Multidimensional Liquid Chromatography: Theory and Applications in Industrial Chemistry and the Life Sciences

Multidimensional Liquid Chromatography (MDLC) is a very powerful separation technique for analyzing exceptionally complex samples in one step. This authoritative reference presents a number of recent contributions that help define the current art and science of MDLC. Topics covered include instrumentation, theory, methods development, and applications of MDLC in the life sciences and in industrial chemistry. With the information to help you perform very difficult separations of complex samples, this reference includes chapters contributed by leading experts or teams of experts.

Table of Contents:
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

About the Author :
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.

Review :
"It is a timely publication and present a valuable resource of scientific information on MDLC…It presents systematically gathered scientific information from a plethora of articles scattered over a wide range of sources. This effort should be appreciated by a wide audience of scientists and researchers who deal with complex separation programs in biomedical, environmental, and natural products; industrial polymers; food and other sources." (Journal of the American Chemical Society, November 12, 2008)