Characterization of low-level water addition for preparative chiral SFC

Supercritical fluid chromatography (SFC) is widely employed for high-efficiency chiral separations in pharmaceutical applications; however, the influence of water as a co-solvent additive in preparative-scale SFC remains underexplored. In this study, our objective was to assess the effect of incorporating 0-7.5% (v/v) water into the co-solvent on the performance of ten preparative chiral columns. We evaluated commonly used stationary phase chemistries (seven polysaccharide-based columns, one Pirkle-type column, and two macrocyclic glycopeptide-based columns) using CO₂ with 10%, 25%, or 40% MeOH containing 0%, 2.5%, 5%, or 7.5% water. Retention times and baseline peak widths of the second-eluting enantiomer were measured for representative chiral analytes on each column. Except for S,S-Whelk-O1, introducing minor quantity of water into the SFC co-solvent for the remaining nine columns led to decreased run times and narrower collection windows (baseline peak widths reduced by approximately 20-40%). To further interpret the results, we investigated the retention mechanisms of the chiral columns under hydrophilic interaction liquid chromatography (HILIC) conditions to rationalize the observed changes in retention time and collection window with increasing water content in SFC across different stationary phases.