Basics of Supercritical Fluid Chromatography

Welcome Back!

Welcome back for this third session on a brief introduction into supercritical fluid chromatography. Hopefully you are now aware of what a supercritical fluid is and how an SFC instrument needs to be adapted in comparison to an LC to work efficiently. The operating parameters can highly influence the success of the chromatographic separation, therefore, it is important to know which variables need to be investigated. In this section, we will look at the effect of stationary phase type.  

The approach for the SFC analytical condition development process is basically the same as for determining analytical conditions for HPLC. HPLC can generally target any component in compounds that can be dissolved in the mobile phase. In contrast, SFC can be used to analyse any compound that is compatible with supercritical carbon dioxide and can be dissolved in an organic solvent. If chromatography is used for quantitative analysis, then optimal analytical conditions should achieve resolution of 1.5 or more. The basic equation for calculating resolution R_s is indicated below

 

The α, k, and N values that affect R_s are all independent factors that vary depending on the column and mobile phase used, where N represents separation efficiency and α selectivity. In particular, separation can be improved by increasing N and α values. The supercritical carbon dioxide used for SFC offers different properties than mobile phase solvents used for HPLC, therefore when a column used for HPLC is used for SFC, it can sometimes result in different elution behaviour (Fig. 1).

Fig. 1 Separation of three types of pharmaceutical ingredients (upper: HPLC, Lower: SFC)

SFC Stationary Phases

In principle, the various columns used for HPLC can also be used for SFC, but in order to ensure the CO₂ is kept in a supercritical fluid state, make sure the column has the pressure capacity necessary for maintaining the required pressure level. SFC columns are generally compatible with fittings used in HPLC systems, but it is important to confirm the pressure capacity and other requirements for actual use with respective column manufacturers. Columns used in SFC must be able to withstand high pressures at both the inlet and outlet due to the back pressure regulators that maintain SFC conditions and keep CO₂ in a supercritical state. Polymer-based columns require special care, as pressure tends to increase during use when the stationary phase expands. In addition, reducing the amount of elastomeric material in the column helps ensure reliable, leak-free performance.

Since supercritical carbon dioxide has a polarity comparable to n-hexane, the main SFC separation modes are expected to resemble those of normal-phase chromatography. However, unlike HPLC, SFC can utilize both normal phase columns (e.g., silica, diol, CN) and reversed phase columns (e.g., C18, cholesteryl, phenyl). Furthermore, the supercritical carbon dioxide used in SFC exhibits high diffusivity and low molecular density, making it more susceptible to secondary interactions than HPLC. As a result, separation behaviour can vary significantly depending on the stationary phase. Therefore, to determine optimal conditions, it is essential to evaluate a wide range of stationary phase types.

Although supercritical carbon dioxide has low polarity similar to n-hexane, as mentioned above, it can be blended with highly polar solvents. This enables normal-phase columns to be replaced with reversed-phase columns without changing the mobile phase type or modifier composition. Figure 2 shows a comparison of the elution order for HPLC and SFC. In HPLC, mobile phases must be tailored to either reversed phase or normal phase separation modes, requiring the system to be purged each time the mode is switched. In contrast, SFC allows the same mobile phase conditions to be used for both separation modes, meaning results can be assessed simply by changing the column.

Fig. 2 Comparison of elution order for HPLC and SFC

Since normal phase separation is the primary mode used in SFC, normal phase columns such as Shimadzu’s UC-Diol and UC-Diol II are widely adopted as the first choice for SFC systems. The next most commonly used are UC-Py columns, valued for their similarity in separation behaviour to ethylpyridine-based columns. In contrast to HPLC, which requires aqueous or non-aqueous mobile phases with significantly different compositions for reversed phase and normal phase analyses respectively, SFC employs the same mobile phase – a mixture of supercritical fluid carbon dioxide and a modifier (a water-soluble organic solvent such as methanol) - regardless of the stationary phase. This means a single mobile phase composition can be applied across multiple column types, enabling seamless serial analysis. Since supercritical carbon dioxide can penetrate tiny pores more easily, it promotes higher interaction with the stationary phase. That means SFC offers the ability of separating isomers or other compounds that are difficult to be separated by HPLC. In particular, columns with unique or multiple interaction mechanisms can further enhance separation performance in SFC. For SFC, no single column offers the broad applicability comparable to the C18 column in HPLC reversed phase chromatography

This is illustrated in Fig. 3, which shows chromatograms obtained using diol group, hydroxyphenyl group, and cholesteryl group columns on an acidic, basic and neutral compounds (ibuprofen, propranolol and indapamide, respectively).

Fig. 3 Differences in retention behaviour for different stationary phases (normal phase for diol, normal phase + static electric interaction for HyP and hydrophobic interaction for Choles)