Acid‐base Equilibria of the Excited Singlet and Triplet States and the Semi‐reduced Form of Acridine Orange

The acid‐base equilibria of the excited singlet and triplet states of acridine orange (AO) were studied by flash‐photolysis and fluorometric methods. The dye is a stronger base in the first excited singlet state (pKs= 13.3) than in the triplet and ground states (pKr= 10.3: pKc, = 10.2); acridine orange follows the trend observed with some other heterocyclic compounds, viz. pKs >pKr= pK,c. At room temperature, an anomalous fluorescence occurs from the dye in basic media: the assignment of this emission is discussed. The semi‐reduced dye was studied as a function of pH. In a large pH range (3–14), only the protolytic equilibrium between the cationic (AOH2+) and the neutral (AOH) radicals was observed; the pK value corresponding to this equilibrium was found to be in the range of pH 5–6.