Comparative Spectroscopy of Basic Fuchsin, Schiff Reagent, and a Formalin-Schiff Derivative in Relation to Dye Structure and Staining

Abstract

Ultraviolet (UV) absorption (200-330 nm) for 0.5 mg/ml aqueous solutions of basic fuchsia unadjusted and those adjusted to pH 0, 1.5, 8.5, and 11 were determined as well as spectra in the visible range (400-675 nm) for solutions with pH 1.9, 2.8, 3.9, 4.7, 5.5, 5.9, 6.5, 7.5, 9.3, 10.4, and 11. The UV absorbance of degassed Schiff reagent containing 1 or 0.5 mg dye/ml, and that of this reagent adjusted to pH 1.5, 2.3, 3.1, 4.5, 6.0, 7.1, and 8.4 were obtained for comparison. The progressive reaction of formalin with degassed Schiff reagent, followed spectrometrically for 2.5 hr, required 2 hr to reach completion. The degassed Schiff reagent contained only traces of-SO₃H as judged from its minimal absorbance between 280 and 295 nm. The UV absorption of this reagent and basic fuchsin in 1 N HCl were found to be identical. The absorbance is that of basic fuchsin reduced by the addition of Cl⁻ or SO₃H⁻ to the central methane carbon and H to the amino groups, therefore the leuco structure of basic fuchsin so reduced shows the fomation-NH₃ groups. Infrared (IR) spectra of basic fuchsin, Schiff crystals, and a crystalline formalin-Schiff reaction product support these observations and indicate that the final colored product is a methylsulfonic acid derivative of basic fuchsin. Identical IR spectra were obtained for two types of crystals derived from Schiff reagents indicating that both are the same chemically, although only one became colored on exposure to air. When these crystals were redissolved and SO₂ added, a Schiff reagent of appropriate pH was produced. Since it is derived from a crystalline product, this type of reagent should be useful in histochemical studies