PH dependence of the absorption and emission behaviour of riboflavin in aqueous solution

Abstract

The absorption and fluorescence behaviour of riboflavin is investigated in aqueous solution over a wide range of pH values from pH=−1.1 to pH=13.4. Absorption spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence signal decays are measured. Riboflavin exists in three different forms depending on the pH of the solutions. There is an equilibrium between the cationic form (RFl oxH 2 +) and the neutral form (RFl oxH) at low pH (p K c=0.4), and between the neutral form and the anionic form (RFl ox −) at high pH (p K a=9.75). The cationic, neutral, and anionic species have different absorption spectra. The fluorescence quantum yields and fluorescence lifetimes of the various forms are separated. The cationic form is non-fluorescent (fluorescence quantum yield φ F<5×10 −5), and the anionic form has a low fluorescence quantum yield of φ F≈1.2×10 −3. The fluorescence quantum yield of the neutral form is around φ F≈0.26 above pH=4 and decreases strongly below pH=4 because of [H +]-dependent excited-state reaction of RFl oxH * to RFl oxH 2 +*. At low pH the fluorescence signal decay is single-exponential since only the neutral form, RFl oxH, is emitting. At high pH a bi-exponential fluorescence decay occurs because RFl oxH and RFl ox − emit with different time constants.