Supercritical fluid chromatography and liquid chromatography for isomeric separation of a multiple chiral centers analyte

The development of a chiral separation strategy has always been a challenge of crucial importance, particularly in the pharmaceutical field. Chromatographic methods have become popular, particularly High Performance Liquid Chromatography and Supercritical Fluid Chromatography from a preparative scale point of view. A bioactive compound bearing three stereogenic centers was entrusted in our laboratory and the aim of this work was to obtain the complete resolution of the eight stereoisomers. Nine different polysaccharide-based columns were tested in SFC under various carbon dioxide-based mobile phases. The use of a single chiral column Lux Cellulose-2 under 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40 degrees C, 120 bar outlet pressure allowed the obtention of eight peaks. To further improve the resolution of the two last isomers, two columns were serially coupled . The results obtained with the six different combinations are discussed. The tandem column supercritical fluid chromatography has demonstrated to be a useful technique to resolve the eight stereoisomers on Lux Cellulose-2//Cellulose-2 tandem of coupled columns with 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40 degrees C and 120 bar outlet pressure, despite a long analysis time. In order to compare the two methods (i.e supercritical and liquid), chiral liquid chromatography under polar aqueous-organic mode, polar organic mode and normal-phase mode, was implemented. The last mode allowed the full baseline resolution of the eight isomers on Cellulose-5 CSP, with 20% 2-PrOH in n-heptane at a flow-rate of 0.8 mL/min, at 25 degrees C, lambda = 220 nm. The limits of detection and of quantification were determined for this method and the best values obtained for isomer 8 were equal to 2.84 and 9.37 nM respectively. Finally, a small-scale preparative separation of the multiple chiral centers compound was implemented on Cellulose-5 CSP within 10% 2-PrOH in n-heptane in order to study the stereoisomer elution order on Cellulose-2, Cellulose-5 and Chiralpak AD-H, under EtOH or 2-PrOH in n-heptane mobile phases, and partial reversal elution orders were observed. (C) 2021 Elsevier B.V. All rights reserved.