A spectroscopic study of dyes: mechanism of detection of nonabsorbing analytes in reverse-phase chromatography with the aid of chromophores

In order to understand the mechanism by which dyes assist chromatographic detection in reverse-phase systems, absorption spectra of brilliant green and methylene blue were investigated. It is shown that dye-assisted chromatographic detection depends on the ability of the analyte to shift the monomer-dimer equilibrium of the dyes toward greater monomer concentrations. Monomers have higher molar absorptivities than dimers. Equilibrium constants and molar absorptivities for monomeric and dimeric forms of the dyes are reported.