A Colorimetric Procedure for the Determination of Small Amounts of Fatty Acid

The basis of the method is the measurement of the color change produced by the reducing action of fatty acid or cholesterol on a sulfuric acid-dichromate mixture. This reaction has been used in several ways for the determination of fatty acids and other organic material. Bang (1) made it the basis of a micromethod by titrating the excess of dichromate with starch-iodine and thiosulfate. In this laboratory (2) the procedure of Bang was modified to make the oxidation complete. Van Slyke and Folch (3) used the reagent by collecting and measuring the carbon dioxide evolved in the oxidation. As far as can be determined, no one has reported the use of the color change in the reagent as a means of measurement of fatty acids or other organic material, and, since it was found that the change was closely proportional to the amount of fatty acid oxidized and that the actual determination was rapid and easy to carry out, a procedure was developed and reported (4). Since the reagent is sensitive to organic matter of all kinds, it is necessary to isolate the fatty acids before measurement. The procedure for the isolation has been worked out by previous investigators and need not be reported here. The change in color cannot be measured in the Duboscq or similar visual calorimeters, but it can be determiped by the photoelectric type of instrument. For greater sensitivity a depth of liquid greater than in the ordinary photoelectric calorimeter was desirable and a special calorimeter was devised and is described below. Reagent-The Nicloux type of sulfuric acid-dichromate reagent was used. It is made by dissolving 10 gm. of AgN03 and 10 gm. of K2Cr20? separately in water, mixing the solutions, and purifying the precipitated silver dichromate by centrifuging and washing the precipitate two or three times with water in the centrifuge. 100 cc. centrifuge tubes were found convenient for mixing and washing. The separated precipitate was dissolved in 500 cc. of concentrated HzS04 and kept in a glass-stoppered bottle. Solve&--Either purified petroleum ether or a mixture of 7 parts of petroleum ether to 1 of chloroform can be used. The latter is more satisfactory, since it does not easily form emulsi&s in extracting lipide from either acid or alkaline solutions.