Multidimensional Liquid Chromatography

Steven A. Cohen, PhD, is Life Sciences Director, RDE of the Proteomics Technology Group at the Waters Corporation in Milford, Massachusetts. MARK R. SCHURE, PhD, is Technical Director of the Computational Chemistry Group and Technical Director of the Theoretical Separation Science Laboratory at the Rohm and Haas Company in Springhouse, Pennsylvania.

Foreword xiii

Preface xv

Contributors xvii

1 Introduction 1

1.1 Previous Literature Which Covers MDLC 4

1.2 How this Book is Organized 5

References 6

Part I Theory 9

2 Elements of the Theory of Multidimensional Liquid Chromatography 11

2.1 Introduction 11

2.2 Peak Capacity 13

2.3 Resolution 17

2.4 Orthogonality 19

2.5 Two-Dimensional Theory of Peak Overlap 21

2.6 Dimensionality, Peak Ordering, and Clustering 23

2.7 Theory of Zone Sampling 24

2.8 Dilution and Limit of Detection 26

2.9 Chemometric Analysis 27

2.10 Future Directions 28

References 30

3 Peak Capacity in Two-Dimensional Liquid Chromatography 35

3.1 Introduction 35

3.2 Theory 37

3.3 Procedures 41

3.4 Results and Discussion 42

3.5 Conclusions 49

Appendix 3A Generation of Random Correlated Coordinates 50

Appendix 3B Derivation of Limiting Correlation Coefficient r 54

References 56

4 Decoding Complex 2D Separations 59

4.1 Introduction 59

4.2 Fundamentals: The Statistical Description of Complex Multicomponent Separations 62

4.3 Decoding 1D and 2D Multicomponent Separations by Using the SMO Poisson Statistics 68

4.4 Decoding Multicomponent Separations by the Autocovariance Function 74

4.5 Application to 2D Separations 78

4.5.1 Results from SMO Method 81

4.5.2 Results from 2D Autocovariance Function Method 84

4.6 Concluding Remarks 88

Acknowledgments 88

References 88

Part II Columns, Instrumentation and Methods Development 91

5 Instrumentation for Comprehensive Multidimensional Liquid Chromatography 93

5.1 Introduction 93

5.2 Heart-Cutting Versus Comprehensive Mode 95

5.3 Chromatographic Hardware 97

5.3.1 Valves 97

5.4 CE Interfaces 104

5.4.1 Gated Interface for HPLC–CE 104

5.4.2 Microfluidic Valves for On-Chip Multidimensional Analysis 105

5.5 Columns and Combinations 106

5.5.1 Column Systems, Dilution, and Splitting 108

5.6 Detection 109

5.7 Computer Hardware and Software 109

5.7.1 Software Development 110

5.7.2 Valve Sequencing 111

5.7.3 Data Format and Storage 113

5.8 Zone Visualization 115

5.8.1 Contour Visualization 115

5.8.2 2D Peak Presentation 117

5.8.3 Zone Visualization in Specific Chemical (pI) Regions 117

5.8.4 External Plotting Programs 117

5.8.5 Difference Plots 118

5.8.6 Multi-channel Data 118

5.9 Data Analysis and Signal Processing 119

5.10 Future Prospects 120

References 121

6 Method Development in Comprehensive Multidimensional Liquid Chromatography 127

6.1 Introduction 127

6.2 Previous Work 128

6.3 Column Variables 130

6.4 Method Development 130

6.4.1 The Cardinal Rules of 2DLC Method Development 132

6.5 Planning the Experiment 143

6.6 General Comments on Optimizing the 2DLC Experiment: Speed–Resolution Trade-off 143

Acknowledgment 144

References 144

7 Monolithic Columns and Their 2D-HPLC Applications 147

7.1 Introduction 147

7.2 Monolithic Polymer Columns 148

7.2.1 Structural Properties of Polymer Monoliths 148

7.2.2 Chromatographic Properties of Polymer Monolithic Columns 150

7.2.3 Two-Dimensional HPLC Using Polymer Monoliths 152

7.3 Monolithic Silica Columns 153

7.3.1 Preparation 154

7.3.2 Structural Properties of Monolithic Silica Columns 154

7.3.3 Chromatographic Properties of Monolithic Silica Columns 156

7.4 Peak Capacity Increase by Using Monolithic Silica Columns in Gradient Elution 158

7.5 2D HPLC Using Monolithic Silica Columns 159

7.5.1 RP-RP 2D HPLC Using Two Different Columns 161

7.5.2 RP–RP 2D HPLC Using Two Similar Columns 164

7.5.3 Ion Exchange–Reversed-Phase 2D HPLC Using a Monolithic Column for the 2nd-D 166

7.5.4 IEX-RP 2D HPLC Using a Monolithic RP Capillary Column for the 2nd-D 168

7.6 Summary and Future Improvement of 2D HPLC 171

References 171

8 Ultrahigh Pressure Multidimensional Liquid Chromatography 177

8.1 Background: MDLC in the Jorgenson Lab 177

8.1.1 Cation Exchange–Size Exclusion 178

8.1.2 Anion Exchange–Reversed Phase 180

8.1.3 Cation Exchange–Reversed Phase 181

8.1.4 Size Exclusion–Reversed Phase 183

8.2 Online Versus Off-Line MDLC 188

8.3 MDLC Using Ultrahigh Pressure Liquid Chromatography: Benefits and Challenges 189

8.3.1 An Introduction to UHPLC 190

8.3.2 UHPLC for LC LC: High Speed Versus High Peak Capacity 191

8.3.3 LC UHPLC for Separations of Intact Proteins 191

8.4 Experimental Details 193

8.4.1 Instrumentation 193

8.4.2 Data Analysis 194

8.4.3 Chromatographic Conditions 195

8.4.4 Samples 196

8.5 Results and Discussion 196

8.6 Future Directions for UHP-MDLC 202

References 203

Part III Life Science Applications 205

9 Peptidomics 207

9.1 State of the Art—Why Peptidomics? 207

9.2 Strategies and Solutions 208

9.3 Summary and Conclusions 218

References 218

10 A Two-Dimensional Liquid Mass Mapping Technique for Biomarker Discovery 221

10.1 Introduction 221

10.2 Methods for Separating and Identifying Proteins 223

10.2.1 pI-Based Methods of Separation 223

10.2.2 Chromatofocusing-A Column Based pH Separation 225

10.2.3 Nonporous Separation of Proteins 226

10.2.4 Electrospray-Time of Flight-Mass Spectrometry 228

10.2.5 MALDI Peptide Mass Fingerprinting 229

10.2.6 Data Analysis and Recombination 230

10.3 Applications 230

10.3.1 Proteomic Mapping and Clustering of Multiple Samples—Application to Ovarian Cancer Cell Lines 230

10.3.2 2D Liquid Mass Mapping of Tumor Cell Line Secreted Samples, Application to Metastasis-Associated Protein Profiles 233

10.3.3 Identification Annotation and Data Correlation in MCF10 Human Breast Cancer Cell Lines 235

10.4 Summary and Conclusions 237

Acknowledgments 238

References 238

11 Coupled Multidimensional Chromatography and Tandem Mass Spectrometry Systems for Complex Peptide Mixture Analysis 243

11.1 Scx-rp/ms/ms 245

11.2 Scx/rp/ms/ms 248

11.3 MudPIT 251

11.4 Alternative First Dimension Approaches 254

11.5 Conclusion 255

References 255

12 Development of Orthogonal 2DLC Methods for Separation of Peptides 261

12.1 Introduction 261

12.2 Previous Work 263

12.3 Developing Orthogonal 2DLC Methods 264

12.3.1 LC Selectivity for Peptides: Experimental Design 264

12.3.2 Investigation of 2DLC Orthogonality for Separation of Peptides 266

12.3.3 Geometric Approach to Orthogonality in 2DLC 271

12.3.4 Practical 2DLC Considerations in Proteome Research 275

12.3.5 Evaluation of Selected 2DLC MS/MS Systems 276

12.3.6 Peak Capacity in 2DLC-MS/MS 280

12.3.7 Considerations of Concentration Dynamic Range 282

12.4 Conclusions 284

Acknowledgment 284

References 284

13 Multidimensional Separation of Proteins with Online Electrospray Time-of-Flight Mass Spectrometric Detection 291

13.1 Introduction 291

13.2 Chromatographic Parameters 293

13.3 Analyte Detection and Subsequent Analysis 293

13.4 Building a Multidimensional Protein Separation 294

13.4.1 Selection of an Ion-Exchange–Reversed-Phase Separation System for Protein-Level Separations 295

13.4.2 Chromatographic Sorbent Considerations 295

13.4.3 Chromatographic Behavior of Proteins 296

13.5 Comprehensive Multidimensional Chromatographic Systems 296

13.6 Coupling 2DLC with Online ESI–MS Detection 299

13.6.1 Interactions between the Two Dimensions of Chromatography (Step Vs. Linear) 304

13.6.2 Recognizing Increased Selectivity in 2DLC Separations 306

13.7 Expanding Multidimensional Separations into a “Middle-Out” Approach to Proteomic Analysis 308

13.8 Future Directions in Protein MDLC 311

13.8.1 Protein Chromatography 312

13.8.2 MS Analysis of Proteins 313

13.8.3 Data Interpretation 314

13.9 Conclusion 314

References 315

14 Analysis of Enantiomeric Compounds Using Multidimensional Liquid Chromatography 319

14.1 Online Achiral-Chiral LC-LC 320

14.2 Applications 323

14.2.1 Analysis of Enantiomers in Plasma and Urine 323

14.3 Amino Acids 328

14.3.1 Physiological Fluids or Tissues 328

14.3.2 In Food, Beverages, and Other Products 333

14.4 Other Applications 334

14.4.1 Analysis of Enantiomers from Plant and Environmental Sources 334

14.5 Miscellaneous Applications 336

14.6 Conclusion 338

References 339

Part IV Multidimensional Separation Using Capillary Electrophoresis 345

15 Two-Dimensional Capillary Electrophoresis for the Comprehensive Analysis of Complex Protein Mixtures 347

15.1 Introduction 347

15.2 Previous Work 348

15.2.1 Miniaturized IEF/SDS-PAGE 348

15.2.2 One-Dimensional Capillary Electrophoresis for Protein Analysis 349

15.3 Two-Dimensional Capillary Separations for Analysis of Peptides and Proteins 352

15.3.1 Capillary Liquid Chromatography Coupled with Capillary Electrophoresis for Analysis of Unlabeled Peptides and Proteins 352

15.3.2 Two-Dimensional Capillary Electrophoresis for Analysis of Proteins 352

15.3.3 High-Speed Two-Dimensional Capillary Electrophoresis 356

15.3.4 The Analysis of a Single Fixed Cell 358

15.4 Conclusions 360

15.5 Abbreviations 360

References 360

16 Two-Dimensional HPLC–CE Methods for Protein/Peptide Separation 365

16.1 Introduction 365

16.2 Off-line Versus Online 366

16.3 HPLC Fractionation 366

16.4 2d Hplc–ce 367

16.5 CE–MS Detection 368

16.6 Applications 370

16.7 Concluding Remarks 380

Acknowledgment 381

References 381

Part V Industrial Applications 385

17 Multidimensional Liquid Chromatography in Industrial Applications 387

17.1 Introduction 387

17.2 Principles of Multidimensional Liquid Chromatography as Applied to Polymer Analysis 390

17.3 Experimental 393

17.4 Analysis of Alkylene Oxide-Based Polymers 395

17.4.1 Amphiphilic Polyalkylene Oxides 395

17.5 Excipients 399

17.6 Polyether Polyols 403

17.7 Analysis of Condensation Polymers 406

17.8 Polyamides 407

17.9 Aromatic Polyesters 414

17.10 Aliphatic Polyesters 417

References 420

18 The Analysis of Surfactants by Multidimensional Liquid Chromatography 425

18.1 Introduction 425

18.2 Analytical Characterization Methods 428

18.2.1 CE and CGE 429

18.2.2 Sec 430

18.2.3 Nplc 431

18.2.4 Rplc 433

18.3 Detection Methods 434

18.4 2dlc 434

18.4.1 RPLC Coupled to SEC 435

18.4.2 NPLC Coupled to RPLC 435

18.5 Conclusions 442

References 443

Index 447