High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model
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...
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Gradient elution demystifiedOf 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 sampleDescribes the best approach for troubleshooting problems with gradient methodsGuides the reader on the equipment used for gradient elutionLists which conditions should be varied first during method development, and explains how to interpret scouting gradientsExplains how to avoid problems in transferring gradient methodsWith 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.
Preface xvGlossary of Symbols and Terms xxiIntroduction 1The "General Elution Problem" and the Need for Gradient Elution 1Other Reasons for the Use of Gradient Elution 4Gradient Shape 7Similarity of Isocratic and Gradient Elution 10Gradient and Isocratic Elution Compared 10The Linear-Solvent-Strength Model 13Computer Simulation 18Sample Classification 19Sample Compounds of Related Structure ("Regular Samples") 19Sample Compounds of Unrelated Structure ("Irregular" Samples) 19Gradient Elution Fundamentals 23Isocratic Separation 23Retention 23Peak Width and Plate Number 24Resolution 25Role of Separation Conditions 27Optimizing Retention [Term a of Equation (2.7)] 27Optimizing Selectivity [alpha] [Term b of Equation (2.7)] 28Optimizing the Column Plate Number N [Term c of Equation (2.7)] 28Gradient Separation 31Retention 32Gradient and Isocratic Separation Compared for "Corresponding" Conditions 34Peak Width 38Resolution 39Resolution as a Function of Values of S for Two Adjacent Peaks ("Irregular" Samples) 42Using Gradient Elution to Predict Isocratic Separation 45Sample Complexity and Peak Capacity 47Effect of Gradient Conditions on Separation 49Gradient Steepness b: Change in Gradient Time 50Gradient Steepness b: Change in Column Length or Diameter 51Gradient Steepness b: Change in Flow Rate 55Gradient Range [delta phi]: Change in Initial Percentage B ([phi subscript 0]) 58Gradient Range [delta phi]: Change in Final Percentage B ([delta subscript f]) 60Effect of a Gradient Delay 63Equipment Dwell Volume 66Effect of Gradient Shape (Nonlinear Gradients) 67Overview of the Effect of Gradient Conditions on the Chromatogram 71Related Topics 72Nonideal Retention in Gradient Elution 72Gradient Elution Misconceptions 72Method Development 74A Systematic Approach to Method Development 74Separation Goals (Step 1 of Fig. 3.1) 75Nature of the Sample (Step 2 of Fig. 3.1) 78Initial Experimental Conditions 79Repeatable Results 79Computer Simulation: Yes or No? 80Sample Preparation (Pretreatment) 81Initial Experiments 81Interpreting the Initial Chromatogram (Step 3 of Fig. 3.1) 85"Trimming" a Gradient Chromatogram 87Possible Problems 88Developing a Gradient Separation: Resolution versus Conditions 90Optimizing Gradient Retention k* (Step 4 of Fig. 3.1) 92Optimizing Gradient Selectivity [alpha]* (Step 5 of Fig. 3.1) 92Optimizing the Gradient Range (Step 6 of Fig. 3.1) 95Changes in Selectivity as a Result of Change in k* 96Segmented (Nonlinear) Gradients (Step 6 of Fig. 3.1 Continued) 100Optimizing the Column Plate Number N* (Step 7 of Fig. 3.1) 102Column Equilibration Between Successive Sample Injections 106Fast Separations 106Computer Simulation 108Quantitative Predictions and Resolution Maps 109Gradient Optimization 111Changes in Column Conditions 112Separation of "Regular" Samples 114Other Features 115Isocratic Prediction (5 in Table 3.5) 115Designated Peak Selection (6 in Table 3.5) 117Change in Other Conditions (7 in Table 3.5) 117Computer-Selection of the Best Multisegment Gradient (8 in Table 3.5) 117"Two-Run" Procedures for the Improvement of Sample Resolution 119Accuracy of Computer Simulation 119Peak Tracking 119Method Reproducibility and Related Topics 120Method Development 121Routine Analysis 122Change in Column Volume 123Additional Means for an Increase in Separation Selectivity 124Orthogonal Separations 127Two-Dimensional Separations 128Gradient Equipment 133Gradient System Design 133High-Pressure vs Low-Pressure Mixing 133Tradeoffs 135Dwell Volume 135Degassing 136Accuracy 137Solvent Volume Changes and Compressibility 137Flexibility 139Independent Module Use 140Other System Components 140Autosampler 140Column 140Detector 141Data System 141Extra-Column Volume 142General Considerations in System Selection 142Which Vendor? 143High-Pressure or Low-Pressure Mixing? 144Who Will Fix It? 144Special Applications 144Measuring Gradient System Performance 145Gradient Performance Test 146Gradient Linearity 146Dwell Volume Determination 147Gradient Step-Test 147Gradient Proportioning Valve Test 148Additional System Checks 149Flow Rate Check 149Pressure Bleed-Down 150Retention Reproducibility 150Peak Area Reproducibility 151Dwell Volume Considerations 151Separation Artifacts and Troubleshooting 153Avoiding Problems 154Equipment Checkout 157Installation Qualification, Operational Qualification, and Performance Qualification 157Dwell Volume 158Blank Gradient 158Suggestions for Routine Applications 158Reagent Quality 159System Cleanliness 159Degassing 159Dedicated Columns 159Equilibration 159Priming Injections 159Ignore the First Injection 160System Suitability 160Standards and Calibrators 160Method Development 160Use a Clean and Stable Column 160Use Reasonable Mobile Phase Conditions 161Clean Samples 162Reproducible Runs 162Sufficient Equilibration 162Reference Conditions 162Additional Tests 162Method Transfer 163Compensating for Dwell Volume Differences 163Injection Delay 163Adjustment of the Initial Isocratic Hold 164Use of Maximum-Dwell-Volume Methods 165Adjustment of Initial Percentage B 165Other Sources of Method Transfer Problems 168Gradient Shape 169Gradient Rounding 169Inter-Run Equilibration 169Column Size 169Column Temperature 169Interpretation of Method Instructions 170Column Equilibration 170Primary Effects 171Slow Equilibration of Column and Mobile Phase 173Practical Considerations and Recommendations 174Separation Artifacts 175Baseline Drift 176Baseline Noise 179Baseline Noise: A Case Study 180Peaks in a Blank Gradient 182Mobile Phase Water or Organic Solvent Impurities 182Other Sources of Background Peaks 185Extra Peaks for Injected Samples 185t[subscript 0] Peaks 185Air Peaks 186Late Peaks 187Peak Shape Problems 188Tailing and Fronting 188Excess Peak Broadening 188Split Peaks 190Injection Conditions 191Sample Decomposition 193Troubleshooting 195Problem Isolation 196Troubleshooting and Maintenance Suggestions 197Removing Air from the Pump 197Solvent Siphon Test 197Premixing to Improve Retention Reproducibility in Shallow Gradients 198Cleaning and Handling Check-Valves 199Replacing Pump Seals and Pistons 200Leak Detection 200Repairing Fitting Leaks 200Cleaning Glassware 201For Best Results with TFA 201Improved Water Purity 201Isolating Carryover Problems 203Troubleshooting Rules of Thumb 204Gradient Performance Test Failures 206Linearity (4.3.1.1) 206Step Test (4.3.1.3) 206Gradient-Proportioning-Valve Test (4.3.1.4) 209Flow Rate (4.3.2.1) 211Pressure Bleed-Down (4.3.2.2) 212Retention Reproducibility (4.3.2.3) 212Peak Area Reproducibility (4.3.2.4) 213Troubleshooting Case Studies 213Retention Variation - Case Study 1 213Retention Variation - Case Study 2 218Contaminated Reagents - Case Study 3 220Baseline and Retention Problems - Case Study 4 224Separation of Large Molecules 228General Considerations 228Values of S for Large Molecules 229Values of N* for Large Molecules 235Conformational State 236Homo-Oligomeric Samples 238Separation of Large Homopolymers 241Proposed Models for the Gradient Separation of Large Molecules 242"Critical Elution Behavior": Biopolymers 246Measurement of LSS Parameters for Large Molecules 247Biomolecules 248Peptides and Proteins 248Sample Characteristics 249Conditions for an Initial Gradient Run 249Method Development 253Segmented Gradients 259Other Separation Modes and Samples 261Hydrophobic Interaction Chromatography 262Ion Exchange Chromatography 264Hydrophilic Interaction Chromatography 266Separation of Viruses 267Separation Problems 271Fast Separations of Peptides and Proteins 274Two-Dimensional Separations of Peptides and Proteins 274Synthetic Polymers 275Determination of Molecular Weight Distribution 277Determination of Chemical Composition 278Preparative Separations 283Introduction 283Equipment for Preparative Separation 285Isocratic Separation 286Touching-Peak Separation 287Theory 287Column Saturation Capacity 289Sample-Volume Overload 292Method Development for Isocratic Touching-Peak Separation 292Optimizing Separation Conditions 295Selecting a Sample Weight for Touching-Peak Separation 297Scale-Up 298Sample Solubility 300Beyond Touching-Peak Separation 301Gradient Separation 302Touching-Peak Separation 306Method Development for Gradient Touching-Peak Separation 306Step Gradients 311Sample-Volume Overload 312Possible Complications of Simple Touching-Peak Theory and Their Practical Impact 312Crossing Isotherms 313Unequal Values of S 314Severely Overloaded Separation 315Is Gradient Elution Necessary? 316Displacement Effects 317Method Development 317Separations of Peptides and Small Proteins 318Column Efficiency 320Production-Scale Separation 320Other Applications of Gradient Elution 323Gradient Elution for LC-MS 324Application Areas 325Requirements for LC-MS 325Basic LC-MS Concepts 326The Interface 326Column Configurations 328Quadrupoles and Ion Traps 328LC-UV vs LC-MS Gradient Conditions 330Method Development for LC-MS 332Define Separation Goals (Step 1, Table 8.2) 332Collect Information on Sample (Step 2, Table 8.2) 334Carry Out Initial Separation (Run 1, Step 3, Table 8.2) 339Optimize Gradient Retention k* (Step 4, Table 8.2) 339Optimize Selectivity [alpha]* (Step 5, Table 8.2) 339Adjust Gradient Range and Shape (Step 6, Table 8.2) 340Vary Column Conditions (Step 7, Table 8.2) 341Determine Inter-Run Column Equilibration (Step 8, Table 8.2) 341Special Challenges for LC-MS 341Dwell Volume 342Gradient Distortion 342Ion Suppression 343Co-Eluting Compounds 345Resolution Requirements 346Use of Computer Simulation Software 347Isocratic Methods 347Throughput Enhancement 347Ion-Exchange Chromatography 349Theory 349Dependence of Separation on Gradient Conditions 356Method Development for Gradient IEC 356Choice of Initial Conditions 356Improving the Separation 357Normal-Phase Chromatography 359Theory 359Method Development for Gradient NPC 360Hydrophilic Interaction Chromatography 361Method Development for Gradient HILIC 361Ternary- or Quaternary-Solvent Gradients 365Theory and Derivations 370The Linear Solvent Strength Model 370Retention 372Gradient and Isocratic Retention Compared 374Small Values of k[subscript 0] 376Peak Width 378Gradient Compression 380Selectivity and Resolution 383Advantages of LSS Behavior 385Second-Order Effects 386Assumptions About [phi] and k 386Incomplete Column Equilibration 386Solvent Demixing 391Nonlinear Plots of log k vs [phi] 393Dependence of V[subscript m] on [phi] 393Nonideal Equipment 393Accuracy of Gradient Elution Predictions 397Gradient Retention Time 397Confirmation of Equation (9.2) 397Computer Simulation 399Peak Width Predictions 399Measurement of Values of S and log k[subscript 0] 400Values of S 401Estimating Values of S from Solute Properties and Experimental Conditions 402Values of N in Gradient Elution 404The Constants Approximation in Gradient Elution 414Estimation of Conditions for Isocratic Elution, Based on an Initial Gradient Run 416Characterization of Reversed-Phase Columns for Selectivity and Peak Tailing 418Solvent Properties Relevant to the Use of Gradient Elution 434Theory of Preparative Separation 436Further Information on Virus Chromatography 445Index 450
\ From the Publisher"This book is clear, well written, and easy to understand despite the complexity of the subject." (Journal of the American Chemical Society, July 2007)\ \ \
