HPLC made to measure: a practical handbook for optimization
Gespeichert in:
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| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
Weinheim, Bergstr [u.a.]
WILEY-VCH
2006
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XXXIII, 753 S. 300 schw.-w. Ill., 40 farb. Ill. 240 mm x 170 mm |
| ISBN: | 352731377X 9783527313778 |
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| 245 | 1 | 0 | |a HPLC made to measure |b a practical handbook for optimization |c ed. by Stavros Kromidas |
| 264 | 1 | |a Weinheim, Bergstr [u.a.] |b WILEY-VCH |c 2006 | |
| 300 | |a XXXIII, 753 S. |b 300 schw.-w. Ill., 40 farb. Ill. |c 240 mm x 170 mm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 650 | 4 | |a High performance liquid chromatography | |
| 650 | 4 | |a High performance liquid chromatography |x Methodology | |
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Contents
Foreword V
Preface IX
List of Contributors XXV
Structure of the Book XXXI
1 Fundamentals of Optimization 1
1.1 Principles of the Optimization of HPLC Illustrated by
RP-Chromatography 3
Stavros Kromidas
1.1.1 Before the First Steps of Optimization 3
1.1.2 What Exactly Do We Mean By Optimization ? 5
1.1.3 Improvement of Resolution ( Separate Better ) 6
1.1.3.1 Principal Possibilities for Improving Resolution 8
1.1.3.2 What has the Greatest Effect on Resolution? 10
1.1.3.3 Which Sequence of Steps is Most Logical When Attempting
an Optimization? 12
1.1.3.4 How to Change k, a, and N 17
1.1.3.4.1 Isocratic Mode 17
1.1.3.4.2 Gradient Mode 18
1.1.3.4.3 Acetonitrile or Methanol? 19
1.1.4 Testing of the Peak Homogeneity 22
1.1.5 Unknown Samples: How Can I Start? ; Strategies and Concepts 35
1.1.5.1 The Two Days Method 36
1.1.5.2 The 5-Step Model 39
1.1.6 Shortening of the Run Time ( Faster Separation ) 48
1.1.7 Improvement of the Sensitivity
( To See More , i.e. Lowering of the Detection Limit) 48
HPLC Made to Measure: A Practical Handbook for Optimization. Edited by Stavros Kromidas
Copyright © 2006 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim
ISBN: 3-527-31377-X
I
1.1.8 Economics in HPLC ( Cheaper Separation ) 48
1.1.9 Final Remarks and Outlook 51
References 57
1.2 Fast Gradient Separations 59
Uwe D. Neue, Yung-Fong Cheng, and Ziling Lu
1.2.1 Introduction 59
1.2.2 Mam Part 59
1.2.2.1 Theory 59
1.2.2.2 Results 61
1..2.2.2.1 General Relationships 61
1.2.2.2.2 Short Columns, Small Particles 62
1.2.2.2.3 An Actual Example 64
1.2.2.3 Optimal Operating Conditions and Limits of Currently Available
Technology 66
1.2.2.4 Problems and Solutions 67
1.2.2.4.1 Gradient Delay Volume 67
1.2.2.4.2 Detector Sampling Rate and Time Constant 68
1.2.2.4.3 Ion Suppression in Mass Spectrometry 69
References 70
1.3 pH and Selectivity in RP-Chromatography 71
Uwe D. Neue, Alberto Mendez, KimVan Iran, and Diane M. Diehl
1.3.1 Introduction 71
1.3.2 Main Section 71
1.3.2.1 Ionization and pH 71
1.3.2.2 Mobile Phase and pH 73
1.3.2.2.1 Buffer Capacity 74
1.3.2.2.2 Changes of pKand pH Value in the Presence of an Organic
Solvent 76
1.3.2.3 Buffers 78
1.3.2.3.1 Classical HPLC Buffers 78
1.3.2.3.2 MS-Compatible pH Control 79
1.3.2.4 Influence of the Samples 79
1.3.2.4.1 The Sample Type: Acids, Bases, Zwitterions 80
1.3.2.4.2 Influence of the Organic Solvent on the Ionization of the
Analytes 81
1.3.3 Application Example 81
1.3.4 Troubleshooting 85
1.3.4.1 Reproducibility Problems 85
1.3.4.2 Buffer Strength and Solubility 86
1.3.4.3 Constant Buffer Concentration 86
1.3.5 Summary 87
References 87
I
1.4 Selecting the Correct pH Value for HPLC 89
Michael McBrien
1.4.1 Introduction 89
1.4.2 Typical Approaches to pH Selection 90
1.4.3 Initial pH Selection 91
1.4.4 Basis of pKj Prediction 92
1.4.5 Correction of pH Based on Organic Content 93
1.4.6 Optimization of Mobile Phase pH Without Chemical Structures 94
1.4.7 A Systematic Approach to pH Selection 96
1.4.8 An Example - Separation of l,4-Bis[(2-pyridin-2-ylethyl)thio]butane-
2,3-diol from its Impurities 97
1.4.9 Troubleshooting Mobile Phase pH 102
1.4.10 The Future 102
1.4.11 Conclusion 103
References 103
1.5 Optimization of the Evaluation in Chromatography 105
Hans-Joachim Kuss
1.5.1 Evaluation of Chromatographic Data - An Introduction 105
1.5.2 Working Range 105
1.5.3 Internal Standard 106
1.5.4 Calibration 107
1.5.5 Linear Regression 107
1.5.6 Weighting Exponent 320
1.5.7 In Real Practise 111
1.5.8 Drug Analysis 111
1.5.9 Measurement Uncertainty 112
1.5.10 Calibration Line Through the Origin 115
References 115
1.6 Calibration Characteristics and Uncertainty - Indicating Starting
Points to Optimize Methods 117
Stefan Schomer
1.6.1 Optimizing Calibration - What is the Objective? 117
1.6.2 The Essential Performance Characteristic of Calibration 118
1.6.3 Examples 118
1.6.3.1 Does Enhanced Sensitivity Improve Methods? 118
1.6.3.2 A Constant Variation Coefficient - Is it Good, Poor or Just an
Inevitable Characteristic of Method Performance? 122
1.6.3.3 How to Prove Effects Due to Matrices - May the Recovery Function
be Replaced? 133
1.6.3.4 Having Established Matrix Effects - Does Spiking Prove Necessary
in Every Case? 136
1.6.3.5 Testing linearity - Does a Calibration Really Need to Fit a Straight
Line? 139
1.6.3.6 Enhancing Accuracy - Obtaining Robust Calibration Functions
with Weighting 143
References 147
2 Characteristics of Optimization in Individual HPLC Modes 149
2.1 RP-HPLC 151
2.1.1 Comparison and Selection of Commercial RP-Columns 151
Stavros Kromidas
2.1.1.1 Introduction 151
2.1.1.2 Reasons for the Diversity of Commercially Available RP-Columns -
First Consequences 151
2.1.1.2.1 On Polar Interactions 156
2.1.1.2.2 First Consequences 156
2.1.1.3 Criteria for Comparing RP-Phases 174
2.1.1.3.1 Similarity According to Physico-chemical Properties 174
2.1.1.3.2 Similarity Based on Chromatographic Behavior;
Expressiveness of Retention and Selectivity Factors 175
2.1.1.3.3 Tests for the Comparison of Columns and Their Expressiveness 181
2.1.1 A Similarity of RP-Phases 195
2.1.1.4.1 Selectivity Maps 196
2.1.1.4.2 Selectivity Plots 200
2.1.1.4.3 Selectivity Hexagons 205
2.1.1.4.4 Chemometric Analysis of Chromatographic Data 229
2.1.1.5 Suitability of RP-Phases for Special Types of Analytes and Proposals
for the Choice of Columns 233
2.1.1.5.1 Polar and Hydrophobic RP-Phases 233
2.1.1.5.2 Suitability of RP-Phases for Different Classes of Substances 237
2.1.1.5.3 Procedure for the Choice of an RP-Column 248
References 253
2.1.2 Column Selectivity in RP-Chromatography 254
Uwe D. Neue, Bonnie A. Alden, and Pamela C. Iraneta
2.1.2.1 Introduction 254
2.1.2.2 Main Section 255
2.1.2.2.1 Hydrophobicity and Silanol Activity (Ion Exchange) 255
2.1.2.2.2 Polar Interactions (Hydrogen Bonding) 259
2.1.2.2.3 Reproducibility of the Selectivity 261
References 263
I
2.1.3 The Use of Principal Component Analysis for the Characterization
of Reversed-Phase Liquid Chromatographic Stationary Phases 264
Melvin R. Euerby and Patrik Petersson
2.1.3.1 Introduction 264
2.1.3.2 Theory of Principal Component Analysis 265
2.1.3.3 PCA of the Database of RP Silica Materials 267
2.1.3.3.1 PCA of Polar Embedded, Enhanced Polar Selectivity, and AQ/Aqua
Phases 269
2.1.3.3.2 PCA of Perfluonnated Phases 270
2.1.3.4 Use of PCA in the Identification of Column/Phase Equivalency 271
2.1.3.5 Use of PCA in the Rational Selection of Stationary Phases for
Method Development 277
2.1.3.5.1 Proposed Solvent/Stationary Phase Optimization Strategy 278
References 279
2.1 A Chemometrics - A Powerful Tool for Handling a Large Number
of Data 280
Cinzia Stella and Jean-Luc Veuthey
2.1 A.I Introduction 280
2.1.4.2 Chromatographic Tests and Their Importance in Column
Selection 280
2.1.4.3 Use of Principal Component Analysis (PCA) in the Evaluation
and Selection of Test Compounds 281
2.1.4.3.1 Physicochemical Properties of Test Compounds 281
2.1.4.3.2 Chromatographic Properties of Test Compounds 284
2.1.4.4 Use of PCA for the Evaluation of Chromatographic Supports 285
2.1 A A.I Evaluation of Chromatographic Supports in Mobile Phases
Composed of pH 7.0 Phosphate Buffer 286
2.1.4.4.2 Evaluation of Chromatographic Supports in Mobile Phases
Composed of pH 3.0 Phosphate Buffer 289
2.1.4.5 How a Chromatographic Test can be Optimized by
Chemometrics 291
2.1.4.5.1 Test Compounds 291
2.1.4.5.2 Mobile Phases 292
2.1.4.5.3 Chromatographic Parameters and Batch (Column)
Reproducibility 292
2.1.4.6 Conclusion and Perspectives 295
References 295
2.1.5 Linear Free Energy Relationships (LFER) - Tools for Column
Characterization and Method Optimization in HPLC? 296
Frank Steiner
2.1.5.1 Characterization and Selection of Stationary Phases for HPLC 296
2.1.5.2 What are LFERs and Why can they be Profitable in HPLC? 297
2.1.5.3 How to Obtain Analyte Descriptors for the Multivariate
Regression 300
2.1.5.4 LFER Procedure Using the Solvation Equation 301
2.1.5.4.1 Comparing Stationary Phases on the Basis of LFER Data 301
2.1.5.4.2 The Influence of the Mobile Phase Expressed in LFER
Parameters 307
2.1.5.4.3 The Prediction of Chromatographic Selectivity from LFER Data 308
2.1.5.5 An Empirical Approach to the Determination of LFER Solute
Parameters (Descriptors) from HPLC Data 310
2.1.5.5.1 How does this Strategy Differ from the Use of Predetermined
Solute Descriptors 310
2.1.5.5.2 The Experimental Plan 311
2.1.5.5.3 Determination of the Five LFER Parameters - A Procedure in
Eight Steps 312
2.1.5.5.4 Variation of the Eluent Conditions 315
2.1.5.5.5 Stationary Phase Characterization with Empirical LFER
Parameters 317
2.1.5.6 Concluding Remarks on LFER Applications in HPLC 319
References 320
2.1.6 Column Selectivity in Reversed-Phase Liquid Chromatography 321
Lloyd R. Snyder and John W. Dolan
2.1.6.1 Introduction 321
2.1.6.2 The Subtraction Model of Reversed-Phase Column Selectivity 323
2.1.6.3 Applications 326
2.1.6.3.1 Selecting Equivalent Columns 326
2.1.6.3.2 Selecting Columns of Very Different Selectivity 330
2.1.6.4 Conclusions 332
References 333
2.1.7 Understanding Selectivity by the Use of Suspended-State
High-Resolution Magic-Angle Spinning NMR Spectroscopy 334
Urban Skogsberg, Heidi Handel, Norbert Welsch, and Klaus Albert
2.1.7.1 Introduction 334
2.1.7.2 Is the Comparison Between NMR and HPLC Valid? 337
2.1.7.3 The Transferred Nuclear Overhauser Effect (trNOE) 340
2.1.7.4 Suspended-State XH HR/MAS T1 Relaxation Measurements 343
2.1.7.5 Where do the Interactions Take Place? 345
I
2.1.7.6 Hydrogen Bonding 345
2.1.7.7 Some Practical Considerations 345
2.1.7.8 Future Aspects 347
References 347
2.2 Optimization in Normal-Phase HPLC 349
Veronika R. Meyer
2.2.1 Introduction 349
2.2.2 Mobile Phases in NP-HPLC 350
2.2.3 Stationary Phases in NP-HPLC 354
2.2.4 Troubleshooting in Normal-Phase HPLC 356
References/Further Reading 357
2.3 Optimization of GPC/SEC Separations by Appropriate Selection
of the Stationary Phase and Detection Mode 359
Peter KHz
2.3.1 Introduction 359
2.3.2 Fundamentals of GPC Separations 360
2.3.2.1 Chromatographic Modes of Column Separation 362
2.3.2.2 GPC Column Selection Criteria and Optimization of GPC
Separations 364
2.3.2.2.1 Selection of Pore Size and Separation Range 364
2.3.2.2.2 Advantages and Disadvantages of Linear or Mixed-Bed
Columns 365
2.3.2.3 Highspeed GPC Separations 367
2.3.3 The Role of Comprehensive Detection in the Investigation
of Macromolecular Materials 369
2.3.3.1 Coupling of Liquid Chromatography with Information-Rich
Detectors 371
2.3.3.2 Copolymer GPC Analysis by Multiple Detection 372
2.3.3.3 Simultaneous Separation and Identification by GPC-FTIR 375
2.3.3.4 Application of Molar Mass-Sensitive Detectors in GPC 377
2.3.3.4.1 Light-Scattering Detection 377
2.3.3.4.2 Viscometry Detection 379
2.3.4 Summary 380
References 381
2.4 Gel Filtration/Size-Exclusion Chromatography (SEC) of Biopolymers -
Optimization Strategies and Troubleshooting 383
Milena Quaglia, Egidijus Machtejevas, Tom Hennessy, and Klaus K. linger
2.4.1 Where Are We Now and Where Are We Going? 383
2.4.2 Theory in Brief 384
2.4.3 SEC vs. HPLC Variants 387
I
2.4.4 Optimization Aspects in SEC of Biopolymers 388
2.4.4.1 Column Selection and Optimal Flow Rate 388
2.4.4.2 Optimization of the Mobile Phase 392
2.4.4.3 Sample Preparation 394
2.4.4.4 Sample Viscosity and Sample Volume - Two Critical Parameters
at Injection 395
2.4.4.5 Detection Methods 396
2.4.5 Applications 397
2.4.5.1 High-Performance SEC 397
2.4.5.2 Determination of Molecular Weight 398
2.4.5.3 Gel Filtration as a Tool to Study Conformational Changes of
Proteins 398
2.4.5.4 Gel Filtration in Preparative and Process Separations
(Downstream Processing) 399
2.4.5.5 SEC Columns Based on the Principle of Restricted Access
and Their Use in Proteome Analysis 400
References 403
2.5 Optimization in Affinity Chromatography 405
Egbert Muller
2.5.1 Introduction to Resm Design and Method Development in
Affinity Chromatography 405
2.5.2 Base Matrix 408
2.5.3 Immobilization Methods 409
2.5.4 Activation Methods 409
2.5.5 Spacer 412
2.5.6 Site-Directed Immobilization 415
2.5.7 Non-Particulate Affinity Matrices 416
2.5.8 Affinity Purification 417
2.5.9 Factorial Design for the Preparation of Affinity Resins 419
2.5.10 Summary of Immobilization 423
References 423
2.6 Optimization of Enantiomer Separations in HPLC 427
Markusjuza
2.6.1 Introduction 427
2.6.2 Basic Principles of Enantioselective HPLC 427
2.6.2.1 Thermodynamic Fundamentals of Enantioselective HPLC 429
2.6.2.2 Adsorption and Chiral Recognition 430
2.6.2.3 Differences to Reversed-Phase and Normal-Phase HPLC 433
2.6.2 A Principles for Optimization of Enantioselective HPLC
Separations 433
2.6.3 Selectors and Stationary Phases 433
2.6.4 Method Selection and Optimization 440
I
2.6.4.1 Cellulose and Amylose Derivatives 441
2.6.4.2 Immobilized Cellulose and Amylose Derivatives 443
2.6.4.3 Stationary Phases Derived from Tartaric Acid 444
2.6.4.4 7i-Acidic and 7i-Basic Stationary Phases 444
2.6.4.5 Macrocyclic Selectors, Cyclodextnns, and Antibiotics 446
2.6.4.6 Proteins and Peptides 450
2.6.4.7 Ruthenium Complexes 450
2.6.4.8 Synthetic and Imprinted Polymers 450
2.6.4.9 Metal Complexation and Ligand-Exchange Phases 451
2.6.4.10 Chiral Ion Exchangers 451
2.6.5 Avoiding Errors and Troubleshooting 452
2.6.5.1 Equipment and Columns - Practical Tips 452
2.6.5.2 Detection 454
2.6.5.3 Mistakes Originating from the Analyte 454
2.6.6 Preparative Enantioselective HPLC 454
2.6.6.1 Determination of the Loading Capacity 455
2.6.6.2 Determination of Elution Volumes and Flow Rates 456
2.6.6.3 Enantiomer Separation using Simulated Moving Bed (SMB)
Chromatography 458
2.6.6.3.1 Principles of Simulated Moving Bed Chromatography 458
2.6.6.3.2 Separation of Commercial Active Pharmaceutical Ingredients by
SMB 459
2.6.7 Enantioselective Chromatography by the Addition of
Chiral Additives to the Mobile Phase in HPLC and Capillary
Electrophoresis 461
2.6.8 Determination of Enantiomeric Purity Through the Formation of
Diastereomers 462
2.6.9 Indirect Enantiomer Separation on a Preparative Scale 462
2.6.10 Enantiomer Separations Under Supercritical Fluid Chromatographic
(SFC) Conditions 462
2.6.11 New Chiral Stationary Phases and Information Management
Software 463
2.6.12 Summary 463
References 464
2.7 Miniaturization 467
2.7.1 mLC/NanoLC - Optimization and Troubleshooting 467
Jurgen Maier-Rosenkranz
2.7.1.1 Introduction 467
2.7.1.2 Sensitivity 467
2.7.1.2.1 Influence of Column Length 467
2.7.1.2.2 Influence of Column Internal Diameter 467
2.7.1.2.3 Influence of Stationary Phase 469
2.7.1.3 Robustness 469
I
2.7.1.3.1 System Choice 469
2.7.1.3.2 Capillary Connections 472
2.7.1.3.3 Precautions Against Blocking 477
2.7.1.3.4 Testing for Leakages 478
2.7.1.3.5 Guard Column Switching and Sample Loading Strategies 478
2.7.1.4 Sensitivity/Resolution 483
2.7.1.4.1 Column Dimensions 483
2.7.1.4.2 Packing Materials/Surface Covering 484
2.7.1.4.3 Detectors 484
References 486
2.7.2 Microchip-Based Liquid Chromatography - Techniques and
Possibilities 487
Jorg P. Kutter
2.7.2.1 Introduction 487
2.7.2.2 Techniques 488
2.7.2.2.1 Pressure-Driven Liquid Chromatography (LC) 488
2.7.2.2.2 Open-Channel Electrochromatography (OCEC) 488
2.7.2.2.3 Packed-Bed Electrochromatography 488
2.7 .2.2.4 Microfabricated Chromatographic Beds (Pillar Arrays) 489
2.7.2.2.5 In Situ Polymerized Monolithic Stationary Phases 489
2.7.2.3 Optimization and Possibilities 490
2.7.2.3.1 Separation Performance 490
2.7.2.3.2 Isocratic and Gradient Elution 491
2.7.2.3.3 Tailor-Made Stationary Phases 492
2.7.2.3.4 Sample Pretreatment and More-Dimensional Separations 492
2.7.2.3.5 Issues and Challenges 492
2.7.2.4 Application Examples 493
2.7.2.5 Conclusions and Outlook 496
References 496
2.7.3 Ultra-Performance Liquid Chromatography 498
Uwe D. Neue, Eric S. Crumbach, Marianna Kele,
Jeffrey R. Mazzeo, and Dirk Sievers
2.7.3.1 Introduction 498
2.7.3.2 Isocratic Separations 499
2.7.3.3 Gradient Separations 502
References 505
I
3 Coupling Techniques 507
3.1 Immunochromatographic Techniques 509
Michael C. Weller
3.1.1 Introduction 509
3.1.2 Binding Molecules 509
3.1.3 Immunoassays 511
3.1.4 Immunochromatographic Techniques 511
3.1.4.1 Affinity Enrichment (Affinity SPE) 513
3.1.4.2 Weak Affinity Chromatography
(True Affinity Chromatography) 519
3.1.4.3 Biochemical Detectors 520
3.1.5 Examples 522
3.1.5.1 Example 1: Affinity Extraction (Affinity SPE) 522
3.1.5.2 Example 2: Weak Affinity Chromatography (WAC) 523
3.1.5.3 Example 3: Biochemical Detection 525
References 525
3.2 Enhanced Characterization and Comprehensive Analyses
by Two-Dimensional Chromatography 527
Peter Kilz
3.2.1 Introduction 527
3.2.2 How Can I Take Advantage? - Experimental Aspects 529
3.2.3 2D Data Presentation and Analysis 533
3.2.4 The State-of-the-Art in 2D Chromatography 535
3.2.5 Summary 539
References 540
3.3 LC/MS - Hints and Recommendations on Optimization and
Troubleshooting 541
Friedrich Mandel
3.3.1 Optimization of the Ionization Process 541
3.3.2 Lost LC/MS Peaks 542
3.3.2.1 Mobile Phase pH at the Edge of the Optimum Range 543
3.3.2.2 Ion-Pairing Agents in the HPLC System 543
3.3.2.3 Ion Suppression by the Sample Matrix or Sample
Contaminants 544
3.3.3 How Clean Should an LC/MS Ion Source Be? 544
3.3.4 Ion Suppression 545
References 549
I
3.4 LC-NMR Coupling 551
Klaus Albert, Manfred Krucker, Karsten Putzbach, and Marc D. Grynbaum
3.4.1 NMR Basics 551
3.4.2 Sensitivity of the NMR Experiment 552
3.4.3 NMR Spectroscopy in Flowing Systems 553
3.4.4 NMR Probes for LC-NMR 553
3.4.5 Practical Realization of Analytical HPLC-NMR and
Capillary-HPLC-NMR 554
3.4.6 Continuous-Flow Measurements 555
3.4.7 Stopped-Flow Measurements 557
3.4.8 Capillary Separations 559
3.4.9 Outlook 560
References 563
4 Computer-Aided Optimization 565
4.1 Computer-Facilitated HPLC Method Development Using DryLab®
Software 567
Lloyd R. Snyder and Loren Wrisley
4.1.1 Introduction 567
4.1.1.1 History 569
4.1.1.2 Theory 570
4.1.2 DryLab Capabilities 570
4.1.2.1 DryLab Operation 570
4.1.2.2 Mode Choices 571
4.1.3 Practical Applications of DryLab® in the Laboratory 572
4.1.4 Conclusions 584
References 585
4.2 ChromSword® Software for Automated and Computer-Assisted
Development of HPLC Methods 587
Sergey Calushko, Vsevolod Tanchuk, Irina Shishkina, Oleg Pylypchenko,
and Wolf Dieter Beinert
4.2.1 Introduction 587
4.2.1.1 Off-Line Mode 587
4.2.1.2 On-line Mode 587
4.2.2 ChromSword® Versions 587
4.2.3 Experimental Set-Up for On-Line Mode 588
4.2.4 Method Development with ChromSword® 588
4.2.4.1 Off-Line Mode (Computer-Assisted Method Development) 588
4.2.4.2 On-Line Mode - Fully Automated Optimization of Isocratic
and Gradient Separations 592
4.2.4.2.1 Software Functions for Automation 597
I
4.2.4.2.2 How Does the System Optimize Separations? 597
4.2.5 Conclusion 600
References 600
4.3 Multifactorial Systematic Method Development and Optimization
in Reversed-Phase HPLC 601
Michael Pfeffer
4.3.1 Introduction and Factorial Viewpoint 601
4.3.2 Strategy for Partially Automated Method Development 603
4.3.3 Comparison of Commercially Available Software Packages with
Regard to Their Contribution to Factorial Method Development 608
4.3.4 Development of a New System for Multifactorial Method
Development 609
4.3.4.1 Selection of Stationary Phases 611
4.3.4.2 Optimizing Methods with HEUREKA 612
4.3.4.3 Evaluation of Data with HEUREKA 618
4.3.5 Conclusion and Outlook 623
References 623
5 User Reports 625
5.1 Nano-LC-MS/MS in Proteomics 627
Heike Schafer, Christiane Lohaus, Helmut E. Meyer, and Katrin Marcus
5.1.1 Proteomics - An Introduction 627
5.1.2 Sample Preparation for Nano-LC 628
5.1.3 Nano-LC 629
5.1.4 On-line LC-ESI-MS/MS Coupling 633
5.1.5 Off-Line LC-MALDI-MS/MS Coupling 635
5.1.5.1 Sample Fractionation 635
5.1.5.2 MALDI-TOF-MS/MS Analyses 635
5.1.6 Data Analysis 637
5.1.7 Application in Practice: Analysis of a-Crystallin A in Mice Lenses 637
References 640
5.2 Verification Methods for Robustness in RP-HPLC 643
Hans Bilke
5.2.1 Introduction 643
5.2.2 Testing Robustness in Analytical RP-HPLC by Means of
Systematic Method Development 643
5.2.3 Robustness Test in Analytical RP-HPLC by Means of Statistical
Experimental Design (DoE) 652
5.2.4 Conclusion 665
References 666
I
5.3 Separation of Complex Sample Mixtures 669
Knut Wagner
5.3.1 Introduction 669
5.3.2 Multidimensional HPLC 670
5.3.3 Techniques for Multidimensional Separations 672
5.3.3.1 Off-Line Technique 672
5.3.3.2 On-Line Technique 672
5.3.4 On-Line Sample Preparation as a Previous Stage of
Multidimensional HPLC 674
5.3.5 Fields of Application of Multidimensional HPLC 675
5.3.5.1 What can be Realized? - A Practical Example 676
5.3.6 Critical Parameters of Multidimensional HPLC 682
References 683
5.4 Evaluation of an Integrated Procedure for the Characterization of
Chemical libraries on the Basis of HPLC-UV/MS/CLND 685
Mario Arangio, Federico R. Sirtori, Katia Marcucci,
Giuseppe Razzano, Maristella Colombo, Roberto Biancardi, and
Vincenzo Rizzo
5.4.1 Introduction 685
5.4.2 Materials and Methods 686
5.4.2.1 Instrumentation 686
5.4.2.2 Chemicals and Consumables 686
5.4.2.3 High-Throughput Platform (HTP1) Method Set-up 688
5.4.2.4 Chromatographic Conditions 688
5.4.2.5 Mass Spectrometer and CLND Conditions 689
5.4.2.6 Data Processing and Reporting 689
5.4.2.7 Multilinear Regression Analysis for the Derivation of CLND
Response Factors 690
5.4.3 Results and Discussion 691
5.4.3.1 Liquid Chromatography and UV Detection 691
5.4.3.2 Mass Spectrometric Method Development 692
5.4.3.3 CLND Set-Up 693
5.4.3.4 Validation with Commercial Standards 693
5.4.3.5 Validation with Proprietary Compounds 695
5.4.4 Conclusions 699
References 700
Appendix 703
Subject Index 729
|
| adam_txt |
I
Contents
Foreword V
Preface IX
List of Contributors XXV
Structure of the Book XXXI
1 Fundamentals of Optimization 1
1.1 Principles of the Optimization of HPLC Illustrated by
RP-Chromatography 3
Stavros Kromidas
1.1.1 Before the First Steps of Optimization 3
1.1.2 What Exactly Do We Mean By "Optimization"? 5
1.1.3 Improvement of Resolution ("Separate Better") 6
1.1.3.1 Principal Possibilities for Improving Resolution 8
1.1.3.2 What has the Greatest Effect on Resolution? 10
1.1.3.3 Which Sequence of Steps is Most Logical When Attempting
an Optimization? 12
1.1.3.4 How to Change k, a, and N 17
1.1.3.4.1 Isocratic Mode 17
1.1.3.4.2 Gradient Mode 18
1.1.3.4.3 Acetonitrile or Methanol? 19
1.1.4 Testing of the Peak Homogeneity 22
1.1.5 Unknown Samples: "How Can I Start?"; Strategies and Concepts 35
1.1.5.1 The "Two Days Method" 36
1.1.5.2 "The 5-Step Model" 39
1.1.6 Shortening of the Run Time ("Faster Separation") 48
1.1.7 Improvement of the Sensitivity
("To See More", i.e. Lowering of the Detection Limit) 48
HPLC Made to Measure: A Practical Handbook for Optimization. Edited by Stavros Kromidas
Copyright © 2006 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim
ISBN: 3-527-31377-X
I
1.1.8 Economics in HPLC ("Cheaper Separation") 48
1.1.9 Final Remarks and Outlook 51
References 57
1.2 Fast Gradient Separations 59
Uwe D. Neue, Yung-Fong Cheng, and Ziling Lu
1.2.1 Introduction 59
1.2.2 Mam Part 59
1.2.2.1 Theory 59
1.2.2.2 Results 61
1.2.2.2.1 General Relationships 61
1.2.2.2.2 Short Columns, Small Particles 62
1.2.2.2.3 An Actual Example 64
1.2.2.3 Optimal Operating Conditions and Limits of Currently Available
Technology 66
1.2.2.4 Problems and Solutions 67
1.2.2.4.1 Gradient Delay Volume 67
1.2.2.4.2 Detector Sampling Rate and Time Constant 68
1.2.2.4.3 Ion Suppression in Mass Spectrometry 69
References 70
1.3 pH and Selectivity in RP-Chromatography 71
Uwe D. Neue, Alberto Mendez, KimVan Iran, and Diane M. Diehl
1.3.1 Introduction 71
1.3.2 Main Section 71
1.3.2.1 Ionization and pH 71
1.3.2.2 Mobile Phase and pH 73
1.3.2.2.1 Buffer Capacity 74
1.3.2.2.2 Changes of pKand pH Value in the Presence of an Organic
Solvent 76
1.3.2.3 Buffers 78
1.3.2.3.1 Classical HPLC Buffers 78
1.3.2.3.2 MS-Compatible pH Control 79
1.3.2.4 Influence of the Samples 79
1.3.2.4.1 The Sample Type: Acids, Bases, Zwitterions 80
1.3.2.4.2 Influence of the Organic Solvent on the Ionization of the
Analytes 81
1.3.3 Application Example 81
1.3.4 Troubleshooting 85
1.3.4.1 Reproducibility Problems 85
1.3.4.2 Buffer Strength and Solubility 86
1.3.4.3 Constant Buffer Concentration 86
1.3.5 Summary 87
References 87
I
1.4 Selecting the Correct pH Value for HPLC 89
Michael McBrien
1.4.1 Introduction 89
1.4.2 Typical Approaches to pH Selection 90
1.4.3 Initial pH Selection 91
1.4.4 Basis of pKj Prediction 92
1.4.5 Correction of pH Based on Organic Content 93
1.4.6 Optimization of Mobile Phase pH Without Chemical Structures 94
1.4.7 A Systematic Approach to pH Selection 96
1.4.8 An Example - Separation of l,4-Bis[(2-pyridin-2-ylethyl)thio]butane-
2,3-diol from its Impurities 97
1.4.9 Troubleshooting Mobile Phase pH 102
1.4.10 The Future 102
1.4.11 Conclusion 103
References 103
1.5 Optimization of the Evaluation in Chromatography 105
Hans-Joachim Kuss
1.5.1 Evaluation of Chromatographic Data - An Introduction 105
1.5.2 Working Range 105
1.5.3 Internal Standard 106
1.5.4 Calibration 107
1.5.5 Linear Regression 107
1.5.6 Weighting Exponent 320
1.5.7 In Real Practise 111
1.5.8 Drug Analysis 111
1.5.9 Measurement Uncertainty 112
1.5.10 Calibration Line Through the Origin 115
References 115
1.6 Calibration Characteristics and Uncertainty - Indicating Starting
Points to Optimize Methods 117
Stefan Schomer
1.6.1 Optimizing Calibration - What is the Objective? 117
1.6.2 The Essential Performance Characteristic of Calibration 118
1.6.3 Examples 118
1.6.3.1 Does Enhanced Sensitivity Improve Methods? 118
1.6.3.2 A Constant Variation Coefficient - Is it Good, Poor or Just an
Inevitable Characteristic of Method Performance? 122
1.6.3.3 How to Prove Effects Due to Matrices - May the Recovery Function
be Replaced? 133
1.6.3.4 Having Established Matrix Effects - Does Spiking Prove Necessary
in Every Case? 136
1.6.3.5 Testing linearity - Does a Calibration Really Need to Fit a Straight
Line? 139
1.6.3.6 Enhancing Accuracy - Obtaining 'Robust' Calibration Functions
with Weighting 143
References 147
2 Characteristics of Optimization in Individual HPLC Modes 149
2.1 RP-HPLC 151
2.1.1 Comparison and Selection of Commercial RP-Columns 151
Stavros Kromidas
2.1.1.1 Introduction 151
2.1.1.2 Reasons for the Diversity of Commercially Available RP-Columns -
First Consequences 151
2.1.1.2.1 On Polar Interactions 156
2.1.1.2.2 First Consequences 156
2.1.1.3 Criteria for Comparing RP-Phases 174
2.1.1.3.1 Similarity According to Physico-chemical Properties 174
2.1.1.3.2 Similarity Based on Chromatographic Behavior;
Expressiveness of Retention and Selectivity Factors 175
2.1.1.3.3 Tests for the Comparison of Columns and Their Expressiveness 181
2.1.1 A Similarity of RP-Phases 195
2.1.1.4.1 Selectivity Maps 196
2.1.1.4.2 Selectivity Plots 200
2.1.1.4.3 Selectivity Hexagons 205
2.1.1.4.4 Chemometric Analysis of Chromatographic Data 229
2.1.1.5 Suitability of RP-Phases for Special Types of Analytes and Proposals
for the Choice of Columns 233
2.1.1.5.1 Polar and Hydrophobic RP-Phases 233
2.1.1.5.2 Suitability of RP-Phases for Different Classes of Substances 237
2.1.1.5.3 Procedure for the Choice of an RP-Column 248
References 253
2.1.2 Column Selectivity in RP-Chromatography 254
Uwe D. Neue, Bonnie A. Alden, and Pamela C. Iraneta
2.1.2.1 Introduction 254
2.1.2.2 Main Section 255
2.1.2.2.1 Hydrophobicity and Silanol Activity (Ion Exchange) 255
2.1.2.2.2 Polar Interactions (Hydrogen Bonding) 259
2.1.2.2.3 Reproducibility of the Selectivity 261
References 263
I
2.1.3 The Use of Principal Component Analysis for the Characterization
of Reversed-Phase Liquid Chromatographic Stationary Phases 264
Melvin R. Euerby and Patrik Petersson
2.1.3.1 Introduction 264
2.1.3.2 Theory of Principal Component Analysis 265
2.1.3.3 PCA of the Database of RP Silica Materials 267
2.1.3.3.1 PCA of Polar Embedded, Enhanced Polar Selectivity, and AQ/Aqua
Phases 269
2.1.3.3.2 PCA of Perfluonnated Phases 270
2.1.3.4 Use of PCA in the Identification of Column/Phase Equivalency 271
2.1.3.5 Use of PCA in the Rational Selection of Stationary Phases for
Method Development 277
2.1.3.5.1 Proposed Solvent/Stationary Phase Optimization Strategy 278
References 279
2.1 A Chemometrics - A Powerful Tool for Handling a Large Number
of Data 280
Cinzia Stella and Jean-Luc Veuthey
2.1 A.I Introduction 280
2.1.4.2 Chromatographic Tests and Their Importance in Column
Selection 280
2.1.4.3 Use of Principal Component Analysis (PCA) in the Evaluation
and Selection of Test Compounds 281
2.1.4.3.1 Physicochemical Properties of Test Compounds 281
2.1.4.3.2 Chromatographic Properties of Test Compounds 284
2.1.4.4 Use of PCA for the Evaluation of Chromatographic Supports 285
2.1 A A.I Evaluation of Chromatographic Supports in Mobile Phases
Composed of pH 7.0 Phosphate Buffer 286
2.1.4.4.2 Evaluation of Chromatographic Supports in Mobile Phases
Composed of pH 3.0 Phosphate Buffer 289
2.1.4.5 How a Chromatographic Test can be Optimized by
Chemometrics 291
2.1.4.5.1 Test Compounds 291
2.1.4.5.2 Mobile Phases 292
2.1.4.5.3 Chromatographic Parameters and Batch (Column)
Reproducibility 292
2.1.4.6 Conclusion and Perspectives 295
References 295
2.1.5 Linear Free Energy Relationships (LFER) - Tools for Column
Characterization and Method Optimization in HPLC? 296
Frank Steiner
2.1.5.1 Characterization and Selection of Stationary Phases for HPLC 296
2.1.5.2 What are LFERs and Why can they be Profitable in HPLC? 297
2.1.5.3 How to Obtain Analyte Descriptors for the Multivariate
Regression 300
2.1.5.4 LFER Procedure Using the Solvation Equation 301
2.1.5.4.1 Comparing Stationary Phases on the Basis of LFER Data 301
2.1.5.4.2 The Influence of the Mobile Phase Expressed in LFER
Parameters 307
2.1.5.4.3 The Prediction of Chromatographic Selectivity from LFER Data 308
2.1.5.5 An Empirical Approach to the Determination of LFER Solute
Parameters (Descriptors) from HPLC Data 310
2.1.5.5.1 How does this Strategy Differ from the Use of Predetermined
Solute Descriptors 310
2.1.5.5.2 The Experimental Plan 311
2.1.5.5.3 Determination of the Five LFER Parameters - A Procedure in
Eight Steps 312
2.1.5.5.4 Variation of the Eluent Conditions 315
2.1.5.5.5 Stationary Phase Characterization with Empirical LFER
Parameters 317
2.1.5.6 Concluding Remarks on LFER Applications in HPLC 319
References 320
2.1.6 Column Selectivity in Reversed-Phase Liquid Chromatography 321
Lloyd R. Snyder and John W. Dolan
2.1.6.1 Introduction 321
2.1.6.2 The "Subtraction" Model of Reversed-Phase Column Selectivity 323
2.1.6.3 Applications 326
2.1.6.3.1 Selecting "Equivalent" Columns 326
2.1.6.3.2 Selecting Columns of Very Different Selectivity 330
2.1.6.4 Conclusions 332
References 333
2.1.7 Understanding Selectivity by the Use of Suspended-State
High-Resolution Magic-Angle Spinning NMR Spectroscopy 334
Urban Skogsberg, Heidi Handel, Norbert Welsch, and Klaus Albert
2.1.7.1 Introduction 334
2.1.7.2 Is the Comparison Between NMR and HPLC Valid? 337
2.1.7.3 The Transferred Nuclear Overhauser Effect (trNOE) 340
2.1.7.4 Suspended-State XH HR/MAS T1 Relaxation Measurements 343
2.1.7.5 Where do the Interactions Take Place? 345
I
2.1.7.6 Hydrogen Bonding 345
2.1.7.7 Some Practical Considerations 345
2.1.7.8 Future Aspects 347
References 347
2.2 Optimization in Normal-Phase HPLC 349
Veronika R. Meyer
2.2.1 Introduction 349
2.2.2 Mobile Phases in NP-HPLC 350
2.2.3 Stationary Phases in NP-HPLC 354
2.2.4 Troubleshooting in Normal-Phase HPLC 356
References/Further Reading 357
2.3 Optimization of GPC/SEC Separations by Appropriate Selection
of the Stationary Phase and Detection Mode 359
Peter KHz
2.3.1 Introduction 359
2.3.2 Fundamentals of GPC Separations 360
2.3.2.1 Chromatographic Modes of Column Separation 362
2.3.2.2 GPC Column Selection Criteria and Optimization of GPC
Separations 364
2.3.2.2.1 Selection of Pore Size and Separation Range 364
2.3.2.2.2 Advantages and Disadvantages of Linear or Mixed-Bed
Columns 365
2.3.2.3 Highspeed GPC Separations 367
2.3.3 The Role of Comprehensive Detection in the Investigation
of Macromolecular Materials 369
2.3.3.1 Coupling of Liquid Chromatography with Information-Rich
Detectors 371
2.3.3.2 Copolymer GPC Analysis by Multiple Detection 372
2.3.3.3 Simultaneous Separation and Identification by GPC-FTIR 375
2.3.3.4 Application of Molar Mass-Sensitive Detectors in GPC 377
2.3.3.4.1 Light-Scattering Detection 377
2.3.3.4.2 Viscometry Detection 379
2.3.4 Summary 380
References 381
2.4 Gel Filtration/Size-Exclusion Chromatography (SEC) of Biopolymers -
Optimization Strategies and Troubleshooting 383
Milena Quaglia, Egidijus Machtejevas, Tom Hennessy, and Klaus K. linger
2.4.1 Where Are We Now and Where Are We Going? 383
2.4.2 Theory in Brief 384
2.4.3 SEC vs. HPLC Variants 387
I
2.4.4 Optimization Aspects in SEC of Biopolymers 388
2.4.4.1 Column Selection and Optimal Flow Rate 388
2.4.4.2 Optimization of the Mobile Phase 392
2.4.4.3 Sample Preparation 394
2.4.4.4 Sample Viscosity and Sample Volume - Two Critical Parameters
at Injection 395
2.4.4.5 Detection Methods 396
2.4.5 Applications 397
2.4.5.1 High-Performance SEC 397
2.4.5.2 Determination of Molecular Weight 398
2.4.5.3 Gel Filtration as a Tool to Study Conformational Changes of
Proteins 398
2.4.5.4 Gel Filtration in Preparative and Process Separations
(Downstream Processing) 399
2.4.5.5 SEC Columns Based on the Principle of Restricted Access
and Their Use in Proteome Analysis 400
References 403
2.5 Optimization in Affinity Chromatography 405
Egbert Muller
2.5.1 Introduction to Resm Design and Method Development in
Affinity Chromatography 405
2.5.2 Base Matrix 408
2.5.3 Immobilization Methods 409
2.5.4 Activation Methods 409
2.5.5 Spacer 412
2.5.6 Site-Directed Immobilization 415
2.5.7 Non-Particulate Affinity Matrices 416
2.5.8 Affinity Purification 417
2.5.9 Factorial Design for the Preparation of Affinity Resins 419
2.5.10 Summary of Immobilization 423
References 423
2.6 Optimization of Enantiomer Separations in HPLC 427
Markusjuza
2.6.1 Introduction 427
2.6.2 Basic Principles of Enantioselective HPLC 427
2.6.2.1 Thermodynamic Fundamentals of Enantioselective HPLC 429
2.6.2.2 Adsorption and Chiral Recognition 430
2.6.2.3 Differences to Reversed-Phase and Normal-Phase HPLC 433
2.6.2 A Principles for Optimization of Enantioselective HPLC
Separations 433
2.6.3 Selectors and Stationary Phases 433
2.6.4 Method Selection and Optimization 440
I
2.6.4.1 Cellulose and Amylose Derivatives 441
2.6.4.2 Immobilized Cellulose and Amylose Derivatives 443
2.6.4.3 Stationary Phases Derived from Tartaric Acid 444
2.6.4.4 7i-Acidic and 7i-Basic Stationary Phases 444
2.6.4.5 Macrocyclic Selectors, Cyclodextnns, and Antibiotics 446
2.6.4.6 Proteins and Peptides 450
2.6.4.7 Ruthenium Complexes 450
2.6.4.8 Synthetic and Imprinted Polymers 450
2.6.4.9 Metal Complexation and Ligand-Exchange Phases 451
2.6.4.10 Chiral Ion Exchangers 451
2.6.5 Avoiding Errors and Troubleshooting 452
2.6.5.1 Equipment and Columns - Practical Tips 452
2.6.5.2 Detection 454
2.6.5.3 Mistakes Originating from the Analyte 454
2.6.6 Preparative Enantioselective HPLC 454
2.6.6.1 Determination of the Loading Capacity 455
2.6.6.2 Determination of Elution Volumes and Flow Rates 456
2.6.6.3 Enantiomer Separation using Simulated Moving Bed (SMB)
Chromatography 458
2.6.6.3.1 Principles of Simulated Moving Bed Chromatography 458
2.6.6.3.2 Separation of Commercial Active Pharmaceutical Ingredients by
SMB 459
2.6.7 Enantioselective Chromatography by the Addition of
Chiral Additives to the Mobile Phase in HPLC and Capillary
Electrophoresis 461
2.6.8 Determination of Enantiomeric Purity Through the Formation of
Diastereomers 462
2.6.9 Indirect Enantiomer Separation on a Preparative Scale 462
2.6.10 Enantiomer Separations Under Supercritical Fluid Chromatographic
(SFC) Conditions 462
2.6.11 New Chiral Stationary Phases and Information Management
Software 463
2.6.12 Summary 463
References 464
2.7 Miniaturization 467
2.7.1 mLC/NanoLC - Optimization and Troubleshooting 467
Jurgen Maier-Rosenkranz
2.7.1.1 Introduction 467
2.7.1.2 Sensitivity 467
2.7.1.2.1 Influence of Column Length 467
2.7.1.2.2 Influence of Column Internal Diameter 467
2.7.1.2.3 Influence of Stationary Phase 469
2.7.1.3 Robustness 469
I
2.7.1.3.1 System Choice 469
2.7.1.3.2 Capillary Connections 472
2.7.1.3.3 Precautions Against Blocking 477
2.7.1.3.4 Testing for Leakages 478
2.7.1.3.5 Guard Column Switching and Sample Loading Strategies 478
2.7.1.4 Sensitivity/Resolution 483
2.7.1.4.1 Column Dimensions 483
2.7.1.4.2 Packing Materials/Surface Covering 484
2.7.1.4.3 Detectors 484
References 486
2.7.2 Microchip-Based Liquid Chromatography - Techniques and
Possibilities 487
Jorg P. Kutter
2.7.2.1 Introduction 487
2.7.2.2 Techniques 488
2.7.2.2.1 Pressure-Driven Liquid Chromatography (LC) 488
2.7.2.2.2 Open-Channel Electrochromatography (OCEC) 488
2.7.2.2.3 Packed-Bed Electrochromatography 488
2.7'.2.2.4 Microfabricated Chromatographic Beds (Pillar Arrays) 489
2.7.2.2.5 In Situ Polymerized Monolithic Stationary Phases 489
2.7.2.3 Optimization and Possibilities 490
2.7.2.3.1 Separation Performance 490
2.7.2.3.2 Isocratic and Gradient Elution 491
2.7.2.3.3 Tailor-Made Stationary Phases 492
2.7.2.3.4 Sample Pretreatment and More-Dimensional Separations 492
2.7.2.3.5 Issues and Challenges 492
2.7.2.4 Application Examples 493
2.7.2.5 Conclusions and Outlook 496
References 496
2.7.3 Ultra-Performance Liquid Chromatography 498
Uwe D. Neue, Eric S. Crumbach, Marianna Kele,
Jeffrey R. Mazzeo, and Dirk Sievers
2.7.3.1 Introduction 498
2.7.3.2 Isocratic Separations 499
2.7.3.3 Gradient Separations 502
References 505
I
3 Coupling Techniques 507
3.1 Immunochromatographic Techniques 509
Michael C. Weller
3.1.1 Introduction 509
3.1.2 Binding Molecules 509
3.1.3 Immunoassays 511
3.1.4 Immunochromatographic Techniques 511
3.1.4.1 Affinity Enrichment (Affinity SPE) 513
3.1.4.2 "Weak Affinity Chromatography"
(True Affinity Chromatography) 519
3.1.4.3 Biochemical Detectors 520
3.1.5 Examples 522
3.1.5.1 Example 1: Affinity Extraction (Affinity SPE) 522
3.1.5.2 Example 2: "Weak Affinity Chromatography" (WAC) 523
3.1.5.3 Example 3: Biochemical Detection 525
References 525
3.2 Enhanced Characterization and Comprehensive Analyses
by Two-Dimensional Chromatography 527
Peter Kilz
3.2.1 Introduction 527
3.2.2 How Can I Take Advantage? - Experimental Aspects 529
3.2.3 2D Data Presentation and Analysis 533
3.2.4 The State-of-the-Art in 2D Chromatography 535
3.2.5 Summary 539
References 540
3.3 LC/MS - Hints and Recommendations on Optimization and
Troubleshooting 541
Friedrich Mandel
3.3.1 Optimization of the Ionization Process 541
3.3.2 Lost LC/MS Peaks 542
3.3.2.1 Mobile Phase pH at the Edge of the Optimum Range 543
3.3.2.2 Ion-Pairing Agents in the HPLC System 543
3.3.2.3 Ion Suppression by the Sample Matrix or Sample
Contaminants 544
3.3.3 How Clean Should an LC/MS Ion Source Be? 544
3.3.4 Ion Suppression 545
References 549
I
3.4 LC-NMR Coupling 551
Klaus Albert, Manfred Krucker, Karsten Putzbach, and Marc D. Grynbaum
3.4.1 NMR Basics 551
3.4.2 Sensitivity of the NMR Experiment 552
3.4.3 NMR Spectroscopy in Flowing Systems 553
3.4.4 NMR Probes for LC-NMR 553
3.4.5 Practical Realization of Analytical HPLC-NMR and
Capillary-HPLC-NMR 554
3.4.6 Continuous-Flow Measurements 555
3.4.7 Stopped-Flow Measurements 557
3.4.8 Capillary Separations 559
3.4.9 Outlook 560
References 563
4 Computer-Aided Optimization 565
4.1 Computer-Facilitated HPLC Method Development Using DryLab®
Software 567
Lloyd R. Snyder and Loren Wrisley
4.1.1 Introduction 567
4.1.1.1 History 569
4.1.1.2 Theory 570
4.1.2 DryLab Capabilities 570
4.1.2.1 DryLab Operation 570
4.1.2.2 Mode Choices 571
4.1.3 Practical Applications of DryLab® in the Laboratory 572
4.1.4 Conclusions 584
References 585
4.2 ChromSword® Software for Automated and Computer-Assisted
Development of HPLC Methods 587
Sergey Calushko, Vsevolod Tanchuk, Irina Shishkina, Oleg Pylypchenko,
and Wolf Dieter Beinert
4.2.1 Introduction 587
4.2.1.1 Off-Line Mode 587
4.2.1.2 On-line Mode 587
4.2.2 ChromSword® Versions 587
4.2.3 Experimental Set-Up for On-Line Mode 588
4.2.4 Method Development with ChromSword® 588
4.2.4.1 Off-Line Mode (Computer-Assisted Method Development) 588
4.2.4.2 On-Line Mode - Fully Automated Optimization of Isocratic
and Gradient Separations 592
4.2.4.2.1 Software Functions for Automation 597
I
4.2.4.2.2 How Does the System Optimize Separations? 597
4.2.5 Conclusion 600
References 600
4.3 Multifactorial Systematic Method Development and Optimization
in Reversed-Phase HPLC 601
Michael Pfeffer
4.3.1 Introduction and Factorial Viewpoint 601
4.3.2 Strategy for Partially Automated Method Development 603
4.3.3 Comparison of Commercially Available Software Packages with
Regard to Their Contribution to Factorial Method Development 608
4.3.4 Development of a New System for Multifactorial Method
Development 609
4.3.4.1 Selection of Stationary Phases 611
4.3.4.2 Optimizing Methods with HEUREKA 612
4.3.4.3 Evaluation of Data with HEUREKA 618
4.3.5 Conclusion and Outlook 623
References 623
5 User Reports 625
5.1 Nano-LC-MS/MS in Proteomics 627
Heike Schafer, Christiane Lohaus, Helmut E. Meyer, and Katrin Marcus
5.1.1 Proteomics - An Introduction 627
5.1.2 Sample Preparation for Nano-LC 628
5.1.3 Nano-LC 629
5.1.4 On-line LC-ESI-MS/MS Coupling 633
5.1.5 Off-Line LC-MALDI-MS/MS Coupling 635
5.1.5.1 Sample Fractionation 635
5.1.5.2 MALDI-TOF-MS/MS Analyses 635
5.1.6 Data Analysis 637
5.1.7 Application in Practice: Analysis of a-Crystallin A in Mice Lenses 637
References 640
5.2 Verification Methods for Robustness in RP-HPLC 643
Hans Bilke
5.2.1 Introduction 643
5.2.2 Testing Robustness in Analytical RP-HPLC by Means of
Systematic Method Development 643
5.2.3 Robustness Test in Analytical RP-HPLC by Means of Statistical
Experimental Design (DoE) 652
5.2.4 Conclusion 665
References 666
I
5.3 Separation of Complex Sample Mixtures 669
Knut Wagner
5.3.1 Introduction 669
5.3.2 Multidimensional HPLC 670
5.3.3 Techniques for Multidimensional Separations 672
5.3.3.1 Off-Line Technique 672
5.3.3.2 On-Line Technique 672
5.3.4 On-Line Sample Preparation as a Previous Stage of
Multidimensional HPLC 674
5.3.5 Fields of Application of Multidimensional HPLC 675
5.3.5.1 What can be Realized? - A Practical Example 676
5.3.6 Critical Parameters of Multidimensional HPLC 682
References 683
5.4 Evaluation of an Integrated Procedure for the Characterization of
Chemical libraries on the Basis of HPLC-UV/MS/CLND 685
Mario Arangio, Federico R. Sirtori, Katia Marcucci,
Giuseppe Razzano, Maristella Colombo, Roberto Biancardi, and
Vincenzo Rizzo
5.4.1 Introduction 685
5.4.2 Materials and Methods 686
5.4.2.1 Instrumentation 686
5.4.2.2 Chemicals and Consumables 686
5.4.2.3 High-Throughput Platform (HTP1) Method Set-up 688
5.4.2.4 Chromatographic Conditions 688
5.4.2.5 Mass Spectrometer and CLND Conditions 689
5.4.2.6 Data Processing and Reporting 689
5.4.2.7 Multilinear Regression Analysis for the Derivation of CLND
Response Factors 690
5.4.3 Results and Discussion 691
5.4.3.1 Liquid Chromatography and UV Detection 691
5.4.3.2 Mass Spectrometric Method Development 692
5.4.3.3 CLND Set-Up 693
5.4.3.4 Validation with Commercial Standards 693
5.4.3.5 Validation with Proprietary Compounds 695
5.4.4 Conclusions 699
References 700
Appendix 703
Subject Index 729 |
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| id | DE-604.BV021287237 |
| illustrated | Illustrated |
| index_date | 2024-07-02T13:48:51Z |
| indexdate | 2024-07-09T20:34:46Z |
| institution | BVB |
| isbn | 352731377X 9783527313778 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-014608157 |
| oclc_num | 181462802 |
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| physical | XXXIII, 753 S. 300 schw.-w. Ill., 40 farb. Ill. 240 mm x 170 mm |
| publishDate | 2006 |
| publishDateSearch | 2006 |
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| publisher | WILEY-VCH |
| record_format | marc |
| spelling | HPLC made to measure a practical handbook for optimization ed. by Stavros Kromidas Weinheim, Bergstr [u.a.] WILEY-VCH 2006 XXXIII, 753 S. 300 schw.-w. Ill., 40 farb. Ill. 240 mm x 170 mm txt rdacontent n rdamedia nc rdacarrier High performance liquid chromatography High performance liquid chromatography Methodology HPLC (DE-588)4072641-1 gnd rswk-swf HPLC (DE-588)4072641-1 s DE-604 Kromidas, Stavros 1954- (DE-588)122124685 edt HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014608157&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | HPLC made to measure a practical handbook for optimization High performance liquid chromatography High performance liquid chromatography Methodology HPLC (DE-588)4072641-1 gnd |
| subject_GND | (DE-588)4072641-1 |
| title | HPLC made to measure a practical handbook for optimization |
| title_auth | HPLC made to measure a practical handbook for optimization |
| title_exact_search | HPLC made to measure a practical handbook for optimization |
| title_exact_search_txtP | HPLC made to measure a practical handbook for optimization |
| title_full | HPLC made to measure a practical handbook for optimization ed. by Stavros Kromidas |
| title_fullStr | HPLC made to measure a practical handbook for optimization ed. by Stavros Kromidas |
| title_full_unstemmed | HPLC made to measure a practical handbook for optimization ed. by Stavros Kromidas |
| title_short | HPLC made to measure |
| title_sort | hplc made to measure a practical handbook for optimization |
| title_sub | a practical handbook for optimization |
| topic | High performance liquid chromatography High performance liquid chromatography Methodology HPLC (DE-588)4072641-1 gnd |
| topic_facet | High performance liquid chromatography High performance liquid chromatography Methodology HPLC |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014608157&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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