Effect of pressure on the chromatographic separation of enantiomers under reversed-phase conditions

Commercially available ultra high pressure liquid chromatography (UHPLC) equipment offers the ability to explore the influence of backpressure on chromatographic separations. However, the influence of pressure on the chromatographic separation of enantiomers on chiral stationary phases remains largely unexplored. In this investigation we surveyed the effects of pressure on the separation of enantiomers using several reversed-phase chiral stationary phases. The experiments were conducted at constant flow rate and column temperature, using isocratic conditions. The only variable parameter was pressure, which was adjusted using a post-column backpressure regulator. For the separation of enantiomers on chiral stationary phases, an increase in pressure from approximately 2000 psi (138 bar) to approximately 8000 psi (552 bar) at constant flow rate and temperature led to an increase of retention factors for some analytes and a decrease for others. Achiral separations on a C-18 stationary phase always led only to an increase of retention factor. Interestingly, changes in pressure led to small changes in enantioselectivity during reversed-phase chiral separation of enantiomers, suggesting that such studies may be of value for better understanding the mechanisms underlying chromatographic enantioseparation.