Supercritical fluid chromatography for Diastereoselective resolution of Cyclopentane Diol core: a scalable approach to chiral purity.

Preparative supercritical fluid chromatography (SFC) method was successfully developed and implemented for the large-scale resolution of four closely related diastereomers of a cis-cyclopentane diol intermediate, a key structural fragment in a lead discovery candidate. The separation leveraged the unique physicochemical properties of supercritical CO₂, whose low viscosity and high diffusivity enable high linear velocities while maintaining column efficiency, thereby facilitating rapid and scalable separations. Diastereomeric resolution was achieved on a polysaccharide-based chiral stationary phase, where differential hydrogen bonding, steric fit, and dipole-dipole interactions between the multiple hydroxyl functionalities of the analytes and the chiral selector governed selectivity. Fine control of the mobile-phase composition-specifically the percentage and nature of the polar organic modifier (e.g., methanol or ethanol), along with basic or acidic additives-proved critical for modulating analyte solubility, peak shape, and resolution. From a scalability perspective, challenges such as sample solubility limits, pressure drop management, and consistent retention behaviour during column scale-up were systematically addressed through solvent strength optimization and loading studies. The resulting preparative SFC process delivered high throughput, excellent recovery, and reproducible stereochemical purity, underscoring the suitability of SFC as a robust and efficient platform for multigram to kilogram-scale diastereomer separations in early pharmaceutical development.