Supercritical Fluid Chromatography: Current Status and Prognosis
Richard D. Smith, Bob W. Wright, and Clement R. Yonker Chemical Methods and Separations Group Chemical Sciences Department Pacific Northwest Laboratory Richland, WA 99352
In the 26 years since its use was first reported (1), supercritical fluid chromatography (SFC) has undergone curious development. Initial interest in SFC was largely supplanted by the introduction, rapid evolution, and success of high-performance liquid chromatography (HPLC). The resurgence of SFC in the early 1980s was prompted by innovations in instrumentation and new developments in column technology. Probably the most important factors were the application of opentubular fused-silica capillary columns to SFC by Lee and co-workers at Brigham Young University (BYU) in 1981 (2) and the introduction of commercial instrumentation by Hewlett Packard (HP) in 1982 (3). Both the BYU and HP groups have been strong advocates in this recent resurgence. Their efforts resulted in a rapid first wave of development, much of which involved researchers with strong interests in column technology, instrumentation, and general methodology rather than in immediate or specific applications. As a result, instrumentation advanced rapidly, especially for the capillary SFC approach. The instrumental orientation of the 0003-2700/87/A360-1323/$01.50/0 © 1988 American Chemical Society
first wave was crucial; it provided the time necessary for SFC to mature to a state whereby it could begin to address real analytical problems. In the early 1980s SFC would have failed in almost any comparison with established methods such as gas chromatography (GC) and HPLC. SFC was sustained through this period because the mass transport properties of supercritical fluids compared with those of liquids provided a
REPORT sure basis for obtaining greater speed or greater numbers of theoretical plates than could be achieved with HPLC. In addition, it was widely anticipated that the solvating properties of supercritical fluids would allow more nonvolatile, labile, and higher molecular weight compounds to be determined than is possible with GC. After being unavailable for two years (HP withdrew its packed-column SFC instrument from the market in 1983), commercial SFC instrumentation was reintroduced in 1985. The resulting second wave of development has focused on methodologies for specific applications. Instrumentation has continued to improve, and advantages of both capillary and packed-column formats have been recognized. (The growth in packed-column applications was probably spurred partly by the
patent position of Lee Scientific with regard to capillary SFC.) A substantial number of analytical problems that are well suited to SFC have now been identified. To date, the greatest number of practitioners and amount of enthusiasm for SFC have come from within the GC community, which may explain the rapid acceptance of the capillary format. SFC clearly provides an approach to problems that cannot be solved by GC. The much greater skepticism of the HPLC community toward SFC, on the other hand, stems from the more nebulous advantages of SFC relative to separation efficiency. (It is a maxim in chromatography that one should never claim that a method gives a faster or higher resolution separation than is possible with other methods; the number of chromatographic variables is sufficiently large that, separation efficiency aside, any such example could be demonstrated as incorrect.) The role of SFC in chemical analysis now depends on whether or not the crucial third wave of wide routine application evolves. Researchers will have to obtain improved precision and will have to address questions such as those related to restrictor performance. (Although generally ignored by those unfamiliar with SFC, the restrictor provides crucial control of SFC fluid flow by decompression through a small orifice, capillary, or porous medium, and constitutes a potential Achilles' heel for SFC [4].) Perhaps more important,
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the further evolution of SFC still depends on developments that will facilitate broader application—the capability to address previously intractable separation problems or those that can be better handled by SFC than by HPLC. In this REPORT we will briefly describe the current status and limitations of SFC, focusing on fundamental constraints caused by the solvating properties of supercritical fluids and on new developments in this area. The prognosis for development of SFC beyond its current niche between GC and HPLC will also be considered. Supercritical fluid solvent properties
The successful development of SFC methodologies benefits greatly from an understanding of fluid properties and their dependence on pressure and temperature. A supercritical fluid is defined as a substance above its critical temperature. A primary advantage of chromatography using supercritical mobile phases results from the mass transfer characteristics of the solute. The increased diffusion coefficients of supercritical fluids compared with liquids can lead to greater speed in separations or greater resolution in complex mixture analyses. Thus the physical properties of supercritical fluids place the optimum efficiencies obtainable with SFC inexorably between GC and HPLC. Another advantage of supercritical fluids compared with gases is that they can solubilize thermally labile and nonvolatile solutes and, upon expansion (decompression) of this solution, introduce the solute into the vapor phase for detection. Although they are sometimes incorrectly thought of as "super solvents," supercritical fluids do not provide any advantages in solvating power over liquids given a similar temperature constraint. (In fact, many unique capabilities of supercritical fluids can be attributed to the poor solvent properties obtained at lower fluid densities.) This solubilization advantage is augmented by the ability to readily vary the solvent power of the fluid by controlling density through changes in pressure or temperature. As density is increased from the gas-phase limit, the dense gas will increasingly exhibit the solvating properties of a liquid under conditions where transport properties are still significantly advantageous compared with liquids. The temperature and density of a particular solvent will govern the extent of solute solubility (5). Nonetheless, high solvent powers are obtainable in supercritical fluids but generally require higher temperatures, and sometimes higher pressures, than those currently used for most SFC. The solvent properties that are most
relevant for SFC are the critical temperature, polarity, any specific solutesolvent intermolecular interactions (such as hydrogen bonding, which can enhance solubility and selectivity in a separation), and any secondary equilibrium in supercritical fluids that can be used to alter selectivity. Nonpolar or low-polarity solvents with moderate critical temperatures (e.g., N2O, CO2, ethane, propane, pentane, xenon, SF 6 , and various fréons) have been well explored for SFC. Carbon dioxide is currently the fluid of choice in many SFC applications because of its low critical temperature (31 °C), nontoxic nature, and lack of interference with most detection methods (particularly the flame ionization detector, or FID). Unfortunately, highly polar or high molecular weight solutes generally have limited solubilities in these fluid systems. Polar fluids (such as NH3) exhibit useful properties above their critical points and continue to attract attention, but the complications resulting from their reactivity have thus far lim-
ited their application. To allow comparison with conventional liquid solvents, the pressuredependent solvating powers of supercritical fluids have been studied using spectroscopic methods such as the solvatochromic method, which uses selected probe molecules to determine the polarity or polarizability of the fluid (6). The solvent strengths for several fluids expressed in the Kamlet-Taft w* scale of solvent polarity/polarizability compared with several conventional liquids are shown in Figure 1. (This ir* scale ignores solute-specific interactions such as hydrogen bonding.) These measurements confirm our expectation that fluid polarity or polarizability will increase as fluid density increases. More polar solvents such as ammonia show a large change in solvent strength as density increases, whereas less polar solvents (e.g., SFe, C2H6) show smaller effects. Because single-component polar solvents such as ammonia continue to pose problems for routine SFC, fluid
1.0
Benzene
EtOH
0.5 NH3
CCI4
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C0 2 Hexane N20
Freon-13
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CfiFl4
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Figure 1. The ττ* solvent polarizability/polarity parameter for various supercritical fluids as a function of reduced density (plpc) at a reduced temperature of 1.03. The 7Γ* values of several liquids are given for comparison.
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modifiers (entrainers) have been used to increase the solvent strength of lowpolarity fluids such as CO2. The ability to tailor the nature and degree of the solute-solvent intermolecular interac tions in binary fluids allows separa tions with enhanced solvent properties and altered selectivities. For example, Figure 2 shows the solvatochromic measurements of the solvent strength of the binary solvent system of pure CO2 and CO2 with different concentra tions of 2-propanol modifier as a func tion of pressure (7). These observations are consistent with the anticipated greater solvent strength for increasing modifier concentration and pressure. A rule of thumb in selecting a solvent modifier is to first try a substance that is a good solvent for the analytes. Ex ploitation of specific interactions that can greatly enhance solubility (outside the scope of the IT* parameter) is also useful in selecting SFC solvent modifi ers. The higher concentrations of 2propanol in Figure 2 show similar sol vatochromic shifts resulting from for mation of a two-phase system at 44 °C (8). Clearly, the phase equilibria be havior for mixed-solvent systems must
be understood before one can fully ex ploit their advantages for SFC. Such phase behavior for mixed fluids is not only of concern for column conditions, but also for conditions relevant to fluid preparation, storage, and sample injec tion. Although a vast amount of data exists on the phase behaviors of such systems in the chemical engineering lit erature, this information is rarely con sulted by analytical chemists. Exciting opportunities for the en hancement of selectivity or the solubi lization of highly polar compounds in supercritical fluids include introducing secondary chemical equilibria using or ganized molecular assemblies. The most recent extension of secondary chemical equilibria for SFC involves the formation and use of reverse mi celles, microemulsions, and metal che lates. Reverse micelles and microemul sions (micelles swollen with water) pro vide new solvent environments for su percritical fluids because of the micellar interface and aqueous core regions (9). The solvent properties are readily varied with water content. Reverse-mi celle SFC has been demonstrated for separation of water-soluble dyes using
Isooctane
Oxazine perchlorate
Malachite green
Inject
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Time (min) 0.25 Figure 3. A reverse-micelle separation using a ternary propane/AOT/water mobile phase and a 5-μηη silica microbore column at 103 °C and 375 bar. 0.15
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Pressure (bar) Figure 2. The π* solvent polarizability/polarity parameter for pure C 0 2 and for C 0 2 with different concentrations of 2-propanol modifier as a function of pressure at 44 °C. Ο: 13.2 mole %; · : 10.6 mole %; • : 5.1 mole %; O: 0.0 mole %. 1326 A · ANALYTICAL CHEMISTRY, VOL. 60, NO. 23, DECEMBER 1, 1988
the surfactant molecule sodium bis(2ethylhexyl)sulfosuccinate (AOT) in propane as the supercritical fluid (10, 11). An example of such a separation is shown in Figure 3. Reverse micelles also present intriguing possibilities for separation of proteins and enzymes. Experiments in our laboratory have demonstrated the extraction of pro teins from aqueous solution into re verse-micelle phases in supercritical fluids (12). In such cases, selection of fluid density and water-to-surfactant ratio can provide significant selectivity in separations of biological macromolecules. Metal chelate complexes (ferrocenes, β-diketonates, oxinates, diethyldithiocarbamidates) have also been separat ed (13) using SFC. The use of complexing agents in supercritical fluids pro vides the basis for high selectivities for metals (14). In broader application to supercritical fluid extraction, these systems could lead to enhanced metal recoveries, more rapid separations (in mass-transfer-limited systems), and greater selectivity than are available using liquids. The introduction of oth er secondary chemical equilibria for SFC using (for example) cyclodextrins,
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crown ethers, and ion-pairing reagents is also being investigated in several lab oratories and has potential for specific applications. SFC gradients in pressure (or densi ty), temperature, and mobile-phase composition, or any combination of these, can be used to alter solvent strength and therefore solute reten tion. Pressure gradients are most wide ly used in SFC for manipulating solute retention (15). Rapid pressure pro gramming with short capillaries can re duce analysis time to a few minutes. Temperature programming methods currently have more limited applica tion. Two potentially useful gradient programming techniques for SFC in volve solvent composition program ming and simultaneous pressuresolvent composition programming. The ability to dynamically change the composition of the eluent allows one to tailor specific solute-solvent interac tions to alter selectivity or enhance the resolution of the separation. The enhanced capabilities obtained using modifier gradients and pressuremodifier gradients for SFC, including more rapid separation times and the ability to effectively elute more polar compounds, have recently been dem onstrated (16-18). Binary and ternary gradient methods involving density, temperature, or mobile-phase compo sition combine the advantages of pro gramming techniques available in GC and liquid chromatography (LC) into a single separation technique. The ready manipulation of chromatographic vari ables is often advantageous for separa tions of complex samples or for initial screening of unknowns, and it provides considerable flexibility in optimizing selectivity. Current status of SFC instrumentation Several years ago, an article in a chro matography trade magazine (79) raised the hackles of the fledgling SFC com munity by defining SFC as "a mixture of GC and LC with the advantages of neither." A more reasonable descrip tion is that SFC combines the instru mental complexities of GC and HPLC with some of the advantages of both. Because the physical properties of the mobile phase define the feasible chro matographic efficiencies, and because the solvating powers of supercritical fluids define the ultimate breadth of application, a successful application will depend on the capabilities of avail able instrumentation. Injection. The ideal injection sys tem for either packed or capillary col umns would allow introduction of a narrow and concentrated analyte band, provide no sample discrimination, ex hibit perfect reproducibility, be nonperturbing to the chromatographic sys tem, and tolerate sufficiently large in
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jection volumes so that adequate sensitivity could be obtained for trace analysis. In typical 1-5-mm i.d. packed columns, large sample volumes can be readily injected. Sample introduction poses a greater challenge for capillar ies, particularly for the 25-75-μηι i.d. columns most commonly used. The dif ficulty in capillary SFC injection re sults from the very small sample vol umes (generally ranging from 1 to 30 nL) that must be introduced into the column to avoid serious band-broaden ing and column overloading. This should not be too surprising; the total volume of a 10 m X 50 μπι i.d. column is only 20 μΐ^. Such a small injection vol ume requires rather concentrated sam ples and makes serious demands on de tector sensitivity. The most widely used injection pro cedure in capillary SFC involves sam ple splitting after ambient temperature injection. Split ratios can be as low as 1:2 or as high as 1:100, depending on the sample, column, and specific de sign. Sample splitting is necessary to obtain a narrow injection band on the column and, in some cases, to prevent overloading the column or generation of an excessively wide solvent peak. Re cent studies have shown that good pre cision (
