A N A L Y T I C A L EDITION PUBLISHED
BY
THE
AMERICAN
CHEMICAL
SOCIETY
HARRISON
E.
HOWE,
EDITOR
Quantitative Spectral Analysis of Fats J. H. RIITCHELL, JR., H. R. KFUYBILL, AND F. P. ZSCHEILE Research Laboratory, American Meat Institute, University of Chicago, Chicago, Ill., and Purdue University Agricultural Experiment Station, Lafayette, Ind.
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The first procedure tried in carrying out the isomerization was to reflux a weighed sample of fat in a 1 to 4 solution of potassium hydroxide in ethylene glycol for 25 minutes, then free the fatty acids with hydrochloric acid and extract them with ether.
H E Kaufmann (6) method of analysis for oleic, linoleic, and linolenic acids in some fats is based on determinations of the iodine number, thiocyanogen number, and saturated acid content of the mixed fatty acids. The analysis for the saturated acids alone involves an iodine number determination of the separated “solid” acids and, depending upon the method used, may also involve a thiocyanogen number determination in order to correct for the unsaturated acids which are not completely separated from the solid acids. Until recently, analyses of fats made by the Kaufmann method were in error, because theoretical values were used for the thiocyanogen numbers of linoleic and linolenic acids. Waterman et al. (15), Riemenschneider and Wheeler ( I d ) , and however, found the values for linoleic and Kass et al. (3, linolenic acids to be empirical. Although thiocyanogen numbers are reliable when determined under carefully controlled conditions, it is desirable to have a more direct method for analysis of the linoleic and linolenic acid content of fats. Prolonged heating of fats during saponification has been observed by Moore (9) to cause nonconjugated double bonds in fatty acids to become conjugated and thereby to absorb radiation in the ultraviolet region. Kassetal. (5)and Miller and Burr (7) reported that it is possible to measure quantitatively, by spectral absorption, the amount of linoleic acid in vegetable oils after heating them with a 1to 4 solution of potassium hydroxide in ethylene glycol. The acids affected by this treatment are the unsaturated ones containing more than one double bond. I n many fats the unsaturated acids consist chiefly of oleic, linoleic, and linolenic acids. Isomerization by alkali has been studied and made the basis for the method of analysis described in this paper.
Consistent results were not obtained by this method, and difficulty was experienced with emulsions in some cases. During heating of ethylene glycol and potassium hydroxide alone as a blank a substance which absorbs ultraviolet radiation is dissolved from the glass vessel or is formed from the reagents, and is extracted during the ether extraction. This, together with the fact that some oxidation of the easily oxidized conjugated double bonds may occur during the manipulation, probably accounts in part for the inability to obtain consistent results by this procedure. Temperature is important in the rate of isomerization. Another difficulty was encountered because the boiling point of ethylene glycol-potassium hydroxide solution varies with the amount of water derived from the potassium hydroxide. The analytical reagent grade of this alkali contains about 10 per cent water. The temperature of reflux varies with the type of condenser used.
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To overcome the latter difficulty, samples were heated at a temperature below the boiling point. I t was found that a much weaker solution of alkaline glycol than the 1 to 4 solution previously used would serve for the isomerization. A concentration of 7.5 grams of potassium hydroxide, assaying 85 per cent potassium hydroxide, per 100 ml. of ethylene glycol, was adopted. This solution was 1.3 N with respect to potassium hydroxide. In preparation of the reagent, the alkaline glycol solution was boiled in an Erlenmeyer flask until the temperature reached 190” C. This removed most of the water, and thus it was possible t o maintain a constant temperature of 180’ C. while heating the samples. The oil bath for heating the samples was maintained at 180 * 0.1” c.
Experimental Procedure The absorption data were obtained from measurements made with a photoelectric spectrophotometer (2,8),employing a Hilger double monochromator with crystal quartz optics. As a source of ultraviolet radiation, a Munch (10) hydrogen discharge tube with a fused quartz window was used. In the discussion of the results, the term “specific alpha” will be used:
It was possible to simplify the method and to obtain consistent results by making absorption measurements directly on the soap solution, using a blank solution in the solvent cell. Thus the troublesome procedure of freeing the fatty acids and extracting them was found unnecessary. A high dilution of the original reaction mixture is required before spectroscopic readings are taken. Ethanol is very suitable for this purpose, since the soaps are completely soluble in this solvent and do not foam when the solution is shaken. Ethanol is easily purified for optical use by distillation over potassium hydroxide and zinc dust.
Io
log,a Specific (Y = cl .. where a = absorption coefficient ZO = intensity of radiation transmitted by the solvent I = intensity of radiation transmitted by the solution c = concentration of solute in grams per 1000 ml. 1, = length in centimeters of solution through which the radiation passes 1
a
2
I
I LINOLEIC
70L
I ',
70!
!
Only one band is present in the isomerized linoleic acid and it is due to diene conjugation. The specific alpha value a t 2340 A. is 87.1. The band owith a maximum of 53.7 at 2680 A. is due to triene conjugatio?, while the maximum of 60.0 at 2340 A. in this same curve is due to diene conjugation in the isomerized linolenic acid. The reproducibility of these values is illustrated in Table I. The determinations were made a t several different times and with alkaline glycol which was made up fresh each time. I n analysis, the intensity of absorption at 2680 A. is a measure of the amount of linolenic acid present, while $he intensity of absorption at 2340 A. is a measure of both linoleic and linolenic acids. I n case both acids are present, a correction is made for the absorption at 2340 A. which is due to diene conjugation resulting from the linolenic acid.
LINOLEIC
2 LlhOLENlC
u
Vol. 15, No. 1
INDUSTRIAL AND ENGINEERING CHEMISTRY
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2 LINOLENIC
c=z
3 LINOLENIC
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