Peculiarities of the determination of the sorption enthalpy of anthocyanins under conditions of reverse-phase HPLC

Abstract

Changes in the thermodynamic parameters of the transfer of some anthocyanins from the mobile phase to the stationary phase were determined using a simplified method. The studies were carried out under conditions of reversed-phase chromatography on a Symmetry C18 column in a mobile phase containing
8 vol.% acetonitrile, 10 vol.% formic acid in water. It was previously found that the retention of anthocyanins depends on the pressure at the inlet to the chromatographic column, therefore, in order to obtain the correct results with a change in temperature, it is necessary to change the flow rate of the mobile phase to achieve the same pressure. In this case, the obtained parameters do not depend on the pressure at which they were obtained at the column inlet. Therefore, in the simplified (faster) method proposed in this study, measurements were carried out only at one pressure at the column inlet. The enthalpies of transfer of 3-glucosides of six main natural anthocyanidins, unambiguously determined from experimental data insignificantly differ from each other: the lowest enthalpy was found for peonidin-3-glucoside (-27.9±0.3 kJ / mol), and the highest enthalpy was determined for delphinidin-3-glucoside (-33.1±0.3 kJ/mol), while the patterns of change in this parameter upon the addition of hydroxy or methoxy groups were not clearly expressed. Entropy change, ΔΔS^o parameters for pelargonidin-3-glucoside showed that enthalpy-entropy compensation is observed for anthocyanins, which explains the difficulties in elucidating the effect of added functional groups on thermodynamic parameters. For the elimination of the effect of this compensation, it was proposed to calculate ΔΔG^o, as a parameter calculated by the difference (for the removal of the parameter depending on the phase ratio) of the natural logarithms of the retention factors of the analyte and the reference substance - pelargonidin-3-glucoside. As expected, in this case the addition of hydrophilic hydroxy groups leads (taking into account the sign) to an increase in ΔΔG^o by ~ 1.7 kJ/mol per group, and the addition of hydrophobic methoxy groups, on the contrary, leads to a decrease in ΔΔG^o by ~ -1.0 kJ/mol per group with good additivity of group contributions (±0.1 kJ/mol). It was also shown that ΔΔG^o increases by 2.6±0.2 kJ/mol during the transition from 3-glucosides to 3,5-diglucosides of five anthocyanidins. Changes of ΔΔG^o for the more complex carbohydrate radical in position 3 were also calculated.