Optimal conditions of ramentaceone and plumbagin separation and isolation from carnivorous plants extracts using normal phase–high performance liquid chromatography (NP-HPLC)

The preparative high – performance liquid chromatography is especially profitable technique of separation and isolation an usable amount of substances from different complicated mixtures. The work presents the optimal conditions of ramentaceone and plumbagin separation and isolation in a way allowing simultaneous separation with maximal selectivity of naphthoquinones and ballast substances in chloroform extracts from plants D. aliciae and D. capensis using NP-HPLC technique and isocratic elution.

Using gradient elution for separation of substances in preparative scale is unprofitable. It enlarges costs and technical complication, in relation to isocratic conditions. Composition of the mobile phase for silica gel as sorbent were systematically optimized. The systems most selective towards components of chloroform extract from plants D. aliciae are mixture n-hexane/methyl- tert–butyl ether 97/3 (v/v), however from plants D. capensis-n-hexane/dioxane 98.5/1.5 (v/v).

Highly polar components of plant extract strongly adsorbed on the stationary phase were removed from the HPLC column by using backflush instead of step elution. Step elution is time consuming as a result of column reconditioning, i.e. stationary phase surface equilibration with the solvent of low elution strength. Consequently, using backflush was far more advantageous, the more so because the mobile phase flow rate can be increased during backflush thus reducing its time.

Optimal conditions of scale up isolation of ramentaceone and plumbagin in crystalline form will be presented. The column productivity and purity of metabolites in maximum level of HPLC column overloading conditions were defined.

Acknowledgements: State Commitee for Scientific Research, Grant No N N523 4531 33

Ministry of Science and Higher Education, Grant No PBZ-KBN 112/P06/2005