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.

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.

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 nomenclature this drastic was not encouraged by attendees at several recent pcSFC conferences in both Europe and the United States. Suffice it to say, a change in nomenclature at this time is not suggested here. Nevertheless, this drastic shift in mindset and practice as suggested by Berger and Geiser during the last decade concerning both stationary phase and mobile phase has been a large reason for the current resurgence of pcSFC technology for problem solving at the industrial and academic levels worldwide. As proof, analytical scale achiral SFC is discussed in Chapter 6 along with ion pair SFC, reversed phase SFC, and HILIC‐SFC.

While SFC has experienced much painful growth and disappointment during its evolution over 50 plus years, the “flame” has never been extinguished in the minds of a core group of separation scientists. A major reason for this mindset has been the near‐annual, well‐attended scientific meetings that have taken place in Europe and the United States over the past 25 years. Initially, the meetings were known as “the International Symposium on Supercritical Fluid Chromatography and Extraction” wherein the focus was almost exclusively on capillary column SFC. Milton Lee at BYU and Karen Markides from University of Uppsala, Sweden served as hosts for the first meeting (1988) in Park City, UT. Subsequent meetings and approximate dates that have mostly been within the United States are listed in Table 0.3. Not shown in the table, but the youngest of us (DP) presented a poster at probably the earliest conference in this series called “SFC‐87, Pittsburgh.” Attendance was approximately 150.

These meetings were terminated soon after 2004 due to a lack of vendor commitment and support and user interest. In 2007, a series of new conferences with a different name (“International Conference on Packed Column Supercritical Fluid Chromatography”) that gave attention to exclusively packed column Supercritical Fluid Chromatography was initiated first by Suprex Corporation, Pittsburgh, PA, then Berger SFC, and later by both Waters Corp. and Agilent. These meetings which now attract primarily industrial scientists, engineers, and academic colleagues from Europe and the United States are currently sponsored by the Green Chemistry Group. During the past 10 years the meetings have occurred annually and have alternated mostly between Europe and the United States (Table 0.4). To gain a greater world‐wide audience the Green Chemistry Group has sponsored pcSFC meetings in China and Japan (i.e. 2016–2017, respectively). Additional meetings are scheduled in 2019 for both China and Japan.

pcSFC during the past 10 years has become a viable chiral chromatographic technique in the areas of pharmaceutical drug discovery and drug development. Chiral separations using carbon dioxide which incorporate a host of normal phase, silica‐based stationary phases with principally ultraviolet and mass spectrometric online detection are now common. Nearly every pharmaceutical company in the United States, Asia, and Europe has multiple pcSFC instruments operating in a variety of laboratories. Interest in India, China, Korea, and the Pacific Rim, for example, is growing.

Currently activity centers around (i) development and application of mass‐directed pcSFC, (ii) enhancement of robustness and sensitivity to meet various regulatory requirements, (iii) production of new polar stationary phases for separation of metabolomics and related biochemicals, and (iv) theoretical modeling of column physical properties dictated by employment of compressible polar modified mobile phase and stationary phase – bonded sub‐2‐μm particles.

There is rapidly growing interest in achiral pcSFC where the separation of highly polar compounds has been demonstrated. Applications to polymeric materials, natural products, water soluble analytes, surfactants, organic salts, fatty acids, lipids, organometallics, etc. are experiencing great success. Depending upon the nature of the stationary and mobile phases employed, a variety of separation mechanisms can be expected such as reversed phase pcSFC, ion pairing pcSFC, and aqueous promoted HILIC‐pcSFC. Each mode of chromatography can be expected to augment the more popular normal phase pcSFC that has been used for decades and employs nonpolar mobile phases.

0.3  Audience for the Book

This book will be of interest to industrial, government, and academic users of pcSFC and is expected to be useful as a chemistry textbook in graduate‐level separations courses. Laboratories looking to adopt SFC as part of their regular analytical tools will find this book useful as they learn fundamental principles behind technology and how pcSFC complements both HPLC and GC.

One's view of SFC today is entirely different from that of 25–30 years ago wherein (i) flow rates and gradient delivery were not reproducible, (ii) analytical UV sensitivity was not acceptable, and (iii) stationary phases were designed for reversed phase chromatography as opposed to normal phase chromatography. Today, SFC is considered to be primarily normal phase chromatography (i.e. a separation technique similar to HPLC) using mostly the same hardware and software developed for HPLC. The mobile phase is a binary or ternary mixture with CO₂ as the main component. The separation is usually performed with gradient elution where the composition of the mobile phase becomes more polar with time. Polar stationary phases such as bare silica, cyanopropylsilica, 3‐aminopropylsilica, and 2‐ethylpyridylsilica are routinely employed. pcSFC has numerous practical advantages relative to reversed phase HPLC such as higher speed, greater throughput, more rapid equilibration, and shorter cycle times. SFC yields lower operating cost and lower column pressure drop, and is orthogonal to reversed phase HPLC. Finally, compounds of interest can be isolated with a relatively small amount of solvent because CO₂ vaporizes away. This feature has become particularly important for preparative applications in which elution volumes can be large.

During this time period, a SFC system was introduced by Waters Corp. (Milford, MA, USA). The system featured the efficient cooling of the CO₂ pump heads by Peltier and the design of a dual stage back pressure regulator that was heated to avoid frost formation. In this case, separations with the Waters instrument were mostly identified as ultrahigh performance supercritical fluid chromatography (UHPSFC). A similar system like Waters was introduced in 2012 by Agilent which was a hybrid that allowed both UHPLC and SFC separations. Shimadzu has more recently introduced hardware that performs similar operations. This combined vendor news reenergized many workers in the SFC community and caused potential users of the technology to re‐investigate the research potential of pcSFC. The instrumentation from these three vendors nowadays appears to represent the current methodology to perform analytical pcSFC which should enhance its acceptability by the separation scientists into the immediate future. UHPSFC via either vendor affords a high throughput approach for profiling analytes such as free fatty acids, acylglycerols, biodiesel, peptides, basic drugs, etc. via light scattering, UV, and Q‐TOF‐MS detection without the waste and uncertainty of sample preparation procedures. This more modern terminology is prevalent throughout this book. The older pcSFC instruments, while still useable in numerous laboratories are no longer being manufactured.

0.4  SFC Today

Being green is a good thing, but most people nowadays seemingly go for pcSFC because of its speed and fast method development rather than its environmental advantages. Experts in the field now readily agree that ultrahigh performance supercritical fluid chromatography (UHPSFC) has established itself as the preferred way of doing chiral and achiral analysis on both analytical and preparative scales. They also say that SFC will become the norm for small‐scale purifications. Increased interest in (i) petrochemical and food industries, (ii) environmental air quality, (iii) biodiesel quality control, and (iv) protein separations can be expected in the not too distant future [6].

Much of the increased experimental capability alluded to above has been made possible by the introduction of pumping systems that deliver enhanced reproducible and accurate flow of CO₂ and modifier. In this case, separations are generally identified as UHPSFC.

Anyone with an interest in analytical and/or preparative scale pcSFC coupled to both spectroscopic and flame‐based detectors will find this book beneficial. Subcritical fluid chromatography and enhanced fluidity chromatography as developed by Susan Olesik at the Ohio State University are also applicable here. Bonafide experience of the separation scientist in analytical or preparative scale SFC is not necessary for reading this book. Some knowledge of chromatographic principles is, however, desirable. With the introduction of more reliable instrumentation and eye‐catching applications, a new generation of separation scientists and engineers are beginning to express much interest in the technology. Because the book is written with teaching in mind, the text could very well be the reference document on the desk of each person who is applying pcSFC.

References

1. 1 Berger, T.A., Fogleman, K., Staats, T. et al. (2000). The development of a semi‐preparatory scale SFC for high‐throughput purification of combi‐chem libraries. Journal of Biochemical and Biophysical Methods 43: 87–111.
2. 2 Mourier, P., Eliot, E., Caude, M. et al. (1985). Super‐ and sub‐ critical fluid chromatography on a chiral stationary phase for the resolution of phosphine oxide enantiomers. Analytical Chemistry 57: 2819–2823.
3. 3 Giddings, J.C., Myers, M.N., McLaren, L.M., and Keller, R.A. (1968). High pressure gas chromatography of nonvolatile species. Science 162: 67–73.
4. 4 Lee, M.L. and Markides, K.E. (1990). Analytical Supercritical Fluid Chromatography and Extraction. Provo, Utah: Chromatography Conferences, Inc.
5. 5 Berger, T.A. (1995). Packed Column SFC. Cambridge: Royal Society of Chemistry.
6. 6 Francotte, E. (2016). Practical advances in SFC for the purification of pharmaceutical molecules. LCGC Europe 29 (4): 194–204.