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Tables of Contents for Practical Hplc Method Development
Chapter/Section Title
Page #
Page Count
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