Reaction of Tertiary Amine, Quaternary Ammonium-, and Inorganic Salts with Citric Acid in Acetic Anhydride Wilson D. Langley Department of Biochemistry, School of Medicine, State University of New York a t Buffalo, N. Y, Many alkaloids, betaines, and inorganic salts give colored solutions when heated with citric acid in acetic anhydride. Conditiotrs are established under which the color can be relatied to the amount of chromogenic substances, whether in aqueous or anhydrous solution. Two reagents are recommended, and the conditions under which each may be used are described. Relatively stable colors are obtained and 5-rg amounts of chromogen may be determined. Color values for a number of substances are reported as absorbance per micromole. The color reaction promises to be useful in many types of study, especially with chromatography.
THE EXPERIMENTS reported herein were done to convert a colorimetric test for tertiary amine- and quaternary ammonium salts reported by Ohkuma ( I ) into a quantitative procedure for use with aqueous solutions. The test, cited by Feigl (2), consists of heating the substance with citric acid in acetic anhydride, with subsequent development of a violet or blue color. cis-Aconitic anhydride rather than citric acid is used by Palumbo (3, 4), Cromwell (3, Sass et al. (6), and Bottcher et al. (7), but only with anhydrous solutions. A number of metal salts give color with citric acid and acetic anhydride, as reported oy Critchfield and Johnson (8), but again, the procedure is only for anhydrous solutions of chromogen. In the present study cis-aconitic anhydride, aconitic acid, and citric acid in acetic anhydride are tested, and citric acid reagents are described which give colors with ether aqueous or anhydrous solutions of many chromogenic substances. According to Sass et al. (6), color is produced by reaction of a cationic amine with cis-aconitic anhydride, probably as a salt with the enol of the anhydride. EXPERIMENTAL
Reagents. High grade free bases and salts are used as purchased, solutions being diluted to give suitable absorbance. The salts are the acetate, acid phthalate, bicarbonate, carbonate, hydrochloride, hydrobromide, gluconate, lactate, and sulfate. Nitrates and flavianates do not give full color development. Analytical grade citric acid often gives extraneous color because of contamination, but repeated recrystallization from 90 acetic acid frees it of chromogen.
