Fluorescence investigation of isochlorotetracycline: ground-state and excited-state acid-base equilibria

The fluorescence emission from isochlorotetracycline is shown to be associated with the hydroxyphthalide portion of the molecule. Neutral, anionic, zwitterionic and cationic forms of the fluorophore are proposed to account for the fluorescent behaviour in aqueous and organic media of different acidities. In strongly acidic aqueous solution intermolecular photoautomerism is observed, while in chloroform intramolecular phototautomerism occurs. In aqueous solution an excited-state pK_a of 3.3 was observed for the fluorophore. From a study of the pH vs. fluorescence profile with excitation at 350 nm, the ground-state microscopic dissociation constants for isochlorotetracycline hydrochloride were calculated. The optimum conditions for the fluorimetric determination of isochlorotetracycline are an alkaline medium (pH > 10) with κ_ex = 350 nm and κ_em = 415 nm.