pH-Zone refining counter-current chromatography of polar catecholamines using di-(2-ethylhexyl)phosphoric acid as a ligand.

The use of di-(2-ethylhexyl)phosphoric acid (DEHPA) as a ligand in the stationary phase effectively increased the partition coefficient of polar catecholamines. pH-Zone refining counter-current chromatography of six components, i.e. five catecholamines and one amino acid (DOPA), was successfully performed using a two-phase solvent system composed of methyl tert.-butyl ether and water by adding DEHPA (20%) and ammonium acetate (200 mM) to the organic phase and HCl (50 mM) to the aqueous mobile phase. DOPA was eluted first as a normal peak followed by the five catecholamines which formed a succession of highly concentrated rectangular peaks associated with sharp impurity peaks at their borders (UV tracing at 280 nm). Both pH and standard partition coefficient of collected fractions indicated minimum overlap between the main peaks. Each component was identified by NMR analysis.