Chromatographic resolution of closely related species: Separation of warfarin and hydroxylated isomers

• Rapid chiral and achiral screening platforms for separation and analysis of complex mixtures.
• 1.8 min achiral SFC method for separation of warfarin and hydroxylated isomers.
• 8.0 min chiral SFC method for resolution of all twelve components.
• 2.6 min achiral RP-UPLC method for separation of warfarin and hydroxywarfarins.
• RP chiral LC–MS method using MS deconvolution for enantioseparation of hydroxywarfarins.

Recent developments in the field of organic synthesis are leading to increasingly complex mixtures of closely related species (positional isomers, regioisomers, diastereomers, etc.) that often prove challenging for chromatographic analysis and separation. In this study we investigate the separation of a representative mixture of warfarin and 5 different monohydroxylation isomers to assess whether conventional techniques are suitable for addressing this separation challenge, or whether ‘next generation’ separation tools such as multidimensional chromatography may be required. In this example, modifications of results obtained from conventional achiral and chiral chromatography method development screening platforms afford rapid separation of all components for both achiral and chiral analysis, with supercritical fluid chromatography showing the best performance in both cases (1.8 min for separation of six components by achiral SFC and 8.0 min for separation of twelve components by chiral SFC). While other more complex mixtures may require additional tools, these results suggest that new applications of existing separation platforms may be useful for creating the chromatographic methods required to support this new area of synthetic chemistry.