Supercritical fluid chromatography (SFC) has been practiced for approximately 50 years. Early studies used packed columns with varying degrees of success (1) and it would be fair to say that SFC did not generate much interest in the separation science community in this period. In the early 1980s interest in SFC exploded when it was reported that SFC could be easily accomplished with wall-coated open tubular (WCOT) columns similar to the columns currently used for gas chromatography (GC).

The columns differed in two respects: SFC columns had smaller inner diameters and the polymeric stationary phase coating the walls was more extensively cross-linked (2). The emergence of SFC with a mobile phase that exhibited appreciable solvating power relative to helium and hydrogen was primarily promoted by users of GC who saw SFC as a way to increase the limited sample base afforded by GC. This technology, which used pure carbon dioxide, flourished for some time but it had several disadvantages for wide-scale acceptance by the analytical community. Specifically, the technique was traditionally limited to relatively nonpolar compounds because 100% carbon dioxide as the mobile phase exhibited poor solvating power similar to hexane; robust sample injection was not feasible; a wide polarity range of stationary phases was not available; high-quality separations required nearly an hour; gradient carbon dioxide pressure and flow rate were coupled such that flow rate changed as mobile phase pressure changed; passive, fragile restrictors at the column outlet often plugged and the separation had to be aborted; and multigram scale-up of a successful separation was not feasible (3).

Polar Analytes Favor Packed Columns

By 1997 it was clear that the future of SFC would focus more on the separation of moderately polar analytes with polar-bonded silica-based stationary phases, modified carbon dioxide and spectroscopic detectors. In other words, practical SFC, which affords a bridge between high performance liquid chromatography (HPLC) and GC would be more HPLC-like than GC-like (5). Many of these newer developments were inspired by the work of Dr. Terry Berger who spent more than a decade systematically undoing many of the misconceptions concerning packed column SFC (pcSFC) that existed in the 1980s (6). Some examples of the Berger findings include very long columns with large pressure drops were feasible for SFC; mobile phase carbon dioxide density and solvent strength were disconnected; the effect of mobile phase additives (that is, secondary modifiers) on peak shape and retention were introduced; packed column SFC was shown to be broadly applicable to small drug-like molecules; and quantitative SFC recovery of solutes without conventional cyclone trapping and aerosol generation were demonstrated.

Solubility Versus Activity

Even with these instrumental improvements, wide acceptance of the technology was not forthcoming because the perception was that highly polar analytes were not soluble in a carbon dioxide–based mobile phase and were, therefore, not separable (7). Historically, carbon dioxide has been treated as a nonpolar solvent, primarily because of its low dielectric constant and zero dipole moment. Carbon dioxide has also been described as a quadrupolar solvent because of its significant quadrupole moment.

As far as its microscopic solvent behaviour is concerned, carbon dioxide has the potential to act as both a weak Lewis acid and Lewis base. Strong theoretical and experimental evidence has indicated that carbon dioxide can participate in conventional or nonconventional hydrogen bonding interactions (8). Nevertheless, researchers initially sought to extend the applicability of SFC by either using more polar pure fluids, such as ammonia, sulfur dioxide, nitrous oxide and Freon-23 or by adding a polar organic solvent that is, "modifier") to carbon dioxide to improve the solvating power of the mobile phase (9). Both these experimental routes, however, did not generally lead to successful separations.

Figure 1

In the 1990s virtually all peak distortions or nonelution events in pcSFC were attributed either to irreversible adsorption of the very polar analyte to the silica support or (unlike HPLC) to poor solubility of the compound in the modified mobile phase. In pcSFC, endcapping, deactivation and polymer-coated supports, however, enjoyed only limited success (10). The use of additives in conjunction with modifiers — as will be shown later — proved to be as highly successful for SFC as it was for HPLC. Although WCOT columns exhibit much less activity, the elution of even weakly polar analytes was considerably more problematic via this SFC mode because delivery of modified fluids was not experimentally feasible (11). Needless to say, separation of both ionizable and fully ionic analytes with a carbon dioxide–based mobile phase during the early 1990s was thought to be highly unlikely. In other words, SFC provided (at the time) a number of advantages over traditional HPLC, such as speed, practical use of longer columns, a normal-phase retention mechanism and reduced use of organic solvents, but the nature of SFC mobile and stationary phases was thought to not allow the elution of highly polar analytes — much less ionic compounds.

Suffice to say, this situation has drastically changed because experimental approaches using additives have been quite successful. Effective additives are generally too polar to be miscible with carbon dioxide. Instead they are added to the primary modifier and then the modifier plus additive is pumped as a single ternary fluid thereby augmenting the solvating power and flow of pressurized carbon dioxide. In general, ionizable analytes cannot be eluted — or else they are eluted with severely distorted peaks without an appropriate additive in the mobile phase. Today, it is apparent that the use of additives dramatically extends the range of solute polarity amenable to carbon dioxide–based SFC. This review focuses on publications that describe the application of packed column SFC to both ionizable and fully ionic compounds.

Additives Enhance Polar Separations

Berger has suggested that additives perform at least four different functions (12): cover active sites on the solid support; change the polarity of the stationary phase; suppress ionization of the analyte; enhance ion pair formation with the analyte; and raise the polarity and solvating power of the mobile phase. The most obvious role for additives is to change the mobile phase polarity and solvent strength. In this regard, solvatochromic dyes have been used to measure the solvent strength of tertiary mobile phases (13). Small concentrations of additive were sometimes (but not always) found to increase the apparent polarity or solvent strength of the mobile phase. Ion suppression is another rather obvious role for additives, especially if the additive is clearly more acidic (or more basic) than the acidic (or basic) solute.

Figure 2

Stationary phase surface coverage by additives is less well defined for conventional columns because the small particle surface is made up of both exposed silica solid support and bonded stationary phase. Surprisingly, carbon dioxide has been shown to strongly adsorb onto column packing. Maximum adsorption of carbon dioxide occurred near the critical temperature and critical pressure (14). Parcher and colleagues have suggested that a thick film of carbon dioxide may act as part of the stationary phase. Modifiers were also shown to strongly adsorb to the stationary phase (15). Because the total amount of adsorbed material increased under these conditions, the adsorption of modifiers was stated to not displace adsorbed carbon dioxide. The quantity of additives that adsorb onto various stationary phases has been measured (16).

Low to moderately polar stationary phases, such as octyl and cyanopropyl, adsorbed only small amounts (0.4–0.6% of a monolayer) of additive. Under the same conditions sulfonic acid and diol columns adsorbed much larger amounts of additive creating surface coverage up to 21% of a monolayer. Finally, certain additives can be very strongly held by the stationary phase thereby changing the stationary phase polarity, vide infra. In this case, additives can cause an increase, a decrease, or no change in solute retention. In these experiments, the sudden introduction of an additive does not result in an immediate change in retention time because the initial process is very similar to a titration with an endpoint.