In an effort to overcome some of the shortcomings of previous methods for determination of cholesterol in wool fat, the znk reaction has been studied. A procedure is presented in which cholesterol is separated as its digitonide with subsequent development of color by the addition of ferric ammonium chloride and sulfuric acid. The method is reproducible and appears to be superior to other methods now in use.
HE spectrophotometric methods currently in T use for the determination of cholesterol in wool fat are for the most part based on the Liebermann-Burchard reaction (1-3). The use of this reaction as a quantitative measure of cholesterol, however, leaves much to be desired due to the dependence of the reaction on temperature, time of color development, and stability of the reagent. The ferric chloride reaction proposed by Zlatkis, Zak, and Boyle (4) for the determination of cholesterol has been shown to be a reliable method for determination of blood cholesterol. Later publications by Zak (5-7) have set forth improvements in the original procedure.
The color produced by the Zak reagent with cholesterol has been found to be reproducible without the necessity for temperature control.
The color reaches a maximum in about 10 minutes and is sufficiently stable to be read in the period of 10-50 minutes after mixing. The reagent, composed of ferric ammonium chloride (FeCt.-2NH1CleH20) in 80% acetic acid is stable for periods up to six months. This new method has, therefore, overcome most of the troublesome aspects of the procedure in which the Liebermann-Burchard reaction is used.
It was the purpose of this research to determine if the Zak reaction could be utilized in the analysis of wool fat and wool fat-containing products for cholesterol content. It appears to the authors that the reaction can be used for this purpose.
EXPERIMENTAL
Equipment.-The Beckman DK-2 recording spectrophotometer was used for determination of the various spectra. The routine colorimetric analyses were run on a Coleman Universal spectrophotometer, using square cuvettes of 1.3 cm. light path.
Materials.-Cholesterol U.S.P.,' after crystallization from alcohol-water and drying at No, had m.p.