Calculation of the Absorption Spectra of Various Anthocyanin Species in an Acidic Medium Using Stopped-Flow Spectroscopy

Equilibrated solutions of anthocyanins in a moderately acidic medium contain quinoidal base, hemiketal, and the cis- and trans-chalcone forms in a reversible, pH-dependent equilibrium. Reverse pH jumps to pH ≤1, monitored by stopped-flow spectroscopy, reveal the stepwise formation of the flavylium cation: initially from the quinoidal base within the mixing time, followed by slower conversion from the hemiketal, and then from the cis-chalcone. Final equilibration through cis-trans isomerization to trans-chalcone was monitored by using a standard spectrophotometer. Exploiting these kinetic differences, we reconstructed the individual absorption spectra of all anthocyanin species. This study demonstrates the utility of reverse pH jumps combined with complementary spectroscopic analysis for resolving transient species in complex chemical networks.