<p>Preface xv</p> <p>About the Editors xvii</p> <p>List of Abbreviations xix</p> <p>Notation xxiii</p> <p><b>1 Introduction </b><b>1<br /></b><i>Henner Schmidt-Traub and Reinhard Ditz</i></p> <p>1.1 Chromatography, Development, and Future Trends 1</p> <p>1.2 Focus of the Book 4</p> <p>1.3 Suggestions on How to Read this Book 4</p> <p>References 6</p> <p><b>2 Fundamentals and General Terminology </b><b>9<br /></b><i>Andreas Seidel-Morgenstern</i></p> <p>2.1 Principles and Features of Chromatography 9</p> <p>2.2 Analysis and Description of Chromatograms 13</p> <p>2.2.1 Voidage and Porosity 13</p> <p>2.2.2 Retention Times and Capacity Factors 16</p> <p>2.2.3 Efficiency of Chromatographic Separations 17</p> <p>2.2.4 Resolution 20</p> <p>2.2.5 Pressure Drop 23</p> <p>2.3 Mass Transfer and Fluid Dynamics 25</p> <p>2.3.1 Principles of Mass Transfer 25</p> <p>2.3.2 Fluid Distribution in the Column 27</p> <p>2.3.3 Packing Nonidealities 28</p> <p>2.3.4 Extra-Column Effects 29</p> <p>2.4 Equilibrium Thermodynamics 29</p> <p>2.4.1 Definition of Isotherms 29</p> <p>2.4.2 Models of Isotherms 31</p> <p>2.4.2.1 Single-Component Isotherms 31</p> <p>2.4.2.2 Multicomponent Isotherms Based on the Langmuir Model 33</p> <p>2.4.2.3 Competitive Isotherms Based on the Ideal Adsorbed Solution Theory 34</p> <p>2.4.2.4 Steric Mass Action Isotherms 37</p> <p>2.4.3 Relation Between Isotherms and Band Shapes 38</p> <p>2.5 Column Overloading and Operating Modes 44</p> <p>2.5.1 Overloading Strategies 44</p> <p>2.5.2 Beyond Isocratic Batch Elution 45</p> <p>References 46</p> <p><b>3 Stationary Phases </b><b>49<br /></b><i>Michael Schulte</i></p> <p>3.1 Survey of Packings and Stationary Phases 49</p> <p>3.2 Inorganic Sorbents 50</p> <p>3.2.1 Activated Carbons 50</p> <p>3.2.2 Synthetic Zeolites 54</p> <p>3.2.3 Porous Oxides: Silica, Activated Alumina, Titania, Zirconia, and Magnesia 54</p> <p>3.2.4 Silica 55</p> <p>3.2.4.1 Surface Chemistry 57</p> <p>3.2.4.2 Mass Loadability 59</p> <p>3.2.5 Diatomaceous Earth 59</p> <p>3.2.6 Reversed Phase Silicas 60</p> <p>3.2.6.1 Silanization of the Silica Surface 60</p> <p>3.2.6.2 Silanization 60</p> <p>3.2.6.3 Starting Silanes 61</p> <p>3.2.6.4 Parent Porous Silica 61</p> <p>3.2.6.5 Reaction and Reaction Conditions 62</p> <p>3.2.6.6 Endcapping 62</p> <p>3.2.6.7 Chromatographic Characterization of Reversed Phase Silicas 63</p> <p>3.2.6.8 Chromatographic Performance 63</p> <p>3.2.6.9 Hydrophobic Properties Retention Factor (Amount of Organic Solvent for Elution), Selectivity 65</p> <p>3.2.6.10 Shape Selectivity 65</p> <p>3.2.6.11 Silanol Activity 67</p> <p>3.2.6.12 Purity 68</p> <p>3.2.6.13 Improved pH Stability Silica 68</p> <p>3.2.7 Aluminum Oxide 69</p> <p>3.2.8 Titanium Dioxide 70</p> <p>3.2.9 Other Oxides 71</p> <p>3.2.9.1 Magnesium Oxide 71</p> <p>3.2.9.2 Zirconium Dioxide 71</p> <p>3.2.10 Porous Glasses 72</p> <p>3.3 Cross-Linked Organic Polymers 73</p> <p>3.3.1 General Aspects 74</p> <p>3.3.2 Hydrophobic Polymer Stationary Phases 77</p> <p>3.3.3 Hydrophilic Polymer Stationary Phases 78</p> <p>3.3.4 Ion Exchange (IEX) 79</p> <p>3.3.4.1 Optimization of Ion-Exchange Resins 81</p> <p>3.3.5 Mixed Mode 88</p> <p>3.3.6 Hydroxyapatite 88</p> <p>3.3.7 Designed Adsorbents 91</p> <p>3.3.7.1 Protein A Affinity Sorbents 91</p> <p>3.3.7.2 Other IgG Receptor Proteins: Protein G and Protein L 96</p> <p>3.3.7.3 Sorbents for Derivatized/Tagged Compounds: Immobilized Metal Affinity Chromatography (IMAC) 96</p> <p>3.3.7.4 Other Tag-Based Affinity Sorbents 101</p> <p>3.3.8 Customized Adsorbents 102</p> <p>3.3.8.1 Low Molecular Weight Ligands 105</p> <p>3.3.8.2 Natural Polymers (Proteins, Polynucleotides) 108</p> <p>3.3.8.3 Artificial Polymers 111</p> <p>3.4 Advective Chromatographic Materials 111</p> <p>3.4.1 Adsorptive Membranes and Grafted-Polymer Membranes 114</p> <p>3.4.2 Adsorptive Nonwovens 115</p> <p>3.4.3 Fiber/Particle Composites 117</p> <p>3.4.4 Area-Enhanced Fibers 117</p> <p>3.4.5 Monolith 118</p> <p>3.4.6 Chromatographic Materials for Larger Molecules 121</p> <p>3.5 Chiral Stationary Phases 121</p> <p>3.5.1 Cellulose- and Amylose-Based CSP 122</p> <p>3.5.2 Antibiotic CSP 128</p> <p>3.5.3 Cyclofructan-Based CSP 128</p> <p>3.5.4 Synthetic Polymers 128</p> <p>3.5.5 Targeted Selector Design 130</p> <p>3.5.6 Further Developments 132</p> <p>3.6 Properties of Packings and Their Relevance to Chromatographic Performance 132</p> <p>3.6.1 Chemical and Physical Bulk Properties 132</p> <p>3.6.2 Morphology 133</p> <p>3.6.3 Particulate Adsorbents: Particle Size and Size Distribution 133</p> <p>3.6.4 Pore Texture 134</p> <p>3.6.5 Pore Structural Parameters 137</p> <p>3.6.6 Comparative Rating of Columns 137</p> <p>3.7 Sorbent Maintenance and Regeneration 138</p> <p>3.7.1 Cleaning in Place (CIP) 138</p> <p>3.7.2 CIP for IEX 140</p> <p>3.7.3 CIP of Protein A Sorbents 140</p> <p>3.7.4 Conditioning of Silica Surfaces 143</p> <p>3.7.5 Sanitization in Place (SIP) 145</p> <p>3.7.6 Column and Adsorbent Storage 145</p> <p>References 146</p> <p><b>4 Selection of Chromatographic Systems </b><b>159<br /></b><i>Michael Schulte</i></p> <p>4.1 Definition of the Task 164</p> <p>4.2 Mobile Phases for Liquid Chromatography 167</p> <p>4.2.1 Stability 168</p> <p>4.2.2 Safety Concerns 172</p> <p>4.2.3 Operating Conditions 172</p> <p>4.2.4 Aqueous Buffer Systems 176</p> <p>4.3 Adsorbent and Phase Systems 178</p> <p>4.3.1 Choice of Phase System Dependent on Solubility 178</p> <p>4.3.2 Improving Loadability for Poor Solubilities 180</p> <p>4.3.3 Dependency of Solubility on Sample Purity 183</p> <p>4.3.4 Generic Gradients for Fast Separations 184</p> <p>4.4 Criteria for Choosing Normal Phase Systems 184</p> <p>4.4.1 Retention in NP Systems 186</p> <p>4.4.2 Solvent Strength in Liquid-Solid Chromatography 188</p> <p>4.4.3 Pilot Technique Thin-Layer Chromatography Using the PRISMA Model 190</p> <p>4.4.3.1 Step (1): Solvent Strength Adjustment 199</p> <p>4.4.3.2 Step (2): Optimization of Selectivity 199</p> <p>4.4.3.3 Step (3): Final Optimization of the Solvent Strength 200</p> <p>4.4.3.4 Step (4): Determination of the Optimum Mobile Phase Composition 200</p> <p>4.4.4 Strategy for an Industrial Preparative Chromatography Laboratory 202</p> <p>4.4.4.1 Standard Gradient Elution Method on Silica 203</p> <p>4.4.4.2 Simplified Procedure 204</p> <p>4.5 Criteria for Choosing Reversed Phase Systems 206</p> <p>4.5.1 Retention and Selectivity in RP Systems 208</p> <p>4.5.2 Gradient Elution for Small Amounts of Product on RP Columns 212</p> <p>4.5.3 Rigorous Optimization for Isocratic Runs 213</p> <p>4.5.4 Rigorous Optimization for Gradient Runs 217</p> <p>4.5.5 Practical Recommendations 222</p> <p>4.6 Criteria for Choosing CSP Systems 223</p> <p>4.6.1 Suitability of Preparative CSP 223</p> <p>4.6.2 Development of Enantioselectivity 224</p> <p>4.6.3 Optimization of Separation Conditions 226</p> <p>4.6.3.1 Determination of Racemate Solubility 226</p> <p>4.6.3.2 Selection of Elution Order 226</p> <p>4.6.3.3 Optimization of Mobile/Stationary Phase Composition, Including Temperature 226</p> <p>4.6.3.4 Determination of Optimum Separation Step 227</p> <p>4.6.4 Practical Recommendations 227</p> <p>4.7 Downstream Processing of mAbs Using Protein A and IEX 231</p> <p>4.8 Size-Exclusion Chromatography (SEC) 236</p> <p>4.9 Overall Chromatographic System Optimization 237</p> <p>4.9.1 Conflicts During Optimization of Chromatographic Systems 237</p> <p>4.9.2 Stationary Phase Gradients 241</p> <p>References 246</p> <p><b>5 Process Concepts </b><b>251<br /></b><i>Malte Kaspereit and Henner Schmidt-Traub</i></p> <p>5.1 Discontinuous Processes 252</p> <p>5.1.1 Isocratic Operation 252</p> <p>5.1.2 Gradient Chromatography 253</p> <p>5.1.3 Closed-Loop Recycling Chromatography 256</p> <p>5.1.4 Steady-State Recycling Chromatography (SSRC) 258</p> <p>5.1.5 Flip-Flop Chromatography 259</p&g

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