A number of chromatographic systems, often automated, have been developed for the separation of amino acid o-phthalaldehyde derivatives. The molarity of the buffer used in the o-phthalaldehyde-2-mercaptoethanol reaction has no effect on the amino acid derivative formation. When physiological fluids such as urine or cerebrospinal fluid are analyzed, high concentrations of nonamino acid amines that consume reagent are often present. The precolumn derivatization of amino acids using an o-phthalaldehyde reagent has many advantageous features, but the instability of the derivatives could hinder acceptance of the method. The effect of pH on the separation of amino acid o-phthalaldehyde derivatives on a C-8 column can be stated quite simply: all amino acids elute at longer times as the pH is decreased, but the effect is much greater for amino acids with a carboxylic side chain. Amino acids and reagent are combined in a mixing tee just before injection onto the column.