Preparative Separation of Picralima Alkaloids using a New Flat-Twisted Tubing in pH-Zone-Refining Countercurrent Chromatography Mode

A novel column for spiral countercurrent chromatography (CCC) has been designed to interrupt the laminar flow of the mobile phase through the tubing and continuously mix the two phases throughout the column [1]. Flat-twisted tubing system was originally introduced for purification of large molecules (proteins and peptides). Its application in the separation of small molecules is presented for the first time. Here pH-zone-refining CCC is successfully applied to the preparative separation of Picralima alkaloids using this new tubing filled in the spiral tubing support rotor (STS, CC Biotech, Rockville MD). The seeds of Picralima nitida (Apocynaceae), popularly known in African ethnomedicine for its antimalarial activity, were extracted successively with solvents of increasing polarity to obtain hexane, dichloromethane (DCM) and methanol (MeOH) extracts. The experiment was performed with a two-phase solvent system composed of t-methyl-butyl ether-n-butanol-acetonitrile-water at an optimized volume ratio of 2:2:1:5 (v/v) where triethylamine (5mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. Two separation runs of 4.0g each of the DCM and MeOH extracts yielded six major indole alkaloids including akuammine, akuammicine, alstonine, picraline, Melinonine A and akuammidine with purities ranging from 90% (Melinonine A) to 98% (akuammine) as determined by HPLC. Overall, the results revealed that the new Flat-twisted tubing system yielded high partition efficiency with a satisfactory level of stationary phase retention in a short elution time.

Reference: [1] Yang Y, Aisa HA, Ito Y (2009)J. Chromatogr A., 1216(27): 5265–71.