Stability constants of copper (II)-calcein blue complexes

Abstract

The acid dissociation constants of calcein blue (H3Cb) were determined potentiometrically and electrophoretically. The pKa, values of the carboxyl, the phenol, and the ammonium group were 2.45, 7.24, and 11.3, respectively. The solutions containing equimolar amounts of Cu (II) and H3Cb were titrated with 0.1 N KOH. The titration curves showed existence of CuHCb and CuCb complex. The acid dissociation constant of CuHCb was given 5.17 by the titration curves, and the stability constants of CuHCb and CuCb- obtained by Bjerrum's formation function were KCuHCb=1.60×1012 and KCuCb=1.88×1014, respectively. The theoretical consideration on the basis of Kcucb disagreed with the experimental quenching effect with Cu (II) on the fluorescence of H3Cb at pH 5.0. This difference was due to the use of potentimetric method in order to determine the equilibrium constants of fluorescent compound. Forster and Weller have shown that the values of Ka for the excited singlet (S1) state of molecules (such as naphthol) are larger than the corresponding ground (S0) state values. Accordingly, the acid dissociation constant of H3Cb and the stability constant (K*Cucb) of CuCb- for S1 state were investigated fluorophotometrically. The pKa, of phenolic proton for S1 state was estimated by Bartok's method, and it was 4.39. K*CuCb obtained by equilibrium concentration method was 1.70×1014. The theoretical quenching effect with Cu (II) derived from K*CuCb agreed with the experimental result. It was suggested that the complexation equilibrium involving fluorescent ligand and its complex should be investigated for S1 state as well as S0 state. © 1980, The Japan Society for Analytical Chemistry. All rights reserved.