A comprehensive study to reveal the potential of a more sustainable ultra-high performance enantioselective reversed-phase chromatography on Pirkle-type stationary phase, with Whelk-O1 as a case study

In this study, we aimed to make enantioselective chromatography more sustainable, more sensitive, and compatible with aqueous formulations analysis and ESI-MS. To achieve this, we examined the effects of transitioning from normal-phase chromatography (which uses hydrocarbon-based solvents) to reversed-phase chromatography (using mobile phases based on water) using broad-spectrum Whelk-O1 columns as a critical study. For the first time, we holistically compared the thermodynamics and kinetics of the two elution modes in order to answer the question of whether same-column chemistry can effectively separate the compounds even in reversed-phase mode and found, unexpectedly, that reversed-phase chromatography using acetonitrile as the organic modifier was competitive from a kinetic standpoint. We also evaluated the effectiveness of three organic modifiers simultaneously on a sample of 11 molecules already resolved in NP conditions with different resolutions and achieved a resolution value of 1.5 for 91% and a resolution value of 2 for 82% of cases. Finally, we separated three racemates (within a k factor of 9) using only 480 µL of solvent per chromatographic run on a millibore column of 1 mm I.D., demonstrating that our approach allows for greener chromatographic separations.