A microcolorimetric method for the determination of manganese in biological materials with 4,4' tetramethyldiaminotriphenylmethane.

In studies of manganese deficiency in the rabbit,1 a method for the determination of this element in small samples of blood serum and other tissues was needed. The most promising of the organic reagents suitable for manganese appeared to be 4,4’-tetramethyldiaminodiphenylmethane, which has been used by Harry (1) in a calorimetric procedure and by Mellan (2) as a spot test reagent. Preliminary tests showed that 4,4’-tetramethyldiaminotriphenylmethane is approximately 10 times as sensitive as the diphenyl compound; consequently this investigation was limited to the triphenyl compound. The method consists of a periodate oxidation of manganese to permanganate with subsequent development of a yellow color by the reaction of permanganate and 4,4’-tetramethyldiaminotriphenylmethane. Factors influencing the reactions involved were found to be the kind and concentration of acid used, the concentration of periodate, and the temperature and time of reaction between the reagent and permanganate. An acid mixture of 0.6 N in HN03 and 3.2 N in H3P04 was found to be satisfactory. The concentration of periodate is kept sufficiently constant by measuring the required amount of sodium periodate with a bowl-shaped glass scoop. Oxidation of the reagent by periodate is appreciable at a temperature of 90-100’ and is slight by permanganate at temperatures of 20-40”. If the reagent is added to the permanganate-periodate solution at 75-85”, the reaction with the periodate is not appreciable, while the reaction with permanganate is sufficient to yield a good color. A temperature of 80° was therefore chosen as most suitable. The measurement of the final color is made by means of a photoelectric calorimeter of the Evelyn type (3) with matched t,est-tubes 18 mm. in diameter. The course of the reaction is followed by noting the galvanometer reading, and the lowest per cent transmittance is taken as the final reading.