On-line high-performance liquid chromatography : Ultraviolet-visible spectroscopy of phenolic compounds in plant extracts using post-column derivatization

High-performance liquid chromatography coupled with ultraviolet-visible spectroscopy, using a photodiode array detector, was applied to the investigation of plant extracts for polyphenolic compounds. Simultaneous detection at different wavelengths and measurement of the UV spectrum of each separated compound during the elution allow an easy and rapid identification of flavonoids and xanthones. However, as some of these compounds have closely related structures, a characterization by their UV spectra is insufficient. Derivatization with reagents inducing a shift of the UV absorption maxima is required and furnishes additional structural information. In the present work, shift reagents were added to the eluate using a post-column derivatization system. The reagents were adapted in order to be compatible with the solvent system used for the chromatographic separation. The technique was applied to the analysis of crude extracts of various Gentiana species.