Behaviour of sulphonated azodyes in ion-pairing reversed-phase high-performance liquid chromatography

Ion-pairing reversed-phase high-performance liquid chromatography (RP-HPLC) operation conditions were studied to obtain information useful for development and optimization of separation methods suitable for HPLC/MS analysis of dyes. The retention of eight sulphonated azodyes with widely differing structures (1–5 acid groups, molecular weight range 350–1220) was measured in mobile phases containing various ion-pairing reagents in aqueous-methanolic mobile phases. The effects of the type and of the concentration of ammonium acetate, tetrabutylammonium hydrogen sulphate and five di- and tri-alkylammonium acetate ion-pairing reagents on the chromatographic behaviour of dyes were compared in mobile phases with varying concentrations of methanol. Structural effects on the retention of dyes were studied in detail. The retention scale based on lipophilic and polar indices can be used for optimization of mobile phase for HPLC/MS of dyes and, on the other hand, may provide some information on the structure of unknown dyes.