SUPERCRITICAL FLUID CHROMATOGRAPHY

1 SUPERCRITICAL FLUID . CHROMATOGRAPHY . Primer SUPERCRITICAL FLUID . CHROMATOGRAPHY . Primer Terry A. Berger This information is subject to change without notice. Agilent Technologies, Inc., 2015. Printed in the USA, July 1, 2015. 5991-5509EN. CONTENTS. Contents III. Foreword VI. About the Author XI. Introduction XIII. Symbols XIV. Abbreviations XIV. 1 I ntroduction to SUPERCRITICAL FLUID CHROMATOGRAPHY 1. What is SFC? 1. Why deploy SFC? 2. What can SFC separate? 12. What's in a name? 16. 2 The Mobile Phase 19. Why use CO 2? 19. Using 100 % CO 2 21.

2 Modifiers or cosolvents 23. Additives 31. Extending solute polarity with added water 34. 3 The Stationary Phase 35. Materials 35. Achiral bonded phases 35. Stationary phase comparisons 40. Relationship between particle diameter and column dimensions 41. Recommended column dimensions 45. Columns for chiral separations 48. III. 4 E ffect of Mobile Phase Variables on Retention and Selectivity 49. Modifier concentration 49. Temperature 50. Pressure 53. Flow 55. Generalizations on effects of control variables on retention and selectivity 57. 5 Method Development 58.

3 Matching solute and phase polarity 58. Polarity windows 60. Getting started 60. Polar solutes 61. Low polarity solutes 64. Multivariate methods 65. 6 Achiral Separations 66. Case Study 1 A typical low-polarity sample 66. Case study 2 A moderately polar sample 71. Case study 3 Sulfonamides 83. Other observations 89. 7 Chiral Separations 90. Background 90. Enantiomeric excess determinations 91. Normal phase for chiral separations 91. Effect of control variables on chiral separations 93. Developing a chiral method 98. IV. 8 Quantification in SFC 103.

4 Stages of Validation 103. Method development for quantification of sorbate, benzoate, and caffeine in beverages and foods 105. Calibration 108. Summary of the quantification of benzoate, sorbate and caffeine 116. Chiral separations 116. 9 Instrumental Considerations 121. Pumping 121. UV detector optimization 124. The doubling gradient 137. Autosampler considerations 138. Miscellaneous 146. Ultrahigh performance with SFC 148. Hybrid systems switching between SFC and HPLC 150. Interfacing to mass spectrometers 154. Other Detectors 156. References 159.

5 V. FOREWORD. The author, Terry Berger has a unique, convoluted history with what we now call SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC), and is uniquely qualified to write this primer. Many of the early interactions were incidental and unfocused, but later meaningful. He brings nearly 40 years of relationships with people associated with SFC, along with over 35 years of direct experience. SFC had first been proposed nearly 60 years ago, in 1958, by Jim Lovelock, who later became a personal friend of the author through the 1980s and 1990s with numerous discussions about SFC.

6 Ernst Klesper demonstrated SFC experimentally in 1962 when he separated metal porphorins with dense clorofluorocarbons as mobile phase. The author later interacted with Professor Klesper on numerous occasions, through the late 1970s to mid-1980s in both the USA and Germany, and again had extensive discussions about SFC. Through the 1960s and 1970s, the technique largely languished with, at most, a dozen groups worldwide making a total of a few sporadic scientific contributions per year. Applications tended to be extensions of gas CHROMATOGRAPHY (GC) to higher molecular weight, higher boiling, relatively nonpolar solutes.

7 In the early 1970s, the author was a graduate student at Purdue University in Indiana. The adjacent laboratory was that of buck Rodgers, a highly respected chromatographer at the time. After attending a scientific symposium in 1971, he came back determined to explore this new form of CHROMATOGRAPHY . Controlling the pressure of the mobile phase was the primary control variable, and pressure programming from low to high pressures was then the norm. All equipment in this period was homemade, with poor instrumental control. The physical and chemical characteristics of the mobile phases were primitively understood, at best.

8 Rodgers rescued ultrahigh pressure hydrogenation pumps from storage, built an oven roughly 4-feet long that looked like a small coffin using 2-inch thick household foam insulation. Columns were 30 inches long or longer, and packed with irregular particles with a wide particle-size distribution. The author watched the progression of these developments and recalls that, at the time, being skeptical that this odd form of CHROMATOGRAPHY was useful. Rodgers results suggested there was a serious problem with density gradients along the length of VI.

9 The column. This, and later work by Milos Novotney, seemed to indicate that the technique was unlikely to produce results as good as the current high-performance liquid CHROMATOGRAPHY (HPLC). This tended to suppress further work on packed-column SFC. The author was trained in HPLC at Imperial College in London where he developed an amperometric electrochemical detector for HPLC, in the early 1970s. After subsequently teaching for a year in Brazil, he worked on a number of projects for instrumentation for spacecraft in Cleveland, Ohio. The author was then hired by Hewlett-Packard in 1979, and joined the research group at the Avondale Division.

10 Dennis Gere, Henk Lauer, Doug McManigill, and Harry Weaver, all scientists or engineers at Hewlett-Packard Labs or the Avondale Division, presented a series of talks on dense gas CHROMATOGRAPHY at that year's Pittsburg Conference (where the author was actually hired). Some of these talks were later published in such journals as Analytical Chemistry, and Science, as well as related application notes. These works resulted in a customer demand that led to the creation of a commercial kit that converted a Hewlett Packard model HP 1084 HPLC instrument into an SFC system.