Ch 01: HPLC Columns and Accessories - MZ-AT

144 Pages·2013·10.35 MB·English

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
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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.
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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
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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
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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
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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
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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
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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.
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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
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