METHOD FOR IDENTIFICATION OF AZO DYES 215 35 of the listed colors. Ponceau SX, Yellow RFS and Yellow RY, are missing because they were unavailable in Spain at the time of testing. EXPERIMENTAL Description of Technique Chromatographic procedures were carried out after an initial purifi- cation process. Since the 35 assayed dyes are all acid colors, they can be fixed on wool from a weakly acid solution and then released into weakly alkaline media, resulting in a high degree of purity. There are, therefore, two stages in the method, purification and chromatography. I. Pur•/icalion Process. Fifteen to 25 ml. of the clye solution (or of the aqueous extract obtained frm a dyed food, drug or cosmetic) was placed in a 100 ml. beaker, and 1-3 g. of tartaric acid was added. The solution was brought to a boil, and a piece of white nonfluorescent wool cloth was added, and kept in the gently boiling solution for one minute. The colored wool was then removed and thoroughly washed with tap water, rinsed with dis- tilled water, and transferred to a new beaker containing 1 per cent ammonia. The alkaline solution was gently boiled until the color was stripped, the wool was then removed and discarded, and the solution was gently evaporated until the color intensity was suitable for chromatography. (For test purposes, a drop was placed on Whatman No. 1 filter paper.) The identical dyeing and extraction procedure was used in the case of dye samples to insure the same conditions as those that prevail during the work-up of finished products. It was noted that the sample solutions pre- pared directly from dyes without parification almost always showed the same chromatographic behavior as the extracts from wool. This fact is interpreted as evidence of the high degree of purity of the dye samples handled, a fact previously noted in the literature (23). I1. Paper Chromatography. Although different techniques did not appear to influence the results significantly, the ascending method was used for separation in all cases. The solvent was allowed to climb through a cylinder of filter paper, using the previously described (25) adaptation of Williams and Kirby's technique (24). This technique is simple and rapid, making it possible to carry out many tests in each chromatogram. The operating method is as follows: Square sheets of Whatman No. 1 filter paper (30-40 cm. length) are cut. At a distance of approximately 2 cm. a fine line is drawn parallel to the base of the papers with a soft pencil. The dye solutions to be tested are then spotted 1.5 to 2 cm. apart on this line with a micropipette, leaving a margin of 2 to 3 cm. from the edge of the papers. After drying, the papers are curled into cylinders, clipped at the ends and center with stainless steel clips and placed into the chromatographic chamber with a 1 cm. deep layer

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