V O L U M E 2 5 , N O . 6, J U N E 1 9 5 3
953
LITERATURE CITED
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1401-20 (1948). ( 3 ) Markham, R., and
Smith, J. D., Nature, 164, 1052 (1949). ( 4 ) lIarshak, A , , and Togel, H. J., Federation Proc.. 9,85-6 (1950). ( 5 ) IIoore, S.,and Stein, W.H., J . Bid. Chem., 176, 367-88 (1948). (6) Stein, W.H., and JIoore, S., Cold Spring Harbor Sy??iposiu C)i((i?it. Riol., 14,179-90 (1949).
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RECEIVED for review January 13, 1953. Accepted March 23, 1953. Presented before the Federation of -4merican Societies of Experimental Biology, I f a r c h 1952. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part b y a grant from the Atomic Energy Commission.
Spectrophotometric Micromethod for Determining Polyunsaturated Fatty Acids S. F. HERB
AND
R . W. RIEMENSCHNEIDER
Eastern Regional Research Laboratory, Philadelphia, Pa.
'There is considerable need for a micromethod of analysis of polyunsaturated fatty acids in fats or lipides, because often more than a few milligrams of sample niay be difficult or impractical to obtain. A spectrophotometric method is described which requires 1 to 10 mg. of fat for determination of acids containing from two to five double bonds. Results of analyses by the micromethod were in agreement with those obtained by macromethods. The micromethod should find greatest application in studies of the changes in lipide composition of 5uids and tissues of living animals and plants owing to the small quantity of sample required for analysis.
I
1IPIIOVEIII:STS in spectrophotometric methods for determining polyunsaturated acids in fats and oils have been discauwd in recent papers ( 6 , 7 ) . Although the methods currently in upc require only 100 m g . of sample, there is a great deal of iiitweut in a niicromet~hod that would require only a fen- milligrams. Sunicrous inquiiiea concerning such an adaptation of thr method have been rweived from persons engaged in biological :inti mediral research, herause often moi'e than a few milligrams of lipide may be difficult or iinprnctical to obtain-for example, i i i :inalysis of hody fluids Liken from living animals. A search of the literature. has furniqhed further evidence of keen illtorest iii spcctrophotornetric micromethods. Considerable \r.ork in this direction has been published (4,9, 10, 1 2 , 12). l-iifortun:ztcl~-, these invcstig:ttors did not have pure, natural, uris:itur:itcd acsiilr :ivailahle as reference standards from which they cmultl determine the correct spectrophotometric con.tants f o r thtir .spc&l ronditions of alkali isomerization. This is particdarly true for witis of greater unsaturation than linolenic acid. Coiiscquently, thrsv woi~lrersdid not recommend their published ronst:iiits, :inti :idvisetl othew to determine their on-n independcntlJ.. 111spit(, of t h r fact th:it no satisfactory micromethod resulted fi~onithis earlier work. the greater amount of conjugation protluwtl hy isomerizing in higher concentrations of potassium hyrlroxitle iii glycol intlicated possibilities for a more sensitive mc.thoti. For examplr. Holman and Burr (9) reported an extincGY tion cocfirient, E:,,",. = (i22, fur tetraene (3000 A , ) from Lie1~t~omiriatiori-ar:ic~liidonic :rcicl isomerized in glycol solution cont,:iiriing 22 to 23 gi':ims of potassium hydroxide per 100 nil. for 8 iuinutes at 178" C. This value is more than twice as great as the value 258, obtained under the conditions used by Beadle and I
