A series of basic experiments has been performed with a set of DNP-amino acids and two-phase solvent system composed of methyl tertiary-butyl ether/water. The retainer acid (TFA) was added to the stationary phase and the eluent base (NH3) to the mobil phase at various concentrations. The results were found to be consistent with the mathematical analyses developed on the hydrodynamic mechanism operating in the present system. The overall results of the studies revealed that the present method bears a close resemblance to displacement chromatography in many aspects: One solute suppresses the ionization or affinity of others to alter the partition coefficient of the latter; in the column a train of sharply defined solute bands is formed and each band moves together at the same rate; impurities are concentrated at the boundaries of each solute bands; etc. However, there is a contrasting difference in the action of the key reagents. In pH-zone-refining CCC, the retainer acid transfers solute molecules from the mobile phase to the stationary phase at the front end of the solute bands whereas in displacement chromatography the displacer displaces solute molecules absorbed onto the stationary phase at the rear end of the solute bands. These mutually reversed actions of the key reagents at the opposite ends of the solute bands produce identical elution profiles. From this viewpoint, the present technique may also be called """"""""Reverse Displacement CCC"""""""". This term further suggests the presence of displacement CCC as well as that of reverse displacement chromatography.
