Introduction in modern liquid chromatography

957 Pages·8.2 MB·english

by  Snyder L.R.

INTRODUCTION TO
MODERN LIQUID
CHROMATOGRAPHY
Third Edition
INTRODUCTION TO
MODERN LIQUID
CHROMATOGRAPHY
Third Edition
LLOYD R. SNYDER
LCResources,Inc.
Orinda,CA
JOSEPH J. KIRKLAND
AdvancedMaterialsTechnology
Wilmington,DE
JOHN W. DOLAN
LCResources,Inc.
Amity,OR
AJohnWiley&Sons,Inc.,Publication
Copyright©2010byJohnWiley&Sons,Inc.Allrightsreserved.
PublishedbyJohnWiley&Sons,Inc.,Hoboken,NewJersey.
PublishedsimultaneouslyinCanada.
No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any
form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise,
except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without
either the prior written permission of the Publisher, or authorization through payment of the
appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers,
MA 01923, [phone], fax [phone], or on the webat www.copyright.com. Requests
to the Publisher for permission should be addressed to the Permission Department, John Wiley &
Sons,Inc.,111RiverStreet,Hoboken,NJ07030,[phone],fax[phone],oronlineat
http://www.wiley.com/go/permission.
LimitofLiability/DisclaimerofWarranty:Whilethepublisherandauthorhaveusedtheirbestefforts
in preparing this book, they make no representations or warranties with respect to the accuracy
or completeness of the contents of this book and specifically disclaim any implied warranties of
merchantabilityorfitnessforaparticularpurpose.Nowarrantymaybecreatedorextendedbysales
representatives or written sales materials. The advice and strategies contained herein may not be
suitable for your situation. You should consult with a professional where appropriate. Neither the
publishernorauthorshallbeliableforanylossofprofitoranyothercommercialdamages,including
butnotlimitedtospecial,incidental,consequential,orotherdamages.
Forgeneralinformationonourotherproductsandservicesorfortechnicalsupport,pleasecontact
ourCustomerCareDepartmentwithintheUnitedStatesat(800)762-2974,outsidetheUnitedStates
at(317)572-3993orfax(317)572-4002.
Wileyalsopublishesitsbooksinavarietyofelectronicformats.Somecontentthatappearsinprint
maynotbeavailableinelectronicformats.FormoreinformationaboutWileyproducts,visitourweb
siteatwww.wiley.com.
LibraryofCongressCataloging-in-PublicationData:
Snyder,LloydR.
Introductiontomodernliquidchromatography/LloydR.Snyder,JosephJ.Kirkland. – 3rd
ed./JohnW.Dolan.
p. cm.
Includesindex.
ISBN978-0-470-16754-0 (cloth)
1. Liquidchromatography. I.Kirkland,J.J.(JosephJack),1925- II.Dolan,JohnW.III.
Title.
QD79.C454S582009
543(cid:2).84–dc22
2009005626
PrintedintheUnitedStatesofAmerica.
10987654321
CONTENTS
PREFACE xxxi
GLOSSARYOFSYMBOLSANDABBREVIATIONS xxxv
1 INTRODUCTION 1
1.1 BackgroundInformation, 2
1.1.1 WhatIsHPLC?, 2
1.1.2 WhatCanHPLCDo?, 4
1.2 AShortHistoryofHPLC, 6
1.3 SomeAlternativestoHPLC, 8
1.3.1 GasChromatography(GC), 8
1.3.2 Thin-LayerChromatography(TLC), 9
1.3.3 SupercriticalFluidChromatography
(SFC), 10
1.3.4 CapillaryElectrophoresis(CE), 11
1.3.5 CountercurrentChromatography, 11
1.3.6 SpecialFormsofHPLC, 12
1.4 OtherSourcesofHPLCInformation, 12
1.4.1 Books, 12
1.4.2 Journals, 13
1.4.3 Reviews, 13
1.4.4 ShortCourses, 13
1.4.5 TheInternet, 13
References, 15
2 BASICCONCEPTSANDTHECONTROLOFSEPARATION 19
2.1 Introduction, 20
2.2 TheChromatographicProcess, 20
2.3 Retention, 24
v
vi
CONTENTS
2.3.1 RetentionFactork andColumnDead-Time
t , 25
0
2.3.2 RoleofSeparationConditionsandSample
Composition, 28
2.3.2.1 IntermolecularInteractions, 30
2.3.2.2 Temperature, 34
2.4 PeakWidthandtheColumnPlateNumberN, 35
2.4.1 DependenceofNonSeparation
Conditions, 37
2.4.1.1 Band-BroadeningProcessesThat
DetermineValuesofN, 39
2.4.1.2 SomeGuidelinesforSelecting
ColumnConditions, 46
2.4.2 PeakShape, 50
2.5 ResolutionandMethodDevelopment, 54
2.5.1 OptimizingtheRetentionFactork, 57
2.5.2 OptimizingSelectivityα, 59
2.5.2.1 ‘‘Regular’’and‘‘Irregular’’
Samples, 60
2.5.3 OptimizingtheColumnPlateNumberN, 61
2.5.3.1 EffectsofColumnConditionson
Separation, 61
2.5.3.2 FastHPLC, 63
2.5.4 MethodDevelopment, 65
2.5.4.1 AssessmentofSample
CompositionandSeparation
Goals, 65
2.5.4.2 SamplePretreatment, 66
2.5.4.3 SelectionofChromatographic
Mode, 66
2.5.4.4 DetectorSelection, 66
2.5.4.5 ChoiceofSeparation
Conditions, 67
2.5.4.6 Anticipation,Identification,and
SolutionofPotential
Problems, 67
2.5.4.7 MethodValidationandSystem
Suitability, 69
2.6 SampleSizeEffects, 69
2.6.1 VolumeOverload:EffectofSampleVolume
onSeparation, 70
2.6.2 MassOverload:EffectofSampleWeighton
Separation, 71
vii
CONTENTS
2.6.3 AvoidingProblemsduetoTooLargea
Sample, 73
2.6.3.1 HigherThanExpectedSample
Concentrations, 73
2.6.3.2 TraceAnalysis, 73
2.7 RELATEDTOPICS, 74
2.7.1 ColumnEquilibration, 74
2.7.2 GradientElution, 75
2.7.3 PeakCapacityandTwo-dimensional
Separation, 76
2.7.4 PeakTracking, 77
2.7.5 SecondaryEquilibria, 78
2.7.6 ColumnSwitching, 79
2.7.7 RetentionPredictionsBasedonSolute
Structure, 80
2.7.7.1 Solvation-ParameterModel, 82
References, 83
3 EQUIPMENT 87
3.1 Introduction, 88
3.2 ReservoirsandSolventFiltration, 89
3.2.1 ReservoirDesignandUse, 90
3.2.2 Mobile-PhaseFiltration, 91
3.3 Mobile-PhaseDegassing, 92
3.3.1 DegassingRequirements, 92
3.3.2 HeliumSparging, 94
3.3.3 VacuumandIn-lineDegassing, 95
3.4 TubingandFittings, 96
3.4.1 Tubing, 96
3.4.1.1 Low-PressureTubing, 96
3.4.1.2 High-PressureTubing, 97
3.4.2 Fittings, 99
3.4.2.1 Low-PressureFittings, 99
3.4.2.2 High-PressureFittings, 101
3.4.2.3 SpecialtyFittings, 103
3.5 PumpingSystems, 104
3.5.1 Reciprocating-PistonPumps, 104
3.5.1.1 Dual-PistonPumps, 108
3.5.1.2 Accumulator-PistonPumps, 108
3.5.1.3 ActiveCheckValve, 109
3.5.2 On-lineMixing, 109
3.5.2.1 High-PressureMixing, 109
3.5.2.2 Low-PressureMixing, 111
3.5.2.3 HybridSystems, 111
viii
CONTENTS
3.5.3 GradientSystems, 112
3.5.4 SpecialApplications, 112
3.5.4.1 Low-Flow(MicroandNano)
Applications, 112
3.5.4.2 High-Flow(Prep)
Applications, 113
3.5.4.3 High-PressureApplications, 113
3.6 Autosamplers, 113
3.6.1 Six-PortInjectionValves, 114
3.6.1.1 Filled-LoopInjection, 114
3.6.1.2 Partial-LoopInjection, 115
3.6.2 AutosamplerDesigns, 116
3.6.2.1 Pull-to-FillAutosamplers, 117
3.6.2.2 Push-to-FillAutosamplers, 118
3.6.2.3 Needle-in-Loop
Autosamplers, 119
3.6.3 Sample-SizeEffects, 119
3.6.3.1 InjectionVolume, 120
3.6.3.2 InjectionSolvent, 121
3.6.4 OtherValveApplications, 122
3.6.4.1 ColumnSwitching, 122
3.6.4.2 FractionCollectors, 123
3.6.4.3 WasteDiversion, 124
3.7 ColumnOvens, 125
3.7.1 Temperature-ControlRequirements, 125
3.7.2 OvenDesigns, 126
3.7.2.1 BlockHeater, 126
3.7.2.2 AirBath, 126
3.7.2.3 PeltierHeater, 126
3.8 DataSystems, 127
3.8.1 ExperimentalAids, 127
3.8.2 SystemControl, 129
3.8.3 DataCollection, 129
3.8.4 DataProcessing, 130
3.8.5 ReportGeneration, 130
3.8.6 RegulatoryFunctions, 130
3.9 Extra-ColumnEffects, 131
3.10 Maintenance, 131
3.10.1 System-PerformanceTests, 131
3.10.1.1 InstallationQualification,
OperationalQualification,and
PerformanceQualification, 132
3.10.1.2 GradientPerformanceTest, 132
3.10.1.3 AdditionalSystemChecks, 135
ix
CONTENTS
3.10.2 PreventiveMaintenance, 138
3.10.2.1 PeriodicMaintenance, 138
3.10.2.2 SuggestionsforRoutine
Applications, 141
3.10.3 Repairs, 143
3.10.3.1 Personnel, 143
3.10.3.2 RecordKeeping, 143
3.10.3.3 SpecificRepair
Recommendations, 144
References, 144
4 DETECTION 147
4.1 Introduction, 148
4.2 DetectorCharacteristics, 149
4.2.1 GeneralLayout, 149
4.2.2 DetectionTechniques, 151
4.2.2.1 BulkPropertyDetectors, 151
4.2.2.2 Sample-SpecificDetectors, 152
4.2.2.3 Mobile-PhaseModification
Detectors, 152
4.2.2.4 HyphenatedTechniques, 152
4.2.3 Signal,Noise,Drift,andAssay
Precision, 152
4.2.3.1 NoiseandDrift, 153
4.2.3.2 Signal-to-NoiseRatio(S/N), 155
4.2.4 DetectionLimits, 157
4.2.5 Linearity, 158
4.3 IntroductiontoIndividualDetectors, 160
4.4 UV-VisibleDetectors, 160
4.4.1 Fixed-WavelengthDetectors, 163
4.4.2 Variable-WavelengthDetectors, 164
4.4.3 Diode-ArrayDetectors, 165
4.4.4 GeneralUV-DetectorCharacteristics, 166
4.5 FluorescenceDetectors, 167
4.6 Electrochemical(Amperometric)Detectors, 170
4.7 RadioactivityDetectors, 172
4.8 ConductivityDetectors, 174
4.9 ChemiluminescentNitrogenDetector, 174
4.10 ChiralDetectors, 175
4.11 RefractiveIndexDetectors, 177
4.12 Light-ScatteringDetectors, 180
4.12.1 EvaporativeLight-ScatteringDetector
(ELSD), 181
x
CONTENTS
4.12.2 CondensationNucleationLight-Scattering
Detector(CNLSD), 182
4.12.3 LaserLight-ScatteringDetectors(LLSD), 183
4.13 Corona-DischargeDetector(CAD), 184
4.14 MassSpectralDetectors(MS), 185
4.14.1 Interfaces, 186
4.14.1.1 ElectrosprayInterface(ESI), 186
4.14.1.2 Atmospheric-Pressure
Chemical-IonizationInterface
(APCI), 187
4.14.1.3 OtherInterfaceDesigns, 188
4.14.1.4 Flow-RateConsiderations, 188
4.14.2 QuadrupolesandIonTraps, 188
4.14.3 OtherMSDetectors, 190
4.15 OtherHyphenatedDetectors, 191
4.15.1 Infrared(FTIR), 191
4.15.2 NuclearMagneticResonance(NMR), 192
4.16 SampleDerivatizationandReactionDetectors, 194
References, 196
5 THECOLUMN 199
5.1 Introduction, 200
5.2 ColumnSupports, 200
5.2.1 ParticleCharacterization, 201
5.2.1.1 ParticleType, 201
5.2.1.2 ParticleSizeandPore
Diameter, 203
5.2.2 SilicaSupports, 203
5.2.2.1 ColumnEfficiency, 205
5.2.2.2 NatureoftheSilicaSurface, 208
5.2.2.3 ParticlePreparation, 211
5.2.3 PorousPolymers, 212
5.2.4 Monoliths, 212
5.2.4.1 Silica-BasedMonoliths, 213
5.2.4.2 Polymer-BasedMonoliths, 214
5.2.5 OtherInorganicParticles, 214
5.2.5.1 Zirconia, 215
5.2.5.2 AluminaandTitania, 217
5.2.5.3 GraphitizedCarbon, 217
5.3 StationaryPhases, 217
5.3.1 ‘‘Bonded’’StationaryPhases, 218
5.3.2 OtherOrganic-BasedStationary
Phases, 223
xi
CONTENTS
5.3.2.1 MechanicallyHeld
Polymers, 223
5.3.2.2 HybridParticles, 223
5.3.2.3 ColumnsforHighlyAqueous
MobilePhases, 224
5.3.3 ColumnFunctionality(LigandType), 225
5.4 ColumnSelectivity, 227
5.4.1 BasisofRPCColumnSelectivity, 227
5.4.1.1 Hyperbolic-Subtraction
Model, 229
5.4.1.2 ShapeSelectivity, 232
5.4.2 ColumnReproducibilityand‘‘Equivalent’’
Columns, 235
5.4.3 OrthogonalSeparation, 236
5.4.4 OtherApplicationsofColumn
Selectivity, 237
5.4.4.1 PeakTailing, 237
5.4.4.2 Stationary-Phase
De-Wetting, 237
5.4.4.3 ColumnDegradation, 238
5.5 ColumnHardware, 238
5.5.1 ColumnFittings, 238
5.5.2 ColumnConfigurations, 239
5.6 Column-PackingMethods, 240
5.6.1 Dry-Packing, 240
5.6.2 Slurry-PackingofRigidParticles, 240
5.6.2.1 SelectionofSlurryLiquid, 241
5.6.2.2 RigidPolymericParticles, 243
5.6.3 SoftGels, 244
5.7 ColumnSpecifications, 244
5.7.1 ManufacturingStandards, 244
5.7.2 ColumnPlateNumber, 245
5.8 ColumnHandling, 246
References, 250
6 REVERSED-PHASECHROMATOGRAPHYFORNEUTRALSAM-
PLES 253
6.1 Introduction, 254
6.1.1 AbbreviatedHistoryofReversed-Phase
Chromatography, 255
6.2 Retention, 256
6.2.1 SolventStrength, 257
6.2.2 Reversed-PhaseRetentionProcess, 259
6.3 Selectivity, 263