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.

While identified by the respective flavylium cation, anthocyanins are much more than this molecule. The flavylium cation (generally appearing only at very acidic pH values) is one of the molecules of a complex sequence of pH dependent molecular species reversibly interconnected by different chemical reactions. These species include the red flavylium cation, purple quinoidal base and blue or bluish anionic quinoidal bases. At the common pH of the vacuoles of simpler anthocyanins, the red flavylium cation is present only at very acidic pH values and at moderately acidic pHs there is no significant colour of the purple quinoidal base. Moreover, the blue or bluish anionic quinoidal base appearing around neutral pH values is not stable. Intermolecular (copigmentation) and intramolecular (in acylated anthocyanins) interactions increase the colour hue and yield bathochromic shifts in the absorption bands, permitting to extend the pH domain of the flavylium cation and increase the mole fraction of the quinoidal bases. Metal complexation is another strategy. In particular, the Al³⁺ cation plays an essential role in the blue colour of hydrangea. The most sophisticated structures are however the metaloanthocyanins, such as the one that gives the blue colour of commelina communis, constituted of six anthocyanins, six flavanones and two metals. In this work we discuss how physical chemical tools are indispensable to account for the chemical behaviour of these complex systems. The experimental procedures and the equations needed to calculate all equilibrium constants of anthocyanins and the consequent pH dependent mole fraction distributions in the absence or presence of copigments are described in detail. Reverse pH jumps monitored by stopped flow have been shown to be an indispensable tool to calculate these parameters.

A four component host–guest system comprising β-cyclodextrin (β-CD), cucurbit[7]uril (CB7) and two chalcone guests was demonstrated to undergo pH-triggered self-sorting in aqueous solution. This phenomenon arises from the contrasting complexation induced pK_a shifts displayed by these guests after complexation with CB7.