Post-column reaction for simultaneous analysis of chromatic and leuco forms of malachite green and crystal violet by high-performance liquid chromatography with photometric detection

The chromatic and leuco forms of malachite green and crystal violet were readily separated and detected by a sensitive and selective high-performance liquid chromatographic procedure. The chromatic and leuco forms of the dyes were separated within 11 min on a C₁₈ column with a mobile phase of 0.05 M sodium acetate and 0.05 M acetic acid in water (19%) and methanol (81%). A reaction chamber, containing 10% PbO₂ in Celite 545, was placed between the column and the spectrophotometric detector to oxidize the leuco forms of the dyes to their chromatic forms. Chromatic and leuco malachite green were quantified by their absorbance at 618 nm; and chromatic and leuco Crystal Violet by their absorbance at 588 nm. Detection limits for chromatic and leuco forms of both dyes ranged from 0.12 to 0.28 ng. A linear range of 1 to 100 ng was established for both forms of the dyes.