Keeping it Simple(r) in SFC

When I talk to analytical chemists about supercritical fluid chromatography (SFC), they often say that SFC is more complex than reversed phase high performance liquid chromatography (rHPLC) because, unlike the almost universal use of C18 in rHPLC, there are “too many” achiral stationary phases for SFC.

As a side note, there are reasons for the proliferation: historically, polar solutes often tailed badly or did not elute when using traditional polar stationary phases (silica, diol, Cyano, amino), so polar additives were added to the mobile phase. While dramatically extending the range of solutes amenable to SFC, additives also caused problems, particularly with MS detection (1). And so the search was on for new achiral stationary phases that could eliminate the need for additives. Some of these new phases have improved elution but, so far, they have not been successful in completely eliminating the need for additives. The search continues, and that’s why recent years have seen the introduction of (too) many new phases for SFC.

Although there are many stationary phases available, choosing one does not have to be a Herculean task. As Lesellier and West point out in their systematic approach to characterizing stationary phases in SFC (2), it is often far simpler to choose – and stick with – one appropriate stationary phase or at least narrow the possibilities to just a few for known solute structures. It’s clear that this good message has not reached all users, some of whom now approach SFC stationary phase selection as if it were chiral method development. In chiral method development, there remains no means to a priori predict the stationary phase that will give any, let alone the best, separation. Thus, many different chiral stationary phases are compared using mobile phases with and without additives.

A similar approach is being used in SFC for achiral stationary phase selection with a generic composition gradient at fixed temperatures and pressures. This “stationary phase-centric” view of method development largely ignores variations in the interactions of the mobile phase with both the solutes and the stationary phase. I believe this approach wastes time and resources evaluating inappropriate stationary phases, while minimizing the probability of finding conditions that get anywhere near optimum.

In SFC, method development involves much more than stationary phase selection. Like the stationary phase, the mobile phase should have chemical characteristics similar to, or at least compatible with, the solutes. Pure CO2 is inappropriate for solvating any but the least polar solutes. Modifiers can dramatically increase the solvent strength of mixed fluids; Snyder’s solvent triangle (3) is a reasonable guide to modifier selection. Solvent strength in SFC largely follows the P scale, which allows the appropriate modifiers to be predicted – provided you know the structures of the solutes. After choosing an appropriate modifier, the next obvious question is whether or not an additive is needed, and can be answered by a few quick injections on the chosen stationary phase. Most of the time, the need for (or nature of) the additive can be predicted (acid versus base).

There is another difference between SFC and HPLC: temperature tends to have little effect on selectivity in HPLC, whereas in SFC relatively modest temperature changes (+/- 5-10°C) can have significant and unpredictable effects. Even almost identical compounds can respond differently to small shifts in temperature, and such shifts can be used to separate peaks that previously overlapped (change selectivity).

Despite significant over-emphasis in the literature, changes in selectivity are not caused by simple bulk density effects. Indeed, much of the recent literature treats density as a major control variable, but this is deceptive. Most of us think of density as a homogeneous property of the mobile phase; however, pressure and temperature variations have much more impact than simple bulk density. Temperature affects the degree of adsorption of mobile phase components and the solutes on the stationary phase. It also affects the composition of the solvent sheath around solute molecules. These major aspects of SFC are almost completely ignored!

Pressure tends to be a secondary control variable in SFC. Unlike temperature, increasing pressure tends to simply decrease retention to a modest degree. There are seldom peak reversals, indicating only modest selectivity changes. Adsorption and modifier clustering around solute molecules appears to be primarily driven by thermal energy, not pressure.

In my view, using a chiral development system to choose the “best” achiral stationary phases for a particular sample with a “universal” gradient is misguided. A little bit of thought goes a long way in choosing an appropriate stationary phase, the most likely modifier, and the nature of the additive, if needed. After that, the most useful instrumental variable is temperature, followed by pressure. In short, I’m positive that most SFC separations could be optimized on a few similar stationary phases using instrumentally controlled variables.