Chirality plays a crucial role in the physical and biological properties of molecules, making the separation of stereoisomers an important and challenging research focus. In this context, supercritical fluid chromatography (SFC), using supercritical carbon dioxide as the primary mobile phase, offers high throughput, environmental friendliness, and improved resolution, especially when multiple columns are coupled to increase separation efficiency and selectivity.

Despite advances, chiral separations are often based on trial and error and require rationalization. This study applies a mathematical model to multi-column systems to predict the retention behavior of warfarin enantiomers. Retention factor, retention time, and selectivity were calculated and experimentally determined on two, three, and four columns coupled in series. The relative deviation for retention time determination ranged from 0.68 to 7.29 % and for selectivity from 0.00 to 4.28 %.