Potential of chiral anion-exchangers operated in various subcritical fluid chromatography modes for resolution of chiral acids

Abstract

Anion-exchange-type chiral stationary phases (CSPs) derived from quinine or quinidine were applied in subcritical fluid chromatography (SFC) for the direct separation of chiral acidic compounds. Employing subcritical (sc) mobile phase modes (CO₂ + methanol as co-solvent and acids and bases as additives) first the influence of type and amount of acidic and basic additives on separation performance was investigated. Secondly, water was tested as a neutral additive and the influence of temperature variation on enantioselectivity was studied. Thirdly, we could chromatographically confirm that the often verbalized "inherent acidity" of sc CO₂ + methanol is manifested by the in situ formation of methylcarbonic acids in the sc mobile phase and thus functioning as acidic additive. Accordingly the dissociated methylcarbonic acid, acting as a counterion, enables an anion exchange mechanism between the cationic CSP and the corresponding acidic analyte. In the absence of a dissociable acid in the mobile phase such an ion exchange mode would not work following a stoichiometric displacement model. This finding is further corroborated by the use of ammonia in methanol as co-solvent thus generating in situ the ammonium salt of methylcarbonic acid. In summary, we report on ion-exchange mediated chromatographic separations in SFC modes by merely using (i) sc CO₂ and MeOH, (ii) sc CO₂ and ammonia in MeOH, and (iii) sc CO₂ and MeOH plus acids and bases as additives. Comparisons to HPLC mode have been undertaken to evaluate merits and limitations. This mode exhibits high potential for preparative chromatography of chiral acids combining pronounced enantioselectivity with high column loadability and avoiding possibly troublesome mobile phase additives, as the in situ formed methylcarbonic acid disintegrates to CO₂ and methanol upon pressure release.