Preface xv ; About the Editors xvii ; List of Abbreviations xix ; Notation xxiii ; 1 Introduction Henner Schmidt-Traub and Reinhard Ditz ; 1.1 Chromatography, Development, and Future Trends 1 ; 1.2 Focus of the Book 4 ; 1.3 Suggestions on How to Read this Book 4 ; References 6 ; 2 Fundamentals and General Terminology Andreas Seidel-Morgenstern ; 2.1 Principles and Features of Chromatography 9 ; 2.2 Analysis and Description of Chromatograms 13 ; 2.2.1 Voidage and Porosity 13 ; 2.2.2 Retention Times and Capacity Factors 16 ; 2.2.3 Efficiency of Chromatographic Separations 17 ; 2.2.4 Resolution 20 ; 2.2.5 Pressure Drop 23 ; 2.3 Mass Transfer and Fluid Dynamics 25 ; 2.3.1 Principles of Mass Transfer 25 ; 2.3.2 Fluid Distribution in the Column 27 ; 2.3.3 Packing Nonidealities 28 ; 2.3.4 Extra-Column Effects 29 ; 2.4 Equilibrium Thermodynamics 29 ; 2.4.1 Definition of Isotherms 29 ; 2.4.2 Models of Isotherms 31 ; 2.4.2.1 Single-Component Isotherms 31 ; 2.4.2.2 Multicomponent Isotherms Based on the Langmuir Model 33 ; 2.4.2.3 Competitive Isotherms Based on the Ideal Adsorbed Solution Theory 34 ; 2.4.2.4 Steric Mass Action Isotherms 37 ; 2.4.3 Relation Between Isotherms and Band Shapes 38 ; 2.5 Column Overloading and Operating Modes 44 ; 2.5.1 Overloading Strategies 44 ; 2.5.2 Beyond Isocratic Batch Elution 45 ; References 46 ; 3 Stationary Phases Michael Schulte ; 3.1 Survey of Packings and Stationary Phases 49 ; 3.2 Inorganic Sorbents 50 ; 3.2.1 Activated Carbons 50 ; 3.2.2 Synthetic Zeolites 54 ; 3.2.3 Porous Oxides: Silica, Activated Alumina, Titania, Zirconia, and Magnesia 54 ; 3.2.4 Silica 55 ; 3.2.4.1 Surface Chemistry 57 ; 3.2.4.2 Mass Loadability 59 ; 3.2.5 Diatomaceous Earth 59 ; 3.2.6 Reversed Phase Silicas 60 ; 3.2.6.1 Silanization of the Silica Surface 60 ; 3.2.6.2 Silanization 60 ; 3.2.6.3 Starting Silanes 61 ; 3.2.6.4 Parent Porous Silica 61 ; 3.2.6.5 Reaction and Reaction Conditions 62 ; 3.2.6.6 Endcapping 62 ; 3.2.6.7 Chromatographic Characterization of Reversed Phase Silicas 63 ; 3.2.6.8 Chromatographic Performance 63 ; 3.2.6.9 Hydrophobic Properties Retention Factor (Amount of Organic Solvent for Elution), Selectivity 65 ; 3.2.6.10 Shape Selectivity 65 ; 3.2.6.11 Silanol Activity 67 ; 3.2.6.12 Purity 68 ; 3.2.6.13 Improved pH Stability Silica 68 ; 3.2.7 Aluminum Oxide 69 ; 3.2.8 Titanium Dioxide 70 ; 3.2.9 Other Oxides 71 ; 3.2.9.1 Magnesium Oxide 71 ; 3.2.9.2 Zirconium Dioxide 71 ; 3.2.10 Porous Glasses 72 ; 3.3 Cross-Linked Organic Polymers 73 ; 3.3.1 General Aspects 74 ; 3.3.2 Hydrophobic Polymer Stationary Phases 77 ; 3.3.3 Hydrophilic Polymer Stationary Phases 78 ; 3.3.4 Ion Exchange (IEX) 79 ; 3.3.4.1 Optimization of Ion-Exchange Resins 81 ; 3.3.5 Mixed Mode 88 ; 3.3.6 Hydroxyapatite 88 ; 3.3.7 Designed Adsorbents 91 ; 3.3.7.1 Protein A Affinity Sorbents 91 ; 3.3.7.2 Other IgG Receptor Proteins: Protein G and Protein L 96 ; 3.3.7.3 Sorbents for Derivatized/Tagged Compounds: Immobilized Metal Affinity Chromatography (IMAC) 96 ; 3.3.7.4 Other Tag-Based Affinity Sorbents 101 ; 3.3.8 Customized Adsorbents 102 ; 3.3.8.1 Low Molecular Weight Ligands 105 ; 3.3.8.2 Natural Polymers (Proteins, Polynucleotides) 108 ; 3.3.8.3 Artificial Polymers 111 ; 3.4 Advective Chromatographic Materials 111 ; 3.4.1 Adsorptive Membranes and Grafted-Polymer Membranes 114 ; 3.4.2 Adsorptive Nonwovens 115 ; 3.4.3 Fiber/Particle Composites 117 ; 3.4.4 Area-Enhanced Fibers 117 ; 3.4.5 Monolith 118 ; 3.4.6 Chromatographic Materials for Larger Molecules 121 ; 3.5 Chiral Stationary Phases 121 ; 3.5.1 Cellulose- and Amylose-Based CSP 122 ; 3.5.2 Antibiotic CSP 128 ; 3.5.3 Cyclofructan-Based CSP 128 ; 3.5.4 Synthetic Polymers 128 ; 3.5.5 Targeted Selector Design 130 ; 3.5.6 Further Developments 132 ; 3.6 Properties of Packings and Their Relevance to Chromatographic Performance 132 ; 3.6.1 Chemical and Physical Bulk Properties 132 ; 3.6.2 Morphology 133 ; 3.6.3 Particulate Adsorbents: Particle Size and Size Distribution 133 ; 3.6.4 Pore Texture 134 ; 3.6.5 Pore Structural Parameters 137 ; 3.6.6 Comparative Rating of Columns 137 ; 3.7 Sorbent Maintenance and Regeneration 138 ; 3.7.1 Cleaning in Place (CIP) 138 ; 3.7.2 CIP for IEX 140 ; 3.7.3 CIP of Protein A Sorbents 140 ; 3.7.4 Conditioning of Silica Surfaces 143 ; 3.7.5 Sanitization in Place (SIP) 145 ; 3.7.6 Column and Adsorbent Storage 145 ; References 146 ; 4 Selection of Chromatographic Systems ; 159 Michael Schulte ; 4.1 Definition of the Task 164 ; 4.2 Mobile Phases for Liquid Chromatography 167 ; 4.2.1 Stability 168 ; 4.2.2 Safety Concerns 172 ; 4.2.3 Operating Conditions 172 ; 4.2.4 Aqueous Buffer Systems 176 ; 4.3 Adsorbent and Phase Systems 178 ; 4.3.1 Choice of Phase System Dependent on Solubility 178 ; 4.3.2 Improving Loadability for Poor Solubilities 180 ; 4.3.3 Dependency of Solubility on Sample Purity 183 ; 4.3.4 Generic Gradients for Fast Separations 184 ; 4.4 Criteria for Choosing Normal Phase Systems 184 ; 4.4.1 Retention in NP Systems 186 ; 4.4.2 Solvent Strength in Liquid-Solid Chromatography 188 ; 4.4.3 Pilot Technique Thin-Layer Chromatography Using the PRISMA Model 190 ; 4.4.3.1 Step (1): Solvent Strength Adjustment 199 ; 4.4.3.2 Step (2): Optimization of Selectivity 199 ; 4.4.3.3 Step (3): Final Optimization of the Solvent Strength 200 ; 4.4.3.4 Step (4): Determination of the Optimum Mobile Phase Composition 200 ; 4.4.4 Strategy for an Industrial Preparative Chromatography Laboratory 202 ; 4.4.4.1 Standard Gradient Elution Method on Silica 203 ; 4.4.4.2 Simplified Procedure 204 ; 4.5 Criteria for Choosing Reversed Phase Systems 206 ; 4.5.1 Retention and Selectivity in RP Systems 208 ; 4.5.2 Gradient Elution for Small Amounts of Product on RP Columns 212 ; 4.5.3 Rigorous Optimization for Isocratic Runs 213 ; 4.5.4 Rigorous Optimization for Gradient Runs 217 ; 4.5.5 Practical Recommendations 222 ; 4.6 Criteria for Choosing CSP Systems 223 ; 4.6.1 Suitability of Preparative CSP 223 ; 4.6.2 Development of Enantioselectivity 224 ; 4.6.3 Optimization of Separation Conditions 226 ; 4.6.3.1 Determination of Racemate Solubility 226 ; 4.6.3.2 Selection of Elution Order 226 ; 4.6.3.3 Optimization of Mobile/Stationary Phase Composition, Including Temperature 226 ; 4.6.3.4 Determination of Optimum Separation Step 227 ; 4.6.4 Practical Recommendations 227 ; 4.7 Downstream Processing of mAbs Using Protein A and IEX 231 ; 4.8 Size-Exclusion Chromatography (SEC) 236 ; 4.9 Overall Chromatographic System Optimization 237 ; 4.9.1 Conflicts During Optimization of Chromatographic Systems 237 ; 4.9.2 Stationary Phase Gradients 241 ; References 246 ; 5 Process Concepts Malte Kaspereit and Henner Schmidt-Traub ; 5.1 Discontinuous Processes 252 ; 5.1.1 Isocratic Operation 252 ; 5.1.2 Gradient Chromatography 253 ; 5.1.3 Closed-Loop Recycling Chromatography 256 ; 5.1.4 Steady-State Recycling Chromatography (SSRC) 258 ; 5.1.5 Flip-Flop Chromatography 259.