LC and LC/MS Columns - analitica PDF
Preview LC and LC/MS Columns - analitica
HPLC Column Selection Flow Chart LC and LC/MS Columns HPLC Column Selection Tousethecolumnselectionguidediagrambelow,simplyfollowthepathforyouranalyteandmobilephase.Atthefarright,followyourfinal columnselectiontothepagesindicated. seepages Normal-PhaseModewithBareSilica 873-874 HexaneSoluble seepages Normal-PhaseModewithBondedPhase 873-874 MeOHandMeOH: HOorACN 2 andACN: OrganicSolvent HOSoluble Reversed-PhaseModewithBondedPhase seepage827 Soluble 2 seepages Gel-Permeation(SmallMolecule) THFSoluble 947-948 MolecularWeight <2000 Non-Ionic Reversed-PhaseModewithBondedPhase seepage827 Reversed-PhaseModewithBondedPhase seepage819 WaterSoluble UsingIonizationControl Reversed-PhaseModewithBondedPhase seepage819 UsingIon-PairAgent Ionic Sample Reversed-PhaseModewithBareSilica seepage877 (HILIC) Ion-ExchangeMode seepage879 OrganicSolvent seepages Gel-PermeationChromatography Soluble 947-948 MolecularWeight >2000 Gel-FiltrationChromatography seepage997 Ion-ExchangeMode WaterSoluble seepage1013 withWide-PoreMaterial Reversed-PhaseMode seepage1016 withWide-PoreMaterial Adaptedwithpermissionfrom"PracticalHPLCMethodologyandApplications,"BrianA.Bidlingmeyer,JohnWiley&Sons,Inc.,NewYork,p.109 LCANDLC/MS 796 www.agilent.com/chem/lccolumns LC and LC/MS Columns Column and Mobile Phase Guidelines: Reversed Phase HPLCcolumnsconsistoftwoparts:thecolumnchemistryandhardware.Forthepropercolumn chemistry,consultthecatalogsectionforeachtypeofbondedphase.Forchoosingcolumn hardwareandparticlesizes,consultthesectiononcolumnsizesandrapidseparations,including AgilentZORBAXRapidResolutionHT,SolventSaver,CapillaryandPrepHTcolumns. Pore Size Selection Chooseacolumnpackingwithsmallpore(60-120Å)ifthesolutemolecularweightislessthan about5000.Otherwise,usecolumnpackingwiththe300Åporesize. Particle Size Selection ThetypicalparticlesizeforHPLCcolumnsis5μmwith3.5μmandsmallernowcommonin methoddevelopment.Ifhigh-speedanalysesorhigherresolutionanalysesarerequired,packing with1.8μmand2-3μmparticlescanbeused.Shortercolumnswiththeseparticlescanproduce fasterhigh-resolutionseparations,withthe1.8μmparticlesizeprovidingthehighestefficiency and2.7μmsuperficiallyporousprovidingsimilarresults.With1.8,2.7,3.5and5μmparticle sizestochoosefrom,startwiththesmallestparticlesizeforyourHPLCorUHPLC–400bar,600 bar,or1200bar–toachievethebestresults. Column Configuration Choosingthebestcolumnsizeformethoddevelopmenthaschangeddramaticallyinthepastfew years.Smaller3.0mmIDor2.1mmIDcolumnsarenowusedmorethan4.6mmIDtolower solventuseandachievecompatibilitywithMSdetectors.Andshorter50,75and100mmlong columnscanbeagreatstartingchoice,withlongercolumnsusedonlywhenmoreresolutionis neededorwhen3.5and5μmparticlesizesareused. LCANDLC/MSCOLUMNS 797 LC and LC/MS Columns Silica Type and Bonded Phase Base Material ThebasematerialforanLCcolumnismostoftenhighpuritysilicamaterialwithtotallyporous particlessuchasthatusedinmostAgilentZORBAXcolumns.However,morechoicesare available,includingpolymermaterialwithhighpHstabilityusedinPLRP-Scolumnsand superficiallyporoussilicaparticlessuchasthoseusedinPoroshell120columns.Thehighpurity TypeBsilicas,includingtheZORBAXRx-SilusedinZORBAXEclipsePlus,andsuperficiallyporous Poroshell120,areanexcellentfirstchoiceformostmethods.TypeAsilicas,suchasZORBAX SIL,usedinOriginalZORBAXcolumns,arestillmanufacturedandusedinmanymethods. Bonded Phase AgoodfirstchoiceforbondedphaseisC18orC8,andtherecommendedstartingcolumn choicesareEclipsePlusC18orPoroshell120EC-C18.Thesetwochoicesprovideexcellentpeak shapeandcanbeusedoverthepHrange2-9,accommodatingmosttypicalLCandLC/MS mobilephases.Ifthesamplesolutesofinterestarenotadequatelyseparatedonthesecolumns, CNandPhenylcolumns–includingPhenyl,Phenyl-HexylandDiphenyl–mayoffersignificant differencesinselectivityfromstraight-chainalkylphasestoeffecttheseparation. pH and Mobile Phase Thechoiceofmobilephaseforareversed-phasesystemstartswithselectingtheorganic modifier.Acetonitrileisthemostcommonlyusedorganicmodifier.However,selectivity differencesandsampleretentionwillvarysignificantlyamongmobilephasescontaining acetonitrile,methanol,andtetrahydrofuran(THF).Samplesolubilityislikelytodifferinsuch solventsanddictateuseofaspecificsolventorsolvents.UVdetectionatcertainwavelengthsis notpossiblewithcertainmodifiers(e.g.,methanolat200nm). Tips&Tools Agilentoffersavarietyofe-Seminarsandon-sitetraining tohelpyoulearnhowtobeamoreeffectivechromatographer. Formoreinformation,visitwww.agilent.com/chem/education LCANDLC/MS 798 www.agilent.com/chem/lccolumns LC and LC/MS Columns HPLCColumn Stationary Column Phase Dimensions Typeof Particle Inner PoreSize Length Surface Size Diameter ChemicalProperties PhysicalProperties ChemicalLifetime/Selectivity Efficiency RetentionFactor Sensitivity Speed ColumnChoiceRelativetoApplicationObjective ObjectiveColumn Application Diameter(mm) Veryhighsensitivity,LC/MS,peptidesandproteins 0.1,0.075 Veryhighsensitivity,limitedsample,LC/MS,peptidesandproteins 0.3,0.5 Highsensitivity,limitedsample,LC/MS 1.0 Savesolvent;speciallow-volumeinstrumentationisavailable 2.1 Specialdetectors,e.g.,massspec 2.1 Highsensitivity,limitedsample 2.1 Savesolvent;standardHPLCequipmentavailable,LC/MS 3.0 Standardseparations 4.6 Small-scale(mg)preparativeseparations 9.4 Large-scalepreparativeseparations(100mg-gram) 21.2 Large-scalepreparativeseparations(upto100mg-gram) 30,50 ConsulttheColumnHardwaresectionforguardcolumnconfigurations LCANDLC/MSCOLUMNS 799 Method Development RecommendedColumnChoicesforMethodDevelopment SmallMolecules LargeMolecules MW<2000 MW>2000 80-120Å PackingPoreSize 300Å Thefirstchoiceofapackingporesizeisbasedonthesizeofmoleculestobeanalyzed.Typicalsmallmoleculescandiffuseeasilyinandoutofstandard 80-120Åporepackings,butpeptidesandproteinsmaynot.Forthisreason,use300Åporepackings(300SB)forisocraticorgradientseparations ofpeptidesandproteins. EclipsePlusC18 StartingColumnBondedPhase StableBond300SB-C18 C18isrecommendedasthestartingcolumnbondedphaseformostsamplessinceitmaximizesretentionformoderatelypolartonon-polarcompounds. ShorterchainphasesshouldbeconsideredifresolutioncannotbeoptimizedwithaC18phaseorifyouareanalyzinglargerproteins. StartingColumnChoices SmallMolecules LargeMolecules StandardAnalysis FastAnalysis StandardAnalysis FastAnalysis EclipsePlusC18 Poroshell120EC-C18 ZORBAX300SB-C18 ZORBAX300SB-C18 4.6x150mm,3.5μm 4.6x100mm,2.7μm 4.6x150mm,5μm 4.6x50mm,3.5μm 959963-902 695975-902 883995-902 865973-902 Poroshell120EC-C18 RapidResolutionHDEclipse Poroshell300SB-C18 PlusC18,1200bar 4.6x75mm,2.7μm 2.1x75mm,5μm 2.1x50mm,1.8μm 697975-902 660750-902 959757-902 LCANDLC/MS 800 www.agilent.com/chem/lccolumns USP Designations USP Designations TheUSPharmacopeia(USP)isastandardsourceformanypharmaceuticalmethodsthatspecifiescolumnsbypackingmaterialsratherthan bymanufacturer.ListedbelowaretherecommendedAgilentTechnologiesHPLCcolumnssuitableformostLCmethodslistedwiththeUSP. USPMethod USPPackingMaterials Column ParticleSize(μm) PageNo. L1 Octadecylsilanechemicallybondedtoporoussilica Poroshell120EC-C18 2.7 822 orceramicmicro-particles,1.5to10μmindiameter Poroshell120SB-C18 2.7 822 ZORBAXEclipsePlusC18 1.8,3.5,5 827 ZORBAXEclipseXDB-C18 1.8,3.5,5,7 831 ZORBAXSB-C18 1.8,3.5,5,7 838 ZORBAXRx-C18 3.5,5 854 ZORBAXExtend-C18 1.8,3.5,5,7 850 ZORBAXODS 3.5,5,7 870 ZORBAXODSclassic 5 870 PursuitXRsC18 3,5,10 862 PursuitC18 3,5,10 860 PolarisC18-A 3,5,10 867 PolarisC18-Ether 3,5 867 SepTechST60C18 10 928 SepTechST150C18 10 928 L2 Octadecylsilanechemicallybondedtoporoussilicagel N/A ofacontrolledsurfaceporositythathasbeenbonded toasolidsphericalcore,30to50μmindiameter L3 Poroussilicaparticles,5to10μmindiameter ZORBAXSIL 5 873 ZORBAXRx-Sil 3.5,5 873 PursuitXRsSi 3,5,10 862 PolarisSi-A 5,10 867 MicroSpherSi 5 Microsorb100Si 5 L4 Silicagelofcontrolledsurfaceporositybondedto asolidsphericalcore,30to50μmindiameter L5 Aluminaofcontrolledsurfaceporositybondedto N/A asolidsphericalcore,30to50μmindiameter L6 Strongcation-exchangepacking:sulfonated N/A fluorocarbonpolymercoatedonasolidsphericalcore, 30to50μmindiameter (Continued) LCANDLC/MSCOLUMNS 801 USP Designations USPMethod USPPackingMaterials Column ParticleSize(μm) PageNo. L7 Octylsilanechemicallybondedtototallyporous Poroshell120EC-C8 2.7 822 microsilicaparticles,1.5to10μmindiameter ZORBAXEclipsePlusC8 1.8,3.5,5 827 ZORBAXEclipseXDB-C8 1.8,3.5,5,7 831 ZORBAXSB-C8 1.8,3.5,5,7 838 ZORBAXRx-C8 1.8,3.5,5,7 854 ZORBAXC8 5 870 PursuitXRsC8 3,5,10 856 PursuitC8 3,5,10 856 PolarisC8-A 3,5 865 PolarisC8-Ether 3,5 865 Microsorb100C8 5 L8 Anessentiallymonomolecularlayerofaminopropylsilane ZORBAXNH2 5 873 chemicallybondedtototallyporoussilicagelsupport, PolarisNH2 5 865 10μmindiameter Microsorb100Amino 5 L9 10μmirregular,totallyporoussilicagelhaving ZORBAXSCX 5spherical 879 achemicallybonded,stronglyacidiccation exchangecoating L10 Nitrilegroupschemicallybondedtoporoussilica ZORBAXCN 5 873 particles,3to10μmindiameter ZORBAXSB-CN 3.5,5 838 ZORBAXEclipseXDB-CN 3.5,5 831 Microsorb100Cyano 5 L13 Trimethylsilanechemicallybondedtoporoussilica ZORBAXTMS 5 particles,3to10μmindiameter L14 Silicagel10μmindiameterwithachemically ZORBAXSAX 5 879 bonded,stronglybasicquaternaryammonium IonoSpherA anionexchangecoating L15 Hexylsilanechemicallybondedtototallyporoussilica MetaSilC6 particles,3to10μmindiameter L16 Dimethylsilanechemicallybondedtototallyporoussilica N/A particles,3to10μmindiameter L17 Strongcationexchangeresinconsistingofsulfonated Hi-PlexH 8 881 cross-linkedstyrene-divinylbenzenecopolymerinthe hydrogenform,7to11μmindiameter L18 Aminoandcyanogroupschemicallybondedtoporous N/A silicaparticles,5to10μmindiameter L19 Strongcationexchangeresinconsistingofsulfonated Hi-PlexCa 8 881 cross-linkedstyrene-divinylbenzenecopolymerinthe Hi-PlexCa(Duo) 8 881 calciumform,9μmindiameter L20 Dihydroxypropanegroupschemicallybondedtoporous LiChrospherDiol 5 silicaparticles,3to10μmindiameter (Continued) LCANDLC/MS 802 www.agilent.com/chem/lccolumns USP Designations USPMethod USPPackingMaterials Column ParticleSize(μm) PageNo. L21 Arigid,sphericalstyrene-divinylbenzenecopolymer, PLgel 3,5,10,20 947 5to10μmindiameter PLRP-S100Å 3,5,8 1027 PLRP-S300Å 3,5,8 1027 PLRP-S1000Å 5,8 1027 L22 Acationexchangeresinmadeofporouspolystyrenegel Hi-PlexH 8 881 withsulfonicacidgroups,about10μminsize L23 Anionexchangeresinmadeofporouspolymethacrylate N/A orpolyacrylategelwithquaternaryammoniumgroups, about10μminsize L24 Asemi-rigidhydrophilicgelconsistingofvinylpolymers N/A withnumeroushydroxylgroupsonthematrixsurface, 32to63μmindiameter L25 Packinghavingthecapacitytoseparatecompounds PLaquagel-OH 5,8 974 withaMWrangefrom1,000to5,000da (asdeterminedbythepolyethyleneoxide),applied toneutral,ionicandcationicwater-solublepolymers L26 Butylsilanechemicallybondedtototallyporoussilica MicroSorbC4 5 particles,5to10μmindiameter L27 Poroussilicaparticles,30to50μmindiameter BondesilSilica 204 L28 Amultifunctionalsupport,whichconsistsofahigh N/A purity,100Å,sphericalsilicasubstratethathasbeen bondedwithanionic(amine)functionalityinaddition toconventionalreversed-phaseC8functionality L29 Gammaalumina,reversedphase,lowcarbon N/A percentagebyweight,alumina-basedpolybutadiene sphericalparticles,5μmdiameterwithaporediameter of80Å L30 Ethylsilanechemicallybondedtoatotallyporoussilica N/A particle,3to10μmindiameter L31 Astronganion-exchangeresin-quaternaryamine N/A bondedonlatexparticlesattachedtoacoreof8.5μm macroporousparticleshavingaporesizeof2000Å andconsistingofethylvinylbenzenecross-linkedwith 55%divinylbenzene L32 Achiralligand-exchangepackingL-prolinecopper N/A complexcovalentlybondedtoirregularlyshapedsilica particles,5to10μmindiameter L33 Packinghavingthecapacitytoseparateproteinsby ZORBAXGF-250 4 1001 molecularsizeoverarangeof4,000to400,000da. BioSEC-3 3 997 Itisspherical,silica-based,andprocessedtoprovide BioSEC-5 5 999 pHstability (Continued) LCANDLC/MSCOLUMNS 803 USP Designations USPMethod USPPackingMaterials Column ParticleSize(μm) PageNo. L34 Strongcationexchangeresinconsistingofsulfonated Hi-PlexPb 8 881 cross-linkedstyrene-divinylbenzenecopolymerinthe leadform,9μmindiameter L35 Azirconium-stabilizedsphericalsilicapacking ZORBAXGF-250 4 1001 withahydrophilic(diol-type)molecularmonolayer ZORBAXGF-450 6 1001 bondedphase L36 L-Phenylgylcine-3,5-dinitrobenzoylon5μmamino N/A propylsilica L37 Polymethacrylategelpackinghavingthecapacity N/A toseparateproteinsbymolecularsizeoverarange of2,000to4,000daMW L38 Methacrylate-basedsizeexclusionpacking N/A forwatersolubles L39 Hydrophilicpolyhydroxymethacrylategeloftotally N/A poroussphericalresin L40 Cellulosetris-3,5-dimethylphenylcarbamatecoated N/A poroussilicaparticles,5to20μmindiameter L41 Immobilizedalpha-acidglyco-proteinonsphericalsilica N/A particles,5μmindiameter L42 Octylsilaneandoctadecylsilanegroupschemically N/A bondedtoporoussilicaparticles L43 Pentafluorophenylgroupschemicallybondedtosilica PursuitPFP 3,5 856 particles5to10μmindiameter L44 Amultifunctionalsupport,whichconsistsofahigh N/A purity,60Åsphericalsilicasubstrate,thathasbeen bondedwithacationicexchanger,sulfonicacid functionalityinadditiontoaconventionalreversed phaseC8functionality L45 Betacyclodextrinbondedtoporoussilicaparticles, ChiraDexChiral 5 915 5to10μmindiameter L46 Polystyrene/divinylbenzenesubstrateagglomerated N/A withquaternaryaminefunctionalizedlatexbeads, 10μmindiameter L47 Highcapacityanionexchangemicroporoussubstrate, N/A fullyfunctionalizedwithatrimethyl-aminegroup, 8μmindiameter L48 Sulfonated,cross-linkedpolystyrenewithanouterlayer N/A ofsubmicron,porous,anion-exchangemicrobeads, 15μmindiameter L49 Amylosetris-3,5-dimethylphenyl-carabamate-coated, N/A porous,spherical,silicaparticles,5to10μmindiameter (Continued) LCANDLC/MS 804 www.agilent.com/chem/lccolumns USP Designations USPMethod USPPackingMaterials Column ParticleSize(μm) PageNo. L50 Astrongcationexchangeresinmadeofporoussilica ZORBAX300SCX 5 879 withsulfopropylgroups,5to10μmindiameter L51 Areversed-phasepackingmadebycoatingathinlayer N/A ofpolybutadieneontosphericalporouszirconia particles,3to10μmindiameter L52 Multifunctionresinwithreversed-phaseretention N/A andstronganion-exchangefunctionalities.Theresin consistsofethylvinyl-benzene,55%cross-linked withdivinylbenzenecopolymer,3to15μmindiameter, andasurfaceareaofnotlessthan350m2/g,substrate iscoatedwithquaternaryammoniumfunctionalized latexparticlesconsistingofstyrenecross-linked withdivinylbenzene. L53 Ananion-exchangeresinconsistingofrigid,spherical BioSAX 3,5,10 1006 styrene-divinylbenzenecopolymerwith trimethylammoniumgroupsataloadingofabout2meq perg,3to29μmindiameter L54 Strongcation-exchangeresinconsistingofsulfonated N/A cross-linkedstyrene-divinylbenzenecopolymerin thesodiumform,about7to11μmdiameter L55 Weakcation-exchangeresinconsistingof N/A ethylvinylbenzene,55%cross-linkedwithdivinylbenzene copolymer,3to15μmdiameter.Substrateissurface graftedwithcarboxylicacidand/orphosphoricacid functionalizedmonomers.Capacitynotlessthan 500μeq/column L56 Propylsilanechemicallybondedtototallyporoussilica SB-C3 3,5 838 particles,3to10μmindiameter L57 Achiral-recognitionprotein,ovomucoid,chemically UltronES-OVM 5 913 bondedtosilicaparticles,about5μmindiameter, withaporesizeof120angstroms L58 Strongcation-exchangeresinconsistingofsulfonated Hi-PlexNa 10 881 cross-linkedstyrene-divinylbenzenecopolymerinthe Hi-PlexNa(Octo) 8 881 sodiumform,about6to30μmindiameter L59 Packinghavingthecapacitytoseparateproteinsby N/A molecularweightovertherangeof5to7000kDa.Itis spherical(5-10μm),silica-based,andprocessedto providehydrophiliccharacteristicsandpHstability L60 Spherical,poroussilicagel,10μmorlessindiameter, Bonus-RP 1.8,3.5,5 846 thesurfaceofwhichhasbeencovalentlymodified PolarisAmide-C18 3,5 865 withalkylamidegroupsandendcapped LCANDLC/MSCOLUMNS 805
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