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5991-1059EN

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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-2àmcolumns.

ã ZoRBaX Rapid Resolution High definition (RRHd) columns 1.8àmcolumnsfeatureimproved
packingprocessestoachievestabilityupto1200barforusewiththeAgilent1290InfinityLCand
otherUHPLCinstrumentsandareavailableinmorethan14phases,plusHILIC.

ã ZoRBaX Eclipse Plus columns C18andC8columnsdeliversuperiorpeakshape,whilethe
phenyl-hexylbondedphaseandC18bondedphaseforPAHseparationsexpandselectivityoptionsfor
moreapplications.AllEclipsePlusphasesareavailableinFastLC/UHPLCRRHDandRRHTcolumns,
1.8àm.Forscalability,theEclipsePlusC18phaseisverysimilartothePoroshell120EC-C18phase.

ã 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

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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

210


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.
Lookforitonlineandviayourmobiledeviceat

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.

212


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

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.

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Phases


CoLuMnS FoR SMaLL MoLECuLE SEPaRaTionS

Fast Columns
for Reversed-Phase HPLC/UHPLC
Thepastdecadehasseenasteadyincreaseintheefficiencyandspeedofchromatography,startingwith
smallerparticlesizes,thatenablehigherresolution,andcontinuingwithnewtechnologicaladvancesin
particledesignsuperficiallyporousparticlesthatenablethesesameresolutionenhancementswith
lowerbackpressure.
Designedespeciallyforhigh-productivityanalysis(FastLC),AgilentZORBAXandPoroshellcolumnsare
thebestfirstchoiceforanyanalysis,becausetheygiveyou:

ã Theproductivityyouneedtostayaheadofyourcompetition:technologicaladvanceslikesub-2àm
particlesandsuperficiallyporousPoroshell120columnsdeliverincreasedspeedandresolution.

ã Flexibilityandmethodscalabilityfromlabtolabandaroundtheworldforsmallmoleculeand
biomoleculeanalyses.

ã Unbeatablechromatographicperformance:ZORBAXsilicathebasesilicausedforallZORBAX
andPoroshell120columnsisultra-pure,verystrong,andhighlyuniformforultimatereliability.

ã Thebroadestrangeofphasesandcolumnconfigurationstosuityourspecificapplicationneeds.
Recommendations for Fast LC Columns
your Lab Situation

agilent Recommends

Rationale

YoureusingbothUHPLC(1000+bar)
andHPLCinstruments
(e.g.Agilent1290InfinityLC
and1260InfinityLC600bar)

1.Poroshell120
2.ZORBAXRRHD1.8àm

Poroshell120isaneasycolumntouseonbothinstrument
types.ZORBAXRRHDwillhelpyouoptimizethecapabilities
ofthe1290InfinityLCforUHPLC.

Only400-600barHPLCs
Agilent1200s,Agilent1100s(400bar)
aswellasthe1220InfinityLC
or1260InfinityLC
(600bar)

1.Poroshell120

WithPoroshell120,youcanenhancetheperformanceofolder
400-barinstruments,andalsogetevenbetterperformancefrom
2.ZORBAXEclipsePlus3.5àmand5àm
newer600barUHPLCinstruments.Forestablishedmethodsthat
youcanttransfer,theZORBAXEclipsePluscolumnwillprovide
exceptionalpeakshapeandperformance.

AmixofUHPLCinstruments
1.ZORBAXRRHD1.8àm
(Agilent1290InfinityLC,
2.Poroshell120
other1000+barinstruments)
andsomeHPLCinstruments(e.g.1200LC)

ZORBAXRRHDcandeliveroptimumperformanceonallthese
instruments.Poroshell120canbeusedonthe600barinstruments
tooptimizetheirperformance.

TiPS & TooLS
AgilentCrossLaboffersarangeofPEEKcapillariesandtubing.Usedincombinationwiththerightfittings,
theyprovideaninertsurfacefortheFastLCofsensitivebiomolecules. Turntopage130.

www.agiLEnT.CoM/CHEM/LC

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