Polymethoxylated Flavones: Dependence of Retention on the Introduction of Methoxy Groups into the Molecular Structure

Abstract

In this study the retention of polymethoxylated flavones (PMF) of black galangal (Kaempferia parviflora) root extracts and orange peel under reversed-phase (RP) HPLC conditions were investigated. These plant sources have non-overlapping sets of different PMFs. The separation of all flavones is better in methanol-based eluents acidified with orthophosphoric acid than in acetonitrile-based eluents, for which several inversions of retention times are observed with changes in the concentration of the organic modifier. The work analyzes changes in electronic absorption spectra – the position on the wavelength scale of the maximum of the absorption band of the so-called band I upon the introduction of methoxy groups into various positions of the flavone. It has been shown that the introduction of methoxy groups into the structure related to the localization of the electronic transition of band I is always accompanied by a bathochromic shift. When analyzing the effect of adding a methoxy group to the flavone structure on retention under RP HPLC conditions, not only the logarithms of the retention factors of flavones with different compositions of mobile phases, depending on these compositions, were used, but also the result of approximating the retention for purely aqueous mobile phases, which more clearly characterize the properties of sorbates. It was found that when adding a methoxy group to the 4' position, retention increases, whereas when adding another methoxy group to such structures to the 3' position, retention always decreases significantly. The first methoxy group (at position 4'), in a parallel orientation relative to the plane of ring B, is involved in hyperconjugation with the aromatic system. If we assume that the adjacent methoxy group is oriented parallel to the plane of the ring in a non-rigid manner, then due to the change in the thickness of the molecule (perpendicular to the conjugation plane), sensitivity of retention in reversed-phase chromatography to the shape of the molecule may be evident. Similarly, the introduction of a methoxy group into ring A at position 6 with positions 5 and 7 occupied is always accompanied by a decrease in retention due to the exit of the methyl group of this methoxy group from the conjugation plane. The presented results indicate the special properties of the methoxy group as a substituent in aromatic systems