No abstract

The acid dissociation constants (pK a) of 7-hydroxyflavone, 3-hydroxyflavone and 5-hydroxyflavone, which are practically insoluble compounds in water but of great biological and physicochemical interest, were determined by UV–visible spectroscopy in ethanol–water and acetonitrile–water solutions, varying the solvent relative permittivity in the interval of 61 to 75, at constant ionic strength (0.050 mol·kg–1) and temperature (298.15 K). The pK a values of the compounds increase as the permittivity of the reaction medium decreases. Correlations were established between the pK a values and empirical parameters of the solvents, such as the relative permittivity and hydrogen-bond donor capacity. These equations allow the interpretation of the solvent effect on the acid–base equilibria and the determination of pK a values at 298.15 K in pure water. The ordering of the pK a  values was 7-hydroxyflavone (7.28) < 3-hydroxyflavone (8.68) < 5-hydroxyflavone (11.75). In addition, the dissociation constants were also calculated by means of DFT methods (B3LYP/6-311+G­(2d,p) level of theory), employing several thermodynamic cycles. The solvent effect on the optimized structures in the gas-phase was evaluated using the polarizable continuum model. A good agreement was observed between the theoretical and experimental pK a values. Finally, the experimental ordering in the acidity of the hydroxyflavones was explained using natural bond orbital analysis.

Two approaches are proposed to measure the effect of different experimental factors (such as the modifier concentration and temperature) on the elution strength and peak shape in reversed-phase liquid chromatography, which quantify the percentage change in the retention factor and peak width (referred to the weakest conditions) per unit change in the experimental factor. The approaches were applied to the separation of a set of flavonoids with aqueous micellar mobile phases of the surfactant Brij-35 (polyoxyethylene(23)dodecanol), in comparison with acetonitrile-water mixtures, using an Eclipse XDB-C18 column. The particular interaction of each flavonoid with the oxyethylene chains of Brij-35 molecules (adsorbed on the stationary phase or forming micelles) changed the elution window, distribution of chromatographic peaks and partitioning kinetics, depending on the hydroxyl substitution in the aromatic rings of flavonoids. At 25 °C, peak shape with Brij-35 mobile phases was significantly poorer with regard to acetonitrile-water mixtures. At increasing temperature, the efficiency of Brij-35 increased, approaching at 80 °C the values obtained at equilibrium conditions, already reached with acetonitrile at 25 °C.