Post-column deprotonation and complexation in HPLC as a tool for identification and structure elucidation of compounds from natural dyes of historical importance

Flavonoids, anthraquinoids and hydroxybenzoic acids were investigated by reversed phase HPLC analysis with UV and fluorescence detection. Post-column deprotonation with a borax buffer faciliates structure elucidation, particularly with respect to obtaining information about the presence of free 4'- and 3-hydroxy groups in flavonoids and identifying hydroxy groups in 1,4 positions in anthraquinone derivatives. Complexation with aluminum(III) and zirconyl ions was applied in order to lower detection limits. Al(III) is a slow post-column complexing agent whereas the zirconyl ion instantaneously forms fluorescent complexes that can be easily observed. Complexation could be useful in preliminary identification of flavonols and in identification of vicinal hydroxy groups in anthraquinones. The zirconyl ion is also superior to Al(III) in enhancement of fluorescence detection of flavonols, mono-and dihydroxybenzoic acids.