Normal phase high-performance liquid chromatography of pneumocandins: in situ modification of silica with L-proline to separate structural analogues

Lipopeptides such as pneumocandin B(0) are often produced by fermentation processes. Many compounds with similar structures (structural analogues), and hence similar physiochemical properties, are coproduced in the fermentation. We employed high performance liquid chromatography using silica gel as the stationary phase and a ternary ethyl acetate/MeOH/water mobile phase to separate pneumocandin B(0) from these structural analogues. Despite extensive efforts to optimize this system, two key structural analogues, pneumocandin E(0) and pneumocandin B(5), continued to be poorly resolved from the main product peak (pneumocandin B(0)). As a result, feed load was restricted and productivity was limited. In situ modification of the silica gel stationary phase with l-proline or other amino acids significantly enhances the resolution of the two key structural analogues from the compound of interest, enabling a two-fold increase in productivity. Results of a systematic study showed that the amine group in l-proline and other amino acids plays a key role in the modification of the surface of the silica gel to mediate the selectivity enhancement.