A new preparative chromatographic technique called """"""""pH-zone refining CCC"""""""" is introduced. The method utilizes the difference in partition coefficient between two molecular species of an ionizable compound, i.e., the hydrophobic protonated form below the pK(a) and hydrophilic deprotonated form above the pK(a). In the separation of compounds with a carboxylic group, an organic acid such as trifluoroacetic acid is added to the sample solution and/or the stationary phase to protonate the compounds while the pH of the mobile phase was raised by adding a base such as ammonium hydroxide to deprotonate the compounds. When the sample is eluted under this particular condition, the major component(s) forms a stable pH zone by saturating the solvent behind the sharp border of the acid gradient zone, which is formed by the acid added to the sample solution or the stationary phase. Within this pH-zone formed by the major component, minor impurities are efficiently excluded either forward or backward depending on their partition coefficient. Any increase in sample size does not affect this purification process except that the separation time is proportionally increased. The capability of the method was demonstrated in the preparative separation of DNP amino acids and various hydroxyxanthene dyes. The present method may be applied to other compounds including many organic acids and bases.
