Practical HPLC Method Development

by Snyder, Lloyd R.; Kirkland, Joseph J.; Glajch, Joseph L.

• ISBN13: 9780471007036
• ISBN10: 047100703X
• Edition: 2nd
• Format: Hardcover
• Copyright: 1997-03-17
• Publisher: Wiley-Interscience
• More Book Details
• Purchase Benefits

Summary

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.

Author Biography

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.

Joseph J. Kirkland, PhD, is Vice President of Research and Development for Advanced Materials Technology, Inc., and coauthor of Introduction to Modern Liquid Chromatography, Second Edition, Practical HPLC Method Development, Second Edition, and Modern Size-Exclusion Liquid Chromatography, Second Edition, all published by Wiley.

Joseph L. Glajch is the author of Practical HPLC Method Development, Second Edition, published by Wiley.

Table of Contents

PREFACE	xix	(4)
GLOSSARY OF SYMBOLS AND TERMS	xxiii
1 Getting Started	1	(20)
1.1 Introduction	1	(2)
1.2 What Is Known Before Starting	3	(3)
1.2.1 Nature of the Sample	3	(2)
1.2.2 Separation Goals	5	(1)
1.3 Sample Pretreatment and Detection	6	(1)
1.4 Developing the Separation	7	(10)
1.4.1 Selecting an HPLC Method and Initial Conditions	7	(3)
1.4.2 Getting Started on Method Development	10	(3)
1.4.3 Improving the Separation	13	(3)
1.4.4 Repeatable Separation	16	(1)
1.5 Completing the HPLC Method	17	(3)
1.5.1 Quantitation and Method Validation	17	(1)
1.5.2 Checking for Problems	18	(1)
1.5.3 Method Ruggedness	19	(1)
References	20	(1)
2 Basics of Separation	21	(38)
2.1 Introduction	21	(1)
2.2 Resolution: General Considerations	22	(5)
2.2.1 Measurement of Resolution	22	(3)
2.2.2 Minimum Resolution	25	(2)
2.3 Resolution as a Function of Conditions	27	(23)
2.3.1 Effect of Solvent Strength	31	(3)
2.3.2 Effect of Selectivity	34	(1)
2.3.2.1 Changes in the Mobile Phase	34	(6)
2.3.2.2 Changes in the Column	40	(1)
2.3.2.3 Changes in Temperature	40	(1)
2.3.3 Effect of Column Plate Number	41	(2)
2.3.3.1 Column Conditions and Separation	43	(3)
2.3.3.2 Plate Number as a Function of Conditions	46	(1)
2.3.3.3 Extra-column Effects	47	(3)
2.4 Sample-Size Effects	50	(7)
2.4.1 Volume Overload: Effect of Sample Volume on Separation	51	(2)
2.4.2 Mass Overload: Effect of Sample Weight on Separation	53	(3)
2.4.3 Avoiding Problems Due to Too Large a Sample Size	56	(1)
2.4.3.1 Higher-Than-Expected Sample Concentrations	56	(1)
2.4.3.2 Trace Analysis	56	(1)
References	57	(2)
3 Detection Sensitivity and Selectivity	59	(41)
3.1 Introduction	59	(1)
3.2 UV Detection	60	(20)
3.2.1 General Considerations	60	(3)
3.2.2 Choice of Wavelength	63	(1)
3.2.2.1 Sample Absorbance as a Function of Molecular Structure	63	(3)
3.2.2.2 Mobile-Phase Absorbance as a Function of Composition	66	(5)
3.2.3 Signal, Noise, and Assay Precision	71	(2)
3.2.4 Maximizing Signal/Noise Ratio for Better Assay Precision	73	(3)
3.2.5 Detector Linearity	76	(1)
3.2.6 Diode-Array UV Detectors	77	(3)
3.3 Other HPLC Detectors	80	(17)
3.3.1 Universal Detection	80	(1)
3.3.2 Fluorescence Detection	81	(3)
3.3.3 Electrochemical Detection	84	(5)
3.3.4 Mass Spectrometer Detection (LC-MS)	89	(2)
3.3.4.1 Mass Analyzers	91	(1)
3.3.4.2 Ionization Methods	92	(3)
3.3.5 Selecting the Mass Spectrometric Detector	95	(1)
3.3.6 Less Common Detectors	96	(1)
References	97	(3)
4 Sample Preparation	100	(74)
4.1 Introduction	101	(2)
4.2 Types of Samples	103	(1)
4.3 Preliminary Processing of Solid and Semi-solid Samples	103	(7)
4.3.1 Reducing Sample Particle Size	103	(5)
4.3.2 Drying the Sample	108	(1)
4.3.3 Filtration	108	(2)
4.4 Sample Pretreatment for Liquid Samples	110	(34)
4.4.1 Liquid-Liquid Extraction	110	(2)
4.4.1.1 Theory	112	(2)
4.4.1.2 Practice	114	(3)
4.4.1.3 Problems	117	(2)
4.4.2 Solid-Phase Extraction	119	(1)
4.4.2.1 SPE vs. LLE	119	(1)
4.4.2.2 SPE vs. HPLC	120	(1)
4.4.2.3 Uses of SPE	120	(1)
4.4.2.4 SPE Devices	121	(4)
4.4.2.5 SPE Apparatus	125	(2)
4.4.2.6 SPE Method Development	127	(12)
4.4.2.7 Column Chromatography for Sample Pretreatment	139	(1)
4.4.3 Membrance Separations	139	(5)
4.5 Sample Pretreatment for Solid Samples	144	(10)
4.5.1 Traditional Extraction Methods	145	(2)
4.5.2 Newer Extraction Methods	147	(1)
4.5.2.1 Supercritical Fluid Extraction	147	(4)
4.5.2.2 Microwave-Assisted Solvent Extraction	151	(2)
4.5.2.3 Accelerated Solvent Extraction	153	(1)
4.5.3 Comparison of Methods for Extraction of Solids	154	(1)
4.6 Column Switching	154	(7)
4.6.1 Principle of Operation	158	(2)
4.6.2 Developing a Column-Switching Method: General Considerations	160	(1)
4.6.3 Examples of Column Switching for Sample Cleanup	160	(1)
4.7 Derivatization	161	(9)
4.7.1 Detectability	163	(2)
4.7.1.1 UV Detection	165	(1)
4.7.1.2 Fluorescence Detection	165	(2)
4.7.2 Pre-and Post-column Derivatization	167	(1)
4.7.2.1 Pre-column Derivatization	167	(1)
4.7.2.2 Post-column Derivatization	168	(1)
4.7.3 Chiral Analysis by Derivatization	169	(1)
References	170	(4)
5 The Column	174	(59)
5.1 Introduction	175	(1)
5.2 Characterstics of Columns and Column Packings	175	(30)
5.2.1 Column-Packing Particles	175	(3)
5.2.1.1 Silica Packing Particles	178	(4)
5.2.1.2 Porous Polymers	182	(2)
5.2.1.3 Other Inorganic Supports	184	(2)
5.2.2 Column Configuration	186	(3)
5.2.3 Stationary Phases	189	(1)
5.2.3.1 Bonded Silances	189	(3)
5.2.3.2 Other Stationary Phases	192	(1)
5.2.3.3 Retention of the Bonded Phase in RPC	192	(1)
5.2.3.4 Stability of Bonded-Phase Columns	193	(10)
5.2.4 Sources of Retention and Selectivity Variability	203	(2)
5.3 Column Specifications	205	(9)
5.3.1 Plate Number	205	(3)
5.3.2 Peak Asymmetry and Peak Tailing	208	(2)
5.3.3 Column Failure: How Long Should a Column Last?	210	(2)
5.3.4 Retention Reproductivity	212	(1)
5.3.5 Pressure Drop	212	(1)
5.3.6 Bonded-Phase Concentration (Coverage)	213	(1)
5.4 Column Problems and Remedies	214	(16)
5.4.1 Retention and Resolution Irreproducibility	214	(5)
5.4.2 Band Tailing	219	(4)
5.4.3 Why Do Columns Die?	223	(1)
5.4.3.1 Column Frit Problems	224	(1)
5.4.3.2 Strongly Held Sample Components	225	(1)
5.4.3.3 Poorly Packed Columns	226	(1)
5.4.3.4 Pressure Effects	226	(1)
5.4.3.5 Chemical Attack	227	(1)
5.4.3.6 Other Factors	227	(2)
5.4.4 Suggested Column for Method Development	229	(1)
References	230	(3)
6 Non-ionic Samples: Reversed-and Normal-Phase HPLC	233	(1)
6.1 Introduction	234	(1)
Part I: Reversed-Phase Chromatography	234	(32)
6.2 Retention in Reversed-Phase Chromatography	235	(7)
6.2.1 Mobile-Phase Effects	236	(1)
6.2.1.1 Choice of % B	237	(2)
6.2.1.2 Mobile-Phase Strength	239	(1)
6.2.2 Column and Temperature Effects	240	(2)
6.3 Selectivity in Reversed-Phase Chromatography	242	(10)
6.3.1 Solvent-Strength Selectivity	242	(2)
6.3.2 Solvent-Type Selectivity	244	(4)
6.3.3 Column-Type Selectivity	248	(3)
6.3.4 Temperature Selectivity	251	(1)
6.4 Optimizing the Separation of Non-ionic Samples in Reversed-Phase Chromatography	252	(12)
6.4.1 Getting Started	253	(1)
6.4.2 Optimizing Selectivity	254	(1)
6.4.2.1 Solvent Strength (% B) Effects	255	(1)
6.4.2.2 Solvent-Type Effects Plus % B Effects	255	(2)
6.4.2.3 Use of Organic Solvent Mixtures	257	(3)
6.4.2.4 Column-Type Effects Plus % B Effects	260	(1)
6.4.2.5 Combined Use of Different Solvents Plus Column Types	260	(4)
6.5 Non-aqucous Reversed-Phase HPLC	264	(2)
Part II: Normal-Phase Chromatography	266	(26)
6.6 Retention and Selectivity in Normal-Phase Chromatography	268	(14)
6.6.1 General Aspects	268	(1)
6.6.1.1 Sample and Solvent Localization	269	(2)
6.6.2 Mobile-Phase Effects	271	(1)
6.6.2.1 Solvent Strength	271	(2)
6.6.2.2 Mobile-Phase Selectivity	273	(3)
6.6.3 Column-Type Effects	276	(2)
6.6.4 Temperature Effects	278	(1)
6.6.5 Use of Aqueous Mobile Phases for Hydrophilic Samples	278	(4)
6.7 Optimizing the Separation of Non-ionic Samples in Normal-Phase Chromatography	282	(7)
6.7.1 Initial Conditions	282	(1)
6.7.1.1 Choice of Column	282	(2)
6.7.1.2 Mobile Phase Solvents	284	(1)
6.7.2 Adjusting Retention	284	(1)
6.7.3 Optimizing Selectivity	285	(2)
6.7.4 Other Considerations	287	(1)
6.7.4.1 Slow Column Equilibration and Solvent Demixing	287	(1)
6.7.4.2 Changes in Stationary-Phase Water Content	288	(1)
References	289	(3)
7 Ionic Samples: Reversed-Phase, Ion-Pair, and Ion-Exchange HPLC	292	(58)
7.1 Introduction	293	(1)
7.2 Acidic and Basic Samples	294	(9)
7.2.1 Acid-Base Equilibria and Reversed-Phase Retention	294	(2)
7.2.2 Choice of Buffers	296	(1)
7.2.2.1 Buffer Capacity	297	(3)
7.2.2.2 Buffer UV Absorbance	300	(1)
7.2.2.3 Other Buffer Properties	300	(1)
7.2.2.4 Preferred Buffers	301	(1)
7.2.3 pKa as a Function of Compound Structure	301	(1)
7.2.3.1 Preferred Mobile-Phase pH	302	(1)
7.2.4 Which HPLC Method is Best for Ionic Samples?	303	(1)
7.3 Optimizing the Reversed-Phase Separation of Ionic Samples	303	(14)
7.3.1 Initial Experiments	303	(1)
7.3.2 Controlling Selectivity	304	(1)
7.3.2.1 pH	305	(2)
7.3.2.2 Solvent Strength (% B)	307	(1)
7.3.2.3 Solvent Type	307	(1)
7.3.2.4 Temperature	308	(1)
7.3.2.5 Buffer Concentration	309	(1)
7.3.2.6 Amine Modifiers	309	(2)
7.3.2.7 Column Type	311	(1)
7.3.3 Special Problems	311	(1)
7.3.3.1 pH Sensitivity	311	(1)
7.3.3.2 Silanol Effects	311	(2)
7.3.3.3 Temperature Sensitivity	313	(1)
7.3.4 Summary	313	(4)
7.4 Ion-Pair Chromatography	317	(24)
7.4.1 Basis of Retention	318	(1)
7.4.1.1 pH and Ion Pairing	318	(2)
7.4.1.2 Ion-Pair Reagent Concentration	320	(2)
7.4.1.3 Ion-Pair Reagent Type	322	(2)
7.4.2 Initial Experiments	324	(3)
7.4.3 Controlling Retention Range and Selectivity: Changes in % B, pH, and Ion-Pair Reagent Concentration	327	(1)
7.4.3.1 Retention Range	327	(1)
7.4.3.2 Selectivity	328	(4)
7.4.4 Other Changes in Selectivity	332	(1)
7.4.4.1 Solvent Strength (% B)	332	(1)
7.4.4.2 Temperature	333	(1)
7.4.4.3 Buffer Concentration	333	(1)
7.4.4.4 Solvent Type	333	(4)
7.4.4.5 Buffer Type or Added Salt	337	(1)
7.4.4.6 Amine Modifiers	337	(1)
7.4.5 Special Problems	337	(1)
7.4.5.1 Artifactual Peaks	337	(1)
7.4.5.2 Slow Column Equilibration	338	(1)
7.4.5.3 Poor Peak Shape	339	(1)
7.4.6 Summary	339	(2)
7.5 Ion-Exchange Chromatography	341	(5)
7.5.1 Basis of Retention	342	(1)
7.5.1.1 pH Effects	343	(1)
7.5.1.2 Salt or Buffer Type	343	(1)
7.5.1.3 Organic Solvents	343	(1)
7.5.1.4 Column Type	343	(1)
7.5.2 Method Development	344	(1)
7.5.3 Mixed-Mode Separations	344	(2)
7.5.4 Silica Columns	346	(1)
References	346	(4)
8 Gradient Elution	350	(52)
8.1 Introduction	351	(1)
8.2 Applications of Gradient Elution	352	(11)
8.2.1 Gradient Elution for Routine Analysis	353	(1)
8.2.1.1 Sample Retention Range	353	(1)
8.2.1.2 High-Molecular-Weight Sample Components	353	(3)
8.2.1.3 Late Eluters	356	(1)
8.2.1.4 Maximizing Detection Sensitivity	356	(1)
8.2.1.5 Dilute Sample Solutions	356	(2)
8.2.1.6 Alternatives to Gradient Elution	358	(1)
8.2.2 Gradient Elution for Method Development	359	(1)
8.2.2.1 Isocratic or Gradient Separation?	359	(3)
8.2.2.2 Estimating the Best Isocratic Conditions	362	(1)
8.2.2.3 Estimating the Best Gradient Conditions	362	(1)
8.3 Principles of Gradient Elution	363	(11)
8.3.1 Gradient vs. Isocratic Elution	365	(2)
8.3.2 Effect of Gradient Steepness	367	(1)
8.3.3 Effect of Gradient Range	367	(5)
8.3.4 Effect of Gradient Shape	372	(1)
8.3.4.1 Homologous or Oligomeric Samples	372	(2)
8.3.4.2 Chromatograms with Peak Bunching	374	(1)
8.4 Developing a Gradient Separation	374	(11)
8.4.1 Selecting Gradient Conditions	376	(1)
8.4.1.1 Gradient Steepness	376	(1)
8.4.1.2 Gradient Range	376	(1)
8.4.1.3 Gradient Shape	377	(1)
8.4.2 Varying Band Spacing	377	(1)
8.4.2.1 Gradient Steepness	377	(3)
8.4.2.2 Solvent Type	380	(1)
8.4.2.3 Other Variables	380	(2)
8.4.3 Adjusting Column Conditions	382	(3)
8.5 Experimental Considerations	385	(12)
8.5.1 Effect of Equipment on Separation: System Dwell Volume	386	(1)
8.5.1.1 Equipment Differences	386	(1)
8.5.1.2 Changes in Separation for Different HPLC Systems	387	(3)
8.5.1.3 Minimizing the Effect of Equipment Dwell Volume	390	(2)
8.5.1.4 Determining the Dwell Volume	392	(2)
8.5.2 Reproducible Separation	394	(1)
8.5.2.1 Column Regeneration	394	(1)
8.5.2.2 Column Equilibration	394	(1)
8.5.2.3 Inaccurate Gradients	395	(1)
8.5.3 Baseline Problems	396	(1)
8.5.3.1 Drift	396	(1)
8.5.3.2 Artifactual Bands	397	(1)
8.6 Summary of Gradient Elution Method Development	397	(3)
8.6.1 Systematic Approach	397	(2)
8.6.2 Computer Simulation	399	(1)
References	400	(2)
9 Systematic Approach to the Reversed-Phase Separation of Regular Samples	402	(37)
9.1 Introduction	403	(7)
9.1.1 Some Guiding Principles	405	(1)
9.1.1.1 Classifying the Sample	406	(1)
9.1.1.2 Initial Separation Conditions: The Column and Flow Rate	406	(1)
9.1.1.3 Initial Separation Conditions: The Mobile Phase	407	(1)
9.1.1.4 Other Initial Separation Conditions	408	(1)
9.1.1.5 Ensuring Accurate Retention Data	408	(1)
9.1.1.6 Confirming Good Column Performance	409	(1)
9.1.1.7 Peak Tracking	410	(1)
9.2 Getting Started	410	(10)
9.2.1 Initial Conditions	410	(1)
9.2.2 Adjusting the Retention Range	411	(1)
9.2.2.1 Isocratic Separation	411	(3)
9.2.2.2 Gradient Separation	414	(2)
9.2.2.3 Early or Late Eluters	416	(1)
9.2.2.4 Very Hydrophobic Cations	416	(1)
9.2.2.5 Complex Samples	417	(1)
9.2.2.6 No Real Peaks	418	(1)
9.2.3 Evaluating Peak Shape and Plate Number	418	(2)
9.3 Completing Isocratic Method Development	420	(11)
9.3.1 Optimizing Retention and Selectivity	420	(2)
9.3.1.1 Sample A: An Easy Separation	422	(1)
9.3.1.2 Sample B: A Typical Separation	422	(2)
9.3.1.3 Sample C: A Difficult Separation	424	(2)
9.3.1.4 Further Improvements in Separation	426	(3)
9.3.1.5 Changing the Method for Later Samples or Applications	429	(1)
9.3.2 Optimizing Column Conditions	430	(1)
9.4 Alternative To Completing Isocratic Method Development	431	(2)
9.5 Completing Gradient Method Development	433	(4)
References	437	(2)
10 Computer-Assisted Method Development	439	(40)
10.1 Introduction	439	(2)
10.1.1 Summary of Commercial Method-Development Software	440	(1)
10.2 Computer-Simulation Software (DryLab)	441	(14)
10.2.1 Isocratic Separation Varying % B and Column Conditions	443	(2)
10.2.1.1 Use of Other Variables for Changing Selectivity	445	(3)
10.2.2 Gradient Separations	448	(4)
10.2.2.1 Segmented Gradients	452	(1)
10.2.2.2 Other Applications	452	(3)
10.3 Software for Solvent-Type Optimization (ICOS, DIAMOND)	455	(3)
10.4 Grid-Search Software (PESOS)	458	(5)
10.5 Structure-Based Predictive Software	463	(4)
10.5.1 ELUEX	463	(2)
10.5.2 CHROMDREAM	465	(1)
10.5.3 Special-Purpose Programs	465	(2)
10.6 Method Ruggedness	467	(3)
10.7 Peak Tracking	470	(5)
10.7.1 Injection of Standards	470	(2)
10.7.2 Retention and Area Comparisons	472	(1)
10.7.3 Trends in Retention	473	(1)
10.7.4 Spectral Identification	473	(2)
10.8 Pitfalls	475	(1)
References	476	(3)
11 Biochemical Samples: Proteins, Nucleic Acids, Carbohydrates, and Related Compounds	479	(58)
11.1 Introduction	480	(17)
11.1.1 Primary Structure	482	(1)
11.1.1.1 Peptides and Proteins	482	(3)
11.1.1.2 Oligonucleotides and Nucleic Acids	485	(3)
11.1.1.3 Modified Oligonucleotides	488	(1)
11.1.2 Special Requirements of Biochemical HPLC	488	(1)
11.1.2.1 Columns	488	(4)
11.1.2.2 Sample Molecular Conformation	492	(2)
11.1.2.3 Sample Recovery: Mass and Bioactivity	494	(1)
11.1.2.4 Sample Handling and Pretreatment	495	(2)
11.1.2.5 Sample Detection	497	(1)
11.2 Separation of Peptide and Protein Samples	497	(22)
11.2.1 Reversed-Phase HPCL	497	(1)
11.2.1.1 Preferred Conditions for an Initial Separation	498	(4)
11.2.1.2 Variables for Changing Selectivity	502	(5)
11.2.1.3 Common Problems and Remedies	507	(2)
11.2.2 Ion-Exchange HPLC	509	(3)
11.2.2.1 Preferred Conditions for an Initial Separation	512	(3)
11.2.2.2 Variables for Changing Selectivity	515	(1)
11.2.2.3 Common Problems and Remedies	515	(1)
11.2.3 Hydrophobic Interaction Chromatography	516	(1)
11.2.3.1 Preferred Conditions for HIC Separation	517	(2)
11.3 Separation of Oligonucleotides	519	(4)
11.3.1 Ion-Pair HPLC	520	(1)
11.3.2 Ion-Exchange HPLC	521	(2)
11.4 Size-Exclusion Chromatography	523	(10)
11.4.1 The Basis of SEC Retention	523	(5)
11.4.2 Applications	528	(2)
11.4.3 Preferred Conditions for an SEC Separation	530	(1)
11.4.4 Common Problems and Remedies	531	(2)
11.4.5 Protein Folding	533	(1)
References	533	(4)
12 Chiral Separations	537	(79)
12.1 Introduction	538	(10)
12.1.1 Chiral Derivatization	540	(1)
12.1.2 Chiral Mobile-Phase Additives	540	(1)
12.1.3 Chiral Stationary Phases	541	(1)
12.1.4 Principles of Chiral Recognition	542	(4)
12.1.5 General Considerations for Chiral HPLC Method Development	546	(1)
12.1.5.1 Sample Information	546	(1)
12.1.5.2 Preparative Separations	547	(1)
12.1.6 Selecting a Chiral Column	547	(1)
12.2 Protein-Derived Chiral Stationary Phases for HPLC	548	(20)
12.2.1 Introduction	548	(1)
12.2.2 Background	548	(2)
12.2.3 Mechanism of Chiral Interactions	550	(1)
12.2.4 Characteristics of Protein-Based Chiral Columns	550	(2)
12.2.5 Adjusting Retention and Selectivity with the Mobile Phase	552	(2)
12.2.5.1 Organic Mobile-Phase Modifiers	554	(1)
12.2.5.2 pH, Ionic Strength, and Ion-Pairing Effects	555	(4)
12.2.6 Experimental Parameters	559	(1)
12.2.6.1 Mobile-Phase Effects	559	(2)
12.2.6.2 Sample Loading and Injection	561	(1)
12.2.6.3 Column Temperature	561	(1)
12.2.6.4 Column Configuration	561	(2)
12.2.6.5 Column Care and Stability	563	(1)
12.2.7 Application and Special Techniques	563	(4)
12.2.8 Systematic Method Development	567	(1)
12.3 Polysaccharide (Carbohydrate) Columns	568	(17)
12.3.1 Introduction	568	(1)
12.3.2 Properties of Commercial Polysaccharide Phases	568	(1)
12.3.2.1 General Characteristics	568	(2)
12.3.2.2 Availability	570	(2)
12.3.3 Mechanism of Chiral Interactions	572	(4)
12.3.4 Experimental Parameters	576	(1)
12.3.4.1 Mobile-Phase Selection	576	(3)
12.3.4.2 Temperature and Pressure Effects	579	(1)
12.3.4.3 Column Configuration and Operation	579	(2)
12.3.4.4 Sample Size	581	(1)
12.3.5 Applications	581	(1)
12.3.6 Strategy for Method Development	581	(4)
12.4 Donor-Acceptor (Pirkle) Columns	585	(15)
12.4.1 Introduction	585	(1)
12.4.2 Properties of Commercial Donor-Acceptor CSPs	586	(2)
12.4.3 Mobile-Phase Conditions	588	(3)
12.4.3.1 Solvents	591	(1)
12.4.4 Method Development with Pirkle CSPs	591	(1)
12.4.4.1 Column	591	(1)
12.4.4.2 Mobile Phase	591	(1)
12.4.4.3 Derivatization	592	(2)
12.4.4.4 Effects of Temperature and Flow Rate	594	(6)
12.5 Cavity-Type Columns	600	(13)
12.5.1 Introduction	600	(4)
12.5.2 Method Development for Separation Using Underivatized CD Columns	604	(1)
12.5.2.1 Separation Modes	604	(1)
12.5.2.2 Reversed-Phase Mode	604	(4)
12.5.2.3 Polar-Organic and Normal-Phase Modes	608	(2)
12.5.3 Method Development with Derivatized Cyclodextrins	610	(3)
References	613	(3)
13 Preparative HPLC Separation	616	(27)
13.1 Introduction	616	(2)
13.2 Developing a Preparative HPLC Separation	618	(9)
13.2.1 General Considerations	618	(3)
13.2.2 Effect of Sample Size: Touching-Band Separation	621	(1)
13.2.3 Optimizing Conditions for Preparative HPLC	622	(3)
13.2.4 Gradient Separations	625	(2)
13.2.5 Trace Recovery	627	(1)
13.3 Practical Aspects of Preparative HPLC	627	(1)
13.3.1 Sample Solubility	627	(1)
13.3.2 Equipment Requirements	628	(1)
13.4 Quantitative Prediction of Preparative HPLC Separation	628	(8)
13.4.1 General Relationships	629	(2)
13.4.2 Column Saturation Capacity	631	(1)
13.4.3 Gradient Elution Separations	631	(1)
13.4.4 Heavily Overloaded Separations	632	(2)
13.4.5 Unusual Isotherm Behavior	634	(2)
13.5 Summary and Example of Method Development for Preparative HPLC	636	(5)
13.5.1 Process-Scale HPLC Separations	640	(1)
References	641	(2)
14 Quantitation (Including Trace Analysis)	643	(42)
14.1 Introduction	643	(4)
14.1.1 Accuracy, Precision, and Linearity	644	(1)
14.1.2 Limits of Detection and Quantitation	645	(2)
14.2 Measurement of Signals	647	(6)
14.2.1 Noise	647	(2)
14.2.2 Peak Height	649	(1)
14.2.3 Peak Area	650	(2)
14.2.4 Peak Height vs. Peak Area for Quantitation	652	(1)
14.3 Quantitation Methods	653	(7)
14.3.1 Normalized Peak Area	654	(1)
14.3.2 External Standard Calibration	655	(2)
14.3.3 Internal Standard Calibration	657	(3)
14.3.4 Method of Standard Addition	660	(1)
14.4 Sources of Error in Quantitation	660	(6)
14.4.1 Sampling and Sample Preparation	662	(1)
14.4.2 Chromatographic Effects	663	(2)
14.4.3 Data System Effects	665	(1)
14.5 Trace Analysis	666	(17)
14.5.1 Sample Preparation	666	(1)
14.5.2 Column Resolution	667	(6)
14.5.3 Sample Injection	673	(3)
14.5.4 Detection	676	(2)
14.5.5 Calibration	678	(2)
14.5.6 General Strategy	680	(3)
References	683	(2)
15 Completing the Method: Validation and Transfer	685	(29)
15.1 Introduction	686	(1)
15.1.1 General Approach to Method Validation	686	(1)
15.2 Accuracy	687	(3)
15.2.1 Comparison to a Standard	688	(1)
15.2.2 Analyte Recovery	688	(1)
15.2.3 Method of Standard Addition	689	(1)
15.3 Precision	690	(1)
15.4 Linearity	691	(3)
15.5 Range	694	(1)
15.6 Limit of Detection and Limit of Quantitation	695	(1)
15.7 Specificity	695	(6)
15.7.1 Spiking of Potential Interferents	697	(1)
15.7.2 Sample Degradation	697	(1)
15.7.3 Peak Collection and Analysis	698	(1)
15.7.4 Additional On-Line Detection	698	(2)
15.7.5 Chromatographic Cross-Check	700	(1)
15.7.6 Changing HPLC Conditions	700	(1)
15.8 Ruggedness	701	(1)
15.9 Robustness	702	(2)
15.10 Stability	704	(1)
15.11 System Suitability	705	(1)
15.12 Documentation of Validation Results and the Final Method	706	(1)
15.13 Interlaboratory Crossover Studies (Transferability)	707	(5)
15.13.1 Determining Equivalence	708	(4)
15.14 Method Validation Protocol	712	(1)
References	712	(2)
Appendix I Plate Number and Resolution	714	(7)
Appendix II Properties of Solvents Used in HPLC	721	(8)
Appendix III Retention in Reversed-Phase and Normal-Phase HPLC as a Function of Sample Molecular Structure	729	(6)
Appendix IV Preparing Buffered Mobile Phases	735	(5)
Appendix V Characterizing the Differences Among C(8) or C(18) Reversed-Phase Columns from Different Suppliers	740	(4)
Appendix VI Adjusting Mobile-Phase Water Content for Normal-Phase HPLC	744	(3)
Index	747