Post-column continuous-flow analysis combined with reversed-phase liquid chromatography and computer-aided photodiode-array detection for the characterization of phloroglucinols by second-derivative difference spectroscopy

Flow-injection analysis coupled with liquid chromatography (LC) and a photodiode-array detector was applied to the investigation of phloroglucinol derivatives (kosins) from Hagenia abyssinica. Because of the structural similarities of the kosins, it was not possible to distinguish between the individual components in the crude mixture by their spectra alone. Post-column pH changes with various reagents, added using a flow-injection system, induced a bathochromic shift of the UV absorption maxima and these were found to furnish additional qualitative information. Addition of 0.3 M potassium hydroxide induced a smaller red shift in the spectrum for α-kosin compared with the shifts for kosotoxin and protokosin. This was confirmed in the normalized difference spectra, ΔA(λ), computed for each kosin by subtracting the normalized spectrum (in eluent at pH 6) from the normalized pH-shifted spectrum (at pH 12). The small pH-induced difference in ΔA(λ) was enhanced in the second-derivative transformation of the difference spectrum. This novel technique for solute recognition permits closely similar pH-sensitive spectra to be discriminated and characterized.