| Preface |
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xv | |
| About the Book |
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xvii | |
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Part I Optimization Strategies for Different Modes and Uses ofHPLC |
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1 | (176) |
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1.1 2D-HPLC - Method Development for Successful Separations |
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3 | (20) |
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1.1.1 Motivations for Two-Dimensional Separation |
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3 | (1) |
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1.1.1.1 Difficult-to-Separate Samples |
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3 | (1) |
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4 | (1) |
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4 | (1) |
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1.1.2 Choosing a Two-Dimensional Separation Mode |
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4 | (1) |
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1.1.2.1 Analytical Goals Dictate Choice of Mode |
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5 | (1) |
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1.1.2.2 Survey of Four 2D Separation Modes |
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5 | (2) |
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1.1.2.3 Hybrid Modes Provide Flexibility |
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7 | (1) |
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1.1.3 Choosing Separation Types/Mechanisms |
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8 | (1) |
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1.1.3.1 Complementarity as a Guiding Principle |
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8 | (1) |
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1.1.3.2 Pirok Compatibility Table |
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9 | (1) |
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1.1.3.3 Measuring the Complementarity of Separation Types |
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9 | (2) |
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1.1.4 Choosing Separation Conditions |
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11 | (1) |
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1.1.4.1 Starting with Fixed First-Dimension Conditions |
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11 | (2) |
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1.1.4.2 Starting from Scratch - Flexible First-Dimension Conditions |
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13 | (1) |
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1.1.4.3 Special Considerations for Comprehensive 2D-LC Methods |
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13 | (1) |
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13 | (1) |
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1.1.5 Method Development Examples |
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14 | (1) |
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1.1.5.1 Example 1 - Use of LC-LC to Identify an Impurity in a Synthetic Oligonucleotide |
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14 | (1) |
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1.1.5.2 Example 2 - Comprehensive 2D-LC Separation of Surfactants |
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14 | (3) |
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1.1.6 Outlook for the Future |
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17 | (1) |
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18 | (1) |
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18 | (5) |
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1.2 Do you HILIC? With Mass Spectrometry? Then do it Systematically |
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23 | (16) |
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1.2.1 Initial Situation and Optimal Use of Stationary HILIC Phases |
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25 | (3) |
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1.2.2 Initial Situation and Optimal Use of the "Mobile" HILIC Phase |
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28 | (1) |
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28 | (3) |
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31 | (2) |
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33 | (2) |
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1.2.3 Further Settings and Conditions Specific to Mass Spectrometric Detection |
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35 | (1) |
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1.2.4 Short Summary on Method Optimization in HILIC |
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36 | (1) |
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36 | (3) |
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1.3 Optimization Strategies in LC-MS Method Development |
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39 | (18) |
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39 | (1) |
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1.3.2 Developing New Methods for HPLC-MS Separations |
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39 | (1) |
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1.3.2.1 Optimizing the LC Separation |
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40 | (1) |
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1.3.2.1.1 Optimizing for Sensitivity and Limit of Detection - Which Column to Take? |
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40 | (1) |
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1.3.2.1.2 Optimizing Resolution vs. Sample Throughput |
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41 | (2) |
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1.3.2.1.3 MS-Compatible Eluent Compositions and Additives |
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43 | (1) |
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1.3.2.2 Optimizing Ion Source Conditions |
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44 | (3) |
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1.3.2.3 Optimizing MS Detection |
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47 | (1) |
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1.3.2.4 Verifying the Hyphenated Method |
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48 | (1) |
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1.3.2.5 Method Development Supported by Software-based Parameter Variation |
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49 | (1) |
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1.3.3 Transferring Established HPLC Methods to Mass spectrometry |
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50 | (1) |
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1.3.3.1 Transfer of an Entire HPLC Method to a Mass Spectrometer |
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51 | (1) |
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1.3.3.2 Selected Analysis of an Unknown Impurity - Solvent Change by Single-/Multi-Heartcut Techniques |
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52 | (2) |
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54 | (1) |
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55 | (2) |
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1.4 Chromatographic Strategies for the Successful Characterization of Protein Biopharmaceuticals |
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57 | (16) |
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1.4.1 Introduction to Protein Biopharmaceuticals |
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57 | (1) |
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1.4.2 From Standard to High-Performance Chromatography of Protein Biopharmaceuticals |
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58 | (4) |
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1.4.3 Online Coupling of Nondenaturing LC Modes with MS |
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62 | (2) |
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1.4.4 Multidimensional LC Approaches for Protein Biopharmaceuticals |
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64 | (2) |
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1.4.5 Conclusion and Future Trends in Protein Biopharmaceuticals Analysis |
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66 | (1) |
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67 | (6) |
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1.5 Optimization Strategies in HPLC for the Separation of Biomolecules |
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73 | (14) |
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1.5.1 Optimizing a Chromatographic Separation |
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73 | (4) |
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1.5.2 Optimizing the Speed of an HPLC Method |
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77 | (2) |
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1.5.3 Optimizing the Sensitivity of an HPLC Method |
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79 | (1) |
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1.5.4 Multidimensional Separations (See also
Chapter 1.1) |
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80 | (1) |
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1.5.5 Considerations for MS Detection (See also
Chapter 1.3) |
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81 | (2) |
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1.5.6 Conclusions and Future Prospects |
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83 | (1) |
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84 | (3) |
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1.6 Optimization Strategies in Packed-Column Supercritical Fluid Chromatography (SFC) |
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87 | (20) |
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1.6.1 Selecting a Stationary Phase Allowing for Adequate Retention and Desired Selectivity |
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88 | (1) |
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1.6.1.1 Selecting a Stationary Phase for Chiral Separations |
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88 | (2) |
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1.6.1.2 Selecting a Stationary Phase for Achiral Separations |
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90 | (3) |
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1.6.2 Optimizing Mobile Phase to Elute all Analytes |
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93 | (1) |
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1.6.2.1 Nature of the Cosolvent |
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93 | (1) |
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1.6.2.2 Proportion of Cosolvent |
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94 | (2) |
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96 | (1) |
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97 | (1) |
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1.6.3 Optimizing Temperature, Pressure, and Flow Rate |
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97 | (1) |
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1.6.3.1 Understanding the Effects of Temperature, Pressure, and Flow Rate on your Chromatograms |
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97 | (2) |
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1.6.3.2 Optimizing Temperature, Pressure, and Flow Rate Concomitantly |
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99 | (1) |
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1.6.4 Considerations on SFC-MS Coupling |
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100 | (1) |
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1.6.5 Summary of Method Optimization |
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101 | (1) |
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1.6.6 SFC as a Second Dimension in Two-Dimensional Chromatography |
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102 | (1) |
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102 | (1) |
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103 | (4) |
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1.7 Strategies for Enantioselective (Chiral) Separations |
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107 | (34) |
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108 | (1) |
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109 | (1) |
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1.7.3 Chiral Polysaccharide Stationary Phases as First Choice |
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110 | (3) |
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1.7.4 Screening Coated and Immobilized Polysaccharide CSPs in Normal-Phase and Polar Organic Mode |
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113 | (3) |
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1.7.5 Screening Coated and Immobilized Polysaccharide CSPs in Reversed-Phase Mode |
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116 | (3) |
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1.7.6 Screening Immobilized Polysaccharide CSPs in Medium-Polarity Mode |
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119 | (1) |
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1.7.7 Screening Coated and Immobilized Polysaccharide CSPs under Polar Organic Supercritical Fluid Chromatography Conditions |
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120 | (5) |
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1.7.8 Screening Immobilized Polysaccharide CSPs in Medium-Polarity Supercritical Fluid Chromatography Conditions |
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125 | (2) |
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127 | (1) |
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1.7.10 Are There Rules for Predicting Which CSP Is Suited for My Separation Problem? |
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127 | (1) |
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1.7.11 Which Are the Most Promising Polysaccharide CSPs? |
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127 | (2) |
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1.7.12 Are some CSPs Comparable? |
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129 | (3) |
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1.7.13 "No-Go's," Pitfalls, and Peculiarities in Chiral HPLC and SFC |
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132 | (1) |
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1.7.14 Gradients in Chiral Chromatography |
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133 | (1) |
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1.7.15 Alternative Strategies to Chiral HPLC and SFC on Polysaccharide CSPs |
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133 | (2) |
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1.7.16 How Can I Solve Enantiomer Separation Problems Without Going to the Laboratory? |
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135 | (1) |
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1.7.17 The Future of Chiral Separations - Fast Chiral Separations (cUHPLC and cSFC)? |
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136 | (2) |
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138 | (3) |
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1.8 Optimization Strategies Based on the Structure of the Analytes |
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141 | (24) |
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Christoph A. Fleckenstein |
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141 | (1) |
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1.8.2 The Impact of Functional Moieties |
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142 | (1) |
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143 | (3) |
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1.8.4 Influence of Water Solubility by Hydrate Formation of Aldehydes and Ketones |
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146 | (2) |
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1.8.5 Does "Polar" Equal "Hydrophilic"? |
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148 | (2) |
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1.8.6 Peroxide Formation of Ethers |
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150 | (1) |
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1.8.7 The pH Value in HPLC |
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151 | (1) |
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1.8.7.1 Acidic Functional Groups |
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152 | (1) |
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1.8.7.2 Basic Functional Groups |
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153 | (2) |
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1.8.8 General Assessment and Estimation of Solubility of Complex Molecules |
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155 | (2) |
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1.8.9 Octanol-Water Coefficient |
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157 | (3) |
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1.8.10 Hansen Solubility Parameters |
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160 | (2) |
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1.8.11 Conclusion and Outlook |
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162 | (1) |
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163 | (1) |
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163 | (2) |
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1.9 Optimization Opportunities in a Regulated Environment |
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165 | (12) |
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165 | (1) |
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165 | (2) |
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167 | (1) |
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167 | (1) |
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1.9.3.1.1 Preliminary Remark |
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167 | (1) |
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168 | (1) |
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168 | (1) |
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1.9.3.2 Improving the Peak Shape |
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169 | (2) |
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1.9.4 Peak-to-Noise Ratio |
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171 | (1) |
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171 | (1) |
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1.9.5 Coefficient of Variation, VC (Relative Standard Deviation, RSD) |
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171 | (5) |
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176 | (1) |
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Part II Computer-aided Optimization |
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177 | (42) |
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2.1 Strategy for Automated Development of Reversed-Phase HPLC Methods for Domain-Specific Characterization of Monoclonal Antibodies |
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179 | (20) |
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179 | (2) |
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2.1.2 Interaction with Instruments |
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181 | (1) |
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182 | (1) |
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2.1.4 Sample Preparation and HPLC Analysis |
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183 | (1) |
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2.1.5 Automated Method Development |
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184 | (1) |
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2.1.5.1 Columns Screening |
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185 | (1) |
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2.1.5.2 Rapid Optimization |
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186 | (2) |
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2.1.5.3 Fine Optimization and Sample Profiling |
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188 | (1) |
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188 | (1) |
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2.1.6.1 Selection of the Variables |
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189 | (1) |
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2.1.6.2 Selection of the experimental design |
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190 | (1) |
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2.1.6.3 Definition of the Different Levels for the Factors |
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191 | (1) |
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2.1.6.4 Creation of the Experimental Set-up |
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191 | (1) |
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2.1.6.5 Execution of Experiments |
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192 | (1) |
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2.1.6.6 Calculation of Effects and Response and Numerical and Graphical Analysis of the Effects |
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192 | (2) |
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2.1.6.7 Improving the Performance of the Method |
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194 | (2) |
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196 | (1) |
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196 | (3) |
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2.2 Fusion QbD* Software Implementation of APLM Best Practices for Analytical Method Development, Validation, and Transfer |
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199 | (20) |
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199 | (1) |
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2.2.1.1 Application to Chromatographic Separation Modes |
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200 | (1) |
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2.2.1.2 Small-and Large-Molecule Applications |
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200 | (1) |
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2.2.1.3 Use for Non-LC Method Development Procedures |
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200 | (1) |
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2.2.2 Overview - Experimental Design and Data Modeling in Fusion QbD |
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201 | (1) |
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2.2.3 Analytical Target Profile |
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201 | (1) |
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2.2.4 APLM Stage 1 - Procedure Design and Development |
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202 | (1) |
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2.2.4.1 Initial Sample Workup |
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202 | (2) |
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2.2.5 Chemistry System Screening |
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204 | (1) |
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2.2.5.1 Starting Points Based on Molecular Structure and Chemistry Considerations |
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205 | (1) |
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2.2.5.2 Trend Responses and Data Modeling |
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205 | (2) |
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2.2.6 Method Optimization |
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207 | (1) |
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2.2.6.1 Optimizing Mean Performance |
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207 | (3) |
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2.2.6.2 Optimizing Robustness In Silico - Monte Carlo Simulation |
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210 | (3) |
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2.2.6.3 A Few Words About Segmented (Multistep) Gradients and Robustness |
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213 | (1) |
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2.2.7 APLM Stage 2 - Procedure Performance Verification |
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214 | (1) |
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2.2.7.1 Replication Strategy |
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214 | (1) |
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2.2.8 The USP <1210> Tolerance Interval in Support of Method Transfer |
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214 | (2) |
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2.2.9 What is Coming - Expectations for 2021 and Beyond |
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216 | (1) |
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217 | (2) |
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Part III Current Challenges for HPLC Users in Industry |
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219 | (92) |
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3.1 Modern HPLC Method Development |
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221 | (12) |
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3.1.1 Robust Approaches to Practice |
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222 | (1) |
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3.1.1.1 Generic Systems for all Tasks |
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222 | (3) |
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3.1.2 The Classic Reverse-phase System |
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225 | (2) |
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3.1.3 A System that Primarily Separates According to ji-ji Interactions |
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227 | (1) |
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3.1.4 A system that Primarily Separates According to Cation Exchange and Hydrogen Bridge Bonding Selectivity |
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227 | (1) |
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3.1.5 System for Nonpolar Analytes |
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228 | (1) |
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3.1.6 System for Polar Analytes |
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228 | (2) |
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230 | (1) |
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3.1.8 The Maximum Peak Capacity |
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230 | (1) |
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231 | (1) |
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231 | (2) |
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3.2 Optimization Strategies in HPLC from the Perspective of an Industrial Service Provider |
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233 | (6) |
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233 | (1) |
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3.2.2 Research and Development |
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233 | (1) |
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234 | (1) |
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3.2.4 Process Control Analytics |
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235 | (2) |
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3.2.5 Decision Tree for the Optimization Strategy Depending on the Final Application Field |
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237 | (2) |
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3.3 Optimization Strategies in HPLC from the Perspective of a Service Provider - The UNTIE® Process of the CUP Laboratories |
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239 | (12) |
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3.3.1 Common Challenges for a Service Provider |
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239 | (1) |
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3.3.2 A Typical, Lengthy Project - How it Usually Goes and How it Should not be Done! |
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239 | (2) |
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3.3.3 How Do We Make It Better? - The UNTIE8 Process of the CUP Laboratories |
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241 | (1) |
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3.3.4 Understanding Customer Needs |
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241 | (1) |
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3.3.5 The Test of an Existing Method |
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242 | (1) |
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3.3.6 Method Development and Optimization |
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243 | (2) |
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3.3.7 Execution of the Validation |
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245 | (3) |
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248 | (1) |
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249 | (1) |
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249 | (2) |
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3.4 Optimization Strategies in HPLC |
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251 | (60) |
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3.4.1 Definition of the Task |
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252 | (1) |
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3.4.2 Relevant Data for the HPLC Analysis of a Substance (see also
Chapter 1.8) |
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252 | (1) |
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252 | (5) |
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3.4.2.2 Acidity Constants (pK) |
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257 | (1) |
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3.4.2.2.1 Polarity of Acidic or Alkaline Substances (see also
Chapter 1.8) |
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257 | (2) |
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259 | (1) |
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3.4.2.2.3 Influence on the Peak Shape |
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259 | (4) |
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3.4.2.2.4 Acid Constant Estimation |
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263 | (1) |
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3.4.2.3 Octanol-Water Partition Coefficient |
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263 | (7) |
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270 | (2) |
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3.4.2.5 Stability of the Dissolved Analyte |
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272 | (6) |
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278 | (1) |
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3.4.3.1 General Method for the Analysis of Active Pharmaceutical Ingredients |
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278 | (1) |
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3.4.3.2 Extensions of the Range of Application |
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279 | (1) |
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3.4.3.3 Limits of this General Method |
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279 | (1) |
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3.4.3.4 Example, Determination of Butamirate Dihydrogen Citrate in a Cough Syrup |
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279 | (1) |
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279 | (1) |
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3.4.3.4.2 Expected Difficulties |
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279 | (1) |
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279 | (1) |
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3.4.3.4.4 Example Chromatogram |
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279 | (1) |
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3.4.4 General Tips for Optimizing HPLC Methods |
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279 | (5) |
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3.4.4.1 Production of Mobile Phases |
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284 | (1) |
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284 | (1) |
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3.4.4.1.2 Vessels and Bottles |
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285 | (1) |
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3.4.4.1.3 Measurement of Reagents and Solvent |
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285 | (1) |
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3.4.4.1.4 Preparation of Buffer Solutions |
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286 | (1) |
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3.4.4.1.5 Filtration of Solvents and Buffer |
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286 | (1) |
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3.4.4.1.6 Degassing of Mobile Phases |
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287 | (1) |
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287 | (1) |
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3.4.4.3 Defining Measurement Wavelengths for UV Detection |
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288 | (1) |
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3.4.4.4 UV Detection at Low Wavelengths |
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288 | (3) |
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291 | (1) |
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3.4.4.4.2 Acids and Buffer Additives |
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292 | (2) |
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3.4.4.4.3 Drift at Solvent Gradients |
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294 | (1) |
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3.4.4.5 Avoidance of Peak Tailing |
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295 | (7) |
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3.4.4.6 Measurement Uncertainty and Method Design |
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302 | (1) |
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3.4.4.6.1 Weighing in or Measuring |
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302 | (1) |
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303 | (1) |
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304 | (1) |
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3.4.4.6.4 Internal Standards |
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305 | (1) |
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3.4.4.7 Column Dimension and Particle Sizes |
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305 | (4) |
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309 | (2) |
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Part IV Current Challenges for HPLC Equipment Suppliers |
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311 | (73) |
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4.1 Optimization Strategies with your HPLC - Agilent Technologies |
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313 | (16) |
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4.1.1 Increase the Absolute Separation Performance: Zero Dead-Volume Fittings |
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314 | (1) |
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4.1.2 Separation Performance: Minimizing the Dispersion |
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314 | (2) |
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4.1.3 Increasing the Throughput - Different Ways to Lower the Turnaround Time |
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316 | (1) |
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4.1.4 Minimum Carryover for Trace Analysis: Multiwash |
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317 | (1) |
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4.1.5 Increase the Performance of What you have got - Modular or Stepwise Upgrade of Existing Systems |
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318 | (1) |
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4.1.6 Increase Automation, Ease of Use, and Reproducibility with the Features of a High-End Quaternary UHPLC Pump |
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319 | (2) |
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4.1.1 Increase Automation: Let your Autosampler do the Job |
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321 | (1) |
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4.1.8 Use Your System for Multiple Purposes: Multimethod and Method Development Systems |
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321 | (1) |
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4.1.9 Combine Sample Preparation with LC Analysis: Online SPE |
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322 | (1) |
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4.1.10 Boost Performance with a Second Chromatographic Dimension: 2D-LC (see also
Chapter 1.1) |
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323 | (1) |
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4.1.11 Think Different, Work with Supercritical C02 as Eluent: SFC - Supercritical Fluid Chromatography (see also
Chapter 1.6) |
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324 | (1) |
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4.1.12 Determine Different Concentration Ranges in One System: High-Definition Range (HDR) HPLC |
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325 | (1) |
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4.1.13 Automize Even Your Method Transfer from other LC Systems: Intelligent System Emulation Technology (ISET) |
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326 | (1) |
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327 | (1) |
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328 | (1) |
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4.2 To Empower the Customer - Optimization Through Individualization |
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329 | (14) |
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329 | (1) |
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4.2.2 Define Your Own Requirements |
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329 | (1) |
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4.2.2.1 Specification Sheet, Timetable, or Catalogue of Measures |
|
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329 | (2) |
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4.2.2.2 Personnel Optimization Helps to make Better Use of HPLC |
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331 | (1) |
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4.2.2.3 Mastering Time-Consuming Method Optimizations in a Planned Manner |
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332 | (1) |
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4.2.2.4 Optimizations at Device Level do not Always have to Mean an Investment |
|
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332 | (1) |
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4.2.3 An Assistant Opens Up Many New Possibilities |
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333 | (1) |
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4.2.3.1 If the HPLC System must Simply be able to do more in the Future |
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333 | (1) |
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4.2.3.2 Individual Optimizations with an Assistant |
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333 | (1) |
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4.2.3.3 Automatic Method Optimization and Column Screening |
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334 | (1) |
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4.2.3.4 A New Perspective at Fractionation, Sample Preparation, and Peak Recycling |
|
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335 | (1) |
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4.2.3.5 Continuous Chromatography, a New Level of Purification |
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336 | (1) |
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4.2.4 The Used Materials in the Focus of the Optimization |
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337 | (1) |
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4.2.4.1 Wetted vs. Dry Components of the HPLC |
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337 | (1) |
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4.2.4.2 Chemical Resistance of Wetted Components |
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338 | (2) |
|
4.2.4.3 Bioinert Components |
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340 | (1) |
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4.2.4.3.1 Material Certification |
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340 | (1) |
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4.2.5 Software Optimization Requires Open-Mindedness |
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340 | (1) |
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341 | (2) |
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4.3 (U)HPLC Basics and Beyond |
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343 | (12) |
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4.3.1 An Evaluation of (U)HPLC-operating Parameters and their Effect on Chromatographic Performance |
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343 | (1) |
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4.3.1.1 Compressibility Settings |
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343 | (3) |
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4.3.1.2 Solvent Composition and Injection Volume |
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346 | (2) |
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4.3.1.3 Photodiode Array Detector: Slit Width |
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348 | (1) |
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4.3.2 "Analytical Intelligence" - AI, M2M, IoT - How Modern Technology can Simplify the Lab Routine |
|
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349 | (1) |
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4.3.2.1 Auto-Diagnostics and Auto-Recovery to Maximize Reliability and Uptime |
|
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349 | (1) |
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4.3.2.2 Advanced Peak Processing to Improve Resolution |
|
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350 | (3) |
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4.3.2.3 Predictive Maintenance to Minimize System Downtime |
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353 | (1) |
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354 | (1) |
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4.4 Addressing Analytical Challenges in a Modern HPLC Laboratory |
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355 | (20) |
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4.4.1 Vanquish Core, Flex, and Horizon - Three Different Tiers, all Dedicated to Specific Requirements |
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356 | (6) |
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4.4.2 Intelligent and Self-Contained HPLC Devices |
|
|
362 | (1) |
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4.4.3 2D-LC for Analyzing Complex Samples and Further Automation Capabilities (see also
Chapter 1.1) |
|
|
363 | (1) |
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4.4.3.1 Loop-based Single-Heart-Cut 2D-LC |
|
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364 | (1) |
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4.4.3.2 Loop-based Multi-Heart-Cut 2D-LC |
|
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364 | (2) |
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4.4.3.3 Trap-based Single-Heart-Cut 2D-LC for Eluent Strength Reduction |
|
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366 | (1) |
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4.4.3.4 Trap-based Single-Heart-Cut 2D LC-MS Using Vanquish Dual Split Sampler |
|
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367 | (1) |
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4.4.4 Software-Assisted Automated Method Development |
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368 | (6) |
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374 | (1) |
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374 | (1) |
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4.5 Systematic Method Development with an Analytical Quality-by-Design Approach Supported by Fusion QbD and UPLC-MS |
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|
375 | (9) |
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| References |
|
384 | (1) |
| Index |
|
385 | |
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