The HPLC Expert II: Find and Optimize the Benefits of HPLC/UHPLC

369 Pages·010.655 MB·English

by  Kromidas, Stavros

TheHPLCExpertII
The HPLC Expert II
FindandOptimizetheBenefitsofyourHPLC/UHPLC
EditedbyStavrosKromidas
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v
Contents
ListofContributors xiii
Foreword xv
TheStructureof“TheHPLC-Expert2” xvii
1 WhenShouldIUseMyUHPLCasaUHPLC? 1
StavrosKromidas
1.1 Introduction 1
1.2 WhatDoIWanttoAchieveandWhatIsaUHPLCCapableof? 2
1.2.1 WhatDoIWanttoAchieve? 2
1.2.2 WhatIsaUHPLCCapableof? 2
1.3 WhatIsRequiredfromanHPLCMethod? 3
1.3.1 SeparateWell 3
1.3.2 SeparateFast 12
1.3.3 ImproveMassSensitivity 13
1.3.4 RobustSeparationsinRoutineUse 15
1.4 TheUHPLCinRoutineUse–ABriefReport 17
1.5 HowCanthePotentialofUHPLCEffectivelyBeFullyExploited?
(SeeAlsoChapters2,3,and9) 20
1.5.1 DeadVolumes 20
1.6 SummaryandOutlook 22
1.6.1 Outlook 24
References 25
PartI HardwareandSoftware,SeparationModes,
Materials 27
2 TheModernHPLC/UHPLCDevice 29
2.1 TheModernHPLC/UHPLCSystem 29
SteffenWieseandTerenceHetzel
2.1.1 Today’sDemandsontheIndividualModules 29
2.1.1.1 Overview 29
2.1.2 UHPLCPumpTechnology 30
2.1.2.1 High-andLow-PressurePumps 30
2.1.2.2 GradientDelayVolume 34
vi Contents
2.1.3 Autosampler 35
2.1.3.1 Fixed-LoopAutosamplers 36
2.1.3.2 Flow-ThroughAutosamplers 38
2.1.3.3 ReviewoftheAdvantagesandDisadvantagesofFixed-Loopand
Flow-ThroughAutosamplers 40
2.1.4 ColumnOven 41
2.1.5 Detectors 44
2.1.6 CapillariesandFittings 47
Acknowledgment 50
References 50
2.2 TheThermostateofColumns–AMinorMatter 52
MichaelHeidornandFrankSteiner
2.2.1 ThermalModesofColumnThermostats 54
2.2.2 TemperatureDifferencesbetweenColumnandMobilePhase 57
2.2.3 FrictionalHeat–JustaPhenomenoninUHPLC? 62
2.2.4 ThermostaticControlinMethodTransfer,MethodSpeed-Up,and
MethodDevelopment 68
Literature 71
3 TheIssueofExternalBandBroadeninginHPLC/UHPLC
Devices 73
MonikaDittmann
3.1 Introduction 73
3.2 TheoreticalBackground 74
3.2.1 EfficiencyandResolutionofModernUHPLCColumns 74
3.2.2 EstimationofColumnPeakVolumes 76
3.3 ExtracolumnDispersionin(U)HPLCSystems 78
3.3.1 SourcesofExternalBandBroadeninginHPLC/UHPLCSystems 78
3.3.1.1 InjectionSystems 79
3.3.1.2 Tubing 80
3.3.1.3 FittingsandConnections 83
3.3.1.4 HeatExchangers 84
3.3.1.5 Detection 85
3.3.2 DeterminationofExternalBandBroadening 88
3.3.2.1 AnalysisofExtracolumnVolumewithoutColumn(ShortCircuit
Method) 88
3.3.2.2 AnalysisofExtracolumnVolumeIncludingaColumn 89
3.4 ImpactofExternalContributionsinDifferentApplicationAreas 90
3.4.1 ImpactonIsocraticSeparations 90
3.4.2 ImpactonGradientSeparations 92
3.5 OptimizationofHPLC/UHPLCSystems 94
3.5.1 TestingofColumnPerformance 95
3.5.2 OtherIsocraticSeparations 95
3.5.3 High-ResolutionGradientSeparations 96
3.5.4 FastGradientSeparations 96
3.6 Conclusions 97
References 98
Contents vii
4 TheGradient;Requirements,OptimalUse,Hints,and
Pitfalls 101
FrankSteiner
4.1 InstrumentalInfluencesinGradientElution–AnOverview 101
4.1.1 TheGradientDelayEffectandtheGradientDwellVolumeofa
System 101
4.1.2 TheRoleandFunctionoftheGradientMixer 103
4.1.3 DeviationsfromIdealBehaviorofGradientGenerationResultingfrom
FundamentalPhysicochemicalPhenomena 106
4.1.4 InstrumentalInfluencesonGradientElutionOutsidethePump 112
4.1.5 TheStressandWearonColumnsinGradientMethods 115
4.2 GradientElutionTechnologyandHowtoSystematicallyCharacterize
GradientInstrumentation 117
4.2.1 PhysicochemicalEffectsofHighPressureonLiquids 117
4.2.2 TheNeedofSolventDegassing 120
4.2.3 TheDifferentTypesofPumpTechnology(Serial,Parallel,CamDrive,
LinearDrive)andTheirSpecificPropertiesandRequirements 122
4.2.4 TheSpecificGradientPumpTypeandItsImplicationsforPractical
Operation 125
4.2.5 HPGPumpsandHowDiscontinuousPumpCyclesResultingfrom
PressurePulsationImpactRetentionTimePrecisioninPractice 127
4.2.6 LPGPumpsandHowTheirImmanentDiscontinuousGenerationof
GradientCompositionMayImpactRetentionTimePrecisionin
Practice 132
4.2.7 ThermalEffectsinGradientPumpsandHowIntelligentInstrument
ControlCanMinimizetheConsequencesonChromatography 134
4.2.8 UltrafastMethodswithVerySteeporBallisticGradients 137
4.2.9 FundamentalConsiderationsontheDeterminationofaGradient
DelayVolume 143
4.2.10 TheMarkerPulseMethodasaQuickWayforGDV
Determination 145
4.2.11 TheDolanTestastheClassicalMethodforGDVOptimization 148
4.2.12 DesignsofMixersandTheirEffectivenessRelativetoTheirGDV
Contribution 150
4.2.13 SystematicCharacterizationoftheMixingEfficiencyandGradient
FormationofaPump 155
4.2.14 OptimizingtheMixingVolumeinDependenceofPumpTypeand
FlowRateforDemandingApplicationsSuchasTFAGradients 162
4.2.15 ExceptionalElutionBehaviorofProteinswithMobile-PhaseMixing
Ripples 168
References 169
5 RequirementsofLC-HardwarefortheCouplingofDifferent
MassSpectrometers 171
TerenceHetzel,ThorstenTeutenberg,ChristophPortner,andJochenTuerk
5.1 Introduction 171
5.2 FromTargetAnalysistoScreeningApproaches 171
viii Contents
5.2.1 TargetAnalysis 171
5.2.2 Suspected-TargetScreening 172
5.2.3 NontargetScreening 172
5.3 WhatShouldBeConsideredforUHPLC/MSHyphenation? 173
5.3.1 TheInterfaceandtheOptimumFlowRate 173
5.3.2 OptimizationofMSParameters 174
5.3.3 OptimizationoftheChromatographicParameters 174
5.3.4 ChoiceoftheSuitableColumnandColumnDimension 176
5.4 TargetAnalysisUsingTriple-QuadrupoleMass
Spectrometry 178
5.5 ScreeningApproachesUsingLC-MS 185
5.6 Miniaturization–LC-MSQuoVadis? 189
References 192
6 2Dchromatography–Opportunitiesandlimitations 193
ThorstenTeutenbergandJuriLeonhardt
6.1 Introduction 193
6.2 WhyTwo-DimensionalHPLC? 193
6.3 PeakCapacityofOne-andTwo-DimensionalLiquid
Chromatography 195
6.3.1 PeakCapacityofOne-DimensionalLiquidChromatography 195
6.3.2 PeakCapacityofTwo-DimensionalLiquidChromatography 196
6.3.2.1 Heart-Cut2DLC(LC-LC) 196
6.3.2.2 Comprehensive2DLC(LC×LC) 197
6.4 Modulation 200
6.4.1 OnlineHeart-Cut2DLC 200
6.4.2 ComprehensiveOnline2DLC 200
6.4.3 Stop-FlowandOfflineLC×LC 202
6.5 PracticalProblemsofOnlineLC×LC 203
6.5.1 CompatibilityoftheSolventSystems 203
6.5.2 Dilution 203
6.5.3 HighFlowRate 203
6.5.4 CompatibilitywithMassSpectrometry 203
6.6 DevelopmentofaMiniaturizedLC×LCSystem 204
6.6.1 TechnicalPlatform 204
6.6.2 SelectionoftheStationaryPhase 204
6.6.3 SelectionoftheMobilePhaseandTemperature 205
6.6.4 ColumnDimensionandModulation 205
6.6.5 GradientProgrammingandOverallAnalysisTime 206
6.6.6 CouplingwithMassSpectrometry 206
6.7 RealApplications 207
6.7.1 MeasurementofaReferenceStandard 207
6.7.2 MeasurementofaRealSample 209
6.8 AdvantagesoftheMS/MSFunctionality 211
6.9 GeneralCommentstoSpecificAspectsofanLC×LCSystem 211
Contents ix
6.9.1 OfflineLC×LCversusOnlineLC×LC 211
6.9.2 Stop-FlowLC×LC 214
6.9.3 MultipleHeart-CutLC-LCandSelectedLC×LC(sLC×LC) 214
6.10 MethodDevelopmentandGradientProgramming 215
6.11 PresentationsoftheInstrumentManufacturers(inAlphabetical
Order) 215
6.11.1 CommerciallyAvailableSolutionsforLC×LC 216
6.11.1.1 Agilent 216
6.11.1.2 Shimadzu 216
6.11.1.3 Thermo/Dionex 216
6.11.2 FurtherSystems 216
6.11.2.1 Sciex 216
6.11.2.2 Waters 216
6.12 2DLC–QuoVadis? 217
6.12.1 Software 217
6.12.2 SystemSetup 217
6.12.3 PeakversusPeakCapacity 218
References 219
7 MaterialsinHPLCandUHPLC–WhattoUseforWhich
Purpose 223
TobiasFehrenbachandSteffenWiese
7.1 Introduction 223
7.2 RequirementsforMaterialsinUHPLC 225
7.2.1 MechanicalStability 225
7.2.2 ChemicalStability 225
7.2.3 AnalyteCompatibility/Biocompatibility 226
7.3 FlowPathsinUHPLCSystems 227
7.3.1 Low-PressureandHigh-PressureFlowPath 227
7.3.2 Mobile-PhaseandSampleFlowPath 228
7.4 Low-PressureFlowPath 229
7.5 High-PressureFlowPath 231
7.5.1 Pumps 231
7.5.1.1 InletandOutletValves 231
7.5.1.2 PumpHead 233
7.5.1.3 PumpPistonsandPistonSeals 236
7.5.1.4 PracticalAspects 237
7.5.2 Autosamplers 238
7.5.2.1 Materials 238
7.5.2.2 SampleNeedles,SampleVials,andClosures 238
7.5.2.3 InjectionValves 239
7.5.2.4 PracticalAspects 241
7.5.3 TubingandFittingSystems 242
7.5.3.1 Outline 242
7.5.3.2 Materials 243
x Contents
7.5.3.3 Tubing 244
7.5.3.4 FittingSystems 246
7.6 WhenandWhyCananInertUHPLCSystemBeRequired? 248
7.6.1 ConceptofInertness 248
7.6.1.1 GeneralInertness 248
7.6.1.2 Analyte-SpecificInertness 249
7.6.2 NatureofthePassiveLayer 249
7.6.2.1 PassiveLayersofChromiumAlloys 251
7.6.2.2 PassiveLayersofTitaniumAlloys 252
7.6.3 RequirementsandInteractions 253
7.6.3.1 MechanicalandPhysicalIntegrityoftheUHPLCSystem 253
7.6.3.2 RequirementoftheDetectionMethod 254
7.6.3.3 InteractionofAnalyteandUHPLCSystem 254
7.6.4 PassivationStrategiesandMethods 258
References 261
PartII ExperienceReports,Trends 269
8 WhataSoftwarehastoPossessinOrdertoUsetheHardware
Optimally 271
ArnoSimon
8.1 FunctionalityandHandling 271
8.1.1 Integration 272
8.1.2 InstrumentControl 273
8.1.3 Useability 274
8.1.4 EaseofUse 274
8.1.5 UserInterface 275
8.1.6 Multilingual 276
8.2 DataExchange 277
8.2.1 ImportandExportofData 278
8.3 FromPCsScalabilitytoGlobalInstallation 278
8.3.1 SoftwarePlacement 278
9 AspectsoftheModernHPLCDevice–ExperienceReportofan
Operator 281
SteffenWieseandTerenceHetzel
9.1 Introduction 281
9.2 DeterminationoftheGradientDelayVolume 281
9.3 High-ThroughputSeparations 285
9.4 MethodTransferbetweenUHPLCSystemsofDifferent
Manufacturers 287
9.5 ApplicationofElevatedTemperatures 290
9.6 Large-VolumeInjection(LVI) 293
9.7 UHPLCSeparationwith1mmIDColumns 296
Acknowledgment 299
References 299
Contents xi
10 ExperiencesofanIndependentServiceEngineer–Hintsand
RecommendationsforanOptimalOperationofAgilentand
Waters-Devices 301
SiegfriedChroustovsky
10.1 Introduction 301
10.2 TheDegasser,Principles 301
10.2.1 DifferentManufacturers,DifferentConcepts 302
10.3 ThePump,Principles 303
10.3.1 DifferentManufacturers,DifferentConcepts 305
10.4 TheAutosampler,Principles 306
10.4.1 DifferentManufacturers,DifferentConcepts 307
10.5 TheUVDetector,Principles 308
10.5.1 DifferentManufacturers,DifferentConcepts 309
11 TheAnalyte,theQuestion,andtheUHPLC–TheUseofUHPLC
inPractice 311
StefanLamotte
11.1 Introduction 311
11.2 WhenDoesItMakeSensetoUseUHPLCandWhenShouldIBetter
UseConventionalHPLC? 311
11.3 DissolutionTestsinPharmaceuticalIndustry 313
11.4 MethodDevelopmentandOptimization 314
11.5 Typical“Classical”LiquidChromatographic
Analysis 314
11.6 Fast(MostSecond)DimensionofMultidimensional
Chromatography 315
11.7 Separationof(Bio)polymers 316
11.8 ProcessAnalysis(PAT) 316
11.9 Conclusion 316
References 316
12 ReportofDeviceManufacturers–ArticlebyAgilent,Shimadzu,
andThermoScientific 319
12.1 AgilentTechnologies 319
JensTrafkowski
References 328
12.2 HPLCCurrentStatusandFutureDevelopment 328
Björn-ThoralfErxleben
12.3 ThermoFisherScientific,Germering 334
FrankSteiner
12.3.1 TotalSystemRequirementsandRelatedKeyExperiences 334
12.3.1.1 NanoLC 335
12.3.1.2 HPLCandUHPLConTwoInstrumentalPlatforms(UltiMate3000,
Vanquish) 336
12.3.1.3 Viper-BasedSystemTubing 338
12.3.2 TheContributionoftheIndividualComponentstotheSuccessofa
System 338