VI. ESTIMATION 289 



the method has been made official by the American Oil Chemists' Society,^'' 

 it is not popular because the preparation of thiocyanogen reagent is tedious, 

 and the method is not applicable to oils containing acids of higher unsatura- 

 tion. The method is empirical in nature, for pure acids do not give the 

 theoretical thiocyanogen uptake. The method has been largely superseded 

 by spectrophotometric analysis for routine work. 



2. POLYBROMIDE METHODS 



An empirical method has been worked out for the determination of 

 linoleic acid in mixed fatty acids by bromination in cold petroleum ether.^ 

 The weight of the insoluble tetrabromides formed from linoleic acid can 

 be related to the linoleic acid content of the sample. The yield of tetra- 

 bromides is affected by sample size and by the presence of other acids. 

 However, if the empirical conditions are strictly followed, the analysis 

 can be made quite accurate for determination of linoleic acid in samples 

 containing no linolenic or more unsaturated acids. 



The hexabromide number of fatty acids can be used to determine the 

 linolenic acid content, if linolenic acid is the most unsaturated acid present.^" 

 The bromination is done in cold ethyl ether, and the weight of insoluble 

 hexabromides precipitated is a measure of the amount of linolenic acid in 

 the sample. The yield is affected by size of sample and the presence of other 

 acids. Application of a similar bromination technique to methyl esters allows 

 the detection and measurement of arachidonate, subject to the same errors 

 mentioned above. ^^ The advantage claimed for these polybromide methods 

 is that only the natural isomers of polyunsaturated acids and thus only 

 the physiologically active forms are measured. These methods employ a 

 sample of the order of 1 g. 



C. ENZYMATIC METHOD 



Soybean lipoxidase has been shown to be specific in attacking only lino- 

 leic, linolenic, and arachidonic acids. ^^^ ^^ The reaction can be carried out 

 under conditions in which the conjugated end product is proportional to the 

 amount of linoleic acid in the sample. The method is applicable to the meas- 

 urement of 10 to 100 7 of linoleic acid. The method has been used to detect 

 and measure linoleic acid in chromatographic eluates.^"* The presence of 

 oleic and saturated acids partially inhibits the enzyme, thus limiting its 



sb Official and Tentative Methods, American Oil Chemists' Society Cd 2-38 (1946). 



« M. F. White and J. B. Brown, /. Am. Oil Chemists' Soc. 26, 385 (1949). 

 10 M. F. White and J. B. Brown, /. Am. Oil Chemists' Soc. 26, 133 (1949). 

 " M. F. White and J. B. Brown, /. Am. Oil Chemists' Soc. 26, 85 (1949). 

 1^ S. Bergstrom and R. T. Holman, Advances in Enzymol. 8, 425 (1948). 

 " R. T. Holman and S. Bergstrom, The Enzymes, Vol. 2, Part 1, Chapter 60. Aca- 

 demic Press, New York, 1951. 

 '^ R. T. Holman and W. T. Williams, /. Am. Chem. Soc. 73, 5285 (1951). 



