LARRY M. MILLER is Principal Scientist at Amgen in Cambridge, MA, and is the author or co-author of more than 30 peer reviewed publications and two book chapters. J. DAVID PINKSTON, PHD, is Technical Services Manager at Archer Daniels Midland in Decatur, IL and the author or co-author of over 60 peer-reviewed publications and book chapters. LARRY T. TAYLOR, PHD, is Emeritus Professor of Chemistry at Virginia Tech in Blacksburg, VA, and the author or co-author of more than 400 peer-reviewed publications, books, and book chapters.

• Preface xiii1 Historical Development of SFC 11.1 Physical Properties of Supercritical Fluids 11.2 Discovery of Supercritical Fluids (1822–1892) 61.3 Supercritical Fluid Chromatography (1962–1980) 81.4 SFC with Open Tubular Columns (1980–1992) 151.5 Rediscovery of pcSFC (1992–2005) 191.6 Modern Packed Column SFC 22References 242 Carbon Dioxide as the Mobile Phase 292.1 Introduction to Carbon Dioxide 292.2 Supercritical Carbon Dioxide 322.3 Solvating Power of Supercritical CO2 352.4 Solvating Power of Modified CO2 452.5 Clustering of CO2 49References 523 Instrumentation for Analytical Scale Packed Column SFC 553.1 Introduction 563.2 Safety Considerations 563.3 Fluid Supply 583.3.1 Carbon Dioxide and Other Compressed Gases 583.3.2 Mobile Phase “Modifiers” and “Additives” 593.4 Fluid Delivery – Pumps and Pumping Considerations 603.4.1 Pump Thermostating 603.4.2 Fluid Pressurization and Metering 603.4.3 Modifier Fluid Pumping 613.4.4 Pressure and Flow Ranges 623.4.5 Fluid Mixing 623.5 Sample Injection and Autosamplers 623.6 Tubing and Connections 643.6.1 Tubing 643.6.1.1 Stainless Steel Tubing 643.6.1.2 Polymeric Tubing 653.6.2 Connections 663.7 Column and Mobile Phase Temperature Control 663.8 Chromatographic Column Materials of Construction 673.9 Backpressure Regulation 683.9.1 Passive Flow Restriction 693.9.2 Active Backpressure Regulation 703.10 Waste Disposal 723.11 Conclusion 72References 724 Detection in Packed Column SFC 774.1 Introduction 784.2 Predecompression Detection (Condensed‐Fluid‐Phase Detection) 784.2.1 UV/VIS Absorbance 784.2.2 Fluorescence Detection 814.2.3 Electrochemical Detection 824.2.4 Other Less Common Condensed Phase Detectors 834.2.4.1 Flow‐Cell Fourier Transform Infra‐Red Absorbance (FTIR) Detection 834.2.4.2 Online Nuclear Magnetic Resonance (NMR) Detection 844.2.4.3 Refractive Index (RI) Detection 854.3 Postdecompression Detection (Gas/Droplet Phase Detection) – Interfacing Approaches 854.3.1 Pre-BPR Flow Splitting 864.3.2 Total Flow Introduction (Post-BPR Detection) 884.3.2.1 BPR Requirements for Total‐Flow Introduction Detection 884.3.2.2 Total Flow Introduction with Mechanical BPR 894.3.2.3 Total Flow Introduction – Pressure‐Regulating‐Fluid (PRF) Interface 894.3.2.4 Total Flow Introduction without Active Backpressure Regulation 914.4 Postdecompression Detection 934.4.1 Flame‐Based Detectors 934.4.2 Evaporative Light Scattering Detection (ELSD) and Charged Aerosol Detection (Corona CAD) 974.4.3 Mass Spectrometric Detection 984.4.3.1 Interfacing and Ionization Approaches 994.4.3.2 Atmospheric Pressure Chemical Ionization (APCI) 1004.4.3.3 Pneumatically Assisted Electrospray Ionization (ESI) 1014.4.3.4 Atmospheric Pressure Photoionization (APPI) 1034.4.4 Postdecompression Detection Using Less Common Approaches – Deposition IR 1034.5 Concluding Remarks 103References 1045 Chiral Analytical Scale SFC – Method Development, Stationary Phases, and Mobile Phases 1175.1 Introduction 1175.2 Chiral Stationary Phases for SFC 1195.3 Chiral SFC vs. Chiral HPLC 1285.4 Method Development Approaches 1305.4.1 Modifiers for Chiral SFC 1325.4.2 Additives for Chiral SFC 1335.4.3 Nontraditional Modifiers 1355.4.4 Method Development Approaches 1375.5 High Throughput Method Development 1395.6 Summary 141References 1426 Achiral Analytical Scale SFC – Method Development, Stationary Phases, and Mobile Phases 1476.1 Introduction 1476.2 The Mixture to Be Separated 1486.2.1 Molecular Interactions 1486.2.2 Molecular “Handles” 1496.3 Achiral SFC Stationary Phases 1506.3.1 Column Safety and Compatibility 1506.3.2 Efficiency 1506.3.3 Retention 1536.3.4 Selectivity 1566.4 Mobile‐Phase Choices 1576.4.1 Primary Mobile‐Phase Component 1586.4.2 Secondary Mobile‐Phase Component – The “Modifier” 1596.4.3 Tertiary Mobile‐Phase Component – “Additives” 1636.5 Influence of Column Temperature on Efficiency and Selectivity 1706.6 Where Do I Go from Here? Method Development Decision Tree and Summary 172References 1747 Instrumentation for Preparative Scale Packed Column SFC 1837.1 Introduction 1837.2 Safety Considerations 1847.3 Fluid Supply 1857.3.1 Carbon Dioxide 1857.3.2 Mobile Phase Modifiers and Additives 1877.3.3 Carbon Dioxide Recycling 1887.4 Pumps and Pumping Considerations 1897.4.1 CO2 and Modifier Fluid Pumping 1897.4.2 Pressures and Flow Ranges 1897.5 Sample Injection 1907.5.1 Injection of Solutions 1907.5.2 Extraction Type Injection 1907.6 Chromatographic Columns 1927.7 Detection 1927.8 Back Pressure Regulation 1937.9 Fraction Collection 1937.9.1 Cyclone Collection 1947.9.2 Open‐Bed Collection 1957.10 Conclusion 197References 1978 Preparative Achiral and Chiral SFC – Method Development, Stationary Phases, and Mobile Phases 1998.1 Introduction 2008.1.1 Advantages and Disadvantages of SFC vs. HPLC for Purification 2018.1.2 Cost Comparison: Preparative HPLC vs. SFC 2028.2 Safety Considerations 2028.3 Developing Preparative Separations 2038.3.1 Linear Scale‐Up Calculations 2098.3.2 Scaling Rule in Supercritical Fluid Chromatography 2108.3.3 Metrics for Preparative Separations 2138.3.4 Options for Increasing Purification Productivity 2148.3.4.1 Closed‐Loop Recycling 2148.3.4.2 Stacked Injections 2148.3.5 Importance of Solubility on Preparative Separations 2148.3.6 Preparative SFC Injection Options 2178.4 Preparative Chiral SFC Purifications 2208.4.1 Chiral Stationary Phases (CSPs) for Preparative SFC 2208.4.2 Method Development for Chiral Purifications 2228.4.3 Preparative SFC Examples 2238.4.3.1 Milligram Scale Chiral Purification 2238.4.3.2 Gram Scale Chiral Purification 2248.4.4 Impact of Solubility on Productivity 2268.4.5 Use of Immobilized Chiral Stationary Phase (CCP) for Solubility‐Challenged Samples 2278.4.5.1 Immobilized CSP Example #1 2278.4.5.2 Immobilized CSP Example #2 2288.4.6 Coupling of Chiral and Achiral Columns for SFC Purifications 2298.5 Preparative Achiral SFC Purifications 2318.5.1 Introduction to Achiral SFC Purifications 2318.5.2 Stationary Phases for Achiral Preparative SFC 2328.5.3 Method Development for Achiral Purifications 2328.5.4 Achiral SFC Purification Examples 2348.5.4.1 Achiral Purification Example #1 2348.5.4.2 Achiral Purification Example #2 2348.5.5 Purifications Using Mass‐Directed SFC 2368.5.6 Impurity Isolation Using Preparative SFC 2378.5.6.1 Impurity Isolation Example 2408.5.7 SFC as Alternative to Flash Purification 2418.6 Best Practices for Successful SFC Purifications 2448.6.1 Sample Filtration and Inlet Filters 2448.6.2 Sample Purity 2468.6.3 Salt vs. Free Base 2478.6.4 Primary Amine Protection to Improve Enantiomer Resolution 2508.6.5 Evaluation of Alternate Synthetic Intermediates to Improve SFC Purification Productivity 2508.7 Summary 254References 2549 Impact and Promise of SFC in the Pharmaceutical Industry 2679.1 Introduction to Pharmaceutical Industry 2679.2 SFC in Pharmaceutical Discovery 2689.2.1 Early Discovery Support 2689.2.2 SFC in Medicinal Chemistry 2699.2.2.1 Analytical SFC 2709.2.2.2 Preparative SFC 2719.2.3 Physiochemical Measurement by SFC 2739.2.4 Use of SFC for Pharmacokinetic and Drug Metabolism Studies 2749.3 SFC in Development and Manufacturing 2769.3.1 Analytical SFC Analysis of Drug Substances and Drug Products 2769.3.2 Preparative SFC in Development and Manufacturing 2829.3.3 Metabolite/PKDM Studies in Development 2839.3.4 SFC in Chemical Process Development 2839.4 SFC for Analysis of Illegal Drugs 2849.5 Summary 286References 28610 Impact of SFC in the Petroleum Industry 29710.1 Petroleum Chemistry 29710.1.1 Crude Refining Processes 29710.1.2 Petrochemical Processes 29810.2 Introduction to Petroleum Analysis 29910.3 Historical Perspective 30110.3.1 Hydrocarbon Analysis via FIA 30110.3.2 SFC Replaces FIA 30110.3.3 Hydrocarbon SFC Analysis via ASTM 5186‐91 30210.4 Early Petroleum Applications of SFC 30410.4.1 Samples with Broad Polymer Distribution 30410.4.2 SFC Purification of Polycyclic Aromatic Hydrocarbons 30510.4.3 Coal Tar Pitch 30510.4.4 Enhanced SFC Performance 30510.4.5 Sulfur Detection in a Petroleum Matrix 30710.5 SFC Replacement for GC and LC 30810.5.1 Simulated Distillation 30810.5.2 Hydrocarbon Group‐Type Separations – PIONA Analysis 31010.6 Biodiesel Purification 31110.7 Multidimensional Separations 31410.7.1 Comprehensive Two‐Dimensional SFC 31410.7.2 SFC‐GC × GC 31510.7.3 Comprehensive – SFC‐Twin‐Two‐Dimensional (GC × GC) 316References 31711 Selected SFC Applications in the Food, Polymer, and Personal Care Industries 32111.1 Introduction 32111.2 Selected Applications in the Foods Industry 32211.2.1 Fats, Oils, and Fatty Acids 32211.2.2 Tocopherols 32511.2.3 Other Vitamins 32711.2.4 Food Preservatives (Other Antioxidants and Antimicrobials) 33011.2.5 Coloring Agents 33011.2.6 Sugars 33111.3 Selected Applications in the Field of Synthetic Polymers 33211.3.1 Molecular Weight Distribution 33211.3.2 Structural Characterization 33411.3.3 “Critical Condition” Group/Block Separations of Complex Polymers Using CO2‐containing Mobile Phases 33411.3.4 Polymer Additives 33511.4 Selected Applications in the Personal Care Industry 33711.4.1 Lipophilic Components of Cosmetics 33711.4.2 Surfactants in Cleaning Mixtures 33711.4.3 Emulsifiers in Personal Care Products 33711.4.4 Preservatives 33811.5 Conclusions 340References 34012 Analysis of Cannabis Products by Supercritical Fluid Chromatography 34712.1 Introduction 34712.1.1 Cannabis History 34812.2 Analytical SFC 35112.2.1 Introduction 35112.2.2 Early SFC of Cannabis Products 35212.2.3 Achiral SFC 35312.2.4 Chiral SFC 35412.2.5 Metabolite Analysis 35712.3 Preparative SFC 35712.4 Summary 360References 36113 The Future of SFC 36513.1 Introduction 36513.2 SFC Publication Record 36613.3 SFC Research in Academia 36813.4 SFC Conferences 36813.5 Anticipated Technical Advances 36913.6 Limits to SFC Expansion 37013.7 Summary 372References 373Index 377

LARRY M. MILLER is Principal Scientist at Amgen in Cambridge, MA, and is the author or co-author of more than 30 peer reviewed publications and two book chapters. J. DAVID PINKSTON, PHD, is Technical Services Manager at Archer Daniels Midland in Decatur, IL and the author or co-author of over 60 peer-reviewed publications and book chapters. LARRY T. TAYLOR, PHD, is Emeritus Professor of Chemistry at Virginia Tech in Blacksburg, VA, and the author or co-author of more than 400 peer-reviewed publications, books, and book chapters.

• Preface xiii1 Historical Development of SFC 11.1 Physical Properties of Supercritical Fluids 11.2 Discovery of Supercritical Fluids (1822–1892) 61.3 Supercritical Fluid Chromatography (1962–1980) 81.4 SFC with Open Tubular Columns (1980–1992) 151.5 Rediscovery of pcSFC (1992–2005) 191.6 Modern Packed Column SFC 22References 242 Carbon Dioxide as the Mobile Phase 292.1 Introduction to Carbon Dioxide 292.2 Supercritical Carbon Dioxide 322.3 Solvating Power of Supercritical CO2 352.4 Solvating Power of Modified CO2 452.5 Clustering of CO2 49References 523 Instrumentation for Analytical Scale Packed Column SFC 553.1 Introduction 563.2 Safety Considerations 563.3 Fluid Supply 583.3.1 Carbon Dioxide and Other Compressed Gases 583.3.2 Mobile Phase “Modifiers” and “Additives” 593.4 Fluid Delivery – Pumps and Pumping Considerations 603.4.1 Pump Thermostating 603.4.2 Fluid Pressurization and Metering 603.4.3 Modifier Fluid Pumping 613.4.4 Pressure and Flow Ranges 623.4.5 Fluid Mixing 623.5 Sample Injection and Autosamplers 623.6 Tubing and Connections 643.6.1 Tubing 643.6.1.1 Stainless Steel Tubing 643.6.1.2 Polymeric Tubing 653.6.2 Connections 663.7 Column and Mobile Phase Temperature Control 663.8 Chromatographic Column Materials of Construction 673.9 Backpressure Regulation 683.9.1 Passive Flow Restriction 693.9.2 Active Backpressure Regulation 703.10 Waste Disposal 723.11 Conclusion 72References 724 Detection in Packed Column SFC 774.1 Introduction 784.2 Predecompression Detection (Condensed‐Fluid‐Phase Detection) 784.2.1 UV/VIS Absorbance 784.2.2 Fluorescence Detection 814.2.3 Electrochemical Detection 824.2.4 Other Less Common Condensed Phase Detectors 834.2.4.1 Flow‐Cell Fourier Transform Infra‐Red Absorbance (FTIR) Detection 834.2.4.2 Online Nuclear Magnetic Resonance (NMR) Detection 844.2.4.3 Refractive Index (RI) Detection 854.3 Postdecompression Detection (Gas/Droplet Phase Detection) – Interfacing Approaches 854.3.1 Pre-BPR Flow Splitting 864.3.2 Total Flow Introduction (Post-BPR Detection) 884.3.2.1 BPR Requirements for Total‐Flow Introduction Detection 884.3.2.2 Total Flow Introduction with Mechanical BPR 894.3.2.3 Total Flow Introduction – Pressure‐Regulating‐Fluid (PRF) Interface 894.3.2.4 Total Flow Introduction without Active Backpressure Regulation 914.4 Postdecompression Detection 934.4.1 Flame‐Based Detectors 934.4.2 Evaporative Light Scattering Detection (ELSD) and Charged Aerosol Detection (Corona CAD) 974.4.3 Mass Spectrometric Detection 984.4.3.1 Interfacing and Ionization Approaches 994.4.3.2 Atmospheric Pressure Chemical Ionization (APCI) 1004.4.3.3 Pneumatically Assisted Electrospray Ionization (ESI) 1014.4.3.4 Atmospheric Pressure Photoionization (APPI) 1034.4.4 Postdecompression Detection Using Less Common Approaches – Deposition IR 1034.5 Concluding Remarks 103References 1045 Chiral Analytical Scale SFC – Method Development, Stationary Phases, and Mobile Phases 1175.1 Introduction 1175.2 Chiral Stationary Phases for SFC 1195.3 Chiral SFC vs. Chiral HPLC 1285.4 Method Development Approaches 1305.4.1 Modifiers for Chiral SFC 1325.4.2 Additives for Chiral SFC 1335.4.3 Nontraditional Modifiers 1355.4.4 Method Development Approaches 1375.5 High Throughput Method Development 1395.6 Summary 141References 1426 Achiral Analytical Scale SFC – Method Development, Stationary Phases, and Mobile Phases 1476.1 Introduction 1476.2 The Mixture to Be Separated 1486.2.1 Molecular Interactions 1486.2.2 Molecular “Handles” 1496.3 Achiral SFC Stationary Phases 1506.3.1 Column Safety and Compatibility 1506.3.2 Efficiency 1506.3.3 Retention 1536.3.4 Selectivity 1566.4 Mobile‐Phase Choices 1576.4.1 Primary Mobile‐Phase Component 1586.4.2 Secondary Mobile‐Phase Component – The “Modifier” 1596.4.3 Tertiary Mobile‐Phase Component – “Additives” 1636.5 Influence of Column Temperature on Efficiency and Selectivity 1706.6 Where Do I Go from Here? Method Development Decision Tree and Summary 172References 1747 Instrumentation for Preparative Scale Packed Column SFC 1837.1 Introduction 1837.2 Safety Considerations 1847.3 Fluid Supply 1857.3.1 Carbon Dioxide 1857.3.2 Mobile Phase Modifiers and Additives 1877.3.3 Carbon Dioxide Recycling 1887.4 Pumps and Pumping Considerations 1897.4.1 CO2 and Modifier Fluid Pumping 1897.4.2 Pressures and Flow Ranges 1897.5 Sample Injection 1907.5.1 Injection of Solutions 1907.5.2 Extraction Type Injection 1907.6 Chromatographic Columns 1927.7 Detection 1927.8 Back Pressure Regulation 1937.9 Fraction Collection 1937.9.1 Cyclone Collection 1947.9.2 Open‐Bed Collection 1957.10 Conclusion 197References 1978 Preparative Achiral and Chiral SFC – Method Development, Stationary Phases, and Mobile Phases 1998.1 Introduction 2008.1.1 Advantages and Disadvantages of SFC vs. HPLC for Purification 2018.1.2 Cost Comparison: Preparative HPLC vs. SFC 2028.2 Safety Considerations 2028.3 Developing Preparative Separations 2038.3.1 Linear Scale‐Up Calculations 2098.3.2 Scaling Rule in Supercritical Fluid Chromatography 2108.3.3 Metrics for Preparative Separations 2138.3.4 Options for Increasing Purification Productivity 2148.3.4.1 Closed‐Loop Recycling 2148.3.4.2 Stacked Injections 2148.3.5 Importance of Solubility on Preparative Separations 2148.3.6 Preparative SFC Injection Options 2178.4 Preparative Chiral SFC Purifications 2208.4.1 Chiral Stationary Phases (CSPs) for Preparative SFC 2208.4.2 Method Development for Chiral Purifications 2228.4.3 Preparative SFC Examples 2238.4.3.1 Milligram Scale Chiral Purification 2238.4.3.2 Gram Scale Chiral Purification 2248.4.4 Impact of Solubility on Productivity 2268.4.5 Use of Immobilized Chiral Stationary Phase (CCP) for Solubility‐Challenged Samples 2278.4.5.1 Immobilized CSP Example #1 2278.4.5.2 Immobilized CSP Example #2 2288.4.6 Coupling of Chiral and Achiral Columns for SFC Purifications 2298.5 Preparative Achiral SFC Purifications 2318.5.1 Introduction to Achiral SFC Purifications 2318.5.2 Stationary Phases for Achiral Preparative SFC 2328.5.3 Method Development for Achiral Purifications 2328.5.4 Achiral SFC Purification Examples 2348.5.4.1 Achiral Purification Example #1 2348.5.4.2 Achiral Purification Example #2 2348.5.5 Purifications Using Mass‐Directed SFC 2368.5.6 Impurity Isolation Using Preparative SFC 2378.5.6.1 Impurity Isolation Example 2408.5.7 SFC as Alternative to Flash Purification 2418.6 Best Practices for Successful SFC Purifications 2448.6.1 Sample Filtration and Inlet Filters 2448.6.2 Sample Purity 2468.6.3 Salt vs. Free Base 2478.6.4 Primary Amine Protection to Improve Enantiomer Resolution 2508.6.5 Evaluation of Alternate Synthetic Intermediates to Improve SFC Purification Productivity 2508.7 Summary 254References 2549 Impact and Promise of SFC in the Pharmaceutical Industry 2679.1 Introduction to Pharmaceutical Industry 2679.2 SFC in Pharmaceutical Discovery 2689.2.1 Early Discovery Support 2689.2.2 SFC in Medicinal Chemistry 2699.2.2.1 Analytical SFC 2709.2.2.2 Preparative SFC 2719.2.3 Physiochemical Measurement by SFC 2739.2.4 Use of SFC for Pharmacokinetic and Drug Metabolism Studies 2749.3 SFC in Development and Manufacturing 2769.3.1 Analytical SFC Analysis of Drug Substances and Drug Products 2769.3.2 Preparative SFC in Development and Manufacturing 2829.3.3 Metabolite/PKDM Studies in Development 2839.3.4 SFC in Chemical Process Development 2839.4 SFC for Analysis of Illegal Drugs 2849.5 Summary 286References 28610 Impact of SFC in the Petroleum Industry 29710.1 Petroleum Chemistry 29710.1.1 Crude Refining Processes 29710.1.2 Petrochemical Processes 29810.2 Introduction to Petroleum Analysis 29910.3 Historical Perspective 30110.3.1 Hydrocarbon Analysis via FIA 30110.3.2 SFC Replaces FIA 30110.3.3 Hydrocarbon SFC Analysis via ASTM 5186‐91 30210.4 Early Petroleum Applications of SFC 30410.4.1 Samples with Broad Polymer Distribution 30410.4.2 SFC Purification of Polycyclic Aromatic Hydrocarbons 30510.4.3 Coal Tar Pitch 30510.4.4 Enhanced SFC Performance 30510.4.5 Sulfur Detection in a Petroleum Matrix 30710.5 SFC Replacement for GC and LC 30810.5.1 Simulated Distillation 30810.5.2 Hydrocarbon Group‐Type Separations – PIONA Analysis 31010.6 Biodiesel Purification 31110.7 Multidimensional Separations 31410.7.1 Comprehensive Two‐Dimensional SFC 31410.7.2 SFC‐GC × GC 31510.7.3 Comprehensive – SFC‐Twin‐Two‐Dimensional (GC × GC) 316References 31711 Selected SFC Applications in the Food, Polymer, and Personal Care Industries 32111.1 Introduction 32111.2 Selected Applications in the Foods Industry 32211.2.1 Fats, Oils, and Fatty Acids 32211.2.2 Tocopherols 32511.2.3 Other Vitamins 32711.2.4 Food Preservatives (Other Antioxidants and Antimicrobials) 33011.2.5 Coloring Agents 33011.2.6 Sugars 33111.3 Selected Applications in the Field of Synthetic Polymers 33211.3.1 Molecular Weight Distribution 33211.3.2 Structural Characterization 33411.3.3 “Critical Condition” Group/Block Separations of Complex Polymers Using CO2‐containing Mobile Phases 33411.3.4 Polymer Additives 33511.4 Selected Applications in the Personal Care Industry 33711.4.1 Lipophilic Components of Cosmetics 33711.4.2 Surfactants in Cleaning Mixtures 33711.4.3 Emulsifiers in Personal Care Products 33711.4.4 Preservatives 33811.5 Conclusions 340References 34012 Analysis of Cannabis Products by Supercritical Fluid Chromatography 34712.1 Introduction 34712.1.1 Cannabis History 34812.2 Analytical SFC 35112.2.1 Introduction 35112.2.2 Early SFC of Cannabis Products 35212.2.3 Achiral SFC 35312.2.4 Chiral SFC 35412.2.5 Metabolite Analysis 35712.3 Preparative SFC 35712.4 Summary 360References 36113 The Future of SFC 36513.1 Introduction 36513.2 SFC Publication Record 36613.3 SFC Research in Academia 36813.4 SFC Conferences 36813.5 Anticipated Technical Advances 36913.6 Limits to SFC Expansion 37013.7 Summary 372References 373Index 377

LARRY M. MILLER is Principal Scientist at Amgen in Cambridge, MA, and is the author or co-author of more than 30 peer reviewed publications and two book chapters. J. DAVID PINKSTON, PHD, is Technical Services Manager at Archer Daniels Midland in Decatur, IL and the author or co-author of over 60 peer-reviewed publications and book chapters. LARRY T. TAYLOR, PHD, is Emeritus Professor of Chemistry at Virginia Tech in Blacksburg, VA, and the author or co-author of more than 400 peer-reviewed publications, books, and book chapters.

• Preface xiii1 Historical Development of SFC 11.1 Physical Properties of Supercritical Fluids 11.2 Discovery of Supercritical Fluids (1822–1892) 61.3 Supercritical Fluid Chromatography (1962–1980) 81.4 SFC with Open Tubular Columns (1980–1992) 151.5 Rediscovery of pcSFC (1992–2005) 191.6 Modern Packed Column SFC 22References 242 Carbon Dioxide as the Mobile Phase 292.1 Introduction to Carbon Dioxide 292.2 Supercritical Carbon Dioxide 322.3 Solvating Power of Supercritical CO2 352.4 Solvating Power of Modified CO2 452.5 Clustering of CO2 49References 523 Instrumentation for Analytical Scale Packed Column SFC 553.1 Introduction 563.2 Safety Considerations 563.3 Fluid Supply 583.3.1 Carbon Dioxide and Other Compressed Gases 583.3.2 Mobile Phase “Modifiers” and “Additives” 593.4 Fluid Delivery – Pumps and Pumping Considerations 603.4.1 Pump Thermostating 603.4.2 Fluid Pressurization and Metering 603.4.3 Modifier Fluid Pumping 613.4.4 Pressure and Flow Ranges 623.4.5 Fluid Mixing 623.5 Sample Injection and Autosamplers 623.6 Tubing and Connections 643.6.1 Tubing 643.6.1.1 Stainless Steel Tubing 643.6.1.2 Polymeric Tubing 653.6.2 Connections 663.7 Column and Mobile Phase Temperature Control 663.8 Chromatographic Column Materials of Construction 673.9 Backpressure Regulation 683.9.1 Passive Flow Restriction 693.9.2 Active Backpressure Regulation 703.10 Waste Disposal 723.11 Conclusion 72References 724 Detection in Packed Column SFC 774.1 Introduction 784.2 Predecompression Detection (Condensed‐Fluid‐Phase Detection) 784.2.1 UV/VIS Absorbance 784.2.2 Fluorescence Detection 814.2.3 Electrochemical Detection 824.2.4 Other Less Common Condensed Phase Detectors 834.2.4.1 Flow‐Cell Fourier Transform Infra‐Red Absorbance (FTIR) Detection 834.2.4.2 Online Nuclear Magnetic Resonance (NMR) Detection 844.2.4.3 Refractive Index (RI) Detection 854.3 Postdecompression Detection (Gas/Droplet Phase Detection) – Interfacing Approaches 854.3.1 Pre-BPR Flow Splitting 864.3.2 Total Flow Introduction (Post-BPR Detection) 884.3.2.1 BPR Requirements for Total‐Flow Introduction Detection 884.3.2.2 Total Flow Introduction with Mechanical BPR 894.3.2.3 Total Flow Introduction – Pressure‐Regulating‐Fluid (PRF) Interface 894.3.2.4 Total Flow Introduction without Active Backpressure Regulation 914.4 Postdecompression Detection 934.4.1 Flame‐Based Detectors 934.4.2 Evaporative Light Scattering Detection (ELSD) and Charged Aerosol Detection (Corona CAD) 974.4.3 Mass Spectrometric Detection 984.4.3.1 Interfacing and Ionization Approaches 994.4.3.2 Atmospheric Pressure Chemical Ionization (APCI) 1004.4.3.3 Pneumatically Assisted Electrospray Ionization (ESI) 1014.4.3.4 Atmospheric Pressure Photoionization (APPI) 1034.4.4 Postdecompression Detection Using Less Common Approaches – Deposition IR 1034.5 Concluding Remarks 103References 1045 Chiral Analytical Scale SFC – Method Development, Stationary Phases, and Mobile Phases 1175.1 Introduction 1175.2 Chiral Stationary Phases for SFC 1195.3 Chiral SFC vs. Chiral HPLC 1285.4 Method Development Approaches 1305.4.1 Modifiers for Chiral SFC 1325.4.2 Additives for Chiral SFC 1335.4.3 Nontraditional Modifiers 1355.4.4 Method Development Approaches 1375.5 High Throughput Method Development 1395.6 Summary 141References 1426 Achiral Analytical Scale SFC – Method Development, Stationary Phases, and Mobile Phases 1476.1 Introduction 1476.2 The Mixture to Be Separated 1486.2.1 Molecular Interactions 1486.2.2 Molecular “Handles” 1496.3 Achiral SFC Stationary Phases 1506.3.1 Column Safety and Compatibility 1506.3.2 Efficiency 1506.3.3 Retention 1536.3.4 Selectivity 1566.4 Mobile‐Phase Choices 1576.4.1 Primary Mobile‐Phase Component 1586.4.2 Secondary Mobile‐Phase Component – The “Modifier” 1596.4.3 Tertiary Mobile‐Phase Component – “Additives” 1636.5 Influence of Column Temperature on Efficiency and Selectivity 1706.6 Where Do I Go from Here? Method Development Decision Tree and Summary 172References 1747 Instrumentation for Preparative Scale Packed Column SFC 1837.1 Introduction 1837.2 Safety Considerations 1847.3 Fluid Supply 1857.3.1 Carbon Dioxide 1857.3.2 Mobile Phase Modifiers and Additives 1877.3.3 Carbon Dioxide Recycling 1887.4 Pumps and Pumping Considerations 1897.4.1 CO2 and Modifier Fluid Pumping 1897.4.2 Pressures and Flow Ranges 1897.5 Sample Injection 1907.5.1 Injection of Solutions 1907.5.2 Extraction Type Injection 1907.6 Chromatographic Columns 1927.7 Detection 1927.8 Back Pressure Regulation 1937.9 Fraction Collection 1937.9.1 Cyclone Collection 1947.9.2 Open‐Bed Collection 1957.10 Conclusion 197References 1978 Preparative Achiral and Chiral SFC – Method Development, Stationary Phases, and Mobile Phases 1998.1 Introduction 2008.1.1 Advantages and Disadvantages of SFC vs. HPLC for Purification 2018.1.2 Cost Comparison: Preparative HPLC vs. SFC 2028.2 Safety Considerations 2028.3 Developing Preparative Separations 2038.3.1 Linear Scale‐Up Calculations 2098.3.2 Scaling Rule in Supercritical Fluid Chromatography 2108.3.3 Metrics for Preparative Separations 2138.3.4 Options for Increasing Purification Productivity 2148.3.4.1 Closed‐Loop Recycling 2148.3.4.2 Stacked Injections 2148.3.5 Importance of Solubility on Preparative Separations 2148.3.6 Preparative SFC Injection Options 2178.4 Preparative Chiral SFC Purifications 2208.4.1 Chiral Stationary Phases (CSPs) for Preparative SFC 2208.4.2 Method Development for Chiral Purifications 2228.4.3 Preparative SFC Examples 2238.4.3.1 Milligram Scale Chiral Purification 2238.4.3.2 Gram Scale Chiral Purification 2248.4.4 Impact of Solubility on Productivity 2268.4.5 Use of Immobilized Chiral Stationary Phase (CCP) for Solubility‐Challenged Samples 2278.4.5.1 Immobilized CSP Example #1 2278.4.5.2 Immobilized CSP Example #2 2288.4.6 Coupling of Chiral and Achiral Columns for SFC Purifications 2298.5 Preparative Achiral SFC Purifications 2318.5.1 Introduction to Achiral SFC Purifications 2318.5.2 Stationary Phases for Achiral Preparative SFC 2328.5.3 Method Development for Achiral Purifications 2328.5.4 Achiral SFC Purification Examples 2348.5.4.1 Achiral Purification Example #1 2348.5.4.2 Achiral Purification Example #2 2348.5.5 Purifications Using Mass‐Directed SFC 2368.5.6 Impurity Isolation Using Preparative SFC 2378.5.6.1 Impurity Isolation Example 2408.5.7 SFC as Alternative to Flash Purification 2418.6 Best Practices for Successful SFC Purifications 2448.6.1 Sample Filtration and Inlet Filters 2448.6.2 Sample Purity 2468.6.3 Salt vs. Free Base 2478.6.4 Primary Amine Protection to Improve Enantiomer Resolution 2508.6.5 Evaluation of Alternate Synthetic Intermediates to Improve SFC Purification Productivity 2508.7 Summary 254References 2549 Impact and Promise of SFC in the Pharmaceutical Industry 2679.1 Introduction to Pharmaceutical Industry 2679.2 SFC in Pharmaceutical Discovery 2689.2.1 Early Discovery Support 2689.2.2 SFC in Medicinal Chemistry 2699.2.2.1 Analytical SFC 2709.2.2.2 Preparative SFC 2719.2.3 Physiochemical Measurement by SFC 2739.2.4 Use of SFC for Pharmacokinetic and Drug Metabolism Studies 2749.3 SFC in Development and Manufacturing 2769.3.1 Analytical SFC Analysis of Drug Substances and Drug Products 2769.3.2 Preparative SFC in Development and Manufacturing 2829.3.3 Metabolite/PKDM Studies in Development 2839.3.4 SFC in Chemical Process Development 2839.4 SFC for Analysis of Illegal Drugs 2849.5 Summary 286References 28610 Impact of SFC in the Petroleum Industry 29710.1 Petroleum Chemistry 29710.1.1 Crude Refining Processes 29710.1.2 Petrochemical Processes 29810.2 Introduction to Petroleum Analysis 29910.3 Historical Perspective 30110.3.1 Hydrocarbon Analysis via FIA 30110.3.2 SFC Replaces FIA 30110.3.3 Hydrocarbon SFC Analysis via ASTM 5186‐91 30210.4 Early Petroleum Applications of SFC 30410.4.1 Samples with Broad Polymer Distribution 30410.4.2 SFC Purification of Polycyclic Aromatic Hydrocarbons 30510.4.3 Coal Tar Pitch 30510.4.4 Enhanced SFC Performance 30510.4.5 Sulfur Detection in a Petroleum Matrix 30710.5 SFC Replacement for GC and LC 30810.5.1 Simulated Distillation 30810.5.2 Hydrocarbon Group‐Type Separations – PIONA Analysis 31010.6 Biodiesel Purification 31110.7 Multidimensional Separations 31410.7.1 Comprehensive Two‐Dimensional SFC 31410.7.2 SFC‐GC × GC 31510.7.3 Comprehensive – SFC‐Twin‐Two‐Dimensional (GC × GC) 316References 31711 Selected SFC Applications in the Food, Polymer, and Personal Care Industries 32111.1 Introduction 32111.2 Selected Applications in the Foods Industry 32211.2.1 Fats, Oils, and Fatty Acids 32211.2.2 Tocopherols 32511.2.3 Other Vitamins 32711.2.4 Food Preservatives (Other Antioxidants and Antimicrobials) 33011.2.5 Coloring Agents 33011.2.6 Sugars 33111.3 Selected Applications in the Field of Synthetic Polymers 33211.3.1 Molecular Weight Distribution 33211.3.2 Structural Characterization 33411.3.3 “Critical Condition” Group/Block Separations of Complex Polymers Using CO2‐containing Mobile Phases 33411.3.4 Polymer Additives 33511.4 Selected Applications in the Personal Care Industry 33711.4.1 Lipophilic Components of Cosmetics 33711.4.2 Surfactants in Cleaning Mixtures 33711.4.3 Emulsifiers in Personal Care Products 33711.4.4 Preservatives 33811.5 Conclusions 340References 34012 Analysis of Cannabis Products by Supercritical Fluid Chromatography 34712.1 Introduction 34712.1.1 Cannabis History 34812.2 Analytical SFC 35112.2.1 Introduction 35112.2.2 Early SFC of Cannabis Products 35212.2.3 Achiral SFC 35312.2.4 Chiral SFC 35412.2.5 Metabolite Analysis 35712.3 Preparative SFC 35712.4 Summary 360References 36113 The Future of SFC 36513.1 Introduction 36513.2 SFC Publication Record 36613.3 SFC Research in Academia 36813.4 SFC Conferences 36813.5 Anticipated Technical Advances 36913.6 Limits to SFC Expansion 37013.7 Summary 372References 373Index 377