Strong cation exchange chiral stationary phase—A comparative study in high-performance liquid chromatography and subcritical fluid chromatography

• The influence of modifier content in scCO2 on the in situ formed acid was evaluated.
• Effect of additives on chromatographic performance in SFC/HPLC is described.
• Step-wise modification of subcritical fluid with a modifier was studied.
• Comparison of in situ formed buffer salt and related buffers in SFC mode is discussed.

The performance of a strong cation exchange-type (SCX) chiral stationary phase (CSP) was evaluated with subcritical fluid chromatography (subFC) and high performance liquid chromatography (HPLC). The chromatographic conditions in subFC were optimized by changing the amount of polar organic modifier, concentration of a basic additive in the modifier, system pressure and temperature. In this way the concentration of in situ formed transient ionic species could be varied. The gradual change of the concentration of the transient buffer, i.e. gradient elution conditions in subFC, was found beneficial for separation of a mixture of racemic compounds. The strength and amount of the in situ formed buffer was estimated on the basis of comparative experiments in subSFC and HPLC.