New Procedure To Calculate All Equilibrium Constants in Flavylium Compounds: Application to the Copigmentation of Anthocyanins

[Image: see text] A new experimental procedure to calculate all equilibrium constants of the multistate of species of anthocyanins and related compounds, including those in basic medium, is reported. The procedure is based on a series of pH jumps monitored by stopped flow from an extended pH range of solutions at pseudo-equilibrium (when there is no significant formation of trans-chalcones) or at equilibrium to pH = 1.0. The experimental procedure is described for the anthocyanin model compound 4′-hydroxyflavylium, which exhibits a peculiar behavior in moderately acidic medium, because the quinoidal base, hemiketal, and cis-chalcone have similar mole fractions at pseudo-equilibrium, permitting good discrimination among these species. The experimental procedure can be extended to the copigmentation phenomenon and allow the calculation of the 1:1 copigmentation constants of the flavylium cation, quinoidal base, hemiketal, and cis- and trans-chalcones (this last from the equilibrium) and their respective ionized forms. The method was applied to calculate the copigmentation constants of the model compound 4′-hydroxyflavylium as well as malvidin-3-glucoside with caffeine. In the last compound, the strongest interaction takes place with the quinoidal base (K = 303 M(–1)) and flavylium cation (K = 134 M(–1)) and, to a lesser extent, with the ionized quinoidal base (K = 43 M(–1)) and cis-chalcone (K = 17 M(–1)). The caffeine interaction with the hemiketal and the other ionized species is negligible.