pH-zone-refining counter-current chromatography: a displacement mode applied to separation of dinitrophenyl amino acids.

A new mode of pH-zone-refining counter-current chromatography operates in a manner analogous to displacement chromatography. The method uses a retainer base (acid) in the stationary phase to retain analytes in the column and a displacer acid (base) to elute the analytes in the decreasing order of pKa and hydrophobicity. The elution produces a train of highly concentrated rectangular solute peaks with minimum overlap. Utility of the method is demonstrated in the separation of dinitrophenyl amino acids in a two-phase solvent system composed of methyl tert.-butyl ether and water. Compared with the original mode of pH-zone-refining counter-current chromatography, the present mode is more amenable to ligand-affinity separation which may cover a broader range of analytes including non-ionizable compounds.