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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 RPChromatography
Stavros Kromidas 		3
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? 11
1.1.3.4 How to Change k, , 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
1.1.8 Economics in HPLC (“Cheaper Separation”) 48
1.1.9 Final Remarks and Outlook 51
References 57
1.2 Fast Gradient Separations
Uwe D. Neue, Yung-Fong Cheng, and Ziling Lu 		59
1.2.1 Introduction 59
1.2.2 Main 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
Uwe D. Neue, Alberto Méndez, KimVan Tran, and Diane M. Diehl 		71
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 pK and 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
1.4 Selecting the Correct pH Value for HPLC
Michael McBrien 		89
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 pKa 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 1,4-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
Hans-Joachim Kuss 		105
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 110
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
Stefan Schömer 		117
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
Stavros Kromidas 		151
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
2.1.1.4 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
Uwe D. Neue, Bonnie A. Alden, and Pamela C. Iraneta 		254
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
2.1.3 The Use of Principal Component Analysis for the Characterization of ReversedPhase Liquid Chromatographic Stationary Phases
Melvin R. Euerby and Patrik Petersson 		264
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 Perfluorinated 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
2.1.4 Chemometrics – A Powerful Tool for Handling a Large Number of Data
Cinzia Stella and Jean-Luc Veuthey 		280
2.1.4.1 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
2.1.4.4.1 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?
Frank Steiner 		296
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
Lloyd R. Snyder and John W. Dolan 		321
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 HighResolution MagicAngle Spinning NMR Spectroscopy
Urban Skogsberg, Heidi Händel, Norbert Welsch, and Klaus Albert 		334
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 1H HR/MAS T1 Relaxation Measurements 343
2.1.7.5 Where do the Interactions Take Place? 345
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
Veronika R. Meyer 		349
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
Peter Kilz 		359
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
Milena Quaglia, Egidijus Machtejevas, Tom Hennessy, and Klaus K. Unger 		383
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
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 Müller
2.5.1 Introduction to Resin 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
Markus Juza 		427
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
2.6.2.4 Principles for Optimization of Enantioselective HPLC Separations 433
2.6.3 Selectors and Stationary Phases 433
2.6.4 Method Selection and Optimization 440
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 -Acidic and -Basic Stationary Phases 444
2.6.4.5 Macrocyclic Selectors, Cyclodextrins, 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
Jürgen Maier-Rosenkranz 		467
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
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
Jörg P. Kutter 		487
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
Uwe D. Neue, Eric S. Grumbach, Marianna Kele, Jeffrey R. Mazzeo, and Dirk Sievers 		498
2.7.3.1 Introduction 498
2.7.3.2 Isocratic Separations 499
2.7.3.3 Gradient Separations 502
References 505
3 Coupling Techniques 507
3.1 Immunochromatographic Techniques
Michael G. Weller 		509
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
Peter Kilz 		527
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
Friedrich Mandel 		541
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
3.4 LC-NMR Coupling
Klaus Albert, Manfred Krucker, Karsten Putzbach, and Marc D. Grynbaum 		551
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
Lloyd R. Snyder and Loren Wrisley 		567
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
Sergey Galushko, Vsevolod Tanchuk, Irina Shishkina, Oleg Pylypchenko, and Wolf-Dieter Beinert 		587
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
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
Michael Pfeffer 		601
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
Heike Schäfer, Christiane Lohaus, Helmut E. Meyer, and Katrin Marcus 		627
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 -Crystallin A in Mice Lenses 637
References 640
5.2 Verification Methods for Robustness in RP-HPLC
Hans Bilke 		643
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
5.3 Separation of Complex Sample Mixtures
Knut Wagner 		669
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
Mario Arangio, Federico R. Sirtori, Katia Marcucci, Giuseppe Razzano, Maristella Colombo, Roberto Biancardi, and Vincenzo Rizzo 		685
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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