Paper Chromatography of Anionic Disazo Dyes, Especially Trypan Blue and Its Red Impurity

Abstract

Twenty-one samples of 10 dyes were studied by paper chromatography, using Whatman #1 paper and developing by the ascending technique with 80% ethanol. Fifteen other Whatman papers were tested. Solvents faster than 80% ethanol (10% pyridine, 50% actone, 50% and 70% ethanol) were not useful. Slower solvents (isoamyl alcohol, isoamyl alcohol saturated with NH₄OH, 90% acetone, acetone, 95% ethanol) were generally unsatisfactory but sometimes useful for extreme sharpness of spots produced in descending chromatograms. Blue and violet (reddish) fractions were found in trypan blue, Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B, and Niagara sky blue 6B. Trypan blue and Evans blue showed traces of additional fractions in acid or alkaline 80% ethanol or on acid-washed papers. Red, orange and yellow fractions were generally present in trypan red. A single red component was in Congo red and vital red and only a violet component in Congo corinth. A special study was made of one sample of trypan blue, attempting to isolate its dye fractions by means of extraction of dye powder, solvent partition, a standard reprecipitation method, and column chromatography. It was relatively easy to obtain from trypan blue at least small amounts of a red impurity (violet fraction) but difficult, except upon cellulose columns, to purify trypan blue itself. Absorption maxima of trypan blue were at 232.5, 315 and 605 mμ and those of the red dye (violet fraction) at 310 and 555-560 mμ.