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 xiiiPreface xvContributors xvii1 Introduction 11.1 Previous Literature Which Covers MDLC 41.2 How this Book is Organized 5References 6Part I Theory 92 Elements of the Theory of Multidimensional Liquid Chromatography 112.1 Introduction 112.2 Peak Capacity 132.3 Resolution 172.4 Orthogonality 192.5 Two-Dimensional Theory of Peak Overlap 212.6 Dimensionality, Peak Ordering, and Clustering 232.7 Theory of Zone Sampling 242.8 Dilution and Limit of Detection 262.9 Chemometric Analysis 272.10 Future Directions 28References 303 Peak Capacity in Two-Dimensional Liquid Chromatography 353.1 Introduction 353.2 Theory 373.3 Procedures 413.4 Results and Discussion 423.5 Conclusions 49Appendix 3A Generation of Random Correlated Coordinates 50Appendix 3B Derivation of Limiting Correlation Coefficient r 54References 564 Decoding Complex 2D Separations 594.1 Introduction 594.2 Fundamentals: The Statistical Description of Complex Multicomponent Separations 624.3 Decoding 1D and 2D Multicomponent Separations by Using the SMO Poisson Statistics 684.4 Decoding Multicomponent Separations by the Autocovariance Function 744.5 Application to 2D Separations 784.5.1 Results from SMO Method 814.5.2 Results from 2D Autocovariance Function Method 844.6 Concluding Remarks 88Acknowledgments 88References 88Part II Columns, Instrumentation and Methods Development 915 Instrumentation for Comprehensive Multidimensional Liquid Chromatography 935.1 Introduction 935.2 Heart-Cutting Versus Comprehensive Mode 955.3 Chromatographic Hardware 975.3.1 Valves 975.4 CE Interfaces 1045.4.1 Gated Interface for HPLC–CE 1045.4.2 Microfluidic Valves for On-Chip Multidimensional Analysis 1055.5 Columns and Combinations 1065.5.1 Column Systems, Dilution, and Splitting 1085.6 Detection 1095.7 Computer Hardware and Software 1095.7.1 Software Development 1105.7.2 Valve Sequencing 1115.7.3 Data Format and Storage 1135.8 Zone Visualization 1155.8.1 Contour Visualization 1155.8.2 2D Peak Presentation 1175.8.3 Zone Visualization in Specific Chemical (pI) Regions 1175.8.4 External Plotting Programs 1175.8.5 Difference Plots 1185.8.6 Multi-channel Data 1185.9 Data Analysis and Signal Processing 1195.10 Future Prospects 120References 1216 Method Development in Comprehensive Multidimensional Liquid Chromatography 1276.1 Introduction 1276.2 Previous Work 1286.3 Column Variables 1306.4 Method Development 1306.4.1 The Cardinal Rules of 2DLC Method Development 1326.5 Planning the Experiment 1436.6 General Comments on Optimizing the 2DLC Experiment: Speed–Resolution Trade-off 143Acknowledgment 144References 1447 Monolithic Columns and Their 2D-HPLC Applications 1477.1 Introduction 1477.2 Monolithic Polymer Columns 1487.2.1 Structural Properties of Polymer Monoliths 1487.2.2 Chromatographic Properties of Polymer Monolithic Columns 1507.2.3 Two-Dimensional HPLC Using Polymer Monoliths 1527.3 Monolithic Silica Columns 1537.3.1 Preparation 1547.3.2 Structural Properties of Monolithic Silica Columns 1547.3.3 Chromatographic Properties of Monolithic Silica Columns 1567.4 Peak Capacity Increase by Using Monolithic Silica Columns in Gradient Elution 1587.5 2D HPLC Using Monolithic Silica Columns 1597.5.1 RP-RP 2D HPLC Using Two Different Columns 1617.5.2 RP–RP 2D HPLC Using Two Similar Columns 1647.5.3 Ion Exchange–Reversed-Phase 2D HPLC Using a Monolithic Column for the 2nd-D 1667.5.4 IEX-RP 2D HPLC Using a Monolithic RP Capillary Column for the 2nd-D 1687.6 Summary and Future Improvement of 2D HPLC 171References 1718 Ultrahigh Pressure Multidimensional Liquid Chromatography 1778.1 Background: MDLC in the Jorgenson Lab 1778.1.1 Cation Exchange–Size Exclusion 1788.1.2 Anion Exchange–Reversed Phase 1808.1.3 Cation Exchange–Reversed Phase 1818.1.4 Size Exclusion–Reversed Phase 1838.2 Online Versus Off-Line MDLC 1888.3 MDLC Using Ultrahigh Pressure Liquid Chromatography: Benefits and Challenges 1898.3.1 An Introduction to UHPLC 1908.3.2 UHPLC for LC LC: High Speed Versus High Peak Capacity 1918.3.3 LC UHPLC for Separations of Intact Proteins 1918.4 Experimental Details 1938.4.1 Instrumentation 1938.4.2 Data Analysis 1948.4.3 Chromatographic Conditions 1958.4.4 Samples 1968.5 Results and Discussion 1968.6 Future Directions for UHP-MDLC 202References 203Part III Life Science Applications 2059 Peptidomics 2079.1 State of the Art—Why Peptidomics? 2079.2 Strategies and Solutions 2089.3 Summary and Conclusions 218References 21810 A Two-Dimensional Liquid Mass Mapping Technique for Biomarker Discovery 22110.1 Introduction 22110.2 Methods for Separating and Identifying Proteins 22310.2.1 pI-Based Methods of Separation 22310.2.2 Chromatofocusing-A Column Based pH Separation 22510.2.3 Nonporous Separation of Proteins 22610.2.4 Electrospray-Time of Flight-Mass Spectrometry 22810.2.5 MALDI Peptide Mass Fingerprinting 22910.2.6 Data Analysis and Recombination 23010.3 Applications 23010.3.1 Proteomic Mapping and Clustering of Multiple Samples—Application to Ovarian Cancer Cell Lines 23010.3.2 2D Liquid Mass Mapping of Tumor Cell Line Secreted Samples, Application to Metastasis-Associated Protein Profiles 23310.3.3 Identification Annotation and Data Correlation in MCF10 Human Breast Cancer Cell Lines 23510.4 Summary and Conclusions 237Acknowledgments 238References 23811 Coupled Multidimensional Chromatography and Tandem Mass Spectrometry Systems for Complex Peptide Mixture Analysis 24311.1 Scx-rp/ms/ms 24511.2 Scx/rp/ms/ms 24811.3 MudPIT 25111.4 Alternative First Dimension Approaches 25411.5 Conclusion 255References 25512 Development of Orthogonal 2DLC Methods for Separation of Peptides 26112.1 Introduction 26112.2 Previous Work 26312.3 Developing Orthogonal 2DLC Methods 26412.3.1 LC Selectivity for Peptides: Experimental Design 26412.3.2 Investigation of 2DLC Orthogonality for Separation of Peptides 26612.3.3 Geometric Approach to Orthogonality in 2DLC 27112.3.4 Practical 2DLC Considerations in Proteome Research 27512.3.5 Evaluation of Selected 2DLC MS/MS Systems 27612.3.6 Peak Capacity in 2DLC-MS/MS 28012.3.7 Considerations of Concentration Dynamic Range 28212.4 Conclusions 284Acknowledgment 284References 28413 Multidimensional Separation of Proteins with Online Electrospray Time-of-Flight Mass Spectrometric Detection 29113.1 Introduction 29113.2 Chromatographic Parameters 29313.3 Analyte Detection and Subsequent Analysis 29313.4 Building a Multidimensional Protein Separation 29413.4.1 Selection of an Ion-Exchange–Reversed-Phase Separation System for Protein-Level Separations 29513.4.2 Chromatographic Sorbent Considerations 29513.4.3 Chromatographic Behavior of Proteins 29613.5 Comprehensive Multidimensional Chromatographic Systems 29613.6 Coupling 2DLC with Online ESI–MS Detection 29913.6.1 Interactions between the Two Dimensions of Chromatography (Step Vs. Linear) 30413.6.2 Recognizing Increased Selectivity in 2DLC Separations 30613.7 Expanding Multidimensional Separations into a “Middle-Out” Approach to Proteomic Analysis 30813.8 Future Directions in Protein MDLC 31113.8.1 Protein Chromatography 31213.8.2 MS Analysis of Proteins 31313.8.3 Data Interpretation 31413.9 Conclusion 314References 31514 Analysis of Enantiomeric Compounds Using Multidimensional Liquid Chromatography 31914.1 Online Achiral-Chiral LC-LC 32014.2 Applications 32314.2.1 Analysis of Enantiomers in Plasma and Urine 32314.3 Amino Acids 32814.3.1 Physiological Fluids or Tissues 32814.3.2 In Food, Beverages, and Other Products 33314.4 Other Applications 33414.4.1 Analysis of Enantiomers from Plant and Environmental Sources 33414.5 Miscellaneous Applications 33614.6 Conclusion 338References 339Part IV Multidimensional Separation Using Capillary Electrophoresis 34515 Two-Dimensional Capillary Electrophoresis for the Comprehensive Analysis of Complex Protein Mixtures 34715.1 Introduction 34715.2 Previous Work 34815.2.1 Miniaturized IEF/SDS-PAGE 34815.2.2 One-Dimensional Capillary Electrophoresis for Protein Analysis 34915.3 Two-Dimensional Capillary Separations for Analysis of Peptides and Proteins 35215.3.1 Capillary Liquid Chromatography Coupled with Capillary Electrophoresis for Analysis of Unlabeled Peptides and Proteins 35215.3.2 Two-Dimensional Capillary Electrophoresis for Analysis of Proteins 35215.3.3 High-Speed Two-Dimensional Capillary Electrophoresis 35615.3.4 The Analysis of a Single Fixed Cell 35815.4 Conclusions 36015.5 Abbreviations 360References 36016 Two-Dimensional HPLC–CE Methods for Protein/Peptide Separation 36516.1 Introduction 36516.2 Off-line Versus Online 36616.3 HPLC Fractionation 36616.4 2d Hplc–ce 36716.5 CE–MS Detection 36816.6 Applications 37016.7 Concluding Remarks 380Acknowledgment 381References 381Part V Industrial Applications 38517 Multidimensional Liquid Chromatography in Industrial Applications 38717.1 Introduction 38717.2 Principles of Multidimensional Liquid Chromatography as Applied to Polymer Analysis 39017.3 Experimental 39317.4 Analysis of Alkylene Oxide-Based Polymers 39517.4.1 Amphiphilic Polyalkylene Oxides 39517.5 Excipients 39917.6 Polyether Polyols 40317.7 Analysis of Condensation Polymers 40617.8 Polyamides 40717.9 Aromatic Polyesters 41417.10 Aliphatic Polyesters 417References 42018 The Analysis of Surfactants by Multidimensional Liquid Chromatography 42518.1 Introduction 42518.2 Analytical Characterization Methods 42818.2.1 CE and CGE 42918.2.2 Sec 43018.2.3 Nplc 43118.2.4 Rplc 43318.3 Detection Methods 43418.4 2dlc 43418.4.1 RPLC Coupled to SEC 43518.4.2 NPLC Coupled to RPLC 43518.5 Conclusions 442References 443Index 447

"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)