Simultaneous determination of methylene violet, halogenated methylene violet and their photoproducts in the presence of DNA by high-performance liquid chromatography using an internal surface reversed-phase column.

A simple and rapid isocratic high-performance liquid chromatographic method for the analysis of methylene violet, a neutral phenothiazine dye, in the presence of DNA has been developed. These chromatographic conditions are also applicable to its N-demethylated, bromo and iodo analogs. The method utilizes an internal surface reversed-phase column and a mobile phase consisting of 20% acetonitrile in 50 mM phosphate buffer (pH 7.0) and detection at 280 nm. Under these conditions all five dyes are well resolved from one another and from the faster migrating DNA. The effects of organic modifiers, ionic strength and pH of the buffer on the capacity factors of the dyes have been investigated. The method has successfully been applied to detect the photoproducts of methylene violet and its bromo analog in the presence of DNA without removing the biopolymer from the reaction mixture.