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.

Author Notes

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	p. xiii
Preface	p. xv
Contributors	p. xvii
1 Introduction	p. 1
1.1 Previous Literature Which Covers MDLC	p. 4
1.2 How this Book is Organized	p. 5
References	p. 6
Part I Theory	p. 9
2 Elements of the Theory of Multidimensional Liquid Chromatography	p. 11
2.1 Introduction	p. 11
2.2 Peak Capacity	p. 13
2.3 Resolution	p. 17
2.4 Orthogonality	p. 19
2.5 Two-Dimensional Theory of Peak Overlap	p. 21
2.6 Dimensionality, Peak Ordering, and Clustering	p. 23
2.7 Theory of Zone Sampling	p. 24
2.8 Dilution and Limit of Detection	p. 26
2.9 Chemometric Analysis	p. 27
2.10 Future Directions	p. 28
References	p. 30
3 Peak Capacity in Two-Dimensional Liquid Chromatography	p. 35
3.1 Introduction	p. 35
3.2 Theory	p. 37
3.3 Procedures	p. 41
3.4 Results and Discussion	p. 42
3.5 Conclusions	p. 49
Appendix 3A Generation of Random Correlated Coordinates	p. 50
Appendix 3B Derivation of Limiting Correlation Coefficient r	p. 54
References	p. 56
4 Decoding Complex 2D Separations	p. 59
4.1 Introduction	p. 59
4.2 Fundamentals: The Statistical Description of Complex Multicomponent Separations	p. 62
4.3 Decoding 1D and 2D Multicomponent Separations by Using the SMO Poisson Statistics	p. 68
4.4 Decoding Multicomponent Separations by the Autocovariance Function	p. 74
4.5 Application to 2D Separations	p. 78
4.5.1 Results from SMO Method	p. 81
4.5.2 Results from 2D Autocovariance Function Method	p. 84
4.6 Concluding Remarks	p. 88
Acknowledgments	p. 88
References	p. 88
Part II Columns, Instrumentation and Methods Development	p. 91
5 Instrumentation for Comprehensive Multidimensional Liquid Chromatography	p. 93
5.1 Introduction	p. 93
5.2 Heart-Cutting Versus Comprehensive Mode	p. 95
5.3 Chromatographic Hardware	p. 97
5.3.1 Valves	p. 97
5.4 CE Interfaces	p. 104
5.4.1 Gated Interface for HPLC-CE	p. 104
5.4.2 Microfluidic Valves for On-Chip Multidimensional Analysis	p. 105
5.5 Columns and Combinations	p. 106
5.5.1 Column Systems, Dilution, and Splitting	p. 108
5.6 Detection	p. 109
5.7 Computer Hardware and Software	p. 109
5.7.1 Software Development	p. 110
5.7.2 Valve Sequencing	p. 111
5.7.3 Data Format and Storage	p. 113
5.8 Zone Visualization	p. 115
5.8.1 Contour Visualization	p. 115
5.8.2 2D Peak Presentation	p. 117
5.8.3 Zone Visualization in Specific Chemical (pI) Regions	p. 117
5.8.4 External Plotting Programs	p. 117
5.8.5 Difference Plots	p. 118
5.8.6 Multi-channel Data	p. 118
5.9 Data Analysis and Signal Processing	p. 119
5.10 Future Prospects	p. 120
References	p. 121
6 Method Development in Comprehensive Multidimensional Liquid Chromatography	p. 127
6.1 Introduction	p. 127
6.2 Previous Work	p. 128
6.3 Column Variables	p. 130
6.4 Method Development	p. 130
6.4.1 The Cardinal Rules of 2DLC Method Development	p. 132
6.5 Planning the Experiment	p. 143
6.6 General Comments on Optimizing the 2DLC Experiment: Speed-Resolution Trade-off	p. 143
Acknowledgment	p. 144
References	p. 144
7 Monolithic Columns and Their 2D-HPLC Applications	p. 147
7.1 Introduction	p. 147
7.2 Monolithic Polymer Columns	p. 148
7.2.1 Structural Properties of Polymer Monoliths	p. 148
7.2.2 Chromatographic Properties of Polymer Monolithic Columns	p. 150
7.2.3 Two-Dimensional HPLC Using Polymer Monoliths	p. 152
7.3 Monolithic Silica Columns	p. 153
7.3.1 Preparation	p. 154
7.3.2 Structural Properties of Monolithic Silica Columns	p. 154
7.3.3 Chromatographic Properties of Monolithic Silica Columns	p. 156
7.4 Peak Capacity Increase by Using Monolithic Silica Columns in Gradient Elution	p. 158
7.5 2D HPLC Using Monolithic Silica Columns	p. 159
7.5.1 RP-RP 2D HPLC Using Two Different Columns	p. 161
7.5.2 RP-RP 2D HPLC Using Two Similar Columns	p. 164
7.5.3 Ion Exchange-Reversed-Phase 2D HPLC Using a Monolithic Column for the 2nd-D	p. 166
7.5.4 IEX-RP 2D HPLC Using a Monolithic RP Capillary Column for the 2nd-D	p. 168
7.6 Summary and Future Improvement of 2D HPLC	p. 171
References	p. 171
8 Ultrahigh Pressure Multidimensional Liquid Chromatography	p. 177
8.1 Background: MDLC in the Jorgenson Lab	p. 177
8.1.1 Cation Exchange-Size Exclusion	p. 178
8.1.2 Anion Exchange-Reversed Phase	p. 180
8.1.3 Cation Exchange-Reversed Phase	p. 181
8.1.4 Size Exclusion-Reversed Phase	p. 183
8.2 Online Versus Off-Line MDLC	p. 188
8.3 MDLC Using Ultrahigh Pressure Liquid Chromatography: Benefits and Challenges	p. 189
8.3.1 An Introduction to UHPLC	p. 190
8.3.2 UHPLC for LC x LC: High Speed Versus High Peak Capacity	p. 191
8.3.3 LC x UHPLC for Separations of Intact Proteins	p. 191
8.4 Experimental Details	p. 193
8.4.1 Instrumentation	p. 193
8.4.2 Data Analysis	p. 194
8.4.3 Chromatographic Conditions	p. 195
8.4.4 Samples	p. 196
8.5 Results and Discussion	p. 196
8.6 Future Directions for UHP-MDLC	p. 202
References	p. 203
Part III Life Science Applications	p. 205
9 Peptidomics	p. 207
9.1 State of the Art-Why Peptidomics?	p. 207
9.2 Strategies and Solutions	p. 208
9.3 Summary and Conclusions	p. 218
References	p. 218
10 A Two-Dimensional Liquid Mass Mapping Technique for Biomarker Discovery	p. 221
10.1 Introduction	p. 221
10.2 Methods for Separating and Identifying Proteins	p. 223
10.2.1 pI-Based Methods of Separation	p. 223
10.2.2 Chromatofocusing-A Column Based pH Separation	p. 225
10.2.3 Nonporous Separation of Proteins	p. 226
10.2.4 Electrospray-Time of Flight-Mass Spectrometry	p. 228
10.2.5 MALDI Peptide Mass Fingerprinting	p. 229
10.2.6 Data Analysis and Recombination	p. 230
10.3 Applications	p. 230
10.3.1 Proteomic Mapping and Clustering of Multiple Samples-Application to Ovarian Cancer Cell Lines	p. 230
10.3.2 2D Liquid Mass Mapping of Tumor Cell Line Secreted Samples, Application to Metastasis-Associated Protein Profiles	p. 233
10.3.3 Identification Annotation and Data Correlation in MCF10 Human Breast Cancer Cell Lines	p. 235
10.4 Summary and Conclusions	p. 237
Acknowledgments	p. 238
References	p. 238
11 Coupled Multidimensional Chromatography and Tandem Mass Spectrometry Systems for Complex Peptide Mixture Analysis	p. 243
11.1 SCX-RP/MS/MS	p. 245
11.2 SCX/RP/MS/MS	p. 248
11.3 MudPIT	p. 251
11.4 Alternative First Dimension Approaches	p. 254
11.5 Conclusion	p. 255
References	p. 255
12 Development of Orthogonal 2DLC Methods for Separation of Peptides	p. 261
12.1 Introduction	p. 261
12.2 Previous Work	p. 263
12.3 Developing Orthogonal 2DLC Methods	p. 264
12.3.1 LC Selectivity for Peptides: Experimental Design	p. 264
12.3.2 Investigation of 2DLC Orthogonality for Separation of Peptides	p. 266
12.3.3 Geometric Approach to Orthogonality in 2DLC	p. 271
12.3.4 Practical 2DLC Considerations in Proteome Research	p. 275
12.3.5 Evaluation of Selected 2DLC MS/MS Systems	p. 276
12.3.6 Peak Capacity in 2DLC-MS/MS	p. 280
12.3.7 Considerations of Concentration Dynamic Range	p. 282
12.4 Conclusions	p. 284
Acknowledgment	p. 284
References	p. 284
13 Multidimensional Separation of Proteins with Online Electrospray Time-of-Flight Mass Spectrometric Detection	p. 291
13.1 Introduction	p. 291
13.2 Chromatographic Parameters	p. 293
13.3 Analyte Detection and Subsequent Analysis	p. 293
13.4 Building a Multidimensional Protein Separation	p. 294
13.4.1 Selection of an Ion-Exchange-Reversed-Phase Separation System for Protein-Level Separations	p. 295
13.4.2 Chromatographic Sorbent Considerations	p. 295
13.4.3 Chromatographic Behavior of Proteins	p. 296
13.5 Comprehensive Multidimensional Chromatographic Systems	p. 296
13.6 Coupling 2DLC with Online ESI-MS Detection	p. 299
13.6.1 Interactions between the Two Dimensions of Chromatography (Step Vs. Linear)	p. 304
13.6.2 Recognizing Increased Selectivity in 2DLC Separations	p. 306
13.7 Expanding Multidimensional Separations into a "Middle-Out" Approach to Proteomic Analysis	p. 308
13.8 Future Directions in Protein MDLC	p. 311
13.8.1 Protein Chromatography	p. 312
13.8.2 MS Analysis of Proteins	p. 313
13.8.3 Data Interpretation	p. 314
13.9 Conclusion	p. 314
References	p. 315
14 Analysis of Enantiomeric Compounds Using Multidimensional Liquid Chromatography	p. 319
14.1 Online Achiral-Chiral LC-LC	p. 320
14.2 Applications	p. 323
14.2.1 Analysis of Enantiomers in Plasma and Urine	p. 323
14.3 Amino Acids	p. 328
14.3.1 Physiological Fluids or Tissues	p. 328
14.3.2 In Food, Beverages, and Other Products	p. 333
14.4 Other Applications	p. 334
14.4.1 Analysis of Enantiomers from Plant and Environmental Sources	p. 334
14.5 Miscellaneous Applications	p. 336
14.6 Conclusion	p. 338
References	p. 339
Part IV Multidimensional Separation Using Capillary Electrophoresis	p. 345
15 Two-Dimensional Capillary Electrophoresis for the Comprehensive Analysis of Complex Protein Mixtures	p. 347
15.1 Introduction	p. 347
15.2 Previous Work	p. 348
15.2.1 Miniaturized IEF/SDS-PAGE	p. 348
15.2.2 One-Dimensional Capillary Electrophoresis for Protein Analysis	p. 349
15.3 Two-Dimensional Capillary Separations for Analysis of Peptides and Proteins	p. 352
15.3.1 Capillary Liquid Chromatography Coupled with Capillary Electrophoresis for Analysis of Unlabeled Peptides and Proteins	p. 352
15.3.2 Two-Dimensional Capillary Electrophoresis for Analysis of Proteins	p. 352
15.3.3 High-Speed Two-Dimensional Capillary Electrophoresis	p. 356
15.3.4 The Analysis of a Single Fixed Cell	p. 358
15.4 Conclusions	p. 360
15.5 Abbreviations	p. 360
References	p. 360
16 Two-Dimensional HPLC-CE Methods for Protein/Peptide Separation	p. 365
16.1 Introduction	p. 365
16.2 Off-line Versus Online	p. 366
16.3 HPLC Fractionation	p. 366
16.4 2D HPLC-CE	p. 367
16.5 CE-MS Detection	p. 368
16.6 Applications	p. 370
16.7 Concluding Remarks	p. 380
Acknowledgment	p. 381
References	p. 381
Part V Industrial Applications	p. 385
17 Multidimensional Liquid Chromatography in Industrial Applications	p. 387
17.1 Introduction	p. 387
17.2 Principles of Multidimensional Liquid Chromatography as Applied to Polymer Analysis	p. 390
17.3 Experimental	p. 393
17.4 Analysis of Alkylene Oxide-Based Polymers	p. 395
17.4.1 Amphiphilic Polyalkylene Oxides	p. 395
17.5 Excipients	p. 399
17.6 Polyether Polyols	p. 403
17.7 Analysis of Condensation Polymers	p. 406
17.8 Polyamides	p. 407
17.9 Aromatic Polyesters	p. 414
17.10 Aliphatic Polyesters	p. 417
References	p. 420
18 The Analysis of Surfactants by Multidimensional Liquid Chromatography	p. 425
18.1 Introduction	p. 425
18.2 Analytical Characterization Methods	p. 428
18.2.1 CE and CGE	p. 429
18.2.2 SEC	p. 430
18.2.3 NPLC	p. 431
18.2.4 RPLC	p. 433
18.3 Detection Methods	p. 434
18.4 2DLC	p. 434
18.4.1 RPLC Coupled to SEC	p. 435
18.4.2 NPLC Coupled to RPLC	p. 435
18.5 Conclusions	p. 442
References	p. 443
Index	p. 447

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