Supercritical fluid chromatography versus high-performance liquid chromatography – Performance, robustness and selectivity in the separation of vitamin A acetate and related substances

Abstract

This study presents the separation of vitamin A acetate (VAA) and other retinoids, as well as their respective stereoisomeric forms, with the help of supercritical fluid chromatography (SFC). The basis for this was a method of reversed-phase high-performance liquid chromatography (HPLC), presented by Schräder et al. (2024). There, the separation of retinoids and VAA stereoisomers was performed on a Chiralpak IG-3 chiral stationary phase and the retention mechanism was investigated via van ‘t Hoff experiments. The presented study aimed for method transfer to SFC and the comparison to HPLC in terms of robustness and selectivity. During the initial method development, peak deformations were observed and investigated with systematic alterations of the chromatographic conditions to find the limits of a robust separation. It was found that the proper desorption of analytes is only possible above a certain system pressure threshold, meaning a robust method required a minimum elution strength of the mobile phase. Comparison of both methods revealed differences in terms of selectivity. Most notable was a reversal in retention order for the retinoids based on their polarity, but not of the stereoisomers within the retinoid groups. With the help of van ‘t Hoff experiments, it was concluded that the separation mode of SFC was more akin to normal-phase, with the non-polar retinoids eluting before the polar retinoids. At the same time, the plots confirmed that the molecular shape recognition capabilities of the utilised stationary phase also applied to the SFC separation of the respective isomers.