Chromoxane cyanine R. I. Physical and chemical properties of the dye and of some of its iron complexes.

Chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) is a valuable biological stain. The dyestuff is supplied as a powder containing a little less than 50% by weight of the monosodium salt of the dye, mixed with colourless crystalline and amorphous fillers. The tetrabasic colour acid was prepared and purified for study of the chemical and spectral properties of the dye. Chromoxane cyanine R is an acid-base indicator, with five different colours corresponding to the colour acid and the four anions. The most conspicuous colour change, from yellow to blue, occurs with ionization of the phenolic hydroxyl group at pH 11-12. The dye is assayed by measuring the absorbance of a strongly alkaline solution at 585 nm, with reference to a standard solution prepared from the purified colour acid. Spectrophotometric evidence has been found for the existence of three dye-metal complexes in solutions of the dye containing added ferric chloride at pH 1.5 (the pH of iron-dye solutions most useful in histological staining). These have the postulated compositions [ Fe2H (dye)]- (red), [ FeH2 (dye)]- (red), and [Fe2(dye)]2- (blue). The first two are probably simple carboxylate complexes of low stability. Increase in pH or molar iron: dye ratio promotes formation of the more stable blue complex, which is a metal chelate. Other blue complexes have been described by other investigators in solutions less acid than those that are useful in microtechnique. The production of blue and various shades of red in tissues stained by solutions containing iron (III) and chromoxane cyanine R probably involves reactions of both the red and the blue complexes of the dye.