Anal. Chem. 2004, 76, 4606-4613
Reviews
Supercritical Fluid and Unified Chromatography T. L. Chester*
The Procter & Gamble Company, Miami Valley Laboratories, 11810 East Miami River Road, Cincinnati, Ohio 45252 J. D. Pinkston
Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, Ohio 45040 Review Contents Theory and Fundamental Measurements Mobile Phases and (Achiral) Stationary Phases Instrumentation, Techniques, and Performance Preparative Separations Detection Applications Food-Related Applications Natural Products Fossil Fuels Synthetic Oligomers, Polymers, and Polymer Additives Achiral Pharmaceuticals and Bioactive Compounds Chiral Applications Literature Cited
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Supercritical fluid chromatography (SFC) and related unified chromatography techniques using compressible, solvating fluids and conventional liquids at high or ultrahigh temperatures continue to grow in use. However, continuing the trend in recent years, there has been a further decrease in fundamental work and in the number of published research articles. Despite this, interest among end users continues to grow, particularly in pharmaceutical applications. Pharmaceutical researchers also continue to lead in publishing applications, perhaps reflecting the higher importance placed on publication in this business than in most others. During this review period, one analytical instrument supplier (for whom SFC was a minor product area) left the business, but another supplier, one fully dedicated to this field, enthusiastically joined. Growth also continues in the preparative SFC area and in materials processing using supercritical and related fluids, and this is expected to continue stimulating analytical applications. As in the past, we have made a very loose interpretation of the term “supercritical” for the purposes of selecting articles to cite (and in keeping with the underlying unified chromatography theme) and have included any solvating fluid that is used in either condensed or supercritical form at a temperature above its normal vaporization temperature or above the usual temperature range of conventional HPLC. We resume from our last report (1) and have examined the literature abstracted in the ISI Current Contents Connect (http://isicc.com/CCC.cgi) database through November, 2003. We found about 200 articles from which we have cited a fraction, representative of recent progress. 4606 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
THEORY AND FUNDAMENTAL MEASUREMENTS Several general reviews appeared that may be useful to people new to the field (2-4). Dominguez et al. presented a revision of the 1993 IUPAC Nomenclature for Chromatography that addresses hold-up volume and the physical meaning of several terms (5). The measurement and meaning of hold-up times and volumes as well as derived parameters, such as solute retention factor in an SFC instrument or other chromatographic system in which the mobile phase is compressible, solvent strength changes with mobile-phase density, and pressure and temperature change longitudinally along the column, will require some additional attention. Amines have been successfully determined by SFC techniques for years, but there has been uncertainty if or when amines react with CO2, if the peaks actually migrating through a column under SFC conditions are the original amines or something else and if this in any way limits the applicability of SFC. Mass spectrometric detection has been inconclusive because the amine adducts could revert back to the original amines when the pressure is reduced from the on-column value when the peak reaches the MS source, and the mass spectrum of the original amines may result, regardless of the form of the solutes during transport through the chromatograph. Fischer et al. (6). investigated this question using on-line NMR to directly look for the unstable reaction products of amines and CO2 under supercritical conditions. They found that the formation of reaction products depends on steric properties, among other parameters. These authors work in what we will call a customer organization, in this case, a pharmaceutical company. We acknowledge and thank these authors and others for researching and freely sharing such fundamentally important work. However, the appearance of such profound work from a customer organization, combined with the general absence of fundamental work in the academic community, causes concern. We feel that both the academics and the funding organizations are missing some needed areas of research. Most customer organizations are unwilling to fund such fundamental work except through the taxes they pay, so most opportunities for fundamental work and further advances will not occur unless some priority changes are made in the academic community and the funding organizations. SFC, and chromatography in general, are mature techniques, but they are far from perfect. We can easily envision improvements 10.1021/ac040088p CCC: $27.50
© 2004 American Chemical Society Published on Web 06/18/2004
of 2× or more in performance criteria for analytical and preparative chromatography by further utilizing fluid properties and by truly understanding the processes. Fundamental work is the platform for rational improvement. When this work is done only by customers and by commercial suppliers, the results too often are kept secret. Effort is needlessly and redundantly repeated by others needing the same knowledge. Single-source products emerge that wreak havoc on customers whenever suppliers make changes or decide to abandon an insufficiently profitable product that happens to be essential to a particular customer. For example, DeltaBond columns, a longtime favorite of many SFC users, were recently discontinued, thus forcing customers to now develop and validate new methods. This criticism is not meant to suggest that we cannot develop and keep trade secrets, but we do suggest that fundamental work in chromatography is not being funded, published, and shared at a sufficient level given the enormous economic impact of these techniques to customer organizations. Zhu et al. used a molecular dynamics method to predict diffusion coefficients in various fluids (7). Gonzalez et al. measured the diffusion coefficients of several new solutes in CO2 using the Taylor-Aris dispersion method (8). Mishima et al. measured the solubilities of undecanolide and pentadecanolactone in CO2 by an SFC method at two temperatures and pressures between 12.2 and 25.3 MPa (9). Tuma et al. measured the solubilities of anthraquinone dyes using an SFC method (10). Roth et al. determined the retention factors of 11 n-alkanes ranging from C21 to C40 plus C60 and C70 fullerenes (11). From this, they derived partial molar volumes, transfer enthalpies, and measures of the short-range interactions between the solutes and CO2. Roth later determined Krichevskii parameters of several n-alkanes in CO2, finding that this parameter varied linearly with carbon number (12). Roth also determined second virial coefficients for a system of CO2 and various n-alkanes (13). The development of economically optimized separation processes requires sufficient knowledge of the effect of various parameters. Jiang et al. determined the effects of pressure, temperature, and modifier (ethanol) concentration on retention and resolution of tocopherols under SFC conditions (14). They found that resolution increased with increasing temperature and with decreasing pressure and that solute retention could be predicted using a five-parameter model derived from unified molecular theory. Poole and Poole described the use of a solvation parameter model in predicting retention and selectivity in various chromatographic systems including SFC (15). Lesellier et al. studied the effects of modifier choice and concentration, temperature, and outlet pressure under SubFC conditions on the retention of alkylbenzene homologues (16). They found that retention was most strongly influenced by modifier concentration and temperature and that changes in phase ratio due to adsorption and desorption of mobile phase components influenced retention of the shorter homologues. This allowed the authors to derive a model that is helpful in predicting retention and resolution. In another report, these authors investigated methylene selectivity of alkylbenzenes as a function of alkyl carbon number using CO2-acetonitrile and CO2-methanol mobile phases (17). This work revealed that short and long homologues
partition differently when acetonitrile is used, but that all the homologues partition similarly when methanol is used. They attributed these differences to the adsorption behavior differences of methanol and acetonitrile used as modifiers. Thompson and Carr studied the ability of several pharmaceutical compounds to survive high-temperature analysis within the time frame of high-temperature (or ultrahigh-temperature) liquid chromatography (18). They showed examples of success at temperature above 100 °C and proposed criteria for rejecting questionable or thermally sensitive solutes. Oswald et al. (19) and Krupcik et al. (20) studied the interconversion of enantiomers under or including SFC conditions. They derived interconversion energy barriers and rate constants. MOBILE PHASES AND (ACHIRAL) STATIONARY PHASES There may be some confusion in the mobile-phase nomenclature encountered in the literature. Strictly speaking, a mobile phase must be used above both its critical temperature (Tc) and critical pressure (Pc) to be supercritical. If either parameter exceeds its critical value, then there is no phase change in the fluid when the other parameter is changed through its critical value. Instead, the distinction between liquid and gas ceases to exist, leaving quite some latitude in permissible values of temperature and pressure for a particular purpose. This makes the practical definition of a supercritical fluid somewhat arbitrary, and users may often operate below one or the other critical parameter without realizing it. For example, in packed-column SFC using modifier gradients, a chromatogram may be started in the supercritical region of the phase diagram for the initial mobile phase, but the values of the critical parameters of the mobile phase will continuously change (increase usually) as the modifier concentration is increased, and at some point, the conditions may no longer be supercritical if the operating temperature and pressure are held constant as the modifier concentration is increased. Since no discontinuous transition of the mobile phase occurs, this change in the formal definition of the mobile phase that may occur in practice is of no interest or consequence to the user, but instead confirms that useful and unusual chromatographic behavior can be achieved with many mobile phases that are quite different from normal liquids and gases. The key difference in practice is that the column outlet must be controlled at a pressure higher than ambient to maintain the desired fluid properties over the entire length of the column. This behavior gives rise to the multiplicity of terms encountered, including subcritical fluid, near-critical fluid, high-temperature liquid, ultra-high-temperature liquid, superheated liquid, etc. These terms can be used interchangeably if the fluid is normally a liquid. The term enhanced-fluidity liquid implies that a viscosity-lowering fluid, such as a soluble condensed gas, has been added to the mobile phase. Solvating gas chromatography may utilize a supercritical fluid, a condensed gas, or a superheated liquid. The pressure is not actively controlled at the column outlet; rather, the tubing downstream from the outlet usually provides enough back pressure against the mobile phase flow to prevent the mobile phase from boiling until the mobile phase reaches, or at least nears, the detector. If a phase transition occurs near the column outlet, it is ignored. Regardless, all these named fluid states occur Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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within the same continuum of fluid behavior and may be performed on the same equipment, thus suggesting the term unified chromatography as the most appropriate for much practical work. Unified chromatography, in this context, implies that temperature, pressure, and composition of the mobile phase are adjustable and are set to specific values to achieve benefits not possible at ambient temperature or pressure. Such mobile phases continuously bridge all fluid behavior and encompass all aspects within the continuum between ordinary gas and liquid chromatography. CO2, with the addition of organic modifiers, is the most frequently used mobile phase with these techniques. A recent review by Wells et al. provides good perspective for people new to the area (21). There has been a great deal of interest in subcritical (or superheated) water recently. Greibrokk and Andersen reviewed the field (22). Fields et al. replaced acetonitrile-water mixtures with superheated water up to 200 °C (23). They investigated the effects of such factors as flow rate and mobile phase preheating in their successful separation of testosterone and other compounds using zirconia- and silica-substituted stationary phases. Yang et al. split the outlet flow of their subcritical water chromatograph to a flame-ionization detector (24). They separated and detected carbohydrates, acids, and amino acids using several different columns. Nakajima et al. used similar capability to separate low-molecular-weight alcohols and to determine ethanol in wine (25). Wu et al. used water above its normal boiling point as mobile phase under conditions they described as solvating gas chromatography (26). They used a capillary column packed with 3-µm polybutadiene-coated zirconia and coupled the column outlet to a flame-ionization detector. They found that at temperatures above 100 °C, the head pressure required to produce a given flow rate dropped rapidly, permitting the use of long columns. Since our last review, subcritical water chromatography combined with flame-ionization detection has been made commercially available. Yang et al. and Kondo and Yang studied temperature effects on peak width and column efficiency in subcritical water chromatography (27, 28). Kondo et al. modified subcritical water with dimethyl sulfoxide and separated mixtures of polyhydroxybenzenes, phenols, BTEX components, and PAHs (29). Tajuddin and Smith directly coupled superheated water extraction with superheated water chromatography and applied this to the determination of several pharmaceuticals and antioxidants from a model matrix (30). They used a polystyrene-divinylbenzene stationary phase and a thermal gradient. Lamm and Yang combined subcritical water extraction with off-line subcritical water chromatography (31). They studied temperature effects and used the technique in several applications, including determining flavones in orange peel. Yarita et al. investigated the stability of polystyrene-divinylbenzene packings and showed they are stable to 150 °C (32). Retention decreased with increasing temperature. He and Yang investigated the long-term stability of several silica-, polystyrenedivinylbenzene-, and zirconia-polystyrene packings using subcritical water up to 150 °C (33). They found the polystyrenedivinylbenzene packing to be the most stable under their test conditions. 4608
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The wide range of mobile-phase options available to researchers utilizing unified chromatography techniques also allows new detector capabilities. For example, Saha et al. used superheated deuterium oxide without an organic modifier as mobile phase in temperature-gradient HPLC of ginger extract with on-line NMR detection (34). They programmed the temperature from 50 to 130 °C in 20 min. Modifiers can still be used along with superheated water, if desired. Jones and Yang used methanol-water mixtures up to 140 °C to separate PAHs, PCBs, and petroleum components (35). Thurbide and Cooke compared supercritical argon and CO2 as mobile phases in packed-column SFC of sulfur-containing analytes in which they used flame photometric detection for sulfur (36). The emission spectrum was overwhelmed with signals from carbon-containing molecular species when CO2 was used, but there was no mobile-phase emission when Ar was used. There have been several reports in recent years of the deleterious effects of using helium headspace to raise the pressure in CO2 cylinders to improve the delivery efficiency of liquid CO2 through siphon tubes in the cylinders. Parcher and Xiong carefully examined the solubility isotherms of He in CO2, the effect of He on the density of CO2, and the phase behavior of He-CO2 mixtures (37). This work was prompted by a need to understand the behavior of He-CO2 mixtures to explain the previous observation by this group of the retention of solutes in empty tubes flowing with He-pressurized CO2. By controlling conditions, Wells et al. (38) and Luo et al. (39) showed that a liquid-vapor phase separation can be induced in He-CO2 mixtures, resulting in the dynamic coating of a tube with a CO2-rich liquid. Gas-liquid chromatography can then be performed by partitioning solutes between these phases. These researchers examined the phase behavior of CO2-methanol mixtures to determine the temperature-pressure composition regions where a dynamic two-phase system can be formed. Coym and Dorsey investigated shape selectivity of two monomeric C-18 stationary phases using CO2-acetonitrile mobile phases (40). Shape selectivity changes with temperature paralleled that expected in reversed-phase HPLC, but the influence of acetonitrile concentration was different in SFC from that in RPHPLC, and mobile-phase density in SFC was also shown to have some influence in shape selectivity. Although several new stationary phases designed specifically to work well with CO2-based mobile phases have been commercialized in the last several years, only a few reports regarding stationary phases were published during this review period. Hanai reviewed the use of carbon columns for the separation of polar solutes and cited examples using superheated water and supercritical CO2 mobile phases (41). Planeta et al. described an optimized process for slurry-packing capillary columns using CO2 as the packing fluid, in which they could prevent the sudden, rapid, initial transport of packing at the beginning of the process (42). They evaluated their columns using LC conditions and achieved a value of 2.2 for the average reduced plate height. Lesellier et al. showed that the high porosity and low pressure drop of monolithic columns allows them to be coupled in subcritical fluid chromatography, and that, unlike with conventional columns, the efficiency of the columns is additive (43). They coupled up to six
monolithic columns to one conventional column to achieve the separation of β-carotene isomers. INSTRUMENTATION, TECHNIQUES, AND PERFORMANCE For many years, the techniques of this review have promised new capabilities, particularly speed and new selectivity, for analysts. However, to be practical alternatives in the workplace, these capabilities must also provide adequate accuracy, precision, and ruggedness. Even though packed-column SFC uses the same injectors and detectors as HPLC, many users find that peak-area precision is worse in SFC than in HPLC. Coym and Chester found that evaporation of mobile phase from the injection loop, leaving the loop filled with gas instead of liquid, as in HPLC, is a major source of loading problems and imprecision (44). Adding steps to the loading sequence to ensure that the gas is purged from the loop prior to loading the sample solution solved this problem and resulted in precision matching that of HPLC. Another promise is speed, as predicted by the much faster diffusion rates in supercritical, superheated, low-viscosity, etc. fluids, as compared to conventional liquids. Bronstrup noted that mass spectrometry coupled with fast, serial HPLC or SFC sample delivery can exceed speeds of 1 sample/min (45). Hoke et al. combined a short, packed-column SFC separation taking only ∼5 s, yet providing a retention factor of 1.5, with MS/MS detection for the analysis of dextromethorphan in plasma (46). Injections could be made approximately every 6 s, and 96 analyses could be completed in ∼10 min. Medvedovici et al. reported an on-line solidphase extraction/SFC analysis of carbaryl capable of detecting low-parts-per-billion concentrations in water samples and cycling in about 35 min (47). Low-viscosity mobile phases also provide the opportunity to use longer columns than in conventional HPLC or to couple columns. Hirata et al. studied the coupling of capillary columns with dissimilar stationary phases, with and without a coupling restrictor (48). They were able to tune the influence of the second column to the first and tune the overall selectivity according to the resistance of the coupling restrictor. Hirata et al. reported a comprehensive, two-dimensional packed-column instrument in which the first column was operated at subcritical conditions in stopped-flow mode and the second column was operated continuously with supercritical conditions (49). The authors applied this to the separation of triglycerides in fats and oils. Simulated moving bed separations utilizing supercritical fluids continue to move forward. Di Giovanni et al. improved performance by tuning the elution strength of the mobile phase and changing the pressure in the four sections of the apparatus (50). Johannsen et al. developed an analytical-scale packed-column SFC separation of binaphthol enantiomers and transferred this to a simulated moving-bed process that they eventually increased to operate at 54 g (feed) per liter of stationary phase per hour (51). PREPARATIVE SEPARATIONS The needs of people interested in preparative separations may influence the further development and application of analyticalscale SFC, particularly since analytical separations are often used to model and develop preparative separations. Interest seems to be focusing on pharmaceuticals, natural products, and polymers.
The greatest interest in preparative separations involving supercritical and related fluids is in the pharmaceutical area. Several general articles give perspective and address capabilities (52-56). Understandably, few specific industrial applications have been published. Cox et al. separated flurbiprofen enantiomers by preparative SFC (57). Toribio et al. semipreparatively separated albendazole sulfoxide enantiomers (58). Shimada et al. used preparative SFC to isolate fractions of individual oligomers of poly(ethylene glycol) with degrees of polymerization ranging from 6 to 40 (59). They went on to use these purified PEGs to study signal generation in MALDI-TOF mass spectrometry and found molecular-mass-dependent differences in sensitivity. Taylor and King used preparative supercritical fluid extraction combined with preparative SFC to obtain extract fractions enriched with free sterols and ferulate-phytosterol esters (60). DETECTION Publications describing advances in detection in SFC have decreased in number compared to previous review periods. This is consistent with the move of SFC from academic and development labs into industrial applications. The majority of publications describing detection revolve around “informative” spectroscopic and spectrometric detectors, which provide structural information about eluted analytes. Evaporative light scattering detection (ELSD) has the potential to work better in SFC-like techniques than in HPLC as a result of the ease of evaporating supercritical, etc., mobile phases. Deschamps et al. separated ceramides under subcritical conditions on an ODS column and reported that ELSD response could be improved by adding equimolar amounts of triethylamine and formic acid to the mobile phase (61). Lesellier et al. used CO2 with methanol modifier to separate skin lipids from a silica column at subcritical temperature (62). Since this column was not compatible with triethylamine and formic acid additives in the mobile phase, they added these agents postcolumn in various solvents to improve ELSD response. Lafosse and Herbreteau reported analyzing carbohydrates by LC- and SFC-ELSD (63). Cheng and Hochlowski reported on analytical SFC-MS in a recent review (64). Recent research reports include work from Garzotti et al., who interfaced an SFC with a hybrid (Q-TOF 2) mass spectrometer (65). They noted that this arrangement provides on-line accurate mass determinations and that the fast acquisition rate of the mass spectrometer is compatible with highspeed MS-directed fraction collection. Bolanos et al. compared quadrupole and TOF mass spectrometers and, not surprisingly, noted improvements in the narrowing of peaks and in resolution when using the faster (TOF) instrument, which was capable of a spectral acquisition time of 0.1 s (66). In some cases, it is advantageous to do off-line analyses of SFC peaks. Planeta et al. reported a device for collecting SFC peaks for analysis by MALDI-TOF and applied it to the characterization of silicone oils (67). Berger noted that when the outlet of a packed-column SFC is split to a mass spectrometer, the split ratio is usually not constant if the pressure is programmed, resulting in variable response factors for mass-sensitive detectors, such as the FID (68). He noted that with conditions of constant mass flow, a simple Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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correction can be made to make quantitative measurements possible. SFC techniques have been used successfully for the elemental analysis of solutes. The specific subject of SFC linked with inductively coupled plasma mass spectrometry was recently reviewed by de Leon et al. (69). Bertoncini et al. split the effluent of an SFC between a microwave-induced plasma detector and other detectors (70). They applied this to the analysis of lubricant additives for N, P, and Zn compounds. APPLICATIONS We have been very selective in the applications included here. We’ve chosen to include only applications that are new or novel or otherwise merit being mentioned. Food-Related Applications. Food-related applications of SFC were included in reviews by Andrikopoulos (71, 72), by Brondz (73), by Ibanez and Cifuentes (74), and by Ahmed (75). The last work in this list was concerned with the analysis of pesticides and their metabolites in foodstuffs, whereas the first in the list were primarily concerned with triglyceride speciation. Fatty acids and triacylglycerols were also the subject of investigations described by Senorans and Ibanez (76), Rezanka and Votruba (77), and Soheili et al. (78). Senorans and Ibanez discussed the use of open-tubular and packed-column SFC for free fatty acids and fatty acid methyl esters and described the best packed-column stationary phases for the elution of free fatty acids with unmodified CO2 (76). The changes upon heating of low-linolenic soybean oil vs partially hydrogenated soybean oil were investigated by Soheili et al. (78). SFC analyses revealed the partially hydrogenated oil to be more stable than the genetically modified low-linolenic oil at a statistically significant level. Two publications described the use of SFC to monitor the products of enzymatic reactions in supercritical CO2 (79, 80). King et al. compared various enzymes for the formation of sterol esters (80). Under the conditions explored, the most efficient enzyme, Chirazyme L-1, yielded over 90% reaction of cholesterol and sitostanol with C8 through C18 fatty acids. In contrast, Rezaei and Temelli studied the hydrolysis of canola oil triglycerides by an immobilized lipase from Mucor miehei (79). They concluded that on-line extraction/reaction was a promising process for producing high-value products from oil seeds. In other publications related to foods, Kamangerpour et al. used SFC/UV to characterize polyphenolic compounds in an SFE extract of grape seed (81). They used methanol containing 1% citric acid as a mobile-phase modifier and two coupled diol columns for the separation. Aro et al. used combined, on-line SFE/ SFC/GC/MS to study semivolatile compounds in fresh, stored, raw, and baked Baltic herring (82). The SFE extract was fractionated by on-line SFC, and the volatile fraction was directed to the GC/MS. Not surprisingly, the fraction of volatiles increased, as did the proportion of short chain acids during storage at 6 °C for up to 9 days. Natural Products. Merfort (83) and Bos et al. (84) included SFC in reviews of techniques for sesquiterpene and valepotriate analysis, respectively. Kaplan et al. used SFC to show that SFE is a promising method for the extraction of feverfew (Tanacetum parthenium) (85). The “tunability”, or selectivity, of SFE extraction at different pressures was highlighted. Polyprenols are complex 4610
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polymers synthesized by rubber-producing plants. Bamba et al. note that the chromatographic resolution of polyprenols with chainlength and geometric-isomer variations is markedly improved with SFC over conventional HPLC separations (86, 87). Individual homologues containing from 10 to 100 oligomeric units were separated, and geometric isomers containing from 13 to 20 units were isolated by SFC fractionation (86). The authors report that, for the first time, all-trans polyprenols with degree of polymerization >10 were shown to exist in nature. Tetrahydrofuran was used as mobile-phase modifier (87). The SFC distributions agreed with matrix-isolation laser desorption/ionization (MALDI) MS and proton NMR results. Fossil Fuels. Albuquerque used SFC with UV detection at 240 nm to determine conjugated dienes in petroleum products (88). The method used two coupled silica columns and pure CO2 mobile phase, as in the group-type analysis of diesel fuels by SFC (ASTM standard method D5186). Conjugated dienes are the only substances eluting in the nonaromatic range that absorb appreciable UV radiation at 240 nm. This method could be coupled to the standard method with the appropriate combination of detectors. Synthetic Oligomers, Polymers, and Polymer Additives. Ethoxylated and propoxylated oligomers are widely used industrial surfactants and lubricants. Berger and Todd demonstrated the versatility of pure CO2 mobile phase for separations of these mixtures using both flame ionization and low-wavelength (191 nm) ultraviolet absorbance detection (89). Ten consecutive separations of trimethylsilyl-derivatized oligomers provided relative standard deviations of 0.2-0.3% for retention times and 0.85-1.3% for peak areas. The UV and FID detectors yielded equivalent results. Separations of ethoxylated alkylphenols by packed-column SFC and HPLC were compared by Hoffman and Taylor (90). Coupled diol and cyano columns produced the best results by SFC. The authors observed that SFC provides resolution similar to HPLC, but with a shorter analysis time. Pinkston et al. used 1-m-long packed columns for high-resolution SFC/MS separations of alkoxylated oligomers (91). The time/intensity/mass-to-charge ratio data were converted to images and analyzed using customized image-analysis software. SFC has long been used for the characterization of polysiloxanes. In the work of Berger and Todd, the molecular weight range of SFC was extended beyond the reach of pure CO2 (estimated at 25 000 for pure CO2) by the addition of hexane as a mobilephase modifier (92). Detection was by ultraviolet absorbance at 191 nm. The power of SFC for both analytical and preparative separations was illustrated in a number of publications that shared a common goal of characterizing polymeric mixtures. Matsunaga et al. used SFC to fractionate polypropylene glycol acrylates, followed by MALDI-MS, GC, and analytical-scale SFC on the resulting fractions (93). These tools provided complementary data that yielded a comprehensive view of the various oligomeric distributions of the polymer. X-ray crystallography was used to determine the structure of poly(vinylidene fluoride) in an impressive paper by Tashiro and Hanesaka (94). SFC fractionation was used to obtain oligomers of uniform molecular weight for the X-ray analysis. Kawai et al. used CO2 modified with a mixture of tetrahydrofuran, chloroform, and ethanol on a silica column to separate styrene-methyl methacrylate copolymers (95). The effects
of modifier composition and column temperature were studied. Open-tubular SFC was one of many tools used to characterize polyisobutylene functionalized with isothiocyanate groups by Buchmann et al. (96). In addition, size-exclusion chromatography, HPLC, and GC/MS were used in this work. Ude et al. (97) and Johnston et al. (98) used the reasonable combination of SFE followed by SFC to extract and analyze plasticizers and low-molecular-weight, reactive components from polymers. Ude and co-workers worked with plasticizers from poly(vinyl butyral) (97). The plasticizers eluted and were separated using either open-tubular or packed-column SFC, but the packedcolumn separations were more rapid, whereas the open-tubular approach provided a higher resolution separation. The low reactivity of pure CO2 was exploited by Johnston and co-workers as they used on-line SFE-SFC to extract and analyze reactive peroxides and hydroquinones from an ethyl and methyl methacrylate copolymer blend (98). The extracted species were concentrated on a C18 solid-phase trap before analysis. Achiral Pharmaceuticals and Bioactive Compounds. The use of SFC has grown rapidly in the pharmaceutical industry during the period covered by this review. This is the point of Harris’s review (99). The greatest area of impact has been in chiral separations (see below), but many achiral applications have also been published. Xu et al. found that bromosulfone (2-bromo-4′(methylsulfonyl) acetophenone) underwent on-column degradation during reversed-phase HPLC separation (100). Not only was the compound stable under SFC conditions; the required separation was also faster. Seven process-related impurities were separated from bromosulfone in 5 min (100). Gyllenhaal and Hulthe reported the direct injection of aqueous sample solutions of isosorbide-5-mononitrate by pcSFC (101). The CO2 mobile phase was modified with 20% 2-propanol, and the stationary phase was a diol-functionalized silica. Imdur tablets were dissolved in gastric media and directly injected. Tablet dissolution and drug release could be monitored by recirculating the dissolution solution through the injection loop. Aqueous injections are unusual in SFC. In this application, the stationary phase was sufficiently polar to allow such injections without significant breakthrough. Ferrieri published a two-part series of papers describing the current state and future potential of supercritical fluids for medical radioisotope processing and chemistry (102, 103). Few descriptions of research in this area have been published, but work in the author’s laboratory shows the potential of SFC, SFE, and related techniques for medical radioisotopes. Dost and Davidson used pcSFC with atmospheric-pressure chemical ionization (APCI) detection for the determination of artemisinin, an anti-malarial agent (104). The relative standard deviation for the retention time (3.54 min) was 0.5%, and the detection limit was 370 pg. In the area of bioactive compounds, Patel and Agrawal investigated the use of pcSFC/UV for the determination of the potential carcinogen benzidine and its acetylated metabolite (105). The limit of quantitation for benzidine was 100 pg/mL for a 1-mL plasma sample. The authors compared the method to the published HPLC/UV method and found that the SFC method was superior with regard to speed, organic solvent usage, sensitivity, specificity, and accuracy. Chiral Applications. Chiral separations continue to be one of the most active applications in SFC. We have therefore
separated these applications into their own section. Ward published a review of the various chiral chromatographic methods and positioned SFC relative to other approaches (106). Two groups used SFC to study and determine the enantiomerization barriers of chiral molecules (107, 108). Trapp et al. (107) included SFC in a discussion of the use of various stopped-flow and dynamic chromatographic methods to determine enantiomerization and isomerization methods. New software tools were described which facilitate on-line determination (107). Oswald et al. (108) evaluated two models, one reversible and the other irreversible, to determine the enantiomerization energy barrier for N-(-p-methoxybenzyl)1,3,2-benzodithiazol-1-oxide using SFC data. The reversible model failed, but the irreversible model was more successful. Juvancz and Szejtli reviewed the many uses of cyclodextrins in chiral chromatographic methods, including SFC (109). Mechanistic options were discussed. In a more applied direction, Cox and Amoss described two new high-performance amylase chiral columns, Chiralpak AS-H and Chiralpak AS-RH (110). Both were designed to be compatible with SFC. Turning toward the mobile phase, Phinney and Sander studied the effects of two basic additives, isopropylamine and triethylamine, on chiral SFC separations using macrocyclic glycopeptide or derivatized polysaccharide columns (111). The additives were added to the methanol modifier. They found that the additives were required for elution of many of the analytes from the glycopeptide column and that increasing the additive concentration resulted primarily in a decrease in retention. The additives had little effect on retention on the polysaccharide phase, but resulted in better peak shapes and resolution. These same authors recognized the importance of monitoring changes in column performance over time and lotto-lot variability in column manufacturing (112). To assess these changes, they proposed a new Standard Reference Material. The Standard consists of solutions of five chiral compounds. The use of these solutions was demonstrated by evaluating the chromatographic behavior of eight commercially available stationary phases in SFC and HPLC mode (112). The power of mass spectrometry as a detector was emphasized by Garzotti and Hamdan (113). They used electrospray (ESI) mass spectrometry to perform high-throughput analyses of complex mixtures containing chiral components. No modification of the interface was required, and the UV and mass spectrometric peak profiles were very similar. Zhao et al. published an excellent description of the use of SFC/MS and chiral SFC to determine, in an automated fashion, the empirically optimized column and conditions for a chiral mixture (114). SFC/MS allows one to monitor chiral synthesis without interferences from chiral or achiral impurities. Enantiomeric excess can also be determined with far lower detection limits than with other common detectors. Analysis times were far shorter with SFC than with normal- or reversed-phase HPLC (114). Chiral SFC was applied to a wide variety of chiral compounds, mostly pharmaceutical agents. Bojarski reviewed the use of various chromatographic methods, including SFC, for the separation of chiral cardiovascular drugs (115). Chen et al. used SFC on a Chiralpak AD column to separate a thiazolbenzenesulfonamide compound (116). They studied the effects of temperature and modifier on the separation and used analogues and precursors to study the impact of functional groups and configuration on the Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
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separation. Chiral SFC was used by Pereillo et al. to separate eight stereoisomers of a human-liver-microsome metabolite of 2-oxoclopidogrel (117). This is, in turn, a metabolite of clopidogrel, a potent antiplatelet drug, which is inactive without these biotransformations. Only one of the stereoisomers was shown to have biological activity, revealing the importance of stereochemistry in this particular pathway. Kasai et al. used chiral SFC to separate thermally unstable stereoisomeric furan derivatives (118). These are often used as intermediates in pharmaceutical synthesis. Stable furan derivatives were studied by GC/MS. Del Nozal et al. investigated the effects of various mobile-phase modifiers on the chiral SFC of triadimefon and triadimenol enantiomers and diasteriomers on a Chiralpak AD column. Methanol and ethanol provided better results than 2-propanol (119). A mixture containing six stereoisomers required only 15 min to resolve. The same group compared Chiralpak AD and Chiralcel OD columns for the enantiomeric separation of albendazole (120). Methanol, ethanol, 2-propanol, and acetonitrile were compared as modifiers. 2-Propanol worked best for the Chiralpak AD column, whereas methanol was best for the Chiralcel OD. Bernal et al. provided a nice comparison of chiral SFC and chiral HPLC for a group of antifungal agents and precursors (121). SFC typically provided higher resolution more rapid analysis, or both. One enantiomeric pair could not be resolved by SFC, but HPLC provided baseline resolution. The speed of chiral SFC separations relative to conventional normal or reversed-phase chiral HPLC was emphasized by Lynam and Stringham (122). They used a Chiralpak AD (R)-H column for amphetamine and methamphetamine enantiomers. Gyllenhaal and Karlsson described an interesting investigation of the use of (L)-(+)-tartaric acid as a chiral mobile phase additive for the separation of several β-adrenoreceptor-blocking agents on a Hypercarb stationary phase (123). The mobile phase also contained CO2 and methanol, to which an aliphatic amine additive was added. Increased concentrations of the tartaric acid additive yielded higher retention and increased enantioselectivity. The authors demonstrated the utility of the method by accomplishing an enantiomeric separation of a batch of N-methylated (S)metoprolol in
