Explains why modern supercritical fluid chromatography (SFC) is the leading "green" analytical and purification separations technology.
Modern supercritical fluid chromatography (SFC) is the leading method used to analyze and purify chiral and achiral chemical compounds, many of which are pharmaceuticals, pharmaceutical candidates, and natural products including cannabis-related compounds. This book covers current SFC instrumentation as it relates to greater robustness, better reproducibility, and increased analytical sensitivity.
Modern Supercritical Fluid Chromatography: Carbon Dioxide Containing Mobile Phases covers the history, instrumentation, method development and applications of SFC. The authors provided readers with an overview of analytical and preparative SFC equipment, stationary phases, and mobile phase choices. Topics covered include: Milestones of Supercritical Fluid Chromatography; Physical Properties of Supercritical Fluids; Instrumentation for SFC; Detection in SFC; Achiral SFC Method Development; Chiral SFC Method Development; and Preparative Scale SFC. The book also includes highlights of modern applications of SFC in the final chapters-namely pharmaceuticals, consumer products, foods, polymers, petroleum-related mixtures, and cannabis-and discusses the future of SFC.
Provides a clear explanation of the physical and chemical properties of supercritical fluids, which gives the reader a better understanding of the basis for improved performance in SFC compared to HPLC and GC
Describes the advantages of SFC as a green alternative to HPLC and GC for the analysis of both polar, water-soluble, and non-polar analytes
Details both achiral and chiral SFC method development, including modifiers, additives, the impact of temperature and pressure, and stationary phase choices
Details why SFC is the premier modern preparative chromatographic technique used to purify components of mixtures for subsequent uses, both from performance and economic perspectives
Covers numerous detectors, with an emphasis on SFC-MS, SFC-UV, and SFC-ELSD (evaporative light scattering detection)
Describes the application of SFC to numerous high-value application areas
Modern Supercritical Fluid Chromatography: Carbon Dioxide Containing Mobile Phases will be of great interest to professionals, students, and professors involved in analytical, bioanalytical, separations science, medicinal, petroleum, and environmental chemistries. It will also appeal to pharmaceutical scientists, natural-product scientists, food and consumer-products scientists, chemical engineers, and managers in these areas.
<b>Explains why modern supercritical fluid chromatography (SFC) is the leading "green" analytical and purification separations technology.</b>
Modern supercritical fluid chromatography (SFC) is the leading method used to analyze and purify chiral and achiral chemical compounds, many of which are pharmaceuticals, pharmaceutical candidates, and natural products including cannabis-related compounds. This book covers current SFC instrumentation as it relates to greater robustness, better reproducibility, and increased analytical sensitivity.
<i>Modern Supercritical Fluid Chromatography: Carbon Dioxide Containing Mobile Phases </i>covers the history, instrumentation, method development and applications of SFC. The authors provided readers with an overview of analytical and preparative SFC equipment, stationary phases, and mobile phase choices. Topics covered include: Milestones of Supercritical Fluid Chromatography; Physical Properties of Supercritical Fluids; Instrumentation for SFC; Detection in SFC; Achiral SFC Method Development; Chiral SFC Method Development; and Preparative Scale SFC. The book also includes highlights of modern applications of SFC in the final chapters-namely pharmaceuticals, consumer products, foods, polymers, petroleum-related mixtures, and cannabis-and discusses the future of SFC.
<ul><li>Provides a clear explanation of the physical and chemical properties of supercritical fluids, which gives the reader a better understanding of the basis for improved performance in SFC compared to HPLC and GC</li><li>Describes the advantages of SFC as a green alternative to HPLC and GC for the analysis of both polar, water-soluble, and non-polar analytes</li><li>Details both achiral and chiral SFC method development, including modifiers, additives, the impact of temperature and pressure, and stationary phase choices</li><li>Details why SFC is the premier modern preparative chromatographic technique used to purify components of mixtures for subsequent uses, both from performance and economic perspectives</li><li>Covers numerous detectors, with an emphasis on SFC-MS, SFC-UV, and SFC-ELSD (evaporative light scattering detection)</li><li>Describes the application of SFC to numerous high-value application areas</li></ul>
<i>Modern Supercritical Fluid Chromatography: Carbon Dioxide Containing Mobile Phases </i>will be of great interest to professionals, students, and professors involved in analytical, bioanalytical, separations science, medicinal, petroleum, and environmental chemistries. It will also appeal to pharmaceutical scientists, natural-product scientists, food and consumer-products scientists, chemical engineers, and managers in these areas.
Preface xiii
<b>1 Historical Development of SFC 1</b>
1.1 Physical Properties of Supercritical Fluids 1
1.2 Discovery of Supercritical Fluids (1822-1892) 6
1.3 Supercritical Fluid Chromatography (1962-1980) 8
1.4 SFC with Open Tubular Columns (1980-1992) 15
1.5 Rediscovery of pcSFC (1992-2005) 19
1.6 Modern Packed Column SFC 22
References 24
<b>2 Carbon Dioxide as the Mobile Phase 29</b>
2.1 Introduction to Carbon Dioxide 29
2.2 Supercritical Carbon Dioxide 32
2.3 Solvating Power of Supercritical CO<sub>2 </sub>35
2.4 Solvating Power of Modified CO<sub>2</sub> 45
2.5 Clustering of CO<sub>2 </sub>49
References 52
<b>3 Instrumentation for Analytical Scale Packed Column SFC 55</b>
3.1 Introduction 56
3.2 Safety Considerations 56
3.3 Fluid Supply 58
3.3.1 Carbon Dioxide and Other Compressed Gases 58
3.3.2 Mobile Phase "Modifiers" and "Additives" 59
3.4 Fluid Delivery - Pumps and Pumping Considerations 60
3.4.1 Pump Thermostating 60
3.4.2 Fluid Pressurization and Metering 60
3.4.3 Modifier Fluid Pumping 61
3.4.4 Pressure and Flow Ranges 62
3.4.5 Fluid Mixing 62
3.5 Sample Injection and Autosamplers 62
3.6 Tubing and Connections 64
3.6.1 Tubing 64
3.6.1.1 Stainless Steel Tubing 64
3.6.1.2 Polymeric Tubing 65
3.6.2 Connections 66
3.7 Column and Mobile Phase Temperature Control 66
3.8 Chromatographic Column Materials of Construction 67
3.9 Backpressure Regulation 68
3.9.1 Passive Flow Restriction 69
3.9.2 Active Backpressure Regulation 70
3.10 Waste Disposal 72
3.11 Conclusion 72
References 72
<b>4 Detection in Packed Column SFC 77</b>
4.1 Introduction 78
4.2 Predecompression Detection (Condensed-Fluid-Phase Detection) 78
4.2.1 UV/VIS Absorbance 78
4.2.2 Fluorescence Detection 81
4.2.3 Electrochemical Detection 82
4.2.4 Other Less Common Condensed Phase Detectors 83
4.2.4.1 Flow-Cell Fourier Transform Infra-Red Absorbance (FTIR) Detection 83
4.2.4.2 Online Nuclear Magnetic Resonance (NMR) Detection 84
4.2.4.3 Refractive Index (RI) Detection 85
4.3 Postdecompression Detection (Gas/Droplet Phase Detection) - Interfacing Approaches 85
4.3.1 Pre-BPR Flow Splitting 86
4.3.2 Total Flow Introduction (Post-BPR Detection) 88
4.3.2.1 BPR Requirements for Total-Flow Introduction Detection 88
4.3.2.2 Total Flow Introduction with Mechanical BPR 89
4.3.2.3 Total Flow Introduction - Pressure-Regulating-Fluid (PRF) Interface 89
4.3.2.4 Total Flow Introduction without Active Backpressure Regulation 91
4.4 Postdecompression Detection 93
4.4.1 Flame-Based Detectors 93
4.4.2 Evaporative Light Scattering Detection (ELSD) and Charged Aerosol Detection (Corona CAD) 97
4.4.3 Mass Spectrometric Detection 98
4.4.3.1 Interfacing and Ionization Approaches 99
4.4.3.2 Atmospheric Pressure Chemical Ionization (APCI) 100
4.4.3.3 Pneumatically Assisted Electrospray Ionization (ESI) 101
4.4.3.4 Atmospheric Pressure Photoionization (APPI) 103
4.4.4 Postdecompression Detection Using Less Common Approaches - Deposition IR 103
4.5 Concluding Remarks 103
References 104
<b>5 Chiral Analytical Scale SFC - Method Development, Stationary Phases, and Mobile Phases 117</b>
5.1 Introduction 117
5.2 Chiral Stationary Phases for SFC 119
5.3 Chiral SFC vs. Chiral HPLC 128
5.4 Method Development Approaches 130
5.4.1 Modifiers for Chiral SFC 132
5.4.2 Additives for Chiral SFC 133
5.4.3 Nontraditional Modifiers 135
5.4.4 Method Development Approaches 137
5.5 High Throughput Method Development 139
5.6 Summary 141
References 142
<b>6 Achiral Analytical Scale SFC - Method Development, Stationary Phases, and Mobile Phases 147</b>
6.1 Introduction 147
6.2 The Mixture to Be Separated 148
6.2.1 Molecular Interactions 148
6.2.2 Molecular "Handles" 149
6.3 Achiral SFC Stationary Phases 150
6.3.1 Column Safety and Compatibility 150
6.3.2 Efficiency 150
6.3.3 Retention 153
6.3.4 Selectivity 156
6.4 Mobile-Phase Choices 157
6.4.1 Primary Mobile-Phase Component 158
6.4.2 Secondary Mobile-Phase Component - The "Modifier" 159
6.4.3 Tertiary Mobile-Phase Component - "Additives" 163
6.5 Influence of Column Temperature on Efficiency and Selectivity 170
6.6 Where Do I Go from Here? Method Development Decision Tree and Summary 172
References 174
<b>7 Instrumentation for Preparative Scale Packed Column SFC 183</b>
7.1 Introduction 183
7.2 Safety Considerations 184
7.3 Fluid Supply 185
7.3.1 Carbon Dioxide 185
7.3.2 Mobile Phase Modifiers and Additives 187
7.3.3 Carbon Dioxide Recycling 188
7.4 Pumps and Pumping Considerations 189
7.4.1 CO<sub>2 </sub>and Modifier Fluid Pumping 189
7.4.2 Pressures and Flow Ranges 189
7.5 Sample Injection 190
7.5.1 Injection of Solutions 190
7.5.2 Extraction Type Injection 190
7.6 Chromatographic Columns 192
7.7 Detection 192
7.8 Back Pressure Regulation 193
7.9 Fraction Collection 193
7.9.1 Cyclone Collection 194
7.9.2 Open-Bed Collection 195
7.10 Conclusion 197
References 197
<b>8 Preparative Achiral and Chiral SFC - Method Development, Stationary Phases, and Mobile Phases 199</b>
8.1 Introduction 200
8.1.1 Advantages and Disadvantages of SFC vs. HPLC for Purification 201
8.1.2 Cost Comparison: Preparative HPLC vs. SFC 202
8.2 Safety Considerations 202
8.3 Developing Preparative Separations 203
8.3.1 Linear Scale-Up Calculations 209
8.3.2 Scaling Rule in Supercritical Fluid Chromatography 210
8.3.3 Metrics for Preparative Separations 213
8.3.4 Options for Increasing Purification Productivity 214
8.3.4.1 Closed-Loop Recycling 214
8.3.4.2 Stacked Injections 214
8.3.5 Importance of Solubility on Preparative Separations 214
8.3.6 Preparative SFC Injection Options 217
8.4 Preparative Chiral SFC Purifications 220
8.4.1 Chiral Stationary Phases (CSPs) for Preparative SFC 220
8.4.2 Method Development for Chiral Purifications 222
8.4.3 Preparative SFC Examples 223
8.4.3.1 Milligram Scale Chiral Purification 223
8.4.3.2 Gram Scale Chiral Purification 224
8.4.4 Impact of Solubility on Productivity 226
8.4.5 Use of Immobilized Chiral Stationary Phase (CCP) for Solubility-Challenged Samples 227
8.4.5.1 Immobilized CSP Example #1 227
8.4.5.2 Immobilized CSP Example #2 228
8.4.6 Coupling of Chiral and Achiral Columns for SFC Purifications 229
8.5 Preparative Achiral SFC Purifications 231
8.5.1 Introduction to Achiral SFC Purifications 231
8.5.2 Stationary Phases for Achiral Preparative SFC 232
8.5.3 Method Development for Achiral Purifications 232
8.5.4 Achiral SFC Purification Examples 234
8.5.4.1 Achiral Purification Example #1 234
8.5.4.2 Achiral Purification Example #2 234
8.5.5 Purifications Using Mass-Directed SFC 236
8.5.6 Impurity Isolation Using Preparative SFC 237
8.5.6.1 Impurity Isolation Example 240
8.5.7 SFC as Alternative to Flash Purification 241
8.6 Best Practices for Successful SFC Purifications 244
8.6.1 Sample Filtration and Inlet Filters 244
8.6.2 Sample Purity 246
8.6.3 Salt vs. Free Base 247
8.6.4 Primary Amine Protection to Improve Enantiomer Resolution 250
8.6.5 Evaluation of Alternate Synthetic Intermediates to Improve SFC Purification Productivity 250
8.7 Summary 254
References 254
<b>9 Impact and Promise of SFC in the Pharmaceutical Industry 267</b>
9.1 Introduction to Pharmaceutical Industry 267
9.2 SFC in Pharmaceutical Discovery 268
9.2.1 Early Discovery Support 268
9.2.2 SFC in Medicinal Chemistry 269
9.2.2.1 Analytical SFC 270
9.2.2.2 Preparative SFC 271
9.2.3 Physiochemical Measurement by SFC 273
9.2.4 Use of SFC for Pharmacokinetic and Drug Metabolism Studies 274
9.3 SFC in Development and Manufacturing 276
9.3.1 Analytical SFC Analysis of Drug Substances and Drug Products 276
9.3.2 Preparative SFC in Development and Manufacturing 282
9.3.3 Metabolite/PKDM Studies in Development 283
9.3.4 SFC in Chemical Process Development 283
9.4 SFC for Analysis of Illegal Drugs 284
9.5 Summary 286
References 286
<b>10 Impact of SFC in the Petroleum Industry 297</b>
10.1 Petroleum Chemistry 297
10.1.1 Crude Refining Processes 297
10.1.2 Petrochemical Processes 298
10.2 Introduction to Petroleum Analysis 299
10.3 Historical Perspective 301
10.3.1 Hydrocarbon Analysis via FIA 301
10.3.2 SFC Replaces FIA 301
10.3.3 Hydrocarbon SFC Analysis via ASTM 5186-91 302
10.4 Early Petroleum Applications of SFC 304
10.4.1 Samples with Broad Polymer Distribution 304
10.4.2 SFC Purification of Polycyclic Aromatic Hydrocarbons 305
10.4.3 Coal Tar Pitch 305
10.4.4 Enhanced SFC Performance 305
10.4.5 Sulfur Detection in a Petroleum Matrix 307
10.5 SFC Replacement for GC and LC 308
10.5.1 Simulated Distillation 308
10.5.2 Hydrocarbon Group-Type Separations - PIONA Analysis 310
10.6 Biodiesel Purification 311
10.7 Multidimensional Separations 314
10.7.1 Comprehensive Two-Dimensional SFC 314
10.7.2 SFC-GC x GC 315
10.7.3 Comprehensive - SFC-Twin-Two-Dimensional (GC x GC) 316
References 317
<b>11 Selected SFC Applications in the Food, Polymer, and Personal Care Industries 321</b>
11.1 Introduction 321
11.2 Selected Applications in the Foods Industry 322
11.2.1 Fats, Oils, and Fatty Acids 322
11.2.2 Tocopherols 325
11.2.3 Other Vitamins 327
11.2.4 Food Preservatives (Other Antioxidants and Antimicrobials) 330
11.2.5 Coloring Agents 330
11.2.6 Sugars 331
11.3 Selected Applications in the Field of Synthetic Polymers 332
11.3.1 Molecular Weight Distribution 332
11.3.2 Structural Characterization 334
11.3.3 "Critical Condition" Group/Block Separations of Complex Polymers Using CO<sub>2</sub>-containing Mobile Phases 334
11.3.4 Polymer Additives 335
11.4 Selected Applications in the Personal Care Industry 337
11.4.1 Lipophilic Components of Cosmetics 337
11.4.2 Surfactants in Cleaning Mixtures 337
11.4.3 Emulsifiers in Personal Care Products 337
11.4.4 Preservatives 338
11.5 Conclusions 340
References 340
<b>12 Analysis of Cannabis Products by Supercritical Fluid Chromatography 347</b>
12.1 Introduction 347
12.1.1<i> Cannabis</i> History 348
12.2 Analytical SFC 351
12.2.1 Introduction 351
12.2.2 Early SFC of Cannabis Products 352
12.2.3 Achiral SFC 353
12.2.4 Chiral SFC 354
12.2.5 Metabolite Analysis 357
12.3 Preparative SFC 357
12.4 Summary 360
References 361
<b>13 The Future of SFC 365</b>
13.1 Introduction 365
13.2 SFC Publication Record 366
13.3 SFC Research in Academia 368
13.4 SFC Conferences 368
13.5 Anticipated Technical Advances 369
13.6 Limits to SFC Expansion 370
13.7 Summary 372
References 373
Index 377
Preface
OUTLINE
0.1 Scope of the Book
Supercritical fluid chromatography (SFC) is more than 50?years old. Chapter 1 entitled "Historical Development of SFC" recaps over a much greater time-frame of the discovery of supercritical fluids and their development as a medium for chromatographic separation of both volatile and nonvolatile analytes. A real interest in SFC using either packed or open tubular columns began in the early 1980s when the first commercial preparative SFC instrument became available [1]. This development led to growing interest in the separation of stereoisomers which started with the pioneering work of Frenchman Marcel Caude and his research group in 1985 [2]. Thus, a wide variety of chiral separations were reported and applied near the turn of the century employing both analytical and preparative packed column (pcSFC) technology. SFC with open tubular columns (otSFC) also peaked in the 1980s but fizzled during the following decade. Interest in pcSFC is currently higher than ever before. For example, the technique is capable of generating peak efficiencies approaching those observed in gas chromatography (GC). On the other hand, pcSFC separations can achieve much higher efficiencies per unit time than in high performance liquid chromatography (HPLC). pcSFC has embraced a critical mass of separation scientists and technicians in terms of the number of workers in the field worldwide. Hundreds of supercritical fluid chromatographs currently are in use. Furthermore, pcSFC is (i) detector and environmentally friendly, (ii) interfaceable with sample preparation, (iii) relatively economical in cost, and (iv) is a superior purification tool. Chapters 3 and 4 provide discussion of these critical developments that earlier had been referred to as dense gas chromatography [3]. Related work in the field currently uses both supercritical and subcritical mobile phase conditions to perform separations as well as purifications.
During the past 20?years, pcSFC has created a bonafide niche for itself as the go-to workhorse in chiral separations. Chapter 6 discusses in detail this topic. It has afforded many advantages for rapid separation of enantiomers over HPLC due to its greater separation efficiency per unit time. The advantages of pcSFC over HPLC which are also discussed later in the book, however, are practical but not fundamental. The greatest difference between pcSFC and pcHPLC is just simply the need to hold the outlet pressure above ambient in separations in order to prevent expansion (i.e. boiling) of the mobile phase fluid.
Enantiomeric separations are more compatible with ambient SFC than with high temperature HPLC because chiral selectivity usually favors decreasing temperature wherein the risk of analyte racemization is minimized. On the other hand, the risk of analyte thermal decomposition as in the GC of cannabis - related components is lessened. Furthermore, the straightforward search (primarily by trial and error) for a highly selective chiral stationary phase is a key step in the development of chiral pcSFC separations that address industrial applications. In this regard, a number of screening strategies that incorporate a wealth of stationary phases are discussed in the book that take advantage of short columns, small particles, high flow rates, and fast gradients.
Upon scale-up of analytical chromatography to preparative supercritical fluid separations as discussed in Chapter 8, the resulting decrease in solvent usage and waste generation relative to preparative scale HPLC is strikingly dramatic. SFC product can be routinely recovered at higher concentration relative to HPLC which greatly reduces the amount of mobile phase that must be evaporated during product isolation. Higher SFC flow rates contribute to higher productivity. The faster SFC process makes the separation cycle time significantly shorter such that it becomes practical as well as feasible to make purification runs by "stacking" small injections in short time windows without compromising throughput. Table 0.1 lists additional advantages of supercritical fluid chromatography.
Table 0.1 Advantages of supercritical fluid chromatography.
- High diffusivity/low viscosity yield greater resolution per unit time.
- Longer packed columns afford greater number of theoretical plates
- Low temperature reduces risk of analyte isomerization
- Scale-up of separation and isolation of fractions are facilitated
pcSFC (as most analytical techniques) has had a tortuous development history, but it appears that analytical and preparative scale chiral SFC are currently on the firmest foundation ever experienced with vendors that are strongly committed to advancing the technology. Extensive, new developments in achiral SFC and a much broader spectrum of applications outside the pharmaceutical area are already happening. Unlike reversed phase HPLC, the identification of the correct column chemistry is critical for the successful application of achiral pcSFC. Very different selectivity can be achieved depending on the column chemistry. Basic, neutral, and acidic compounds are well eluted on most columns that indicates the suitability of pcSFC for a broad range of chemical functionalities. The number of "SFC" columns for achiral purifications has also grown rapidly in the past three years. Activity in (i) agricultural and clinical research, (ii) environmental remediation, (iii) food and polymer science, (iv) petrochemicals, and (v) biological chemistry immediately come to mind. Additional Chapters 9-12 have been introduced into the book since writing began that reflect numerous additional applications of pcSFC such as pharmaceuticals, petroleum, food, personal care products, and cannabis. Additional advantages of SFC are listed in Table 0.2.
0.2 Background for the Book
While there have been numerous books published concerning SFC as both monographs and edited volumes, there appear to be only two texts that have had teaching as a major emphasis. One, published in 1990, was edited by Milton L. Lee (Brigham Young University) and Karin E. Markides (Uppsala University, Sweden) and written by a committee of peers is entitled "Analytical Supercritical Fluid Chromatography and Extraction" [4]. For chromatographic discussion, this book focused almost entirely on wall coated open tubular capillary column SFC (otSFC), which is not widely performed today having been replaced almost 100% by packed column SFC (pcSFC).
In the early days, otSFC and pcSFC coevolved and vigorously competed with each other as described in Chapter 1. otSFC lost ground and eventually faded away, mainly as a result of poor chromatographic reproducibility issues in terms of flow rate, gradient delivery, pressure programming, and sample injection. The early systems were costly and not user friendly, which resulted in the technique being marginalized as too expensive and inefficient. While otSFC was capable of outstanding feats such as the separation of nonvolatile polymeric mixtures and isomeric polyaromatic hydrocarbons, most workers in the field would agree nowadays that the approaches used in otSFC are among the worst parameters to test with pcSFC.
Table 0.2 Additional advantages using pcSFC.
- No pre-derivatization to achieve solubility and/or volatility
- Shorter cycle time with gradual gradient elution
- Faster separation facilitated by higher fluid diffusivity
- Reduced column diameter/particle size via lower fluid viscosity
- Less extreme chromatography conditions
- Routine normal phase chromatography
Another book entitled "Packed Column SFC," published by the Royal Society of Chemistry and authored by Terry A. Berger [5] was published in 1995. Given that over 20?years have elapsed since the publication of Berger's book, the book presented here today provides ample references that reflect the current state-of-the-art as understood today. We have written our book that incorporates a more pedagogical style with the explicit intention of providing a sound education in pcSFC. Relatively new users of SFC in the early days were largely forced to rely on concepts developed for either HPLC (in the case of packed columns) or GC (in the case of open tubular columns), which were often inappropriate or misleading when applied to both otSFC and pcSFC. Our book addresses these deficiencies.
In this regard, a detailed discussion of current SFC instrumentation as it relates to greater robustness, better reproducibility, and enhanced analytical sensitivity is a focus of the book (Chapter 3). Originally, SFC was thought to be solely for low molecular weight, nonpolar compounds. Today, we know that SFC spans a much larger polarity and molecular mass range. Even though modern pcSFC books may be more adequately described as either "Carbon Dioxide-Based HPLC" (as Terry Burger once suggested) or "Separations Facilitated by Carbon Dioxide" (as suggested by Fiona Geiser) than "Packed Column Supercritical Fluid Chromatography," a change in...
