Microestimation of citric acid; a new colorimetric reaction for pentabromoacetone.

In an earlier publication’ a method was described for the determination of citric acid in blood serum for amounts as low as 5 y. While this method permitted determinations on from 0.5 to 1.0 ml. of serum, a need was felt for a method which would be suitable for smaller amounts for children. This need was felt most acutely when samples were drawn at frequent intervals from the same child. The extinction coefficient for the sodium sulfide color with pentabromoacetone is relatively low, E:y&. = 92. The development of a new colorimetric reaction with a higher extinction coefficient was of critical importance in our studies. Investigation of various substances available containing sulfur indicated that thiourea and substituted thiourea compounds form colored complexes with pentabromoacetone. For example, if a petroleum ether solution of pentabromoacetone is added to an alcoholic KOH solution containing thiourea, acetylthiourea, ethylthiourea, allylthiourea, sym-diethylthiourea, or sym-dimethylthiourea, yellow to greenish yellow colors are observed in the alcohol layer. In this strongly alkaline solution the colors slowly fade at room temperature. Thiourea was chosen for study because of its solubility in water, ease of purification, and availability. A 4 per cent thiourea solution dissolved in water exhibits a pH of 6.9. When this solution is shaken with a petroleum ether solution of pentabromoacetone for 15 to 30 minutes, a pinkish color, resembling a dilute permanganate solution, develops in the aqueous phase. This color has maximum absorption at 510 rnp. Addition of acid such as dilute acetic acid destroys the color. If the pink solution is now made alkaline to pH 11, a yellow color develops which reaches its maximum intensity after 7 to 15 minutes, with a maximum absorption at 460 mp. Both colors are stable for at least an hour at room temperature. If the pentabromoacetone solution is too concentrated, a precipitate of the pink complex will form.