Contemporary SFC: Accomplishments and Limitations Peter R. Griffiths Department of Chemistry University of California, Riverside Riverside, Calif. 92521
Supercritical fluid chromatography (SFC) can now be said to be in its early adolescence. Born in 1962, when Ernst Klesper made the first report of the separation of metal porphyrins using dense gas chromatography, SFC struggled for acceptance through the 1970s and then started to flex its muscles in the early part of this decade. Excitement over SFC was generated by the reported high-resolution separations of large molecules using open tubular columns of the type used for capillary gas chromatography (GC) with supercritical carbon dioxide as the mobile phase. This technique was used by Milton Lee's group at Brigham Young University (BYU) and Milos Novotny's group at Indiana University. Subsequent reports on capillary SFC indicated the potential of this technique for the highresolution separation of nonvolatile or thermally labile compounds and prompted the commercial development of chromatographs by such companies as Brownlee Laboratories (now part of Applied Biosystems), Computer Chemical Systems, Lee Scientific, and Suprex. After the number of SFC practitioners had increased to several hundred, the time was deemed right by two 0003-2700/88/0360-593A/S01.50/0 © 1988 American Chemical Society
groups to hold meetings at which the state of the art of SFC could be reviewed and discussed. The first of these (SFC '87, or the First International Conference on SFC) was sponsored by the Chromatography Forum of the Society for Analytical Chemists of Pittsburgh and was held last October. The second (1988 Workshop on Supercritical Fluid Chromatography) was organized by Milton Lee and Karin Markides at BYU and took place Jan. 9-11, 1988, at Park City, Utah. Although SFC development is probably not at a sufficiently advanced state to justify two meetings held within a few months of each other, the strengths and weak-
FOCUS nesses of this technique were well presented at both conferences. Those fortunate enough to attend both meetings certainly were exposed to the excitement and the pitfalls of contemporary SFC. The hazards of holding a technical meeting at a ski resort in January were quickly revealed to those participants of the Park City workshop who encountered a severe storm on the morning of their arrival. By the following morning, the skies had returned to the color pictured in the brochures, and, in theory at least, the mountain setting should
have lent a relaxing aura to this meeting. The organizers had other ideas, however, and kept the conferees occupied with a schedule that left little time for personal discussions; formal sessions were held each morning, afternoon, and evening. Under normal circumstances, this frenetic pace would have left even the most seasoned meeting-goer gasping for breath. The meeting was saved, however, by the fact that after each session, in which three to five papers of 20-minute duration were presented, a one-hour discussion was held on preannounced topics. These discussions proved particularly fruitful, although the hour was usually up before all views on the particular subject had been aired. Most meeting organizers usually leave far too little time for open discussion, and I felt the Park City workshop discussion periods were appropriate for a subject such as SFC, which has not yet reached a state of full maturity. Each of the sessions covered individual topics such as the effect of the mobile phase; columns and stationary phases; injection techniques; programming methods; extraction with supercritical fluids; detectors; and hyphenated techniques, including the interfacing of SFC with mass spectrometry (MS) and Fourier transform infrared (FT-IR) spectrometry. Finally, a variety of applications of SFC and supercritical fluid extraction were discussed. In previous reports, researchers con-
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FOCUS eluded that the reproducibility of peak heights and areas were not as good in SFC as in GC and in high-performance liquid chromatography (HPLC). Through the use of such injection tech niques as time splitting, Tom Chester of Procter and Gamble, Tyge Greibrokk of the University of Oslo, and Bruce Richter of Lee Scientific showed that the repeatability of capillary SFC could be as good as ± 1 % and even bet ter than this when an internal standard was included. Another bone of contention among practitioners of SFC that had been raised at many recent meetings and symposia concerned the relative merits of capillary and packed columns. Al though much of the impetus toward the acceptance of SFC in the 1980s was generated by the excitement over the potentially high resolution achievable through the use of open tubular col umns, many excellent separations have been demonstrated using packed col umns, and this trend continued at SFC '88. For example, Yukio Hirata of the Toyohashi University of Technology demonstrated remarkably high resolu tion through the use of packed fusedsilica capillaries. The use of packed col umns certainly provides faster separa tions than open tubular columns operated at the same resolution. Other workers showed that very high-resolu tion separations can be achieved by us ing open tubular columns provided that the linear velocity of the mobile phase is very low, but this necessitates separation times of several hours. Re searchers are beginning to appreciate the strengths and weaknesses of each type of column, and the rancor that was exhibited at previous symposia over whether packed or capillary columns are preferable appears to have been largely dissipated. The logical consen sus that both techniques have a place in the analytical laboratory has finally been reached. Carbon dioxide is, of course, by far the most popular mobile phase in SFC, although the addition of polar modifi ers such as methanol and propylene carbonate is often necessary when packed columns are employed. Robert Denyszyn of Scott Specialty Gases re ported on the solubilities of several commonly used modifiers and showed that their solubility is often lower than had been previously believed. He stressed that care should be exercised when modifier concentrations of much more than 5% are specified, especially if the gases are stored at subambient temperature where the solubility can be reduced. The effect of modifiers at low concentration on separations made
with packed columns is much greater than is observed using capillary col umns, presumably because of the blockage of active sites—especially silanol groups—by the modifier. Thus programming of the modifier concen tration is an effective way to extend the range of compounds that can be sepa rated in a single run. Factors affecting gradient separa tions were discussed by Ernst Klesper of the Technische Hochschule (Aa chen) and by Daido Ishii of Nagoya University, both of whom stressed that the elution of compounds in SFC de pends both on their volatility and their solubility. Thus temperature and sol vent programming can effectively be used to supplement density program ming in SFC. (It is noteworthy that Ernst Klesper was presented with a plaque honoring his pioneering work in SFC at the workshop.) Although supercritical CO2 is able to dissolve compounds of remarkably high polarity, molecules with extensive
The consensus that both capillary and packed columns have a place in the analytical laboratory has finally been reached. hydrogen-bonding networks (especial ly many biopolymers) are usually quite insoluble. One approach to solubilizing molecules of this type that elicited con siderable interest at the Park City workshop involved the addition of mol ecules capable of forming reverse mi celles in supercritical CO2. Richard Smith of Battelle's Pacific Northwest Laboratory reported the solubilization of a 200,000-Dalton protein in this way. However, few chromatographic separa tions using reverse micelles have yet been reported. The role of the stationary phase was also discussed in several papers. Larry Taylor of Virginia Tech showed the en hancement of packed-column SFC through the use of deactivated station ary phases. Pierre Macaudiere of ESPCI (Paris, France) reported on the separation of enantiomers using com mercially available chiral stationary phases. The selection of stationary phases in capillary SFC was discussed by Brian Jones of Lee Scientific. Jones
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concluded that silicones with highly polarizable substituents such as biphenyl are particularly appropriate for separating chemically similar mole cules. Karen Chang of BYU then showed how liquid crystalline station ary phases enhanced the separation of isomeric polycyclic aromatic hydrocar bons, steroids, and other planar mole cules through the dependence of reten tion time on the length-to-breadth ra tio of the analytes. The fact that so many separations are performed using unmodified CO2 has enabled many different types of de tectors to be used for SFC. Although the flame ionization detector is by far the most common, selective detectors are also starting to make their presence felt. For example, Lennart Mathiasson of the University of Lund reported on the analysis of amines using a nitrogenselective thermionic detector. Al though ultraviolet (UV) absorption de tection is commonly employed for packed-column SFC, its use for capil lary separations is less common be cause of the low dead volume required. Darryl Bornhop of Lee Scientific re ported that the cell volume of UV de tectors for capillary SFC should be no greater than 25 nL. He showed that observation through stripped fused-silica tubing of 250-μπι i.d. gave adequate results in many cases. Most of the par ticipants agreed that the development of an electron-capture detector for SFC would represent an important contri bution, but such a detector has not yet been reported. Several other techniques by which the selectivity of SFC detectors could be increased were also described. For example, Kiyokatsu Jinno of Toyoha shi University of Technology reported element-selective detection using on line inductively coupled plasma atomic emission spectrometry. Herbert Hill of Washington State University demon strated the use of ion mobility spec trometry for detecting and identifying picogram quantities of eluites from capillary columns. The use of FT-IR spectrometry for the detection and identification of SFC eluites was re ported by several workers. Richard Wiebolt of Nicolet Instruments and Philippe Morin of ENSIA (Massy, France) reported on the on-line mea surement of infrared spectra obtained using a flow-through cell after capillary and packed SFC, respectively. My group at the University of California, Riverside demonstrated that the mini mum identifiable quantity could be re duced if the mobile phase were elimi nated prior to the measurement of the FT-IR spectrum. Keith Bartle of the
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FOCUS University of Leeds showed impressive spectra of more than 20 antioxidants measured in this way after separation by capillary SFC. The usefulness of GC and HPLC for solving a wide variety of analytical problems has necessitated the develop ment of interfaces with several differ ent types of rapid-scanning spectrom eters, but no technique has proved to be of greater importance for the identi fication or characterization of eluites from gas and liquid chromatographs than MS. Certainly the greatest num ber of reports at Park City on any topic concerned research directed toward the hyphenation of supercritical fluid chromatographs (equipped with both packed and capillary columns) with several types of mass spectrometers. Descriptions of SFC/MS interfaces were given by David Miller (University of North Dakota), Jack Henion (Cor nell University), David Pinkston (Procter and Gamble), Tony Berry (University College, Cardiff), Stephen Lane (Glaxo Group Research), Eric Huang (BYU), and Bob Wright (Pacif ic Northwest Laboratory). From these reports it became apparent that these SFC/MS interfaces usually bear more similarity to current designs for LC/ MS than GC/MS. The facility with which the mobile phase in SFC can be eliminated makes SFC/MS a some what more tractable proposition than LC/MS, however. It seems only a mat ter of time before a versatile, sensitive SFC/MS interface becomes commer cially available. The material presented at Park City emphasized instrumental develop ments, but several authors described applications that would have been dif ficult or impossible with GC or HPLC. Particularly promising results were shown in two areas: the separation of biochemically important molecules and the chemical-class-dependent sep arations of fossil fuels. In the first area, Pat Sandra of the Research Institute for Chromatography (Wevelgem, Bel gium) compared the separation of lip ids and phospholipids by capillary GC and SFC. He showed that GC was gen erally preferable to SFC because of its significantly higher resolution in cases where the lipids could be eluted with out degradation. In cases for which GC is not applicable, SFC provides an ac ceptable substitute. Vernon Reinhold of the Harvard School of Public Health reported several impressive separa tions of oligosaccharides and their glycoconjugates with MS detection. Milos Novotny demonstrated the separation of polar steroids and their conjugates. Citing some earlier work by Chester as
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well as data from his own laboratory, Novotny stressed the need to block ac tive sites in biochemically important molecules by the formation of suitable derivatives. In the petroleum field, Joseph Levy of BP America demonstrated that packed columns could be used to achieve chromatography based on chemical class, such as the separation of paraffins, olefins, naphthenes, and aromatic compounds in fuel oil distil lates. By heart cutting and injection of
SFC will continue to be actively developed over the next two to three years. each fraction into a gas chromatograph, Levy's group achieved high-res olution separations and thus remark ably detailed characterization of fuels. More than 300 chromatograms illus trating the wide variety of applications of SFC were collected by Milton Lee and Karin Markides and were made available to the registrants in the form of a book. Because the important oper ating parameters are listed along with each chromatogram, this book will un doubtedly prove to be of much more lasting utility than the collection of ab stracts that is conventionally produced for technical meetings. The workshop organizers are to be congratulated for this innovative idea. Another deviation from the format of many technical symposia was the inclusion of a few pa pers from relative neophytes who de scribed the difficulties that may be en countered by chemists entering the field of SFC. In this respect, I particu larly appreciated the talk by Alan Nichols of Olin Corporation on the pit falls of quantitative method develop ment. Although SFC '88 was primarily con cerned with chromatographic separa tions, several participants described extractions using supercritical fluids. Jerry King of the USDA Agricultural Research Center (Peoria, 111.) showed several applications of supercritical fluid extraction (SFE) and stressed the importance of being able to use solubil ity parameters to calculate the extractability of components from their natu ral matrix. Steven Yocklovich of Com puter Chemical Systems showed the practicality of using SFE/SFC for qual ity control. Perhaps the paper that gen-
FOCUS erated the most excitement among conferees was the report by Steven Hawthorne (University of North Dakota) of small-scale supercritical fluid extractions in which the extract was trapped at the head of a capillary GC column. Analytes could be extracted from matrices ranging from NBS urban dust standards to slivers of telephone poles and separated by GC in total times of
