-9998EN
Application Note
Using Poroshell 300SB-C18 for High-Sensitivity,
High-Throughput Protein Analysis on the Agilent LC/MSD
Poroshell 300-C18
Protein analysis
5988-7031EN
Application Note
Analysis of Albumin Proteins using ProSEC 300S Columns
ProSEC 300S
Protein analysis
5990-7852EN
Application Note
Analysis of Complex Bacterial Cell Division Proteins by
Size Exclusion Chromatography (SEC)
ProSEC 300S
Protein analysis
5990-8143EN
Application Note
Analysis of Globulins using ProSEC 300S Columns
ProSEC 300S
Protein analysis
5990-7851EN
Application Note
Analysis of Hsp47, a Collagen Chaperone, by Size
Exclusion Chromatography (SEC)
ProSEC 300S
Protein analysis
5990-8142EN
Application Note
Analysis of Various Globular Proteins using
ProSEC 300S Columns
ProSEC 300S
Protein analysis
5990-7850EN
Application Note
Effect of pH on Protein Size Exclusion Chromatography
ProSEC 300S
Protein analysis
5990-8138EN
Application Note
Globular Proteins and the Calibration of
ProSEC 300S Columns
ProSEC 300S
Protein analysis
5990-7767EN
Application Note
(Continued)
480
COLUMNS FOR BIOMOLECULE SEPARATIONS
BioHPLC Columns Literature
Title
Column/Product
Application
Reduce Tubing Volume to Optimize Column Performance
Small diameter columns
Optimizing instrument 5990-4964EN
performance
Publication Number Publication Type
Application Note
Using the High-pH Stability of ZORBAX Poroshell
300Extend-C18 to Increase Signal-to-Noise in LC/MS
ZORBAX 300 Extend-C18
Optimizing instrument 5989-0683EN
performance
Application Note
Increase Sensitivity with Microbore Polymeric HPLC
Columns from Agilent
PLRP-S (Microbore)
Peptide hormone,
5990-8666EN
small proteins, small
molecules
Technical Overview
Decreasing Analysis Time Using Poroshell 300SB-C18 in
Analysis of a Protein Digest
Poroshell 300
Peptide mapping
5988-6081EN
Application Note
Rapid Peptide Mapping Method with High Resolution
using a sub 2-µm Column
ZORBAX 300SB-C18
Peptide mapping
5990-4712EN
Application Note
Increased Peak Capacity for Peptide Analysis with the
Agilent 1290 Infinity LC System
ZORBAX Eclipse Plus
Peptide mapping
5990-6313EN
Application Note
Trypsin-Digested Monoclonal Antibody and BSA using
Agilent ZORBAX RRHD 300SB-C18
ZORBAX RRHD 300SB-C18
Peptide mapping
5990-8244EN
Application Note
Preparative Scale Purification of Bradykinin by
Concentration Overload
PLRP-S
Peptide purification
5990-7736EN
Application Note
Preparative Scale Purification of Bradykinin
by Volume Overload
PLRP-S
Peptide purification
5990-7741EN
Application Note
Preparative Scale Purification of Depherelin by
Concentration Overload
PLRP-S
Peptide purification
5990-7742EN
Application Note
Preparative Scale Purification of Leuprolide by
Concentration Overload
PLRP-S
Peptide purification
5990-7735EN
Application Note
Superior Resolution of Peptides on SepTech ST150
10-C18 using Acetonitrile-Free Gradient Elution
SepTech
Peptide purification
5990-7761EN
Application Note
Agilent PLRP-S Media for HPLC Analysis of Peptides
PLRP-S
Peptides
5990-8667EN
Technical Overview
(Continued)
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481
COLUMNS FOR BIOMOLECULE SEPARATIONS
BioHPLC Columns Literature
Title
Column/Product
Application
Publication Number Publication Type
Light Scattering Analysis of BSA
with ProSEC 300S Columns
ProSEC 300S
Protein analysis
5990-7766EN
Application Note
Static Light Scattering Analysis of Globular Proteins
with Agilent ProSEC 300S Columns
ProSEC 300S
Protein analysis
5990-7939EN
Application Note
LC Handbook and Compliance Guide to Recombinant
Protein Characterization
N/A
Protein analysis
5990-8561EN
Primer
Agilent ZORBAX 300SB-C18 1.8µm Rapid Resolution
High Definition Columns for Proteins
ZORBAX 300SB-C18
Protein analysis
5990-7989EN
Technical Overview
Analysis of Oxidized Insulin Chains using
Reversed-Phase Agilent ZORBAX RRHD 300SB-C18
ZORBAX RRHD 300SB-C18
Protein analysis
5990-7988EN
Application Note
Fast Separation of Recombinant Human Erythropoietin
using Reversed-Phase Agilent ZORBAX RRHD
300SB-C18, 1.8 µm
ZORBAX RRHD 300SB-C18
Protein analysis
5990-9248EN
Application Note
ACN-free HPLC Analysis and Prep Purification
of ACP Fragment
PLRP-S
Protein purification
5990-7762EN
Application Note
Isocratic Purification of Synthetic Acyl Carrier
Protein Fragment 65-74
PLRP-S
Protein purification
5990-7737EN
Application Note
Agilent PL-SAX Anion-Exchange Media for
Amyloglucosidase Purification and Analysis
PL-SAX
Protein purification
5990-8664EN
Technical Overview
Progressive Denaturation of Globular Proteins in Urea
ProSEC 300S
Protein purification
5990-8141EN
Application Note
Optimizing Protein Separations
with Agilent Weak Cation-Exchange Columns
Bio IEX
Protein separation
5990-9628EN
Application Note
Faster Separations Using
Agilent Weak Cation-Exchange Columns
Bio IEX
Protein separation
5990-9931EN
Application Note
Optimum Pore Size for Characterizing Biomolecules
with Agilent Bio SEC Columns
Bio SEC
Protein separation
5990-9894EN
Application Note
Separation of High MW Fibrous Proteins
PLRP-S
Protein separation
5990-8137EN
Application Note
(Continued)
482
COLUMNS FOR BIOMOLECULE SEPARATIONS
BioHPLC Columns Literature
Title
Column/Product
Application
Publication Number Publication Type
Fast Protein Separations Using Agilent Poroshell 300
Poroshell 300
Protein separation
5989-9899EN
Application Note
Fast Separation of Large and Heterogeneous Proteins
using ZORBAX Poroshell C18, C8, and C3 Phases
Poroshell 300
Protein separation
5989-0015EN
Application Note
Protein Identification and Impurity Profiling using
Wide-Pore Reversed-Phase HPLC/UHPLC
Poroshell 300
Protein separation
5991-0625EN
Brochure
Use of Temperature to Increase Resolution in the
Ultrafast HPLC Separation of Proteins with
ZORBAX Poroshell 300SB-C8 HPLC Columns
Poroshell 300-C8
Protein separation
5989-0589EN
Application Note
The Effect of NaCl Concentration on Protein Size
Exclusion Chromatography
ProSEC 300S
Protein separation
5990-8139EN
Application Note
The Effect of Temperature on Protein Size Exclusion
Chromatography
ProSEC 300S
Protein separation
5990-8140EN
Application Note
Infinitely Better for Bio-Molecule Analysis
Agilent 1260 Infinity
Bio-inert Quaternary
LC System
Proteins
5990-6220EN
Brochure
Defining the Optimum Parameters for Efficient Size
Separations of Proteins
Bio SEC
Proteins
5990-8832EN
Technical Poster
Defining the Optimum Parameters for Efficient Size
Separations of Proteins
Bio SEC
Proteins
5990-8895EN
Application Note
Compliance for Biopharmaceutical Laboratories
Many
Proteins
5990-7001EN
Primer
Gradient Purification of Synthetic Acyl Carrier Protein
Fragment 65-74
PLRP-S
Proteins
5990-7738EN
Application Note
Fast Agilent HPLC for Large Biomolecules
PLRP-S, PL-SAX, PL-SCX
Proteins
5990-8663EN
Technical Overview
Agilent Anion-Exchange Media for Proteins –
Loading vs Resolution – Effect of Flow Rate
and Example Protein Separations
PL-SAX
Proteins
5990-8777EN
Technical Overview
Purity Assessment Following Affinity Separation
PL-SAX
Proteins
5990-8436EN
Technical Overview
Agilent PL-SCX Cation-Exchange Media
for Large Biomolecules
PL-SCX
Proteins
5990-8665EN
Technical Overview
(Continued)
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483
COLUMNS FOR BIOMOLECULE SEPARATIONS
BioHPLC Columns Literature
Title
Column/Product
Application
Publication Number Publication Type
Poroshell 300SB-C18 for Fast, High Protein Separation
Poroshell 300
Proteins
5988-2100ENUS
Progressive Denaturation of Globular Proteins in Urea
ProSEC 300S
Proteins
5990-8141EN
Application Note
ProSEC 300S Columns Protein Characterization Columns
ProSEC 300S
Proteins
5990-7468EN
Flyer
Static Light Scattering Analysis of Globular Proteins
with Agilent ProSEC 300S Columns
ProSEC 300S
Proteins
5990-7939EN
Application Note
Confidently Separate and Characterize Biomolecules
with Agilent BioHPLC Columns
Bio SEC, Bio IEX, Bio MAb
Proteins
5990-5195EN
Brochure
Increase your Productivity
with Agilent ZORBAX RRHD 300Å 1.8 µm Columns
ZORBAX RRHD 300SB-C18, Proteins, Peptides
C8
5990-8124EN
Flyer
High Purity, High Recovery, High Throughput –
Agilent Technologies Offers Two New Lines of
Preparative HPLC Columns
Agilent Prep HT
Purification/Prep
5989-2350EN
Brochure
Biomolecule Purification – Purification Columns
and Media for Peptides, Oligonucleotides, and Proteins
PLRP-S, PL-SAX, PL-SCX
Purification/Prep
5990-8335EN
Brochure
The Influence of Silica Pore Size on Efficiency, Resolution
and Loading in Reversed-Phase HPLC
SepTech
Purification/Prep
5990-8298EN
Application Note
Analysis of Protein Primary Structure when using
Wide-Pore sub-2-µm Particles and UHPLC
ZORBAX RRHD 300SB-C18
Purification/Prep
5990-8830EN
Technical Poster
Polyethylene Glycol/Oxide Standards and the
Calibration of Agilent ProSEC 300S Columns
ProSEC 300S
SEC
5990-8147EN
Application Note
TIPS & TOOLS
For the latest application notes
and new product information, go to
www.agilent.com/chem/library
484
Brochure
COLUMNS FOR BIOMOLECULE SEPARATIONS
Citations
ZORBAX 300 Citations
Magherini, F. et al. (2009) Different carbon sources affect lifespan and
protein redox state during Saccharomyces cerevisiae chronological
ageing. Cellular and Molecular Life Sciences, 66(5): 933-947
Portelius, E. et al. (2007) Characterization of Amyloid b Peptides in
Cerebrospinal Fluid by an Automated Immunoprecipitation Procedure
Followed by Mass Spectrometry. Journal of Proteome Research, 6
(11): 4433-4439
Montes-Bayon, M. et al. (2006) Direct comparison of capillary
electrophoresis and capillary liquid chromatography hyphenated to
collision-cell inductively coupled plasma mass spectrometry for the
investigation of Cd-, Cu- and Zn-containing metalloproteins. Journal of
Chromatography A, 1114(1): 138-144
Zahariev, S. et al. (2006) Synthesis of ‘difficult’ peptides free of
aspartimide and related products, using peptoid methodology.
Tetrahedron Letters, 47(25): 4121-4124
Cao H. & Xu S-Y. (2008) Purification and characterization of type II
collagen from chick sternal cartilage. Food Chemistry, 108(2): 439-445
Chen, X. et al. (2010) Quantitative Proteomics Analysis of Cell Cycleregulated Golgi Disassembly and Reassembly. The Journal of Biological
Chemistry, 285: 7197-7207
Trusch, M. et al. (2010) Application of displacement chromatography
for the analysis of a lipid raft proteome. Journal of Chromatography B,
878(3-4): 309-314
Amoresano, A. et al. (2008) A Rapid and Selective Mass Spectrometric
Method for the Identification of Nitrated Proteins. Methods in
Molecular Biology, 477(I): 15-29
Kohler, M. et al. (2009) Identification of Human Pituitary Growth
Hormone Variants by Mass Spectrometry. Journal of Proteome
Research, 8(2): 1071-1076
Berg, M. et al. (2006) Reproducibility of LC-MS-based protein
identification. Journal of Experimental Botany, 57(7): 1509-1514
Kodali, VP., Das, S. & Sen, R. (2009) An exopolysaccharide from a
probiotic: Biosynthesis dynamics, composition and emulsifying activity.
Food Research International, 42(5-6): 695-699
Esteban-Fernández, D. et al. (2008) Atomic (HPLC-ICP-MS) and
molecular mass spectrometry (ESI-Q-TOF) to study cis-platin
interactions with serum proteins. Journal of Analytical Atomic
Spectrometry, 23: 378-384
Loeber, R. et al. (2008) Cross-Linking of the DNA Repair Protein O6Alkylguanine DNA Alkyltransferase to DNA in the Presence of
Antitumor Nitrogen Mustards. Chemical Research in Toxicology, 21(4):
787-795
Everberg, H. et al. (2006) Aqueous Two-Phase Partitioning for
Proteomic Monitoring of Cell Surface Biomarkers in Human Peripheral
Blood Mononuclear Cells. Journal of Proteome Research, 5(5): 11681175
Millette, M. et al. (2008) Purification and identification of the pediocin
produced by Pediococcus acidilactici MM33, a new human intestinal
strain. Journal of Applied Microbiology, 104(1): 269-275
Chen, X. et al. (2009) Quantitative Organellar Proteomics Analysis of
Rough Endoplasmic Reticulum from Normal and Acute Pancreatitis Rat
Pancreas. Journal of Proteome Research, 9(2): 885-896
Portelius, E. et al. (2010) Identification of novel N-terminal fragments
of amyloid precursor protein in cerebrospinal fluid. Experimental
Neurology, 223(2): 351-358
Ahrends, R. et al. (2009) Metal-Coded Affinity Tag Labeling: A
Demonstration of Analytical Robustness and Suitability for Biological
Applications. Analytical Chemistry, 81(6): 2176-2184
Ameri, M., Daddona, P. & Maa, YF. (2009) Demonstrated Solid-State
Stability of Parathyroid Hormone PTH(1–34) Coated on a Novel
Transdermal Microprojection Delivery System. Pharmaceutical
Research, 26(11): 2454-2463
Bíliková, K. et al. (2009) Towards functional proteomics of minority
component of honeybee royal jelly: The effect of post-translational
modifications on the antimicrobial activity of apalbumin2. Proteomics,
9(8): 2131-2138
Gianazza, E. et al. (2010) Different expression of Fibrinopeptide A and
related fragments in serum of type 1 diabetic patients with
nephropathy. Journal of Proteomics, 73(3): 593-601
Schwab, K. et al. (2011) Adaptation of proteomic techniques for the
identification and characterization of protein species from murine heart.
Amino Acids, 41(2): 401-414
Yuan, W. et al. (2008) Two New Holostan-Type Triterpene Glycosides
from the Sea Cucumber Bohadschia marmorata JAEGER. Chemical &
Pharmaceutical Bulletin, 56(8): 1207
Perreault, A. et al. (2009) A Methyltransferase-independent Function
for Rmt3 in Ribosomal Subunit Homeostasis. The Journal of Biological
Chemistry, 284: 15026-15037
WWW.AGILENT.COM/CHEM/LC
485
COLUMNS FOR BIOMOLECULE SEPARATIONS
Poroshell 300 Citations
PLRP-S Citations
Matilainen, L. et al. (2008) The effect of cyclodextrins on chemical and
physical stability of glucagon and characterization of glucagon/g-CD
inclusion complexes. Journal of Pharmaceutical Sciences, 97(7):
2720-2729
Nakagawa, M., Tojo, H. & Fujii, S. (2011) A Glycan of Y-Factor from
Dictyostelium discoideum Contains a Bisecting-GlcNAc, an
Intersecting-GlcNAc, and a Core a-1,6-Fucose. Bioscience,
Biotechnology, and Biochemistry, 75(10):1964-1970
Astarita, G., Ahmed, F. & Piomelli, D. (2008) Identification of
biosynthetic precursors for the endocannabinoid anandamide in the rat
brain. The Journal of Lipid Research, 49: 48-57
Della Donna, L. et al. (2011) Mass spectrometric measurements of the
apolipoproteins of bovine (Bos taurus) HDL. Comparative Biochemistry
and Physiology Part D: Genomics and Proteomics
Matilainen, L. et al. (2008) The stability and dissolution properties of
solid glucagon/g-cyclodextrin powder. European Journal of
Pharmaceutical Sciences, 36(4-5): 412-420
Vondenhoff, GHM. et al. (2011) Characterization of Peptide Chain
Length and Constituency Requirements for YejABEF-Mediated Uptake
of Microcin C Analogues. The Journal of Bacteriology, 193(14):
3618-3623
Wang, Y. et al. (2009) Comparison of Methods for Extracting Kafirin
Proteins from Sorghum Distillers Dried Grains with Solubles. Journal of
Agricultural & Food Chemistry, 57(18): 8366-8372
Guerrera, IC. et al. (2009) A Novel Lipidomic Strategy Reveals Plasma
Phospholipid Signatures Associated with Respiratory Disease Severity
in Cystic Fibrosis Patients. PLoS ONE, 4(11): e7735
Yung, KM. et al. (2011) An amyloid b42-dependent deficit in
anandamide mobilization is associated with cognitive dysfunction in
Alzheimer's disease. Neurobiology of Aging
Reid, CQ. et al. (2010) Rapid whole monoclonal antibody analysis by
mass spectrometry: An Ultra scale-down study of the effect of
harvesting by centrifugation on the post-translational modification
profile. Biotechnology and Bioengineering, 107(1): 85-95
Heinig, K. & Wirz, T. (2009) Determination of Taspoglutide in Human
and Animal Plasma Using Liquid Chromatography − Tandem Mass
Spectrometry with Orthogonal Column-Switching. Analytical Chemistry,
81(10): 3705-3713
Rossi, EA. et al. (2010) A Bispecific Antibody-IFNa2b Immunocytokine
Targeting CD20 and HLA-DR Is Highly Toxic to Human Lymphoma and
Multiple Myeloma Cells. Cancer Research, 70: 7600
Murphy, MP. et al. (2010) Changes in Cognition and Amyloid-b
Processing with Long Term Cholesterol Reduction using Atorvastatin in
Aged Dogs. Journal of Alzheimer's Disease, 22(1): 135-150
Šotkovský, P. et al. (2011) A new approach to the isolation and
characterization of wheat flour allergens. Clinical & Experimental
Allergy, 41(7): 1031-1043
Laabs, E. et al. (2011) Optical Imaging of CCK2/Gastrin ReceptorPositive Tumors With a Minigastrin Near-Infrared Probe. Investigative
Radiology, 46(3): 196-201
Rickert, KW. et al. (2011) Structural Basis for Selective Small Molecule
Kinase Inhibition of Activated c-Met. The Journal of Biological
Chemistry, 286: 11218-11225
Stuknyte, M. et al. (2011) Potential immunomodulatory activity of
bovine casein hydrolysates produced after digestion with proteinases
of lactic acid bacteria. International Dairy Journal, 21(10): 63-769
Nakano, E. et al. (2011) Riboflavin Depletion Impairs Cell Proliferation
in Adult Human Duodenum: Identification of Potential Effectors.
Digestive Diseases and Sciences, 56(4): 1007-1019
Webb, K. et al. (2011) The ribosomal L1 protuberance in yeast is
methylated on a lysine residue catalyzed by a seven beta-strand
methyltransferase. The Journal of Biological Chemistry, M110. 200410
Uehata, T. et al. (2011) Serum hepcidin-25 levels and anemia
in non-dialysis chronic kidney disease patients: a cross-sectional study.
Nephrology Dialysis Transplantation, 27(3): 1076-1083
Lohman, G, Chen, L. & Evans Jr, T. (2011) Kinetic characterization
of single strand break ligation in duplex DNA by T4 DNA Ligase.
The Journal of Biological Chemistry, 286: 44187-44196
(Continued)
486
COLUMNS FOR BIOMOLECULE SEPARATIONS
PLRP-S Citations
PL-SAX Citations
Hudak, J., Yu, H. & Bertozzi, C. (2011) Protein Glycoengineering
Enabled by the Versatile Synthesis of Aminooxy Glycans and the
Genetically Encoded Aldehyde Tag. Journal of the American Chemical
Society, 133(40): 16127-16135
Pratto, F. et al. (2008) Streptococcus pyogenes pSM19035 requires
dynamic assembly of ATP-bound ParA and ParB on parS DNA during
plasmid segregation. Nucleic Acids Research, 3 (11): 3676-3689
Schneider, N. et al. (20111) Prevalence and stability of lysozyme in
cheese. Food Chemistry, 128(1): 145-151
Sendovski, M. et al. (2010) Crystallization and preliminary X-ray
crystallographic analysis of a bacterial tyrosinase from Bacillus
megaterium. Acta Crystallographica, 66(9): 1101-1103
Yan, B. & Boyd, D. (2011) Breaking the Light and Heavy Chain Linkage
of Human Immunoglobulin G1 (IgG1) by Radical Reactions.
The Journal of Biological Chemistry, 286: 24674-24684
Bunger, MK. et al. (2008) Automated Proteomics of E. coli via
Top-Down Electron-Transfer Dissociation Mass Spectrometry. Analytical
Chemistry, 80(5): 1459-1467
Landau, M. et al. (2011) Towards a Pharmacophore for Amyloid. PLoS
Biology, 9(6): e1001080
Vantourout, P. et al. (2009) Specific Requirements for Vg9Vd2 T Cell
Stimulation by a Natural Adenylated Phosphoantigen. The Journal of
Immunology, 183(6): 3848-3857
Kerkaert, B. et al. (2011) Use of lysozyme as an indicator of protein
cross-contact in fresh-cut vegetables via wash waters. Food Research
International, 45(1): 39-44
Schneider, N., Werkmeister, K. & Pischetsrieder, M. (2011) Analysis of
nisin A, nisin Z and their degradation products by LCMS/MS. Food
Chemistry, 127(2): 847-854
Quenee, L. et al. (2011) Prevention of pneumonic plague in mice, rats,
guinea pigs and non-human primates with clinical grade rV10, rV10-2
or F1-V vaccines. Vaccine, 29(38): 6572-6583
Scaboo, AM. et al. (2009) Confirmation of Molecular Markers
and Agronomic Traits Associated with Seed Phytate Content in
Two Soybean RIL Populations. Crop Science, 49(2): 426-432
PL-SCX Citations
Zhang, W. & Czupryn, M. (2003) Analysis of isoaspartate in a
recombinant monoclonal antibody and its charge isoforms. Journal of
Pharmaceutical and Biomedical Analysis, 30(5): 1479-1490
Collinge, J. et al. (2005) Differential Proteomics via Probabilistic
Peptide Identification Scores. Analytical Chemistry, 77(2): 596-606
Schönleben, S. et al. (2007) Proteome analysis of Apis mellifera royal
jelly. Analytical and Bioanalytical Chemistry, 389(4): 1087-1093
Lohaus, C. et al. (2007) Multidimensional Chromatography: a Powerful
Tool for the Analysis of Membrane Proteins in Mouse Brain. Journal of
Proteome Research, 6(1): 105-113
Zahedi, RP. et al. (2007) Phosphoproteome of Resting Human
Platelets. Journal of Proteome Research, 7(2): 526-534
Boehm, A. et al. (2007) Precise protein quantification based on peptide
quantification using iTRAQ™. BMC Bioinformatics, 8: 214
Heller, M. et al. (2003) Trypsin catalyzed 16O-to-18O exchange for
comparative proteomics: tandem mass spectrometry comparison using
MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers. Journal of
the American Society for Mass Spectrometry, 14(7): 704-718
WWW.AGILENT.COM/CHEM/LC
487
GPC/SEC ColumnS and STandardS
GPC/SEC Columns and Standards
• A full portfolio of products for analysis of synthetic and natural polymers
• A wide selection of polymer standards to cover the range of applications in organic and
water based solvents
• PL aquagel-OH-series, for aqueous SEC separations, and PLgel, for organic polymer applications,
are available in mixed and individual pore sizes across a range of particle sizes, to cover the full
spectrum of molecular weights (MW)
• Prep scale columns are available, along with narrow bore columns and columns designed for
specific applications
Gel permeation chromatography (GPC) and size exclusion chromatography (SEC) are names applied to the
most popular technique for measuring the molecular weight distribution (MWD) of natural and synthetic
polymers, a property that affects many of the physical parameters of materials such as strength, toughness
and chemical resistance. GPC and SEC are liquid chromatographic techniques that separate individual
polymer chains on the basis of their size in solution and not on their chemistry. Gel permeation
chromatography (GPC) is the name used to describe the analysis of polymers in organic solvents, such as
tetrahydrofuran. Size exclusion chromatography (SEC) is the name used to describe the analysis of polymers
in water and water-based solvents, such as buffer solutions. GPC/SEC is the only established method for
obtaining a comprehensive understanding of a polymer’s molecular weight distribution.
TiPS & ToolS
For information on SEC columns
for proteins, turn to pages 416-417.
488
GPC/SEC ColumnS and STandardS
Table of Contents
LC and LC/MS Columns for GPC/SEC
GPC/SEC Columns ............................................................ 488
ResiPore ..............................................................................518
PLgel GPC............................................................................496
MesoPore............................................................................520
PLgel MIXED.......................................................................498
OligoPore.............................................................................521
PLgel MIXED-LS.................................................................502
PL aquagel-OH SEC ...........................................................523
PLgel MiniMIX....................................................................504
PL aquagel-OH SEC Analytical ........................................525
PLgel Individual Pore Size ................................................505
PL aquagel-OH SEC Preparative......................................528
PLgel Preparative...............................................................506
GPC Column Accessories.................................................529
EnviroPrep ...........................................................................507
Polymer Standards for GPC/SEC .................................... 530
PLgel Olexis ........................................................................508
EasiVial ................................................................................532
PL HFIPgel...........................................................................509
EasiCal .................................................................................536
PL Rapide ............................................................................510
Polystyrene .........................................................................537
PolarGel ...............................................................................512
Polymethylmethacrylate ...................................................539
PlusPore ..............................................................................514
Polyethylene Glycol/Oxide...............................................541
PolyPore ..............................................................................516
Polysaccharides .................................................................543
Polyacrylic Acid..................................................................545
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489
GPC/SEC ColumnS and STandardS
GPC/SEC Columns
The key to successful GPC/SEC separations is the correct choice of columns. The comprehensive range of
Agilent products for GPC/SEC has been designed to cover virtually all polymer analysis application areas,
and to make selection for the correct column, solvent, and calibration standard fast and reliable.
Agilent’s PLgel GPC series of columns are for polymer applications using organic solvents. PLgel is a highly
cross-linked, porous polystyrene/divinylbenzene matrix, which is recognized as a market leader in GPC
column technology. PLgel materials have high pore volume and high-efficiency to maximize resolution.
Their unequalled solvent compatability makes for easy transfer between polar and non-polar eluents,
and outstanding physical rigidity provides extended lifetimes that maximize downtime. For more
information and full ordering details, see pages 496-497.
Agilent’s PL aquagel-OH series of columns provide a chemically and physically stable matrix for reliable
aqueous SEC separations. The columns are packed with macroporous copolymer beads with an extremely
hydrophilic polyhydroxyl functionality. The “neutral” surface and the capability to operate across a wide
range of eluent conditions provide for high performance analyses of compounds with neutral, ionic, and
hydrophobic moieties, alone or in combination. PL aquagel-OH is available for analytical and preparative
applications. For more information and full ordering details, see page 523.
Polymer standards for GPC/SEC
Agilent manufactures the highest quality polymer standards with extremely narrow polydispersity and the
widest molecular weight range commercially available. These quality polymer standards are supplied with
extensive characterization data utilizing a variety of independent techniques (e.g. light scattering and
viscometry) and high performance GPC to verify polydispersity and assign the peak molecular weight (Mp).
EasiVial – for organic and aqueous calibration. EasiVial is the fastest and most convenient method to deliver
an accurate 12-point column calibration. EasiVial eliminates tedious weight procedures for improved
calibration accuracy and reduces solvent dispensing to limit risks associated with handling solvents.
EasiCal – for organic solvents. EasiCal packs are pre-prepared for a no-fuss process. Two different combs,
each with ten detachable spatulas, support a mixture of five polymer standards. The cost-effective format is
designed to save money.
Individual standards and kits – an extensive range of polymer standard kits of different chemistries designed
to match specific column sets are available, as well as individual standards in various pack sizes. For more
details about Agilent’s calibration standards for GPC/SEC, see page 530.
490
GPC/SEC ColumnS and STandardS
how GPC/SEC works:
• Polymer molecules dissolve in solution to form spherical coils with size dependent on molecular weight
• The polymer coils are introduced to eluent flowing through the column
• Columns are packed with insoluble porous beads with well-defined pore structure
• The size of pores is similar to that of the polymer coils
• The polymer coils diffuse in and out of the pores
• Result is elution based on size – large coils first, smaller coils last
• Size separation converted to molecular weight separation by use of a calibration curve constructed
by the use of polymer standards
Key
Smaller coils can access many pores
Larger coils can access few pores
Very large coils access very few pores
Mechanisms of GPC and SEC
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491
GPC/SEC ColumnS and STandardS
recommendations for setting up a GPC/SEC system
The following questions will help you find the recommended columns and standards for any given application, as well as system parameters such as
injection volumes.
Choosing an eluent for GPC/SEC
Question
answer
1. What is the sample soluble in?
Water or water buffer
Agilent PL aquagel-OH Best choice for water-based applications
with up to 50% methanol
but cannot accommodate organics apart from
methanol up to 50%
Many polymers are only soluble in a small
number of solvents. This is the key question
when developing methods for analyzing polymers.
The solvents mentioned here are all common
eluents employed in GPC/SEC.
Typical organic solvent
such as THF,
chloroform, toluene
TiPS & ToolS
More information on GPC/SEC
instrumentation and systems is a click
away. We have a variety of application
notes, data sheets and brochures
available from Agilent for free.
To learn more, visit
www.agilent.com/chem/gpc
492
recommendation
Agilent PLgel or
Agilent PlusPore
Organic/water mixtures or Agilent PolarGel
polar organics such as,
DMF, NMP
Comments
PLgel are the workhorse columns, PlusPore columns
are an alternative
PolarGel is a smaller column range than PLgel or
PL aquagel-OH columns but is suited to mixtures
of organics and water
GPC/SEC ColumnS and STandardS
Choosing a column for GPC/SEC
Columns shown in bold are the best initial choice
Question
answer
2. What is the expected
molecular weight?
High
Aqueous solvents
(up to several millions) Pl aquagel-oh miXEd-h 8 µm
or combination of
PL aquagel-OH 40 and 60 15 µm
It may seem strange to
ask this question, but in
GPC/SEC the resolution of
a column is related to the
resolving range. Knowing
something of the expected
molecular weight of a
sample helps to choose the
best column that will give
optimum results.
Intermediate (up to
hundreds of
thousands)
Low (up to tens of
thousands)
Very low (a few
thousand)
Unknown
recommendation
Comments
The 15 µm column combination is best only where
sample viscosity is very high, otherwise 8 µm columns
give greater resolution
Organic solvents
Plgel 10 µm miXEd-B or
PLgel 20 µm MIXED-A
The PLgel MIXED-A column resolves higher than the PLgel
MIXED-B but at lower efficiency due to larger particle size
Mixed solvents
PolarGel
No PolarGel column available for this molecular weight range.
Contact your local GPC/SEC expert for advice
Aqueous solvents
Pl aquagel-oh miXEd-m 8 µm
A wide-ranging column that covers most
water-soluble polymers
Organic solvents
Plgel 5 µm miXEd-C or
PLgel 5 µm MIXED-D, PolyPore
or ResiPore
The PLgel columns are the most widely applicable for
the majority of applications; PolyPore and ResiPore columns
are alternatives
Mixed solvents
PolarGel-m
Covers most applications
Aqueous solvents
Combination of Pl aquagel-oh 40
and Pl aquagel-oh 30 8 µm
These two columns in a combined set cover the low end
of the molecular weight range
Organic solvents
Plgel 3 µm miXEd-E
or MesoPore
The PLgel column provides high resolution and is designed
for low molecular weight applications; the MesoPore column
is an alternative
Mixed solvents
PolarGel-l
For low molecular weight applications
Aqueous solvents
Pl aquagel-oh 20 5 µm
This high-performance column gives high resolution at low
molecular weight
Organic solvents
oligoPore or PLgel 3 µm 100Å
The OligoPore column is less prone to dispersion than the
PLgel column, but both work well
Mixed solvents
Plgel
No PolarGel column covers this range so use PLgel columns
as alternatives
Aqueous solvents
Pl aquagel-oh miXEd-m 8 µm
Covers the molecular weight ranges of most polymer samples
Organic solvents
Plgel 5 µm miXEd-C or PolyPore
This PLgel column is the most widely applicable for the
majority of applications
Mixed solvents
PolarGel-m
Covers the majority of applications
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493
GPC/SEC ColumnS and STandardS
Setting up the GPC/SEC system
Question
answer
recommendation
Comments
3. How many columns to use?
Depends on the particle
size of the columns
Particle size 20 µm use 4 columns
Increased number of columns required
for large particle sizes to make up for
low efficiencies
The greater the particle size of the
media in the column (which is
dependent on the expected
molecular weight of the samples), the
lower the resolution and the more
columns are required to maintain the
quality of the results. For higher
molecular weight samples, larger
particles are necessary to reduce the
danger of shear degradation of
samples during analysis.
Particle size 13 µm use 3 columns
Particle size 10 µm use 3 columns
Particle size 8 µm use 2 columns
Particle size 5 µm use 2 columns
Particle size 3 µm use 2 columns
Question
answer
recommendation
Comments
4. What size injection volume?
Depends on the particle
size of the columns
Particle size 20 µm use 200 µL injection
Particle size 13 µm use 200 µL injection
Smaller particle sizes require smaller
loops to minimize band broadening
The injection volume required is
dependent on the particle size of the
column – smaller particles need
lower injection volumes to minimize
dead volume. Larger injection
volumes allow the introduction of
high molecular weight samples at
lower concentrations, reducing
viscosity and ensuring a quality
chromatogram is obtained.
Particle size 10 µm use 200 µL injection
Particle size 5 µm use 100 to 200 µL injection
Particle size 3 µm use 20 µL injection
what standards should i use?
Standards shown in bold are the best initial choice
Question
answer
recommendation
5. What is the eluent?
Water or water buffer with up to 50%
methanol
Polyethylene glycol (PEG)/oxide These standards perform in all water(PEo) or polysaccharides (SAC)
based systems, PEG/PEO in
convenient Agilent EasiVial format
Comments
Standards are polymers, so the
choice of standard mainly reflects
solubility in the chosen eluents.
Typical organic solvent such as THF,
chloroform, toluene
Polystyrene (PS) or
polymethylmethacrylate (PMMA)
Polystyrene is the most commonly
used standard in convenient
EasiVial format
Organic/water mixtures or polar
organics such as DMF, NMP
Polyethylene glycol/oxide or
polymethylmethacrylate
Polar standards perform well
(Continued)
494
GPC/SEC ColumnS and STandardS
what standards should i use?
Question
answer
6. What format of standards
are recommended?
For the quickest and simplest approach Easiest option – EasiVial or EasiCal
where accurate concentrations
are not required
recommendation
Comments
Simple to use, EasiVial preferred
before EasiCal because of the wider
choice of polymer types
Different formats of standards
are available depending on
customer preference.
If accurate concentrations are required Accurate concentrations required –
EasiVial or individual standards
Both formats allow accurate sample
concentrations, EasiVials are simpler
to use
Typical polymer molecular weights
If you are unsure of the molecular weight of your sample, the table below shows some approximate molecular weight ranges for common polymers, which will
help you select the right column for your application.
Polymer Type
Polymers from free radical synthesis
Polymers from ionic synthesis
Polymers from addition synthesis
Polymers from controlled radical polymerization
High (up to several million)
Intermediate (up to hundreds of thousands)
Intermediate (up to hundreds of thousands)
Low (up to tens of thousands)
Intermediate (up to hundreds of thousands)
Low (up to tens of thousands)
Low (up to tens of thousands)
Very low (a few thousand)
Intermediate (up to hundreds of thousands)
Polyolefins
High (up to several million)
Intermediate (up to hundreds of thousands)
Acrylates
High (up to several million)
Small molecule additives
Pre-polymers
Very low (a few thousand)
Low (up to tens of thousands)
Very low (a few thousand)
Low (up to tens of thousands)
Resins
Very low (a few thousand)
Natural biopolymers such as polysaccharides
Intermediate (up to hundreds of thousands)
High (up to several million)
Intermediate (up to hundreds of thousands)
Rubbers
High (up to several million)
Biodegradable polymers
1 Polydispersity
Typical molecular weight of polymer
Intermediate (up to hundreds of thousands)
Low (up to tens of thousands)
Typical polydispersity1
of polymer
~2
~ 1.01
~2
~ 1.1 to 1.5
~ 2 to 200
~2
1
~ 2 to 10
~ 2 to 10
~ 2 to 10
~ 2 to 10
~ 1.1 to 2
is a measure of the distribution of molecular mass of a polymer. Polydispersity index (PDI) = Mw/Mn.
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495
GPC/SEC ColumnS and STandardS
Organic GPC
Plgel GPC Columns
• Robust performance under the most exacting conditions
• Temperature stability up to 220 °C
• Solvent compatibility allows easy and rapid transfer between solvents of varying polarity
PLgel materials have high pore volume and high efficiency to maximize resolution. Their unequalled solvent
compatibility makes for easy transfer between polar and non-polar eluents, and outstanding physical rigidity
provides extended lifetimes that minimize downtime.
The key to successful GPC separations is the correct choice of columns. The comprehensive range of PLgel
products has been designed to cover virtually all organic solvent-based polymer analysis application areas,
and to make selection of the correct column, solvent, and calibration standard fast and reliable.
PLgel is a highly cross-linked, porous polystyrene/divinylbenzene matrix, which is recognized as a
market leader in GPC column technology. PLgel is manufactured to ISO 9001:2000 and benefits
from comprehensive QC/QA for total reproducibility, batch-to-batch and column-to-column.
496
GPC/SEC ColumnS and STandardS
Solvent Compatibility
PLgel columns are routinely supplied in ethyl benzene* but you can easily and rapidly transfer between
solvents of varying polarity. In organic GPC, sample to column interaction may occur occasionally and eluent
modification can be used to eliminate these effects. PLgel columns are the ideal choice for such analyses, as
they easily tolerate eluents in the pH range 1-14, as well as up to 10% water in a miscible organic solvent.
Plgel is compatible with all of these solvents
Solvent Polarity
Solvent
6.0
Perfluoroalkane
7.3
Hexane
8.2
Cyclohexane
8.9
Toluene
9.1
Ethyl acetate
9.1
Tetrahydrofuran (THF)
9.3
Chloroform
9.3
Methyl ethyl ketone (MEK)
9.7
Dichloromethane
9.8
Dichloroethene
9.9
Acetone
10.0
o-Dichlorobenzene (o-DCB)
10.0
Trichlorobenzene (TCB)
10.2
m-Cresol
10.2
o-Chlorophenol (o-CP)
10.7
Pyridine
10.8
Dimethyl acetamide (DMAc)
11.3
n-Methyl pyrolidone (NMP)
12.0
Dimethyl sulfoxide (DMSO)
12.1
Dimethyl formamide (DMF)
*We also provide a custom packing service in which columns can be shipped in specific solvents to provide extra
convenience to our customers.
Plgel Frit Porosity
media Type
PLgel 3 µm
Porosity (µm)
2
PLgel 5 µm
2
PLgel 10 µm
5
PLgel 20 µm
10
For Plgel column accessories ordering information please see page 529
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497
GPC/SEC ColumnS and STandardS
Plgel miXEd Columns
The PLgel MIXED range greatly simplifies column selection for easy decision making. By using these mixed columns, you can eliminate mismatched column
sets and spurious peaks for more reliable results. Every column contains a mixture of individual pore size materials, accurately blended to cover a specified
broad range of molecular weight with a linear calibration to eliminate column mismatch. Simply add extra columns for even greater resolution.
Column Specifications
498
Column
linear mw
operating range
(g/mol)
Guaranteed
Typical
Column Efficiency Pressure
maximum
Flow rate
maximum
Pressure
maximum
Temperature
PLgel MIXED-A
2,000-40,000,000
> 17,000 p/m
1 mL/min (7.5 mm id):
≈ 3 bar (44 psi) per 300 mm
0.3 mL/min (4.6 mm id):
≈ 2.4 bar (35 psi) per 250 mm
(THF @ 20 °C, TCB @ 140 °C)
7.5 mm id:
1.5 mL/min
4.6 mm id:
0.5 mL/min
150 bar
(2175 psi)
220 °C
PLgel MIXED-B
500-10,000,000
> 35,000 p/m
1 mL/min (7.5 mm id):
≈ 10 bar (145 psi) per 300 mm
0.3 mL/min (4.6 mm id):
≈ 8 bar (116 psi) per 250 mm
(THF @ 20 °C, TCB @ 140 °C)
7.5 mm id:
1.5 mL/min
4.6 mm id:
0.5 mL/min
150 bar
(2175 psi)
220 °C
PLgel MIXED-C
200-2,000,000
> 50,000 p/m
1 mL/min (7.5 mm id):
≈ 30 bar (435 psi) per 300 mm
0.3 mL/min (4.6 mm id):
≈ 24 bar (348 psi) per 250 mm
(THF @ 20 °C, TCB @ 140 °C)
7.5 mm id:
1.5 mL/min
4.6 mm id:
0.5 mL/min
150 bar
(2175 psi)
150 °C
PLgel MIXED-D
200-400,000
> 50,000 p/m
1 mL/min (7.5 mm id):
≈ 30 bar (435 psi) per 300 mm
0.3 mL/min (4.6 mm id):
≈ 24 bar (348 psi) per 250 mm
(THF @ 20 °C, TCB @ 140 °C)
7.5 mm id:
1.5 mL/min
4.6 mm id:
0.5 mL/min
150 bar
(2175 psi)
150 °C
PLgel MIXED-E
up to 30,000
7.5 x 300 mm:
> 80,000 p/m
4.6 x 250 mm:
> 70,000 p/m
1 mL/min (7.5 mm id):
≈ 50 bar (725 psi) per 300 mm
0.3 mL/min (4.6 mm id):
≈ 42 bar (609 psi) per 250 mm
(THF @ 20 °C)
7.5 mm id:
1.5 mL/min
4.6 mm id:
0.5 mL/min
180 bar
(2611 psi)
110 °C
GPC/SEC ColumnS and STandardS
Plgel miXEd Column Selection Guide
uhmw polymer distributions
PLgel MIXED-A, 20 µm
high mw polymers, demanding eluents
PLgel MIXED-B, 10 µm
mid range mw polymers, high resolution
PLgel MIXED-C, 5 µm
resins, condensation polymers
PLgel MIXED-D, 5 µm
low mw resins, prepolymers
PLgel MIXED-E, 3 µm
102
MW
107
Plgel miXEd Gel Calibration Curves
MIXED gel calibration curves are designed to be linear over a specified molecular weight range, ensuring that the same degree of resolution is achieved
across the full operating range of the column. The particle size of the packing and porosity of a particular MIXED gel column are carefully matched to the
MW range and application, thus optimizing performance and eliminating the effects of shear degradation. Resolution in GPC is controlled by the slope of
the calibration curve and the particle size of the packing material. Agilent has scientifically determined the minimum number of MIXED gel columns required
to perform accurate MWD determinations based on specific resolution (Rsp). Thus you can have complete confidence in the accuracy and precision of the
calculated data.
Key
10 M
PLgel MIXED-A, 20 µm
PLgel MIXED-B, 10 µm
Polystyrene Molecular Weight
PLgel MIXED-C, 5 µm
PLgel MIXED-D, 5 µm
PLgel MIXED-E, 3 µm
1K
4
Elution Volume (mL)
10
VLC0014
Reference: Meehan, E. (1998) Size exclusion chromatography columns from Polymer Laboratories. In:
Chi-San Wu (Ed.) Column Handbook for Size Exclusion Chromatography. Academic Press, New York, USA.
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499
GPC/SEC ColumnS and STandardS
Plgel miXEd Columns
description
Size (mm)
Part no.
PLgel 20 µm MIXED-A
7.5 x 300
PL1110-6200
PLgel 10 µm MIXED-B
7.5 x 300
PL1110-6100
PLgel 5 µm MIXED-C
7.5 x 300
PL1110-6500
PLgel 5 µm MIXED-D
7.5 x 300
PL1110-6504
PLgel 3 µm MIXED-E
7.5 x 300
PL1110-6300
Plgel miXEd Guards
Size (mm)
Starches
Column:
4 x Plgel 20 µm miXEd-a
Pl1110-6200
7.5 x 300 mm
Mobile Phase:
DMSO + 5 mM NaNO3
Flow Rate:
1.0 mL/min
Temperature:
80 °C
Detector:
RI
Polyphenylene Sulfides
500
Column:
3 x Plgel 10 µm miXEd-B
Pl1110-6100
7.5 x 300 mm
Mobile Phase:
o-Chloronaphthalene
Flow Rate:
1.0 mL/min
Temperature:
210 °C
Detector:
RI
Particle Size (µm)
Part no.
7.5 x 50
20
PL1110-1220
7.5 x 50
10
PL1110-1120
7.5 x 50
5
PL1110-1520
7.5 x 50
3
PL1110-1320
GPC/SEC ColumnS and STandardS
Plasticized PVC
Column:
3 x Plgel 5 µm miXEd-C
Pl1110-6500
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Detector:
RI
Epoxy resin
Column:
3 x Plgel 5 µm miXEd-d
Pl1110-6504
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Detector:
RI
Polyol
Column:
Plgel 3 µm miXEd-E
Pl1110-6300
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Detector:
UV, 254 nm
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501
GPC/SEC ColumnS and STandardS
Plgel miXEd-lS Columns
• Obtain an instant improvement in data quality
• No need for conditioning, saving time and solvent costs
• Maximize the potential of light scattering detectors
The PLgel MIXED-LS series is a PS/DVB packing using an innovative proprietary suspension polymerization
technique to virtually eliminate nano-particle leakage. A startling improvement is achieved immediately in
the quality of light scattering data obtained with PLgel MIXED-LS columns in place of conventional GPC
columns. The light scattering chromatograms shown here were obtained after flushing the columns for
one hour in THF at 1 mL/min. A polystyrene standard (Mp 210,000) was injected at 1 mg/mL in order
to illustrate the dramatic improvement in signal-to-noise with the PLgel MIXED-LS column.
The performance of PLgel MIXED-LS columns has been matched to PLgel 20 µm MIXED-A and PLgel 10 µm
MIXED-B columns in terms of calibration, column efficiency, wide solvent compatibility, and operating
temperature. MIXED-LS are also ideal for online viscosity detection, minimizing the risk of capillary blockage,
and can be used with regular PLgel guard columns that are packed with rigid low pore size gels with no
particle bleed.
Plgel miXEd-lS Columns
description
502
Size (mm)
PLgel 10 µm MIXED-B LS
7.5 x 300
PLgel 10 µm guard
7.5 x 50
PLgel 20 µm MIXED-A LS
7.5 x 300
PLgel 20 µm guard
7.5 x 50
linear mw
Guaranteed
operating range Efficiency
(g/mol) (PS)
(p/m)
500-10,000,000
>35,000
Part no.
PL1110-6100LS
PL1110-1120
2,000-40,000,000
>17,000
PL1110-6200LS
PL1110-1220
GPC/SEC ColumnS and STandardS
Conventional GPC column
Column:
Conventional GPC column
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Detector:
LS
60
min
70
Light scattering detection with a conventional GPC column – noise due to particulate bleed.
Plgel lS column
Column:
Plgel 10 µm miXEd-B lS
Pl1110-6100lS
7.5 x 300 mm, 10 µm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Detector:
LS
60
min
70
VLC0015
Light scattering detection with a PLgel LS column – minimal particulate bleed gives greatly
improved baseline.
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503
GPC/SEC ColumnS and STandardS
Plgel minimiX Columns
• Use about 70% less solvent and save money
• Store less solvent and increase operator safety
• High performance comparable to Agilent's conventional id columns
For reduced solvent cost and consumption, use industry standard PLgel MiniMIX mixed gel columns in
250 x 4.6 mm narrow bore dimensions. These narrow bore columns offer high performance, excellent
solvent compatibility and mechanical stability. PLgel MiniMIX columns can be used with conventional
GPC equipment.
To maintain the same linear velocity through the column, the volumetric flow rate must be reduced to
0.3 mL/min in line with the column cross sectional area, resulting in significantly lower solvent consumption.
Sample loading should also be scaled down in line with reduced column volume, and system dead volume
should be minimized to avoid excessive band broadening.
Plgel minimiX Columns
Size
(mm)
linear mw
Guaranteed
operating range Efficiency
(g/mol) (PS)
(p/m)
PLgel 20 µm MiniMIX-A
4.6 x 250
2,000-40,000,000
PLgel 20 µm MiniMIX-A guard
4.6 x 50
description
504
PLgel 10 µm MiniMIX-B
4.6 x 250
Plgel 10 µm MiniMIX-B guard
4.6 x 50
PLgel 5 µm MiniMIX-C
4.6 x 250
PLgel 5 µm MiniMIX-C guard
4.6 x 50
PLgel 5 µm MiniMIX-D
4.6 x 250
PLgel 5 µm MiniMIX-D guard
4.6 x 50
PLgel 3 µm MiniMIX-E
4.6 x 250
PLgel 3 µm MiniMIX-E guard
4.6 x 50
> 17,000
Part no.
PL1510-5200
PL1510-1200
500-10,000,000
> 35,000
PL1510-5100
PL1510-1100
200-2,000,000
> 50,000
PL1510-5500
PL1510-1500
200-400,000
> 50,000
PL1510-5504
up to 30,000
> 70,000
PL1510-5300
PL1510-1504
PL1510-1300
GPC/SEC ColumnS and STandardS
Plgel individual Pore Size Columns
• Very high efficiency improves productivity
• Choose the optimum column for a perfect match of performance and application
• Fast analysis with fewer columns saves time and money
Individual pore size GPC columns offer high resolution over a specific molecular weight range. The linear
portion of the calibration curve, where the slope is at its shallowest, defines the MW region over which
optimum resolution will be achieved.
Plgel individual Pore Size Columns
Particle
Size
(µm)
Pore Size
(Å)
linear mw
operating range
(g/mol) (PS)
Guaranteed
Efficiency
(p/m)
7.5 x 300
3
100
up to 4,000
> 100,000
PL1110-6320
7.5 x 300
5
50
up to 2,000
> 60,000
PL1110-6515
7.5 x 300
5
100
up to 4,000
> 60,000
PL1110-6520
7.5 x 300
5
500
500-30,000
> 60,000
PL1110-6525
7.5 x 300
5
103
500-60,000
> 50,000
PL1110-6530
7.5 x 300
5
104
10,000-600,000
> 50,000
PL1110-6540
7.5 x 300
5
105
60,000-2,000,000
> 50,000
PL1110-6550
7.5 x 300
10
50
up to 2,000
> 35,000
PL1110-6115
7.5 x 300
10
100
up to 4,000
> 35,000
PL1110-6120
7.5 x 300
10
500
500-30,000
> 35,000
PL1110-6125
500-60,000
> 35,000
PL1110-6130
10,000-600,000
> 35,000
PL1110-6140
Size (mm)
Part no.
7.5 x 300
10
103
7.5 x 300
10
104
7.5 x 300
10
105
60,000-2,000,000
> 35,000
PL1110-6150
7.5 x 300
10
106
600,000-10,000,000
> 35,000
PL1110-6160
PLgel Guard Column information can be found on page 500
Calibrant:
Polystyrene
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
10 M
106Å
Polystyrene Molecular Weight
Calibration curves
105Å
104Å
103Å
500Å
100Å
50Å
1K
4
Elution Volume (mL)
10
VLC0016
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505
GPC/SEC ColumnS and STandardS
Plgel Preparative Columns
• Excellent column efficiency provides optimum resolution
• High loading can isolate mg amounts for further study
• Over 10 times scale up permits efficient quantification
Preparative GPC is generally employed to fractionate polymers, isolate components in a polymer
formulation or simplify mixtures of relatively small molecules in complex matrices. Mixtures of materials
are easily separated on the basis of size, preferably in a low boiling organic solvent. They are then collected
as a series of discrete fractions and isolated by simple evaporation of the solvent.
PLgel preparative columns are packed with the same rigid, high performance media as the analytical
columns. The 10 µm particle provides high column efficiency (> 25,000 p/m) for optimum resolution and
loading characteristics. PLgel 25 mm id preparative columns offer over 10 times scale-up compared to the
7.5 mm analytical columns. The increased id and column volume permit even higher loading. With low
molecular weight materials, sample concentration can also be significantly increased, enabling production
of milligram quantities of very pure material. The actual loading is ultimately controlled by the sample and its
molecular weight.
Plgel Preparative Columns
Size (mm)
linear mw
operating range
(g/mol) (PS)
Part no.
25 x 300
10
50
up to 2,000
PL1210-6115
25 x 300
10
100
up to 4,000
PL1210-6120
25 x 300
10
500
500-30,000
PL1210-6125
25 x 300
10
103
500-60,000
PL1210-6130
25 x 300
10
104
10,000-600,000
PL1210-6140
25 x 300
10
105
60,000-2,000,000
PL1210-6150
25 x 300
10
106
600,000-10,000,000
PL1210-6160
MIXED-B
25 x 300
10
500-10,000,000
PL1210-6100
MIXED-D
25 x 300
10
200-400,000
PL1210-6104
Prep guard
25 x 25
506
Particle Size (µm)
Pore Size
(Å)
PL1210-1120
GPC/SEC ColumnS and STandardS
Columns for Special GPC/SEC Applications
EnviroPrep
• High sample loading ensures effective trace analysis
• Simple clean-up procedure saves sample preparation costs
• Optimized particle size distribution provides high resolution
EnviroPrep columns permit a simple, one stage clean-up as part of a methodology to determine pesticides
in many organic matrices. The higher molecular weight fractions such as lipids, polymers, natural resins
and dispersed high molecular weight components are easily eliminated in the GPC analysis.
Preparative GPC for soil extract clean-up is described in EPA Method 3640A using 300 x 25 mm
and 150 x 25 mm columns to give higher sample loading and fraction yields, which is particularly
useful for low levels of pollutants. Low pore size EnviroPrep columns are ideal for this method.
The columns have 10 µm particles with 100Å pore sizes for high resolution, with an exclusion limit
of 4000 g/mol. The preparative columns offer good resolution and high loading through optimization
of the particle size distribution.
EnviroPrep
Size (mm)
Part no.
21.2 x 150
PL1E10-3120EPA
25 x 150
PL1210-3120EPA
21.2 x 300
PL1E10-6120EPA
25 x 300
PL1210-6120EPA
Columns for sample clean-up
Column:
EnviroPrep
Pl1210-6120EPa
25 x 300 mm
Column:
EnviroPrep
Pl1210-3120EPa
25 x 150 mm
Mobile Phase:
DCM
Flow Rate:
10 mL/min
Detector:
UV, 254 nm
1.
2.
3.
4.
5.
1
Corn oil, 25,000 mg/L
Bis(2-ethylhexyl) phthalate, 1,000 mg/L
Methoxychlor, 200 mg/L
Perylene, 20 mg/L
Sulfur, 80 mg/L
3
2
4
0
min
5
22
VLC0017
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507
GPC/SEC ColumnS and STandardS
Plgel olexis
• Optimized design for polyolefin analysis
• High temperature capability
• High resolution with no damage from sample shear provides clean separations
PLgel Olexis is designed for the analysis of very high molecular weight polymers, specifically polyolefins.
The column resolves up to 100,000,000 g/mol (polystyrene in THF), and is packed with 13 µm particles to
optimize efficiency and resolution without the risk of sample shear degradation during analysis. The packing
of PLgel Olexis has the mechanical stability and robustness expected from a PLgel column, and so it is able
to operate up to 220 °C for the analysis of highly crystalline materials.
Plgel olexis
description
Size (mm)
Part no.
PLgel Olexis
7.5 x 300
PL1110-6400
PLgel Olexis guard
7.5 x 50
PL1110-1400
Plgel olexis reveals true modalities
across the range of polyolefins
3 x Plgel olexis
Pl1110-6400
7.5 x 300 mm
Mobile Phase:
Trichlorobenzene + 0.0125% BHT
Flow Rate:
1.0 mL/min
Injection Volume: 200 µL
Temperature:
160 °C
Detector:
PL-GPC 220 (RI)
1
dw/dlog M
Column:
0
3
Log M
7
VLC0018
508
GPC/SEC ColumnS and STandardS
Pl hFiPgel
• Optimized separation range delivers high performance with no artifacts
• Highly durable packing prolongs column lifetime
• Low operating pressure reduces system wear and unnecessary downtimes
Hexafluoroisopropanol (HFIP) is used as a solvent in GPC for the analysis of important industrial polymers
such as polyesters, polyamides and polylactide/glycolide copolymers. For greatly improved performance in
extremely polar solvents such as HFIP and trifluoroethanol, we have developed novel "multipore"
technology to produce PL HFIPgel, a PS/DVB packing featuring a monodisperse particle size, high pore
volume, and high resolution.
Using PL HFIPgel avoids issues associated with conventional packings and HFIP, such as excessive
curvature of calibration curves, dislocations/shoulders on peaks for polydisperse samples, and poor
resolution in the low MW region.
Column efficiency is guaranteed > 30,000 p/m and the columns are very durable, with a maximum
operating pressure of 145 bar (2030 psi). They are packed and tested in methanol but shipped
ready-to-use in HFIP.
PL HFIPgel columns with 7.5 mm id normally operate at 1 mL/min. However, the 4.6 mm id columns run at
0.3 mL/min, providing a 70% reduction in solvent consumption with consequent savings in the cost of
buying and disposing of solvents.
MW range for PL HFIPgel columns is 2,000,000 g/mol (PMMA in THF).
Pl hFiPgel
description
Size (mm)
Part no.
PL HFIPgel
4.6 x 250
PL1514-5900HFIP
PL HFIPgel
7.5 x 300
PL1114-6900HFIP
PL HFIPgel guard
7.5 x 50
PL1114-1900HFIP
PL HFIPgel guard
4.6 x 50
PL1514-1900HFIP
Polyamides
Column:
2 x Pl hFiPgel
Pl1114-6900hFiP
7.5 x 300 mm
Mobile Phase:
HFIP + 20mM NaTFAc
Flow Rate:
1.0 mL/min
Temperature:
40 °C
Detector:
PL-GPC 50 (RI)
nylon 66
nylon 6
PBT
5
min
22
VLC0019
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509
GPC/SEC ColumnS and STandardS
Pl rapide
• Analysis in less than ten minutes saves time
• Significantly increased sample throughput improves efficiency
• Reduced solvent consumption and disposal costs save money
• Available in L, M, and H versions for low, medium, and high molecular weights; available in F version
for flow injection analysis
Rapid GPC is an excellent tool for screening polymer MWD for trend analysis. Short PL Rapide columns
reduce analysis times while maintaining the excellent solvent compatibility and mechanical stability of all
GPC columns from Agilent.
PL Rapide columns are ideal for high speed applications such as high throughput screening, process
monitoring, or tracking changes in MW distributions, where time is the most critical factor in the analysis.
Packed with high quality gels, these columns cover the complete spectrum of molecular weights and are
available for the analysis of both organic and water soluble polymers. Key features include high pore
volume, high resolution packing materials, no special system requirements, choice of molecular weight
resolving range, wide solvent compatibility, and excellent mechanical stability.
510
GPC/SEC ColumnS and STandardS
Pl rapide
mw range
(g/mol)
Guaranteed
Efficiency (p/m) Part no.
description
Size (mm)
PL Rapide H
7.5 x 150
10 x 100
500-10,000,000
> 35,000
PL1113-3100
PL1013-2100
PL Rapide M
7.5 x 150
10 x 100
200-2,000,000
> 60,000
PL1113-3500
PL1013-2500
PL Rapide L
7.5 x 150
10 x 100
200-400,000
> 80,000
PL1113-3300
PL1013-2300
PL Rapide F
7.5 x 150
10 x 100
up to 4,500
up to 4,500
> 55,000
> 40,000
PL1113-3120
PL1013-2120
PL Rapide Aqua H
7.5 x 150
10 x 100
100-10,000,000
> 35,000
PL1149-3800
PL1049-2800
PL Rapide Aqua L
7.5 x 150
10 x 100
100-30,000
> 35,000
PL1120-3830
PL1020-2830
resin analysis by rapid GPC
Column:
Pl rapide l
Pl1013-2300
10 x 100 mm
Sample:
Epoxy resin
Mobile Phase:
THF
Flow Rate:
1.0 , 2.0 and 3.0 mL/min
Detector:
UV, 254 nm
1.0 ml/min
2.0 ml/min
3.0 ml/min
0
min
6
VLC0020
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511
GPC/SEC ColumnS and STandardS
PolarGel
• Medium polarity surface and high mechanical stability
• Operate in a wide range of solvents and solvent combinations
• Available in two resolving ranges, PolarGel-L and PolarGel-M
The PolarGel range is ideal for use with polar solvents, such as dimethyl formamide (DMF) and dimethyl
sulfoxide (DMSO), and for solvent combinations such as tetrahydrofuran with water. These eluents are very
useful in GPC/SEC to separate polar materials, such as polar resins, modified polysaccharides or complex
polar polymers that are difficult to analyze in traditional SEC solvents, such as tetrahydrofuran alone.
PolarGel-L is used for low molecular weight polar polymers and PolarGel-M for high MW polar polymers.
With polar polymers, highly polar groups can lead to non-specific interactions and secondary separation
mechanisms when using polar solvents and traditional non-polar styrene/divinylbenzene columns.
Additives and/or column conditioning are normally required to reduce these interactions. PolarGel
has no need for these interventions, and also avoids the interactions and secondary effects that produce
chromatogram distortions.
These PolarGel "mixed bed" columns have a medium polarity surface and high mechanical stability. They are
capable of operating in a wide range of solvents and solvent combinations, greatly enhancing your ability to
analyze polar polymers that are not necessarily water soluble. PolarGel is available in two resolving ranges
to meet your precise requirements.
PolarGel
description
Size (mm)
mw range
(g/mol) (PEG/PEo)
Part no.
PolarGel-L
7.5 x 300
Up to 30,000
PL1117-6830
PolarGel-L guard
7.5 x 50
PL1117-1830
PolarGel-L repair gel
7.5 x 300
PolarGel-M guard
7.5 x 50
PolarGel-M repair gel
512
PL1417-0830
PolarGel-M
Up to 2,000,000
PL1117-6800
PL1117-1800
PL1417-0800
GPC/SEC ColumnS and STandardS
Two samples of melamine resin
analyzed by PolarGel-l
Column:
2 x PolarGel-l, 300 x 7.5 mm
Pl1117-6830
Mobile Phase:
Dimethyl acetamide + 0.1% LiBr
Flow Rate:
1.0 mL/min
Injection Volume: 100 µL
Detector:
Agilent PL-GPC 220 (RI)
0
min
22
VLC0021
Excellent separation of two phenol
formaldehyde resins with PolarGel-m
Column:
2 x PolarGel-m, 300 x 7.5 mm
Pl1117-6800
Mobile Phase:
0.2% (w/v) DMF & 0.1% LiBr to reduce sample
aggregation
Flow Rate:
1.0 mL/min
Injection Volume: 100 µL
Detector:
Agilent PL-GPC 50 (RI)
0
min
22
VLC0022
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513
GPC/SEC ColumnS and STandardS
PlusPore
The PlusPore range has an increased pore volume that provides high resolution for specific applications.
The high stability media permits the use of a wide range of organic solvents with accuracy and precision
so that there is no distortion of the MW distribution shape.
The PlusPore series of columns has been specifically designed for high resolution GPC, and represents
the very latest in GPC column technology. These novel packing materials are based on the industry
standard, highly cross-linked polystyrene/divinylbenzene (PS/DVB), for the widest applicability and solvent
compatibility. Each is made using a novel polymerization process to produce particles that exhibit a specific,
controlled pore structure for optimum GPC performance. Typical applications include resins, condensation
polymers, prepolymers, and oligomers.
For high resolution polymer analysis, the PolyPore, ResiPore, MesoPore, and OligoPore columns of the
PlusPore product series exhibit a wide pore size distribution with near linear calibration curves covering an
extended molecular weight range. These so-called "multipore" structures have increased pore volume
compared to regular PS/DVB packing materials. This results in very high resolution GPC columns designed
for specific application areas. The highly cross-linked porous particles provide excellent chemical and
physical stability and permit easy transfer across the full range of organic solvents with little change in the
shape of the calibration curve or the efficiency of the columns. As this multipore column technology does
not require the combination of individual pore size packing materials, the result is high accuracy and
precision without any artifacts in the shape of the molecular weight distribution.
514
GPC/SEC ColumnS and STandardS
PlusPore calibration curves
Key
6.5
PolyPore
ResiPore
MesoPore
Log MW
OligoPore
2
Retention time/min
4
10
VLC0023
PlusPore Selection Guide
Column
mw range
(g/mol) (PS)
nominal
Particle Size (µm)
Typical Efficiency
(p/m)
recommended Calibrants
Frit Porosity (µm)
PolyPore
200-2,000,000
5
> 60,000
EasiCal PS-1or EasiVial PS-H
2
ResiPore
200-400,000
3
> 80,000
EasiCal PS-2 or EasiVial PS-M
2
MesoPore
up to 25,000
3
> 80,000
Polystyrene S-L-10 Kit
2
OligoPore
up to 4,500
6
> 55,000
Polystyrene S-L2-10 Kit
2
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515
GPC/SEC ColumnS and STandardS
PolyPore
• Routine polymer analysis with very high resolution
• Wide operating range simplifies column choice
• Low particle size extracts maximum information from the analyte
PolyPore columns have been specifically developed to give unrivaled resolution for the analysis of polymers
with broad molecular weight distributions. With a wide operating range covering many decades of
molecular weight, PolyPore columns combine a 5 µm particle size with extremely high pore volume to give
the highest possible resolution, ensuring the most detailed information possible from your analysis.
PolyPore
description
516
Size (mm)
Part no.
PolyPore
7.5 x 300
PL1113-6500
PolyPore guard
7.5 x 50
PL1113-1500
GPC/SEC ColumnS and STandardS
Comparison of PolyPore with conventional
individual pore size GPC columns
Column a:
2 x PolyPore
Pl1113-6500
7.5 x 300 mm
Column B:
Plgel 103Å
7.5 x 300 mm, 5 µm
Column C:
Plgel 105Å
7.5 x 300 mm, 5 µm
Sample:
High MW Resin
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
individual Pore Size Columns (B & C)
Injection Volume 100 µL
Detector:
Agilent PL-GPC 50 (RI)
PolyPore (a)
5
Retention time/min
20
VLC0024
Polymethylmethacrylate in dmF
Column:
2 x PolyPore
Pl1113-6500
7.5 x 300 mm
Sample:
Commercial PMMA
Mobile Phase:
DMF + 0.1% LiBr
Flow Rate:
1.0 mL/min
Temperature:
80 °C
Injection Volume: 100 µL
Detector:
Agilent PL-GPC 50 (RI)
0
Retention time/min
24
VLC0025
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517
GPC/SEC ColumnS and STandardS
resiPore
• Efficient separation of complex molecular weight distributions
• Reveals oligomer content to provide a true representation of the sample
• High pore volume extracts maximum information from the analyte
ResiPore columns are the ideal choice for the analysis of resins and condensation polymers with complex
molecular weight distributions that include oligomer content. By combining a 3 µm particle size and high
pore volume, high efficiency ResiPore columns offer maximum resolution of these intermediate molecular
weight polymers.
resiPore
description
518
Size (mm)
Part no.
ResiPore
7.5 x 300
PL1113-6300
ResiPore guard
7.5 x 50
PL1113-1300
GPC/SEC ColumnS and STandardS
alkyd resin
Column:
2 x resiPore
Pl1113-6300
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Injection Volume: 20 µL
Detector:
UV, 254 nm
5
Retention time/min
20
VLC0026
Polyester
Column:
2 x resiPore
Pl1113-6300
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Injection Volume: 20 µL
Detector:
UV, 254 nm
5
Retention time/min
20
VLC0027
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519
GPC/SEC ColumnS and STandardS
mesoPore
• Full solvent compatibility with no detrimental effect on efficiency
• Low particle size extracts maximum information from the analyte
• No MWD dislocations so the distribution is an accurate representation of the sample
MesoPore columns have been specifically designed to provide optimum results in the analysis of
prepolymers, i.e. polymeric materials with a large oligomeric component. By combining a 3 µm particle size
with high pore volume, MesoPore columns give the highest resolution separations for the analysis of low
molecular weight polymers, such as prepolymers, resins, polyols, and siloxanes.
mesoPore
description
Size (mm)
Part no.
MesoPore
7.5 x 300
PL1113-6325
MesoPore guard
7.5 x 50
PL1113-1325
Polyurethanes
Column:
2 x mesoPore
Pl1113-6325
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Injection Volume: 20 µL
Detector:
Agilent PL-GPC 50 (RI)
5
Retention time/min
22
VLC0028
Polyesterimide
Column:
2 x mesoPore
Pl1113-6325
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Injection Volume: 20 µL
Detector:
Agilent PL-GPC 50 (RI)
5
Retention time/min
17
VLC0029
520
GPC/SEC ColumnS and STandardS
oligoPore
• Near linear calibration curve for best accuracy and precision
• Very stable media allows for a wide choice of solvents
• Isolation of individual fractions reveals more information from whole samples
OligoPore columns have been developed from an innovative new media that exhibits significantly increased
pore volumes compared to conventional low pore size GPC columns. The outcome is higher resolution in the
oligomeric region. The 300 x 25 mm preparative column offers high resolution at greatly increased loading
for effective isolation of individual components. Oligomer fractions collected from the OligoPore preparative
column can then be re-injected on analytical columns to check for the purity of the fractions and for
comparison with the whole sample.
oligoPore
description
Size (mm)
Part no.
OligoPore
25 x 300
PL1213-6520
OligoPore
7.5 x 300
PL1113-6520
OligoPore guard
7.5 x 50
PL1113-1320
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521
GPC/SEC ColumnS and STandardS
analysis of low molecular weight polystyrene
and oligomer fractions collected from
oligoPore preparative columns
Column:
whole polymer
2 x oligoPore
Pl1113-6520
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Detector:
UV
F9
F8
F7
F6
F5
F4
F3
F2
F1
10
Retention time/min
20
VLC0030
analytical separation of
low molecular weight polystyrene
Column:
2 x oligoPore
Pl1213-6520
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Loading:
0.2%, 100 mL
Detector:
UV
5
Retention time/min
5
Retention time/min
20
Preparative separation of
low molecular weight polystyrene
522
Column:
2 x oligoPore
Pl1213-6520
25 x 300 mm
Mobile Phase:
THF
Flow Rate:
10.0 mL/min
Loading:
2.0%, 2 mL
Detector:
UV
20
VLC0031
GPC/SEC ColumnS and STandardS
Aqueous SEC of Polymers
Pl aquagel-oh SEC
Aqueous size exclusion chromatography (SEC) is widely used for the determination of molecular weight
distributions of a variety of synthetic and naturally occurring water-soluble polymers, and separations of
oligomers and small molecules. The requirement to eliminate ionic and hydrophobic effects makes aqueous
SEC very demanding.
The PL aquagel-OH series provides a chemically and physically stable matrix for reliable aqueous SEC
separations. The columns are packed with macroporous copolymer beads with an extremely hydrophilic
polyhydroxyl functionality. The "neutral" surface and the capability to operate across a wide range of eluent
conditions provide for high performance analyses of compounds with neutral, ionic, and hydrophobic
moieties, alone or in combination. PL aquagel-OH is available for analytical and preparative applications.
Optimizing Conditions for Aqueous SEC
with PL aquagel-OH Columns
Due to the complex nature of water-soluble polymers, it is often necessary to modify the eluent in order
to avoid sample-to-sample and sample-to-column interactions which can result in poor aqueous SEC
separations. The excellent stability of the PL aquagel-OH packing material allows the eluent to be tailored
to suit the polymer, while retaining the high column efficiency. For ionic interactions, the eluent can
be modified by the addition of salt and/or the adjustment of pH. For water soluble polymers with a
hydrophobic character, only the addition of a weak organic solvent (methanol) is required to inhibit
hydrophobic interactions.
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523
GPC/SEC ColumnS and STandardS
Guide to eluent selection for Pl aquagel-oh applications
water soluble polymer
Polymer Chemistry
Suggested Eluent
Neutral
Anionic
Cationic
Pure Water
0.1-0.3 M NaNO3
pH 7-9
0.01 M NaH2PO4
0.2-0.8 M NaNO3
pH 2-7
0.01 M NaH2PO4
hydrophobic Polymers
Addition of up to 50% methanol
Pl aquagel-oh Column Selection Guide
524
Sample Type
Typical applications
recommended Column Sets
Low MW polymers and oligomers
Surfactants, oligosaccharides, PEGs,
lignosulfonates, polyacrylates
2 or 3, 30, 20
PL aquagel-OH 8 µm, or
PL aquagel-OH 20 5 µm, or
PL aquagel-OH MIXED-M 8 µm
Polydisperse synthetic or naturally
occurring polymers
Polysaccharides, PVA, cellulose derivatives,
PEO, polyacrylic acid
2 or 3
PL aquagel-OH MIXED-H 8 µm, or
PL aquagel-OH 60/50/40 8 µm
Very high MW polymers
Polyacrylamides, hyaluronic acids,
CMC, starches, gums
PL aquagel-OH 60/50/40 15 µm in series
GPC/SEC ColumnS and STandardS
Pl aquagel-oh analytical
• Highly stable matrix ensures reliable separations, even with modified eluents
• MIXED columns cover a wide range of molecular weights, simplifying column selection
• Highly versatile for neutral, polar, anionic and cationic samples
The PL aquagel-OH analytical series has a pH range of 2-10, compatibility with organic solvent
(up to 50% methanol), mechanical stability up to 140 bar (2030 psi) and low column operating pressures.
Pl aquagel-oh analytical
mw range
(g/mol) (PEG/PEo)
Guaranteed
Efficiency
(p/m)
description
Size (mm)
Part no.
PL aquagel-OH 20 5 µm
7.5 x 300
100-20,000
> 5,000
PL1120-6520
PL aquagel-OH 20 8 µm
7.5 x 300
100-20,000
> 35,000
PL1149-6820
PL aquagel-OH 30 8 µm
7.5 x 300
100-30,000
> 35,000
PL1120-6830
PL aquagel-OH 40 8 µm
7.5 x 300
10,000-200,000
> 35,000
PL1149-6840
PL aquagel-OH 40 15 µm
7.5 x 300
10,000-200,000
> 15,000
PL1149-6240
PL aquagel-OH 50 8 µm
7.5 x 300
50,000-1,000,000
> 35,000
PL1149-6850
PL aquagel-OH 50 15 µm
7.5 x 300
50,000-1,000,000
> 15,000
PL1149-6250
PL aquagel-OH 60 8 µm
7.5 x 300
200,000-> 10,000,000
> 35,000
PL1149-6860
PL aquagel-OH 60 15 µm
7.5 x 300
200,000-> 10,000,000
> 15,000
PL1149-6260
PL aquagel-OH MIXED-H 8 µm
7.5 x 300
100-10,000,000
> 35,000
PL1149-6800
PL aquagel-OH MIXED-M 8 µm
7.5 x 300
Up to 600,000
> 35,000
PL1149-6801
PL aquagel-OH 10 µm guard
25 x 25
PL1249-1120
PL aquagel-OH 5 µm guard
7.5 x 50
PL1149-1530
PL aquagel-OH 8 µm guard
7.5 x 50
PL1149-1840
TiPS & ToolS
Buffers in a stored column may
crystallize and cause damage.
Flush the column with water
containing a small amount of
sodium azide to prevent
biological growth.
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525
GPC/SEC ColumnS and STandardS
Polyvinyl alcohol
Column:
3 x Pl aquagel-oh miXEd-h 8µm
Pl1149-6800
7.5 x 300 mm
Mobile Phase:
0.2 M NaNO3, 0.01 M NaH2PO4, pH 7
Flow Rate:
1.0 mL/min
Detector:
Agilent PL-GPC 50 (RI)
10
Retention time/min
33
VLC0032
heparin
Column:
2 x Pl aquagel-oh 30 8 µm
Pl1120-6830
7.5 x 300 mm
Mobile Phase:
0.2 M NaNO3, 0.01 M NaH2PO4, pH 7
Flow Rate:
1.0 mL/min
Detector:
Agilent PL-GPC 50 (RI)
5
Retention time/min
22
VLC0033
526
GPC/SEC ColumnS and STandardS
hyaluronic acid
Column:
Pl aquagel-oh 60 15 µm
Pl1149-6260
7.5 x 300 mm
and
Pl aquagel-oh 40 15 µm
Pl1149-6240
7.5 x 300 mm
Mobile Phase:
Chromatogram
1.0 mL/min
Agilent PL-GPC 50 (RI)
18
1
dw/dlog M
Flow Rate:
Detector:
min
5
0.2 M NaNO3, 0.01 M NaH2PO4, pH 7
Mw 1,800,00 g/mol
0
5
Log M
7
VLC0034
differences in composition of two alkyl
naphthalene sulfonates
Column:
2 x Pl aquagel-oh 20 5µm
Pl1120-6520
7.5 x 300 mm
Mobile Phase:
0.25 M ammonium formate in water
Flow Rate:
1.0 mL/min
8.0
1.0
Cum.Ht
ELS (neb=30 °C, evap=30 °C, gas=1.4 SLM)
dw/Log M
Injection Volume: 20 µL
Detector:
0.0
0.0
1.0
Log M
3.5
VLC0035
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527
GPC/SEC ColumnS and STandardS
Pl aquagel-oh Preparative
• Up to 10 times scale-up maximizes yield
• High loading maximizes sample throughput
• Carefully chosen particle size provides optimum resolution
Preparative SEC is used for the fractionation of a wide variety of water-soluble samples based on their size
in solution. The technique is applied to the fractionation of disperse polymers or to isolate components in a
polymer formulation.
Preparative PL aquagel-OH columns and associated guard columns enable rapid and convenient scale-up
from analytical separations. The 25 mm id prep column offers at least a 10 times scale-up in loading from
the 7.5 mm id analytical columns. Typically, a 10 mL/min flow rate results in a separation time of ten
minutes with a 300 mm column. The columns are packed with the same robust macroporous particles as
the analytical column range. The 8 µm particle size provides optimum resolution and loading characteristics
with column efficiency > 20,000 plates/m.
Pl aquagel-oh Preparative
description
Size (mm)
PL aquagel-OH 30 8 µm
25 x 300
mw range
(g/mol) (PEG/PEo)
100-30,000
Part no.
PL1220-6130
PL aquagel-OH 40 8 µm
25 x 300
10,000-200,000
PL1249-6140
PL aquagel-OH 50 8 µm
25 x 300
50,000-1,000,000
PL1249-6150
PL aquagel-OH MIXED 8 µm
25 x 300
100-10,000,000
PL aquagel-OH 10 µm guard
25 x 25
PL1249-6100
PL1249-1120
Polyvinyl alcohol
Column:
Pl aquagel-oh 40 8 µm
Pl1249-6140
25 x 300 mm
Mobile Phase:
0.2 M NaNO3, 0.01 M NaH2PO4, pH 7
Flow Rate:
10.0 mL/min
Loading:
10 mg/mL, 2 mL
Detector:
Agilent PL-GPC 50 (RI)
0
Retention time/min
15
VLC0036
528
GPC/SEC ColumnS and STandardS
GPC Column accessories
description
unit
Part no.
Frit removal tool for threaded columns only
1/pk
PL1310-0001
2 µm frit kit for threaded columns, 7.5 mm id
5/pk
PL1310-0002
5 µm frit kit for threaded columns, 7.5 mm id
5/pk
PL1310-0012
10 µm frit kit for threaded columns, 7.5 mm id
5/pk
PL1310-0036
PLgel column repair gel, 10 µm
1/pk
PL1410-0101
PLgel column repair gel, 5 µm
1/pk
PL1410-0501
Column connecting nuts, 1/16 in tube
5/pk
PL1310-0007
Tubing ferrules, 1/16 in tube
5/pk
PL1310-0008
Connecting tubing, 10 cm length, 0.01 in id
10/pk
PL1310-0048
LDV intercolumn stainless steel connector
1/pk
PL1310-0005
PLgel column repair gel, 3 µm
1/pk
PL1410-0301
PLgel Olexis column repair gel
1/pk
PL1410-0200
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529
GPC/SEC ColumnS and STandardS
Polymer Standards for GPC/SEC
Polymer standards from Agilent are the ideal reference materials for generating accurate, reliable GPC/SEC
column calibrations, with the assurance of the ISO 9001:2000 quality standard. Additional applications for
our highly characterized homopolymers exhibiting unique characteristics are used as model polymers for
research and analytical method development.
Agilent manufactures the highest quality polymer standards with extremely narrow polydispersity and the
widest molecular weight range commercially available. These quality polymer standards are supplied with
extensive characterization data utilizing a variety of independent techniques (e.g. light scattering and
viscometry) and high performance GPC to verify polydispersity and assign that all important peak molecular
weight (Mp).
Our comprehensive range of EasiVial, EasiCal, and traditional calibration kits has been specifically designed
to cover all molecular weight ranges for organic and aqueous GPC/SEC applications. We provide you with
the widest choice to maximize your specific characterization needs. In addition, we supply other polymers as
individual molecular weights, and broad distribution polymers for system validation or broad standard
calibration procedures.
530
GPC/SEC ColumnS and STandardS
Calibration Kits
Agilent offers a wide range of polymer standards kits for conventional GPC/SEC column calibration or for
calibrating light scattering and viscometry detectors. The kits are in boxed sets of ten different polymer
standards covering a particular molecular weight range, to be used with organic and aqueous, medium
polarity, and polar solvents. Every individual polymer has its own Certificate of Analysis of the analytical
conditions and values, such as Mp needed for constructing a calibration plot. The polymers are chosen to
give equidistant calibration points on a logarithmic MW scale, providing a more uniform calibration curve.
individual Polymer molecular weights
We design our individual standards to have the narrowest molecular weight distribution commercially
available. Additionally, they cover the widest molecular weight range, from 162-15 million MW. The current
polystyrene nominal molecular weight of 15 million MW has a polydispersity ≤1.10. These standards are
generally available in 1, 5 and 10 g quantities, and each comes with its own Certificate of Analysis detailing
analysis conditions and relevant data.
GPC/SEC Standards Selection Guide
Polymer Type
individual Calibration
mw
Kits
EasiCal EasiVial Type of GPC/SEC
Polystyrene
3
3
Polymethylmethacrylate
3
3
3
3
3
Organic
Polyethylene glycol (PEG)
3
3
3
Organic/Aqueous
Polyethylene oxide (PEO)
3
3
3
Pullulan polysaccharide
3
3
Organic/Aqueous
Polyacrylic acid Na salt
3
3
Aqueous
Organic
Organic/Aqueous
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531
GPC/SEC ColumnS and STandardS
EasiVial
• Eliminates tedious weighing procedures to improve calibration accuracy
• Reduces solvent dispensing to limit risks associated with handling solvents
• For conventional and multi detector GPC to maximize applicability
For organic and aqueous GPC/SEC column calibration, this premier product is the quickest and most
convenient method to deliver an accurate 12-point column calibration.
The key to achieving baseline separation from polymer mixtures, and therefore eliminating doubt and errors,
is in selecting only the narrowest polydispersity polymers. This is where Agilent polymer standards excel and
deliver, as shown in the chromatograms.
The EasiVial standards kit is a pre-prepared, time saving product for rapid and reliable GPC column
calibration. EasiVial kits contain three vials, each with a mixture of four accurately pre-weighed polymer
standards, providing a 12-point GPC calibration in just three injections. The mass of each polymer in the vial
is accurately known, so that upon addition of a fixed volume of eluent, the solution is prepared at a precise
concentration. EasiVial is ideal for both conventional and multi detector GPC calibration. Simply prepare and
manually inject, or transfer to autosampler vials, or place directly into a compatible autosampler.
Every EasiVial kit contains 30 vials (ten of each type) that are color-coded for easy identification and are
available in 4 or 2 mL vials making them suitable for most autosamplers. The kits are available for
polystyrene (PS), polymethylmethacrylate (PMMA), polyethylene glycol/oxide (PEG/PEO) and polyethylene
glycol (PEG). For added value, a Tri-Pack (90 vials) is offered, extending reproducibility.
532
GPC/SEC ColumnS and STandardS
EasiVial PS-h
Column:
3 x Plgel 10 µm miXEd-B
Pl1110-6100
7.5 x 300 mm
Mobile Phase:
Flow Rate:
1.0 mL/min
40 °C
Detector:
PL-GPC 220 (RI)
4
3
2
1
THF
Temperature:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
7
6
8
5
11
10
6,035,000
483,000
19,720
1,260
3,053,000
84,900
8,450
580
915,000
60,450
3,370
162
12
9
10
min
33
VLC0037
Polystyrene calibration generated with EasiVials
LogM
1.0e7
1.0e2
15
Retention time/min
29
VLC0044
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533
GPC/SEC ColumnS and STandardS
Specifications
EasiVial Color
EasiVial PS-h
EasiVial PS-m
1,300
780
Red
Yellow
Green
description Key
PS: Polystyrene
PM: Polymethylmethacrylate
PEG/PEO: Polyethylene Glycol/Oxide
H: High
M: Medium
L: Low
534
EasiVial PS-l
EasiVial Pm
nominal mp (g/mol)
580
2,000
EasiVial PEG/PEo
EasiVial PEG
600
282
20,000
6,000
3,000
30,000
12,000
1,000
500,000
50,000
10,000
300,000
125,000
6,000
6,000,000
400,000
40,000
2,000,000
1,200,000
35,000
580
370
370
1,000
200
194
8,500
2,500
2,000
13,000
4,000
600
185,000
25,000
6,000
150,000
60,000
3,750
3,000,000
200,0001
25,000
800,000
1,000,000
21,000
162
162
162
600
100
106
3,400
1,500
1,000
5,700
1,500
420
60,000
11,000
4,000
80,000
25,000
2,000
900,000
100,000
16,000
470,000
460,000
12,000
GPC/SEC ColumnS and STandardS
EasiVial Pre-weighed Calibration Kits
range of nominal mp
(g/mol)
Vial Volume
(ml)
EasiVial PEG/PEO
100-1,200,000
2
EasiVial PEG/PEO
description
unit
Part no.
30/pk PL2080-0201
100-1,200,000
4
30/pk PL2080-0200
EasiVial PEG
106-35,000
2
30/pk PL2070-0201
EasiVial PEG
106-35,000
4
30/pk PL2070-0200
EasiVial PM
600-2,000,000
2
30/pk PL2020-0201
EasiVial PM
600-2,000,000
4
30/pk PL2020-0200
EasiVial PS-H
162-6,000,000
2
30/pk PL2010-0201
EasiVial PS-H
162-6,000,000
4
30/pk PL2010-0200
EasiVial PS-M
162-400,000
2
30/pk PL2010-0301
EasiVial PS-M
162-400,000
4
30/pk PL2010-0300
EasiVial PS-L
162-40,000
2
30/pk PL2010-0401
EasiVial PS-L
162-40,000
PEG/PEO Tri-Pack
4
30/pk PL2010-0400
2
90/pk PL2080-0202
PEG/PEO Tri-Pack
4
90/pk PL2080-0203
PEG Tri-Pack
2
90/pk PL2070-0202
PEG Tri-Pack
4
90/pk PL2070-0203
PMMA Tri-Pack
2
90/pk PL2020-0202
PMMA Tri-Pack
4
90/pk PL2020-0203
PS-H Tri-Pack
2
90/pk PL2010-0202
PS-H Tri-Pack
4
90/pk PL2010-0203
PS-L Tri-Pack
2
90/pk PL2010-0402
PS-L Tri-Pack
4
90/pk PL2010-0403
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535
GPC/SEC ColumnS and STandardS
EasiCal
• Easy three-step process with no fuss
• Cost-effective format saves money
• Only two injections for improved productivity
The EasiCal system for organic solvents consists of two different combs, each with ten detachable spatulas,
supporting a mixture of five polymer standards. The thin film of polymer (approximately 5 mg) on the tip of
the PTFE spatulas rapidly dissolves when immersed in eluent to provide two GPC/SEC calibration solutions.
A single pack provides ten spatulas of each type, with MWs selected to provide equidistant calibration
points for greater accuracy.
EasiCal Pre-prepared Polystyrene Kits
description
range of nominal mp (g/mol)
Polystyrene PS-1
580-7,500,000
Polystyrene PS-2
580-400,000
unit
Part no.
1/pk
PL2010-0501
5/pk
PL2010-0505
1/pk
PL2010-0601
5/pk
PL2010-0605
Column calibration for GPC/SEC is as easy as 1, 2, 3...
1. Place one spatula of each type into appropriate volume of solvent.
Log MW
7
3
13
Retention time/min
25
VLC0038
5
30
5
30
2. Chromatograph each solution; only two injections required
536
3. Generate a 10-point calibration
GPC/SEC ColumnS and STandardS
Polystyrene
• Compatible with most organic solvents
• Certificate of Analysis meets international protocols
• Calibration capability for virtually all applications
Polystyrene standards are the first choice for many organic solvents, either for conventional GPC column
calibration or for calibrating light scattering and viscosity detectors. Our organic polymers cover a range
from 162-15 million MW, with MWs selected to provide equidistant calibration points for greater accuracy.
Every kit contains 0.5 g of ten different molecular weight standards.
Calibration Kits, (all Kits 10 x 0.5 g)
S-h-10
S-h2-10
S-m-10
Part no.
Part no.
Part no.
Pl2010-0103 Pl2010-0104
Pl2010-0100
Constituent Polymer nominal mp (g/mol)
S-m2-10
Part no.
Pl2010-0102
S-l-10
Part no.
Pl2010-0101
S-l2-10
Part no.
Pl2010-0105
300,000
1,000
580
580
162
162
460,000
3,000
1,450
1,400
580
370
700,000
8,600
4,000
2,400
900
580
1,100,000
25,000
10,000
4,750
1,400
800
1,700,000
73,000
27,000
9,500
2,200
1,000
2,600,000
210,000
66,000
19,000
3,400
1,500
4,000,000
600,000
180,000
38,000
5,100
1,900
6,200,000
1,780,000
460,000
75,000
8,100
2,500
9,500,000
5,000,000
1,190,000
150,000
12,800
3,200
15,000,000
15,000,000
3,000,000
300,000
20,000
4,500
description Key
H: High
M: Medium
L: Low
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537
GPC/SEC ColumnS and STandardS
individual Polymer molecular weights
Polymer nominal mp (g/mol)
162
1g
nominal mw/mn Part no.
1.00
5g
Part no.
10 g
Part no.
PL2012-1001 PL2012-1005 PL2012-1010
370
1.11
PL2012-0001 PL2012-0005 PL2012-0010
580
1.11
PL2012-2001 PL2012-2005 PL2012-2010
1,000
1.09
PL2012-3001 PL2012-3005 PL2012-3010
1,300
1.07
PL2012-4001 PL2012-4005 PL2012-4010
2,000
1.05
PL2012-5001 PL2012-5005 PL2012-5010
3,000
1.04
PL2012-6001 PL2012-6005 PL2012-6010
5,000
1.03
PL2012-7001 PL2012-7005 PL2012-7010
7,000
1.04
PL2012-8001 PL2012-8005 PL2012-8010
10,000
1.02
PL2012-9001 PL2012-9005 PL2012-9010
20,000
1.02
PL2013-1001 PL2013-1005 PL2013-1010
30,000
1.02
PL2013-2001 PL2013-2005 PL2013-2010
50,000
1.03
PL2013-3001 PL2013-3005 PL2013-3010
70,000
1.03
PL2013-4001 PL2013-4005 PL2013-4010
100,000
1.02
PL2013-5001 PL2013-5005 PL2013-5010
130,000
1.01
PL2013-6001 PL2013-6005 PL2013-6010
200,000
1.05
PL2013-7001 PL2013-7005 PL2013-7010
300,000
1.03
PL2013-8001 PL2013-8005 PL2013-8010
500,000
1.03
PL2013-9001 PL2013-9005 PL2013-9010
700,000
1.03
PL2014-0001 PL2014-0005 PL2014-0010
1,000,000
1.05
PL2014-1001 PL2014-1005 PL2014-1010
1,500,000
1.04
PL2014-2001 PL2014-2005 PL2014-2010
2,000,000
1.04
PL2014-3001 PL2014-3005 PL2014-3010
2,500,000
1.05
PL2014-4001 PL2014-4005 PL2014-4010
4,000,000
1.04
PL2014-6001 PL2014-6005 PL2014-6010
7,000,000
1.04
PL2014-7001 PL2014-7005 PL2014-7010
10,000,000
1.06
PL2014-8001 PL2014-8005 PL2014-8010
15,000,000
1.06
PL2014-9001 PL2014-9005 PL2014-9010
Polystyrene standards
Column:
2 x oligoPore
Pl1113-6520
7.5 x 300 mm
Mobile Phase:
THF
Flow Rate:
1.0 mL/min
Detector:
Agilent PL-GPC 50 (RI)
1,270
580
0
min
20
VLC0039
538
GPC/SEC ColumnS and STandardS
Polymethylmethacrylate
• Many solvent options increase applicability
• Stringent quality control improves performance
• Proprietary manufacturing methods ensure consistent supply
Polymethylmethacrylate (PMMA) standards are extremely versatile as they can be used for organic
GPC with a wide range of medium polarity eluents, such as tetrahydrofuran, toluene, methyl ethyl ketone,
and ethyl acetate. They also work well with more polar organic eluents, for example dimethylformamide,
dimethylacetamide, and hexafluoroisopropanol. The MWs are selected to provide equidistant calibration
points for greater accuracy, covering from 500-1.5 million MW. Every kit contains 0.5 g of ten different
molecular weight standards.
Calibration Kits, (all Kits 10 x 0.5 g)
m-l-10
Part no.
Pl2010-0100
Constituent Polymer nominal mp (g/mol)
m-m-10
Part no.
Pl2020-0101
600
1,000
840
2,200
1,400
5,000
2,350
11,200
3,900
25,500
6,400
58,000
10,800
130,000
18,000
290,000
30,000
660,000
50,000
1,500,000
description Key
M: Medium
L: Low
www.aGilEnT.Com/ChEm/lC
539
GPC/SEC ColumnS and STandardS
individual Polymer molecular weights
Polymer nominal mp (g/mol)
nominal mw/mn
1g
Part no.
5g
Part no.
500
1.19
PL2022-2001 PL2022-2005 PL2022-2010
1,000
1.26
PL2022-3001 PL2022-3005 PL2022-3010
2,000
1.08
PL2022-5001 PL2022-5005 PL2022-5010
3,000
1.08
PL2022-6001 PL2022-6005 PL2022-6010
5,000
1.09
PL2022-7001 PL2022-7005 PL2022-7010
7,000
1.08
PL2022-8001 PL2022-8005 PL2022-8010
10,000
1.03
PL2022-9001 PL2022-9005 PL2022-9010
13,000
1.03
PL2023-0001 PL2023-0005 PL2023-0010
20,000
1.03
PL2023-1001 PL2023-1005 PL2023-1010
30,000
1.02
PL2023-2001 PL2023-2005 PL2023-2010
50,000
1.02
PL2023-3001 PL2023-3005 PL2023-3010
70,000
1.02
PL2023-4001 PL2023-4005 PL2023-4010
100,000
1.02
PL2023-5001 PL2023-5005 PL2023-5010
130,000
1.05
PL2023-6001 PL2023-6005 PL2023-6010
200,000
1.02
PL2023-7001 PL2023-7005 PL2023-7010
300,000
1.02
PL2023-8001 PL2023-8005 PL2023-8010
500,000
1.06
PL2023-9001 PL2023-9005 PL2023-9010
700,000
1.03
PL2024-0001 PL2024-0005 PL2024-0010
1,000,000
1.09
PL2024-1001 PL2024-1005 PL2024-1010
1,500,000
1.09
PL2024-2001 PL2024-2005 PL2024-2010
Polymethylmethacrylate standards
Column:
2 x Pl hFiPgel
Pl1114-6900hFiP
7.5 x 300 mm
Mobile Phase:
HFIP + 20 mM NaTFAc
Flow Rate:
1.0 mL/min
1.
2.
3.
4.
5.
3
1
4
2
Temperature:
40 °C
Detector:
Agilent PL-GPC 50 (RI)
790,000
144,000
28,900
5,720
1,020
5
5
Retention time/min
22
VLC0040
540
10 g
Part no.
GPC/SEC ColumnS and STandardS
Polyethylene Glycol/oxide
• Simple-to-use kit form
• Combines glycols and oxides to extend the MW range and cover more applications
• MWs selected to provide equidistant calibration points for greater accuracy
These hydrophilic polymers are suitable for both aqueous SEC and organic GPC using the majority of polar
organic solvents. The oxides are available in high molecular weights, while the glycols cover the lower
molecular weight range. The two types are chemically similar so they can be used together across a wider
molecular weight range, with aqueous and organic polymers from 106-1 million MW. Every kit contains
0.2 g or 0.5 g of ten different molecular weight standards.
Calibration Kits
PEG-10 (10 x 0.5 g)
Part no.
Pl2070-0100
Constituent Polymer nominal mp (g/mol)
PEo-10 (10 x 0.2 g)
Part no.
Pl2080-0101
106
20,000
194
30,000
400
50,000
600
70,000
1,000
100,000
2,000
200,000
4,000
300,000
7,000
400,000
13,000
700,000
20,000
1,000,000
www.aGilEnT.Com/ChEm/lC
541
GPC/SEC ColumnS and STandardS
individual Polymer molecular weights
Polymer nominal mp (g/mol)
1g
nominal mw/mn Part no.
5g
Part no.
10 g
Part no.
Polyethylene Glycol
106
1.00
PL2070-1001 PL2070-1005 PL2070-1010
194
1.00
PL2070-2001 PL2070-2005 PL2070-2010
238
1.00
PL2071-2001 PL2071-2005 PL2071-2010
282
1.00
PL2071-3001 PL2071-3005 PL2071-3010
420
1.09
PL2070-3001 PL2070-3005 PL2070-3010
600
1.06
PL2070-4001 PL2070-4005 PL2070-4010
1,000
1.04
PL2070-5001 PL2070-5005 PL2070-5010
1,500
1.04
PL2070-6001 PL2070-6005 PL2070-6010
4,000
1.03
PL2070-7001 PL2070-7005 PL2070-7010
7,000
1.04
PL2070-8001 PL2070-8005 PL2070-8010
10,000
1.05
PL2070-9001 PL2070-9005 PL2070-9010
13,000
1.07
PL2071-0001 PL2071-0005 PL2071-0010
20,000
1.07
PL2071-1001 PL2071-1005 PL2071-1010
Polyethylene oxide
20,000
1.05
PL2083-1001 PL2083-1005 PL2083-1010
30,000
1.07
PL2083-2001 PL2083-2005 PL2083-2010
50,000
1.05
PL2083-3001 PL2083-3005 PL2083-3010
70,000
1.05
PL2083-4001 PL2083-4005 PL2083-4010
100,000
1.06
PL2083-5001 PL2083-5005 PL2083-5010
130,000
1.07
PL2083-6001 PL2083-6005 PL2083-6010
200,000
1.07
PL2083-7001 PL2083-7005 PL2083-7010
300,000
1.07
PL2083-8001 PL2083-8005 PL2083-8010
500,000
1.06
PL2083-9001 PL2083-9005 PL2083-9010
700,000
1.07
PL2084-0001 PL2084-0005 PL2084-0010
1,000,000
1.12
PL2084-1001 PL2084-1005 PL2084-1010
1,500,000
1.13
PL2084-2001 PL2084-2005 PL2084-2010
Polyethylene Glycol/oxide standards
Column:
3
Pl aquagel-oh miXEd-h 8 µm
Pl1149-6800
7.5 x 300 mm
Mobile Phase:
Water
Flow Rate:
1.0 mL/min
Detector:
Agilent PL-GPC 50 (RI)
1.
2.
3.
4.
5.
4
2
5
1
1
Retention time/min
11
VLC0041
542
1,702,000
120,000
12,600
1,470
106
GPC/SEC ColumnS and STandardS
Polysaccharides
• Comprehensive format provides full MW range in one handy kit
• Also available as individual standards
The pullulan polysaccharides kit consists of several simple sugars with relatively narrow polydispersity linear
macromolecules of maltotriose units.
Calibration Kits
SaC-10 (10 x 0.2 g)
Part no.
Pl2090-0100
Constituent Polymer nominal mp (g/mol)
180
738
5,000
10,000
20,000
50,000
100,000
200,000
400,000
700,000
www.aGilEnT.Com/ChEm/lC
543
GPC/SEC ColumnS and STandardS
individual Polymer molecular weights
Polymer nominal mp (g/mol)
unit
Part no.
1,500
0.2 g
PL2091-2000
2,000
0.2 g
PL2091-3000
3,000
0.2 g
PL2091-4000
5,000
0.5 g
PL2090-1000
20,000
0.5 g
PL2090-3000
50,000
0.5 g
PL2090-4000
100,000
0.5 g
PL2090-5000
200,000
0.5 g
PL2090-6000
700,000
0.5 g
PL2090-8000
1,660,000
0.2 g
PL2091-1000
4
Pullulan polysaccharide standards
Column:
1
5
1.
2.
3.
4.
5.
3 x Pl aquagel-oh miXEd-h 8 µm
Pl1149-6800
7.5 x 300 mm
Mobile Phase:
0.2 M NaNO3, 0.01 M NaH2PO4, pH 7
Flow Rate:
1.0 mL/min
Detector:
Agilent PL-GPC 50 (RI)
8
544
2
3
Retention time/min
30
VLC0042
788,000
212,000
47,300
11,800
667
GPC/SEC ColumnS and STandardS
Polyacrylic acid
• Compatible with all aqueous columns for wide applicability
• Aqueous polymers 1,000-2 million MW
• Well-characterized Mp values ensure wide utility
Calibration Kits
Paa-10 (10 x 0.2 g)
Part no.
Pl2140-0100
Constituent Polymer nominal mp (g/mol)
1,000
3,000
7,000
13,000
30,000
70,000
100,000
300,000
700,000
1,000,000
individual Polymer molecular weights
Polymer nominal mp (g/mol)
0.2 g
Part no.
1g
Part no.
1,000
PL2142-3000
PL2142-3001
2,000
PL2142-5000
3,000
PL2142-6000
PL2142-6001
5,000
PL2142-7000
PL2142-7001
7,000
PL2142-8000
PL2142-8001
13,000
PL2143-0000
PL2143-0101
30,000
PL2143-2000
PL2143-2001
50,000
PL2143-3000
PL2143-3001
70,000
PL2143-4000
PL2143-4001
100,000
PL2143-5000
PL2143-5001
130,000
PL2143-6000
PL2143-6001
200,000
PL2143-7000
PL2143-7001
300,000
PL2143-8000
PL2143-8001
500,000
PL2143-9000
PL2143-9001
700,000
PL2144-0000
PL2144-0101
1,000,000
PL2144-1000
PL2144-1001
1,500,000
PL2144-2000
PL2144-2001
2,000,000
PL2144-3000
PL2144-3001
www.aGilEnT.Com/ChEm/lC
545
PRODUCT INDEX
Product Index
Agilent Supplies
Autosamplers
Injection Valves.........................................................61
Kits..........................................................................66-67
Loop Capillaries.........................................................64
Maintenance Schedule ...........................................61
Metering Device........................................................64
Needles and Needle Seats...............................62-63
Trays.............................................................................65
Bio-inert Quaternary
Autosampler ....................................................101-102
Column Compartment ...........................................102
Detectors ..................................................................102
Fittings.......................................................................102
Pumps .......................................................................101
Valves ........................................................................102
Capillaries
1200 and 1100 Prep LC Systems ..........................26
1220/1120 Infinity Series LC Systems ................33
1260 Bio-inert LC System.......................................30
1260/1200/1100 Infinity Series LC Systems.....23
1290 Infinity Series LC Systems ...........................24
1290 Valve Head..................................................25-27
Loop .............................................................................32
Miscellaneous .....................................................27-29
PEEK Coated Fused Silica Capillaries –
100 µL/min Flow ......................................................32
PEEK Coated Fused Silica Capillaries –
20 µL/min Flow.........................................................31
PEEK Coated Fused Silica Capillaries
for Nano LC................................................................31
Chip LC ........................................................................100
Detectors
80 nL and 500 nL Flow Cell..............................95-96
Diode Array Detector (DAD)/
Multiple Wavelength Detector (MWD)...............93
Evaporative Light Scattering Detector (ELSD).....98
Fluorescence Detector (FLD) .................................99
Lamps ..........................................................................90
Maintenance..............................................................89
Maintenance Kits......................................................97
Preparative Flow Cells.............................................96
Refractive Index Detector (RID) ............................98
Variable Wavelength Detector (VWD)...........91-92
Fittings and Unions
Capillary and Fittings Kits .................................36-39
Fittings...................................................................40-41
How to Align Connection Properly.......................45
How to Prepare Connection...................................44
How to Tighten Correctly........................................43
Fraction Collectors
Capillary Kits and Needles .....................................70
Collecting Tubes and Trays.....................................69
G1364D Micro Fraction Collector..........................71
Maintenance Schedule ...........................................68
Well Plate Trays ........................................................69
In-Line Filters ...............................................................12
LC/MS
Chemicals.................................................................107
Cleaning Supplies...................................................108
Foreline Pump .........................................................107
644
Gas Purifiers ............................................................108
Instrument Supplies.......................................105-106
Maintenance Schedule.........................................103
Preventive Maintenance Kit ................................104
Quiet Cover ..............................................................109
Standards
Analyzer Kit............................................................110
Calibrant Mixes.....................................................110
Kits...........................................................................110
Tools...........................................................................108
Loop Capillaries ...........................................................32
Pumps
1290 Infinity LC Pump Supplies ............................51
Frits and Adapters ....................................................54
Inlet Valves.................................................................49
Kits..........................................................................59-60
Outlet Valves..............................................................49
Pistons and Seals.....................................................50
Purge Valves ..............................................................48
Routine Maintenance ..............................................47
Safety Caps ................................................................55
Seal Wash ..................................................................52
Solvent Filters............................................................54
Solvent Reservoir......................................................53
Vacuum Degassers...................................................58
Rack for LC Systems..................................................14
Solvent Filters/Degassers ........................................13
Standards......................................................................15
Thermostatted Column Compartment
Capillary Kits ........................................................83-88
Tools ...............................................................................11
Troubleshooting.................................................546-549
Tubing
Accessories................................................................35
PEEK.............................................................................34
Rigid Capillary Tubing ..............................................35
Unions............................................................................42
Valves
Injection Valves.........................................................73
Maintenance Notes .................................................72
Manual Injection Valves
with Position Sensing Switches .....................76-77
Sample Loops ......................................................78-79
Switching Valves
External Switching Valve......................................74
Internal Switching Valve.......................................75
Syringes for Manual Injection
Fitted Plungers........................................................80
PTFE-Tipped Plungers............................................81
Applications
BioPharmaceutical ...................................................552
Chemical/Industrial .................................................580
Environmental............................................................583
Food and Consumer Products ...............................594
Pharmaceutical..........................................................611
CE and CE/MS
Alignment Interface .................................................190
CE/MS Accessories
Adapter Kit ...............................................................194
Capillaries.................................................................195
Sprayer Kit................................................................195
Capillaries
Alignment Interface ...............................................190
Bulk Fused-Silica ....................................................181
CEP Coated ..............................................................185
Capillary Cassette...................................................190
Capillary Electrochromatography (CEC)........188-189
Cross-linked and Bonded µSIL....................186-187
Extended Light Path
(Bubble Cell) Bare Fused-Silica ..................179-180
Polyvinyl Alcohol (PVA) Coated..................182-184
Standard Bare Fused-Silica..................................178
Universal Bare Fused-Silica .................................181
Capillary Cassette.....................................................190
High Sensitivity Detection Cell......................191-192
Instrument Supplies.................................................199
Solution Kits
Cation ........................................................................173
Forensics ..................................................................175
Inorganic Anion...............................................171-172
Organic Acids ..........................................................174
µPAGE ...............................................................176-177
Standards & Reagents
CZE Buffers for Charged Analytes .....................196
CZE Buffers for Proteins.......................................197
Capillary Conditioning Solutions.........................196
MEKC Buffers for Neutral
and Charged Analytes...........................................197
Plating Bath Analysis Buffer ...............................197
Ultra Pure CE Water ..............................................196
µPAGE Buffer Solutions and Oligo Standards...197
System Start-up and Test Kits...............................198
Troubleshooting.................................................201-203
Vial Rack .....................................................................200
Vials and Caps...........................................................199
Window Etching Tool...............................................200
Columns for Biomolecule Separations
Affinity Chromatography
Agilent Bio-Monolith Protein A ..................434-435
Multiple Affinity Removal System..............438-440
Multiple Affinity Removal System Starter Kits.....441
Protein Fractionation System ..............................437
Proteomic Reagents ..............................................442
mRP-C18 High-Recovery Protein ................442-443
Analysis Scale
Capillary and Nano.................................................452
MicroBore (1.0 mm id)..........................................461
ZORBAX Bio-SCX Series II...................................458
Citations
PL-SAX ......................................................................487
PL-SCX ......................................................................487
PLRP-S...............................................................486-487
Poroshell 300...........................................................486
ZORBAX 300............................................................485
PRODUCT INDEX
Column Selection
Amino Acid Analysis.............................................362
Broad Distribution..................................................363
DNA and RNA Oligonucleotide..........................360
Natural and Synthetic Peptides .........................358
Peptide Mapping....................................................356
Protein ......................................................................353
Ion-Exchange Chromatography
Agilent Bio IEX .......................................................402
Agilent Bio MAb ....................................................399
Agilent Bio-Monolith ....................................412-413
PL-SAX Strong Anion-Exchange........................406
PL-SCX Strong Cation-Exchange .......................410
Literature............................................................477-484
Method Development
Bio Ion-Exchange Column ...................................447
High Sensitivity Capillary Column......................451
Reversed-Phase LC/MS Column.......................446
SEC Column ....................................................449-450
ZORBAX Columns..................................................444
Purification - Prep HPLC
PL-SAX and PL-SCX......................................472-473
PLRP-S..............................................................467-468
Peptide Purification
VariPure IPE ..........................................................476
VariTide RPC .........................................................475
ZORBAX PrepHT ....................................................466
Reversed-Phase HPLC
PLRP-S .....................................................................387
Poroshell 120 ..........................................................385
Poroshell 300 ..................................................380-381
ZORBAX 300Extend-C18 ......................................376
ZORBAX 300StableBond......................................367
ZORBAX Eclipse
Amino Acid Analysis (AAA)........................394-395
ZORBAX RRHD 300-Diphenyl .............................374
Size Exclusion Chromatography (SEC)
Agilent Bio SEC-3 ..................................................418
Agilent Bio SEC-5 ..................................................424
ProSEC 300S ...........................................................428
ZORBAX GF-250 and GF-450 ..............................431
Columns for Small Molecule
Separations
Column Selection
Column and Mobile Phase Guidelines........211-213
Method Development...................................220-221
Quick Guide to Reversed-Phase.................207-209
Small Molecule Overview............................218-219
Fast Columns for Reversed-Phase HPLC/UHPLC
Fast Guards for UHPLC ........................................246
Poroshell 120 ..........................................................228
ZORBAX Rapid Resolution
High Definition (RRHD) 1.8 µm...........................233
ZORBAX Rapid Resolution
High Throughput (RRHT) 1.8 µm .......................239
Guard Columns.................................................223-224
Kits for Analytical HPLC
ZORBAX Method Development Kits.........284-285
ZORBAX Method Validation Kits ...............286-288
Oligo Solutions
StratoSpheres DNA Cartridges ..........................347
TOP Cartridges ...............................................348-349
Other HPLC Techniques
Hi-Plex for Carbohydrate Analysis.....................340
ZORBAX HILIC Plus ..............................................324
ZORBAX Ion-Exchange.........................................333
ZORBAX Normal-Phase ...............................326-328
Preparative HPLC
Bulk Materials ........................................................321
Load & Lock ............................................................322
Polaris Prep .............................................................321
Prep LC.............................................................311-312
Pursuit and Pursuit XRs Prep .............................319
ZORBAX PrepHT ....................................................314
Reversed-Phase HPLC
HC-C18(2).........................................................304-305
PLRP-S .....................................................................306
Polaris.......................................................................298
Pursuit ..............................................................287-288
TC-C18(2) .........................................................304-305
ZORBAX 80Å Extend-C18 ....................................274
ZORBAX 80Å StableBond....................................264
ZORBAX Bonus-RP ...............................................278
ZORBAX Eclipse PAH ...........................................254
ZORBAX Eclipse Plus ...........................................248
ZORBAX Eclipse XDB...........................................256
ZORBAX Original....................................................283
ZORBAX Rx .............................................................272
USP Designations ............................................343-346
CrossLab Supplies
Autosampler Maintenance....................................119
Autosampler Syringes.....................................116-118
CTC HPLC Autosamplers
Autosampler Syringes ..........................................169
Capillaries and Tubing.............................................130
Detector Lamps ........................................................113
Dionex HPLC Systems
Autosampler Supplies...........................................158
Autosampler Syringes ..........................................157
Buffer Loops............................................................160
Capillaries ........................................................166-168
Detector Lamps......................................................157
Fittings, Ferrules and Unions ..............................168
In-Line Filters ..........................................................168
Performance Maintenance Kits .................161-165
Pump Maintenance Procedure...........................126
Pump Supplies................................................158-159
Sample Loops .........................................................160
Tubing .......................................................................168
Valve Replacement Parts.....................................159
Fittings ........................................................................131
In-Line Filters ............................................................132
Performance Maintenance Kits............................129
Pump Maintenance .................................................124
Pump Supplies..................................................122-123
Sealing Mats .............................................................115
Shimadzu HPLC Systems
Autosampler Syringes ..........................................150
Capillaries ................................................................154
Detector Lamps......................................................150
Fittings, Ferrules and Unions ..............................155
In-Line Filters ..........................................................156
Performance Maintenance Kits .........................154
WWW.AGILENT.COM/CHEM/LC
Pump Maintenance Procedure...........................125
Pump Supplies................................................151-152
Sample Loops .........................................................153
Tubing .......................................................................155
Valve Replacement Parts.............................152-153
Troubleshooting................................................133-136
Valve Supplies...................................................127-128
Waters HPLC Systems
Autosampler Syringes ..................................137-138
Capillaries ................................................................148
Detector Lamps......................................................137
Detector Supplies ..................................................142
Fittings, Ferrules and Unions ..............................149
Performance Maintenance Kits .................144-158
Pump Maintenance Procedure...........................124
Pump Supplies................................................139-142
Sample Loops .........................................................143
Valve Replacement Parts.....................................142
Well Plates.................................................................115
GPC/SEC Columns and Standards
Aqueous
PL aquagel-OH Analytical....................................525
PL aquagel-OH Preparative .................................528
Column Accessories................................................529
Organic
PLgel Individual Pore Size ...................................505
PLgel MIXED...................................................498-501
PLgel MIXED-LS.....................................................502
PLgel MiniMix.........................................................504
PLgel Preparative...................................................506
Polymer Standards
EasiCal......................................................................536
EasiVial.....................................................................532
Polyacrylic Acid......................................................545
Polyethylene Glycol/Oxide..........................541-542
Polymethylmethacrylate...............................539-540
Polysaccharides .............................................543-544
Polystyrene......................................................537-538
Setting Up a GPC/SEC System ....................491-495
Special Application
EnviroPrep................................................................507
MesoPore ................................................................520
OligoPore .................................................................521
PL HFIPgel ...............................................................509
PL Rapide.........................................................510-511
PLgel Olexis.............................................................508
PlusPore...........................................................514-515
PolarGel....................................................................512
PolyPore ...................................................................516
ResiPore...................................................................518
645
PART NUMBER INDEX
Part Number Index
0100-0549 ..........................................107
0100-0900.............................................42
0100-0969 ..........................................105
0100-1259.............................................41
0100-1516................40, 92, 96, 99, 106
0100-1543 ..........................................195
0100-1597 ..........................................107
0100-1631.............................................40
0100-1710......................................11, 58
0100-1847.............................................42
0100-1849......................................61, 73
0100-1850......................................75, 77
0100-1851 ........61, 73-75, 77, 101-102
0100-1852.............................................75
0100-1853......................................61, 73
0100-1854....................................75, 108
0100-1855...........................75, 104, 108
0100-1859.............................................77
0100-1860.............................................77
0100-1921.............................................79
0100-1922.............................................79
0100-1923.............................................79
0100-1924.............................................79
0100-2051..................................104, 108
0100-2086.............................................41
0100-2087.............................................75
0100-2088......................................61, 73
0100-2089.............................................75
0100-2175.............................................40
0100-2231......................................61, 73
0100-2233.............................................75
0100-2298.............................................42
0100-2304 ..........................................106
0100-2410.............................................35
0100-2441.............................................42
0101-0376.............................................79
0101-0377.............................................79
0101-0378.............................................79
0101-0379.............................................79
0101-0620.............................................77
0101-0623.............................................77
0101-0921......................................61, 73
0101-1050......................................61, 73
0101-1219.............................................79
0101-1226.............................................79
0101-1227.............................................79
0101-1228.............................................79
0101-1229.............................................79
0101-1230.............................................79
0101-1231.............................................77
0101-1232.............................................77
0101-1233.............................................77
0101-1234.............................................79
0101-1235.............................................79
0101-1236.............................................79
0101-1237.............................................79
0101-1238.............................................79
0101-1239.............................................79
0101-1240.............................................79
0101-1241.............................................79
0101-1242.............................................79
0101-1243.............................................79
0101-1244.............................................79
0101-1245.............................................79
0101-1246.............................................79
0101-1247.............................................79
0101-1248.............................................79
0101-1249.............................................79
646
0101-1250.............................................79
0101-1251.............................................79
0101-1252.............................................79
0101-1253.............................................77
0101-1254.............................................77
0101-1255.............................................77
0101-1258.............................................75
0101-1267......................................61, 73
0101-1268......................................61, 73
0101-1288.......................................74-75
0101-1360.............................................75
0101-1362.............................................75
0101-1385......................................61, 73
0101-1409 .......................74-75, 77, 102
0101-1415.......................................74-75
0101-1416.............................61, 73, 101
0101-1417 ................................74-75, 77
0101-1421.......................................74-75
0101-1422......................................61, 73
0101-2415......................................61, 73
01018-22707 ........................................48
01018-23702 ........................................11
01018-60025 ........................................54
01018-68722 ........................................59
01048-87302 ........................................28
01078-87302..................................32, 64
01078-87305 ........................................28
01080-68702 ........................................15
01080-68704 ........................................15
01080-83202 ........................................40
01090-27609 ........................................12
01090-68702 ........................................12
01090-68703 ........................................12
01090-87304 ........................................28
01090-87306..................................23, 33
01090-87610 ........................................23
01090-87611 ........................................23
01100-68700 ........................................11
05971-80103......................................106
05980-60051......................................108
0890-1727 ..........................................107
0890-1761.............................................34
0890-1762.............................................34
0890-1763....................................34, 102
0890-1915....................................34, 106
0905-1163 ..........................................199
0905-1175.............................................52
0905-1192.............................................48
0905-1294.............................................64
0905-1420.............................................51
0905-1516.............................................54
0905-1599.............................................64
0905-1717.............................................64
0905-1718.............................................52
0905-1719.............................................51
0905-1731 ..........................................101
12102300............................................349
12102301............................................349
126-1012.............................................187
126-1013.............................................187
126-1713.............................................187
127-1012.............................................187
127-1712.............................................187
127-1713.............................................187
1400-0563 ..........................................107
14251921............................................349
1460-2571 .........................104-105, 108
1520-0401 ..........................................195
1535-4045 ................................74-75, 77
1535-4046.............................................77
1535-4860.............................................79
1535-4970..................................104, 107
1535-5045.............................................77
1535-5082.............................................77
160-2644-5 .........................................181
160-2650-5 .........................................181
160-2660-5 .........................................181
190-0131.............................................181
190-0231.............................................181
190-0331.............................................181
190-0431.............................................181
191-1311.............................................177
191-3211.............................................177
191-5211.............................................177
192-1311.............................................176
192-3211.............................................176
192-5211.............................................176
194-8111.............................................186
196-7203.............................................186
197-7202.............................................186
199-2602.............................................186
204310 ................................................442
2140-0585 ..........................................199
2140-0590.............................................90
2140-0600.............................................99
2140-0813.............................................90
2140-0820.............................................90
280959-904 ..........................................12
280959-907 ..........................................12
3150-0509.............................................13
3150-0576.............................................13
3150-0577.............................................13
3150-0619 ..........................................199
3150-0944.............................................54
3162-0178 ..........................................106
3162-1056 ..........................................107
3162-1057 ..........................................107
400510 ................................................442
410910-101 ........................................313
410910-102 ........................................313
410910-301 ........................................313
410910-302 ........................................313
410910-501 ........................................313
410910-502 ........................................313
413910-101 ........................................313
413910-102 ........................................313
413910-301 ........................................313
413910-302 ........................................313
413910-501 ........................................313
413910-502 ........................................313
419910-301 ........................................313
419910-302 ........................................313
419910-501 ........................................313
419910-502 ........................................313
420212-901 ........................................313
420212-902 ........................................313
420420-901 ........................................313
420910-901 ........................................313
420910-902 ........................................313
440905-901 ........................................313
440905-902 ........................................313
440910-901 ........................................313
440910-902 ........................................313
443905-101 ........................................313
443905-102 ........................................313
443905-901 ........................................313
443905-902 ........................................313
443910-901 ........................................313
443910-902 ........................................313
446905-101 ........................................313
446905-102 ........................................313
446905-301 ........................................313
446905-302 ........................................313
446905-901 ........................................313
446905-902 ........................................313
446910-102 ........................................313
449905-101 ........................................313
449905-102 ........................................313
449905-301 ........................................313
449905-302 ........................................313
449905-501 ........................................313
449905-502 ........................................313
449905-901 ........................................313
449905-902 ........................................313
449910-902 ........................................313
5001-3702.............................................82
5001-3726.............................................14
5001-3743.............................................52
5021-1816.............................................28
5021-1817.............................................28
5021-1818.............................................28
5021-1819.......................................28-29
5021-1820.............................................27
5021-1821.............................................27
5021-1822.............................................27
5021-1823......................................27, 92
5021-1845..................................262, 270
5021-1866.............................................11
5022-2133.............................................42
5022-2141 ..........................................195
5022-2144.............................................42
5022-2145.............................................42
5022-2146.............................................95
5022-2155.............................................58
5022-2159.............................................27
5022-2165.............................................12
5022-2166.............................................12
5022-2175.............................................64
5022-2184......................................42, 95
5022-2185.............................................12
5022-2188.............................................51
5022-2192......................................48, 54
5022-6503.............................................82
5022-6509.............................................29
5022-6510.............................................29
5022-6531.............................................69
5022-6532.............................................69
5022-6533.............................................69
5022-6534.............................................69
5022-6536.............................................41
5022-6538......................................65, 69
5022-6539......................................65, 69
5022-6541.............................................71
5022-6542.............................................71
5022-6543.............................................71
5022-6544.............................................71
5022-6546.............................................71
5023-0208.............................................71
5023-0209.............................................71
5023-0213.............................................71
5023-0214.............................................71
5023-0215.............................................71
5023-0238.............................................71
5023-0271.............................................12
PART NUMBER INDEX
5023-0282.............................................11
5023-1803.............................................42
5041-2168....................................54, 199
5042-1385.............................................69
5042-1386.............................................69
5042-1388.............................................69
5042-1389.............................................69
5042-6454.............................................69
5042-6458.............................................69
5042-6459.............................................69
5042-6461.............................................34
5042-6462.............................................34
5042-6463.............................................34
5042-6470.............................................69
5042-6476.............................................15
5042-6478 ..........................................199
5042-6491 ..........................................199
5042-6500.............................................40
5042-8502.............................................69
5042-8507.............................................52
5042-8517......................................42, 71
5042-8518......................................42, 71
5042-8519......................................42, 71
5042-8922.............................................58
5042-8954.............................................52
5042-8957.............................................41
5042-9954.............................................11
5042-9967.............................................11
5043-0221.............................................56
5043-0222.............................................56
5043-0223.............................................56
5043-0224.............................................56
5043-0225.............................................56
5043-0226.............................................56
5043-0227.............................................56
5043-0228.............................................57
5043-0229.............................................57
5043-0230.............................................57
5043-0231.............................................57
5043-0232.......................................56-57
5043-0233.............................................57
5043-0234.............................................56
5043-0235.............................................57
5043-0236.............................................57
5043-0237.............................................57
5043-0238.............................................57
5043-0239.............................................57
5043-0242.............................................56
5043-0243.............................................56
5043-0255.............................................57
5043-0272.............................................56
5043-0300.............................................56
5043-0828.............................................56
5043-0829.............................................56
5043-0830.............................................56
5043-0831.............................................56
5043-0832.............................................56
5061-3303......................................40, 99
5061-3304.............................................39
5061-3315.............................................39
5061-3327.............................................99
5061-3328.............................................99
5061-3329.............................................99
5061-3330 ..........................................395
5061-3331 ..........................................395
5061-3332 ..........................................395
5061-3333 ..........................................395
5061-3334 ..........................................395
5061-3335 ..........................................395
5061-3337 ..........................................395
5061-3339 ..........................................395
5061-3361.............................................35
5061-3362.............................................35
5062-2418.............................................40
5062-2461.............................................23
5062-2462....................23-24, 33-34, 99
5062-2463......................................34, 99
5062-2478 ..........................................395
5062-2479 ..........................................395
5062-2483......................................34, 58
5062-2484.............................................52
5062-2486.............................................11
5062-8517....................................54, 199
5062-8522......................................23, 92
5062-8524..................................172, 175
5062-8529 ..........................................192
5062-8534.............................................58
5062-8535 ...............................23, 33, 92
5062-8541.............................................40
5062-8544 ..........................................199
5062-8562.............................................49
5062-8571 ..........................................196
5062-8572 ..........................................196
5062-8573 ..........................................196
5062-8574 ..........................................197
5062-8575..................................172, 196
5062-8576.........................172, 174, 196
5062-8577 ..........................................196
5062-8578 .........................172-175, 196
5062-8587.............................................64
5062-8588.............................................82
5063-6502.............................................77
5063-6506.............................................63
5063-6510 ..........................................174
5063-6511 ..........................................172
5063-6512 ..........................................188
5063-6513 ..........................................188
5063-6514 ..........................................198
5063-6515 ..........................................198
5063-6520 ..........................................198
5063-6526.............................................82
5063-6531.............................................53
5063-6535 ..........................................188
5063-6536 ..........................................188
5063-6540 ..........................................188
5063-6541 ..........................................188
5063-6544 ..........................................188
5063-6586......................................50, 64
5063-6589......................................51, 64
5063-6591.............................................40
5063-6592.............................................95
5063-6593......................................41, 95
5063-6597......................................15, 99
5063-6598.............................................58
5063-6599.............................................58
5064-8203 ..........................................173
5064-8205 ..........................................173
5064-8206 ..........................................173
5064-8208 ..........................................175
5064-8209 ..........................................175
5064-8211.............................................11
5064-8220.............................................15
5064-8236 ..........................................197
5064-8253 ..........................................271
5064-8254 ..........................................271
5064-8255 ..........................................271
5064-8256 ..........................................271
5064-8257 ..........................................271
5064-8258..................................271, 460
5064-8259..................................373, 460
5064-8260 ..........................................271
5064-8261 ..........................................271
5064-8262 ..........................................271
5064-8263..................................373, 460
5064-8264..................................373, 460
5064-8265..................................373, 460
5064-8266..................................373, 460
5064-8267..................................373, 460
5064-8268..................................373, 460
5064-8269 ..........................................263
5064-8270..................................373, 460
5064-8271 ..........................................263
5064-8273.............................................12
5064-8286 ..........................................263
5064-8287 ..........................................263
5064-8288 ..........................................263
5064-8291 ..........................................263
5064-8293.............................................64
5064-8294..................................373, 460
5064-8295..................................373, 460
5064-8296 ..........................................263
5064-8297 ..........................................263
5064-8298 ..........................................263
5064-8300..................................373, 460
5065-4402.............................................69
5065-4410.............................................41
5065-4420.............................................15
5065-4421.............................................53
5065-4422......................................41, 95
5065-4423.............................................41
5065-4426.............................................40
5065-4427 ..........................................460
5065-4454.............................................40
5065-4459..................................373, 460
5065-4460..................................373, 460
5065-4461..................................373, 460
5065-4462..................................373, 460
5065-4463..................................373, 460
5065-4464..................................379, 460
5065-4465..................................379, 460
5065-4466..................................379, 460
5065-4467..................................379, 460
5065-4468..................................384, 460
5065-4498.............................................66
5065-4499.............................................60
5065-4500.............................................12
5065-9901.............................................12
5065-9908 ..........................................110
5065-9910..................................373, 460
5065-9911..................................373, 460
5065-9912..................................458, 460
5065-9913..................................373, 460
5065-9914..................................373, 460
5065-9915..................................373, 460
5065-9922.............................................60
5065-9923..................................373, 460
5065-9924..................................373, 460
5065-9926.............................................29
5065-9927.............................................29
5065-9931.............................................28
5065-9932.............................................28
5065-9933.......................................28-29
5065-9935.............................................27
5065-9937.............................................36
5065-9938.............................................36
5065-9939.............................................37
5065-9942..................................458, 460
5065-9947......................................37, 82
5065-9948......................................35, 41
5065-9950......................................35, 41
5065-9952.............................................52
5065-9963.............................................28
5065-9964.............................................27
5065-9967.............................................40
5065-9971......................................34, 42
5065-9976.............................................34
WWW.AGILENT.COM/CHEM/LC
5065-9978......................................34, 52
5067-1540.............................................41
5067-1547.............................................41
5067-1551.............................................12
5067-1553.............................................12
5067-1555.............................................12
5067-1557.............................................41
5067-1558.............................................41
5067-1562.............................................12
5067-1565.............................................54
5067-1581.............................................77
5067-1582 ..........................................100
5067-1584 ..........................................100
5067-1585 ..........................................100
5067-1595.............................................84
5067-1596.............................................85
5067-1597.............................................85
5067-4104.............................................77
5067-4105.............................................77
5067-4107.............................................75
5067-4108.............................................75
5067-4111.............................................75
5067-4112.............................................75
5067-4113.............................................75
5067-4114......................................61, 73
5067-4117.......................................74-75
5067-4118.......................................74-75
5067-4121.............................................75
5067-4124.............................................51
5067-4131....................................74, 101
5067-4132.......................................74-75
5067-4134.......................................74-75
5067-4137.............................................75
5067-4141.............................................77
5067-4142.......................................74-75
5067-4144.......................................74-75
5067-4146.......................................74-75
5067-4148.......................................74-75
5067-4158.............................................77
5067-4159..............................74-75, 102
5067-4170.............................................75
5067-4174.............................................48
5067-4202.............................................77
5067-4601.......................................83-85
5067-4607.............................................29
5067-4608.......................................28-29
5067-4609.............................................29
5067-4633.............................................37
5067-4638.............................................12
5067-4646 ...............................38, 84, 86
5067-4647.............................................25
5067-4648.............................................28
5067-4649.............................................25
5067-4650.............................................25
5067-4651.............................................25
5067-4653.............................................26
5067-4657.............................................24
5067-4658.............................................24
5067-4659.............................................24
5067-4660.............................................24
5067-4661.............................................51
5067-4662.............................................67
5067-4669.............................................24
5067-4670.............................................24
5067-4678.............................................50
5067-4682......................................38, 86
5067-4684.............................................25
5067-4685.............................................28
5067-4686.............................................25
5067-4687.............................................25
5067-4688.............................................28
5067-4689.............................................26
647
PART NUMBER INDEX
5067-4695.....................50, 64, 101-102
5067-4697.............................................51
5067-4699.............................................59
5067-4703......................................32, 64
5067-4710......................................32, 64
5067-4716.............................................51
5067-4717.............................................51
5067-4728.......................................48-49
5067-4729......................................39, 87
5067-4730.............................................87
5067-4733.............................................40
5067-4735.............................................25
5067-4737.............................................28
5067-4738.............................................40
5067-4739.............................................40
5067-4741....................................42, 102
5067-4744.............................................25
5067-4745.............................................25
5067-4746.............................................26
5067-4767.............................................88
5067-4769.............................................88
5067-4777.............................................29
5067-4778.............................................29
5067-4779.............................................29
5067-4780.............................................29
5067-4781.............................................29
5067-4782.............................................29
5067-4798.............................................58
5067-4800.............................................87
5067-5103......................................38, 88
5067-5104.............................................25
5067-5106.............................................25
5067-5107.............................................25
5067-5109.............................................26
5067-5110.............................................26
5067-5111.............................................26
5067-5112.............................................26
5067-5113.............................................26
5067-5120.............................................28
5067-5189.............................................39
5067-5378.............................................58
5067-5380.............................................58
5067-5383.............................................58
5068-0001.............................................75
5068-0002.............................................75
5068-0004.............................................48
5068-0005.............................................48
5068-0006.......................................74-75
5068-0007......................................61, 73
5068-0008.......................................74-75
5068-0011.......................................74-75
5068-0012.......................................74-75
5068-0040..............................74-75, 102
5068-0041..............................74-75, 102
5068-0044.......................................74-75
5068-0045..............................74-75, 102
5068-0052.............................................77
5068-0053.............................................77
5068-0060 ...............74-75, 77, 101-102
5068-0067.......................................74-75
5068-0076.......................................74-75
5068-0077.......................................74-75
5068-0082.............................................77
5068-0093.......................................74-75
5068-0095.......................................74-75
5068-0097.......................................74-75
5068-0115.............................................75
5068-0116.............................................75
5068-0122.............................................48
5068-0123.............................................48
5069-3635 ..........................................412
5069-3636 ..........................................412
648
5069-3637 ..........................................412
5069-3639 ..........................................435
5080-5400 ..........................................108
5133001 ..............................................349
5133005 ..............................................349
5180-4108......................................40, 99
5180-4114......................................40, 99
5181-1507 ..........................................199
5181-1512 ..........................................199
5181-1513 ..........................................199
5181-1541.............................................71
5181-8836 ..........................................200
5182-0567 ..........................................199
5182-1530.............................................90
5182-9697 ..........................................199
5182-9710 ..........................................106
5183-2021 ..........................................285
5183-2022 ..........................................285
5183-4619 ..........................................199
5183-4623 ..........................................199
5183-4624 ..........................................285
5183-4625 ..........................................285
5183-4626 ..........................................285
5183-4627 ..........................................285
5183-4669 ..........................................200
5183-4670 ..........................................200
5185-5807 ..........................................285
5185-5808 ..........................................285
5185-5809 ..........................................285
5185-5810 ..........................................285
5185-5920.........................269, 372, 463
5185-5921 ..........................................261
5185-5922 ..........................................282
5185-5923 ..........................................277
5185-5968..................................384, 463
5185-5984 ..........................................439
5185-5985 ..........................................439
5185-5986 ..........................................441
5185-5987 ..........................................441
5185-5988 ..........................................441
5185-5990 ..........................................441
5185-5991 ..........................................441
5188-2743.............................................13
5188-2744.............................................13
5188-2745.............................................13
5188-2746.............................................13
5188-5217 ..........................................439
5188-5218 ..........................................439
5188-5230 ..........................................439
5188-5231 ..........................................442
5188-5249 ..........................................441
5188-5250 ..........................................441
5188-5251 ..........................................441
5188-5252 ..........................................441
5188-5253 ..........................................441
5188-5254 ..........................................441
5188-5289 ..........................................439
5188-5321.............................................69
5188-5322.............................................69
5188-5332 ..........................................439
5188-5333 ..........................................439
5188-5334 ..........................................439
5188-5336 ..........................................439
5188-5341 ..........................................439
5188-6408 ..........................................439
5188-6409 ..........................................439
5188-6410 ..........................................439
5188-6411 ..........................................439
5188-6510 ..........................................442
5188-6511 ..........................................442
5188-6523 ..........................................110
5188-6529.............................................15
5188-6557 ..........................................439
5188-6558 ..........................................439
5188-6559 ..........................................439
5188-6560 ..........................................439
5188-6562 ..........................................439
5188-8283 ..........................................441
5188-8825 ..........................................439
5188-8826 ..........................................439
5190-0443 ..........................................110
5190-0469 ..........................................110
5190-0488.............................................15
5190-0551 ..........................................110
5190-0554 ..........................................110
5190-0555 ..........................................110
5190-0556 ..........................................110
5190-0917....................................90, 199
5190-0924.............................................11
5190-1401 ..........................................108
5190-1431 ..........................................284
5190-1432 ..........................................284
5190-1433 ..........................................284
5190-1434 ..........................................284
5190-1435 ..........................................284
5190-1436 ..........................................284
5190-1443 ..........................................104
5190-1480.............................................80
5190-1484.............................................80
5190-1485.............................................80
5190-1486.......................................80-81
5190-1492.............................................81
5190-1494.............................................80
5190-1499.............................................81
5190-1501.............................................80
5190-1505.............................................81
5190-1508.............................................80
5190-1512.............................................81
5190-1515.............................................80
5190-1520.............................................81
5190-1522.............................................80
5190-1526.............................................81
5190-1558.............................................81
5190-1560.............................................81
5190-1561.............................................81
5190-1562.............................................81
5190-1564.............................................81
5190-1571.............................................81
5190-2401 ..........................................401
5190-2402 ..........................................401
5190-2403 ..........................................401
5190-2404 ..........................................401
5190-2405 ..........................................401
5190-2406 ..........................................401
5190-2407 ..........................................401
5190-2408 ..........................................401
5190-2411 ..........................................401
5190-2412 ..........................................401
5190-2413 ..........................................401
5190-2414 ..........................................401
5190-2415 ..........................................401
5190-2416 ..........................................401
5190-2419 ..........................................401
5190-2420 ..........................................401
5190-2421 ..........................................405
5190-2422 ..........................................405
5190-2423 ..........................................405
5190-2424 ..........................................405
5190-2425 ..........................................405
5190-2426 ..........................................405
5190-2427 ..........................................405
5190-2428 ..........................................405
5190-2431 ..........................................405
5190-2432 ..........................................405
5190-2433 ..........................................405
5190-2434 ..........................................405
5190-2435 ..........................................405
5190-2436 ..........................................405
5190-2439 ..........................................405
5190-2440 ..........................................405
5190-2441 ..........................................405
5190-2442 ..........................................405
5190-2443 ..........................................405
5190-2444 ..........................................405
5190-2445 ..........................................405
5190-2446 ..........................................405
5190-2447 ..........................................405
5190-2448 ..........................................405
5190-2451 ..........................................405
5190-2452 ..........................................405
5190-2453 ..........................................405
5190-2454 ..........................................405
5190-2455 ..........................................405
5190-2456 ..........................................405
5190-2459 ..........................................405
5190-2460 ..........................................405
5190-2461 ..........................................405
5190-2462 ..........................................405
5190-2463 ..........................................405
5190-2464 ..........................................405
5190-2465 ..........................................405
5190-2466 ..........................................405
5190-2467 ..........................................405
5190-2468 ..........................................405
5190-2471 ..........................................405
5190-2472 ..........................................405
5190-2473 ..........................................405
5190-2474 ..........................................405
5190-2475 ..........................................405
5190-2476 ..........................................405
5190-2479 ..........................................405
5190-2480 ..........................................405
5190-2481 ..........................................405
5190-2482 ..........................................405
5190-2483 ..........................................405
5190-2484 ..........................................405
5190-2485 ..........................................405
5190-2486 ..........................................405
5190-2487 ..........................................405
5190-2488 ..........................................405
5190-2491 ..........................................405
5190-2492 ..........................................405
5190-2493 ..........................................405
5190-2494 ..........................................405
5190-2495 ..........................................405
5190-2496 ..........................................405
5190-2499 ..........................................405
5190-2500 ..........................................405
5190-2501 ..........................................423
5190-2502 ..........................................423
5190-2503 ..........................................423
5190-2504 ..........................................423
5190-2505 ..........................................423
5190-2506 ..........................................423
5190-2507 ..........................................423
5190-2508 ..........................................423
5190-2509 ..........................................423
5190-2510 ..........................................423
5190-2511 ..........................................423
5190-2512 ..........................................423
5190-2513 ..........................................423
5190-2514 ..........................................423
5190-2515 ..........................................423
5190-2516 ..........................................427
5190-2517 ..........................................427
5190-2518 ..........................................427
PART NUMBER INDEX
5190-2519 ..........................................427
5190-2520 ..........................................427
5190-2521 ..........................................427
5190-2522 ..........................................427
5190-2523 ..........................................427
5190-2524 ..........................................427
5190-2525 ..........................................427
5190-2526 ..........................................427
5190-2527 ..........................................427
5190-2528 ..........................................427
5190-2529 ..........................................427
5190-2530 ..........................................427
5190-2531 ..........................................427
5190-2532 ..........................................427
5190-2533 ..........................................427
5190-2534 ..........................................427
5190-2535 ..........................................427
5190-2536 ..........................................427
5190-2537 ..........................................427
5190-2538 ..........................................427
5190-2539 ..........................................427
5190-2540 ..........................................427
5190-2541 ..........................................427
5190-2542 ..........................................427
5190-2543 ..........................................427
5190-2544 ..........................................427
5190-2545 ..........................................427
520518-904 ........................................305
520518-905 ........................................305
588905-902 ........................................305
588915-902 ........................................305
588925-902 ........................................305
588935-902 ........................................305
590-3003.............................................200
590-4000....................................177, 197
590-4001....................................177, 197
590-4005....................................177, 197
59980-20134......................................107
59987-20033......................................107
59987-20040......................................105
6040-0798 ..........................................107
6040-0834..................................104, 107
660750-902 ...............................210, 384
660750-906 ........................................384
660750-909 ........................................384
661750-902 ...............................384, 463
661750-906 ...............................384, 463
661750-909 ...............................384, 463
670750-902 ........................................384
671750-902 ...............................384, 463
681775-902 ........................................229
681975-302 ........................................229
681975-902 ........................................229
683775-902 ...............................229, 386
683775-906 ........................................229
683775-914 ........................................229
683975-302 ...............................229, 386
683975-306 ........................................229
683975-314 ........................................229
683975-902 ...............................229, 386
683975-906 ........................................229
683975-914 ........................................229
685775-902 ...............................229, 386
685775-906 ........................................229
685775-914 ........................................229
685975-302 ...............................229, 386
685975-306 ........................................229
685975-314 ........................................229
685975-902 ...............................229, 386
685975-906 ........................................229
685975-914 ........................................229
687775-902 ........................................229
687975-302 ........................................229
687975-902 ........................................229
689775-902 ........................................229
689775-906 ........................................229
689775-914 ........................................229
689975-302 ........................................229
689975-306 ........................................229
689975-314 ........................................229
689975-902 ........................................229
689975-906 ........................................229
689975-914 ........................................229
691775-902 ........................................229
691775-906 ........................................229
691975-302 ........................................229
691975-306 ........................................229
691975-902 ........................................229
691975-906 ........................................229
693768-901 ........................................229
693775-901 ........................................325
693775-902 ...............................229, 386
693775-906 ........................................229
693775-912 ........................................229
693968-301 ........................................229
693968-901 ........................................229
693975-301 ........................................325
693975-302 ...............................229, 386
693975-306 ........................................229
693975-312 ........................................229
693975-901 ........................................325
693975-902 ...............................229, 386
693975-906 ........................................229
693975-912 ........................................229
695768-901 ........................................229
695775-901 ........................................325
695775-902 ...............................229, 386
695775-906 ........................................229
695775-912 ........................................229
695968-301 ........................................229
695968-901 ........................................229
695975-302 ...............................229, 386
695975-306 ........................................229
695975-312 ........................................229
695975-901 ........................................325
695975-902 ......................210, 229, 386
695975-906 ........................................229
695975-912 ........................................229
697775-902 ........................................229
697775-906 ........................................229
697975-302 ........................................229
697975-306 ........................................229
697975-902 ...............................210, 229
697975-906 ........................................229
699768-901 ........................................229
699775-901 ........................................325
699775-902 ........................................229
699775-906 ........................................229
699775-912 ........................................229
699968-301 ........................................229
699968-901 ........................................229
699975-301 ........................................325
699975-302 ........................................229
699975-306 ........................................229
699975-312 ........................................229
699975-901 ........................................325
699975-902 ........................................229
699975-906 ........................................229
699975-912 ........................................229
722975-902 ........................................245
724700-902 ...............................243, 277
724975-302 ...............................243, 277
724975-902 ...............................243, 276
726700-902 ...............................243, 277
726975-302 ...............................243, 277
726975-902 ...............................243, 276
727700-902 ...............................243, 277
727975-302 ...............................243, 277
727975-902 ...............................243, 276
728700-902 ...............................243, 277
728975-302 ...............................243, 277
728975-902 ...............................243, 276
735700-902 ........................................277
735953-902 ........................................276
735954-302 ........................................276
746450-902 ........................................276
75400001............................................349
75700001............................................349
7571901C ...........................................349
75719025............................................349
75719050............................................349
7572915B ...........................................349
7572915C ...........................................349
7573915B ...........................................349
7573915C ...........................................349
757700-302 ...............................238, 277
757700-902 ...............................238, 277
758700-302 ...............................238, 277
758700-902 ...............................238, 277
759700-302 ........................................238
759700-902 ...............................238, 277
760450-902 ........................................277
761600-902 ........................................277
761753-902 ........................................277
761775-902 ........................................379
761973-902 ........................................379
763600-902 ........................................277
763750-902 ........................................379
763953-902 ........................................276
763954-302 ........................................276
763973-902 ........................................379
764953-302 ........................................276
764953-902 ........................................276
765600-902 ........................................277
765750-902 ........................................379
765973-902 ........................................379
766953-902 ........................................276
770050-902 ...............................277, 318
770100-902 ...............................277, 318
770150-902 ...............................277, 318
770450-302 ........................................276
770450-902 ........................................276
770995-902 ........................................379
773450-302 ........................................276
773450-902 ........................................276
773700-902 ........................................277
773995-902 ........................................379
79835-87638 ........................................29
79841-87610 ........................................27
7995108-344......................................262
7995108-585......................................262
7995108-595......................................262
7995118-344......................................262
7995118-504 .............................262, 270
7995118-585......................................262
7995118-595......................................262
7995208-344......................................270
7995208-585......................................270
7995208-595......................................270
7995218-344......................................270
7995218-585..............................270-271
7995218-595......................................270
7995230-344......................................262
8001-0401 ..........................................150
8001-0402 ..........................................150
8001-0403 ..........................................150
WWW.AGILENT.COM/CHEM/LC
8001-0405 ..........................................150
8001-0406 ..........................................150
8001-0501 ..........................................151
8001-0502 ..........................................151
8001-0503 ..........................................151
8001-0504 ..........................................151
8001-0506 ..........................................152
8001-0509 ..........................................154
8001-0510 ..........................................152
8001-0511 ..........................................152
8001-0512 ..........................................152
8001-0513 ..........................................152
8001-0514 ..........................................151
8001-0515 ..........................................151
8001-0516 ..........................................152
8001-0517 ..........................................154
8001-0519 ..........................................152
8001-0520 ..........................................152
8001-0521 ..........................................152
8001-0522 ..........................................151
8001-0527 ..........................................152
8001-0528 ..........................................152
8001-0529 ..........................................152
8001-0530 ..........................................152
8001-0531 ..........................................152
8001-0532 ..........................................152
8001-0533 ..........................................151
8001-0534 ..........................................151
8001-0535 ..........................................152
8001-0601 ..........................................153
8001-0603 ..........................................153
8001-0604 ..........................................153
8001-0607 ..........................................153
8001-0608 ..........................................153
8001-0609 ..........................................153
8001-0610 ..........................................154
8001-0612 ..........................................153
8001-0613 ..........................................153
8001-0614 ..........................................153
8001-0615 ..........................................152
8001-0701 ..........................................150
8001-0702 ..........................................150
8001-0703 ..........................................150
8001-0704 ..........................................150
8001-0705 ..........................................150
8001-0801 ..........................................153
8001-0802 ..........................................153
8001-0803 ..........................................155
8001-0805 ..........................................155
8001-0806 ..........................................155
8001-0807 ..........................................155
8001-0808 ..........................................156
8001-0809 ..........................................153
8001-0810 ..........................................154
8001-0812 ..........................................153
8001-0813 ..........................................155
8001-0814 ..........................................153
8001-0816 ..........................................155
8001-0817 ..........................................155
8001-0818 ..........................................154
8001-0819 ..........................................155
8001-0821 ..........................................154
8001-0822 ..........................................154
8001-0823 ..........................................155
8001-0824 ..........................................155
8001-8020 ..........................................154
8002-0401 ..........................................157
8002-0402 ..........................................157
8002-0403 ..........................................157
8002-0404 ..........................................157
8002-0405 ..........................................157
8002-0406 ..........................................157
649
PART NUMBER INDEX
8002-0407 ..........................................157
8002-0408 ..........................................157
8002-0412 ..........................................157
8002-0413 ..........................................157
8002-0414 ..........................................157
8002-0415 ..........................................157
8002-0501 ..........................................158
8002-0502 ..........................................158
8002-0515 ..........................................158
8002-0516 ..........................................159
8002-0517 ..........................................159
8002-0601 ..........................................158
8002-0602 ..........................................159
8002-0603 ..........................................159
8002-0604 ..........................................159
8002-0605 ..........................................159
8002-0607 ..........................................159
8002-0608 ..........................................159
8002-0610 ..........................................165
8002-0611 ..........................................165
8002-0701 ..........................................157
8002-0702 ..........................................157
8002-0703 ..........................................157
8002-0704 ..........................................157
8002-0705 ..........................................157
8002-0706 ..........................................157
8002-0802 ..........................................168
8002-0803 ..........................................168
8002-0805 ..........................................168
8002-0806 ..........................................168
8002-0808 ..........................................158
8002-0809 ..........................................158
8002-0810 ..........................................158
8002-0811 ..........................................160
8002-0815 ..........................................167
8002-0816 ..........................................167
8002-0817 ..........................................167
8002-0818 ..........................................166
8002-0819 ..........................................166
8002-0820 ..........................................166
8002-0821 ..........................................166
8002-0822 ..........................................166
8002-0823 ..........................................166
8002-0824 ..........................................166
8002-0825 ..........................................166
8002-0826 ..........................................166
8002-0831 ..........................................166
8002-0832 ..........................................166
8002-0833 ..........................................166
8002-0834 ..........................................166
8002-0835 ..........................................167
8002-0837 ..........................................166
8002-0856 ..........................................160
8002-0857 ..........................................160
8002-0858 ..........................................160
8002-0859 ..........................................160
8002-0860 ..........................................160
8002-0901 ..........................................164
8002-0902 ..........................................164
8002-0903 ..........................................164
8002-0904 ..........................................163
8002-0905 ..........................................164
8002-0906 ..........................................163
8002-0907 ..........................................161
8002-0908 ..........................................161
8002-0909 ..........................................162
8002-0910 ..........................................162
8002-0911 ..........................................161
8002-0912 ..........................................162
8002-0913 ..........................................163
8002-0915 ..........................................165
8002-0916 ..........................................165
650
8002-0917 ..........................................161
8002-0918 ..........................................162
8002-0919 ..........................................162
8002-0921 ..........................................168
8002-0923 ..........................................164
8002-0924 ..........................................164
8005-0414 ..........................................138
8005-0416 ..........................................138
8005-0417 ..........................................138
8005-0418 ..........................................138
8005-0419 ..........................................138
8005-0420 ..........................................138
8005-0422 ..........................................138
8005-0423 ..........................................138
8005-0508 ..........................................141
8005-0512 ..........................................142
8005-0513 ..........................................141
8005-0514 ..........................................139
8005-0515 ..........................................140
8005-0516 ..........................................140
8005-0523 ..........................................140
8005-0524 ..........................................141
8005-0525 ..........................................141
8005-0526 ..........................................141
8005-0527 ..........................................140
8005-0528..................................142, 145
8005-0529 ..........................................140
8005-0530 ..........................................140
8005-0531 ..........................................140
8005-0532 ..........................................142
8005-0533 ..........................................140
8005-0535 ..........................................139
8005-0536 ..........................................139
8005-0537 ..........................................141
8005-0538 ..........................................139
8005-0539 ..........................................140
8005-0540 ..........................................140
8005-0541 ..........................................139
8005-0601 ..........................................142
8005-0602 ..........................................142
8005-0603 ..........................................142
8005-0604 ..........................................142
8005-0605 ..........................................142
8005-0702 ..........................................137
8005-0704 ..........................................137
8005-0705 ..........................................137
8005-0812 ..........................................148
8005-0822 ..........................................148
8005-0823 ..........................................148
8005-0824 ..........................................148
8005-0825 ..........................................148
8005-0826 ..........................................148
8005-0835 ..................................148-149
8005-0836 ..........................................149
8005-0837 ..........................................149
8005-0838.........................143, 149, 156
8005-0839 ..........................................143
8005-0840 ..........................................143
8005-0841 ..........................................143
8005-0842 ..........................................143
8005-0843 ..........................................143
8005-0844 ..........................................143
8005-0845 ..........................................143
8005-0846 ..........................................143
8005-0901 ..........................................146
8005-0902 ..........................................146
8005-0903 ..........................................146
8005-0904 ..........................................146
8005-0905 ..........................................147
8005-0906 ..........................................147
8005-0907 ..........................................146
8005-0908 ..........................................147
8005-0911 ..........................................145
8005-0912 ..........................................145
8005-0913 ..........................................144
8005-0914 ..........................................146
8005-0915 ..........................................144
8005-0916 ..........................................144
8005-0925 ..........................................145
8005-0926 ..........................................145
8005-0927 ..........................................146
8005-0928 ..........................................144
8005-0929 ..........................................145
8005-0930 ..........................................144
8010-0440 ..........................................169
8010-0441..................................137, 169
8010-0442..................................137, 169
8010-0443..................................137, 169
8010-0444..................................137, 169
8010-0445..................................137, 169
8010-0446..................................137, 169
8010-0448..................................137, 169
8010-0449 ..........................................169
8010-0450..................................137, 169
8010-0455..................................137, 169
8010-0456..................................137, 169
8010-0457..................................137, 169
8010-0458..................................137, 169
8010-0459..................................137, 169
8010-0460..................................137, 169
8010-0467..................................137, 169
8010-0468..................................137, 169
820212-911 ........................................433
820212-914 ...............................273, 318
820212-915 ......................269, 273, 317
820212-918 ......................317, 373, 466
820212-919 ...............................318, 329
820212-920 ...............................269, 317
820212-921 ......................317, 373, 466
820212-924 ......................317, 373, 466
820212-925 ...............................261, 318
820212-926 ...............................261, 318
820212-928 ...............................282, 318
820212-930 ...............................277, 318
820212-933 ...............................269, 317
820385-901 ........................................318
820400-901 ............261, 269, 273, 277,
282-283, 313, 317-318,
329-330, 373, 433, 466
820444-901 ............261, 269, 273, 277,
282, 313, 317-318,
329-330, 373, 433, 466
820555-901 ......................245, 263, 270
820675-111 ...............................330, 433
820675-112 ........................................261
820675-115 ......................269, 273, 283
820675-119 ................................329-330
820675-124 ......................269, 283, 372
820700-902 ...............................244, 269
820700-905 ...............................244, 269
820700-906 ...............................244, 269
820700-912 ...............................244, 269
820750-901 ......................243, 246, 252
820750-902 ......................244, 246, 267
820750-903 ......................243, 246, 260
820750-904 ......................244, 246, 267
820750-911 ...............................229, 246
820750-912 ...............................229, 246
820750-913 ...............................229, 246
820750-914 ...............................229, 246
820950-901 ........................................330
820950-902 ........................................283
820950-905 ...............................283, 330
820950-906 ........................................283
820950-908 ........................................330
820950-911 ........................................433
820950-912 ........................................283
820950-913 ........................................273
820950-914 ........................................273
820950-915 ........................................269
820950-916 ........................................269
820950-917 ........................................269
820950-918 ........................................372
820950-919 ........................................329
820950-920 ........................................269
820950-921 ........................................372
820950-922 ........................................269
820950-923 ........................................372
820950-924 ...............................283, 372
820950-925 ........................................261
820950-926 ........................................261
820950-927 ........................................261
820950-928 ........................................282
820950-930 ........................................277
820950-931 ........................................394
820950-932 ........................................379
820950-933 ........................................269
820950-935 ...............................261, 329
820950-936 ........................................253
820950-937 ........................................253
820950-938 ........................................253
820950-939 ...............................253, 255
820999-901 ............225, 253, 255, 261,
269, 273, 277, 282-283,
305, 329-330, 334, 372,
379, 384, 394, 433
821075-918 ........................................384
821075-920 ........................................384
821075-924 ........................................384
821125-915 ...............................269, 273
821125-918 ........................................372
821125-919 ........................................329
821125-924 ...............................269, 372
821125-926 ........................................261
821125-928 ........................................282
821125-930 ........................................277
821125-932 ........................................379
821125-933 ........................................269
821125-935 ...............................261, 329
821125-936 ........................................253
821125-937 ........................................253
821125-938 ........................................253
821125-939 ...............................253, 255
821700-902 ...............................245, 270
821700-932 ...............................245, 270
821725-901 .............238, 243, 246, 253
821725-902 .............238, 244, 246, 269
821725-903 .............238, 243, 246, 261
821725-904 .............238, 244, 246, 269
821725-911 ...............................229, 246
821725-912 ...............................229, 246
821725-913 ...............................229, 246
821725-914 ...............................229, 246
821975-902 ...............................245, 270
821975-932 ...............................245, 270
822700-902 ........................................270
822700-932 ........................................270
822975-902 .......................244-245, 270
822975-906 ...............................245, 270
822975-932 ........................................270
823700-902 ...............................245, 270
823700-932 ...............................245, 270
823750-901 .............238, 243, 246, 253
823750-902 .............238, 244, 246, 268
823750-903 .............238, 243, 246, 260
823750-904 .............238, 244, 246, 268
PART NUMBER INDEX
823750-911 ...............................229, 246
823750-912 ...............................229, 246
823750-913 ...............................229, 246
823750-914 ...............................229, 246
823975-902 ...............................245, 270
823975-932 ...............................245, 270
824700-902 ...............................244, 269
824700-905 ...............................244, 269
824700-906 ...............................244, 269
824700-912 ...............................244, 269
824700-914 ...............................244, 269
824975-302 ...............................244, 268
824975-305 ...............................244, 268
824975-306 ...............................244, 268
824975-902 ...............................244, 267
824975-905 ...............................244, 267
824975-906 ...............................244, 267
824975-912 ...............................244, 267
824975-914 ...............................244, 267
825700-902 ...............................245, 270
825700-932 ...............................245, 270
825975-902 ...............................245, 270
825975-932 ...............................245, 270
826700-902 ...............................244, 269
826700-906 ...............................244, 269
826975-302 ...............................244, 268
826975-306 ...............................244, 268
826975-902 ...............................244, 267
826975-906 ...............................244, 267
827668-301 ...............................244, 281
827668-901 ...............................244, 281
827700-901 ...............................244, 329
827700-902 ...............................244, 269
827700-905 ...............................244, 269
827700-906 ...............................244, 269
827700-912 ...............................244, 269
827700-914 ...............................244, 269
827768-901 ...............................244, 282
827975-301 ...............................244, 329
827975-302 ...............................244, 268
827975-305 ...............................244, 268
827975-306 ...............................244, 268
827975-312 ...............................244, 268
827975-314 ...............................244, 268
827975-901 ........................................244
827975-902 ......................244, 267, 329
827975-905 ...............................244, 267
827975-906 ...............................244, 267
827975-912 ...............................244, 267
827975-914 ...............................244, 267
828668-301 ...............................244, 281
828668-901 ...............................244, 281
828700-901 ...............................244, 329
828700-902 ...............................244, 269
828700-905 ...............................244, 269
828700-906 ...............................244, 269
828700-912 ...............................244, 269
828700-914 ...............................244, 269
828768-901 ...............................244, 282
828975-301 ...............................244, 329
828975-302 ...............................244, 268
828975-305 ...............................244, 268
828975-306 ...............................244, 268
828975-309 ........................................268
828975-312 ...............................244, 268
828975-314 ...............................244, 268
828975-901 ...............................244, 329
828975-902 ...............................244, 267
828975-905 ...............................244, 267
828975-906 ...............................244, 267
828975-912 ...............................244, 267
828975-914 ...............................244, 267
829975-302 ...............................244, 268
829975-305 ...............................244, 268
829975-306 ...............................244, 268
829975-312 ...............................244, 268
829975-902 ...............................244, 267
829975-905 ...............................244, 267
829975-906 ...............................244, 267
829975-912 ...............................244, 267
829975-914 ...............................244, 267
830668-901 ...............................244, 281
830975-906 ...............................244, 267
830990-902 ........................................268
830990-906 ........................................268
830990-914 ........................................268
831975-902 ........................................270
831975-906 ........................................270
831975-932 ........................................270
831975-936 ........................................270
832975-902 ........................................267
832975-906 ........................................267
833975-902 ........................................270
833975-906 ........................................270
833975-912 ........................................270
833975-932 ........................................270
833975-936 ........................................270
834975-902 ........................................267
834975-906 ........................................267
835668-901 ........................................281
835975-902 ........................................267
835975-905 ........................................267
835975-906 ........................................267
835975-912 ........................................267
835975-914 ........................................267
840140-901 ............261, 269, 273, 283,
329-330, 372, 433
840300-908 ........................................330
843300-908 ........................................330
846952-704 ........................................334
846975-202 ........................................267
846975-902 ........................................267
846975-906 ........................................267
846975-914 ........................................267
8500-1867 ..........................................107
8500-2236 ..........................................107
8500-4410 ..........................................195
8500-6762.............................................15
8500-6782 ..........................................196
8500-6785 ..........................................174
8500-6786 ..........................................197
8500-6787 ..........................................197
8500-6797 ..........................................172
8500-6900 ..........................................174
8500-6917 ..........................................110
857700-302 ...............................238, 268
857700-305 ...............................238, 268
857700-306 ...............................238, 268
857700-312 ...............................238, 268
857700-314 ........................................238
857700-902 ...............................238, 268
857700-905 ...............................238, 268
857700-906 ...............................238, 268
857700-912 ...............................238, 268
857700-914 ........................................238
857750-902 ........................................372
857750-906 ........................................372
857750-909 ........................................372
857750-944 ...............................372, 375
857768-901 ...............................238, 281
858700-302 ...............................238, 268
858700-305 ...............................238, 268
858700-306 ...............................238, 268
858700-312 ........................................268
858700-314 ........................................238
858700-902 ...............................238, 268
858700-905 ...............................238, 268
858700-906 ...............................238, 268
858700-912 ...............................238, 268
858700-914 ........................................238
858750-902 ........................................372
858750-906 ........................................372
858750-909 ........................................372
858750-944 .......................372, 374-375
858768-901 ...............................238, 281
859700-302 ...............................238, 268
859700-306 ...............................238, 268
859700-902 ...............................238, 268
859700-905 ...............................238, 268
859700-906 ...............................238, 268
859700-912 ...............................238, 268
859700-914 ........................................238
859768-901 ...............................238, 281
860700-304 ........................................334
860700-704 ........................................334
860700-708 ........................................330
860950-902 ........................................372
860950-905 ........................................372
860950-906 ........................................372
860950-909 ........................................372
860975-902 ........................................268
860975-905 ........................................268
860975-906 ........................................268
860975-909 ........................................268
860975-912 ........................................268
860975-914 ........................................268
861600-902 ........................................269
861600-906 ........................................269
861608-901 ........................................282
861630-902 ...............................372, 463
861700-901 ........................................282
861753-902 ........................................268
861753-905 ........................................268
861753-906 ........................................268
861753-912 ........................................268
861753-914 ........................................268
861767-902 ........................................273
861768-901 ........................................282
861775-902 ........................................372
861775-906 ........................................372
861953-902 ........................................267
861953-905 ........................................267
861953-906 ........................................267
861953-912 ........................................267
861953-914 ........................................267
861954-302 ........................................267
861954-305 ........................................267
861954-306 ........................................267
861954-309 ........................................267
861954-312 ........................................267
861954-314 ........................................267
861967-302 ........................................273
861967-902 ........................................273
861971-901 ........................................281
861973-306 ........................................372
861973-902 ........................................372
861973-906 ........................................372
863600-902 ........................................269
863600-905 ........................................269
863600-906 ........................................269
863608-901 ........................................282
863630-902 ...............................372, 463
863630-906 ...............................372, 463
863668-301 ........................................281
863668-901 ........................................281
863700-901 ........................................282
WWW.AGILENT.COM/CHEM/LC
863750-906 ........................................372
863953-902 ........................................267
863953-905 ........................................267
863953-906 ........................................267
863953-912 ........................................267
863953-914 ........................................267
863954-302 ........................................267
863954-305 ........................................267
863954-306 ........................................267
863954-312 ........................................267
863954-314 ........................................267
863967-302 ........................................273
863967-902 ........................................273
863973-902 ...............................210, 372
863973-905 ........................................372
863973-906 ........................................372
863973-909 ........................................372
863974-302 ........................................372
863974-306 ........................................372
863974-309 ........................................372
864668-301 ........................................281
864668-901 ........................................281
865600-902 ........................................269
865600-906 ........................................269
865608-901 ........................................282
865630-902 ...............................372, 463
865630-906 ...............................372, 463
865750-902 ........................................372
865750-906 ........................................372
865973-902 ...............................210, 372
865973-905 ........................................372
865973-906 ........................................372
865973-909 ........................................372
8660-0827 ..........................................108
866668-901 ........................................281
866735-902 ........................................268
866953-302 ........................................267
866953-902 ........................................267
866953-905 ........................................267
866953-906 ........................................267
866953-912 ........................................267
866953-914 ........................................267
866967-902 ........................................273
868050-901 ...............................282, 318
868100-901 ...............................282, 318
868150-901 ...............................282, 318
870050-902 ...............................269, 317
870050-906 ...............................269, 317
870050-914 ...............................269, 317
870100-902 ...............................269, 317
870100-906 ...............................269, 317
870100-914 ...............................269, 317
870150-902 ...............................269, 317
870150-906 ...............................269, 317
870150-914 ...............................269, 317
8710-0004 ..........................................108
8710-0510....................................99, 108
8710-0806 ..........................................108
8710-1534.............................................95
8710-1615 ..........................................108
8710-1622 ..........................................108
8710-1924......................................11, 48
8710-1930 ......................11, 35, 58, 108
8710-1931......................................11, 35
8710-2699 ..........................................108
871700-902 ........................................268
871700-906 ........................................268
871700-914 ........................................268
872700-902 ........................................268
872700-906 ........................................268
873700-902 ........................................270
873700-906 ........................................270
651
PART NUMBER INDEX
873700-932 ........................................270
873700-936 ........................................270
874700-902 ........................................268
874700-906 ........................................268
875700-902 ........................................270
875700-906 ........................................270
875700-932 ........................................270
875700-936 ........................................270
877150-102 ...............................269, 317
877150-106 ...............................269, 317
877150-114 ...............................269, 317
877250-101 ...............................318, 329
877250-102 ...............................269, 317
877250-105 ...............................269, 317
877250-106 ...............................269, 317
877250-112 ...............................269, 317
877250-114 ...............................269, 317
877952-101 ...............................317, 330
877952-102 ...............................283, 317
877952-105 ......................283, 317, 330
877952-106 ...............................283, 317
877952-108 ...............................317, 330
877967-102 ...............................273, 318
877974-901 ........................................433
877974-910 ........................................433
878150-101 ...............................282, 318
878250-101 ...............................282, 318
880668-301 ........................................281
880668-901 ........................................281
880952-201 ........................................330
880952-202 ........................................283
880952-203 ........................................334
880952-204 ........................................334
880952-205 ........................................330
880952-206 ........................................283
880952-208 ........................................330
880952-302 ........................................283
880952-701 ........................................330
880952-702 ........................................283
880952-703 ........................................334
880952-704 ........................................334
880952-705 ........................................330
880952-706 ........................................283
880952-708 ........................................330
880952-710 ........................................283
880952-712 ........................................283
880952-714 ........................................334
880967-201 ...............................267, 273
880967-202 ........................................273
880967-302 ........................................273
880967-901 ........................................273
880967-902 ........................................273
880975-201 ........................................329
880975-202 ........................................267
880975-205 ........................................267
880975-209 ........................................267
880975-212 ........................................267
880975-302 ........................................267
880975-305 ........................................267
880975-306 ........................................267
880975-309 ........................................267
880975-312 ........................................267
880975-314 ........................................267
880975-901 ........................................329
880975-902 ........................................267
880975-905 ........................................267
880975-906 ........................................267
880975-909 ........................................267
880975-912 ........................................267
880975-914 ........................................267
880995-202 ........................................372
880995-205 ........................................372
652
880995-206 ........................................372
880995-209 ........................................372
880995-902 ........................................372
880995-905 ........................................372
880995-906 ........................................372
880995-909 ........................................372
881750-902 ........................................372
883668-301 ........................................281
883668-901 ........................................281
883700-704 ........................................334
883700-714 ........................................334
883700-901 ........................................329
883700-902 ........................................273
883700-905 ........................................268
883700-906 ........................................268
883700-909 ........................................268
883700-912 ........................................268
883700-922 ........................................268
883725-901 ........................................281
883750-902 ........................................372
883750-905 ........................................372
883750-906 ........................................372
883750-909 ........................................372
883952-302 ........................................283
883952-701 ........................................330
883952-702 ........................................283
883952-703 ........................................334
883952-704 ........................................334
883952-705 ........................................330
883952-706 ........................................283
883952-708 ........................................330
883952-710 ........................................283
883952-712 ........................................283
883952-714 ........................................334
883967-302 ........................................273
883967-901 ........................................273
883967-902 ........................................273
883975-202 ........................................267
883975-302 ........................................267
883975-305 ........................................267
883975-306 ........................................267
883975-309 ........................................267
883975-312 ........................................267
883975-314 ........................................267
883975-901 ........................................329
883975-902 ........................................267
883975-905 ........................................267
883975-906 ........................................267
883975-909 ........................................267
883975-912 ........................................267
883975-914 ........................................267
883995-902 ...............................210, 372
883995-905 ........................................372
883995-906 ........................................372
883995-909 ........................................372
884950-507 ........................................283
884950-526 ........................................283
884950-543 ........................................283
884950-567 ........................................267
884950-577 ........................................281
884973-701 ........................................433
884973-901 ........................................433
884973-902 ........................................433
884975-202 ........................................267
895050-902 ......................317, 373, 466
895050-906 ......................317, 373, 466
895050-909 ......................317, 373, 466
895100-902 ......................317, 373, 466
895100-906 ......................317, 373, 466
895100-909 ......................317, 373, 466
895150-902 ......................317, 373, 466
895150-906 ......................317, 373, 466
895150-909 ......................317, 373, 466
897150-102 ......................317, 373, 466
897150-106 ......................317, 373, 466
897150-109 ......................317, 373, 466
897250-102 ......................317, 373, 466
897250-105 ......................317, 373, 466
897250-106 ......................317, 373, 466
897250-109 ......................317, 373, 466
921700-902 ...............................245, 263
921700-932 ...............................245, 263
921975-902 ...............................245, 263
921975-932 ...............................245, 263
922700-902 ...............................245, 262
922700-932 ...............................245, 262
922975-902 ......................243, 245, 262
922975-906 ...............................245, 262
922975-932 ...............................245, 262
923700-902 ...............................245, 263
923700-932 ...............................245, 263
923975-902 ...............................245, 262
923975-932 ...............................245, 262
924700-902 ...............................243, 261
924700-906 ...............................243, 261
924975-302 ...............................243, 260
924975-306 ...............................243, 260
924975-902 ...............................243, 260
924975-906 ...............................243, 260
925700-902 ...............................245, 263
925700-932 ...............................245, 263
925975-902 ...............................245, 262
925975-932 ...............................245, 262
926700-902 ...............................243, 261
926700-906 ...............................243, 261
926975-302 ...............................243, 260
926975-306 ...............................243, 260
926975-902 ...............................243, 260
926975-906 ...............................243, 260
927700-902 ...............................243, 261
927700-906 ...............................243, 261
927975-302 ...............................243, 260
927975-306 ...............................243, 260
927975-902 ...............................243, 260
927975-906 ...............................243, 260
928700-902 ...............................243, 261
928700-906 ...............................243, 261
928975-302 ...............................243, 260
928975-306 ........................................260
928975-902 ...............................243, 260
928975-906 ........................................260
9300-1747 ..........................................199
9300-1748 ..........................................199
9301-0407.............................................99
9301-0656.............................................53
9301-0722 ..........................................200
9301-0895.............................................13
9301-0978 ..........................................199
9301-1291 ..........................................106
9301-1337.............................................58
9301-1420.............................................53
9301-1421.............................................53
9301-1446.............................................99
9301-1450.............................................53
9301-6341.............................................53
9301-6342.............................................53
930990-902 ........................................260
930990-906 ........................................260
931975-902 ........................................262
931975-906 ........................................262
931975-932 ........................................262
931975-936 ........................................262
932967-902 ........................................260
932967-906 ........................................260
933975-902 ........................................262
933975-906 ........................................262
933975-932 ........................................262
933975-936 ........................................262
934967-902 ........................................260
934967-906 ........................................260
935967-902 ........................................260
935967-906 ........................................260
935967-912 ........................................260
946975-902 ........................................260
946975-906 ........................................260
959701-902 ........................................253
959701-906 ........................................253
959701-912 ........................................253
959701-918 ...............................253, 255
959731-902 ...............................243, 253
959731-906 ...............................243, 253
959731-912 ...............................243, 253
959733-902 ........................................253
959733-906 ........................................253
959733-912 ........................................253
959741-902 ...............................243, 253
959741-906 ...............................243, 253
959741-912 ...............................243, 253
959741-918 ......................243, 253, 255
959743-901 ........................................325
959743-902 ........................................253
959743-906 ........................................253
959743-912 ........................................253
959746-902 ........................................253
959746-906 ........................................253
959757-301 ...............................238, 325
959757-302 ...............................238, 252
959757-306 ...............................238, 252
959757-312 ........................................238
959757-901 ...............................238, 325
959757-902 ......................210, 238, 253
959757-906 ...............................238, 253
959757-912 ........................................238
959758-301 ...............................238, 325
959758-302 ...............................238, 252
959758-306 ...............................238, 252
959758-312 ........................................238
959758-901 ...............................238, 325
959758-902 ...............................238, 253
959758-906 ...............................238, 253
959758-912 ........................................238
959759-302 ...............................238, 252
959759-306 ...............................238, 252
959759-901 ...............................238, 325
959759-902 ...............................238, 253
959759-906 ...............................238, 253
959759-912 ........................................238
959763-902 ........................................253
959763-906 ........................................253
959763-912 ........................................253
959764-902 ...............................243, 253
959764-906 ...............................243, 253
959764-912 ...............................243, 253
959764-918 ......................243, 253, 255
959790-918 ...............................253, 255
959793-901 ........................................325
959793-902 ........................................253
959793-906 ........................................253
959793-912 ........................................253
959793-918 ...............................253, 255
959794-902 ........................................243
959931-902 ...............................243, 252
959931-906 ...............................243, 252
959931-912 ...............................243, 252
959931-918 ......................243, 252, 255
959933-902 ........................................252
PART NUMBER INDEX
959933-906 ........................................252
959933-912 ........................................252
959936-902 ........................................252
959936-906 ........................................252
959936-912 ........................................252
959941-302 ...............................243, 252
959941-306 ...............................243, 252
959941-312 ...............................243, 252
959941-902 ...............................243, 252
959941-906 ...............................243, 252
959941-912 ...............................243, 252
959941-918 ......................243, 252, 255
959943-901 ........................................325
959943-902 ........................................252
959943-906 ........................................252
959943-912 ........................................252
959943-918 ...............................252, 255
959946-902 ........................................252
959946-906 ........................................252
959951-902 ...............................243, 252
959961-302 ........................................252
959961-306 ........................................252
959961-312 ........................................252
959961-901 ........................................325
959961-902 ........................................252
959961-906 ........................................252
959961-912 ........................................252
959961-918 ...............................252, 255
959963-302 ........................................252
959963-306 ........................................252
959963-312 ........................................252
959963-902 ...............................210, 252
959963-906 ........................................252
959963-912 ........................................252
959963-918 ...............................252, 255
959964-302 ...............................243, 252
959964-306 ...............................243, 252
959964-312 ...............................243, 252
959964-902 ...............................243, 252
959964-906 ...............................243, 252
959964-912 ...............................243, 252
959964-918 ......................243, 252, 255
959990-318 ...............................252, 255
959990-902 ........................................252
959990-906 ........................................252
959990-912 ........................................252
959990-918 ...............................252, 255
959993-302 ........................................252
959993-306 ........................................252
959993-902 ........................................252
959993-906 ........................................252
959993-912 ........................................252
959993-918 ...............................252, 255
959994-902 ........................................243
959996-902 ........................................252
959996-906 ........................................252
959996-912 ........................................252
959996-918 ...............................252, 255
960967-902 ........................................260
960967-905 ........................................260
960967-906 ........................................260
960967-912 ........................................260
961400-302 ........................................394
961600-902 ........................................261
961600-906 ........................................261
961753-902 ........................................261
961753-905 ........................................261
961753-906 ........................................261
961967-302 ........................................260
961967-306 ........................................260
961967-312 ........................................260
961967-902 ........................................260
961967-905 ........................................260
961967-906 ........................................260
963400-902 ........................................394
963600-902 ........................................261
963600-906 ........................................261
963954-302 ........................................260
963954-305 ........................................260
963954-306 ........................................260
963954-312 ........................................260
963967-902 ........................................260
963967-905 ........................................260
963967-906 ........................................260
963967-912 ........................................260
965600-902 ........................................261
965600-906 ........................................261
966400-902 ........................................394
966735-902 ........................................261
966954-302 ........................................260
966967-902 ........................................260
966967-905 ........................................260
966967-906 ........................................260
966967-912 ........................................260
970050-902 ...............................261, 318
970050-906 ...............................261, 318
970100-902 ...............................261, 318
970100-906 ...............................261, 318
970150-902 ...............................261, 318
970150-906 ...............................261, 318
971700-902 ........................................261
971700-906 ........................................261
972700-902 ........................................261
972700-906 ........................................261
973700-902 ........................................262
973700-906 ........................................262
973700-932 ........................................262
973700-936 ........................................262
974700-902 ........................................261
974700-906 ........................................261
975700-902 ........................................262
975700-906 ........................................262
975700-932 ........................................262
975700-936 ........................................262
977150-102 ...............................261, 318
977150-106 ...............................261, 318
977250-102 ...............................261, 318
977250-106 ...............................261, 318
981757-302 ...............................238, 260
981757-902 ...............................238, 261
981758-302 ...............................238, 260
981758-902 ...............................238, 261
981759-302 ...............................238, 260
981759-902 ...............................238, 261
990967-202 ........................................260
990967-206 ........................................260
990967-302 ........................................260
990967-305 ........................................260
990967-306 ........................................260
990967-312 ........................................260
990967-902 ........................................260
990967-905 ...............................260, 329
990967-906 ........................................260
990967-912 ........................................260
993400-902 ........................................394
993700-902 ........................................260
993700-905 ...............................260, 329
993700-906 ........................................260
993700-912 ........................................260
993967-302 ........................................260
993967-305 ........................................260
993967-306 ........................................260
993967-312 ........................................260
993967-902 ........................................260
993967-905 ...............................260, 329
993967-906 ........................................260
993967-912 ........................................260
A2000020X020..................................301
A2000030X020..................................301
A2000030X046..................................300
A2000050X020..................................301
A2000050X030..................................301
A2000050X046..................................300
A2000100C020..................................302
A2000100C030..................................302
A2000100C046..................................302
A2000100R030..................................302
A2000100R046..................................302
A2000100T030..................................302
A2000100T046..................................302
A2000100X020..................................301
A2000100X030..................................301
A2000100X046..................................300
A2000100X212..................................300
A2000100X300..................................300
A2000125X040..................................301
A2000150C046..................................302
A2000150R030..................................302
A2000150R046..................................302
A2000150T030..................................302
A2000150T046..................................302
A2000150X020..................................301
A2000150X030..................................301
A2000150X040..................................301
A2000150X046..................................300
A2000150X212..................................300
A2000200X046..................................300
A2000250C030..................................302
A2000250C046..................................302
A2000250R046..................................302
A2000250T046..................................302
A2000250X020..................................301
A2000250X030..................................301
A2000250X040..................................301
A2000250X046..................................300
A2000250X100.........................300, 321
A2000250X212.........................300, 321
A2000MG...........................................303
A2000MG2.........................................303
A2001020X020..................................301
A2001030X020..................................301
A2001030X030..................................301
A2001030X046..................................301
A2001050C020..................................302
A2001050R020..................................302
A2001050T020..................................302
A2001050X020..................................301
A2001050X030..................................301
A2001050X046..................................301
A2001075X046..................................300
A2001100C020..................................302
A2001100R030..................................302
A2001100R046..................................302
A2001100T030..................................302
A2001100T046..................................302
A2001100X020..................................301
A2001100X030..................................301
A2001100X046..................................300
A2001150C020..................................302
A2001150C046..................................302
A2001150R020..................................302
A2001150R046..................................302
A2001150T020..................................302
A2001150T046..................................302
A2001150X020..................................301
A2001150X030..................................301
WWW.AGILENT.COM/CHEM/LC
A2001150X046..................................300
A2001200X030..................................301
A2001250C046..................................302
A2001250X020..................................301
A2001250X030..................................301
A2001250X046..................................300
A2001MG...........................................303
A2001MG1.........................................303
A2002250X046..................................300
A2002250X212.........................300, 321
A2002250X500..................................300
A2002MG...........................................303
A2003020X020.........................301, 332
A2003030X020.........................301, 332
A2003050X020.........................301, 332
A2003050X030.........................301, 332
A2003050X046.........................300, 332
A2003050X212.........................300, 331
A2003100X020.........................301, 332
A2003100X030.........................301, 332
A2003100X046.........................300, 332
A2003125X040.........................301, 332
A2003150X020.........................301, 332
A2003150X030.........................301, 332
A2003150X040.........................301, 332
A2003150X046.................300, 331-332
A2003250X020.........................301, 332
A2003250X030.........................301, 332
A2003250X040.........................301, 332
A2003250X046.........................300, 332
A2003250X212................300, 321, 331
A2003MG..................................303, 332
A2003MG2................................303, 332
A2004250X212................300, 321, 331
A2004250X500.........................300, 331
A2004MG..................................303, 332
A2004MG2................................303, 332
A2005020X020.........................301, 332
A2005030X020.........................301, 332
A2005050X020.........................301, 332
A2005050X030.........................301, 332
A2005050X046.........................301, 332
A2005100X020.........................301, 332
A2005100X030.........................301, 332
A2005100X046.........................300, 332
A2005150X020.........................301, 332
A2005150X030.........................301, 332
A2005150X046.........................300, 332
A2005250X020.........................301, 332
A2005250X046.................300-301, 332
A2005MG..................................303, 332
A2005MG2................................303, 332
A2006030X020..................................301
A2006050X020..................................301
A2006050X046..................................300
A2006100X020..................................301
A2006100X030..................................301
A2006100X046..................................300
A2006150X020..................................301
A2006150X030..................................301
A2006150X046..................................300
A2006250X020..................................301
A2006250X030..................................301
A2006250X046..................................300
A2006250X100.........................300, 321
A2006250X212..................................300
A2006MG...........................................303
A2006MG2.........................................303
A2007030X020..................................301
A2007030X030..................................300
A2007050X020..................................301
A2007050X030..................................301
653
PART NUMBER INDEX
A2007050X046..................................301
A2007100X020..................................301
A2007100X030..................................301
A2007100X046..................................300
A2007150X020..................................301
A2007150X030..................................301
A2007150X046..................................300
A2007250X020..................................301
A2007250X030..................................301
A2007250X046..................................300
A2007MG...........................................303
A2007MG2.........................................303
A2008250X100..................................300
A2008250X212..................................300
A2008MG2.........................................303
A2010050X020..................................301
A2010100X030..................................301
A2010100X046..................................300
A2010125X040..................................301
A2010150X020..................................301
A2010150X030..................................301
A2010150X040..................................301
A2010150X046..................................300
A2010250X030..................................301
A2010250X040..................................301
A2010250X046..................................300
A2010250X100..................................321
A2010250X212.........................300, 321
A2010MG...........................................303
A2010MG2.........................................303
A2011030X030..................................301
A2011050X020..................................301
A2011075X046..................................300
A2011100X046..................................300
A2011150X020..................................301
A2011150X046..................................300
A2011250X020..................................301
A2011MG...........................................303
A2011MG2.........................................303
A2013020X020.........................301, 332
A2013030X020.........................301, 332
A2013050X020.........................301, 332
A2013050X046.........................300, 332
A2013100X020.........................301, 332
A2013100X030.........................301, 332
A2013100X046.........................300, 332
A2013125X040.........................301, 332
A2013150X020.........................301, 332
A2013150X030.........................301, 332
A2013150X040.........................301, 332
A2013150X046.........................300, 332
A2013250X020.........................301, 332
A2013250X030.........................301, 332
A2013250X040.........................301, 332
A2013250X046.........................300, 332
A2013250X100................300, 321, 331
A2013250X212................300, 321, 331
A2013MG..................................303, 332
A2013MG2................................303, 332
A2014020X020.........................301, 332
A2014030X020.........................301, 332
A2014050X020.........................301, 332
A2014050X030.........................301, 332
A2014050X046.........................301, 332
A2014100X020.........................301, 332
A2014100X030.........................301, 332
A2014100X046.........................300, 332
A2014150X020.........................301, 332
A2014150X030.........................301, 332
A2014150X046.........................300, 332
A2014250X020.........................301, 332
A2014250X030.........................301, 332
654
A2014250X046.........................300, 332
A2014MG..................................303, 332
A2014MG2................................303, 332
A2020050X020..................................301
A2020050X046..................................300
A2020100X030..................................301
A2020125X040..................................301
A2020150X020..................................301
A2020150X030..................................301
A2020150X040..................................301
A2020150X046..................................300
A2020250X020..................................301
A2020250X030..................................301
A2020250X040..................................301
A2020250X046..................................300
A2020250X100.........................300, 321
A2020250X212..................................300
A2020MG...........................................303
A2020MG2.........................................303
A2021050X020..................................301
A2021050X030..................................301
A2021050X046..................................301
A2021050X100..................................300
A2021075X020..................................301
A2021100X020..................................301
A2021150X020..................................301
A2021150X030..................................301
A2021250X020..................................301
A2021250X046..................................300
A2021MG...........................................303
A2021MG2.........................................303
A2030050X020..................................301
A2030100X030..................................301
A2030125X040..................................301
A2030150X020..................................301
A2030150X030..................................301
A2030150X040..................................301
A2030150X046..................................300
A2030250X020..................................301
A2030250X030..................................301
A2030250X040..................................301
A2030250X046..................................300
A2030250X100.........................300, 321
A2030250X212.........................300, 321
A2030MG...........................................303
A2031050X020..................................301
A2031050X030..................................301
A2031050X046..................................301
A2031100X020..................................301
A2031150X020..................................301
A2031250X020..................................301
A2031250X046..................................300
A2031MG2.........................................303
A3000020X020..................................292
A3000030X020..................................292
A3000050X020..................................292
A3000050X046..................................291
A3000100C020..................................293
A3000100C030..................................293
A3000100C046..................................293
A3000100R030..................................294
A3000100T030..................................294
A3000100X020..................................292
A3000100X030..................................291
A3000100X046..................................291
A3000125X040..................................291
A3000150C020..................................293
A3000150C030..................................293
A3000150C046..................................293
A3000150R030..................................294
A3000150R046..................................294
A3000150T030..................................294
A3000150T046..................................294
A3000150X020..................................292
A3000150X030..................................291
A3000150X039..................................291
A3000150X046..................................291
A3000150X100..................................291
A3000150X212..................................292
A3000250C020..................................293
A3000250C030..................................293
A3000250C046..................................293
A3000250X020..................................292
A3000250X030..................................291
A3000250X040..................................291
A3000250X046..................................291
A3000250X100.........................291, 320
A3000250X212..................................292
A3000300X039..................................291
A3000MG...........................................294
A3000MG1.........................................294
A3000MG2.........................................294
A3001020X020..................................292
A3001030X020..................................292
A3001030X046..................................291
A3001050C020..................................293
A3001050C046..................................293
A3001050R046..................................294
A3001050T046..................................294
A3001050X020..................................292
A3001050X030..................................292
A3001050X046..................................291
A3001100C020..................................293
A3001100C030..................................293
A3001100C046..................................293
A3001100R030..................................294
A3001100T030..................................294
A3001100X020..................................292
A3001100X030..................................291
A3001100X046..................................291
A3001150C020..................................293
A3001150C030..................................293
A3001150C046..................................293
A3001150R030..................................294
A3001150T030..................................294
A3001150X020..................................292
A3001150X030..................................291
A3001150X046..................................291
A3001250X020..................................292
A3001250X030..................................291
A3001250X046..................................291
A3001MG...........................................294
A3001MG2.........................................294
A3002100X046..................................291
A3002150X046..................................291
A3002150X212..................................292
A3002250X046..................................291
A3002250X100..................................291
A3002250X212..................................292
A3002250X500..................................292
A3002300X039..................................291
A3002MG...........................................294
A3002MG2.........................................294
A3030050X020..................................292
A3030100C046..................................293
A3030100X020..................................292
A3030100X046..................................291
A3030150C020..................................293
A3030150C046..................................293
A3030150R046..................................294
A3030150T046..................................294
A3030150X020..................................292
A3030150X046..................................291
A3030250C046..................................293
A3030250X046..................................291
A3030250X100.........................291, 320
A3030MG...........................................294
A3030MG2.........................................294
A3031030X020..................................292
A3031050R020..................................294
A3031050T020..................................294
A3031050X020..................................292
A3031100C046..................................293
A3031100X020..................................292
A3031100X046..................................291
A3031150C046..................................293
A3031150R046..................................294
A3031150T046..................................294
A3031150X020..................................292
A3031150X046..................................291
A3031250C046..................................293
A3031250X046..................................291
A3031MG...........................................294
A3031MG2.........................................294
A3032100X046..................................291
A3032150X046..................................291
A3032250X046..................................291
A3032250X100.........................291, 320
A3032250X212.........................292, 320
A3032250X500..................................292
A3040030X020..................................292
A3040050X020..................................292
A3040050X046..................................291
A3040100X020..................................292
A3040100X046..................................291
A3040100X212..................................292
A3040150X020..................................292
A3040150X030..................................291
A3040150X046..................................291
A3040250X030..................................291
A3040250X046..................................291
A3040250X100..................................320
A3040MG...........................................294
A3040MG1.........................................294
A3040MG2.........................................294
A3041020X020..................................292
A3041030X020..................................292
A3041050X020..................................292
A3041050X030..................................292
A3041050X046..................................291
A3041100X020..................................292
A3041100X030..................................291
A3041100X046..................................291
A3041150X020..................................292
A3041150X030..................................291
A3041150X046..................................291
A3041200X020..................................292
A3041250X020..................................292
A3041250X046..................................291
A3041MG...........................................294
A3041MG1.........................................294
A3041MG2.........................................294
A3050020X020..................................292
A3050030X020..................................292
A3050050X020..................................292
A3050050X046..................................291
A3050100X020..................................292
A3050100X030..................................291
A3050100X046..................................291
A3050150X030..................................291
A3050150X046..................................291
A3050150X100..................................291
A3050150X212..................................292
A3050250X046..................................291
A3050250X100.........................291, 320
A3050250X212..................................292
PART NUMBER INDEX
A3050MG...........................................294
A3050MG2.........................................294
A3051020X020..................................292
A3051030X020..................................292
A3051050X020..................................292
A3051050X030..................................292
A3051050X046..................................291
A3051100X020..................................292
A3051100X030..................................291
A3051100X046..................................291
A3051150X020..................................292
A3051150X030..................................291
A3051150X046..................................291
A3051250X046..................................291
A3051MG...........................................294
A3051MG2.........................................294
A6000030X020..................................296
A6000030X212..................................296
A6000050X020..................................296
A6000050X046..................................295
A6000050X100..................................295
A6000050X212.........................296, 320
A6000050X300..................................296
A6000100X020..................................296
A6000100X030..................................295
A6000100X046..................................295
A6000100X212.........................296, 320
A6000100X300.........................296, 320
A6000150X020..................................296
A6000150X030..................................295
A6000150X040..................................295
A6000150X046..................................295
A6000150X100..................................295
A6000150X212.........................296, 320
A6000150X300.........................296, 320
A6000250X020..................................296
A6000250X030..................................295
A6000250X040..................................295
A6000250X046..................................295
A6000250X100..................................295
A6000250X212.........................296, 320
A6000250X300.........................296, 320
A6000MG...........................................297
A6000MG2.........................................297
A6001020X020..................................296
A6001030X030..................................295
A6001030X046..................................295
A6001050X020..................................296
A6001050X030..................................295
A6001050X046..................................295
A6001100X010..................................296
A6001100X020..................................296
A6001100X030..................................295
A6001100X046..................................295
A6001150X010..................................296
A6001150X020..................................296
A6001150X030..................................295
A6001150X046..................................295
A6001250X020..................................296
A6001250X046..................................295
A6001MG...........................................297
A6001MG2.........................................297
A6002050X046S ...............................295
A6002150X300..................................320
A6002250X046..................................295
A6002250X100.........................295, 320
A6002250X212.........................296, 320
A6002250X300..................................320
A6002250X500.........................296, 320
A6002MG...........................................297
A6004250X046.........................295, 331
A6004250X100.........................295, 331
A6004250X212................296, 320, 331
A6004250X300................296, 320, 331
A6004250X500................296, 320, 331
A6004MG...........................................297
A6005050X020.........................296, 331
A6005050X046.........................295, 331
A6005100X046.........................295, 331
A6006050X046.........................295, 331
A6006100X021.........................296, 331
A6006100X046.........................295, 331
A6010050X020..................................296
A6010100X020..................................296
A6010100X030..................................295
A6010100X046..................................295
A6010100X212..................................320
A6010150X020..................................296
A6010150X030..................................295
A6010150X040..................................295
A6010150X046..................................295
A6010150X212..................................320
A6010250X030..................................295
A6010250X040..................................295
A6010250X046..................................295
A6010250X300..................................320
A6010MG...........................................297
A6010MG2.........................................297
A6011050X020..................................296
A6011050X030..................................295
A6011050X046..................................295
A6011100X020..................................296
A6011100X030..................................295
A6011100X046..................................295
A6011150X020..................................296
A6011150X030..................................295
A6011MG...........................................297
A6011MG2.........................................297
A6012250X212..................................296
A6020050X020..................................296
A6020050X046..................................295
A6020100X030..................................295
A6020100X046..................................295
A6020100X212.........................296, 320
A6020150X020..................................296
A6020150X030..................................295
A6020150X046..................................295
A6020150X300..................................296
A6020250X020..................................296
A6020250X030..................................295
A6020250X046..................................295
A6020250X100..................................295
A6020250X212.........................296, 320
A6020MG...........................................297
A6020MG2.........................................297
A6021030X020..................................296
A6021030X046..................................295
A6021050X020..................................296
A6021050X030..................................295
A6021050X046..................................295
A6021100X010..................................296
A6021100X020..................................296
A6021100X030..................................295
A6021100X046..................................295
A6021150X020..................................296
A6021150X030..................................295
A6021150X046..................................295
A6021150X100..................................295
A6021250X020..................................296
A6021250X046..................................295
A6021MG...........................................297
A6021MG2.........................................297
A6022250X500..................................320
A7000100C030..................................293
A7000100R030..................................294
A7000100T030..................................294
A7000150C046..................................293
A7000150R046..................................294
A7000150T046..................................294
A7000150X046..................................291
A7000250C046..................................293
A7000250R046..................................294
A7000250T046..................................294
A7000250X046..................................291
A7000MG3.........................................297
A7001100C046..................................293
A7001100R030..................................294
A7001100R046..................................294
A7001100T030..................................294
A7001100T046..................................294
A7001100X020..................................292
A7001100X030..................................291
A7001100X046..................................291
A7001150C046..................................293
A7001150R046..................................294
A7001150T046..................................294
A7001MG3.........................................297
A7501030X020..................................297
A7501050X020..................................297
A7501100X020..................................297
A7501100X030..................................297
A7501150X020..................................297
A7501150X030..................................297
A7511030X020..................................297
A7511050X020..................................297
A7511100X020..................................297
A7511150X030..................................297
A7521030X020..................................297
A7521050X020..................................297
A7521100X020..................................297
BHT-4 .........................................104, 108
BMT-4 .................................................108
G1103-60001........................................90
G1156-68711........................................83
G1156-68712........................................83
G1156-68713........................................83
G1156-68714........................................83
G1160-68706........................................83
G1310-68730........................................59
G1310-68731........................................59
G1310-68741........................................59
G1310-68742........................................59
G1311-60003........................................53
G1311-60006........................................12
G1311-60009........................................48
G1311-68705........................................60
G1311-68710........................................59
G1311-68711........................................59
G1312-60020........................................49
G1312-60025........................................49
G1312-60061........................................48
G1312-60066........................................49
G1312-60067........................................49
G1312-67305........................................23
G1312-67500........................................23
G1312-68711........................................59
G1312-68716........................................53
G1312-68726........................................60
G1312-68730........................................60
G1312-68741........................................59
G1312-68755........................................60
G1312-87303........................................23
G1312-87304........................................23
G1312-87305........................................28
G1312-87306........................................28
G1312-87330........................................54
WWW.AGILENT.COM/CHEM/LC
G1313-27302........................................65
G1313-43204........................................63
G1313-43216........................................35
G1313-44510........................................65
G1313-44512........................................65
G1313-44513........................................65
G1313-60004........................................65
G1313-68709........................................66
G1313-68711........................................67
G1313-68719........................................66
G1313-68730........................................66
G1313-87102........................................62
G1313-87201........................................62
G1313-87202........................................62
G1313-87203........................................62
G1313-87300........................................34
G1313-87303.................................32, 64
G1313-87304.................................23, 82
G1313-87305 ..........................23, 33, 82
G1314-60081..................................91-92
G1314-60082..................................91-92
G1314-60083..................................91-92
G1314-60086..................................91-92
G1314-60087..................................91-92
G1314-60100........................................90
G1314-60101........................................90
G1314-60182..................................91-92
G1314-60183..................................91-92
G1314-60186..................................91-92
G1314-60187..................................91-92
G1314-65052.................................91, 97
G1314-65054.................................91, 97
G1314-65056........................................97
G1314-65061.................................91, 97
G1314-87301........................................92
G1314-87302........................................92
G1315-27705........................................96
G1315-45003.................................11, 95
G1315-60011..................................94-95
G1315-60012..................................94-95
G1315-60015..................................94-95
G1315-60016.................................94, 96
G1315-60017.................................94, 96
G1315-60018.................................94, 96
G1315-60022..................................94-95
G1315-60024..................................94-95
G1315-60025..................................94-95
G1315-67301........................................96
G1315-67302........................................96
G1315-68703........................................31
G1315-68708..................................31-32
G1315-68712........................................97
G1315-68713.................................91, 97
G1315-68715..................................96-97
G1315-68716.................................94, 96
G1315-68724.................................94, 96
G1315-68725..................................96-97
G1315-80001........................................96
G1315-80002........................................96
G1315-80003........................................96
G1315-80004........................................96
G1315-87101........................................96
G1315-87302........................................95
G1315-87303........................................23
G1315-87305........................................96
G1315-87306........................................95
G1315-87311 ...................23, 33, 92, 99
G1315-87312........................................23
G1315-87313........................................96
G1315-87318........................................96
G1315-87319........................................95
G1315-87321........................................95
655
PART NUMBER INDEX
G1315-87323........................................96
G1315-87325........................................95
G1315-87328........................................96
G1315-87333........................................96
G1315-87338........................................96
G1315-87339........................................95
G1316-27301........................................28
G1316-60001........................................82
G1316-67005........................................75
G1316-67006........................................75
G1316-67007........................................75
G1316-67009........................................75
G1316-68708........................................83
G1316-68710........................................83
G1316-68711........................................83
G1316-68716.................................38, 82
G1316-68721........................................83
G1316-68744........................................82
G1316-80002........................................82
G1316-80003........................................82
G1316-80004........................................82
G1316-83200........................................82
G1316-87300 ..........................23, 33, 82
G1316-87303.................................27, 82
G1316-87305........................................27
G1316-87306........................................28
G1316-87309........................................23
G1316-87312........................................27
G1316-87313........................................27
G1316-87314........................................27
G1316-87316........................................27
G1316-87317........................................27
G1316-87318........................................27
G1316-87319........................................27
G1316-87321........................................28
G1316-87323........................................28
G1321-60005........................................99
G1321-60007........................................99
G1321-60015........................................99
G1322-67300........................................58
G1322-68705.................................58, 60
G1328-87600........................................23
G1329-60011........................................65
G1329-68718........................................67
G1329-68727........................................67
G1329-68736........................................67
G1329-68737........................................67
G1329-80001........................................62
G1329-87012........................................62
G1329-87017........................................62
G1329-87101........................................62
G1329-87103........................................62
G1329-87300........................................23
G1329-87302.................................32, 64
G1353-68750........................................75
G1361-22402........................................50
G1361-23204........................................54
G1361-23205........................................54
G1361-60012........................................49
G1361-60022........................................53
G1361-67302........................................26
G1361-68707........................................60
G1361-68710........................................60
G1362-68706........................................98
G1362-68709........................................98
G1362-87300........................................98
G1362-87301........................................98
G1364-27107........................................71
G1364-60021........................................71
G1364-68706........................................71
G1364-68711........................................70
G1364-68712........................................70
656
G1364-68723........................................70
G1364-81701........................................71
G1364-83205........................................71
G1364-84516........................................69
G1364-84521........................................69
G1364-84522........................................69
G1364-84523........................................69
G1364-84524........................................69
G1364-84525........................................69
G1364-84531........................................69
G1364-84532........................................69
G1364-86711........................................71
G1364-87201........................................70
G1364-87202........................................70
G1364-87304..................................70-71
G1364-87305..................................70-71
G1364-87306..................................70-71
G1367-60001........................................69
G1367-68730........................................66
G1367-68734........................................66
G1367-68741........................................66
G1367-87012........................................63
G1367-87017........................................63
G1367-87101........................................62
G1367-87102........................................62
G1367-87200.................................62, 70
G1367-87201........................................62
G1367-87202........................................63
G1367-87300.................................32, 64
G1367-87304........................................28
G1375-87301........................................31
G1375-87302........................................31
G1375-87303.................................32, 64
G1375-87304........................................31
G1375-87305........................................32
G1375-87306........................................32
G1375-87308........................................32
G1375-87309..................................31-32
G1375-87310........................................31
G1375-87311........................................32
G1375-87312........................................32
G1375-87315.................................32, 64
G1375-87320........................................31
G1375-87321........................................31
G1375-87322........................................31
G1375-87323........................................31
G1375-87324........................................31
G1375-87325........................................31
G1375-87326........................................34
G1375-87327........................................31
G1376-60003........................................53
G1376-60005........................................59
G1376-68705........................................60
G1376-68707........................................60
G1376-68710........................................60
G1377-44900........................................63
G1377-87000........................................63
G1377-87001........................................63
G1377-87002........................................63
G1377-87201........................................63
G1377-87300.................................32, 64
G1377-87310.................................32, 64
G1379-67310........................................58
G1600-23223 .....................................199
G1600-60002 .....................................190
G1600-60007 .....................................199
G1600-60013 .....................................194
G1600-60027 .....................................192
G1600-60033 .....................................199
G1600-60132 .....................................180
G1600-60150 .....................................190
G1600-60210 .....................................190
G1600-60211 .....................................178
G1600-60230 .....................................190
G1600-60232 .....................................180
G1600-60233 .....................................180
G1600-60310 .....................................190
G1600-60311 .....................................178
G1600-60330 .....................................190
G1600-60332 .....................................180
G1600-60400 .....................................194
G1600-60411 .....................................178
G1600-60419 .....................................184
G1600-61132 .....................................180
G1600-61211 .....................................178
G1600-61219 .....................................184
G1600-61232 ............................173, 180
G1600-61239 .....................................184
G1600-61311 .....................................178
G1600-61332 .....................................180
G1600-61411 .....................................178
G1600-61419 .....................................184
G1600-62132 .....................................180
G1600-62211 ............................172, 178
G1600-62232 .....................................180
G1600-62311 ............................174, 178
G1600-62318 .....................................185
G1600-62332 .....................................180
G1600-62402 .....................................199
G1600-62411 .....................................178
G1600-62700 .....................................190
G1600-63200 .....................................192
G1600-63211 .....................................178
G1600-63311 .....................................178
G1600-63411 .....................................178
G1600-64211 ............................175, 178
G1600-64232 .....................................180
G1600-64311 .....................................178
G1600-64332 .....................................180
G1600-64411 .....................................178
G1600-67201 .....................................199
G1600-67219 ............................184, 195
G1600-67220 .....................................195
G1600-67311 .............................194-195
G1600-67312 .....................................195
G1600-67319 ............................184, 195
G1600-68319 ............................184, 192
G1600-68714 .....................................192
G1600-68715 .....................................192
G1600-68716 .....................................192
G1600-68723 .....................................192
G1603A...............................................194
G1607-20030 .....................................195
G1607-60000 .....................................195
G1607-60001 .....................................195
G1607-60041 .....................................195
G1607A...............................................195
G160U-60419.....................................184
G160U-61219.....................................184
G160U-61239.....................................184
G160U-61419.....................................184
G1946-00034 .....................................107
G1946-20215 .....................................108
G1946-20301 .....................................105
G1946-60037 .....................................105
G1946-60098 .....................................105
G1946-60157 .....................................108
G1946-60180 .....................................108
G1946-80009 .....................................105
G1946-80019 .....................................106
G1946-80049 .....................................108
G1946-80054 .....................................108
G1946-85004 .....................................107
G1946-85021 .....................................107
G1947-20029 .....................................105
G1947-60103 .....................................105
G1956-20302 .....................................106
G1956-80000 .....................................106
G1958-60098 .....................................105
G1958-60136 .....................................105
G1960-80039 .....................................107
G1960-80060 .....................................105
G1969-20302 .....................................106
G1969-60086 .....................................108
G1969-85000 .....................................110
G1969-85001 .....................................107
G1969-85003...............................15, 107
G1969-85010 .....................................110
G1969-85020 .....................................110
G1969-85026 .....................................107
G1972-60025........................................71
G1978-85000...............................15, 107
G1982-85001........................................15
G1982-85002........................................15
G1982-85003........................................15
G2228-68700........................................59
G2250-04500........................................65
G2250-04501........................................65
G2250-04502........................................65
G2250-04503........................................65
G2250-04504........................................65
G2255-68700.................................65, 69
G2255-68709........................................65
G2255-68710........................................65
G2255-68720........................................65
G2255-68730........................................65
G2258-23201........................................54
G2258-60003........................................64
G2258-60011........................................65
G2258-68710........................................63
G2258-87102........................................63
G2258-87307........................................34
G2258-87310........................................34
G2258-87311........................................34
G2258-87312........................................34
G2258-87313........................................34
G2258-87314........................................34
G2258-87315........................................34
G2258-87316........................................34
G2260-68711.................................32, 64
G2260-87101........................................63
G2260-87201........................................63
G2260-87300........................................26
G2260-87301........................................26
G2421-60001 .....................................110
G2423A...............................................107
G2424A...............................................107
G2425A...............................................107
G2426A...............................................107
G2427A...............................................105
G2428A...............................................105
G2431A...............................................110
G2432A...............................................110
G2441-80010 .....................................106
G2453-85050 .....................................107
G2453-85060 .....................................107
G2455-85001........................................15
G2571-80103 .....................................106
G3199B...............................................109
G4203-68708........................................11
G4204-40000........................................51
G4204-40005........................................51
G4204-60004........................................51
G4204-60022........................................49
G4208-68700........................................11
G4212-60007........................................94
PART NUMBER INDEX
G4212-60008........................................94
G4212-60011........................................94
G4212-60022........................................51
G4212-60032........................................94
G4212-60038........................................94
G4212-68001........................................51
G4216-68711........................................67
G4218-20000........................................98
G4218-20001........................................98
G4218-20002........................................98
G4218-20003........................................98
G4218-20004........................................98
G4218-40000........................................98
G4218-40010........................................98
G4218-40011........................................98
G4218-40100........................................98
G4218-40110........................................98
G4218-40130........................................98
G4218-40150........................................98
G4218-40220........................................98
G4218-60100........................................98
G4218-68010........................................98
G4218-85000.................................15, 98
G4220-20012........................................11
G4220-24013........................................52
G4220-26210........................................52
G4220-60006........................................51
G4220-60007........................................53
G4220-60012........................................51
G4220-60015........................................51
G4220-60016........................................51
G4220-60022........................................49
G4220-60028........................................49
G4220-60035........................................51
G4220-63010........................................52
G4220-63015........................................52
G4226-60021........................................65
G4226-60310.................................32, 64
G4226-67001........................................67
G4226-68735........................................66
G4226-87012........................................63
G4226-87020........................................63
G4226-87201........................................63
G4240-23705 .....................................100
G4240-25206 .....................................100
G4240-43200 .....................................100
G4240-87300 .....................................100
G4240-87301 .....................................100
G4240-87302 .....................................100
G4240-87303 .....................................100
G4240-87304 .....................................100
G4240-87309 .....................................100
G4240-87310 .....................................100
G4280-60031........................................48
G4280-60033........................................48
G4280-60061........................................48
G4280-68710.................................60, 66
G4280-68730.................................60, 66
G4280-68750.................................60, 66
G4280-68770.................................60, 66
G4296-68715........................................11
G5611-21503................51, 64, 101-102
G5611-26210...............................52, 101
G5611-60020...............................49, 101
G5611-60025...............................49, 101
G5611-60061...............................48, 101
G5611-60067...............................49, 101
G5611-60500........................................30
G5611-60501........................................30
G5611-60502........................................30
G5611-60503........................................30
G5611-63010 .....................................101
G5611-67300........................................30
G5611-67301........................................30
G5611-68710........................................37
G5611-68741...............................59, 101
G5615-60005 .....................................102
G5615-60017 .....................................102
G5615-60018 .....................................102
G5615-60022 .....................................102
G5616-60050...............................30, 102
G5664-86703 .....................................102
G5664-86706 .....................................102
G5667-40500 .....................................102
G5667-60310........................................32
G5667-60320...............................64, 102
G5667-60500........................................30
G5667-60501........................................30
G5667-60502........................................30
G5667-60503........................................30
G5667-60504........................................30
G5667-60505........................................30
G5667-87017...............................63, 102
G5667-87200...............................63, 101
G6011A...............................................109
G6012A...............................................109
G6013A...............................................109
G6014A...............................................109
G7100-60002 .....................................190
G7100-60007 .....................................199
G7100-60033 .....................................199
G7100-60150 .....................................190
G7100-60210 .....................................190
G7100-60230 .....................................190
G7100-60310 .....................................190
G7100-60330 .....................................190
G7100-60400 ............................190, 194
G7100-62700 .....................................190
G7100-68705 .....................................200
G7100-68723 .....................................192
PCG931AAKIT...................................322
PCG932AAKIT...................................322
PCG933AAKIT...................................322
PCG93LL500X25...............................322
PCG93LL500X25WJ ........................322
PCG93LL500X50...............................322
PCG93LL500X50WJ ........................322
PCG93LL500X75...............................322
PCG93LL500X75WJ ........................322
PCG93LLSTAND123 ........................322
PL1012-5A05.....................................475
PL1013-2100......................................511
PL1013-2120......................................511
PL1013-2300......................................511
PL1013-2500......................................511
PL1020-2830......................................511
PL1049-2800......................................511
PL1110-1120.............................500, 502
PL1110-1220.............................500, 502
PL1110-1320......................................500
PL1110-1400......................................508
PL1110-1520......................................500
PL1110-6100......................................500
PL1110-6100LS.................................502
PL1110-6115......................................505
PL1110-6120......................................505
PL1110-6125......................................505
PL1110-6130......................................505
PL1110-6140......................................505
PL1110-6150......................................505
PL1110-6160......................................505
PL1110-6200......................................500
PL1110-6200LS.................................502
PL1110-6300......................................500
PL1110-6320......................................505
PL1110-6400......................................508
PL1110-6500......................................500
PL1110-6504......................................500
PL1110-6515......................................505
PL1110-6520......................................505
PL1110-6525......................................505
PL1110-6530......................................505
PL1110-6540......................................505
PL1110-6550......................................505
PL1111-3500.............................310, 393
PL1113-1300......................................518
PL1113-1320......................................521
PL1113-1325......................................520
PL1113-1500......................................516
PL1113-3100......................................511
PL1113-3120......................................511
PL1113-3300......................................511
PL1113-3500......................................511
PL1113-6300......................................518
PL1113-6325......................................520
PL1113-6500......................................516
PL1113-6520......................................521
PL1114-1900HFIP.............................509
PL1114-6900HFIP.............................509
PL1117-1800......................................512
PL1117-1830......................................512
PL1117-6800......................................512
PL1117-6830......................................512
PL1120-3830......................................511
PL1120-6520......................................525
PL1120-6830......................................525
PL1145-1802......................................474
PL1145-1803......................................474
PL1147-1501......................................428
PL1147-6501......................................428
PL1149-1530......................................525
PL1149-1840......................................525
PL1149-3800......................................511
PL1149-6240......................................525
PL1149-6250......................................525
PL1149-6260......................................525
PL1149-6800......................................525
PL1149-6801......................................525
PL1149-6820......................................525
PL1149-6840......................................525
PL1149-6850......................................525
PL1149-6860......................................525
PL1151-1802......................................474
PL1151-1803......................................474
PL1151-3802......................................474
PL1151-3803......................................474
PL1170-1810......................................342
PL1170-1820......................................342
PL1170-1830......................................342
PL1170-1840......................................342
PL1170-1850......................................342
PL1170-1860......................................342
PL1170-2820......................................342
PL1170-2823......................................342
PL1170-6810......................................342
PL1170-6820......................................342
PL1170-6830......................................342
PL1170-6840......................................342
PL1170-6860......................................342
PL1171-1140......................................342
PL1171-6140......................................342
PL1210-1120......................................506
PL1210-3120EPA ..............................507
PL1210-6100......................................506
PL1210-6104......................................506
PL1210-6115......................................506
WWW.AGILENT.COM/CHEM/LC
PL1210-6120......................................506
PL1210-6120EPA ..............................507
PL1210-6125......................................506
PL1210-6130......................................506
PL1210-6140......................................506
PL1210-6150......................................506
PL1210-6160......................................506
PL1212-1102......................................470
PL1212-1103......................................470
PL1212-3100......................................470
PL1212-3101......................................470
PL1212-3702......................................470
PL1212-3703......................................470
PL1212-3800......................................470
PL1212-3801......................................470
PL1212-6100......................................470
PL1212-6101......................................470
PL1212-6200......................................470
PL1212-6201......................................470
PL1212-6400......................................470
PL1212-6401......................................470
PL1212-6800......................................470
PL1212-6801......................................470
PL1213-6520......................................521
PL1220-6130......................................528
PL1245-1102......................................474
PL1245-1103.............................411, 474
PL1245-3102......................................474
PL1245-3103.............................411, 474
PL1245-3702.............................411, 474
PL1245-3703.............................411, 474
PL1249-1120.............................525, 528
PL1249-6100......................................528
PL1249-6140......................................528
PL1249-6150......................................528
PL1251-1102.............................409, 474
PL1251-1103.............................409, 474
PL1251-3102.............................409, 474
PL1251-3103.............................409, 474
PL1251-3702.............................409, 474
PL1251-3703.............................409, 474
PL1310-0001......................................529
PL1310-0002......................................529
PL1310-0005......................................529
PL1310-0007......................................529
PL1310-0008......................................529
PL1310-0012......................................529
PL1310-0016....................310, 342, 393
PL1310-0036......................................529
PL1310-0048......................................529
PL1312-1300.............................393, 463
PL1312-1301......................................463
PL1312-1500.............................393, 463
PL1312-1501......................................463
PL1312-1502.............................393, 463
PL1312-1503......................................463
PL1312-1802.............................393, 463
PL1312-1803......................................463
PL1312-3300.............................393, 463
PL1345-1502.............................411, 463
PL1345-1503.............................411, 463
PL1351-1502.............................409, 463
PL1351-1503.............................409, 463
PL1410-0101......................................529
PL1410-0200......................................529
PL1410-0301......................................529
PL1410-0501......................................529
PL1412-4100......................................471
PL1412-4101......................................471
PL1412-4102......................................471
PL1412-4103......................................471
PL1412-4200......................................471
657
PART NUMBER INDEX
PL1412-4201......................................471
PL1412-4400......................................471
PL1412-4401......................................471
PL1412-4702......................................471
PL1412-4703......................................471
PL1412-4A05.....................................475
PL1412-4K00 .....................................471
PL1412-4K01 .....................................471
PL1412-4K02 .....................................471
PL1412-6100......................................471
PL1412-6101......................................471
PL1412-6102......................................471
PL1412-6103......................................471
PL1412-6200......................................471
PL1412-6201......................................471
PL1412-6400......................................471
PL1412-6401......................................471
PL1412-6702......................................471
PL1412-6703......................................471
PL1412-6800......................................471
PL1412-6801......................................471
PL1412-6A05.....................................475
PL1412-6K00 .....................................471
PL1412-6K01 .....................................471
PL1412-6K02 .....................................471
PL1417-0800......................................512
PL1417-0830......................................512
PL1445-4102.............................411, 474
PL1445-4103......................................474
PL1445-4702.............................411, 474
PL1445-4703.............................411, 474
PL1445-6102.............................411, 474
PL1445-6103.............................411, 474
PL1445-6702.............................411, 474
PL1445-6703.............................411, 474
PL1451-4102.............................409, 474
PL1451-4103.............................409, 474
PL1451-4702.............................409, 474
PL1451-4703.............................409, 474
PL1451-6102.............................409, 474
PL1451-6103.............................409, 474
PL1451-6702.............................409, 474
PL1451-6703.............................409, 474
PL1510-1100......................................504
PL1510-1200......................................504
PL1510-1300......................................504
PL1510-1500......................................504
PL1510-1504......................................504
PL1510-5100......................................504
PL1510-5200......................................504
PL1510-5300......................................504
PL1510-5500......................................504
PL1510-5504......................................504
PL1512-1300.............................310, 393
PL1512-1301.............................310, 393
PL1512-1500.............................310, 393
PL1512-1501.............................310, 393
PL1512-1502.............................310, 393
PL1512-1503.............................310, 393
PL1512-1801.............................310, 393
PL1512-1802.............................310, 393
PL1512-1803.............................310, 393
PL1512-3100......................................470
PL1512-3101......................................470
PL1512-3102......................................470
PL1512-3103......................................470
PL1512-3200......................................470
PL1512-3201......................................470
PL1512-3300.............................310, 393
PL1512-3301.............................310, 393
PL1512-3401......................................470
PL1512-3501.............................310, 393
658
PL1512-3702......................................470
PL1512-3703......................................470
PL1512-3800....................310, 393, 470
PL1512-3801....................310, 393, 470
PL1512-3802.............................310, 393
PL1512-3803.............................310, 393
PL1512-5100......................................470
PL1512-5101......................................470
PL1512-5102......................................470
PL1512-5103......................................470
PL1512-5200......................................470
PL1512-5201......................................470
PL1512-5400......................................470
PL1512-5401......................................470
PL1512-5500.............................310, 393
PL1512-5501.............................310, 393
PL1512-5702......................................470
PL1512-5703......................................470
PL1512-5800....................310, 393, 470
PL1512-5801....................310, 393, 470
PL1512-5802.............................310, 393
PL1512-5A05.....................................475
PL1514-1900HFIP.............................509
PL1514-5900HFIP.............................509
PL1545-1502......................................411
PL1545-1503......................................411
PL1545-1802......................................411
PL1545-1803......................................411
PL1545-3102.............................411, 474
PL1545-3103.............................411, 474
PL1545-3702.............................411, 474
PL1545-3703.............................411, 474
PL1545-3802......................................411
PL1545-3803......................................411
PL1545-5102.............................411, 474
PL1545-5103.............................411, 474
PL1545-5702......................................474
PL1545-5703.............................411, 474
PL1547-1501......................................428
PL1547-5501......................................428
PL1551-1502......................................409
PL1551-1503......................................409
PL1551-1802......................................409
PL1551-1803......................................409
PL1551-3102.............................409, 474
PL1551-3103.............................409, 474
PL1551-3702.............................409, 474
PL1551-3703.............................409, 474
PL1551-3802......................................409
PL1551-3803......................................409
PL1551-5102.............................409, 474
PL1551-5103.............................409, 474
PL1551-5702.............................409, 474
PL1551-5703.............................409, 474
PL1570-5810......................................342
PL1612-1801.............................310, 393
PL1670-0810......................................342
PL1670-0820......................................342
PL1670-0830......................................342
PL1670-0840......................................342
PL1670-0850......................................342
PL1670-0860......................................342
PL1671-0140......................................342
PL1712-3100......................................470
PL1712-3101......................................470
PL1712-3102......................................470
PL1712-3103......................................470
PL1712-3200......................................470
PL1712-3201......................................470
PL1712-3400......................................470
PL1712-3401......................................470
PL1712-3702......................................470
PL1712-3703......................................470
PL1712-3800......................................470
PL1712-3801......................................470
PL1712-6800......................................470
PL1712-6801......................................470
PL1745-3102......................................474
PL1745-3103.............................411, 474
PL1745-3702......................................474
PL1745-3703.............................411, 474
PL1751-3102.............................409, 474
PL1751-3103.............................409, 474
PL1751-3702.............................409, 474
PL1751-3703.............................409, 474
PL1812-3102......................................470
PL1812-3103......................................470
PL1812-6100......................................470
PL1812-6101......................................470
PL1812-6200......................................470
PL1812-6201......................................470
PL1812-6400......................................470
PL1812-6401......................................470
PL1812-6800......................................470
PL1812-6801......................................470
PL1845-2102......................................474
PL1845-2103.............................411, 474
PL1845-3102.............................411, 474
PL1845-3103.............................411, 474
PL1851-2102.............................409, 474
PL1851-2103.............................409, 474
PL1851-3102.............................409, 474
PL1851-3103.............................409, 474
PL1912-1300.............................310, 393
PL1912-1301.............................310, 393
PL1912-1500.............................310, 393
PL1912-1501.............................310, 393
PL1912-1502.............................310, 393
PL1912-1503.............................310, 393
PL1912-1801.............................310, 393
PL1912-1802.............................310, 393
PL1912-1803.............................310, 393
PL1912-3300.............................310, 393
PL1912-3301.............................310, 393
PL1912-3500.............................310, 393
PL1912-3501.............................310, 393
PL1912-3801.............................310, 393
PL1912-3802.............................310, 393
PL1912-3803.............................310, 393
PL1912-5500.............................310, 393
PL1912-5501.............................310, 393
PL1912-5801.............................310, 393
PL1945-1502......................................411
PL1945-1503......................................411
PL1945-1802......................................411
PL1945-1803......................................411
PL1945-3802......................................411
PL1945-3803......................................411
PL1951-1502......................................409
PL1951-1503......................................409
PL1951-1802......................................409
PL1951-1803......................................409
PL1951-3802......................................409
PL1951-3803......................................409
PL1C12-2502 .....................................393
PL1E10-3120EPA..............................507
PL1E10-6120EPA..............................507
PL1E12-5A05.....................................475
PL1F70-6830......................................342
PL1F70-6850......................................342
PL2010-0100......................................537
PL2010-0101......................................537
PL2010-0102......................................537
PL2010-0103......................................537
PL2010-0104......................................537
PL2010-0105......................................537
PL2010-0200......................................535
PL2010-0201......................................535
PL2010-0202......................................535
PL2010-0203......................................535
PL2010-0300......................................535
PL2010-0301......................................535
PL2010-0400......................................535
PL2010-0401......................................535
PL2010-0402......................................535
PL2010-0403......................................535
PL2010-0501......................................536
PL2010-0505......................................536
PL2010-0601......................................536
PL2010-0605......................................536
PL2012-0001......................................538
PL2012-0005......................................538
PL2012-0010......................................538
PL2012-1001......................................538
PL2012-1005......................................538
PL2012-1010......................................538
PL2012-2001......................................538
PL2012-2005......................................538
PL2012-2010......................................538
PL2012-3001......................................538
PL2012-3005......................................538
PL2012-3010......................................538
PL2012-4001......................................538
PL2012-4005......................................538
PL2012-4010......................................538
PL2012-5001......................................538
PL2012-5005......................................538
PL2012-5010......................................538
PL2012-6001......................................538
PL2012-6005......................................538
PL2012-6010......................................538
PL2012-7001......................................538
PL2012-7005......................................538
PL2012-7010......................................538
PL2012-8001......................................538
PL2012-8005......................................538
PL2012-8010......................................538
PL2012-9001......................................538
PL2012-9005......................................538
PL2012-9010......................................538
PL2013-1001......................................538
PL2013-1005......................................538
PL2013-1010......................................538
PL2013-2001......................................538
PL2013-2005......................................538
PL2013-2010......................................538
PL2013-3001......................................538
PL2013-3005......................................538
PL2013-3010......................................538
PL2013-4001......................................538
PL2013-4005......................................538
PL2013-4010......................................538
PL2013-5001......................................538
PL2013-5005......................................538
PL2013-5010......................................538
PL2013-6001......................................538
PL2013-6005......................................538
PL2013-6010......................................538
PL2013-7001......................................538
PL2013-7005......................................538
PL2013-7010......................................538
PL2013-8001......................................538
PL2013-8005......................................538
PL2013-8010......................................538
PL2013-9001......................................538
PL2013-9005......................................538
PART NUMBER INDEX
PL2013-9010......................................538
PL2014-0001......................................538
PL2014-0005......................................538
PL2014-0010......................................538
PL2014-1001......................................538
PL2014-1005......................................538
PL2014-1010......................................538
PL2014-2001......................................538
PL2014-2005......................................538
PL2014-2010......................................538
PL2014-3001......................................538
PL2014-3005......................................538
PL2014-3010......................................538
PL2014-4001......................................538
PL2014-4005......................................538
PL2014-4010......................................538
PL2014-6001......................................538
PL2014-6005......................................538
PL2014-6010......................................538
PL2014-7001......................................538
PL2014-7005......................................538
PL2014-7010......................................538
PL2014-8001......................................538
PL2014-8005......................................538
PL2014-8010......................................538
PL2014-9001......................................538
PL2014-9005......................................538
PL2014-9010......................................538
PL2020-0200......................................535
PL2020-0201......................................535
PL2020-0202......................................535
PL2020-0203......................................535
PL2022-2001......................................540
PL2022-2005......................................540
PL2022-2010......................................540
PL2022-3001......................................540
PL2022-3005......................................540
PL2022-3010......................................540
PL2022-5001......................................540
PL2022-5005......................................540
PL2022-5010......................................540
PL2022-6001......................................540
PL2022-6005......................................540
PL2022-6010......................................540
PL2022-7001......................................540
PL2022-7005......................................540
PL2022-7010......................................540
PL2022-8001......................................540
PL2022-8005......................................540
PL2022-8010......................................540
PL2022-9001......................................540
PL2022-9005......................................540
PL2022-9010......................................540
PL2023-0001......................................540
PL2023-0005......................................540
PL2023-0010......................................540
PL2023-1001......................................540
PL2023-1005......................................540
PL2023-1010......................................540
PL2023-2001......................................540
PL2023-2005......................................540
PL2023-2010......................................540
PL2023-3001......................................540
PL2023-3005......................................540
PL2023-3010......................................540
PL2023-4001......................................540
PL2023-4005......................................540
PL2023-4010......................................540
PL2023-5001......................................540
PL2023-5005......................................540
PL2023-5010......................................540
PL2023-6001......................................540
PL2023-6005......................................540
PL2023-6010......................................540
PL2023-7001......................................540
PL2023-7005......................................540
PL2023-7010......................................540
PL2023-8001......................................540
PL2023-8005......................................540
PL2023-8010......................................540
PL2023-9001......................................540
PL2023-9005......................................540
PL2023-9010......................................540
PL2024-0001......................................540
PL2024-0005......................................540
PL2024-0010......................................540
PL2024-1001......................................540
PL2024-1005......................................540
PL2024-1010......................................540
PL2024-2001......................................540
PL2024-2005......................................540
PL2024-2010......................................540
PL2070-0200......................................535
PL2070-0201......................................535
PL2070-0202......................................535
PL2070-0203......................................535
PL2070-1001......................................542
PL2070-1005......................................542
PL2070-1010......................................542
PL2070-2001......................................542
PL2070-2005......................................542
PL2070-2010......................................542
PL2070-3001......................................542
PL2070-3005......................................542
PL2070-3010......................................542
PL2070-4001......................................542
PL2070-4005......................................542
PL2070-4010......................................542
PL2070-5001......................................542
PL2070-5005......................................542
PL2070-5010......................................542
PL2070-6001......................................542
PL2070-6005......................................542
PL2070-6010......................................542
PL2070-7001......................................542
PL2070-7005......................................542
PL2070-7010......................................542
PL2070-8001......................................542
PL2070-8005......................................542
PL2070-8010......................................542
PL2070-9001......................................542
PL2070-9005......................................542
PL2070-9010......................................542
PL2071-0001......................................542
PL2071-0005......................................542
PL2071-0010......................................542
PL2071-1001......................................542
PL2071-1005......................................542
PL2071-1010......................................542
PL2071-2001......................................542
PL2071-2005......................................542
PL2071-2010......................................542
PL2071-3001......................................542
PL2071-3005......................................542
PL2071-3010......................................542
PL2080-0200......................................535
PL2080-0201......................................535
PL2080-0202......................................535
PL2080-0203......................................535
PL2083-1001......................................542
PL2083-1005......................................542
PL2083-1010......................................542
PL2083-2001......................................542
PL2083-2005......................................542
PL2083-2010......................................542
PL2083-3001......................................542
PL2083-3005......................................542
PL2083-3010......................................542
PL2083-4001......................................542
PL2083-4005......................................542
PL2083-4010......................................542
PL2083-5001......................................542
PL2083-5005......................................542
PL2083-5010......................................542
PL2083-6001......................................542
PL2083-6005......................................542
PL2083-6010......................................542
PL2083-7001......................................542
PL2083-7005......................................542
PL2083-7010......................................542
PL2083-8001......................................542
PL2083-8005......................................542
PL2083-8010......................................542
PL2083-9001......................................542
PL2083-9005......................................542
PL2083-9010......................................542
PL2084-0001......................................542
PL2084-0005......................................542
PL2084-0010......................................542
PL2084-1001......................................542
PL2084-1005......................................542
PL2084-1010......................................542
PL2084-2001......................................542
PL2084-2005......................................542
PL2084-2010......................................542
PL2090-1000......................................544
PL2090-3000......................................544
PL2090-4000......................................544
PL2090-5000......................................544
PL2090-6000......................................544
PL2090-8000......................................544
PL2091-1000......................................544
PL2091-2000......................................544
PL2091-3000......................................544
PL2091-4000......................................544
PL2142-3000......................................545
PL2142-3001......................................545
PL2142-5000......................................545
PL2142-6000......................................545
PL2142-6001......................................545
PL2142-7000......................................545
PL2142-7001......................................545
PL2142-8000......................................545
PL2142-8001......................................545
PL2143-0000......................................545
PL2143-0101......................................545
PL2143-2000......................................545
PL2143-2001......................................545
PL2143-3000......................................545
PL2143-3001......................................545
PL2143-4000......................................545
PL2143-4001......................................545
PL2143-5000......................................545
PL2143-5001......................................545
PL2143-6000......................................545
PL2143-6001......................................545
PL2143-7000......................................545
PL2143-7001......................................545
PL2143-8000......................................545
PL2143-8001......................................545
PL2143-9000......................................545
PL2143-9001......................................545
PL2144-0000......................................545
PL2144-0101......................................545
PL2144-1000......................................545
PL2144-1001......................................545
WWW.AGILENT.COM/CHEM/LC
PL2144-2000......................................545
PL2144-2001......................................545
PL2144-3000......................................545
PL2144-3001......................................545
PL3540-C603VP................................476
PL3540-D603VP................................476
PL3540-P603VP ................................476
PL3549-3603VP ................................476
PL3554-1602dAbz ............................347
PL3554-1602dCac ............................347
PL3554-1602dCbz.............................347
PL3554-1602dGdmf .........................347
PL3554-1602dGibu...........................347
PL3554-1602dT.................................347
PL3554-4602dAbz ............................347
PL3554-4602dCac ............................347
PL3554-4602dCbz.............................347
PL3554-4602dGdmf .........................347
PL3554-4602dGibu...........................347
PL3554-4602dT.................................347
RMSN-2..............................................108
RMSN-4..............................................108
659
APPLICATION TITLE INDEX
Application Title Index
12 phenols analyzed
using a longer (4.6 x 100 mm)
Agilent Poroshell 120 EC-C18 column.................231
A
Adrenocorticosteroids
on Pursuit PFP and C18 ..........................................290
Agilent Bio SEC-3
column length comparison, 150 mm...................422
Agilent Bio SEC-3
column length comparison, 300 mm...................422
Alberta Peptide Institute test mix................308, 390
Alcohols and aliphatic compounds......................582
Alkyd resin..................................................................519
Amino acid standard separation
on Eclipse Plus C18..................................................564
Amitrol in water by LC/MS, 0.05 ppb .................585
An overlay of the original ZORBAX
Eclipse Plus 5 µm method and Agilent
Poroshell 120 method. All 11 peaks on
Poroshell 120 are resolved by the time
the first peak elutes on the original 5 µm
ZORBAX Eclipse Plus method ..............................595
Analgesics......................................................................618
Analgesics: Non-steroidal anti-inflammatory
drugs: Narrow bore separation ................................622
Analysis of Biocides in Hand Sanitizer ...............580
Analysis of Bovine Serum Albumin by light
scattering using ProSEC 300S columns..............429
Analysis of carbohydates
on Hi-Plex H columns .............................................340
Analysis of choline kinase on PL-SAX 4000Å.....407
Analysis of ciprofloxacin
and ciprofloxacin metabolites................................643
Analysis of diazepam on Rx-C18...........................272
Analysis of fruit juice...............................................338
Analysis of low molecular weight polystyrene
and oligomer fractions collected from
OligoPore preparative columns ................................522
Analysis of oxidized insulin chains.........................553
Analysis of pesticide residues in green tea.........594
Analysis of representative whey proteins............408
Analysis of sugars with high sodium matrix .......341
Analysis of sweeteners on Hi-Plex Ca columns.....340
Analysis of water soluble vitamins
in multivitamin tablets......................................................612
Analytical separation of
low molecular weight polystyrene .......................522
Anesthetics, local: Bonded phase selectivity....619
Anilines, substituted: Rapid separation ..............585
Anthocyanins from blueberries:
High-efficiency high-speed separation................598
Antibiotics: High speed separation ......................620
Antibodies: Fast separation of IgM
and IgG antibodies ...................................................565
Antidepressants, tricyclic:
Comparative separation ..........................................636
Antifungal medications ...........................................621
Antifungals ........................................................290, 621
Antihistamines: Fast separations
on RRHT Extend-C18 ...............................................617
660
Antibiotics: Lincomycin and Clindamycin
by LC-APCI-MS LC-TIC ............................................620
Aromatic acids/benzoic acids:
Selectivity differences .............................................623
Aromatics II................................................................599
Aspartame: Metabolites and applications..........599
Aspirin and cough remedy .....................................628
B
BSA tryptic digest on RRHT...................................577
Barbiturates................................................................643
Baseline expansion of a separation
of protein standards.................................................414
Basic antihistamines
on Extend-C18 at high pH.......................................275
Benzoic acid/sorbic acid........................................609
Bio-Monolith DEAE column monitors
phage production during fermentation................415
Blueberry anthocyanin analysis ...........................594
Brij 35 ..........................................................................581
C
Calibration curves.....................................................505
Calibration of the ProSEC 300S
column with globular proteins ..............................429
Capillary columns for HPLC
analyses with UV and MS detection ...................455
Carbohydrates in colas............................................601
Carbohydrates in juices...........................................601
Carbohydrates in milk..............................................602
Carbohydrates: Carbohydrate standards.............600
Carbohydrates:
Effect of mobile phase strength............................600
Carbohydrates: Sugar alcohols..............................601
Catecholamines.........................................................578
Catecholamines/biogenic amines:
Rapid separation using ion-pair reagents...........623
Charge isoform analysis
of monoclonal antibodies.......................................401
Chiral ethiazide (diuretic drug) separation .........624
Chiral separation of S- and R-Norfluoxetine......626
Chiral separation of atenolol..................................627
Chiral separation of
fluoxetine enantioners (Prozac) ............................624
Chiral separation of hexobarbitol..........................625
Chiral separation of salbutamol ............................626
Chiral separation of tolperison enantiomers .....627
Cocaine and metabolites ........................................627
Column reproducibility – 200 injections of
reduced monoclonal antibody using an
Agilent ZORBAX RRHD 300SB-C3 column........555
Column stability testing at pH 3 and 60 °C........257
Column stability testing at pH 7.0 ........................258
Columns for sample clean-up................................507
Comparing HILIC and RPLC of morphine
using Agilent ZORBAX RRHD columns
with UHPLC/MS ......................................................631
Comparison of Aβ peptide RP-HPLC
separations at low and high pH............................573
Comparison of Agilent Bio SEC-3
and competitor column in the analysis
of a monoclonal antibody.......................................420
Comparison of PolyPore
with conventional individual
pore size GPC columns ...........................................517
Comparison of phenols separation
with Poroshell 120....................................................584
Components of green tea separated
on Rapid Resolution StableBond SB-C8.............625
Consistent ion-exchange
MAb separation ...............................................400, 562
Corn syrup, Hi-Plex...................................................339
Cough formula mixture:
Fast and efficient separation .................................628
Cough/cold remedies on ZORBAX 300SCX ......334
Crude bradykinin prep load ....................................469
Crude peptide screen...............................................476
D
DNPH: Derivatized Aldehydes
obtained from air.......................................................584
Deoxynucleosides: Using rapid resolution
3.5 µm columns.........................................................577
Determination of anthocyanins in blueberries .....597
Dexamethasone, USP method: Rapid analysis.....639
Differences in composition of
two alkyl naphthalene sulfonates ........................527
Dimethyl-C18/amide, Bonus-RP...........................280
E
EasiVial PS-H .............................................................533
Eclipse Plus C18 vs. C8 ...........................................250
Eclipse Plus C8 is less retentive
than Eclipse Plus C18..............................................251
Eliminate tailing and maximize resolution
with Eclipse Plus Columns.....................................250
Environmental phenols on Poroshell 120...........230
Epoxy resin ................................................................501
Excellent separation of two phenol
formaldehyde resins with PolarGel-M.................513
Exceptional Lot-to-lot Reproducibility..................426
Exceptional separating power ...............................403
Exploiting chemical stability –
NH4OH concentration....................................308, 390
Explosives and related compounds:
Qualitative and quantitative analysis...................586
Explosives from soil extract ...................................587
Extend-C18 provides good peak shape
at low pH ....................................................................276
F
Fast analysis 11 common compounds
found in analgesics .................................................615
Fast analysis of cefepime
and related impurities ....................................217, 611
Fast analysis of Pindolol .........................................642
Fast analysis of sulfa drugs...................................596
Fast and ultra-fast analysis
of basic compounds........................................251, 617
APPLICATION TITLE INDEX
Fast LC/MS/MS analysis of group 4
pharmaceuticals from EPA 1694 ..........................583
Fast method for ginseng analyses
scaled from a traditional method.........................612
Fast separation of recombinant
human erythropoietin..............................................553
Fast separation of reduced
monoclonal antibody...............................................375
Fast vitamin E analysis
on Rapid Resolution HT ..........................................608
Fast, high-resolution separation of
peptides and proteins with
Poroshell 300SB-C18 ...............................................572
Faster analysis of USP Method
for simvastatin tablet...............................................615
Faster separation of sulfa drugs ...........................616
Faster separations using Agilent
weak cation-exchange columns...........................560
Fat-soluble vitamins on
ZORBAX Eclipse XDB-C8........................................605
Five different bonded phases
provide selectivity options......................................266
Flavoring agents........................................................602
Food colors, FD&C....................................................602
Four different 300SB
bonded phases optimize separation
of large polypeptides................................................371
Fraction analysis –
the concentration overload purification .............469
G
Glycosylated proteins: Large Molecules
on Poroshell 300SB-C18 and 300SB- ..................565
Goldenseal and related alkaloids on
Rapid Resolution Eclipse XDB-C18 ......................625
Good peak shape over a wide pH range
with ZORBAX Eclipse XDB ....................................257
Gradient optimizations for ultra-fast
analysis of reduced monoclonal antibody .........556
Guaifenesin: USP analysis of guaifenesin..........629
High sensitivity with capillary columns ..............453
Higher resolution of intact monoclonal antibody...368
Higher resolution of oxidation study..........354, 368
Hormones/steroids..................................................633
Human serum: Low abundance
protein isolation and identification
from 1-D gel band by LC/MS .......................454, 566
Hyaluronic acid..........................................................527
Hydrophilic purine/pyrimidine separation..........579
I
Ibuprofen: Optimizing selectivity
with RRHT Columns.................................................618
Improved peak shape of basic
compounds using Bonus-RP .................................279
Improved reproducibility
of monoclonal antibodies.......................................369
Improved resolution with smaller particle size
with Agilent weak cation-exchange columns.....559
Increase peak capacity with RRHT columns.....240
Increased resolution for peptide mapping....357, 369
Intact MAb monomer
and dimer separation.............................355, 419, 563
L
LC/MS analysis of angiotensin
on Extend-C18 ...........................................................377
LC/MS performance test mix
for Polaris C8-A .........................................................299
Lamotrigine ................................................................642
Large fibrous proteins.....................................309, 392
Liquid chromatography phase
test mixture (LPTM) on Pursuit C8 ......................290
Local anesthetics......................................................619
Long life at high pH with 300Extend-C18...........378
Long life at high pH with Extend-C18..................275
Long lifetime of RRHT columns
at elevated temperatures........................................241
H
M
HPLC of 25 bp DNA ladder ..........................307, 388
HPLC separation of 12 phenols perfomed
in just 5 minutes – and under 400 bar – using
an Agilent Poroshell 120 EC-C18 column .............231
HSA tryptic digest on
ZORBAX Rapid Resolution HT 1.8 µm.................566
Heparin........................................................................526
Herbicide/pesticide standards:
Effect of bonded phase ...........................................588
Herbicides on different bonded phases ..............587
Herbicides: Rapid separation .................................591
High Resolution of 24 Amino Acids
Using ZORBAX Eclipse AAA Protocol.................396
High purity and high recovery
with ZORBAX PrepHT columns ............................315
High resolution and fast analysis
on RRHT Eclipse PAH column...............................254
High resolution normal-phase separation
of octylphenoxy ethanol surfactant
on ZORBAX CN.........................................................327
High resolution separation of a
Poly-T-Oligonucleotide size standard
spiked with 10-mer, 15-mer, 30-mer
and 50-mer (main peaks)........................................408
N
Mechanical stability of Pursuit XRs.....................289
Metronidazole: Updating USP methods..............629
Microbore HPLC for sensitive peptide analysis ...462
MicroBore Poroshell 300 columns
provide maximum sensitivity for LC/MS............382
Mixture of beta blockers ........................................613
Monoclonal IgG1 chains:
Separation on Poroshell 300SB-C8 .............383, 567
Morphine and metabolites:
Extracted blood plasma sample separation.......630
Naproxen analysis ...................................................611
Natural products – capsaicin and
dihydrocapsaicin on Pursuit XRs C18 .................319
Neutraceuticals: Extract from green tea.............603
Neutraceuticals:
Hypericin separation in St. John's Wort.............632
New levels of sensitivity and resolution.............235
Nucleosides, purines and pyrimidines ................564
Nucleosides: Separation of deoxy
and ribonucleosides.................................................568
Nucleotides: Separation of mononucleotides ...568
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O
Oligosaccharides.......................................................610
Opiates (drugs of abuse) by LC/MS....................630
Optimize separations with
Eclipse XDB selectivity options.............................259
Optimizing protein separations with
Agilent weak cation-exchange columns............558
Organic acid analysis...............................................338
Organic acids separated on ZORBAX SB-Aq.......581
Overlay of UV and Light Scattering 90° for a
Sample of y-globulins, Illustrating Monomer,
Dimer, Trimer, and Aggregate Peaks....................430
Overlay of UV and light scattering 90° for a
sample of γ-globulins, illustrating monomer,
dimer, and trimer peaks ..........................................430
P
pH gradient elution for improved separation
of monoclonal antibody charged variants..........561
PLgel LS column .......................................................503
PLgel Olexis reveals true modalities
across the range of polyolefins.............................508
Parabens: High speed separation.........................604
Peak shape and efficiency are better
with ZORBAX Eclipse Plus.....................................249
Peptide RP-HPLC/ESI-MS using NH4OH
mobile phase yields both positive and
negative ion spectra................................................573
Peptides/proteins:
Effect of elevated temperature.....................370, 571
Peptides/proteins:
Equivalent gradient separations............................571
Peptides: Effect of TFA concentration........370, 569
Peptides: Separation of antiotensins
I, II, III with TFA and NH4OH ................................570
Peptides: Separation of antiotensins
I, II, III with TFA and NH4OH ................................632
Phenols, substituted ................................................592
Phenoxyacid herbicides ..........................................591
Plant hormones: Rapid gradient
elution separation.....................................................593
Plasticized PVC.........................................................501
Polyamides .................................................................509
Polycyclic aromatic hydrocarbons
according to EPA Method 610...............................589
Polyester .....................................................................519
Polyesterimide...........................................................520
Polyethylene Glycol/Oxide standards .................542
Polyethylene glycols .......................................307, 388
Polymethylmethacrylate in DMF...........................517
Polymethylmethacrylate standards......................540
Polyol...........................................................................501
Polyphenylene Sulfides...........................................500
Polystyrene standards .............................................538
Polyurethanes............................................................520
Polyvinyl alcohol ..............................................526, 528
Pore size choice: Mouse IgG ................................421
Pore size choice: Proteins......................................421
Poroshell 120 EC-C18 for fast
UHPLC separations ..................................................232
Poroshell 300 columns separate
proteins and peptides in seconds ........................381
661
APPLICATION TITLE INDEX
Preparative fractionation of a culture
filtrate containing amyloglucosidases
on Agilent PL-SAX 4000Å......................................473
Preparative scale purification of
Leuprolide by concentration overload.................359
Preparative separation of low
molecular weight polystyrene ...............................522
Protein digest analysis............................................552
Protein elution pattern on
ZORBAX Poroshell 300SB-C8 ...............................384
Proteins in a complex sample by
2-D HPLC with Nano HPLC columns...................457
Proteins: Effect of bonded phase .........................575
Proteins: Effect of bonded phase, RP..................575
Pullulan polysaccharide standards.......................544
Purification of a 25-mer trityl-off
oligonucleotide and analytical quantitation of
the fraction using PLRP-S 100Å, 4.6 x 50 mm ....469
Purification of a large oligonucleotide.................473
Q
Quantification and qualification of
vitamin C and citric acid in fresh
grapefruit juice ..........................................................609
R
Rapid analysis of an analgesic tablet,
selectivity differences at pH 2.7 and pH 7 .........250
Rapid method development for 18 compounds
with an Agilent RRHD Eclipse PAH column......590
Rapid Resolution HT (RRHT) provides double
the efficiency of Rapid Resolution columns ......240
Reduce analysis time dramatically
with Rapid Resolution HT columns......................241
Reduce peptide map analysis time
by 90% with Poroshell 300SB ...............................382
Resin analysis by rapid GPC ..................................511
Rose wine...................................................................610
S
SB-CN optimizes retention and resolution.........266
Scale-up from analytical to prep ..........................321
Selectivity changes for basic compounds
with Eclipse XDB and StableBond.......................258
Selectivity comparison of TFA and NH4OH
for peptide RP-HPLC\ESI-MS analysis...............574
Selectivity comparison of TFA and
NHSelectivity comparison: C18 columns............236
Selectivity comparison: Phenyl columns ............237
Selectivity for urea pesticides ...............................259
Selectivity in peptide RP-LC...................................389
Selectivity test mix for Polaris columns..............299
Separation of 20 PAHs on Eclipse PAH ..............591
Separation of 8 steroids .........................................613
Separation of azo dye degradation products ....583
Separation of Azo Dyes...........................................598
Separation of EPA 610 PAH Mix...........................589
Separation of a tryptic digest
on ZORBAX MicroBore 300SB-C18 .....................461
Separation of basic peptides on
Bonus-RP versus traditional Alkyl phase ...........569
Separation of charge variants of
human IgG1 with pH gradient ...............................355
662
Separation of group 4 analytes in
EPA 1694 on ZORBAX HILIC Plus column .........325
Separation of heated, stressed MAb...................563
Separation of highly basic antidepressants
above their pKa in free base form
(pKa 9.5-9.7) ...............................................................635
Separation of licorice root on RRHD columns ..234
Separation of pharmaceutical cardiac drugs.....616
Separation of polypeptides in under 1 minute ..572
Separation of protein standards
on Agilent 3 μm ion-exchange columns
by cation-exchange chromatography..................403
Separation of recombinant human
erythropoietin (rEPO)...............................................562
Separation of small molecule anorectics ...........622
Separation of vitamin D2/D3.................................605
Separation optimization for ultra-fast analysis
of reduced and alkylated monoclonal antibody.....554
Several ZORBAX RRHD 1.8 µm selectivities
facilitate method development .............................614
Short-chain ZORBAX 300SB-C3
is stable at low pH, high temperature.................371
Shorter chain ZORBAX SB-CN
is also stable at low pH (pH 2.0, 50 °C)..............265
Space ...........................................................................265
Sports drink................................................................610
StableBond SB-C18 shows excellent
stability at low pH and high temperature...........265
Standard ion-exchange
protein separation............................................407, 576
Standard protein separation ..................................410
Standard proteins by reversed-phase..................576
Starches......................................................................500
Steroids .......................................................................634
Steroids: Easy scalability
using Agilent Prep columns...................................312
Steroids: Separation.................................................633
Sub 1 minute separations
with RRHD columns.................................................235
Sugars..........................................................................604
Sugars in plain and milk chocolate......................604
Sulfa drugs .................................................................641
Sulfonamides – Fast analysis
with RRHT columns .................................................640
Sunscreen ingredients: Perform
conventional, fast and ultra-fast
separations on the same column family ............607
Superior loadability on Agilent Prep C18
with basic compounds ............................................312
T
Temperature as a tool to enhance mass
transfer and improve resolution of
oligonucleotides in ion-pair
reversed-phase HPLC .....................................391, 579
Ten cardiac drugs on
Rapid Resolution HT SB-C18 .................................640
Theobromine in beverages.....................................608
Tocopherols by LC/MS with APPI .......................603
Triamcinolone – USP
analysis of triamcinolone........................................635
Triazine pesticides on Bonus-RP
and Alkyl C8 phase...................................................592
Tricyclic antidepressants.........................................636
Tricyclic antidepressants
and benzodiazepines................................................289
Tricyclic antidepressants
and metabolites: Effect of pore size ....................637
Triton X-114: Decreasing run-time
by changing bonded phase ....................................580
Two samples of melamine resin
analyzed by PolarGel-L ............................................513
U
UHPLC efficiency at HPLC pressures ..................230
Ultra-high speed and high resolution
of intact monoclonal antibodies...........................557
USP analysis of tetracyclines ................................639
USP method: Glyburide and internal
standard, progesterone ...........................................638
USP method for sorbitol.........................................341
USP methods for sugar alcohols ..........................339
Ulcer treatment drugs at intermediate pH .........637
Urine, LSD analysis by LC/MS..............................638
Use ZORBAX Extend-C18 for
alternate selectivity at high pH ....................378, 567
V
VX nerve agent metabolites by
LC/MS-IS standard (C13 labeled) ........................593
Virtually eliminate retention time variations .......400
W
Warfarin: USP chromatographic
purity method using Eclipse XDB-CN..................640
Water-soluble B vitamins
separated on ZORBAX SB-Aq ...............................607
Water-soluble vitamins ...........................................605
Water-soluble vitamins using
the USP 23 method ..................................................606
Water-soluble vitamins:
High speed separation using ion-pairing............606
Weak cation-exchange chromatography
for P128 therapeutic protein sample on the
Agilent 1260 Bio-inert Quaternary LC system
using different cation-exchange columns..........404
Whey proteins in dairy samples – milk......391, 578
X
Xanthines: Higher resolution,
same selectivity with RRHT...................................617
Z
ZORBAX Bio-SCX Series II provides
more retention of small peptides..........................459
ZORBAX Bonus-RP is stable
at low and mid pH....................................................279
ZORBAX Bonus-RP
provides unique selectivity.....................................280
ZORBAX Eclipse Plus: Best peak shape
in the industry without tailing ...............................249
ZORBAX Nano columns for high sensitivity
protein digest analysis by LC/MS........................453
COMPOUND INDEX
Compound Index
A
Acebutolol..................................................................613
Acenaphthene...................................................589-591
Acenaphthylene................................................589-591
Acephate ....................................................................594
Acetaldehyde – DNPH ............................................584
Acetamide ..................................................................615
2-Acetamidophenol.........................................235, 615
Acetaminophen.....................235, 250, 615, 618, 628
Acetanilide ........................................................235, 618
Acetate................................................................174-175
Acetic Acid.................................................................581
Acetone ......................................................................582
Acetone – DNPH......................................................584
Acetophenone...........................................................599
6-Acetylmorphine .....................................................628
Acetylsalicylic acid........................235, 250, 618, 628
Acrolein – DNPH......................................................584
Adenine..............................................................564, 579
Adenosine .........................................................564, 568
Adonitol ......................................................................339
Adrenaline..................................................................578
Alanine...............................................................396, 564
Alanine-3-Glycine-4.........................................308, 390
Albuterol ............................................................325, 583
Alprenolol ...................................................................613
2-Amino-5-azotoluene .............................................183
4-Aminobenzoic acid ......................................290, 621
p-Aminobenzoic acid ...............................................609
3-Amino-benzonitrile ...............................................585
7-Aminoclonazepam ................................................289
2-Amino-4,6-dinitrotoluene ............................586-587
4-Amino-2,6-dinitrotoluene ....................................587
4-Amino-4,6-dinitrotoluene ....................................586
7-Aminoflunitrazepam .............................................289
2-Aminonaphthalene ...............................................183
2-Amino-4-nitrotoluene...................................586-587
2-Amino-6-nitrotoluene...................................586-587
4-Amino-2-nitrotoluene...........................................586
Amitriptyline ....................................258, 289, 635-637
cis-10-OH -Amitriptyline..........................................637
trans-10-OH-Amitriptyline.......................................637
Ammonium ................................................................173
Amylbenzene.............................................................299
Amylobarbitone.........................................................643
Anadamine.................................................................236
Androstadiene 3,17 dione ......................................613
Anethole .....................................................................602
Angiotensin I .................308, 377, 389-390, 462, 570
Angiotensin II ..........................194, 308, 370, 377, 381,
389-390, 462, 570-572
Angiotensin III........................308, 377, 389-390, 570
Aniline .......................................................183, 583, 598
p-Cl-Aniline.................................................................183
Anisidine.....................................................................598
p-Anisidine .................................................................585
Anthracene ...............................................189, 589-591
a-1-Antichymotrypsin.....................................454, 566
Antithrombin-III................................................454, 566
Apomyoglobin ...........................................................572
Aprotinin.....................................................................403
Arabinose ...................................................................341
Arabitol........................................................................339
2-Arachinoylglycerol ................................................381
Arginine .............................................................396, 564
Arsenate .....................................................................175
Arsenite ......................................................................175
Ascorbic acid.............................................................612
Asparagine........................................................396, 596
Aspartame.......................................232, 290, 299, 599
Aspartic acid.....................................................396, 596
Aspartic acid-phenylalanine dipeptide................599
Atenolol ......................................................................613
Atrazine......................................................587-588, 591
Azide..........................................................172, 175, 421
B
Barberine....................................................................625
Barbital........................................................................258
Barbitone....................................................................643
Beclomethasone.......................................................290
Bendroflumethiazide................................................189
Bentazon ....................................................................587
Benzaldehyde – DNPH ...........................................584
Benz(e)pyrene ..........................................................591
Benzidine..................................................183, 583, 598
1,2-Benzisothiazol-3(2H)-one ................................580
Benzisothiiazol-3(2H)-one ......................................235
Benzo(a)anthracene .......................................589-591
Benzo(a)pyrene.................................................589-591
Benzo(b)fluoranthene......................................589-591
Benzocaine ................................................................619
Benzo(g,h,i)perylene........................................589-591
Benzoic acid....................................232, 290, 389, 580,
602, 609, 621, 629
Benzo(k)fluoeanthene.....................................589-591
Benzophenone ..........................................................618
Benzoylecgonine ......................................................627
Benzthiazuron ...........................................................588
n-6-Benzyl adenine ..................................................593
5-Benzyl-3,6-dioxo-2-piperazineacetic acid........599
Berberine...........................................................251, 617
Biosynthetic human insulin...................................384
Biotin (B7)..................................................................612
Biphenyl......................................................................189
Bovine carbonic anhydrase ..........................407, 576
Bromide ......................................................................172
BSA...................................382, 425-426, 432, 576-577
Bumetanide ...............................................................189
Buspirone ...................................................................616
Butacaine ..........................................................266, 619
tert-butanol ................................................................582
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2-Butanone (MEK) – DNPH...................................584
Butylbenzene.............................................................299
Butylparaben ...........................................189, 241, 604
n-Butyraldehyde – DNPH.......................................584
C
Caffeine.........................232, 235, 250, 258, 276, 299,
316, 334, 599, 608, 615
Calcium .......................................................................173
Calmodulin .................................................................571
Canadine.....................................................................625
Carbamazepine.................................................618, 632
Carbaryl.......................................................................594
Carbendazim ............................................235, 580, 594
Carbonate...................................................................175
Carbonic Anhydrase ......................370-371, 381-382,
408, 571-572
Carvone.......................................................................602
Catalase ......................................................................432
Catechol...................................230-231, 584, 603, 625
Cefazolin .....................................................................620
Cefepime.....................................................................217
Cefotaxime .................................................................620
Ceftazidime ................................................................620
Celecoxib ....................................................................235
Cephaclor ...................................................................280
Cephalexin..................................................................280
Cephoxitin ..................................................................280
Cephuroxime..............................................................280
Chlorate.......................................................................172
Chloride......................................................172, 174-175
Chloroaniline.....................................................583, 598
m-Chloroaniline.........................................................585
o-Chloroaniline..........................................................585
p-Chloroaniline..........................................................585
2-Chlorobenzoic ........................................................623
Chlorocaine................................................................619
4-Chloro-3-methylphenol ........................................592
5-Chloro-2-methyl-4-isothiazolin-3-one...............235
2-Chlorophenol..........................................................592
o-Chlorophenoxyacetic acid..........................591, 593
p-Chlorophenoxyacetic acid..........................591, 593
o-Chlorophenoxy proprionic acid..........................593
Chlorothiazide............................................................189
Chlorpheniramine ..........................275, 279, 617, 628
Chlorthalidone...........................................................189
Chrysene.............................................................589-591
Chymotrypsinogen A ...............................................410
Cimetidine ................................................325, 583, 637
d-Cinchonine.....................................................266, 619
Cinnamaldehyde .......................................................599
Ciprofloxacin..............................................................620
Citrate..........................................................................174
Citric Acid................................338, 396, 581, 609-610
Clindamycin ...............................................................620
Clomipramine ............................................................289
663
COMPOUND INDEX
Clonazepam ...............................................................289
Clotrimazole ...............................................................621
Cobalamin (B12) .......................................................612
Cocaine ..............................................................619, 627
Codeine.......................................................................630
Corticosterone...........................................................290
Cortisone ...........................................................290, 299
Cortisone acetate .....................................................290
m-Cresol......................................................................590
o-Cresol.............................................230-231, 584, 592
p-Cresol......................................................230-231, 584
Crotonaldehyde – DNPH ........................................584
Cyanide .......................................................................175
Cyanidin .............................................................237, 597
Cyanocobalamin (B12)............................................605
p-Cymene ...................................................................599
Cyprodinil....................................................................594
Cysteine ......................................................................396
Cytidine .......................................................................568
Cytochrome C ................183, 382, 370-371, 400, 403,
410, 558-560, 571, 572, 576
Cytodine......................................................................564
Cytosine .............................................................564, 579
D
Daidzen .......................................................................266
Dehydroacetic acid ..................................................232
Delphinidin ........................................................237, 597
2' Deoxycytidine .......................................................568
2' Deoxyguanosine ..................................................568
2' Deoxyinosine ........................................................568
Desethylatrazine .......................................................588
Desethyldesisopropylatrazine................................588
Desipramine...............................................................289
Dextromethorphan ...................................................628
Diazepam...........................................................272, 289
Dibenzo(a,h)anthracene..................................589-591
Dichlorobenzidine............................................583, 598
3,3-Dichlorobenzidine..............................................183
2,4-Dichlorophenol ...................................................592
2,3-Dichlorophenoxyacetic acid............................591
2,4-Dichlorophenoxyacetic acid............................591
Diclofenac.................................................235, 615, 618
Dienestrol ...................................................................633
Diethylstilbestrol.......................................................265
Diflusinal............................................................235, 615
Dihydroxy benzylamine ...........................................623
Dihydroxyphenyl acetic acid..................................623
Dihydroxyphenyl alanine.........................................623
Diltiazen ......................................................................616
Dimethoxybenzidine.................................................598
3,3'-Dimethoxybenzidine ........................................583
Dimethylbenzidine....................................................583
2,3-Dimethyl phenol................................230-231, 530
2,4-Dimethyl phenol.................................................592
2,5-Dimethyl phenol................................230-231, 530
3,4-Dimethyl phenol........................................231, 584
1,3-Dimethyluric acid...............................................617
Dimethylxanthine .....................................................276
664
1,7-Dimethylxanthine...............................................617
3,7-Dimethylxanthine (theobromine)...................617
1,3-Dinitrobenzene ...........................................586-587
2,4-Dinitrophenol ......................................................592
2,4-Dinitrotoluene.............................................586-587
2,6-Dinitrotoluene.............................................586-587
Dioctyl phthalate..............................................290, 299
Diphenhydramine ...................................275, 617, 632
Diphenylamine ..........................................................586
Dipropyl phthalate....................................................241
Dipropylthalate..........................................................250
Dipyridamole..............................................................616
Disopyramide.............................................................640
Diuron................................................259, 587-588, 591
Dopamine ..........................................................578, 623
Doxepin.............................................258, 289, 635-636
Doxycycline................................................................639
Doxylamine ..............................................275, 279, 617
Dulcitol........................................................................339
E
Epinephrine................................................................623
Ecgonine methylester..............................................627
Econazole ...................................................................621
Eletriptan ....................................................................616
Epagallocatechin ......................................................603
Epicatechin........................................................603, 625
Epicatechin gallate..........................................603, 625
Epigallocatechin........................................................625
Epigallocatechin gallate.................................603, 625
Estradiol......................................................................633
b -Estradiol.................................................................613
Estriol ..........................................................................266
Estrone........................................................................613
Ethanol ...............................................................582, 610
Ethinylestradiol .........................................................633
Ethoprophos...............................................................594
Ethyl cinnamate........................................................599
2-Ethylhexyl trans-4-methoxycinnamate ............607
bis-(2-Ethylhexyl) phthalate ...................................507
Ethylhexyl salicylate ................................................259
2-Ethylhexyl salicylate.............................................607
Ethylparaben............................................189, 272, 604
Eugenol .......................................................................599
Excipent ......................................................................606
F
Famotidine..................................................................637
Fenfluramine..............................................................622
Fenoprofen .................................................................622
Fenuron.......................................................................259
Fibrinogen..........................................................309, 392
Flunitrazepam............................................................289
Fluocinolone acetonide...........................................290
Fluoranthene.............................................189, 589-591
Fluorene.....................................................189, 589-591
Fluoride ..............................................................172, 175
2-Fluorobenzoic.........................................................623
3-Fluorobenzoic.........................................................623
Fluorocytosine...........................................................579
Folic acid....................................................605-606, 612
Formaldehyde – DNPH ...........................................584
Formate.......................................................................174
Fructose ..................................338, 340, 600, 604, 610
Fumaric Acid..............................................................581
Furosemide ................................................................616
G
Galactose....................................................................340
Genistein ....................................................................266
g-Globulin ...................................................................421
Glucagon.....................................................................384
Glucose ...........................338, 340-341, 600, 604, 610
Glutamine ..........................................................396, 564
Glutamate...................................................................175
Gly3-Gly4 (Na-acetylated).............................308, 390
Glyburide.....................................................................638
Glycerol .......................................................................610
Glyceryl Guaicolate ..................................................334
Gluconate ..........................................................396, 564
Glycine.......................................................389, 396, 564
Guaifenesin................................................................629
Guanine..............................................................564, 579
Guanosine .........................................................564, 568
H
Heptabarbitone .........................................................643
Hexaldehyde – DNPH..............................................584
Hexazinon...................................................................588
Hexogen (RDX) .................................................586-587
Hexyl............................................................................586
Histidine.............................................................396, 564
Homocyclonite ..........................................................586
Homovanillic acid .....................................................623
Holotransferrin ..........................................................572
Hydrastine ..................................................................625
Hydrochlorothiazide .................................................189
Hydrocortisone........................................312, 613, 635
Hydroflumethiazide ..................................................189
Hydroquinone ...........................................230-231, 584
Hydroxyindoleaacetic acid .....................................623
2-Hydroxy-4-methoxybenzophenone ...................607
4-Hydroxyropivacaine ..............................................619
Hydroxyproline .................................................396, 564
5- Hydroxytryptamine ..............................................623
I
Ibuprofen ..................................................235, 396, 618
IgA................................................................................421
IgG2a, I HOPC-1........................................................565
IgM, MOPC-104E......................................................565
Imazalil........................................................................594
Imidacloprid ...............................................................594
Imipramine ...............................................289, 632, 635
Indeno(1,2,3-c,d)pyrene ..................................589-591
COMPOUND INDEX
Indole...........................................................................642
3-Indole acetic acid..................................................593
3-Indole butyric acid ................................................593
3-Indole proprionic acid ..........................................593
Inosine.........................................................................568
Insulin.......................370-371, 381-382, 462, 571-572
Iodide...........................................................................172
Iso-erythritol...............................................................339
Isoleucine ...................................................................564
Isomaltose..................................................................600
Isopropanol ................................................................582
Isoproturon.................................................................588
K
Kathon 1A ..................................................................580
Kathon 1B...................................................................580
Ketoprofen.........................................................235, 615
Kinetin .........................................................................593
Kresoxim-methyl.......................................................594
L
a-Lactalbumin.........................................391, 408, 578
Lactate ................................................................174-175
Lactic Acid ...............................................581, 338, 610
Lactoglobulin A.........................................................183
Lactoglobulin B.........................................................183
b -Lactoglobulin (A chain) ...................391, 408, 578
b -Lactoglobulin (B chain)...........391, 408, 432, 578
Lactose......................................................340, 600, 602
Lamotrigine ................................................................642
Leucine...............................................................396, 564
Leucine Enkephalin........................194, 370, 571-572
Lidocaine ..................................................266, 619, 640
Lincomycin.................................................................620
Linuron ...............................................................259, 588
Lysine .................................................................396, 564
Lysozyme...............183, 370-371, 381-382, 400, 403,
410, 558-560, 571-572, 576
M
Magnesium................................................................173
Malate.........................................................................174
Maleate.......................................................................275
Maleic acid.................................................................628
Malic acid...................................................................610
Maltose.......................................................................600
Maltotriose.................................................................340
Malvidin .............................................................237, 597
Mannitol ....................................................339-341, 604
Mefanamic acid ........................................................241
Mepivacaine ..............................................................619
Metacycline ...............................................................639
Metazachlor...............................................................588
Met-Enkephalin........................................194, 571-572
Metformin..........................................................325, 583
Methabenzthiazuron................................................588
Methacrolein –DHCP...............................................584
Methanol ....................................................................582
3-Methaxytyrosine ...................................................623
Methionine........................................................396, 564
Methoxyaniline .........................................................583
4-Methoxybenzenesulfonamide............................289
Methoxychlor, 200 mg/L........................................507
Methyl-3-aminothiophene-2-carboxylate............289
2-Methyl-4,6-dinitrophenol.....................................592
4,4-Methylene-bis-2-chloroaniline........................183
2-Methyl-4-isothiazolin-3-one ...............................235
1-Methyl naphthalene ............................................591
2-Methyl naphthalene ............................................591
2-Methyl-5-nitroaniline............................................183
Methyl paraben..............................189, 235, 580, 604
Methyl prednisolone................................................290
Methyl salicylate ......................................................602
1-Methylxanthine .....................................................617
Metobromuron ..........................................................588
Metolachlor........................................................587-588
Metoprolol..................................................................613
Metoxuron..................................................................588
Miconazole.................................................................621
Molybdate (VI) ..........................................................172
Monolinuron .....................................................259, 588
Monuron.....................................................................259
Morphine............................................................630-631
Morphine-6-glucuronide .........................................630
Morphine-3-glucuronide ........................................630
Myoglobin ......................370-371, 381-382, 407, 410,
421, 432, 571-572, 576
N
Nadolol...............................................................613, 640
Naphthalene ....................................189, 241, 589-591
1-Naphthol ................................................230-231, 584
1-Naphthyl acetamide.............................................593
1-Naphthyl acetic acid ............................................593
Naphthylamine.................................................583, 598
Naproxen...........................................................241, 618
Neurotensin..............................................381, 571-572
Niacin (B3)................................................605-606, 612
Niacinamide...............................................................607
Nicotinic Acid............................................................607
Nifedipine ..........................................................315, 640
Nimodipine........................................................315, 640
Nisoldipine ........................................................315, 640
Nitrate ................................................................172, 175
Nitrite .................................................................172, 175
2-Nitrobenzoic...........................................................623
3-Nitrobenzoic...........................................................623
2-Nitrophenol...................................230-231, 584, 592
4-Nitrophenol...................................230-231, 584, 592
2-Nitrotoluene ...................................................586-587
3-Nitrotoluene ...................................................586-587
4-Nitrotoluene ...................................................586-587
Noradrenaline............................................................578
Nordiazepam..............................................................289
Nordoxepin.................................................................289
Norepinephrine .........................................................623
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Norethindrone ...........................................................633
Norethindrone acetate............................................613
Normorphine..............................................................631
Nortriptyline............................250, 258, 289, 635-637
cis-10-OH-Nortriptyline ...........................................637
trans-10-OH-Nortriptyline.......................................637
Norvaline ...........................................................396, 564
O
Octogen (HMX).........................................................587
Octylmethoxycinnamate .........................................250
Oleoylethanolamide (OEA)............................328, 614
Orotic Acid .................................................................568
Ovalbumin ......................................381, 407, 412, 421,
558-560, 572, 576
Oxalate ........................................................................174
Oxalic acid.........................................................338, 389
Oxybenzone.......................................................250, 259
Oxytetracycline..........................................................639
Oxytocin......................................................................462
P
Padimate-O .......................................................259, 607
Palatinose...................................................................600
Palmatine ...................................................................625
Palmitoylethanolamide...................................236, 614
Pantothenic acid..............................................605, 612
Parvalbumin...............................................................371
Penconazole ..............................................................594
Pencycuron ................................................................259
Pentachlorophenol...................................................592
iso-Pentane................................................................565
Pentylparaben ...........................................................189
Peonidin.............................................................237, 597
Perphenazine.............................................................632
Perylene......................................................................507
Petunidin ...........................................................237, 597
Phenacetin ......................................235, 615, 618, 622
Phenanthrene...........................................189, 589-591
Phenobarbitone.........................................................643
Phenol.......................................................231, 240, 249,
299, 584, 592
Phenoxyacetic acid..................................................591
2-Phenoxyethanol............................................235, 580
Phentermine ..............................................................622
Phenylalanine (PHE) ..............................396, 564, 599
Phenylbutazone ........................................................622
Phenylephrine ...........................................................334
Phosphate ..........................................................174-175
Phthalic acid..............................................................623
p-hydroxybenzoic acid.............................................232
Picric acid...................................................................586
Pindolol .....................................................613, 640, 642
Pioglitazone ...............................................................616
Pirenzepine ................................................................637
Piroxicam...........................................................235, 615
Poly-DL-alanine.........................................................425
Potassium...................................................................173
665
COMPOUND INDEX
Prednisolone.....................................................290, 632
Prednisolone acetate...............................................290
Procainamide....................................................616, 640
Procaine....................................................266, 619, 640
Progesterone ....................................................613, 638
Promethazine.............................................................632
Prometon....................................................................587
Prométryne........................................................587, 591
Propanil..............................................................587, 591
n-Propanol..................................................................582
Propanolol ..................................................................636
Propazine...................................................587-588, 591
Propionaldehyde – DNPH ......................................584
Propoxur .....................................................................594
Propranolol......................................241, 613, 636, 640
Propylparaben ..................................................189, 604
Protriptyline..............................................396, 564, 632
Pseudoephedrine....................................275, 617, 628
Pymetrozine ...............................................................594
Pyrene .................................................................589-591
Pyridine ..............................................................249, 389
Pyridoxine...........................................................605-607
Pyridoxyl phosphate (B6) .......................................612
Pyrilamine...................................................................334
Pyroglutamate ...........................................................174
Pyruvate......................................................................174
Q
Quercetin....................................................................266
R
Raffinose...................................................340, 600, 604
Ranitidine...........................................................325, 583
Reserpine...........................................................290, 299
Resorcinol..................................................230-231, 584
Retinol .........................................................................605
Retinol acetate..........................................................605
Retinol palmitate ......................................................605
Rhamnose ..................................................................600
Riboflavin (B2)..........................................605-606, 612
Ribonuclease (RNase)...................371, 381, 571-572
Ribonuclease A.......................370, 400, 403, 425-426,
558-560, 571-572, 576
Ribose..........................................................................600
Ropivacaine................................................................619
S
Saccharin....................................................................232
Saccharose ................................................................600
Salicylic acid ...................................235, 290, 618, 621
Sarcosine...........................................................396, 564
Scopolamine .....................................................275, 617
Sebutylazine...............................................................588
Selenate......................................................................175
Serine .................................................................396, 564
Simazine .............................................................587-588
Sodium ........................................................................173
666
Sorbic acid................................................290, 609, 621
Sorbitol.......................................................338-341, 604
Stachyose..........................................................340, 604
Succinate....................................................................174
Succinic Acid...........................................338, 581, 610
Sucrose ....................................338, 340-341, 600, 604
Sulfachloropyridazine ..............................................616
Sulfadiazine......................................249, 616, 640-641
Sulfadimethoxine.............................................616, 641
Sulfamerazine..................................249, 616, 640-641
Sulfamethazine ........................................616, 640-641
Sulfamethizole..................................................616, 641
Sulfamethoxazole ..........................249, 258, 616, 632
Sulfamethoxypyridazine..........................................616
Sulfanilamide............................................249, 640-641
Sulfanilic acid ............................................................641
Sulfapyridine..............................................................616
Sulfate........................................................172, 174-175
Sulfathiazole ............................................249, 616, 640
Sulfisoxazole..............................................................641
Sulfmethazine............................................................249
Sulfur ...........................................................................507
Sulindac .............................................................235, 615
o-Toluidine ................................................183, 583, 598
Trehalulose.................................................................600
Triamcinolone ...................................................290, 635
Triamcinolone acetonide ........................................290
2,4,6-Trichlorophenol................................................592
2,4,5-Trichlorophenoxyacetic acid.........................591
3,4,5-Trichlorophenoxyacetic acid.........................593
2,4,5-Trichlorophenoxypropionic acid (Silvex)....591
3,4,5-Trichlorophenoxyproprionic acid .................593
Triflupromazine..........................................................632
Trimipramine ...........................258, 289, 632, 635-636
1,3,5-Trinitrobenzene........................................586-587
2,4,6-Trinitrotoluene .........................................586-587
Tripelennamine..........................................................632
Triphenylene...............................................................299
Triprolidine................................................275, 279, 617
Tryptophan (TRP)....................................396, 459, 564
Tyrosine (TYR) .........................................396, 459, 564
Tébuthiuron................................................................591
U
Uracil...............240-241, 258, 299, 425-426, 564, 579
Uridine................................................................564, 568
T
Talbarbitone ...............................................................643
Tartarate......................................................................175
Tartaric acid.......................................................338, 610
Tebuthiuron ................................................................587
Terbutylazine..............................................................588
Terphenyl-d14 ...........................................................591
o-Terphenyl.................................................................299
Testosterone .....................................................312, 613
Tetracaine..........................................................266, 619
2,3,4,6-Tetrachlorophenol........................................592
Tetryl ....................................................................586-587
Theobromine............................................276, 316, 608
Theophylline ............................................276, 316, 334
Thiabendazole ...........................................................594
Thiamine pyrophosphate (TPP) ............................594
Thiamine (B1)...................................................607, 612
Thiocyanate ...............................................................172
Thiosulfate .................................................................172
Thiourea.............................................................189, 590
Threonine (THR) ..............................................396, 564
Thymidine...................................................................564
Thymine .............................................................564, 579
Thymol ........................................................................602
Thyroglobulin...................................421, 425-426, 432
a-Tocopherol.....................................................605, 608
b-Tocopherol ..............................................................608
g-Tocopherol......................................................605, 608
Tocopherol acetate...................................................605
o-Tolidine.....................................................................598
Tolmetin ...........................................235, 615, 618, 622
m-Tolualdehyde – DNPH.........................................584
Toluene..............................................240, 299, 589-591
m-Toluic.......................................................................623
m-Toluidine.................................................................585
V
Valeraldehyde – DNPH ...........................................584
Valine..................................................................396, 564
Valine3-Glyine4 (Na-acetylated) .................308, 390
Valine3-Valine4 (Na-acetylated)..................308, 390
Valine-tyrosine-valine ..............................................572
Vitamin A....................................................................605
Vitamin B12 ...............................................................421
Vitamin C...................................................605-606, 609
Vitamin D2..................................................................605
Vitamin D3..................................................................605
Vitamin E (a-VE)........................................................605
W
Warfarin .....................................................................640
X
Xanthosine-5’-monophosphate (XMP) ...............568
Xylitol...........................................................................339
Xylose..........................................................................600
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