HPLC HPLC for Small Molecules 2 Back-PressureRegulators ....................................... 97 StainlessSteelUnions,Tubing ................................... 97 Supelco,APioneerinChromatography ............................ 2 PEEKTubing,PEEKsil™Tubing,PTFETubing ........................ 98 HPLCColumnSelection ........................................ 2 SaturatorColumnKits ......................................... 99 Ascentis®Express2.7Micron ..................................... 5 TubingCutters .............................................. 100 Ascentis®Express5Micron ..................................... 15 ColumnHardware ........................................... 101 Ascentis® ................................................... 18 apHera™-ForHigherpHApplications ............................ 26 TSKgel®HardwareandAccessories .............................. 102 Whatman®Syringe&SyringelessFilters .......................... 102 HamiltonCompanyHPLCColumns .............................. 28 SyringeTipFilters ........................................... 103 HamiltonCompanyHPLCGuardCartridge ......................... 28 Rheodyne®Injectors ......................................... 104 Discovery® .................................................. 29 Rheodyne®HighPressureValves ................................ 109 Discovery®Zirconia ........................................... 33 SUPELCOSIL™ ................................................ 35 Rheodyne®RheBuild®Kits,Fittings,Tools ......................... 109 SUPELCOGEL™ ............................................... 45 PrimingValves,GaugeKits,SSI™Valves,PulseDamper .............. 110 MobilePhaseDegassing/Filtration .............................. 111 Kromasil®HPLCColumns ....................................... 48 InletFilters,Debubbler ....................................... 113 GuardCartridgeAccessories .................................... 54 VacuumPumps ............................................. 114 LegacyColumns ............................................. 56 TemperatureControl ......................................... 115 HPLC for Large Molecules 58 High-FlowBasePlatePurifierSystemsforLC-MSNitrogen ........... 117 SeparationTechniquesforBiomolecules .......................... 58 PumpReplacementParts ..................................... 118 ColumnSelectionforBiomoleculeSeparations ..................... 59 LC-MSPostColumnFlowSplitters .............................. 121 Discovery®BIO ............................................... 59 PostcolumnReactors ......................................... 121 Reversed-PhaseChromatography ................................ 63 ASIStaticMixers ............................................ 121 GelFiltrationChromatography(GFC) ............................. 68 PostcolumnReactor .......................................... 122 IonExchangeChromatography ................................. 74 PHRED:PhotochemicalReactorEnhancedDetection ................ 123 HydrophobicInteractionChromatography(HIC) .................... 77 Solvents and Reagents 124 AffinityChromatography ....................................... 78 CHROMASOLV®Solvents ...................................... 124 HydrophilicInteractionChromatography(HILIC) .................... 78 LC-MSUltraCHROMASOLV®GradeSolventsandAdditivesforUHPLC .. 124 GelPermeationChromatography(GPC) ........................... 79 LC-MSCHROMASOLV®Solvents ................................ 125 TSKgel®HardwareandAccessories ............................... 81 CHROMASOLV®GradientSolvents ............................... 126 HPLC Column Test Mixes 82 LC-MSCHROMASOLV®Pre-BlendedMobilePhaseSolvents .......... 127 PerformanceEvaluation ........................................ 82 LC-MSMobilePhaseAdditives ................................. 127 CustomTestMixes ........................................... 83 RinsingSolutions ............................................ 128 SystemDiagnostics ........................................... 83 CHROMASOLV®PlusSolvents .................................. 128 GradientGradeSolvents ...................................... 131 HPLC Accessories 84 OtherCHROMASOLV®Solvents ................................. 131 Supel™ConnectHighPerformanceFittings ......................... 84 HPLCDerivatizationReagents .................................. 136 UpchurchScientificFittingsandAccessories ....................... 86 HPLCBuffers ............................................... 140 OptimizeTechnologies® ....................................... 90 IonPairReagents ............................................ 142 HPLCDispersionMeasurement .................................. 91 PEEKHPLCFittings ........................................... 91 StainlessSteelFittings ......................................... 93 In-LineFilters ................................................ 95 For all of your analytical needs, visit us at sigma-aldrich.com/analytical 2 HPLC HPLC for Small Molecules Supelco, A Pioneer in Chromatography HPLC for Small Molecules Supelco, A Pioneer in Chromatography Supelcohasbeenaleaderinchromatographyforover30years. Thefirstbasedeactivatedcolumn,SUPELCOSILLC-18-DBwasintroducedin1982andprovidedimprovedpeakshapeforbasiccompoundsthattailedwith earlygenerationcolumns.Theembeddedpolargroup(EPG)columnwasalsofirstlaunchedintheearly1980sandhasevolvedthroughseveralgenerationsto includeAscentisExpressRP-Amide,thenewestEPGcolumnintroducedtothemarket. TheinnovativeAscentisExpressColumn,basedonFused-Coretechnology,wasintroducedin2007andprovideshighspeedandresolutionatlow backpressures.Furthermore,expansionintothechiralchromatographymarketoccurredwiththeacquisitionofAstecchiralchromatographyproducts. Supelco,alongwithSigma,Aldrich,andFlukaprovidesthousandsofchromatographyrelatedproductsthroughworldwideresearchanddevelopment, manufacturing,distributionandsalesoperations. HPLC Column Selection How to Choose an HPLC Column Reversed-Phase HPLC Column Selection Flow Chart This flow chart provides information for choosing an initial column for reversed-phase method development. Packing Pore Size The size of the molecules to be analyzed will determine the packing pore size. Small molecules can diffuse in and out of 80 to 120 Å pore packings (Ascentis® Express, Ascentis®, and Discovery HS), but peptides and proteins may not. For this reason, it is recommended to use 200 to 300 Å pore-size packings (Discovery and Discovery Biowide) for separations of peptides and proteins. Column Diameter Column diameter defines how much material one can inject onto a column. Conventional analytical HPLC uses 4.6 mm internal diameter. However, smaller diameter columns (2.1 and 3.0 mm) provide increased sensitivity and therefore are commonly used in limited sample applications and LC-MS. Larger diameter columns (10 mm and 21.2 mm) allow for high sample loading which is beneficial for sample collection. Particle Size For conventional analytical HPLC, the standard particle size is 5 μm. However, smaller particles provide higher efficiency and resolution but at a higher backpressure on the HPLC system. The latest particle technology, Fused Core, combines the benefits of higher resolution and lower backpressure into a single column format. Larger particles provide lower backpressure making them amenable to large columns such as those used in preparative chromatography. Column Length The conventional column length for analytical HPLC is 150 mm or 250 mm for high resolution. For fast analysis, shorter columns (30 to 50 mm) should be used. Column Bonded Phase A C18 phase is often utilized as a starting point for many separations. C18 will provide enough retention for most non-polar and moderately polar molecules. Analysis of polar molecules or molecules that are highly functionalized will often be enhanced by using a more polar stationary phase such as Phenyl, RP-Amide, F5, or Cyano. Custom-prepared HPLC Columns Ifthecolumnofyourchoiceisnotlistedasastockproductinourcatalog,Supelcomaybeabletoprepareitforyouonacustombasis.Inordertodiscussa specificrequestandobtainaquoteonpriceanddelivery,pleasecontactTechnicalService. Delivery:Supelcotypicallyshipscustom-preparedanalyticalHPLCcolumnswithin5to7businessdaystoanywhereintheworld.Largersizesandspecial requestsmaytakelonger. Performancetesting:Supelcotestscustom-preparedcolumnsforefficiencyandsymmetry.Pleaseletusknowifyouhavespecialtestcriteria. Assistancewithmethoddevelopment:OurTechnicalServicechemistsareavailabletoassistyouwithyourmethoddevelopmentneeds.Theymaybeableto providerecommendations,technicaldataandin-housetestinginsupportofyourtestingneeds. sigma-aldrich.com/analytical Viewourcurrentseminarandtradeshowscheduleatsigma-aldrich.com/analytical-events HPLC 3 HPLC for Small Molecules HPLC Column Selection: Supelco HPLC Columns conform to USP standards Supelco HPLC Columns conform to USP standards HPLCPackings USPCode Description RecommendedPacking L1 Octadecylsilanechemicallybondedtoporousornon-poroussilicaorceramicmicro-particles, Ascentis®C18 1.5to10μmindiameter,oramonolithicrod. Ascentis®ExpressC18 Discovery®C18 Discovery®HSF5 Discovery®BIOWidePoreC18 SUPELCOSIL™LC-18 SUPELCOSIL™LC-18-DB SUPELCOSIL™LC-318 L3 Poroussilicaparticles,1.5to10μmindiameter,oramonolithicsilicarod. Ascentis®ExpressHILIC Ascentis®Si SUPELCOSIL™LC-Si SUPELCOSIL™LC-3Si L7 Octylsilanechemicallybondedtototallyorsuperficiallyporoussilicaparticles,1.5to10μmindiameter, Ascentis®C8 oramonolithicsilicarod. Ascentis®ExpressC8 Discovery®C8 Discovery®BIOWidePoreC8 SUPELCOSIL™LC-8,SUPELCOSIL™LC-8-DB SUPELCOSIL™LC-308 L8 Anessentiallymonomolecularlayerofamino-propylsilanechemicallybondedto SUPELCOSIL™LC-NH2 totallyporoussilicagelsupport,1.5to10μmindiameter. SUPELCOSIL™LC-NH2-NP L9 Irregularorspherical,totallyporoussilicagelhavingachemicallybonded,stronglyacidic SUPELCOSIL™LC-SCX cation-exchangecoating,3to10μmindiameter. L10 Nitrilegroupschemicallybondedtoporoussilicaparticles,1.5to10μmindiameter. Ascentis®ESCyano Discovery®Cyano SUPELCOSIL™LC-CN SUPELCOSIL™LC-PCN L11 Phenylgroupschemicallybondedtoporoussilicaparticles,1.5to10μmindiameter. Ascentis®Phenyl Ascentis®ExpressPhenyl-Hexyl SUPELCOSIL™LC-DP SUPELCOSIL™LC-3DP L13 Trimethylsilanechemicallybondedtoporoussilicaparticles,3to10μmindiameter. SUPELCOSIL™LC-1 L14 Silicagelhavingachemicallybonded,stronglybasicquaternaryammoniumanion-exchangecoating, SUPELCOSIL™SAX1 5to10μmindiameter. L17 Strongcation-exchangeresinconsistingofsulfonatedcross-linkedstyrene-divinylbenzenecopolymerint SUPELCOGEL™C-610H hehydrogenform,6to12μmindiameter. SUPELCOGEL™H L19 Strongcation-exchangeresinconsistingofsulfonatedcross-linkedstyrene-divinylbenzenecopolymer SUPELCOGEL™Ca inthecalciumform,about9μmindiameter. L20 Dihydroxypropanegroupschemicallybondedtoporoussilicaorhybridparticles,1.5to10μmindiameter. Kromasil®Diol SUPELCOSIL™LC-Diol L21 Arigid,sphericalstyrene-divinylbenzenecopolymer,3to10μmindiameter. PRP-1 L22 Acation-exchangeresinmadeofporouspolystyrenegelwithsulfonicacidgroups,about10μminsize. PRP-X200 PRP-X300 SUPELCOGEL™C-160H SUPELCOGEL™H L23 Ananion-exchangeresinmadeofporouspolymethacrylateorpolyacrylategelwithquaternary Discovery®BIOPolyMA-WAX ammoniumgroups,7to12μminsize. L26 Butylsilanechemicallybondedtototallyporoussilicaparticles,1.5to10μmindiameter. SUPELCOSIL™LC-304 L27 Poroussilicaparticles,30to50μmindiameter. Discovery®DSC-Si Supelclean™LC-Si Pelliguard™LC-Si L32 Achiralligand-exchangeresinpacking-L-prolinecoppercomplexcovalentlybondedtoirregularlyshaped Astec™CLC-D silicaparticles,5to10μmindiameter. Astec™CLC-L L34 Strongcation-exchangeresinconsistingofsulfonatedcross-linkedstyrene-divinylbenzenecopolymerinthe SUPELCOGEL™Pb leadform,about7to9μmindiameter. L40 Cellulosetris-3,5-dimethylphenylcarbamatecoatedporoussilicaparticles,5to20μmindiameter. Astec™CelluloseDMP Kromasil®CelluCoat® L41 Immobilizeda1-acidglycoproteinonsphericalsilicaparticles,5μmindiameter. Chiral-AGP L43 Pentafluorophenylgroupschemicallybondedtosilicaparticlesbyapropylspacer,5to10μmindiameter. Ascentis®ExpressF5 Discovery®HSF5 L45 Betacyclodextrinbondedtoporoussilicaparticles,5to10μmindiameter. Astec™CYCLOBOND®I 2000Series L47 Highcapacityanion-exchangemicroporoussubstrate,fullyfunctionalizedwithatrimethylaminegroup, PRP-X100 8μmindiameter. L49 Areversed-phasepackingmadebycoatingathinlayerofpolybutadieneontosphericalporous Discovery®Zr-PBD zirconiaparticles,3to10μmindiameter. L52 Astrongcationexchangeresinmadeofporoussilicawithsulfopropylgroups,5to10μmindiameter. SUPELCOSIL™LC-SCX Forhazardousproductinformation,visitsigma-aldrich.com/safetycenter 4 HPLC HPLC for Small Molecules HPLC Column Selection: Supelco HPLC Columns conform to USP standards HPLCPackings USPCode Description RecommendedPacking L59 Packingforthesize-exclusionseparationsofproteins(separationbymolecularweight)overtherangeof Discovery®BIOGFC100 5to7,000kDa.Itisspherical(1.5to10μm),silicaorhybridpackingwithahydrophiliccoating. Discovery®BIOGFC150 Discovery®BIOGFC300 Discovery®BIOGFC500 Discovery®BIOGFC1000 Discovery®BIOGFC2000 L60 Spherical,poroussilicagel,10μmorlessindiameter,thesurfaceofwhichhasbeencovalentlymodified Ascentis®RP-Amide withalkylamidegroupsandendcapped. Ascentis®ExpressRP-Amide Discovery®RP-AmideC16 SUPELCOSIL™ABZ+PLUS SUPELCOSIL™LC-ABZ L63 Glycopeptideteicoplaninlinkedthroughmultiplecovalentbondstoa100Åunitssphericalsilica. Astec™CHIROBIOTIC®T Astec™CHIROBIOTIC®T2 Astec™CHIROBIOTIC®TAG L67 PorousvinylalcoholcopolymerwithaC18alkylgroupattachedtothehydroxylgroupofthepolymer, apHera™C18 2to10μmindiameter. L68 Spherical,poroussilica,10μmorlessindiameter,thesurfaceofwhichhasbeencovalentlymodifiedwith SuplexpKb-100 alkylamidegroupsandnotendcapped. Helpful Hints Properties of Organic Solvents Commonly Used in HPLC Miscible with UV Refractive Index Solvent Strength, Viscosity Solvent Polarity Water? Cutoff1 20 ˚C ε0 (silica) at 20 ˚C, C P Hexane nonpolar no 200 1.3750 0.00 0.33 Isooctane no 200 1.3910 0.01 0.50 Carbon tetrachloride no 263 1.4595 0.14 0.97 Chloroform no 245 1.4460 0.31 0.57 Methylene chloride no 235 1.4240 0.32 0.44 Tetrahydrofuran yes 215 1.4070 0.35 0.55 Diethyl ether no 215 1.3530 0.29 0.23 Acetone yes 330 1.3590 0.43 0.32 Ethyl acetate poorly 260 1.3720 0.45 0.45 Dioxane yes 215 1.4220 0.49 1.54 Acetonitrile yes 190 1.3440 0.50 0.37 2-Propanol yes 210 1.3770 0.63 2.30 Methanol yes 205 1.3290 0.73 0.60 Water polar yes − 1.3328 >0.73 1.00 1Typical values Helpful Hints Relative Strengths for Different Solvents The graph provides for the interconversion of ACN/H O 2 reversed-phase mobile phases having the same strength. Vertical lines in this figure intersect mobile phases having the same strength. For example, 40% Acetonitrile has the MeOH/H O same strength as 50% Methanol or 30% THF. 2 ACN = Acetonitrile MeOH = Methanol THF = Tetrahydrofuran THF/H O 2 sigma-aldrich.com/analytical Accessouranalyticalliteratureandnewslettersatsigma-aldrich.com/literature HPLC 5 HPLC for Small Molecules Ascentis® Express 2.7 Micron Ascentis® Express 2.7 Micron Four Benefits of Ascentis® Express 1.DoubletheSpeed Ascentis Express Particle Totally Porous Particle • Designedforhighflowrates h at • Halfthebackpressureofsub-2μmparticles 0.5µm 0.5µmP n o usi1.5 µm HPLCAnalysisofHyper-FastSeparationsonAscentis®Expressat Diff HalfthePressuresofsub-2μmColumnsonAscentis®Express 1.7 µm C18 2..7 µm applicationforHPLC column........ AscentisExpressC18,10cm×2.1mmI.D.,2.7μmparticlesandsub-2μmparticle column,5cm×2.1mmI.D.(53823-U) mobilephase.................................................................................................................... 55:45or54:46,water:acetonitrile flowrate.................................................................................................................................................................................. 0.2mL/min columntemp.............................................................................................................................................................................. ambient detector............................................................................................................................................................................. UVat200nm injection.................................................................................................................................................................................................... 1μL ApplicationNo..................................................................................................................................................................... G003973 Ascentis Express C18 1. Estradiol 10 cm length 2. β-Estradiol 3. Impurity 2 4. Estrone Ascentis® Express - A Breakthrough in HPLC 5. Estrone degradant 1400 psi 4 Performance BasedoninnovativeFused-Core®particletechnology,AscentisExpress 1 providesthehighspeedandhighefficiencyofsub-2μmparticles,butat TWICE THE approximatelyhalfthebackpressureforthesamecolumnlength.Thislower RESOLUTION pressuremeansthatAscentisExpresscanberunonconventionalHPLCand AT EQUAL LC-MSsystems,aswellasmid-pressure,UPLC®andotherultra-highpressure PRESSURE systems.Lowerpressurealsomeanslongercolumnscanbeusedfor additionalresolvingpower.AscentisExpressoffersthesebenefitsoversub-2 μmparticles,alongwithexcellentcolumnlifetime. 5 TheFused-CoreAdvantage 3 AttheheartofAscentisExpressisthe2.7μmFused-Coreparticlewhich comprisesa1.7μmsolidcoreanda0.5μmporousshell.Comparedto totallyporousparticles,theFused-Coreparticleshaveamuchshorter 1.0 2.0 3.0 4.0 5.0 6.0 diffusionpathbecauseofthesolidcore.Thispartialporosityreducesaxial Min dispersionofsolutesandminimizespeakbroadening.Otherfeatures,such asaverytightparticlesizedistributionandhighpackingdensity,resultin AscentisExpresscolumnsthatarecapableof240,000N/mandhigher: C18 Sub-2 µm comparabletotheefficiencyofsub-2μmparticlecolumnsandnearlytwice 5 cm length theefficiencypossiblewith3μmparticles. WhiletheAscentisExpressefficiencyisashighassub-2μmcolumns,the 1650 psi 1 largerparticlesizedeliversapproximatelyhalfthebackpressureforthesame columndimensionsandconditions.ThisallowsAscentisExpresstoturnany 2 HPLCsystemintoanextremeperformanceworkhorseforyourlab. 4 5 3 1.0 2.0 3.0 4.0 Min Viewourtechnicalpresentationsatsigma-aldrich.com/analytical-videos 6 HPLC HPLC for Small Molecules Ascentis® Express 2.7 Micron: Four Benefits of Ascentis® Express 2.DoubletheEfficiency HPLCAnalysisHigherEfficiencyofAscentis®ExpressCompared • Shortanalytediffusionpath to3μmParticlesGivesBetterSensitivityonAscentis®Express • Longercolumnspermitdoublingtheplatesoversub-2μmparticles C18 • Twicetheefficiencyof3μmparticles applicationforHPLC column..AscentisExpressC18,5cm×2.1mmI.D.,2.7μmparticlesandC18,5cm×2.1mmI.D.,3 HighEfficiencyResolutiononAscentis®ExpressVersus3μm μmparticles(53822-U) Particles mobilephase.................... 35:0:65or35:4:61,25mMdibasicammoniumphosphate(pH7.0):water: acetonitrile applicationforHPLC flowrate.................................................................................................................................................................................. 0.2mL/min columntemp..................................................................................................................................................................................... 35°C column.. AscentisExpressC18,15cm×4.6mmI.D.,2.7μmparticlesandC18,15cm×4.6mmI. D.,3μmparticles(53829-U) detector............................................................................................................................................................................. UVat220nm mobilephase.............................................................................................................. 35:65or27.5:72.5,water:acetontrile injection.................................................................................................................................................................................................... 1μL flowrate.................................................................................................................................................................................. 1.5mL/min ApplicationNo..................................................................................................................................................................... G003977 columntemp.............................................................................................................................................................................. ambient detector............................................................................................................................................................................. UVat220nm 1 Ascentis Express C18 injection.................................................................................................................................................................................................... 2μL 0 1. Quinidine ApplicationNo..................................................................................................................................................................... G003979 8 2. Fluoxetine C18, 3 µm Sensitivity 3. Diphenhydramine Ascentis Express C18 136,800 1. Naphthalene 60 Gap 2. p-Xylene 1 3 3. Biphenyl 2 NEARLY TWICE THE mV40 EFFICIENCY 2 3 3 5 ,70 20 0 3 3 7,1 0 0 20 0.0 1.0 2.0 3.0 4.0 Min 4.Extendedcolumnlifetimecomparedtoboth3μmandsub-2μm columns 0 2 4 • Narrowparticlesizedistributitionallowsuseof2μmfrits Min • Denseparticlesformorestablebed C18, 3µm 2 1 ,7 0 0 1 2 1 ,1 0 0 3 2 1 ,6 0 0 2 0 2 4 Min 3.Highsampleloadingcapacityandsignal/noisefortraceanalysis • Highsampleloadingfromthick,porousshelllayer • HighcolumnefficiencyforhighS/N sigma-aldrich.com/analytical Toorder,visitsigma-aldrich.com/order HPLC 7 HPLC for Small Molecules Ascentis® Express 2.7 Micron: Improving HPLC Sample Throughput Improving HPLC Sample Throughput ThedemandforincreasedsamplethroughputandspeedofresultshasdrivenHPLCuserstosearchforbreakthroughsinHPLCinstrumentsandcolumn technology.Althoughimprovementshavebeenrealized,setbackshavebeenencountered.Reductionincolumnruggedness,costlyreplacementsofexisting instrumentation,anddifficultiesintransferringmethodstonewsystemshaveoftenmadethesepastimprovementsunappealingtoanalysts. TheFused-Core®HPLCparticletechnologybehindAscentisExpresspermits4-to6-foldreductioninanalysistime,withasubsequentincreaseinsample throughputcomparedtoconventionalHPLCcolumns,withoutsacrificingresolutionorcolumnruggednessandwithouttheneedtochangesystemsor sampleprepprocedures. HPLCAnalysisofSampleThroughputonStandardC18versusAscentis®ExpressC18 applicationforHPLC column......................................................................................................................................................................................................... C18,25cm×4.6mmI.D.,5μmparticlesandAscentisExpressC18,10cm×4.6mmI.D.,2.7μmparticles mobilephase..................................................................................................................................................................................................................................................................................................................................................................................................... 65:35,water:acetonitrile flowrate................................................................................................................................................................................................................................................................................................................................................................................................................................................. 1mL/min columntemp......................................................................................................................................................................................................................................................................................................................................................................................................................................... ambient detector........................................................................................................................................................................................................................................................................................................................................................................................................................................ UVat254nm ApplicationNo................................................................................................................................................................................................................................................................................................................................................................................................................................ G004039 C18 1. Oxazepam:FW = 286 2. Alprazolam: FW = 311 1X 3. Cloazepam: FW = 315 41.5 Min. 4. N-desmethyldiazepam: FW = 270 5. Diazepam: FW = 284 0 10 20 30 40 Min Ascentis Express C18 11XX 22XX 33XX 44XX 9.5 Min. 19 Min. 28.5 Min. 38 Min. 0 10 0 10 0 10 0 10 Forhazardousproductinformation,visitsigma-aldrich.com/safetycenter 8 HPLC HPLC for Small Molecules Ascentis® Express 2.7 Micron: Do More Work in Less Time Without Changing your Method Do More Work in Less Time Without Changing your Method AscentisExpressistheidealchoiceforHPLCanalystsinterestedinincreasingsamplethroughputwhilemaintainingorevenimprovingresolution.Byreducing solutedispersion,theuniqueFused-CoretechnologygivesAscentisExpressanadvantageoverconventionalparticles.Itslowbackpressurecomparedtosub-2 μmparticlesmeansthatAscentisExpresscanachieveUHPLC-likeperformanceonconventionalHPLCsystems.UnderUHPLCconditions,AscentisExpresscan exceedtheefficiencypossibleonsub-2μmcolumnsbecauselongercolumnscanbeused. Ultra-High Resolution HPLC: Column Coupling ColumncouplinginHPLCisgaininginterestsinceLCsystemsarebeing designedtowithstandcolumnbackpressuresofupto15,000psi.Column couplingisasimpleandpracticalwaytoincreaseresolutionbysimply increasingcolumnlength.BecauseAscentisExpressHPLCcolumnsprovide higherefficienciesatanypressurecomparedto3μmandsub-2μm particles,thecouplingofAscentisExpresscolumnsenablessignificantly Ascentis® Express C18 higherresolutionthananyothercolumnonanycommercialHPLCsystem. HPLCAnalysisofBenzeneandDeuteratedBenzeneonAscentis® Ascentis®ExpressC18,2.7MicronHPLCColumn ExpressC18 AscentisExpressHPLCcolumns,throughtheuseofFused-Core®particle applicationforHPLC technology,canprovideyouwithboththehighspeedandhighefficiencies ofsub-2μmparticleswhilemaintaininglowerbackpressures.The column......................................................................................................... AscentisExpressC18,15cm×4.6mmI.D. mobilephase.......................................................................................................................................... 55:45,acetonitrile:water combinationofhighefficiencyandlowbackpressurebenefitsUPLC®(or flowrate.................................................................................................................................................................................. 1.0mL/min otherultrahighpressuresystem)users,aswellasconventionalHPLCusers. columntemp..................................................................................................................................................................................... 50°C VisittheAscentisExpresshomepageformoreinformationonthisnew detector............................................................................................................................................................................................ 254nm columntechnology. injection................................................................................................................................................................................................. 10μL ApplicationNo..................................................................................................................................................................... G004046 Watcha5-minutepresentationthatexplainshowAscentisExpresscolumns canhelpMaximizeSampleThroughput. D suitableforL1perUSP D D particleplatform .............................................................................................................................................................. Fused-Core 5. Toluene = 101,281 plates metals..................................................................................................................................................................................................<5ppm 4. Benzene = 104,463 plates endcapped................................................................................................................................................................................................ Yes 32.. DA6c eBteonpzheenneo =n e1 0=1 ,182502, 0p4l5a tpelsates D D H poresize....................................................................................................................................................................................................90Å Pressure = 480 bar (7,000 psi) D H H operatingpH....................................................................................................................................................................................... 2-9 temp.range......................................................................................................................................................................................... 60°C Ref:1.EthanR.Badman,RichardL.Beardsley,ZhenminLiang,SurendraBansal,Acceleratinghigh H H qualitybioanalyticalLC/MS/MSassaysusingfusedcorecolumnsJ.Chromatogr.B.Analyt.Technol. H Biomed.LifeSci.878,2307-2313(2010) 2.AhmedAbrahim,MohammadAl-Sayah,PeterSkrdla,YuriBereznitski,YadanChen,NaijunWu, Practicalcomparisionof2.7μmfused-coresilicaparticlesandporoussub-2μmparticlesforfast separationsinpharmaceuticalprocessdevelopmentJ.Pharm.Biomed.Anal.51,131-137(2010) I.D.(mm) L(cm) Cat.No. Qty particlesize2.7μm 2.1 2 53799-U 1ea 2.1 3 53802-U 1ea 2.1 7.5 53804-U 1ea 2.1 5 53822-U 1ea 2.1 10 53823-U 1ea 0 2 4 6 8 10 12 14 16 18 2.1 15 53825-U 1ea Min 3.0 3 53805-U 1ea 3.0 5 53811-U 1ea 3.0 7.5 53812-U 1ea 3.0 10 53814-U 1ea 3.0 15 53816-U 1ea 4.6 3 53818-U 1ea 4.6 5 53826-U 1ea 4.6 7.5 53819-U 1ea 4.6 10 53827-U 1ea 4.6 15 53829-U 1ea 1.0 5 582711-U 1ea 10 15 53793-U 1ea Ascentis®ExpressC18,2.7MicronValidationPack AnAscentisExpressC18ValidationPackmakesiteasytodemonstrate methodreproducibilityon3differentlots.Thevalidationpackcontainsakit with3columns-1fromeachof3lotsofbondedphase.AndwithAscentis Expresscolumns,youcanbeassuredthatallthreecolumnswillmeetyour expectations. sigma-aldrich.com/analytical Forallofyouranalyticalneeds,visitusatsigma-aldrich.com/analytical HPLC 9 HPLC for Small Molecules Ascentis® Express 2.7 Micron: Ascentis® Express C18 I.D.(mm) L(cm) Cat.No. Qty Ascentis® Express C8 particlesize2.7μm 2.1 5 53994-U 3ea Ascentis®ExpressC8,2.7MicronHPLCColumn 4.6 10 53995-U 3ea 4.6 15 53996-U 3ea AscentisExpressHPLCcolumns,throughtheuseofFused-Core®particle technology,canprovideyouwithboththehighspeedandhighefficiencies Ascentis®ExpressC18,2.7MicronGuardCartridge ofsub-2μmparticleswhilemaintaininglowerbackpressures.The combinationofhighefficiencyandlowbackpressurebenefitsUPLC®(or AscentisExpressGuardColumnsprovidephysical(filtration)andchemical otherultrahighpressuresystem)users,aswellasconventionalHPLCusers. protectionforcostlyanalyticalcolumnswithoutcompromisingthevery VisittheAscentisExpresshomepageformoreinformationonthisnew highperformanceofAscentisExpresscolumns.TheseAscentisExpress columntechnology. guardcolumnsarecapableofcontinuoususeatpressuresupto9000psi (600bar)withonlyhand-tightening.Guardcartridgesareeasilyreplaced Watcha5-minutepresentationthatexplainshowAscentisExpresscolumns withoutremovingtheguardcolumnholderfromtheflowpath.The canhelpMaximizeSampleThroughput. cartridgesarepackedwithAscentisExpressFused-Core®particles.Order suitableforL7perUSP guardcolumnholderseparately. particleplatform .............................................................................................................................................................. Fused-Core metals..................................................................................................................................................................................................<5ppm Holder endcapped................................................................................................................................................................................................ Yes poresize....................................................................................................................................................................................................90Å operatingpHrange........................................................................................................................................................................ 2-9 temp.range......................................................................................................................................................................................... 60°C I.D.(mm) L(cm) Cat.No. Qty Guard Cartridge particlesize2.7μm 2.1 2 53795-U 1ea Particle 2.1 3 53839-U 1ea Size(μm) L×I.D.(mm) Cat.No. Qty 2.1 5 53831-U 1ea 2.7 5×2.1 53501-U 3ea 2.1 7.5 53843-U 1ea 2.7 5×3.0 53504-U 3ea 2.1 10 53832-U 1ea 2.7 5×4.6 53508-U 3ea 2.1 15 53834-U 1ea 3.0 3 53844-U 1ea Ascentis®ExpressGuardCartridgeHolder 3.0 5 53848-U 1ea Guardcartridgenotincludedwithholder 3.0 7.5 53849-U 1ea forusewithAscentisExpressGuardColumns 3.0 10 53852-U 1ea 3.0 15 53853-U 1ea 53500-U 1ea 4.6 3 53857-U 1ea 4.6 5 53836-U 1ea 4.6 7.5 53858-U 1ea 4.6 10 53837-U 1ea 4.6 15 53838-U 1ea Ascentis®ExpressC8,2.7MicronGuardCartridge AscentisExpressGuardColumnsprovidephysical(filtration)andchemical protectionforcostlyanalyticalcolumnswithoutcompromisingthevery highperformanceofAscentisExpresscolumns.TheseAscentisExpress guardcolumnsarecapableofcontinuoususeatpressuresupto9000psi (600bar)withonlyhand-tightening.Guardcartridgesareeasilyreplaced withoutremovingtheguardcolumnholderfromtheflowpath.The cartridgesarepackedwithAscentisExpressFused-Core®particles.Order guardcolumnholderseparately. Particle Size(μm) L×I.D.(mm) Cat.No. Qty 2.7 5×2.1 53509-U 3ea 2.7 5×3.0 53511-U 3ea 2.7 5×4.6 53512-U 3ea Ascentis®ExpressGuardCartridgeHolder Guardcartridgenotincludedwithholder forusewithAscentisExpressGuardColumns 53500-U 1ea Fortechnicalassistance,visitsigma-aldrich.com/techinfo 10 HPLC HPLC for Small Molecules Ascentis® Express 2.7 Micron: Alternate Selectivity with Ascentis® Express RP-Amide Alternate Selectivity with Ascentis® Express RP-Amide Ascentis® Express RP-Amide WhiletheAscentisExpressC18providesclassicreversed-phaseselectivity, Ascentis®ExpressRP-Amide,2.7MicronHPLCColumn theAscentisExpressRP-Amideprovidesincreasedselectivityforpolar compounds,especiallythosethatcanactasahydrogen-bonddonor.Other Watcha6-minutepresentationontheeffectiveuseofphasechemistryto attributesoftheRP-Amideincludeimprovedpeakshapeforbases,100% alterretentioninthisBeverageAnalysisApplication. aqueouscompatibility,andlowbleedforLC-MSapplications. suitableforL60perUSP particleplatform .............................................................................................................................................................. Fused-Core Separationofphenolics-acomparativeevaluation metals..................................................................................................................................................................................................<5ppm ApplicationNo..................................................................................................................................................................... G004390 endcapped................................................................................................................................................................................................ Yes poresize....................................................................................................................................................................................................90Å operatingpHrange........................................................................................................................................................................ 2-9 temp.range......................................................................................................................................................................................... 60°C I.D.(mm) L(cm) Cat.No. Qty Resorcinol Catechol particlesize2.7μm 2.1 3 53910-U 1ea 2.1 5 53911-U 1ea 2 1 2.1 7.5 53912-U 1ea 1. Resorcinol 5. 2,5-Dimethylresorcinol 2. Catechol 6. 3-Methylcatechol 2.1 10 53913-U 1ea 3. 2-Methylresorcinol 7. 4-Nitrocatechol 2.1 15 53914-U 1ea 4. 4-Methylcatechol 3.0 3 53915-U 1ea Ascentis Express RP-Amide, 2.7 µm 3.0 5 53916-U 1ea pressure: 4400 psi 3.0 7.5 53917-U 1ea 4 3.0 10 53918-U 1ea 3 6 3.0 15 53919-U 1ea 4.6 3 53921-U 1ea 7 5 4.6 5 53922-U 1ea 4.6 7.5 53923-U 1ea 4.6 10 53929-U 1ea 4.6 15 53931-U 1ea 1 2 3 4 5 Min 2.1 2 53797-U 1ea 1 2 Ascentis®ExpressRP-Amide,2.7MicronGuardCartridge Ascentis Express C18, 2.7 µm AscentisExpressGuardColumnsprovidephysical(filtration)andchemical pressure: 4400 psi protectionforcostlyanalyticalcolumnswithoutcompromisingthevery highperformanceofAscentisExpresscolumns.TheseAscentisExpress guardcolumnsarecapableofcontinuoususeatpressuresupto9000psi (600bar)withonlyhand-tightening.Guardcartridgesareeasilyreplaced 4 3 withoutremovingtheguardcolumnholderfromtheflowpath.The 7 6 cartridgesarepackedwithAscentisExpressFused-Core®particles.Order 5 guardcolumnholderseparately. Particle Size(μm) L×I.D.(mm) Cat.No. Qty 2.7 5×2.1 53514-U 3ea 1 2 Min 3 4 5 2.7 5×3.0 53516-U 3ea 2.7 5×4.6 53519-U 3ea 1 2 Ascentis®ExpressGuardCartridgeHolder Waters BEH Shield RP18, 1.7 µm pressure: 8250 psi Guardcartridgenotincludedwithholder forusewithAscentisExpressGuardColumns 53500-U 1ea 4 3 6 7 5 1 2 Min 3 4 5 sigma-aldrich.com/analytical Viewourcurrentseminarandtradeshowscheduleatsigma-aldrich.com/analytical-events