Changes in pyranine absorption and emission spectra arising from its complexation to 2,2′-azo-bis(2-amidinopropane)

2,2′-Azo-bis(2-amidinopropane) dihydrochlorid (AAPH) is probably the most extensively employed water soluble free radical source. In the present work, we show that it readily forms a 1:1 complex with pyranine. The complex present a red shifted absorption spectra and a reduced fluorescence yield, without significant changes in the fluorescence band shape. These changes are associated to a decreased value of the phenolyc pKa in the complex. Time resolved fluorescence measurements reported a short lived component (due to the complexed pyranine) and a long lived component, due to free pyranine, whose lifetime decreases when AAPH concentration increases. The complex formation was quantitatively evaluated from the changes elicited in the absorption spectra. The formation of this complex is extremely dependent of the medium ionic strength. In 10 mM phosphate buffer, pH 7.0, the association constant of the process Py4−+AAPH2+→(complex)−2 is 730±70 M−1.