High Performance Liquid Chromatography and Capillary Electrophoresis : Principles and Practices

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ISBN: 9780121366407 | 0121366405
Cover: Hardcover
Copyright: 4/1/1997

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HPLC and CE: Principles and Practice presents the latest information on the most powerful separation techniques available: high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Fundamental theory, instrumentation, modes of operation, and optimization of separations are presented in a concise, non-technical style to help the user in choosing the appropriate technique quickly and accurately. Well- illustrated and containing convenientend-of-chapter summaries of the major concepts, the book provides in-depth coverage of trouble-shooting, improvement of resolution, data manipulation, selectivity, and sensitivity. Graduate students, technicians, and researchers who must use separations with little or no background in analytical chemistry can overcome separation anxiety and get started in obtaining the best possible separations in minimal time. The book will alsobe useful to analytical chemists who need a better understanding of theory and processes.

Preface

xiii

CHAPTER 1 High-Performance Liquid Chromatography

(23)

1.1 Introduction

(1)

1.2 Classification of Liquid Chromatographic Methods

(5)

1.2.1 Classification According to the Mechanism of Retention

(1)

1.2.2 Classification According to Operating Method

(4)

1.3 Basic Concepts of HPLC

(13)

1.3.1 Capacity Factor

(1)

1.3.2 Selectivity

(2)

1.3.3 Resolution

(1)

1.3.4 Band Broadening and Column Efficiency

(8)

1.4 Summary of Major Concepts

(1)

References

(3)

CHAPTER 2 Separations in High-Performance Liquid Chromatography

(47)

2.1 Introduction

(1)

2.2 Normal-Phase Chromotography

(5)

2.2.1 Mechanism of Retention

(1)

2.2.2 Stationary Phases for Normal-Phase Chromatography

(1)

2.2.3 Mobile Phases for Normal-Phase Chromatography

(1)

2.2.4 Practical Considerations

(1)

2.2.5 Applications for Normal-Phase Chromatography

(1)

2.3 Reversed-Phase Chromatography

(9)

2.3.1 Mechanism of Retention

(1)

2.3.2 Stationary Phases for Reversed-Phase Chromatography

(1)

2.3.3 Mobile Phases for Reversed-Phase Chromatography

(1)

2.3.4 Practical Considerations

(5)

2.3.5 Applications for Reversed-Phase Chromatography

(1)

2.4 Hydrophobic-Interaction Chromatography

(1)

2.5 Ion-Exchange Chromatography

(8)

2.5.1 Mechanism of Retention

(1)

2.5.2 Stationary Phases for Ion-Exchange Chromatography

(2)

2.5.3 Mobile Phases for Ion-Exchange Chromatography

(1)

2.5.4 Practical Considerations

(1)

2.5.5 Applications for Ion-Exchange Chromatography

(2)

2.6 Size-Exclusion Chromatography

(4)

2.6.1 Mechanism of Retention

(1)

2.6.2 Stationary Phases for Size-Exclusion Chromatography

(1)

2.6.3 Mobile Phases for Size-Exclusion Chromatography

(1)

2.6.4 Practical Considerations

(1)

2.6.5 Applications for Size-Exclusion Chromatography

(1)

2.7 Affinity Chromatography

(7)

2.7.1 Mechanism of Retention

(1)

2.7.2 Stationary Phases for Affinity Chromatography

(3)

2.7.3 Mobile Phases for Affinity Chromatography

(1)

2.7.4 Practical Considerations

(1)

2.7.5 Applications for Affinity Chromatography

(1)

2.8 Chiral Separations

(5)

2.8.1 Mechanism of Retention

(1)

2.8.2 Stationary Phases for Chiral Chromatography

(3)

2.8.3 Mobile Phases for Chiral Chromatography

(1)

2.9 Summary of Major Concepts

(5)

References

(4)

CHAPTER 3 Instrumentation for High-Performance Liquid Chromatography

(63)

3.1 Introduction

(1)

3.2 Solvent Delivery Systems

(7)

3.2.1 Pumps

(4)

3.2.2 Minimization of Pump Pulsations

(1)

3.2.3 Optimization of Flow Reproducibility

(1)

3.3 Solvent Degassing

(2)

3.3.1 External Vacuum Degassing

(1)

3.3.2 Helium Sparging

(1)

3.3.3 On-line Degassing

(1)

3.4 Isocratic versus Gradient Pumping Systems

(3)

3.4.1 Isocratic Pumping Systems

(1)

3.4.2 Gradient Pumping Systems

(3)

3.5 Sample Introduction

(2)

3.6 Columns

(5)

3.6.1 Stationary Phases

(2)

3.6.2 Pore Size

(1)

3.6.3 Particle Size

(1)

3.6.4 Internal Diameter of the Column

(1)

3.6.5 Column Length

(1)

3.6.6 Construction Materials of the Column

(1)

3.6.7 Column Care and Use

(1)

3.7 Detectors

(19)

3.7.1 Detector Properties

(1)

3.7.2 Absorbance Detectors

(7)

3.7.3 Fluorescence Detectors

(4)

3.7.4 Electrochemical Detectors

102

(3)

3.7.5 Mass Spectrometric Detectors

105

(3)

3.7.6 Other Detectors

108

(1)

3.8 Sample Preparation

109

(8)

3.8.1 Off-line Sample Preparation Techniques

109

(6)

3.8.2 On-line Sample Preparation Techniques

115

(2)

3.9 Troubleshooting

117

(12)

3.10 Summary of Major Concepts

129

(1)

References

130

(4)

CHAPTER 4 Capillary Electrophoresis

134

(20)

4.1 Introduction

134

(1)

4.2 Classification of Electrophoretic Modes

134

(2)

4.2.1 Classification According to the Electrolyte System

135

(1)

4.2.2 Classification According to the Contribution of the Electroosmotic Flow

136

(1)

4.3 Basic Concepts of Capillary Electrophoresis

136

(14)

4.3.1 Electrophoretic Mobility

137

(1)

4.3.2 Velocity

138

(1)

4.3.3 Electroosmotic Flow

138

(5)

4.3.4 Separation Efficiency

143

(7)

4.3.5 Resolution

150

(1)

4.4 Summary of Major Concepts

150

(2)

References

152

(2)

CHAPTER 5 Separation in Capillary Electrophoresis

154

(31)

5.1 Introduction

154

(1)

5.2 Capillary Zone Electrophoresis

155

(6)

5.2.1 Mechanism of Separation

155

(1)

5.2.2 Buffer Systems

155

(2)

5.2.3 Practical Considerations

157

(4)

5.3 Micellar Electrokinetic Capillary Chromatography

161

(4)

5.3.1 Mechanism of Separation

161

(2)

5.3.2 Buffers

163

(1)

5.3.3 Practical Considerations

164

(1)

5.4 Capillary Gel Electrophoresis

165

(5)

5.4.1 Mechanism of Separation

166

(2)

5.4.2 Sieving Media

168

(1)

5.4.3 Practical Considerations

169

(1)

5.5 Capillary Electrochromatography

170

(2)

5.5.1 Mechanism of Separation

170

(1)

5.5.2 Buffer Systems

171

(1)

5.5.3 Practical Considerations

171

(1)

5.6 Chiral Capillary Electrophoresis

172

(2)

5.6.1 Mechanism of Separation

172

(1)

5.6.2 Buffer Systems

172

(2)

5.6.3 Practical Considerations

174

(1)

5.7 Capillary Isoelectric Focusing

174

(4)

5.7.1 Mechanism of Separation

174

(3)

5.7.2 Buffer Systems

177

(1)

5.7.3 Practical Considerations

177

(1)

5.8 Capillary Isotachophoresis

178

(4)

5.8.1 Mechanism of Separation

179

(1)

5.8.2 Buffer Systems

179

(2)

5.8.3 Practical Considerations

181

(1)

5.9 Summary of Major Concepts

182

(1)

References

183

(2)

CHAPTER 6 Instrumentation of Capillary Electrophoresis

185

(29)

6.1 Introduction

185

(1)

6.2 High-Voltage Power Supply

186

(1)

6.3 Sample Injection

187

(3)

6.3.1 Hydrostatic Injection

187

(1)

6.3.2 Electromigration Injection

187

(2)

6.3.3 Sample Concentration

189

(1)

6.4 Capillaries

190

(3)

6.4.1 Untreated Fused Silica Capillaries

191

(1)

6.4.2 Functionalized Fused Silica Capillaries

192

(1)

6.4.3 Extended Path-Length Capillaries

192

(1)

6.5 Detection

193

(12)

6.5.1 Detector Properties

194

(1)

6.5.2 Absorbance Detectors

194

(2)

6.5.3 Fluorescence Detectors

196

(3)

6.5.4 Electrochemical Detectors

199

(3)

6.5.5 Mass Spectrometry Detectors

202

(3)

6.6 Fraction Collection

205

(1)

6.7 Sample Preparation

205

(1)

6.8 Troubleshooting

206

(4)

6.9 Summary of Major Concepts

210

(1)

References

211

(3)

CHAPTER 7 Data Manipulation

214

(26)

7.1 Introduction

214

(1)

7.2 Identification of Peaks

214

(8)

7.2.1 Use of Retention Data

214

(2)

7.2.2 Standard Addition or Spiking

216

(1)

7.2.3 Internal Standard

216

(1)

7.2.4 Isotopic Labeling

217

(1)

7.2.5 Enzyme Peak Shift

217

(3)

7.2.6 Use of Ultraviolet and Mass Spectometry Libraries

220

(2)

7.3 Quantitation

222

(17)

7.3.1 Integration

222

(10)

7.3.2 Calculation

232

(4)

7.3.3 Statistical Treatment of Data

236

(3)

7.4 Summary of Major Concepts

239

(1)

References

240

(2)

CHAPTER 8 Miniaturization

242

(33)

8.1 Introduction

242

(1)

8.2 Classification of Columns

242

(3)

8.3 Theoretical and Practical Considerations

245

(9)

8.3.1 Theoretical Considerations

245

(3)

8.3.2 Instrumental Considerations

248

(6)

8.4 Applications of Miniaturized Liquid Chromatography

254

(5)

8.5 Separations on a Chip

259

(11)

8.5.1 Fabrication of Planar Devices

259

(3)

8.5.2 Sample Introduction

262

(5)

8.5.3 Synchronized Cyclic Capillary Electrophoresis

267

(2)

8.5.4 Separation Efficiency

269

(1)

8.6 Summary of Major Concepts

270

(1)

References

271

(4)

Index

275

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High Performance Liquid Chromatography and Capillary Electrophoresis : Principles and Practices

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