Copigmentation of anthocyanins with copigments possessing an acid-base equilibrium in moderately acidic solutions

The fact that many copigments such as cinnamic acids exhibit an acid-base equilibrium at moderately acidic pH values have not been taken in consideration in the copigmentation models and respective equations. In this work, the copigmentation constants of malvidin-3-O-glucoside and sinapic acid (pK_a = 4.5) is reported. The flavylium cation (AH⁺) in moderately acid medium (pH ≤ 6) can be considered a monoprotic acid in equilibrium with its conjugated base (CB) equal to the sum of the concentrations of quinoidal base (A), hemiketal (B), cis and trans chalcones (Cc and Ct), with equilibrium constant K'_a. Based on this behavior a new mathematical expression to account for the copigmentation of AH⁺ and CB was deduced. The system was also studied by a series of reverse pH jumps monitored by stopped flow and a previously reported procedure was extended to consider the acid-base equilibrium of the copigment. The copigmentation constant of the flavylium cation with the acidic form of the copigment, CP, is 243 M⁻¹, and no copigmentation with the basic form of the copigment, CP(−), was observed. This was explained by the very small superposition of these two species in the pH domain. The copigmentation constants of CB with the acidic and basic forms of the copigment, calculated from the UV–Vis measurements are respectively K_CBCP = 53 M⁻¹ and K_CBCP(−) = 78 M⁻¹. The reverse pH jumps monitored by stopped flow allowed the calculation of the copigmentation constants of the acidic form of the copigment (CP) with quinoidal base (A), hemiketal (B) and cis-chalcone (Cc), respectively K_ACP = 531 M⁻¹, K_BCP = 17, K_CcCP = 41 M⁻¹ as well as with the basic form of the copigment K_ACP(−) = 196 M⁻¹, K_BCP(−) = 79, K_CcCP(−) = 94 M⁻¹. The reverse pH jumps followed by a standard spectrophotometer allows for determination of K_CtCP = 55 M⁻¹ and K_CcCP(−) = 80 M⁻¹ and K_CBCP = 54 M⁻¹ and K_CBCP(−)=89 M⁻¹. The estimated error for all copigmentation constants is circa 10%.