Practical HPLC Method Development, Second Edition

Description

From the Publisher This revision brings the reader completely up to date on the evolving methods associated with increasingly more complex sample types analyzed using high-performance liquid chromatography, or HPLC. The book also incorporates updated discussions of many of the fundamental components of HPLC systems and practical issues associated with the use of this analytical method. This edition includes new or expanded treatments of sample preparation, computer assisted method development, as well as biochemical samples, and chiral separations. From the Back Cover Thoroughly revised and greatly expanded, the Second Edition of this landmark work brings scientists completely up to date on method development for HPLC, one of the main analytical techniques in use today. The authors, widely recognized as preeminent authorities in the field, offer the most far-reaching treatment available, taking into account advances in chromatography and the increasingly complex sample types that have become significant in the past decade. CONTENTS......Page 6 PREFACE......Page 20 GLOSSARY OF SYMBOLS AND TERMS......Page 24 1.1 Introduction,......Page 28 1.2.1 Nature of the Sample,......Page 30 1.2.2 Separation Goals,......Page 32 1.3 Sample Pretreatment and Detection,......Page 33 1.4.1 Selecting an HPLC Method and Initial Conditions,......Page 34 1.4.2 Getting Started on Method Development,......Page 37 1.4.3 Improving the Separation,......Page 40 1.4.4 Repeatable Separation,......Page 43 1.5.1 Quantitation and Method Validation,......Page 44 1.5.2 Checking for Problems,......Page 45 1.5.3 Method Ruggedness,......Page 46 References,......Page 47 2.1 Introduction,......Page 48 2.2.1 Measurement of Resolution,......Page 49 2.2.2 Minimum Resolution,......Page 52 2.3 Resolution as a Function of Conditions,......Page 54 2.3.1 Effect of Solvent Strength,......Page 58 2.3.2.1 Changes in the Mobile Phase,......Page 61 2.3.2.3 Changes in Temperature,......Page 67 2.3.3 Effect of Column Plate Number,......Page 68 2.3.3.1 Column Conditions and Separation,......Page 70 2.3.3.2 Plate Number as a Function of Conditions,......Page 73 2.3.3.3 Extra-column Effects,......Page 74 2.4 Sample-Size Effects,......Page 77 2.4.1 Volume Overload: Effect of Sample Volume on Separation,......Page 78 2.4.2 Mass Overload: Effect of Sample Weight on Separation,......Page 80 2.4.3.2 Trace Analysis,......Page 83 References,......Page 84 3.1 Introduction,......Page 86 3.2.1 General Considerations,......Page 87 3.2.2.1 Sample Absorbance as a Function of Molecular Structure,......Page 90 3.2.2.2 Mobile-Phase Absorbance as a Function of Composition,......Page 93 3.2.3 Signal, Noise, and Assay Precision,......Page 98 3.2.4 Maximizing Signal/Noise Ratio for Better Assay Precision,......Page 100 3.2.5 Detector Linearity,......Page 103 3.2.6 Diode-Array UV Detectors,......Page 104 3.3.1 Universal Detection,......Page 107 3.3.2 Fluorescence Detection,......Page 108 3.3.3 Electrochemical Detection,......Page 111 3.3.4 Mass Spectrometer Detection (LC-MS),......Page 116 3.3.4.1 Mass Analyzers,......Page 118 3.3.4.2 Ionization Methods,......Page 119 3.3.5 Selecting the Mass Spectrometric Detector,......Page 122 3.3.6 Less Common Detectors,......Page 123 References,......Page 124 4 Sample Preparation......Page 127 4.1 Introduction,......Page 128 4.3.1 Reducing Sample Particle Size,......Page 130 4.3.3 Filtration,......Page 135 4.4.1 Liquid-Liquid Extraction,......Page 137 4.4.1.1 Theory,......Page 139 4.4.1.2 Practice,......Page 141 4.4.1.3 Problems,......Page 144 4.4.2.1 SPE vs. LLE,......Page 146 4.4.2.3 Uses of SPE,......Page 147 4.4.2.4 SPE Devices,......Page 148 4.4.2.5 SPE Apparatus,......Page 152 4.4.2.6 SPE Method Development,......Page 154 4.4.3 Membrane Separations,......Page 166 4.5 Sample Pretreatment for Solid Samples,......Page 171 4.5.1 Traditional Extraction Methods,......Page 172 4.5.2.1 Supercritical Fluid Extraction,......Page 174 4.5.2.2 Microwave-Assisted Solvent Extraction,......Page 178 4.5.2.3 Accelerated Solvent Extraction,......Page 180 4.6 Column Switching,......Page 181 4.6.1 Principle of Operation,......Page 185 4.6.3 Examples of Column Switching for Sample Cleanup,......Page 187 4.7 Derivatization,......Page 188 4.7.1 Detectability,......Page 190 4.7.1.2 Fluorescence Detection,......Page 192 4.7.2.1 Pre-column Derivatization,......Page 194 4.7.2.2 Post-column Derivatization,......Page 195 4.7.3 Chiral Analysis by Derivatization,......Page 196 References,......Page 197 5 The Column......Page 201 5.2.1 Column-Packing Particles,......Page 202 5.2.1.1 Silica Packing Particles,......Page 205 5.2.1.2 Porous Polymers,......Page 209 5.2.1.3 Other Inorganic Supports,......Page 211 5.2.2 Column Configuration,......Page 213 5.2.3.1 Bonded Silanes,......Page 216 5.2.3.3 Retention of the Bonded Phase in RPC,......Page 219 5.2.3.4 Stability of Bonded-Phase Columns,......Page 220 5.2.4 Sources of Retention and Selectivity Variability,......Page 230 5.3.1 Plate Number,......Page 232 5.3.2 Peak Asymmetry and Peak Tailing,......Page 235 5.3.3 Column Failure: How Long Should a Column Last?,......Page 237 5.3.5 Pressure Drop,......Page 239 5.3.6 Bonded-Phase Concentration (Coverage),......Page 240 5.4.1 Retention and Resolution Irreproducibility,......Page 241 5.4.2 Band Tailing,......Page 246 5.4.3 Why Do Columns Die?,......Page 250 5.4.3.1 Column Frit Problems,......Page 251 5.4.3.2 Strongly Held Sample Components,......Page 252 5.4.3.4 Pressure Effects,......Page 253 5.4.3.6 Other Factors,......Page 254 5.4.4 Suggested Column for Method Development,......Page 256 References,......Page 257 6 Non-ionic Samples: Reversed- and Normal-Phase HPLC......Page 260 Part I: Reversed-Phase Chromatography......Page 261 6.2 Retention in Reversed-Phase Chromatography,......Page 262 6.2.1 Mobile-Phase Effects,......Page 263 6.2.1.1 Choice of % B,......Page 264 6.2.1.2 Mobile-Phase Strength,......Page 266 6.2.2 Column and Temperature Effects,......Page 267 6.3.1 Solvent-Strength Selectivity,......Page 269 6.3.2 Solvent-Type Selectivity,......Page 271 6.3.3 Column-Type Selectivity,......Page 275 6.3.4 Temperature Selectivity,......Page 278 6.4 Optimizing the Separation of Non-ionic Samples in Reversed-Phase Chromatography,......Page 279 6.4.1 Getting Started,......Page 280 6.4.2 Optimizing Selectivity,......Page 281 6.4.2.2 Solvent-Type Effects Plus % B Effects,......Page 282 6.4.2.3 Use of Organic Solvent Mixtures,......Page 284 6.4.2.5 Combined Use of Different Solvents Plus Column Types,......Page 287 6.5 Non-aqueous Reversed-Phase HPLC,......Page 291 Part II: Normal-Phase Chromatography......Page 293 6.6.1 General Aspects,......Page 295 6.6.1.1 Sample and Solvent Localization,......Page 296 6.6.2.1 Solvent Strength,......Page 298 6.6.2.2 Mobile-Phase Selectivity,......Page 300 6.6.3 Column-Type Effects,......Page 303 6.6.5 Use of Aqueous Mobile Phases for Hydrophilic Samples,......Page 305 6.7.1.1 Choice of Column,......Page 309 6.7.2 Adjusting Retention,......Page 311 6.7.3 Optimizing Selectivity,......Page 312 6.7.4.1 Slow Column Equilibration and Solvent Demixing,......Page 314 6.7.4.2 Changes in Stationary-Phase Water Content,......Page 315 References,......Page 316 7 Ionic Samples: Reversed-Phase, Ion-Pair, and Ion-Exchange HPLC......Page 319 7.1 Introduction,......Page 320 7.2.1 Acid-Base Equilibria and Reversed-Phase Retention,......Page 321 7.2.2 Choice of Buffers,......Page 323 7.2.2.1 Buffer Capacity,......Page 324 7.2.2.3 Other Buffer Properties,......Page 327 7.2.3 pKa as a Function of Compound Structure,......Page 328 7.2.3.1 Preferred Mobile-Phase pH,......Page 329 7.3.1 Initial Experiments,......Page 330 7.3.2 Controlling Selectivity,......Page 331 7.3.2.1 pH,......Page 332 7.3.2.3 Solvent Type,......Page 334 7.3.2.4 Temperature,......Page 335 7.3.2.6 Amine Modifiers,......Page 336 7.3.3.2 Silanol Effects,......Page 338 7.3.4 Summary,......Page 340 7.4 Ion-Pair Chromatography,......Page 344 7.4.1.1 pH and Ion Pairing,......Page 345 7.4.1.2 Ion-Pair Reagent Concentration,......Page 347 7.4.1.3 Ion-Pair Reagent Type,......Page 349 7.4.2 Initial Experiments,......Page 351 7.4.3.1 Retention Range,......Page 354 7.4.3.2 Selectivity,......Page 355 7.4.4.1 Solvent Strength (%B),......Page 359 7.4.4.4 Solvent Type,......Page 360 7.4.5.1 Artifactual Peaks,......Page 364 7.4.5.2 Slow Column Equilibration,......Page 365 7.4.6 Summary,......Page 366 7.5 Ion-Exchange Chromatography,......Page 368 7.5.1 Basis of Retention,......Page 369 7.5.1.4 Column Type,......Page 370 7.5.3 Mixed-Mode Separations,......Page 371 References,......Page 373 8 Gradient Elution......Page 377 8.1 Introduction,......Page 378 8.2 Applications of Gradient Elution,......Page 379 8.2.1.2 High-Molecular-Weight Sample Components,......Page 380 8.2.1.5 Dilute Sample Solutions,......Page 383 8.2.1.6 Alternatives to Gradient Elution,......Page 385 8.2.2.1 Isocratic or Gradient Separation?,......Page 386 8.2.2.3 Estimating the Best Gradient Conditions,......Page 389 8.3 Principles of Gradient Elution,......Page 390 8.3.1 Gradient vs. Isocratic Elution,......Page 392 8.3.3 Effect of Gradient Range,......Page 394 8.3.4.1 Homologous or Oligomeric Samples,......Page 399 8.4 Developing a Gradient Separation,......Page 401 8.4.1.2 Gradient Range,......Page 403 8.4.2.1 Gradient Steepness,......Page 404 8.4.2.3 Other Variables,......Page 407 8.4.3 Adjusting Column Conditions,......Page 409 8.5 Experimental Considerations,......Page 412 8.5.1.1 Equipment Differences,......Page 413 8.5.1.2 Changes in Separation for Different HPLC Systems,......Page 414 8.5.1.3 Minimizing the Effect of Equipment Dwell Volume,......Page 417 8.5.1.4 Determining the Dwell Volume,......Page 419 8.5.2.2 Column Equilibration,......Page 421 8.5.2.3 Inaccurate Gradients,......Page 422 8.5.3.1 Drift,......Page 423 8.6.1 Systematic Approach,......Page 424 8.6.2 Computer Simulation,......Page 426 References,......Page 427 9 Systematic Approach to the Reversed-Phase Separation of Regular Samples......Page 429 9.1 Introduction,......Page 430 9.1.1 Some Guiding Principles,......Page 432 9.1.1.2 Initial Separation Conditions: The Column and Flow Rate,......Page 433 9.1.1.3 Initial Separation Conditions: The Mobile Phase,......Page 434 9.1.1.5 Ensuring Accurate Retention Data,......Page 435 9.1.1.6 Confirming Good Column Performance,......Page 436 9.2.1 Initial Conditions,......Page 437 9.2.2.1 Isocratic Separation,......Page 438 9.2.2.2 Gradient Separation,......Page 441 9.2.2.4 Very Hydrophobie Cations,......Page 443 9.2.2.5 Complex Samples,......Page 444 9.2.3 Evaluating Peak Shape and Plate Number,......Page 445 9.3.1 Optimizing Retention and Selectivity,......Page 447 9.3.1.2 Sample B: A Typical Separation,......Page 449 9.3.1.3 Sample C: A Difficult Separation,......Page 451 9.3.1.4 Further Improvements in Separation,......Page 453 9.3.1.5 Changing the Method for Later Samples or Applications,......Page 456 9.3.2 Optimizing Column Conditions,......Page 457 9.4 Alternative To Completing Isocratic Method Development,......Page 458 9.5 Completing Gradient Method Development,......Page 460 References,......Page 464 10.1 Introduction,......Page 466 10.1.1 Summary of Commercial Method-Development Software,......Page 467 10.2 Computer Simulation Software (DryLab),......Page 468 10.2.1 Isocratic Separation Varying % B and Column Conditions,......Page 470 10.2.1.1 Use of Other Variables for Changing Selectivity,......Page 472 10.2.2 Gradient Separations,......Page 475 10.2.2.2 Other Applications,......Page 479 10.3 Software for Solvent-Type Optimization (ICOS, DIAMOND),......Page 482 10.4 Grid-Search Software (PESOS),......Page 485 10.5.1 ELUEX,......Page 490 10.5.3 Special-Purpose Programs,......Page 492 10.6 Method Ruggedness,......Page 494 10.7.1 Injection of Standards,......Page 497 10.7.2 Retention and Area Comparisons,......Page 499 10.7.4 Spectral Identification,......Page 500 10.8 Pitfalls,......Page 502 References,......Page 503 11 Biochemical Samples: Proteins, Nucleic Acids, Carbohydrates, and Related Compounds......Page 506 11.1 Introduction,......Page 507 11.1.1.1 Peptides and Proteins,......Page 509 11.1.1.2 Oligonucleotides and Nucleic Acids,......Page 512 11.1.2.1 Columns,......Page 515 11.1.2.2 Sample Molecular Conformation,......Page 519 11.1.2.3 Sample Recovery: Mass and Bioactivity,......Page 521 11.1.2.4 Sample Handling and Pretreatment,......Page 522 11.2.1 Reversed-Phase HPLC,......Page 524 11.2.1.1 Preferred Conditions for an Initial Separation,......Page 525 11.2.1.2 Variables for Changing Selectivity,......Page 529 11.2.1.3 Common Problems and Remedies,......Page 534 11.2.2 Ion-Exchange HPLC,......Page 536 11.2.2.1 Preferred Conditions for an Initial Separation,......Page 539 11.2.2.3 Common Problems and Remedies,......Page 542 11.2.3 Hydrophobie Interaction Chromatography,......Page 543 11.2.3.1 Preferred Conditions for HIC Separation,......Page 544 11.3 Separation of Oligonucleotides,......Page 546 11.3.1 Ion-Pair HPLC,......Page 547 11.3.2 Ion-Exchange HPLC,......Page 548 11.4.1 The Basis of SEC Retention,......Page 550 11.4.2 Applications,......Page 555 11.4.3 Preferred Conditions for an SEC Separation,......Page 557 11.4.4 Common Problems and Remedies,......Page 558 References,......Page 560 12 Chiral Separations......Page 564 12.1 Introduction,......Page 565 12.1.2 Chiral Mobile-Phase Additives,......Page 567 12.1.3 Chiral Stationary Phases,......Page 568 12.1.4 Principles of Chiral Recognition,......Page 569 12.1.5.1 Sample Information,......Page 573 12.1.6 Selecting a Chiral Column,......Page 574 12.2.2 Background,......Page 575 12.2.4 Characteristics of Protein-Based Chiral Columns,......Page 577 12.2.5 Adjusting Retention and Selectivity with the Mobile Phase,......Page 579 12.2.5.1 Organic Mobile-Phase Modifiers,......Page 581 12.2.5.2 pH, Ionic Strength, and Ion-Pairing Effects,......Page 582 12.2.6.1 Mobile-Phase Effects,......Page 586 12.2.6.4 Column Configuration,......Page 588 12.2.7 Application and Special Techniques,......Page 590 12.2.8 Systematic Method Development,......Page 594 12.3.2.1 General Characteristics,......Page 595 12.3.2.2 Availability,......Page 597 12.3.3 Mechanisms of Chiral Interactions,......Page 599 12.3.4.1 Mobile-Phase Selection,......Page 603 12.3.4.3 Column Configuration and Operation,......Page 606 12.3.6 Strategy for Method Development,......Page 608 12.4.1 Introduction,......Page 612 12.4.2 Properties of Commercial Donor-Acceptor CSPs,......Page 613 12.4.3 Mobile-Phase Conditions,......Page 615 12.4.4.2 Mobile Phase,......Page 618 12.4.4.3 Derivatization,......Page 619 12.4.4.4 Effects of Temperature and Flow Rate,......Page 621 12.5.1 Introduction,......Page 627 12.5.2.2 Reversed-Phase Mode,......Page 631 12.5.2.3 Polar-Organic and Normal-Phase Modes,......Page 635 12.5.3 Method Development with Derivatized Cyclodextrins,......Page 637 References,......Page 640 13.1 Introduction,......Page 643 13.2.1 General Considerations,......Page 645 13.2.2 Effect of Sample Size: Touching-Band Separation,......Page 648 13.2.3 Optimizing Conditions for Preparative HPLC,......Page 649 13.2.4 Gradient Separations,......Page 652 13.3.1 Sample Solubility,......Page 654 13.4 Quantitative Prediction of Preparative HPLC Separation,......Page 655 13.4.1 General Relationships,......Page 656 13.4.3 Gradient Elution Separations,......Page 658 13.4.4 Heavily Overloaded Separations,......Page 659 13.4.5 Unusual Isotherm Behavior,......Page 661 13.5 Summary and Example of Method Development for Preparative HPLC,......Page 663 13.5.1 Process-Scale HPLC Separations,......Page 667 References,......Page 668 14.1 Introduction,......Page 670 14.1.1 Accuracy, Precision, and Linearity,......Page 671 14.1.2 Limits of Detection and Quantitation,......Page 672 14.2.1 Noise,......Page 674 14.2.3PeakArea,650......Page 0 14.2.4 Peak Height vs. Peak Area for Quantitation,......Page 679 14.3 Quantitation Methods,......Page 680 14.3.1 Normalized Peak Area,......Page 681 14.3.2 External Standard Calibration,......Page 682 14.3.3 Internal Standard Calibration,......Page 684 14.4 Sources of Error in Quantitation,......Page 687 14.4.1 Sampling and Sample Preparation,......Page 689 14.4.2 Chromatographie Effects,......Page 690 14.4.3 Data System Effects,......Page 692 14.5.1 Sample Preparation,......Page 693 14.5.2 Column Resolution,......Page 694 14.5.3 Sample Injection,......Page 700 14.5.4 Detection,......Page 703 14.5.5 Calibration,......Page 705 14.5.6 General Strategy,......Page 707 References,......Page 710 15 Completing the Method: Validation and Transfer......Page 712 15.1.1 General Approach to Method Validation,......Page 713 15.2 Accuracy,......Page 714 15.2.2 Analyte Recovery,......Page 715 15.2.3 Method of Standard Addition,......Page 716 15.3 Precision,......Page 717 15.4 Linearity,......Page 718 15.5 Range,......Page 721 15.7 Specificity,......Page 722 15.7.2 Sample Degradation,......Page 724 15.7.4 Additional On-Line Detection,......Page 725 15.7.6 Changing HPLC Conditions,......Page 727 15.8 Ruggedness,......Page 728 15.9 Robustness,......Page 729 15.10 Stability,......Page 731 15.11 System Suitability,......Page 732 15.12 Documentation of Validation Results and the Final Method,......Page 733 15.13 Interlaboratory Crossover Studies (Transferability),......Page 734 15.13.1 Determining Equivalence,......Page 735 References,......Page 739 Appendix I Plate Number and Resolution......Page 741 Appendix II Properties of Solvents Used in HPLC......Page 748 Appendix III Retention in Reversed-Phase and Normal-Phase HPLC as a Function of Sample Molecular Structure......Page 756 Appendix IV Preparing Buffered Mobile Phases......Page 762 Appendix V Characterizing the Differences Among C8 or C18 Reversed-Phase Columns from Different Suppliers......Page 767 Appendix VI Adjusting Mobile-Phase Water Content for Normal-Phase HPLC......Page 771 Index......Page 773