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

Abstract

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.

Exploring the Applications of the Photoprotective Properties of Anthocyanins in Biological Systems

“…In addition, oxygen deleterious action can be as big as the influence of pH on anthocyanins and occurs either by direct action or by the formation of radicals [ 30 ]. There are also strategies to enhance anthocyanins chromatic stability such as the formation of aggregates at higher concentrations and their complexation with non-colored compounds such as caffeine [ 31 ], hydroxycinnamic acids, flavonols, and some metal ions [ 32 , 33 ]. Both of these strategies appear to potentiate the color yield of these compounds in solution, prompting a higher stability of their quinoidal structures.…”

Evolution of Flavylium-Based Color Systems in Plants: What Physical Chemistry Can Tell Us

“…The most accurate method to determine the equilibrium constants reported in Scheme 3 is based on the reverse pH jumps (defined by the addition of acid to ensure pH ≤ 1 of equilibrated solutions, or pseudo-equilibrated, at higher pH values) [ 22 ] monitored by stopped flow [ 23 ]. In Figure 2 a, the trace of a typical reverse pH jump is shown.…”

“…Normalization of these amplitudes to the unit directly yields the mole fraction distribution of these species at the pseudo-equilibrium as shown in Figure 2 b. The experimental data obtained for kuromanin in Figure 2 and Figure 3 was used to calculate the equilibrium constants reported in Figure 1 according to the mathematical expressions derived in [ 23 ]. Once these constants were obtained, the energy level diagram can be constructed considering that Δ G ° = RT ln K , where Δ G ° is the Gibbs free energy, R is the ideal gas constant, T is the absolute temperature (Kelvin degrees), and K is the equilibrium constant of the chemical reaction [ 18 , 19 ].…”

Anthocyanin Color Stabilization by Host-Guest Complexation with p-Sulfonatocalix[n]arenes

“…Amongst the CB species only the quinoidal base (A) is colored, but its contribution to CB is minor. In the specific case of the most common anthocyanins, B is often the most abundant species of CB, with A, Cc, and Ct present in significantly lower amounts [7]. Furthermore, because the formation of the trans-chalcone (Ct) in anthocyanins is a very slow process, it is possible to define a pseudo-equilibrium between AH + and CBˆ, with [CBˆ] = [A] + [B] + [Cc], where no significant amounts of Ct are formed (pKˆa) [8].…”