Eosin staining of gelatine

Summary The mechanism of gelatine staining with four selected fluorone derivative dyes (eosin y, ethyl eosin, methyl eosin, uranin) was investigated. Gelatine films were stained in dye-buffer-ethanol solutions at varying pH and in the presence of NaCl and urea. Dye binding was recorded spectrophotometrically. Ionization constants of auxochromic phenolic groups were determined from pH-absorbance curves of dye-buffer-ethanol solutions. Dyebinding was greatest at pH below pK_OH and decreased with increasing pH. The addition of NaCl reduced dye binding slightly below pK_OH but markedly above pK_OH. The addition of 8 M urea decreased dyebinding regardless of pH. Comparing the pH dependence of dyebinding for eosin y and esterified eosins with ionization constants revealed that ionic bonding is unlikely to occur at the carboxyl group as well as at the phenolic group. Dye binding is intimately related to the presence of Br-groups. These results are discussed in conjunction with the functional structure of the dye ions and current concepts of dyebinding mechanisms.