Supercritical Fluid Chromatography - ACS Publications - American

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Anal. Chem. 2008, 80, 4285–4294

Supercritical Fluid Chromatography Larry T. Taylor Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061-0212 Review Contents Reviews Analytical Scale Chiral Separations Analytical Scale Achiral Separations Simulated Moving Bed Preparative Separations Supercritical Fluid Chromatography-Mass Spectrometry Natural Products Open Tubular Column SFC Miscellaneous Literature Cited

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The inaugural fundamental review in Analytical Chemistry devoted solely to supercritical fluid chromatography (SFC) was published in 1990 (1). Selected articles from the 1988 and 1989 publication years of Chemical Abstracts were considered. It was also during this time that the Journal of Supercritical Fluids first appeared, but the journal during the intervening 20 years has been more focused on materials research than upon separation science. In 1990, SFC was no longer considered a curiosity, but in many analytical laboratories it was recognized as a necessity. Unfortunately, the nomenclature has often been loosely applied in this area, but what was generally called SFC then (and even today) employs carbon dioxide above or near its critical temperature of 31 °C and pressure of 73 bar, combined with an organic modifier such as methanol or ethanol. The differences in SFC, subcritical fluid chromatography, enhanced fluidity chromatography, and high performance liquid chromatography (HPLC) have been overstated in the past. Each chromatography represents a part of a continuum of increasing mobile phase solvating coupled with increasing mobile phase viscosity and decreasing mobile phase diffusivity. In later years, the topic of analytical supercritical fluid extraction (SFE) was added to the Analytical Chemistry biannual review. In 2004, the review dealt with supercritical fluid and unified chromatography (2) even though prior years had seen a decrease in fundamental work and in the number of published research articles devoted to SFC primarily because users found that instrumentation was difficult to operate and poorer chromatographic performance than HPLC was obtained. In other words, vendors could not deliver the sensitivity, precision, and reproducibility required in industrial applications. Today, difficulties with back-pressure regulation, consistent flow rates, modifier addition, sample injection, automation, stationary phases, etc. have been successfully overcome principally by Terry Berger and Berger Instruments Inc. who pioneered new developments in the field in the mid-1990s and by other vendors who later followed him. It is safe to say that SFC is solving lots of problems in 2008, and the field is technologically stronger than it has ever been. 10.1021/ac800482d CCC: $40.75  2008 American Chemical Society Published on Web 04/22/2008

In 2006, the review topic was supercritical fluid chromatography, pressurized liquid extraction, and supercritical fluid extraction (3). Well over 300 papers were published in the two year period ending November, 2005. Application papers made up a majority of the published work. Pharmaceutical applications involving moderately polar analytes were a strong theme concerning SFC. Food and natural products along with environmental applications were the prime focus of SFE. Most of the environmental work dealt with nonpolar compounds such as polyaromatic hydrocarbons and polychlorinated biphenyls wherein nonpolar supercritical CO2 yielded high extraction efficiencies. Cosolvent systems which combined CO2 with one or more modifiers and extended the utility of supercritical CO2 to more polar analytes were attractive for solving pharmaceutical separation problems. At that time, new reasons were rediscovered which favored the use of SFC such as an increased demand for environmentally friendly processes and the elimination of organic solvents that lead to ozone depletion. In this biannual review, the field of SFC is again solely examined since over 100 publications have appeared during the stated time of review. The published literature in the period from January 1, 2006 to December 31, 2007 has been considered. The searched database was compiled by SciFinder Scholar which contained non-English journals and patents, books, abstracts of regional and national American Chemical Society meeting presentations, and journals in English. The search keywords were “SFC” and “supercritical fluid chromatography”. The total number of hits was 144. During this review period as in other previous periods, SFC vendors which featured analytical scale separations and quantitative analysis both left and entered the business. Mettler-Toledo Autochem, Inc. is the latest one to leave having been purchased July, 2007 by Thar Instruments Inc., Pittsburgh, PA. Jasco, Inc., Easton, MD and Selerity Inc. Salt Lake City, UT currently complete the U.S. market. The absence of Mettler-Toledo Autochem, Inc. follows a long line of now missing vendors: Brownlee Laboratories, Dionex, Suprex, Hewlett-Packard, Gilson, and Berger Instruments, for example. On the other hand, one preparative SFC vendor who focused on qualitative and purification work, AccelaPure (Newark, DE), left the business during the same period; thus leaving Thar Instruments and NovaSep, Inc. as the prime preparative scale vendors in the U.S. The papers that have been published during the past two years continue a disturbing trend insofar as the fundamental development of SFC is concerned. Specifically, many of the manuscripts are application oriented and authored by customer organizations such as pharmaceutical companies. These companies and authors should be commended for researching and sharing freely such Analytical Chemistry, Vol. 80, No. 12, June 15, 2008

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fundamentally important work. However, the general absence of fundamental SFC-related work in the academic community causes concern from the standpoint of both advancing and globally applying the science and the training of graduate students and future workers in the field. SFC has continued to evolve as a more mature technique comparable to gas chromatography (GC) and liquid chromatography (HPLC). In this regard, it should be noted that most of the advances in instrumentation have been both vendor promoted and financed as opposed to being federally sponsored and funded. Nevertheless, the advantages of packed column SFC relative to more seemingly mature HPLC methodologies are clear: (a) lower viscosity and higher diffusivity supercritical mobile phases relative to liquids which lead to both faster, more efficient separations per unit time, and shorter turn-around time between injections and (b) an inert, environmentally “green”, more volatile carbon dioxide-based mobile phase for large scale separations and energy efficient isolation of the desired product. In the 1980s, however, SFC was thought to be more GC-like with emphasis on open tubular columns and flame-based detectors. This unfortunate diversion no doubt has delayed in part the maturation process and acceptance of SFC. Nowadays, packed column SFC is widely accepted. It uses the same injector and packed column configurations as in LC. It is more robust, more adaptable to a broader spectrum of compound classes, and thus more useful for routine operation than open tubular column SFC. Furthermore, the hyphenation of packed column SFC to mass spectrometry and ultraviolet detectors is experimentally more straightforward than for open tubular column SFC. Chromatographers readily agree that SFC is normal phase chromatography, and as such its orthogonality to reversed phase liquid chromatography is an attractive asset for purity assessment. In the last 10 years, the acceptance and implementation of packed column SFC-mass spectrometry (SFC-MS) to drug applications has gained momentum because of its broad applicability to such areas as high-throughput analysis, purity assessment, structure characterization, and purification. Until recently, SFC instrument design had been based on HPLC designs with some specific modifications (i.e., SFC was treated as a “byproduct” of HPLC). Today, SFC hardware integrates the design of the pumping system, modifier module, postcolumn nozzle, cyclone separator, detector, and associated universal, operational software for generation of a more robust, reliable technique for both analytical and preparative separations. Now, SFC is rapidly becoming the separation of choice for preparative scale multigram processes. Preparative SFC can be viewed as a “greener” alternative to classical preparative chromatography since intensive use of organic solvents is not required. Nevertheless, preparative HPLC is historically used most frequently for the kilogram-scale separations that are needed to support industrial development and organic synthesis, although preparative GC has been successfully implemented for some applications concerning low-molecular weight and/or volatile compounds The use of SFC for preparative separations, however, has received considerable attention during the current review period as a tool supporting preclinical development in the pharmaceutical industry. The resulting decrease in solvent use and waste generation offers a green advantage with an economic 4286

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bonus that makes preparative SFC especially attractive for providing purified materials on even the kilogram scale. The SFC product is recovered in a more concentrated form relative to HPLC, thereby greatly reducing the amount of solvent that must be evaporated and gives rise to considerable savings in labor, time, and energy costs. The higher SFC flow rates also contribute to higher productivity relative to HPLC methods. In summary, SFC (as most analytical techniques) has had a tortuous development history, but it appears that analytical and preparative scale SFC are currently on the strongest foundation ever with vendors that are strongly committed to advancing the technology. New developments and a broader spectrum of applications in the field are, however, anticipated in the future. In addition to reviewing the major SFC contributions over the past 2 years via brief synopses of the major English publications in 2006-2007, SFC references to work published in non-English journals can be found in Table 1. In addition, Table 2 lists the SFC abstracts of ACS meeting presentations during 2006-2007 that were made a part of our search. REVIEWS Today SFC stands tall as both the primary method for chiral separations in drug discovery and potentially the replacement to conventional liquid-based chromatography all the way through the drug pipeline (4). Numerous reviews have been published in 2006-2007 by pharmaceutical chemists that outline recent developments in separation and purification in their company. SFC when coupled with an atmospheric pressure chemical ionization source has proven to be extremely popular (5). The SFC-APCI/ MS method was reviewed during the period and shown to have a shorter analysis time than GC/MS methods without the need for derivatization prior to analysis and to be faster than HPLC methods with better resolution. Terfloth has written a chapter which reviews preparative isolation of impurities (6). The chapter covers a range of chromatographic techniques on a scale from micrograms to multigrams including aspects of automation, efficient isolation, scale-up of chromatographic processes, and determination of the most efficient conditions using HPLC and SFC. The approach taken was loosely based on the input-processoutput meta-model utilized to transform a problem statement into a functional process. A more general review has outlined the attributes of SFC that differentiate it from other chromatographic techniques (7). In addition, it described some of the important operating parameters that should be considered when utilizing this technique for chiral separations. Two approaches to ruggedness and robustness of an analytical method employing several chromatographic protocols including SFC and HPLC have been described (8). Case studies were critically reviewed and discussed. Most examples concerned pharmaceutical assays. Analytical and preparative scale packed column SFC of lipids have also been reviewed (9). Separations of triacylglycerols, free fatty acids, and glyceride mixtures were considered. A review of chromatographic methods that incorporated SFC for determination of carbamate pesticides in environmental samples appeared during the period (10). ANALYTICAL SCALE CHIRAL SEPARATIONS Enantiomeric separation of several chiral sulfoxides belonging to the family of substituted benzimidazoles has been reported with

Table 1. References to SFC in Non-English Journals for the Period 2006-2007

Table 2. Abstracts of SFC Papers Presented at ACS Meetings for the Period 2006-2007

1. Interconversion of stereochemically unstable chiral drugs: Utilization of chromatographic techniques for the study of enantiomerization. Part II: Practical applications. Ceska a Slovenska Farmacie 2006, 55 (1), 12-17. 2. Application of SFC and mass spectrometry in analysis of oil and fat. Zhongguo Youzhi 2006, 31 (9), 48-52. 3. Preparation methods for chiral drugs. Yiyao Daobao 2006, 25 (4) 325-327. 4. Application of chromatographic techniques in quality control of traditional Chinese medicine. Zhongcaoyao 2006, 37 (10), F11-F12. 5. Advances in residue analysis of pesticide in Chinese herbal medicine and new technological development. Zhongguo AhongyaoZazhi 2006, 31 (22), 1841-1846. 6. Chiral separation by SFC. Fain Kemikaru 2007, 36 (1), 30-37. 7. Application of SFC in preparative-scale separation of enantiomers. Jingxi Yu Zhuanyong Huaxuepii 2006, 12 (22), 1-4, 8. 8. Application of chiral stationary phases of chromatography on resolution of chiral drugs. Zhongguo Xinyao Zazhi 2006, 15 (24), 2099-2102. 9. A novel technique of extractiing oil from Chinese herbal medicine-subcritical water extraction. Huaxue Gongcheng 2006, 34 (8), 59-62. 10. Supercritical fluids in analytical chemistry. III. Supercritical fluid chromatography. Quimica Nova 2006, 29 (4), 790-795. 11. Clinical application of enantiomers of chiral drugs. Xibei Guofang Yixue Zaz 2006, 27 (2), 132-134. 12. .Laboratory automation with modular concepts. Efficiency increase in the search for active ingredients and new materials. GIT Labor-Fachzeitschrift 2007, 51 (4), 288-289. 13. Analysis of hydrophobic metabolites by SFC. Journal of the Mass Spectrometry Society of Japan 2007, 55 (3), 193-199. 14. Chromatographic analysis of triglycerides-why and how? Part II. Contribution of chromatography in liquid and supercritical phases. Journal de la Societe Quest-Africaine de Chimie 2006, 11 (22), 1-11. 15. Research development on technology in determining pesticide residue in fruits and vegetables. Tianjin Huagong 2006, 20 (3), 19-22. 16. Separation of solanesol by preparative-scale SFC. Huagong Jinzhan 2007, 26 (10). 17. Isolation of protopine and tetrahydropalmatine in Corydalis yanhusuo by packed-column SFC. Tianran Chanjibu Yu Kaifa 2007, 19 (2), 283-285. 18. Isolation of protopine and tetrahydropalmatine in Corydalis decumbens by series packed-column SFC. Huaxi Yaoxu Zazhi 2007, 22 (2), 133-135. 19. Determination of the nonaromatic and aromatic content of diesel fuels by SFC. Jasco Report 2007, 49 (1), 23-24. 20. Effects of loading and flow rate on the preparation of EPA-EE and DHA-EE by SFC. Gaoxiao Huaxue Gongcheng Xuebao 2007, 21 (2), 189-193. 21.SFC used for separation of optical isomers. Farumashia 2006, 42 (4), 343-345.

1. Separating chiral and achiral compounds: Applications for SFC and SFC-MS. National Meeting of the American Chemical Society, Atlanta, GA, March 26-30, 2006. 2. Increase the efficiency and quality of candidate selection via implementation of SFC. National Meeting of the American Chemical Society, Atlanta, GA, March 26-30, 2006. 3. High purity purification of lead compounds by SFC for AMES testing. National Meeting of the American Chemical Society, San Francisco, CA, September 10-14, 2006. 4. SFC reduces back exchange after solution-phase hydrogen/ deuterium exchange. National Meeting of the American Chemical Society, San Francisco, CA, September 10-14, 2006. 5. SFC vs. LC: No contest? National Meeting of the American Chemical Society, San Francisco, CA, September 10-14, 2006. 6. SFC: A solution to the growing chiral problem. National Meeting of the American Chemical Society, San Francisco, CA, September 10-14, 2006. 7. The new reality of SFC-MS: Increase efficiency and reduce analysis time by incorporating a new technology without learning more software. National Meeting of the American Chemical Society, San Francisco, CA, September 10-14, 2006. 8. SFC with long packed columns. National Meeting of the American Chemical Society, Chicago, IL, March 25-29, 2007. 9. Chiral SFC for analysis of active pharmaceutical ingredients. American Chemical Society meeting, Cookeville, PA, 2007. 10. SFC as green technology development for drug discovery. American Chemical Society meeting, Cookeville, PA, 2007. 11. Comprehensive fluid-based separation techniques (LC × LC, SFC × LC) for the analysis of small molecules. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 12. Packed column SFC: A fast separation technique for the clinical laboratory. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 13. Preparative chromatography in a pharmaceutical discovery laboratory: The need for speed. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 14. Prep SFC columns: A study of safe, high-pressure columns and their efficiencies for the purification laboratory. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 15. SFC benefits over HPLC for the purification of pharmaceutical chiral and achiral molecular targets. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 16. Preparative SFC in high throughput purification. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 17. Chiral chromatographic screens in pharmaceutical analysis and purification. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 18. SFC with tandem mass spectrometry to evaluate the absorption and delivery of individual stereo-isomers of drug candidates. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 19. Chiral SFC and HPLC to support “high throughput” process research. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007. 20. Enhanced fluidity liquid chromatography of polar compounds. Central Regional American Chemical Society meeting, Covington, KY, May 20-22, 2007. 21. Moving prep SFC to the benchtop: Chiral separations to MS triggered purification. National Meeting of the American Chemical Society, Boston, MA, August 19-23, 2007.

a Chiralpak AD stationary phase and methanol modified CO2 mobile phase (11). Resolutions higher than 2 and separation times shorter than 10 min were observed. A reversal of elution order upon change of the modifier to 2-propanol was only observed for omeprazole. The effect of change of temperature on enantioresolution was only minor compared to the change of organic modifier in the mobile phase. In a related study, entioseparation of bifonazole by SFC on Chiralpak AD was studied (12). Ethanol, 2-propanol, and acetonitrile were examined as modifiers. Enantioseparation was possible with all three modifiers, and resolution was higher than 5 in all cases. The isoelution temperature was below the working temperature when using methanol but above it with ethanol and 2-propanol where coelution of enantiomers occurred.

Four new synthetic polymeric chiral selectors were developed, bonded to 5 µm silica and tested with supercritical CO2 plus an alcohol modifier which contained 0.2% (v/v) trifluoroacetic acid during the review period (13). The four columns were tested with a set of 88 chiral compounds. All 88 analytes were separated on one or more of the related polymeric chiral stationary phases in less than 5 min. Eight examples were given by Staskiewicz et al. Analytical Chemistry, Vol. 80, No. 12, June 15, 2008

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during the period of chiral SFC applications for radiolabeled compound synthesis (13). It was noted that SFC is very desirable since a primary requirement in the production of tracers for drug metabolism is speed of analysis. It was reported that it is more “time efficient” to synthesize racemic mixtures and then chromatographically separate the isomers than to develop entiomeric syntheses of the drug candidates of interest. Chiral resolution of four antifungal compounds, three imidazoles, and one triazole, using SFC on Chiralpak AD with methanolmodified CO2 has been published (14). Enantiomeric separation of the three imidazoles with resolution higher than 2 and analysis times lower than 10 min was achieved. Analysis time of the triazole on the other hand was higher than 80 min. Resolution of the four stereoisomers was achieved only partially with modifier mixtures of ethanol and 2-propanol. Welch et al. have published a tool for improved tandem column chiral SFC (15). Method development screening was performed by modification of a commercial analytical SFC instrument with two different software-controllable, six position high pressure column selection valves, each controlling a bank of five different columns and a pass through line. The resulting instrument was reported to have the ability to screen 10 different individual columns and 25 different tandem column arrangements. Mukherjee has reported the development of a direct assay of a chiral acidic drug compound in a 100% aqueous formulation and its quantification following ICH and FDA guidelines on analytical validation (16). A Chiralpak AD-H column was employed. The assay quantification limit was 5 µg/mL which the author suggested could be lower by optimizing the chromatography. Calibration response was linear between 1.0 and 0.0025 mg/mL. Separation of enantiomers of 1-phenyl-1-propanol by SFC on a chiral stationary phase which consisted of cellulose tris(3,5-dimethylphenylcarbamate) coated on a silica support (Chiralcel-OD) was studied under overload and nonlinear chromatographic conditions (17). Pulsed experiments were performed at a temperature of 30 °C using CO2 modified with methanol as a mobile phase. Parameters of the binary Langmuir adsorption isotherm were determined by the inverse method, and then they were compared via experimental and simulated peak responses. Isotherm parameters were derived for modifier concentrations between 1 and 5% (w/w) and operating pressures between 125 and 185 bar. Omeprazole has been enantiomerically separated at the semipreparative scale on a polysaccharide based chiral stationary phase by SFC (18). A modular SFC was adapted to work at the semipreparative scale. Two organic modifiers (ethanol and 2-propanol) as well as different injection volumes and concentrations of the omeprazole racemic mixture were evaluated in order to obtain both high purities and high production rates. In terms of production rates, the best result for S(-)-omeprazole at an enantiomeric purity higher than 99.9% was achieved with sample concentrations of 10 g/L and injection volumes of 2 mL. In the case of R(+)-omeprazole, the results were better using a 4 mL injection volume. In another report, a feasibility study was conducted using SFC for the assay of a chiral drug compound AZM in 100% aqueous formulations at various pHs (19). The response was linear between 0.5 and 0.005 mg/mL at pH 3 with a correlation coefficient of 0.9999. The assay limit of quantification was 2.5 µg/mL. The results indicated that this approach has the 4288

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potential to significantly reduce the typical processing time prior to analysis. The method was reproducible, linear over a wide range, and sensitive enough to detect the minor enantiomeric impurity in the chiral drug compound investigated here. A separation of two demethylated nobiletin metabolites, 3′demethyl-NOB and 4′-demthyl-NOB, via SFC/UV with a chiral stationary phase was found by Wang et al. to be superior to both normal phase and reversed-phase HPLC (20). The chiral phase was always better than the achiral phase for this study. By comparing the SFC profile of synthesized standards, the major nobiletin metabolite in mouse urine was identified as 4′-demethylNOB with a concentration of 28.9 µg/mL. A Chiralpak AD column provided a 10 min retention time difference between the nobiletin regio-isomers. An orthogonal approach to chiral method development screening has been published (21). The screening program focused on four separation technologies: (a) liquid chromatography, (b) SFC, (c) capillary electrophoresis, and (d) GC. An overview of the results from each of the screens, future directions, and a final unified strategy for chiral method development screening was presented. Acceptable chiral resolution was obtained for all 20 compounds in the screening library. SFC should be considered for use with all compounds following the methanol solubility ruleof-thumb. For routine generic analytical chiral SFC, the AD, OD, AS, and OJ should be the first tier of chiral stationary phase screening. A 5-40% modifier gradient mobile phase should be used. The modifiers in the first tier should be 0.1% isopropylamine in methanol, 0.1% isopropylamine plus 0.1% trifluoroacetic acid in methanol, and 0.1% ethansulfonic acid in ethanol. Armstrong, et al. have introduced a new, highly unusual chiral selector termed boromycin (22). The authors noted that this was the first effective chiral selector that contains boron. The boron is a stereogenic center and is essential for chiral molecular recognition. The phase strongly retains and selectively separates enantiomers of a wide variety of primary protonated and unprotonated amine-containing compounds with SC CO2 plus methanol. ANALYTICAL SCALE ACHIRAL SEPARATIONS Metoprolol and a number of related amino alcohols have been chromatographed by Salomonsson et al.on aminopropyl and ethylpyridine silica columns (23). The mobile phase was CO2 with methanol as a modifier, but no amine additive was present. Part of the study included design of experiment as a tool in order to find optimal conditions with respect to retention and selectivity for each column. On the aminopropyl silica, the analytes were more spread out; whereas on the ethylpyridine silica, retention and selectivity were closer. A non-end capped aminopropyl silica column was also used for some experiments, and it showed that end capping was not critical for this column. In a related study, varied types of polar stationary phases, namely, silica gel, cyano, aminopropyl, propanediol, poly(ethyleneglycol), and poly(vinyl alcohol), were investigated in subcritical fluid mobile phase (24). The effect of the nature of the stationary phase on interactions between solute and stationary phase and between solute and CO2 were of interest. Modified mobile phase was studied by use of a linear solvation energy relationship termed the salvation parameter model. The polymeric stationary phases provided the most accurate models, possibly due to their better surface homogeneity. During the period, the same author sys-

tematically studied in a similar fashion the chromatographic behavior of different stationary phases in a subcritical fluid mobile phase (25). Porous graphitic carbon, polystyrene-divinylbenzene, and aromatic-bonded silica stationary phases were examined. In another study, the author using combined SFC methods has characterized octadecylsiloxane-bonded stationary phases of all sorts: classical, protected against silanophilic interactions or not, and containing polar groups such as end capping groups or embedded groups (26). Characterization of stationary phases in supercritical fluids with the solvation parameter model was also reported by the author (27). The properties of numerous basedeactivated octadecyl-bonded phases were also studied by Lesellier et al (28). incorporating a simple test consisting in the injection of carotenoid pigments in subcritical fluid chromatography. The molecules used and the nature of the mobile phase allowed the determination of hydrophobicity, polar site accessibility and type, and bonding density of the stationary phase. The effect of column type on the resolution of a mixture of estrogen metabolites has been studied (29). Two packing materials, cyanopropyl silica (CPS) and Betasil diol silica, were selected. The columns were connected in series to create varying percentages of stationary phase as follows: 100.0% CPS, 37.5% CPS/62.5% Diol, 50% CPS/50% Diol, 62.5% CPs/32.5% Diol, and 100.0% Diol. The results indicated that connecting two columns having the same dimensions (i.e., 1/1, CPS/Diol) in series gave the best resolution. There was no effect on retention time or resolution when the two columns were reversed. A pSFC-UV method for the determination of sodium stearyl fumarate aqueous suspension has been described: commercial tartaric acid network polymeric column (tert-butylbenzoyl), 214 nm detection, (S)-naproxen internal standard (30). Less than 5 min was required for the separation with a resolution of about 3 relative to the internal standard. With some modification of the chromatographic conditions, water samples could also be analyzed. Ion-pair SFC of metoprolol and related amino alcohols has been separated on diol silica by Gyllenhaal et al. (31). Conditions were 10% methanol containing 24 mM acid and 18 mM triethylamine in CO2. The effects on selectivity were stronger with trifluoroacetic acid than with ethanesulfonic acid. The stability of the column showed negligible changes. The authors claimed that this procedure provided a way to tune the selectivity of SFC systems when amines are analyzed without the need to change stationary phase for the chromatographic system. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) have been separated by SFC coupled with evaporative light scattering and mass spectrometric detection (32). Four different silica based stationary phases were investigated: diol, cyanopropyl, 2-ethylpyridine, and 4-ethylpyridine. No additive was required to effect the elution of PC and PE which differed only in the polar headgroup. When the two ethyl pyridine phases were used, near baseline resolution of two species in each lipid case that differed in fatty acid content were seen. PS and PI could be successfully eluted from each phase when isopropylamine was added to the mobile phase. All separations were reproducible and required less than 15 min. Taylor et al. reported that one secondary amine and two quaternary amine salts were successfully eluted from a Deltabond cyano-bonded silica column with the addition of sodium alkylsul-

fonate to the methanol-modified, CO2-based mobile phase (33). A possible ion-pairing interaction between the positively charged analytes and the anionic part of the sulfonate additive was proposed. In another set of experiments, the three amine salts readily eluted from both ethylpyridine-bonded silica and aminobonded silica phases without the need of additive, although peak shapes were less than desirable. The addition of sulfonate salt to the mobile phase again sharpened the peaks. It was suggested that in the presence of methanol and CO2, these stationary phases are positively charged. For the first time, the employment of a strong silica-based anion exchange column for SFC of cationic species was achieved. Separation as the ion-pair was proposed with an ionic additive in the mobile phase; whereas separation of the intact amine salt from the positively charged stationary phases was suggested to be operational without the ionic additive. SIMULATED MOVING BED The establishment of a solvent cycle with a possibility for modifier control in a preparative SFC simulated moving bed (SMB) plant has been reported (34). Fourier transform-infrared spectroscopy and flame ionization detection were investigated for measuring alcohol concentrations in subcritical CO2. The applicability of the developed control concept for usage of 2-propanol in supercritical CO2 was verified by an experimental test series involving an eight column SFC-SMB plant. Different process variations were measured in terms of energy and solvent consumption. Savings of almost all CO2 and 76% of the modifier could be achieved. A comparison between the batch-SFC process and the simulated moving bed process has been presented (35). The separation systems were optimized primarily in terms of their specific productivity. For three of the four investigated model systems, the specific productivity of the SMB process was significantly higher than the productivity of the batch process. Comparison of the two processes was done from an economic point of view considering the minimum product price that has to be realized to fulfill the defined economic aim. It was found that although the optimized specific productivity of the SMB process was significantly higher than the productivities of the batch process, the batch process was the more profitable process for the investigated production rate range. Simulated moving columns (SMC) is a unique column switching technique previously developed for LC (36). The technique is significantly enhanced through the use of SFC. SMC uses two to three short chiral columns connected in series, which enables the unresolved enantiomers to separate repeatedly and exclusively through each column until sufficient resolution is obtained. SFC afforded a dramatic increase in the number of SMC cycles with significant less band broadening compared with LC. Zhang et al. demonstrated enantioselective separation by increasing the column from an actual 20 cm length to a 0.5 m virtual length with remarkably enhanced efficiency (320 00 plates). SMC-SFC was compared with SMC-LC (36). No increase in column back pressure was observed since the total length of the columns remains constant. SMC can be envisaged as a movement of two to three columns in the direction of the mobile phase to keep enantiomers recycling in the columns until sufficient resolution is achieved. SMC worked independent of the pumps. Analytical Chemistry, Vol. 80, No. 12, June 15, 2008

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PREPARATIVE SEPARATIONS There has been considerable activity during the past two years concerning the use of preparative scale packed column SFC for drug discovery and development. For example, the impact of SFC in drug discovery as an analytical and preparative tool has been recently discussed (37). With automated stacked injections and fraction collection, SFC offered a faster and more direct method in chiral resolutions than more standard methods such as crystallization and LC. A case study was published by Welch et al. featuring the intensive use of preparative chiral SFC in the synthesis of an early development drug candidate (38). Semipreparative SFC was employed for gram quantities and large scale equipment for kilogram scale. The authors suggested that different strategies for preparative chromatography should be used at different stages of development with productivity and efficiency being less of a concern at early stages of development and becoming increasingly important as the project moves closer to commercialization. In another brief report, the impact of chiral SFC in a particular drug discovery study from analytical to multigram scale was described (39). The first direct multigram purification of all four isomers of the unnatural amino acid (β-methylphenylalanine) using SFC with stacked injection was reported (40). The carboxy termini were protected by conversion to methyl esters using standard methods. Following esterification, the amino termini were protected by conversion to N-benzylcarbamate derivatives. A Daicel Chiralpak AD-H column (20 mm × 250 mm) using 50:50 methanol/ethanol as the organic modifier resulted in purification of over 3.4 g of material in 6.25 h with >90% recovery. In another study by Wang et al., the imidazoline compound, nutlin-3, is a promising small molecule antagonist of the MDM2-p53 interaction (41). The compound was synthesized as a racemic mixture, and one enantiomer proved to be 100-200-fold more active than the other enantiomer. The chiral SFC method based on Chiralcel OD showed superior separation in terms of selectivity and efficiency. High throughput preparative scale purification ultimately gave from a 5 g racemic mixture in 75 min, a recovery rate above 92%. It was suggested that the daily throughput could be as high as 48 g of each enantiomer at one-third the time and expense compared to the LC method. A supercritical fluid extract of rosemary has been fractionated by using a preparative SFC system (42). The selective isolation of the compounds responsible for both antioxidant and antimicrobial activities was of interest. Two cyclones were employed to collect the fractions which were subsequently characterized by HPLC-DAD, GC, and in vitro antioxidant and antimicrobial assays. By a careful selection of the separation conditions it was possible to obtain two different fractions, one enriched in antioxidant and antimicrobial compounds collected in cyclone no. 2 with no residual rosemary aroma and a second fraction (cyclone no. 1) that contained the essential oil. A method for efficient and relatively large-scale isolation of four polymethoxyflavones from sweet orange (Citrus sinensis) peel by employing SFC has appeared (43). The flavones were nobiletin, tangeretin, 3,5,6,7,8,3′,4′heptamethoxyflavone, and 5,6,7,4′-tetramethoxyflavone. SFC technology had a dramatic advantage over other separation methods and was found to be well suited for providing large amounts of polymethoxyflavones from orange peel extract. 4290

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Steady state recycling (SSR) and SFC were used for the enantiomeric resolution of three pharmaceutical intermediates at various sample scales (44). The separation conditions, the sample purities and yields, the productivities, and the solvent consumptions were discussed in three case studies. The SSR process was used for a low selectivity resolution of 2.0 kg of intermediate. Productivity using SSR techniques increased by a factor of 4.5, while solvent usage decreased by a factor of 4.1 when compared to the productivity and solvent usage of batch HPLC. The SSR column had an inner diameter of 10.3 cm and was packed with 2.0 kg of ChiralPak-AD to a length of 40 cm. The column and mobile phase were thermostatted at 30 °C. The mobile phase was 100% methanol. The SFC process when compared with HPLC was more effective in terms of an increase in productivity and a reduction in solvent usage. The flow rate was 170 g of CO2/min. Operating pressure was 100 bar. The prepacked column (3 cm i.d. × 25 cm, 100 g of Chiralcel-OD-H, 5 µm) was thermostatted at 40 °C. Methanol (20%) was used as a cosolvent. In as less optimized mode, Anderson et al. discovered that SFC was not appropriate as a preparative scale separation technique for omeprazole and related benzimidazoles due to instability of the chromatographic process (45). This was mainly thought to be due to the acidic CO2 that facilitates degradation of the sulfoxide moiety in these compounds to predominantly the corresponding sulfenamide. SUPERCRITICAL FLUID CHROMATOGRAPHY-MASS SPECTROMETRY The usefulness of SFC in the analysis of hydrophobic metabolites has been described (46). Individual homologues from 10 to 100 mers were separated under optimized SFC conditions. When a cyanopropylated silica gel packed column was used for the separation, 14 lipids were successfully detected via MS and the analysis time was less than 15 min. The use of an octadecylsilylated column resulted in the separation of unsaturated fatty acid side chains. Another published study described packed column SFC coupled to an APCI source and a tandem MS/MS (positive ion mode) for rapid and simultaneous determination of clozapine, ondansetron, tolbutamide, and primidone in in vitro (rat microsomes) samples (47). The method was developed in support of metabolic stability experiments. The metabolic stability results obtained by pSFC-MS/MS methods were in good agreement with those obtained by a fast HPLC-MS/MS method. The development of a mass-directed SFC purification system has been reported by Goetzinger et al. (48). Good peak shape and signal were achieved in the MS trace which allowed accurate peak detection and reliable fraction collection. Simple modifications to a commercially available fraction collector enabled fractionation at atmospheric pressure with high recovery. The SFC-MS purification system was used in support of highthroughput library purification, and it was proven to be a valuable tool in complementing a RP-HPLC based technology platform. In another report, the separation of cytarabine (ara-C) from the endogenous compounds in mouse plasma by pSFC was achieved on a bare silica stationary phase with an isocratic mobile phase composed of CO2/methanol solvent with addition of ammonium acetate (49). The pSFC was interfaced with an APCI source and a tandem MS. The pSFC-MS/MS approach required about 2.5 min/sample for the determination of ara-C in mouse plasma. The

methodology was partially validated with respect to stability, linearity, and reproducibility. The mouse plasma levels of ara-C were found to be consistent with those determined by various RP-HPLC methods using a porous graphite carbon column in conjunction with an ion pairing agent coupled to tandem MS. SFC-MS with two different columns was used to achieve a shorter analysis time for the separation between the highly toxic positional isomers quinocide and primaquine in primaquine diphosphate which is used for causative treatment of malaria (50). The study was designed to elucidate additional information about differences in SFC-MS fragmentation patterns which were not possible with LC-MS. With the use of a Chiralpak AD-H chiral column, it was possible to both resolve the enantiomers in primaquine and to separate quinocide from those enantiomers. A fast bioanalytical method for R/S-warfarin in human plasma was developed during the review period employing pSFC and APCIMS/MS (51). During the work, the authors also developed a semiautomated liquid extraction SFC-MS/MS method. The standard curve range was 13.6-2500 ng/mL. Precision of quality control concentrations from four validation runs was 7.0% for R-warfarin and 6.0% for S-warfarin. pSFC coupled to an APCI and a tandem MS system with minimum sample pretreatment has been explored for the rapid and enantioselective determination of (R,S)-propranolol in small volume mouse blood samples (52). Determination at low nanogram per milliliter levels required approximately 3 min/sample. The method was used to support a pharmacokinetic study. Matrix ionization suppression, one of the common concerns when developing new MS based methods, was also investigated. The addition of basic additives to the mobile phase had a strong impact on the ionization efficiency using the APCI source. pSFC-MS/ MS was also used for the separation of 15 estrogen metabolites (53). A gradient of methanol in CO2 with a cyanopropyl silica column connected in series with a diol column packed with 5 µm spherical silica-based particles resulted in separation and quantification in less than 10 min. The limit of detection and limit of quantification was determined to be 0.5 (S/N ) 3) and 5 pg, respectively. Compared with RP-HPLC-MS/MS analysis of the same mixture, the LOD and LOQ were comparable but the analysis time was slower being near 70 min. Examination of the peak widths obtained by each technique revealed that the baseline width in pSFC was 20-30 s; while in LC, the baseline width was in minutes (∼1.8 min). A directly coupled achiral/chiral SFC-MS method has been developed for the purpose of profiling a three-step stereoselective synthesis of cinnamonitrile and hydrocinnamonitrile intermediates (54). The coupled columns were found to significantly enhance the separation of both enantiomers and diastereomers without adding significantly to the overall analysis time. The methodology was said to be useful for systems where there was more than one chiral center. Pinkston et al. have compared the LC-MS and SFC-MS of a diverse library of druglike compounds (55). Over 70% of the library compounds were eluted and detected by SFC-MS; while 79.4% were eluted and detected by LC-MS. The only compound class that appeared to be consistently detected by LC-MS but not SFC-MS under the conditions was compounds containing phosphate, phosphonate, or bisphosphonate.

The APCI source required less cleaning during SFC-MS than it did during LC-MS. Analytical scale SFC-MS saw the publication of more fundamental studies during the period (56). Results of ion-neutral reactions in the gas phase which take place when mixtures of polar modifiers (e.g., methanol, ethanol, and 2-propanol) are introduced into the CO2 mobile phase and expand through the direct fluid interface have been presented. The structure of the reagent ions was studied by collision induced dissociation and deuterium labeling. Three main reaction series were found for this system: (a) ionization followed by R-cleavage, (b) formation of clusters, and (c) formation of protonated dialkyl ethers from the corresponding alcohol. SFC-MS has been used to probe surface exposure in a protein (57). The single biggest problem with solution phase hydrogen/deuterium (H/D) exchange in a protein is back exchange of H for D after the initial H/D exchange has been quenched. SFC replaced LC as the desalting/separation technique prior to mass analysis, thus providing a dramatic reduction in back-exchange compared to fast, cold LC methods. A fully automated, user independent parallel four-column (150 mm × 2.1 mm) SFC-MS system to initially perform highthroughput, enantioselective chromatographic method development has been reported (58). Optimization of the separation of enantiomers was achieved on a single chiral column. To facilitate the process, custom software monitored each of the runs in realtime, processed each data set, and by incorporating user-defined criteria, selected the next set of experiments and automatically optimized the enantioseparation. NATURAL PRODUCTS The chemical nature of davanone isolated from natural davana oil via pSFC with a CO2-based mobile phase has been defined (59). Various analytical-scale, silica-based stationary phases were tested. A semipreparative separation which yielded three fractions was consequently developed on a 2-ethylpyridine phase (250 mm × 10 mm, 5 µm). The davanone fraction was nearly 100% optically pure. The results indicated that fractionation of davana oil with supercritical fluids near room temperature had little effect on the optical integrity of the sample. Two chromatographic methods have been described for standardization of bacoside A3 and bacopaside II, the major triterpenoid saponins present in Bacopa monnieri extract, in order to improve a commercial formulation (60). The first method was reversed phase high performance thin-layer chromatography and the second was pSFC with photodiode-array detection. The effect of temperature on SFC separation of the saponins was studied in detail. Van’t Hoff plots for retention and selectivity were found to be linear. The data revealed that separation of bacoside A3 was enthalpically favored in the range of temperatures investigated; whereas entropy-controlled separation was observed for bacopaside II. Validation of injection precision, repeatability, reproducibility, and sample stability as recommended in the ICH guidelines was performed. In a separate study, parthenolide (1 of 30 sesquiterpene lactones) in feverfew was measured by SFC after extraction in an ultrasonic bath with methanol for 30 min (61). A specially designed column for fractionation of the supercritical fluid extract of rosemary using preparative fraction collection has been reported (62). The column was prepared using a new packing method consisting of a combination of slurry and Analytical Chemistry, Vol. 80, No. 12, June 15, 2008

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supercritical CO2 with commercial silica particles coated with a stationary phase commonly used in GC such as SE-54. The new procedure provided columns with reasonable efficiencies and high stability at high pressure. A 25 cm × 10 mm i.d. column packed with silica particles coated with 3% SE-54 was prepared, and its separation power was tested for isolating fractions with high antioxidant and/or antimicrobial activity from a SF rosemary extract. In another publication, a study was carried out to investigate the presence of coenzyme Q10 (i.e., 10 times more powerful antioxidant than vitamin E) in crude palm oil and palm fiber oil by SFC with UV detection and methanol-modified CO2 using a Metaphase RP C18 (4.6 mm × 250 mm) column (63). Thurbide et al. have reported a direct analysis of the monomeric and four double helical dimeric conformations of gramicidin (i.e., linear hydrophobic polypeptide antibiotic) using pSFC (64). The experimental parameters were PRP-1 polymeric column (50 mm × 4.1 mm, 5 µm particles), 40 °C, 25 MPa, 35% n-pentanol modifier, and 220 nm. All confomers were readily separated. The dynamic behavior of the four double helical dimers with respect to solvent, incubation time, concentration, and temperature was successfully demonstrated. A report concerning the SFC separation of long-chain hydrocarbons and mink oils by using pressure programming has been published (65). Relatively large peptides (at least 40 mers) containing a variety of acidic and basic residues have been eluted via SFC. Trifluoroacetic acid was used as an additive in a CO2/ethanol mobile phase to suppress deprotonation of peptide carboxylic acid groups and to protonate peptide amino groups. A 2-ethylpyridine bonded silica column was used for the majority of the work. The relatively simple mobile phase was compatible with mass spectrometric detection (66). A method for the isolation of individual carotenes in palm oil using SFC was reported (67). SFC offered more powerful and efficient separation properties as well as being clean, hygienic, and nontoxic. A study on the behavior of the maximum wavelength of the individual carotenes in SC CO2 was carried out. It was found that the data obtained in the past for identification of individual carotenes dissolved in hexane could not be applied in a SC environment. OPEN TUBULAR COLUMN SFC A porous layer open tubular silica gel column has been used together with subcritical CO2 as the mobile phase to effect the group separation of polar oxygenated compounds (68). It was demonstrated to be valuable as the first dimension of a comprehensive two-dimensional SFC × GC analysis. With this combination, many of the oxygenated compounds routinely found in petroleum samples could successfully be separated and identified. The presence of tert-amyl methyl ether in a commercial lead-free gasoline sample was demonstrated. Recent advances were published during the review period concerning simulated distillation using SFC of heavy conversion fractions and vacuum residues of crude oils (69). Compared to GC, SFC with capillary columns extended the range of this application up to nC126 hydrocarbons, and it also made possible calibration up to nC200 or around 850 °C as the equivalent boiling point. It was suggested that SFC could be the tool of choice for better determination of conversion in heavy petroleum fraction processing. A method for determining total biodiesel methyl and ethyl ester content in diesel fuels by SFC-FID was developed (70). A silica 4292

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column typically used for determining aromatics in conventional fuels by ASTM D5186 was back-flushed after separation of the hydrocarbons to allow elution of the various esters as a single”total biodiesel”distinct peak. The modification concurrently allowed the determination of total aromatic hydrocarbons and their distribution as mono- and polynuclear compounds. Normalized percent quantification using a hydrocarbon response factor of 1.00 and an ester response factor of 1.19 provided an average percentage error of 1.8% when measuring actual biodiesel/hydrocarbon fuel blends. The ester response factor was the average of the response factors of 10 pure ester compounds. In another study, fatty acid methyl esters were separated on a packed capillary SFC column and detected by a UV detector at 206 nm (71). The temperature controlled restrictor was a 20 cm length of deactivated fused silica of 7 µm i.d. which was held immediately after the UV detector. Another temperature-controlled restrictor for UV detection in capillary SFC was reported (72). Polyaromatic hydrocarbons were separated and detected by a UV detector set at 280 nm. The passive restrictor was a 20 cm length of deactivated fused silica 7 µm i.d. The temperature of the fused silica tube could be varied from 100 to 350 °C in order to maintain a constant flow rate with pressure programming. In a separate report, the chromatographic impulse response technique with a polymer coated capillary column was applied to measurements of infinite dilution, binary diffusion coefficients, and retention factors in SC CO2 (73). Hexane solutions of three unsaturated fatty acids such as linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were injected. A comprehensive two-dimensional capillary SFC in stop-flow mode with synchronized pressure programming was developed (74). The interface consisted of a 10-port valve, a capillary trap, and two fused silica restrictors. The pressure of the system was controlled with a single pump. All of the operations were automated using in-house software. The use of synchronized pressure programming allowed the sampling duration and/or the second dimension separation time to be changed without affecting the separation pattern. Limiting partition coefficients of a selection of low-to-medium volatility solutes between both phases in a biphasic trihexyltetradecylphosphonium chloride ([thtdp][Cl]) ionic liquid-SC CO2 system were obtained by capillary column chromatography (74, 75). [thtdp][Cl] served as the stationary liquid and SC CO2 as the carrier fluid. It was shown that SFC can be used to probe the partitioning behavior of solutes in biphasic systems even when the ionic liquid has nonzero solubility in the carrier fluid. Relative partition coefficients of solutes at a particular temperature and density of CO2 can be correlated within Abraham’s linear salvation energy relationships. MISCELLANEOUS A relatively convenient and effective alternative method of restoring column efficiency loss due to pressure drops in pSFC has been described (76). Under low-density conditions where efficiency loss occurs, using a small coil of resistively heated wire to mildly heat the column inlet area was found to increase the plate number for octadecane by a factor of 8. A similar improvement could be realized by placing a water cooled coil around the outlet area of the column. An investigation into the fundamental limitations and sources of noise in the SFC-UV approach has

been published (77). Identification of the thermal, electronic, and mechanical sources of noise within the UV flow cell as contributing to reduced sensitivity was described. Improvements in the system allowed sufficient sensitivity and accuracy to carry out GMP release testing for enantiopurity analysis using SFC. Two approaches for decreasing diesel hydrocarbon group-type separation times by SFC have been compared. Short (10-15 cm) columns with small 3 µm diameter packing were compared with monolith bare silica columns at high CO2 flow rates approaching 5 mL/min (78). Short packed columns with higher surface area and retention characteristics offerred higher resolutions compared with Chromolith columns. Diesel samples were separated into saturates, mono-, di-, tri-, and polyaromatics in as little as 2 min on a 10 cm packed silica column. SFE has been coupled to comprehensive two-dimensional pSFC (79). Three modes of operation were feasible: SFE-SFC for polymer additives, SFC × SFC for a mixture of benzene derivatives with different polarities, and SFE-SFC × SFC for styrene oligomers in polystyrene which normally require various pretreatments. The binary diffusion of 1,2-diethylbenzene, 1,4-diethylbenzene, 5-tert-butyl-m-xylene, and phenylacetylene at infinite dilution in supercritical CO2 were measured between 15.0 and 35.0 MPa and in the temperature range of 313.16 to 333.16 K by the Taylor-Aris chromatographic method (80). The solutes were chosen in an attempt to give insight into different types of molecular-scale interactions in solution. The applicability of mass transport equations that are commonly used to describe the diffusion in supercritical CO2 was investigated. In a related study, the binary diffusion coefficients, D12, and retention factors for myristoleic acid and its methyl ester at infinite dilution were measured by the chromatographic impulse response technique in SC CO2at temperatures of 313.2, 333.2, and 343.2 K and pressures from 9.2 to 30 MPa for the acid and from 8.0 to 14 MPa for the ester (81). Coupling of a corona-charged aerosol detector (CAD) to packed column SFC by connecting the outlet of the back pressure regulator (BPR) directly to the inlet of the detector has been described Sandra et al. (82). Limits of detection ranged from 3 to 11.5 ng loaded on column. To reduce differences in response at different mobile phase compositions, mobile phase flow compensation was performed by placing a T-piece before the BPR. Performance of the pSFC-CAD combination was illustrated by the analysis of selected pharmaceutically related compounds. In addition, a comparison with UV detection was made. The feasibility of the separation of low-molecular weight lubricant additives using various packed columns and CO2to enable implementation of flame ionization as a universal detector has been determined by Thiebaut (83). Comparison of stationary phases that were supposed to provide hydrocarbon group type separation (i.e., alumina and silica) was a focus of the paper. Separations on alkyl-bonded silica in a nonaqueous mode of selected classes of additives in a test mixture or in base stocks were reported. Adsorption chromatography allowed partial separation of additives from the base stocks; while the direct elution of test additives could only be obtained on reversed phase supports having very efficient silanol group protection. A novel detector based on the frequency of acoustic emissions from an oscillating premixed hydrogen/oxygen flame has been characterized for use in SFC (84). The steady pitch of the acoustic

flame detector (AFD) increased proportionally to the carbon content of the molecule. The detector sensitivity was uniform for all analytes and did not change when using either pure or methanol modified CO2 as a mobile phase. Density gradients caused the baseline to shift due to the changing flow rate encountered when using a passive restrictor. ACKNOWLEDGMENT Thanks are extended to Negin Nazem for her generous assistance concerning the preparation of this review. Larry T. Taylor holds B.S. and Ph.D. degrees from Clemson University. He joined the Virginia Tech chemistry faculty in 1965. From 1998 to 2004 he served as Chair of the Department of Chemistry. He currently holds the title of Emeritus Professor of Chemistry. He has authored over 350 peer reviewed publications. He currently sits on the editorial board of the Journal of Chromatographic Science, Journal of Supercritical Fluids, Journal of Agricultural and Food Chemistry, and Chromatographia. He currently coteaches short courses on packed column supercritical fluid chromatography with applications directed toward the pharmaceutical industry.

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