High-Performance Gradient Elution
Wiley-Interscience (Verlag)
978-0-471-70646-5 (ISBN)
Gradient elution demystified
Of the various ways in which chromatography is applied today, few have been as misunderstood as the technique of gradient elution, which presents many challenges compared to isocratic separation. When properly explained, however, gradient elution can be less difficult to understand and much easier to use than often assumed.
Written by two well-known authorities in liquid chromatography, High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model takes the mystery out of the practice of gradient elution and helps remove barriers to the practical application of this important separation technique. The book presents a systematic approach to the current understanding of gradient elution, describing theory, methodology, and applications across many of the fields that use liquid chromatography as a primary analytical tool.
This up-to-date, practical, and comprehensive treatment of gradient elution:
* Provides specific, step-by-step recommendations for developing a gradient separation for any sample
* Describes the best approach for troubleshooting problems with gradient methods
* Guides the reader on the equipment used for gradient elution
* Lists which conditions should be varied first during method development, and explains how to interpret scouting gradients
* Explains how to avoid problems in transferring gradient methods
With a focus on the use of linear solvent strength (LSS) theory for predicting gradient LC behavior and separations by reversed-phase HPLC, High-Performance Gradient Elution gives every chromatographer access to this useful tool.
LLOYD R. SNYDER, PHD, is a Principal at LC Resources in Walnut Creek, California. He is the author or coauthor of several books including An Introduction to Separation Science, Introduction to Modern Liquid Chromatography, Second Edition, and the bestselling Practical HPLC Method Development, Second Edition, all published by Wiley. JOHN W. DOLAN, PHD, is a Principal at LC Resources. He is author of the popular " LC Troubleshooting" column in LCGC Magazine and coauthor with Lloyd Snyder of Troubleshooting LC Systems.
Preface xv
Glossary of Symbols and Terms xxi
1 Introduction 1
1.1 The “General Elution Problem” and the Need for Gradient Elution 1
1.2 Other Reasons for the Use of Gradient Elution 4
1.3 Gradient Shape 7
1.4 Similarity of Isocratic and Gradient Elution 10
1.4.1 Gradient and Isocratic Elution Compared 10
1.4.2 The Linear-Solvent-Strength Model 13
1.5 Computer Simulation 18
1.6 Sample Classification 19
1.6.1 Sample Compounds of Related Structure (“Regular Samples”) 19
1.6.2 Sample Compounds of Unrelated Structure (“Irregular” Samples) 19
2 Gradient Elution Fundamentals 23
2.1 Isocratic Separation 23
2.1.1 Retention 23
2.1.2 Peak Width and Plate Number 24
2.1.3 Resolution 25
2.1.4 Role of Separation Conditions 27
2.1.4.1 Optimizing Retention [Term a of Equation (2.7)] 27
2.1.4.2 Optimizing Selectivity a [Term b of Equation (2.7)] 28
2.1.4.3 Optimizing the Column Plate Number N [Term c of Equation (2.7)] 28
2.2 Gradient Separation 31
2.2.1 Retention 32
2.2.1.1 Gradient and Isocratic Separation Compared for “Corresponding” Conditions 34
2.2.2 Peak Width 38
2.2.3 Resolution 39
2.2.3.1 Resolution as a Function of Values of S for Two Adjacent Peaks (“Irregular” Samples) 42
2.2.3.2 Using Gradient Elution to Predict Isocratic Separation 45
2.2.4 Sample Complexity and Peak Capacity 47
2.3 Effect of Gradient Conditions on Separation 49
2.3.1 Gradient Steepness b: Change in Gradient Time 50
2.3.2 Gradient Steepness b: Change in Column Length or Diameter 51
2.3.3 Gradient Steepness b: Change in Flow Rate 55
2.3.4 Gradient Range ∆Φ: Change in Initial Percentage B (Φ0) 58
2.3.5 Gradient Range ∆Φ: Change in Final Percentage B (Φf) 60
2.3.6 Effect of a Gradient Delay 63
2.3.6.1 Equipment Dwell Volume 66
2.3.7 Effect of Gradient Shape (Nonlinear Gradients) 67
2.3.8 Overview of the Effect of Gradient Conditions on the Chromatogram 71
2.4 Related Topics 72
2.4.1 Nonideal Retention in Gradient Elution 72
2.4.2 Gradient Elution Misconceptions 72
3 Method Development 74
3.1 A Systematic Approach to Method Development 74
3.1.1 Separation Goals (Step 1 of Fig. 3.1) 75
3.1.2 Nature of the Sample (Step 2 of Fig. 3.1) 78
3.1.3 Initial Experimental Conditions 79
3.1.4 Repeatable Results 79
3.1.5 Computer Simulation: Yes or No? 80
3.1.6 Sample Preparation (Pretreatment) 81
3.2 Initial Experiments 81
3.2.1 Interpreting the Initial Chromatogram (Step 3 of Fig. 3.1) 85
3.2.1.1 “Trimming” a Gradient Chromatogram 87
3.2.1.2 Possible Problems 88
3.3 Developing a Gradient Separation: Resolution versus Conditions 90
3.3.1 Optimizing Gradient Retention k* (Step 4 of Fig. 3.1) 92
3.3.2 Optimizing Gradient Selectivity a* (Step 5 of Fig. 3.1) 92
3.3.3 Optimizing the Gradient Range (Step 6 of Fig. 3.1) 95
3.3.3.1 Changes in Selectivity as a Result of Change in k* 96
3.3.4 Segmented (Nonlinear) Gradients (Step 6 of Fig. 3.1 Continued) 100
3.3.5 Optimizing the Column Plate Number N* (Step 7 of Fig. 3.1) 102
3.3.6 Column Equilibration Between Successive Sample Injections 106
3.3.7 Fast Separations 106
3.4 Computer Simulation 108
3.4.1 Quantitative Predictions and Resolution Maps 109
3.4.2 Gradient Optimization 111
3.4.3 Changes in Column Conditions 112
3.4.4 Separation of “Regular” Samples 114
3.4.5 Other Features 115
3.4.5.1 Isocratic Prediction (5 in Table 3.5) 115
3.4.5.2 Designated Peak Selection (6 in Table 3.5) 117
3.4.5.3 Change in Other Conditions (7 in Table 3.5) 117
3.4.5.4 Computer-Selection of the Best Multisegment Gradient (8 in Table 3.5) 117
3.4.5.5 “Two-Run” Procedures for the Improvement of Sample Resolution 119
3.4.6 Accuracy of Computer Simulation 119
3.4.7 Peak Tracking 119
3.5 Method Reproducibility and Related Topics 120
3.5.1 Method Development 121
3.5.2 Routine Analysis 122
3.5.3 Change in Column Volume 123
3.6 Additional Means for an Increase in Separation Selectivity 124
3.7 Orthogonal Separations 127
3.7.1 Two-Dimensional Separations 128
4 Gradient Equipment 133
4.1 Gradient System Design 133
4.1.1 High-Pressure vs Low-Pressure Mixing 133
4.1.2 Tradeoffs 135
4.1.2.1 Dwell Volume 135
4.1.2.2 Degassing 136
4.1.2.3 Accuracy 137
4.1.2.4 Solvent Volume Changes and Compressibility 137
4.1.2.5 Flexibility 139
4.1.2.6 Independent Module Use 140
4.1.3 Other System Components 140
4.1.3.1 Autosampler 140
4.1.3.2 Column 140
4.1.3.3 Detector 141
4.1.3.4 Data System 141
4.1.3.5 Extra-Column Volume 142
4.2 General Considerations in System Selection 142
4.2.1 Which Vendor? 143
4.2.2 High-Pressure or Low-Pressure Mixing? 144
4.2.3 Who Will Fix It? 144
4.2.4 Special Applications 144
4.3 Measuring Gradient System Performance 145
4.3.1 Gradient Performance Test 146
4.3.1.1 Gradient Linearity 146
4.3.1.2 Dwell Volume Determination 147
4.3.1.3 Gradient Step-Test 147
4.3.1.4 Gradient Proportioning Valve Test 148
4.3.2 Additional System Checks 149
4.3.2.1 Flow Rate Check 149
4.3.2.2 Pressure Bleed-Down 150
4.3.2.3 Retention Reproducibility 150
4.3.2.4 Peak Area Reproducibility 151
4.4 Dwell Volume Considerations 151
5 Separation Artifacts and Troubleshooting 153
5.1 Avoiding Problems 154
5.1.1 Equipment Checkout 157
5.1.1.1 Installation Qualification, Operational Qualification, and Performance Qualification 157
5.1.2 Dwell Volume 158
5.1.3 Blank Gradient 158
5.1.4 Suggestions for Routine Applications 158
5.1.4.1 Reagent Quality 159
5.1.4.2 System Cleanliness 159
5.1.4.3 Degassing 159
5.1.4.4 Dedicated Columns 159
5.1.4.5 Equilibration 159
5.1.4.6 Priming Injections 159
5.1.4.7 Ignore the First Injection 160
5.1.4.8 System Suitability 160
5.1.4.9 Standards and Calibrators 160
5.1.5 Method Development 160
5.1.5.1 Use a Clean and Stable Column 160
5.1.5.2 Use Reasonable Mobile Phase Conditions 161
5.1.5.3 Clean Samples 162
5.1.5.4 Reproducible Runs 162
5.1.5.5 Sufficient Equilibration 162
5.1.5.6 Reference Conditions 162
5.1.5.7 Additional Tests 162
5.2 Method Transfer 163
5.2.1 Compensating for Dwell Volume Differences 163
5.2.1.1 Injection Delay 163
5.2.1.2 Adjustment of the Initial Isocratic Hold 164
5.2.1.3 Use of Maximum-Dwell-Volume Methods 165
5.2.1.4 Adjustment of Initial Percentage B 165
5.2.2 Other Sources of Method Transfer Problems 168
5.2.2.1 Gradient Shape 169
5.2.2.2 Gradient Rounding 169
5.2.2.3 Inter-Run Equilibration 169
5.2.2.4 Column Size 169
5.2.2.5 Column Temperature 169
5.2.2.6 Interpretation of Method Instructions 170
5.3 Column Equilibration 170
5.3.1 Primary Effects 171
5.3.2 Slow Equilibration of Column and Mobile Phase 173
5.3.3 Practical Considerations and Recommendations 174
5.4 Separation Artifacts 175
5.4.1 Baseline Drift 176
5.4.2 Baseline Noise 179
5.4.2.1 Baseline Noise: A Case Study 180
5.4.3 Peaks in a Blank Gradient 182
5.4.3.1 Mobile Phase Water or Organic Solvent Impurities 182
5.4.3.2 Other Sources of Background Peaks 185
5.4.4 Extra Peaks for Injected Samples 185
5.4.4.1 t0 Peaks 185
5.4.4.2 Air Peaks 186
5.4.4.3 Late Peaks 187
5.4.5 Peak Shape Problems 188
5.4.5.1 Tailing and Fronting 188
5.4.5.2 Excess Peak Broadening 188
5.4.5.3 Split Peaks 190
5.4.5.4 Injection Conditions 191
5.4.5.5 Sample Decomposition 193
5.5 Troubleshooting 195
5.5.1 Problem Isolation 196
5.5.2 Troubleshooting and Maintenance Suggestions 197
5.5.2.1 Removing Air from the Pump 197
5.5.2.2 Solvent Siphon Test 197
5.5.2.3 Premixing to Improve Retention Reproducibility in Shallow Gradients 198
5.5.2.4 Cleaning and Handling Check-Valves 199
5.5.2.5 Replacing Pump Seals and Pistons 200
5.5.2.6 Leak Detection 200
5.5.2.7 Repairing Fitting Leaks 200
5.5.2.8 Cleaning Glassware 201
5.5.2.9 For Best Results with TFA 201
5.5.2.10 Improved Water Purity 201
5.5.2.11 Isolating Carryover Problems 203
5.5.2.12 Troubleshooting Rules of Thumb 204
5.5.3 Gradient Performance Test Failures 206
5.5.3.1 Linearity (4.3.1.1) 206
5.5.3.2 Step Test (4.3.1.3) 206
5.5.3.3 Gradient-Proportioning-Valve Test (4.3.1.4) 209
5.5.3.4 Flow Rate (4.3.2.1) 211
5.5.3.5 Pressure Bleed-Down (4.3.2.2) 212
5.5.3.6 Retention Reproducibility (4.3.2.3) 212
5.5.3.7 Peak Area Reproducibility (4.3.2.4) 213
5.5.4 Troubleshooting Case Studies 213
5.5.4.1 Retention Variation – Case Study 1 213
5.5.4.2 Retention Variation – Case Study 2 218
5.5.4.3 Contaminated Reagents – Case Study 3 220
5.5.4.4 Baseline and Retention Problems – Case Study 4 224
6 Separation of Large Molecules 228
6.1 General Considerations 228
6.1.1 Values of S for Large Molecules 229
6.1.2 Values of N* for Large Molecules 235
6.1.3 Conformational State 236
6.1.4 Homo-Oligomeric Samples 238
6.1.4.1 Separation of Large Homopolymers 241
6.1.5 Proposed Models for the Gradient Separation of Large Molecules 242
6.1.5.2 “Critical Elution Behavior”: Biopolymers 246
6.1.5.3 Measurement of LSS Parameters for Large Molecules 247
6.2 Biomolecules 248
6.2.1 Peptides and Proteins 248
6.2.1.1 Sample Characteristics 249
6.2.1.2 Conditions for an Initial Gradient Run 249
6.2.1.3 Method Development 253
6.2.1.4 Segmented Gradients 259
6.2.2 Other Separation Modes and Samples 261
6.2.2.1 Hydrophobic Interaction Chromatography 262
6.2.2.2 Ion Exchange Chromatography 264
6.2.2.3 Hydrophilic Interaction Chromatography 266
6.2.2.4 Separation of Viruses 267
6.2.3 Separation Problems 271
6.2.4 Fast Separations of Peptides and Proteins 274
6.2.5 Two-Dimensional Separations of Peptides and Proteins 274
6.3 Synthetic Polymers 275
6.3.1 Determination of Molecular Weight Distribution 277
6.3.2 Determination of Chemical Composition 278
7 Preparative Separations 283
7.1 Introduction 283
7.1.1 Equipment for Preparative Separation 285
7.2 Isocratic Separation 286
7.2.1 Touching-Peak Separation 287
7.2.1.1 Theory 287
7.2.1.2 Column Saturation Capacity 289
7.2.1.3 Sample-Volume Overload 292
7.2.2 Method Development for Isocratic Touching-Peak Separation 292
7.2.2.1 Optimizing Separation Conditions 295
7.2.2.2 Selecting a Sample Weight for Touching-Peak Separation 297
7.2.2.3 Scale-Up 298
7.2.2.4 Sample Solubility 300
7.2.3 Beyond Touching-Peak Separation 301
7.3 Gradient Separation 302
7.3.1 Touching-Peak Separation 306
7.3.2 Method Development for Gradient Touching-Peak Separation 306
7.3.2.1 Step Gradients 311
7.3.3 Sample-Volume Overload 312
7.3.4 Possible Complications of Simple Touching-Peak Theory and Their Practical Impact 312
7.3.4.1 Crossing Isotherms 313
7.3.4.2 Unequal Values of S 314
7.4 Severely Overloaded Separation 315
7.4.1 Is Gradient Elution Necessary? 316
7.4.2 Displacement Effects 317
7.4.3 Method Development 317
7.4.4 Separations of Peptides and Small Proteins 318
7.4.5 Column Efficiency 320
7.4.6 Production-Scale Separation 320
8 other Applications of Gradient Elution 323
8.1 Gradient Elution for LC-MS 324
8.1.1 Application Areas 325
8.1.2 Requirements for LC-MS 325
8.1.3 Basic LC-MS Concepts 326
8.1.3.1 The Interface 326
8.1.3.2 Column Configurations 328
8.1.3.3 Quadrupoles and Ion Traps 328
8.1.4 LC-UV vs LC-MS Gradient Conditions 330
8.1.5 Method Development for LC-MS 332
8.1.5.1 Define Separation Goals (Step 1, Table 8.2) 332
8.1.5.2 Collect Information on Sample (Step 2, Table 8.2) 334
8.1.5.3 Carry Out Initial Separation (Run 1, Step 3, Table 8.2) 339
8.1.5.4 Optimize Gradient Retention k* (Step 4, Table 8.2) 339
8.1.5.5 Optimize Selectivity a* (Step 5, Table 8.2) 339
8.1.5.6 Adjust Gradient Range and Shape (Step 6, Table 8.2) 340
8.1.5.7 Vary Column Conditions (Step 7, Table 8.2) 341
8.1.5.8 Determine Inter-Run Column Equilibration (Step 8, Table 8.2) 341
8.1.6 Special Challenges for LC-MS 341
8.1.6.1 Dwell Volume 342
8.1.6.2 Gradient Distortion 342
8.1.6.3 Ion Suppression 343
8.1.6.4 Co-Eluting Compounds 345
8.1.6.5 Resolution Requirements 346
8.1.6.6 Use of Computer Simulation Software 347
8.1.6.7 Isocratic Methods 347
8.1.6.8 Throughput Enhancement 347
8.2 Ion-Exchange Chromatography 349
8.2.1 Theory 349
8.2.2 Dependence of Separation on Gradient Conditions 356
8.2.3 Method Development for Gradient IEC 356
8.2.3.1 Choice of Initial Conditions 356
8.2.3.2 Improving the Separation 357
8.3 Normal-Phase Chromatography 359
8.3.1 Theory 359
8.3.2 Method Development for Gradient NPC 360
8.3.3 Hydrophilic Interaction Chromatography 361
8.3.3.1 Method Development for Gradient HILIC 361
8.4 Ternary- or Quaternary-Solvent Gradients 365
9 Theory and Derivations 370
9.1 The Linear Solvent Strength Model 370
9.1.1 Retention 372
9.1.1.1 Gradient and Isocratic Retention Compared 374
9.1.1.2 Small Values of k 0 376
9.1.2 Peak Width 378
9.1.2.1 Gradient Compression 380
9.1.3 Selectivity and Resolution 383
9.1.4 Advantages of LSS Behavior 385
9.2 Second-Order Effects 386
9.2.1 Assumptions About Φ and k 386
9.2.1.1 Incomplete Column Equilibration 386
9.2.1.2 Solvent Demixing 391
9.2.1.3 Nonlinear Plots of log k vs Φ 393
9.2.1.4 Dependence of V m on Φ 393
9.2.2 Nonideal Equipment 393
9.3. Accuracy of Gradient Elution Predictions 397
9.3.1 Gradient Retention Time 397
9.3.1.1 Confirmation of Equation (9.2) 397
9.3.1.2 Computer Simulation 399
9.3.2 Peak Width Predictions 399
9.3.3 Measurement of Values of S and log k 0 400
9.4 Values of S 401
9.4.1 Estimating Values of S from Solute Properties and Experimental Conditions 402
9.5 Values of N in Gradient Elution 404
Appendix I The Constant-S Approximation In Gradient Elution 414
Appendix II Estimation of Conditions for Isocratic Elution, Based on An Initial Gradient Run 416
Appendix III Characterization of Reversed-phase Columns for Selectivity and Peak Tailing 418
Appendix IV Solvent Properties Relevant to the Use of Gradient Elution 434
Appendix V Theory Of Preparative Separation 436
Appendix Vi Further Information On Virus Chromatography 445
Index 450
| Erscheint lt. Verlag | 2.1.2007 |
|---|---|
| Zusatzinfo | Charts: 5 B&W, 0 Color; Drawings: 9 B&W, 0 Color; Tables: 33 B&W, 0 Color; Graphs: 192 B&W, 0 Color |
| Sprache | englisch |
| Maße | 160 x 236 mm |
| Gewicht | 862 g |
| Themenwelt | Naturwissenschaften ► Chemie ► Analytische Chemie |
| ISBN-10 | 0-471-70646-9 / 0471706469 |
| ISBN-13 | 978-0-471-70646-5 / 9780471706465 |
| Zustand | Neuware |
| Haben Sie eine Frage zum Produkt? |
aus dem Bereich
