Application of tandem coupling of columns in supercritical fluid chromatography for stereoisomeric separation: Optimization and simulation

Abstract

Chromatographic separation of compounds with more than one chiral center is challenging, requiring high resolution methods. Owing to the low viscosity of the mobile phase, Supercritical Fluid Chromatography (SFC) enables the tandem coupling of columns which increases resolution compared over a single column and can be effective in resolving stereoisomers. Enantioseparation of a dihydropyridone derivative with two chiral centers, synthetic API, was here studied using SFC. Six polysaccharide-based, chiral stationary phases with a mobile phase consisting of a carbon dioxide/methanol mixture (80:20 v:v) were investigated at 40 °C and a flow-rate of 3 mL/min, but only incomplete separation of the four expected stereoisomers was observed. We then examined different combinations of columns in tandem. It was found that, among the thirteen successful tandems, the OJ-H//AD-H system gave complete baseline resolution of the four stereoisomers with 4.98, 5.63, 6.06 and 6.89 as retention times and 2.97, 1.83 and 3.54 as resolution values. The conditions were further optimized to obtain the best resolution in the shortest elution time. The best conditions were transposed to semi-preparative scale to obtain the pure isomers, with yield increased by using stacked injections. The four fractions allowed the attribution of elution order on all tandem performed previously. The column order itself had no impact on the stereoisomeric elution order but the type of stationary phase and column order strongly influenced the resolution. In parallel, a previously reported mathematical model was used to predict the retention times of the four stereoisomers on each of the six polysaccharide-based, chiral stationary phase column tandems. This mathematical model was successfully applied to predict separation the dihydropyridone derivative’s isomers on two columns with chlorinated stationary phases.