Purification of Arsenazo III,a Ca2+-sensitive dye

Commercial preparations of Arsenazo III. (o-(1,8 dihydroxy-3,6-disulfonaphthylene-2,7-bisazo)-bisbenzenearsonic acid), a dye useful for detection of micromolar concentrations of ionized Ca²⁺, have been found to be grossly and variably contaminated with colored impurities and Ca²⁺. In this paper, methods are described for detection of the colored impurities using DEAE-cellulose thinlayer chromatography in semiorganic solvents, along with methods for purification of the dye using DEAE-cellulose column chromatography and Chelex-100 ion-exchange resin. The impurities from one commercial lot are shown to exhibit significant absorbances over the range 400–725 nm; some of the impurities exhibit spectral shifts in the presence of Ca²⁺, implying Ca²⁺ binding. Thus, use of unpurified dye to quantitate ionized Ca²⁺ might yield data which vary with the source and batch of dye. The chromatographically pure Arsenazo III obtained from column chromatography is characterized with respect to Na⁺, Ca²⁺, and dye content.