Intermolecular Copigmentation between Delphinidin 3- O-Glucoside and Chlorogenic Acid: Taking into Account the Existence of Neutral and Negatively Charged Forms of the Copigment

Stopped flow corroborated by UV-vis measurements allowed for the calculation of the copigmentation constants of delphinidin 3-O-glucoside with the neutral (CP) and negatively charged CP(-) forms of chlorogenic acid. Solutions of delphinidin 3-O-glucoside in the absence and presence of the copigment were equilibrated at several pH values in the acidic region, pH < 6, and reverse pH jumps monitored by stopped flow were carried out by adding sufficient acid to give flavylium cation at pH ≤ 1. This procedure allows for the separation of three contributions: (i) all flavylium cation and quinoidal base species, (ii) all hemiketal species, and (iii) all cis-chalcone species. Reverse pH jumps can also be performed at fixed pH versus copigment addition. The contribution of trans-chalcone, minor species in the present system, requires reverse pH jumps from the equilibrium followed by a common spectrophotometer. The system was also studied by UV-vis as a function of the copigment addition at different pH values. A global fitting of all experimental data allowed for determination of the copigmentation constants with flavylium cation, KAH+CP = 167 M-1, KAH+CP(-) = 338 M-1 and quinoidal base, KACP = 1041 M-1, KACP(-)= 221 M-1. No significant copigmentation was observed for hemiketal and chalcones. Computational calculations confirm different geometries for the interactions of flavylium cation and quinoidal base with the neutral or the negatively charged forms of the copigment as well as predict identical relative order for the binding energies of the four adducts.