Mixed Mode Chromatography Principles, Methods, and

Applications

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For Hong, Rose, Andrew, and Ada
Preface

Since the FDA approved the first monoclonal antibody drug in 1986, the therapeutic
antibody drug market has experienced explosive growth. New antibody drugs have
been approved to treat a variety of human diseases, including many cancers,
autoimmune, metabolic, and infectious diseases. The manufacture of therapeutic
antibody drugs usually involves a lengthy and expensive purification process based
on protein A affinity chromatography. However, protein A chromatography has two
main problems: ligands are easily cleaved by proteases present in cell lysates, and
purified antibodies tend to aggregate under the low pH conditions required for
elution. Therefore, considerable efforts have been made to mitigate these adverse
effects.
Mixed-mode chromatography has emerged to simulate protein A chromatogra-
phy for antibody purification. Synthetic ligands with multiple functional groups are
used to replace natural biomolecules and bind to target molecules through a combi-
nation of hydrophobic, electrostatic, and hydrogen bonding interactions. Binding
affinity and selectivity can be optimized by following the principles of molecular
recognition, such as multivalency, complementarity, and geometric fitting. This
book tends to introduce the principles, methods, and applications of mixed-mode
chromatography. It starts from a historical perspective covering the development of
chromatography in general and mixed-mode chromatography in particular. Then,
theoretical considerations are provided, focusing on the mass transfer and band
broadening in the packed bed, and the different separation modes and packing
materials are described in detail. The following chapters deal with ligand design,
retention mechanisms, stationary phases, and mobile phases in mixed-mode chro-
matography. A large number of examples are used to explain and illustrate the
method development and application in biopharmaceutical manufacturing.
I would like to thank Tianjin University for its continuous strong support and the
Springer Nature book project team, especially Lewis Liu and Kavitha Jayakumar, for
their suggestions during the writing of this book.

Tianjin, China Qian-Hong Wan

July 08, 2021
vii
Contents

1 Historical Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Separation Technology Before Chromatography . . . . . . . . . . . 2
1.3 Birth of Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.3.1 Adsorption Chromatography . . . . . . . . . . . . . . . . . . . 5
1.3.2 Partition Chromatography . . . . . . . . . . . . . . . . . . . . . 8
1.4 Development of Chromatography . . . . . . . . . . . . . . . . . . . . . . 9
1.4.1 Thin-Layer Chromatography . . . . . . . . . . . . . . . . . . . 9
1.4.2 Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 11
1.4.3 Size Exclusion Chromatography . . . . . . . . . . . . . . . . 11
1.4.4 Ion Exchange Chromatography . . . . . . . . . . . . . . . . . 12
1.4.5 Affinity Chromatography . . . . . . . . . . . . . . . . . . . . . 13
1.4.6 Chiral Chromatography . . . . . . . . . . . . . . . . . . . . . . 13
1.5 High-Performance Liquid Chromatography . . . . . . . . . . . . . . . 14
1.6 Mixed-Mode Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 16
1.6.1 Mixed-Mode Chromatography of Nucleic Acids . . . . . 18
1.6.2 Mixed-Mode Chromatography of Proteins . . . . . . . . . 22
1.6.3 Mixed-Mode Chromatography of Small Molecules . . . 28
1.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2 Theoretical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.2 Chromatographic Process . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.3 Solute Retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.4 Solute Zone Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.5 Flow Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.5.1 Packing Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.5.2 Permeability of the Packing Structure . . . . . . . . . . . . 47
2.5.3 Capillary Model of Seepage in Porous Media . . . . . . . 48

ix
x Contents

2.5.4 Flow Channels in Porous Media . . . . . . . . . . . . . . . . 49

2.5.5 Flow Velocity Distribution . . . . . . . . . . . . . . . . . . . . 50
2.6 Molecular Diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.6.1 Diffusion in Solution . . . . . . . . . . . . . . . . . . . . . . . . 52
2.6.2 Diffusion in Porous Media . . . . . . . . . . . . . . . . . . . . 56
2.7 Chromatographic Peak Spreading . . . . . . . . . . . . . . . . . . . . . . 60
2.7.1 Plate Height Equation . . . . . . . . . . . . . . . . . . . . . . . . 61
2.7.2 Reduced Plate Height Equation . . . . . . . . . . . . . . . . . 66
2.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3 Separation Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
3.2 Normal Phase Chromatography . . . . . . . . . . . . . . . . . . . . . . . 71
3.3 Reversed-Phase Chromatography . . . . . . . . . . . . . . . . . . . . . . 76
3.4 Size Exclusion Chromatography . . . . . . . . . . . . . . . . . . . . . . 85
3.5 Ion Exchange Chromatography . . . . . . . . . . . . . . . . . . . . . . . 88
3.6 Hydrophobic Interaction Chromatography . . . . . . . . . . . . . . . 92
3.7 Hydrophilic Interaction Chromatography . . . . . . . . . . . . . . . . 97
3.8 Coordination Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 100
3.9 Affinity Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
3.10 Chiral Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
3.11 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4 Chromatographic Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
4.2 Silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.2.1 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.2.2 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.2.3 Structural Characterization and Evaluation . . . . . . . . . 142
4.2.4 Surface Modification and Functionalization . . . . . . . . 143
4.3 Hydroxyapatite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.3.1 Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
4.3.2 Crystal Structure and Properties . . . . . . . . . . . . . . . . . 149
4.3.3 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
4.4 Porous Glass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.4.1 Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.4.2 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4.4.3 Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
4.5 Synthetic Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
4.5.1 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
4.5.2 Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
4.5.3 Surface Functionalization . . . . . . . . . . . . . . . . . . . . . 161
4.5.4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Contents xi

4.6 Agarose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

4.6.1 Structure and Performance . . . . . . . . . . . . . . . . . . . . 165
4.6.2 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
4.7 Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
4.7.1 Dissolution and Regeneration . . . . . . . . . . . . . . . . . . 168
4.7.2 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
4.8 Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4.8.1 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4.9 Surface Functionalization of Polysaccharide Matrix . . . . . . . . . 171
4.9.1 Direct Coupling Method . . . . . . . . . . . . . . . . . . . . . . 171
4.9.2 Activation Coupling Method . . . . . . . . . . . . . . . . . . . 172
4.9.3 Spacer Coupling Method . . . . . . . . . . . . . . . . . . . . . 174
4.10 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
5 Ligand Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
5.2 Fragment Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
5.3 Combinatorial Peptide Library . . . . . . . . . . . . . . . . . . . . . . . . 189
5.4 Ugi Multicomponent Reaction . . . . . . . . . . . . . . . . . . . . . . . . 192
5.5 Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
5.6 Oligonucleotide Aptamer by SELEX . . . . . . . . . . . . . . . . . . . 202
5.7 Rational Ligand Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
5.7.1 Molecular Docking . . . . . . . . . . . . . . . . . . . . . . . . . . 207
5.7.2 Molecular Dynamics Simulation . . . . . . . . . . . . . . . . 210
5.7.3 Rational Design of Ligands . . . . . . . . . . . . . . . . . . . . 212
5.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
6 Retention Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
6.2 Retention Mechanism I: Cox Model . . . . . . . . . . . . . . . . . . . . 225
6.2.1 Ion Exchange Equilibrium of Silanol Groups . . . . . . . 225
6.2.2 Coupled Ionic and Hydrophobic Interaction . . . . . . . . 226
6.2.3 Experimental Verification . . . . . . . . . . . . . . . . . . . . . 229
6.3 Retention Mechanism II: Horvath Model . . . . . . . . . . . . . . . . 234
6.3.1 Electrostatic Interaction Energy . . . . . . . . . . . . . . . . . 235
6.3.2 Hydrophobic Interaction Energy . . . . . . . . . . . . . . . . 236
6.3.3 Coupling of Electrostatic and Hydrophobic
Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
6.3.4 The Effect of pH on the Charge Density of the
Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
6.3.5 Experimental Verification . . . . . . . . . . . . . . . . . . . . . 239
6.4 Retention Mechanism III: Carr Model . . . . . . . . . . . . . . . . . . 241
6.4.1 “Pure” Ion Exchange (IEX) Retention . . . . . . . . . . . . 242
xii Contents

6.4.2 “Pure” Reversed-Phase (RP) Retention . . . . . . . . . . . 243

6.4.3 Single-Point Model for Mixed-Mode Retention . . . . . 243
6.4.4 Two-Point Model for Mixed-Mode Retention . . . . . . . 244
6.4.5 Analysis of Plots of log k vs. 1/[C+]m . . . . . . . . . . . . 245
6.4.6 Experimental Verification . . . . . . . . . . . . . . . . . . . . . 246
6.5 Retention Mechanism IV: Wan Model . . . . . . . . . . . . . . . . . . 251
6.5.1 Multivalency and Cooperativity . . . . . . . . . . . . . . . . . 252
6.5.2 Experimental Verification . . . . . . . . . . . . . . . . . . . . . 256
6.6 Retention Mechanism V: Hydrophilic Ion Exchange Model . . . 262
6.7 Retention Mechanism VI: Repulsion Hydrophilic Interaction
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
6.8 Retention Mechanism VII: Coordination Ion Exchange
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
6.9 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
7 Stationary Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
7.2 Design Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
7.2.1 Multivalent Binding . . . . . . . . . . . . . . . . . . . . . . . . . 280
7.2.2 Functional Complementarity . . . . . . . . . . . . . . . . . . . 281
7.2.3 Geometric Fit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
7.2.4 Ligand-Solute Reciprocity . . . . . . . . . . . . . . . . . . . . 282
7.3 Classification of Ligands Used in Mixed-Mode
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
7.3.1 Binary Mixed-Mode Chromatography Ligands . . . . . . 283
7.3.2 Multiple Mixed-Mode Ligands . . . . . . . . . . . . . . . . . 286
7.4 Silica-Based Stationary Phases . . . . . . . . . . . . . . . . . . . . . . . . 287
7.5 Polysaccharide-Based Stationary Phases . . . . . . . . . . . . . . . . . 301
7.6 Commercial Mixed-Mode Chromatography Stationary
Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
7.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
8 Mobile Phases and Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
8.2 The Composition of the Mobile Phase . . . . . . . . . . . . . . . . . . 322
8.2.1 Polar Organic Solvents . . . . . . . . . . . . . . . . . . . . . . . 322
8.2.2 Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
8.2.3 Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
8.2.4 Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
8.3 Effect of Mobile Phase Parameters on Binding and Elution . . . 337
8.3.1 Effect of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
8.3.2 Effect of Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . 339
8.3.3 Effect of Organic Solvent . . . . . . . . . . . . . . . . . . . . . 341
8.3.4 Effect of Additive . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Contents xiii

8.4 Washing and Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

8.4.1 Elution by pH Change . . . . . . . . . . . . . . . . . . . . . . . 350
8.4.2 Elution by Ionic Strength Change . . . . . . . . . . . . . . . 353
8.4.3 Elution by Polarity Change . . . . . . . . . . . . . . . . . . . . 354
8.4.4 Elution by Additives . . . . . . . . . . . . . . . . . . . . . . . . . 355
8.4.5 Specific Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
8.4.6 Elution by Selective Displacement
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
8.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
9 Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
9.2 Method Development in Hydroxyapatite Chromatography . . . . 372
9.2.1 General Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
9.2.2 Examples of Method Development . . . . . . . . . . . . . . 376
9.3 Method Development in Hydrophobic Charge Induction
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
9.3.1 Factors That Influence Protein Adsorption
and Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
9.3.2 Examples of Method Development . . . . . . . . . . . . . . 391
9.4 Method Development in Multimodal Ion Exchange
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
9.4.1 General Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
9.4.2 Examples of Method Development . . . . . . . . . . . . . . 405
9.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
10 Biopharmaceutical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 423
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
10.2 Separation and Purification of Oligonucleotide Drugs . . . . . . . 424
10.3 Separation and Purification of Plasmid DNA . . . . . . . . . . . . . . 428
10.3.1 Topological Structure of Plasmid DNA . . . . . . . . . . . 431
10.3.2 Industrial Scale Preparation of Plasmid DNA . . . . . . . 433
10.3.3 Separation and Purification of Plasmid DNA . . . . . . . 434
10.4 Separation and Purification of Minicircle DNA . . . . . . . . . . . . 445
10.5 Separation and Purification of Antibody Drugs . . . . . . . . . . . . 451
10.5.1 Purification of Antibody Drugs . . . . . . . . . . . . . . . . . 453
10.6 Separation and Purification of Antibody Fragments . . . . . . . . . 465
10.7 Purification of Recombinant Protein Drugs . . . . . . . . . . . . . . . 465
10.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
Chapter 1
Historical Perspectives

Abstract Chromatography is a technique that uses the difference in the partition

coefficient of a compound between the stationary phase and the mobile phase to
separate the mixture. The stationary phase is a solid-phase carrier that is immobilized
during the separation process and its ligands loaded on the surface of the carrier. The
mobile phase is a fluid that moves relative to the stationary phase during the
separation process and is mainly used to carry solute molecules through the station-
ary phase. In the course of more than 100 years of development, chromatographic
technology has formed two major branches of liquid chromatography and gas
chromatography. The mobile phase of the former is liquid and the latter is gas.
Liquid chromatography can be divided into normal phase chromatography, reverse-
phase chromatography, ion exchange chromatography, and affinity chromatography
according to the separation mechanism. In recent years, the rapid development of
mixed-mode chromatography has generated considerable interest because it has a
different mechanism of action and application characteristics than other chromato-
graphic modes. This chapter will review the birth and development of chromatog-
raphy focusing on mixed-mode chromatography.

1.1 Introduction

Chromatography is a technique that uses the difference in the partition coefficient of

a compound between the stationary phase and the mobile phase to separate the
mixture. The so-called stationary phase refers to the solid-phase carrier that is
immobile during the separation process and its ligands loaded on the surface of the
carrier. The mobile phase refers to the fluid that moves relative to the stationary
phase during the separation process, which can be liquid or gas. According to the
state of the fluid used, chromatography can be divided into three important branches:
liquid chromatography, gas chromatography, and supercritical fluid chromatogra-
phy. According to the type of interaction involved in the separation process,
chromatography can be divided into normal phase chromatography, reverse-phase
chromatography, ion exchange chromatography, affinity chromatography, and so
on. According to the way the separation process is realized, chromatography can be

© Springer Nature Singapore Pte Ltd. 2021 1

Q.-H. Wan, Mixed-Mode Chromatography,
https://doi.org/10.1007/978-981-16-5485-5_1
2 1 Historical Perspectives

divided into packed-column chromatography, open-tubular column chromatogra-

phy, electrochromatography, thin-layer chromatography, continuous chromatogra-
phy, multidimensional chromatography, and so on. According to the scale of
application, chromatography can be divided into two major branches: analytical
chromatography and preparative chromatography. According to the application
field, chromatography can be divided into ion chromatography, chiral chromatogra-
phy, protein chromatography, nucleic acid chromatography, and so
on. Chromatography can also be used in conjunction with other analytical tech-
niques such as mass spectrometry to achieve component separation and structural
characterization of analytical samples in one run. In short, the chromatographic
technology has a wide variety, convenience, flexibility, and adaptability, making it
unique among many separation technologies.
Compared with older separation techniques such as crystallization, solvent
extraction, and distillation, chromatography has many advantages. First of all,
chromatography is a powerful separation technique. It can separate and purify one
or more components of interest from complex multicomponent mixtures without
knowing the type, quantity, or relative quantity of the substances present. Secondly,
chromatography has a wide range of applications. It can not only deal with mole-
cules of different sizes, from the smallest molecules, which contain only two atoms
of hydrogen in the molecule, to viruses composed of millions of atoms, but also deal
with molecules of different properties, from simple gaseous molecules to complex
and chargeable biological macromolecules such as proteins, antibodies, and nucleic
acids; in addition, it can handle materials from trace amounts to bulk quantities.
Some forms of analytical chromatography can detect substances present at the level
of attogram (10 18 g), which makes this method an excellent trace analysis tech-
nique widely used to detect residual pesticides in biological materials and the
environment, forensic science and detection of therapeutic drugs and drugs of
abuse. Preparative chromatography is the main method of downstream processing
in the biopharmaceutical process. It is used to separate and purify the recombinant
protein produced by genetic engineering to make its purity meet the strict require-
ments of the pharmaceutical regulatory authority. In the foreseeable future, chroma-
tography will continue to dominate separation science and technology due to its
irreplaceable separation capabilities and will play an important role in many indus-
tries including pharmaceutical, food, biotechnology, and chemical industry.
In this chapter, we will briefly review the development of chromatography,
especially mixed-mode chromatography, and learn from pioneers in the field how
to overcome obstacles and create new things.

1.2 Separation Technology Before Chromatography

The separation process is a very old technology, which has already begun almost
since human beings entered the civilized age. We know that most elements or
compounds in nature are in an impure state, mostly in the form of mixtures, and
1.2 Separation Technology Before Chromatography 3

cannot be used directly without separation. The alchemy or metallurgy created by the
ancients was to obtain pure precious metals such as gold from ore raw materials
through the calcination process. In the traditional wine making process, the fermen-
tation products are distilled to increase the alcohol content. In the water taken from
the pond or river, alum is added and stirred to precipitate impurities and obtain clear
and clean drinking water. These examples show that since ancient times, humans
have learned to use separation technology to separate raw materials before they can
be put into production or used to meet daily needs. This makes separation technol-
ogy important not only for industrial production but also for daily life.
Industrial separation is usually used for production purposes and is carried out on
a large scale by using techniques such as precipitation, crystallization, and distilla-
tion to extract effective ingredients from the original mixture. Separation can also be
used for analysis and determination, using a small amount of samples to concentrate
or to remove impurities in order to determine some of the components. In some
cases, separation requires complete purification, such as electrolytic refining of
bauxite in aluminum metal ore, crystallization and purification of antibiotic products
in fermentation broth, and separation and purification of recombinant protein
secreted in cell supernatant. But in other cases, only partial separation is required.
A good example of this is the separation of plasma proteins. The Cohn process
developed by Edwin J. Cohn is a series of purification steps to extract albumin from
plasma, an excellent substitute for human plasma. Using the characteristics of
different solubility of plasma protein at different pH, ethanol concentration, temper-
ature, ionic strength, and protein concentration, the plasma protein is divided into
several fractions by adjusting the experimental parameters, and each fraction con-
tains several proteins. The cryogenic ethanol precipitation method separates human
plasma and produces human serum albumin, serum gamma globulin, fibrinogen,
thrombin, and blood group globulin (Surgenor 2002).
Another example of incomplete separation is the refining of crude oil (Gary and
Handwerk 1984). In nature, crude oil exists as a mixture of various hydrocarbons
and impurities. The refining process breaks down this mixture into other more
valuable mixtures, such as natural gas, gasoline, and chemical raw materials. None
of them are pure substances, but each must be separated from crude oil. In both
cases, a series of separations are required to obtain the desired final product. In the
case of oil refining, crude oil is subjected to a series of separate distillation steps,
each of which produces different products or intermediates.
Precipitation is the formation of solid precipitates in a liquid solution through
reaction. These precipitates aggregate and settle under the action of gravity and
separate from the solution (Zumdahl 2005). The chemical substance that causes
the solid to form is called a “precipitating agent.” There is not enough gravity
(sedimentation) to gather the solid particles together, and the sediment stays in
suspension. In this situation, centrifugal force can be used to compress it into a
compact mass to accelerate the settlement. An important stage of the precipitation
process is the beginning of nucleation. Some energy based on the relative surface
energy of the solid and the solution is required for the formation of an interface.
4 1 Historical Perspectives

Without this energy, and without a suitable nucleation surface, oversaturation will
occur.
Distillation is the process of separating components or substances from a liquid
mixture by using selective boiling and condensation. Distillation can result in a
substantially complete separation (almost pure components), or possibly a partial
separation, thereby increasing the concentration of selected components in the
mixture (Seader and Henley 1998). In either case, the process takes advantage of
the difference in the relative volatility of the components of the mixture. In the
chemical industry, distillation is actually a unit operation of universal importance,
but it is a physical separation process, not a chemical reaction. Distillation is only
suitable for the purification of volatile components. In real life, we have to deal
with many substances, which are nonvolatile and cannot be separated and purified
by distillation technology, such as many polar molecules such as salts, sugars,
and amino acids. In this case, we usually use another purification technique that is
crystallization to separate and purify them.
Crystallization is the process of forming solids or crystals (natural or artificial), in
which atoms or molecules are highly organized into structures called crystals.
Some ways of crystal formation are precipitation from solution, freezing, or rarely
directly from gas (McCabe and Smith 2000). The properties of the resulting
crystals are largely dependent on factors such as temperature and air pressure,
as well as the time for the fluid to evaporate.
Crystallization is a solid–liquid chemical separation technique in which solutes are
transferred from a liquid solution to a pure solid crystalline phase. In chemical
engineering, crystallization occurs in the crystallizer. Therefore, crystallization is
related to precipitation, although the result is not amorphous or disordered, but
crystalline.
Recrystallization is a technique used to purify chemical substances. By dissolving
both impurities and compounds in an appropriate solvent, the desired compound
or impurity can be removed from the solution while the other is retained. It gets its
name from the crystals that often form when the compound precipitates. In
addition, recrystallization can refer to the natural growth process of large ice
crystals, and smaller ice crystals form large ice crystals through the process of
dissolution and recrystallization.
Filtration is a physical, biological, or chemical operation that can separate solids
and fluids from the mixture through a filter medium with a complex structure
(Sparks and Chase 2015). The fluid that can pass through the filter medium is
called filtrate. Oversized particles that cannot pass through the filter media may
form a filter cake on the top of the filter and be recovered. Solid–liquid separation
is a convenient and fast unit operation, the main purpose is to remove solid
particles from the solution. The disadvantage of this method is that the recovered
solids may be contaminated by some fluids and the filtrate will contain fine
particles (depending on the pore size, filter thickness, and biological activity).
Filtration occurs both in the natural environment and in engineering systems.
There are biological, geological, and industrial forms.
1.3 Birth of Chromatography 5

Distillation and crystallization are the most commonly used separation techniques
by organic chemists, and many applications have been found in both daily life and
industrial processes. However, they also have some obvious shortcomings, for
example, time-consuming operation, long separation cycle, and limited applicability.
Obviously, with the rapid development of phytochemistry and biochemistry at the
end of the nineteenth century, traditional separation and purification techniques such
as distillation and crystallization were incapable of meeting the increasing demands
of scientific research, prompting scientists to find more efficient separation and
purification methods.

1.3 Birth of Chromatography

At the end of the nineteenth century and the beginning of the twentieth century,
natural sciences developed rapidly, especially phytochemistry, food chemistry, and
biochemistry were in a stage of rapid advancement. Many biologically active
compounds with complex structures, strong hydrophilicity, thermal instability, and
large molecular weight were discovered. There was an urgent need for better
techniques than traditional distillation and crystallization for the complete charac-
terization of these substances. Looking back on history, it is not difficult to find that
it is the difficult problems of separating pigments in plants and amino acids from
protein degradation products in wool that lead to the birth of adsorption chromatog-
raphy and partition chromatography, respectively (Wixom 2001).

1.3.1 Adsorption Chromatography

Long before the discovery of chromatography, dye chemists used capillary phenom-
ena to separate dyes. They tested their dye mixtures by dipping string or cloth strips
or filter paper into dye buckets. On the inserted material, the dye solution undergoes
differential migration through capillary action, dividing the dye components into
different colored strips. In the nineteenth century, several German chemists deliber-
ately designed experiments to explore this phenomenon. They observed that by
dropping a solution of an inorganic compound into the center of a piece of filter
paper, concentric colored circles were formed. This method is named “capillary
analysis” (Wixom 2001).
However, the discovery of chromatography is usually attributed to Russian
phytochemist Mikhail S. Tswett. Because he first realized the physical and chemical
basis of chromatographic separation in 1901 and applied this principle to the
separation of complex plant pigments, especially the separation of carotenoids and
chlorophyll, which was a major challenge faced by chemists at that time. Tswett’s
research topic is about the physical and chemical structure of chlorophyll. He needed
to separate chlorophyll in a form that is as close to its natural state as possible. In this
6 1 Historical Perspectives

work, he discovered a strange phenomenon. The chlorophyll in plant leaves can be

extracted by polar solvents (such as ethanol), but it cannot be extracted by nonpolar
solvents (such as petroleum ether). The chlorophyll isolated from the plant is readily
soluble in nonpolar solvents. After thinking about this confusing problem, Tswett
came to the conclusion that this behavior of chlorophyll is not due to a simple
solubility problem, nor is it caused by the chemical change of chlorophyll in ethanol
into a “soluble” form, but it is because of the molecular adsorption force on the plant
tissue that the chlorophyll is adsorbed and fixed on the surface of the plant tissue. In
order to extract chlorophyll from plant tissues, one need not only to solve the
problem of chlorophyll dissolution but also to overcome the adsorption of plant
tissues to chlorophyll. The latter may be the main reason for the different extraction
abilities of the solvents.
In subsequent work, Tswett studied in detail the interactions of plant pigments
with more than 100 organic and inorganic powdery particles that may be used as
adsorbents and their relative solvent strength, in order to determine the general
adsorption and elution behavior of these substances. Ultimately, these studies led
to a new method of separating chlorophyll and carotenoids through stepwise selec-
tive adsorption and elution. He named the newly developed separation method
chromatography. The packed-column chromatography technique described by
Tswett in 1906 is almost identical to the column chromatography technique still
widely used today in organic chemistry or phytochemistry laboratories. He packed a
vertical glass column with absorbent solid materials (such as calcium carbonate,
alumina, silica gel, or powdered sugar), added a plant pigment solution to the top of
the column, and then used an organic solvent as an eluent to pass through the
column. The pigment is divided into a series of discrete color bands on the chro-
matographic column, separated by areas that are completely free of pigment. Since
Tswett processed colored substances and formed ribbons on the columns, he called it
chromatography (derived from Greek, meaning color writing). However, Tswett also
specifically pointed out that chromatography is also suitable for the separation of
compounds that do not contain chromophores.
Regrettably, the chromatographic method invented by Tswett did not attract
enough attention from the scientific community. It may be because his work was
mainly published in German and Russian botany journals, which was not easily
understood by English-speaking scientists. Another important reason is that Tswett’s
research work was interrupted by the outbreak of the First World War, and Tswett
was transferred to several different universities. In this disturbing period, his health
was deteriorating. He died in Voronezh on June 26, 1919, at the age of 47 (Fig. 1.1).
Soon after Tswett invented adsorption chromatography and reported the success-
ful separation of chlorophyll, carotenoids, and lutein from plant leaves, American
scientist L. S. Palmer entered the field of food chemistry. The question he faced was
why the color of butter made in summer and winter is different. Presumably it is
caused by the change of plant pigments in dairy cow feed. The German scientists
R. Willstätter and W. Mieg just recently determined the basic composition of
“carotene” and “lutein” through a multistep crystallization purification method, but
the chemical structure of these compounds was not figured out until the late 1920s.
1.3 Birth of Chromatography 7

Fig. 1.1 Mikhail S. Tswett

(1872–1919). (Reproduced
from Wikipedia at https://en.
wikipedia.org/wiki/
Mikhail_Tsvet)

Therefore, Palmer had to make a choice in 1910, whether to use the traditional
multistep crystallization process or the newly created adsorption chromatography to
purify plant pigments. Relying on Tswett’s adsorption chromatography, Palmer
found carotenoids in his butter extract, which explained the color change of butter
caused by cow’s diet from summer to winter. His research may represent the first
practical application of chromatography in the American continent in addition to
Tswett’s own work.
Then, it was the excellent work of Richard Kuhn and his colleagues that really
made Tswett’s adsorption chromatography out of the laboratory and into the public
eye. Richard Kuhn is an Austrian–German biochemist. In 1929, he became the head
of the Institute of Chemistry of the newly established Kaiser Wilhelm Institute of
Medicine (the Max Planck Institute of Medicine in Heidelberg since 1950). Kuhn’s
research fields include theoretical issues of organic chemistry (stereochemistry of
aliphatic and aromatic compounds, synthesis of polyenes and cumene, composition
and color, acidity of hydrocarbons), and a wide range of biochemistry (carotenoid,
flavins, research on vitamins and enzymes). Specifically, he carried out important
work in vitamin B2 and anti-dermatitis vitamin B6. Kuhn, Winterstein, and Lederer
published an important paper on the purification of lutein on a CaCO3 adsorption
chromatography column in 1931. R. Kuhn was awarded the 1938 Nobel Prize in
Chemistry for his “research on carotenoids and vitamins.”
In the early and mid-1930s, Paul Karrer (1889–1971) was actively engaged in the
research of natural products at the University of Zurich in Switzerland. After
learning the results of Kuhn–Lederer, he used adsorption chromatography exten-
sively in his research and introduced the application of chromatography in natural