turn. The openings of the vertical holes and of the horizontal slots were closed by a lid and cover glass, respectively, t o exclude dirt and extraneous light. The front face of the comparator (Figure 2) was painted a flat, light, neutral gray to facilitate color matching. RESULTS A N D DISCUSSION
A series of standard samples of benzene, the thiophene contents of which had previously been determined b y the variation of ( d ) , was analyzed for thiophene by several operators using the modified isatin procedure and the comparator. These analyses were repeated at intervals over 9 weeks (Table 11). The visual colorimetric method yields results which are in error [by comparison with the variation of ( d ) ] b y -0.06 p.p.m. of thiophene when the artificial color standards are 1 month old, and by -0.09 p.p.m. of thiophene when the standards are 2 months old. The maximum error observed was hO.1 p.p.m., or less, when the standards were less than 2 months old. The maximum
statistical range of any set of ratings of a standard benzene sample b y several operators was 0.04 p.p.m. of thiophene; the average statistical range was approximately 0.02 p.p.m. of thiophene. The artificial color standards should be replaced after 1 month. The India ink is probably the least stable standard, and some more stable substitute (a black dye ?) might be found. As suggested b y French ( S ) , colored glass or plastic standards would be more stable but also more difficult to prepare and to change in hue. Standards for the analysis of benzene samples from a particular source should be prepared individually to match the indophenin-acid solutions from similar samples. Slight variations must be anticipated in the colors of the indophenin solutions from benzene samples produced at one plant from time to time, or a t different plants. If desired, samples of benzene containing somewhat less than 0.8 p.p.m. of thiophene can be handled b y increasing the sample size; samples containing more than 1.1 p.p.m. of thiophene can
be handled by decreasing the sample size or diluting with thiophene-free benzene. Although only the application of the modified procedure to oleum-washed benzene has been demonstrated here, the principle of the method should apply equally well to benzene refined by other processrs. LITERATURE CITED
(1) Am. SOC. Testing Materials, Philadelphia, Pa., “1955 Book of ASTM
Standards,” Part 5, BSTM D 931-50. ( 2 ) Am. Soc. Testing Materials, “Tentative Method of Test for Traces of Thiophene in Benzene Using Isatin-and Spectrophotometry,’’ ASTPll D 168559T, 1959. (3) French, K. H. V., A N . ~ L .CHEM.20, 301-3 (1948). (4) Meyer, V., “Die Thiophengruppe,” Fnedrich Vieweer und Sohn. Braunschweig, 1888.
-
RECEIVEDfor review March 31, 1959. Resubmitted February 16, 1960. .Accepted February 16, 1960. Tenth Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 2, 1959.
Determination of Trisatu rated Glycerides in Fats with Mercaptoacetic Acid 1. R. ESHELMAN, E. Y. MANZO, S. J. MARCUS, A. E. DECOTEAU, and E. G. HAMMOND Department o f Dairy and Food Industry, Iowa State University, Ames, Iowa
b A method for the determination of trisaturated glycerides in fats is based on the reaction of the unsaturated glycerides wth mercaptoacetic acid. The mercaptoacetic-glycerides that are formed are separated from the neutral trisaturated glycerides by extraction of the ammonium salts and ion exchange treatment. The method has been applied to a number of commercial fats and oils. The results are reproducible and recovery experiments indicate that the recovery of the trisaturated glyceride i s nearly quantitative.
A
trisaturated glycerides occur in many fats and oils, isolation from their host of structurally similar compounds is difficult. Three methods have been reported for the determination of the trisaturated glyceride fraction in fats. The first developed by Hilditch and Lea (IO) depends on oxidation of the fat with potassium permanganate and the subsequent sepa844
LTHOUGH
ANALYTICAL CHEMISTRY
ration of the neutral trisaturated glycerides from the acidic azelaoglycerides formed in the oxidation of the unsaturated portion of the fat. The second method developed by Hilditch and coworkers (9) depends on the great difference in the solubility of long-chain saturated and unsaturated glycerides. The method of Reiser and Dieckert (20) is based on isotope dilution. The potassium permanganate method has several disadvantages as a quantitative analytical technique. Hilditch ( 8 ) has pointed out the difficulties with emulsions in the separation of the neutral and acidic glycerides. Ting (22) attempted to use this method with butterfat and concluded it was not reproducible. Kartha ( I S ) has claimed that considerable hydrolysis of the ester groups of a fat occurs during potassium permanganate oxidation. He recommended carrying out the oxidation in the presence of acetic acid to neutralize the alkaline materials that are generated. Recently Eshelman and Hammond (6) have confirmed that ester hydrolysis may occur. They also
showed that if the oxidation was carried out in the presence of acetic acid, synthesis of new ester groups occurred and a neutral side product was formed. This has also been confirmed by Lakshminarayana and Rebello ( I @ , n-ho believed the neutral product mas an acetic acid ester of a keto1 formed from partial oxidation of the double bonds. Thus it appears that the permanganate oxidation technique is liable to sereral sources of error and map not give an accurate estimate of the trisaturated glycerides in fat. The crystallization method appears to give reliable results with some fats (5). However, it cannot be applied to the analysis of fats such as butterfat in which there is a great variation in the chain length of the saturated fatty acids that are present. Also the trisaturated glycerides cannot be completely isolated by this method. It is necessary to assume that any unsaturated material concentrated with the trisaturated glycerides is monounsaturated-disaturated glyceride, and that the saturated acids of the trisaturated glycerides are present
in the same proportion in the contarninating unsaturated glyceride. Error may also be introduced by the method that is used to determine tlie proportion of unsaturated fatty acid present. The isotope dilution method (20) uses labeled tripalmitin. Trisaturated glycerides were isolated from the fats by crystallization from acetone. It was assumed that the trisaturated glyceride behaved as a single component whose solubility was the same as that of the tripalmitin. Trisaturated glycerides that were completely free from unsaturated glycerides could not be obtained and a correction had to be applied. This technique would be 1iniitc.d to fats in which the saturated acids show little variation in chain length. This paper describes a method based on the reaction of mercaptoacetic acid with the unsaturated fatty acids of the fat. The reaction is as follow: -C=CH H
+ HSCHZCOOH
+
H H
-c-c-
H SCH2COOH
Hoog and Eichwald ( I I ) have proposed that this reaction might be used to fractionate saturated and unsaturated hydrocarbons. Asberg and Holmberg ( 1 ) have claimed that the reaction is quantitative enough to be used for the determination of iodine value (1.V.) in fats. Recently Koenig and Swern (15) and Fore, O'Connor, and Goldblatt ( 7 ) have studied the addition of mercapto:icetic acid to oleate and linoleate. REAGENTS
Technical grade mercaptoacetic acid (Eastman) was mixed with an equal volume of benzene, and the benzene was distilled t o dehydrate the mercaptoacetic acid. The residue was heated to 100" C. to remove most of the benzene. The mercaptoacetic acid was then distilled a t about 5-mni. pressure and a fraction boiling from 88" to 90" C. was collected. This was stored in closed bottles a t 3' C. until used. N,iV-Diet h y l a n i i n o e t h y l c e 11 u 1o s e (DEAE-cellulose) was prepared hy the method of Peterson and Sober ( I S ) . The petroleum ether (boiling iange 40" to 60" C.) was distilled over potassium carbonate. The fats and oils used were obtained fresh from the processors and refrigerated until analyzed. The triolein was prepared by transesterification of methyl oleate and triacetin using a sodium methoxide catalyst. The methyl oleate was prepared by the procedure of Knight, Jordan, and Roe (14). The triolein was crystallized from acetone and Skellysolve B. It had I.V. 86.2 (theory 86.0); Sap. V. 192.9 (theory 190.1); ny 1.4619 (corr.); reported 1.4621 (23). The trilaurin was prepared by trans-
esterification of methyl laurate with sodium niethoxide. The methyl laurate was prepared from commercial lauric acid. The trilaurin was crystallized from Skellysolve 13 and gave ny 1.4401, reported 1.44039 (19); Sap. V. 260.4 (theory 263.5); m.p. 46.2' C., reported 46.4 ' C. (8). PROCEDURE
An amount of fat was taken for analysis such t h a t a t least 50 mg. of trisaturated glyceride was obtained if this did not require more than a &gram sample. About 5 grams was the maximum amount t h a t could be handled conveniently. The values of the trisaturated glycerides of many fats that are reported in the literature provide a means of estimating sample size. The amount of mercaptoacetic acid was determined as follows: For fats with a n iodine value above 80 Grams of mercaptoacetic acid
=
0.01089 X iodine value X weight of fat
For fats with a n iodine value below 80 Grams of mercaptoacetic acid = 0.8 X weight of fat The larger proportion of mercaptoacetic acid is used with the more saturated fats t o obtain a homogeneous reaction mixture. Special precautions for cleaning the glassware were necessary to obtain reproducible results. The glassware in which the reaction was carried out was soaked 30 minutes in alcoholic sodium hydroxide, washed B ith distilled water, soaked in 1 N ammonium hydroxide overnight, and dried in an oven a t 100' C. The pipets used to handle the mercaptoacetic acid and fat were cleaned in the same way. Glassware should not be cleaned with ouidizinp acids. The fat and mercaptoacetic acid were reacted in 250-ml. centiifuge bottles by shaking the reaction mixture overnight at 50' C. in a Warburg bath, They formed two phases at the beginning of the reaction, but as the reaction proceeded they formed a single phase. Shaking ensured the mixing of the phases and speeded the reaction. The reaction mixture was dissolved in 21 ml. of ethyl alcohol and 50 ml. of petroleum ether. The resulting solution was neutralized to the phenolphthalein end point with concentrated ammonium hydroxide. Enough water was then added to bring the total water present to 70 ml. The phases were mixed by swirling and the bottles were centrifuged a t 1800 r.p.m. to separate the phases. The loiq*er phase mas removed by an 8-mm. glass tube drawn out t o a fine tip and connected through a stopcock to a water pump. The extraction procedure removed tlie excess mercaptoacetic acid and some of the mercaptoacetic-glycerides. Some fats formed three layers when subjected to the extraction procedure. I n these cases, the lower layer was removed and the two upper layers were extracted with 50 ml. of 30y0 ethyl alcohol made
alkaline with a few drops of concentrated ammonium hydroxide. The mixture now formed two phases and the usual procedure 17 as followed. The petroleum ether layer recovered from the extraction procedure n as passed through a column of DEAEcellulose. I n these experiments the columns were 14, 20, and 30 in outside diameter and were filled to 18. 19, and 30 cm., respectively, with cellulose. These nere referred to as small, medium, and large columns. The size was determined empirically and depended on the amount and kind of fat being analyzed. I n general, the more saturated the fat and the longer the chain length of the saturated fatty acids, the larger the column that m-as required. The columns were prepared a s follows: Cellulose, which was stored as a n alcohol-damp powder, was made into a slurry with petroleum ether and poured into the columns The petroleum ether was drained from the column and as the cellulose settled it was packed firmly with a glass rod but not so hard t h a t a flow rate of less than 4 drops per second resulted. The column was washed with about one and one half times the column volume of petroleum ether. This step is important t o remove material which will otherwise contaminate the trisaturated glyceride. After the petroleum ether layer from the eutraction mixture was passed through the column, the column was washed with one and one half times the column volume of petroleum ether. The petroleum ether was evaporated in a weighed flask on a steam plate a t about 110' C. The residue which consisted of trisaturated glyceride plus some unreacted fat had a titer of less than 0.015 meq. when sufficient cellulose was used. The amount of trisaturated glyceride in the fat can be approximated by determining the iodine value of the residue and correcting the percentage of residue by the formula:
% GSa
%R '%R X I.V. X (320.5 f 2 h1.W.)
=
25382
where GS, is the trisaturated glycerides, R is the residue, I.V. is the iodine value of the residue, and h1.W. is the average molecular weight of the saturated fatty acids in the original fat. This formula assumes that the contaminating glyceride is a dieaturated mono-olein and that the saturated fatty acids in the monounsaturated glyceride are in the same ratios as in the original fat. The molecular weight of the saturated fattjacids can be obtained from analyses in the literature in many cases. A more accurate determination of the trisaturated glyceride was obtained by recycling the residue through the procedure two, three, or four times. I n these experiments about 0.8 gram of mercaptoacetic acid was used with the VOL. 32, NO. 7, JUNE 1960
e
845
Table I.
Fat
%
C0l.0 Residue SOY S 0.43 S 1.31 Randomized soy S 2.52 Cottonseed Randomized cottonseed S 4.52 S 0.34 Safflower S 0.49 Linseed 31 1.60 Peanut Corn S 0.24 Lard M 6.72 Oleo oil LI 10.2 Coconut 1 11 75.2 Coconut 2 M 88.6 Cocoa butter L 19.7 Butter 1 nI 34.7 Butter 2 %I 35.4 Triolein S 0.03 a S-small, M-medium, L-large column. a large column. n.d., not detected.
residues to be recycled; otherwise the procedure was the same. The iodine value was reduced a t each cycle. I n some cases a minimum iodine value was reached which could not be further reduced. I n other cases very low iodine values could be obtained. The above formula was used t o correct for the residual value or a plot of iodine us. percentage residue was extrapolated to zero iodine value, to estimate the trisaturated glyceride. The DEAE-cellulose could be regenerated and re-used indefinitely b y removing most of the excess petroleum ether and allowing the cellulose to stand overnight in 1.V sodium hydroxide. The next day the cellulose was filtered through cheesecloth on a Buchner funnel and washed with distilled water until the washings were neutral to phenolphthalein. The cellulose was washed with 95% and then with 100% ethyl alcohol to dehydrate it. The ethyl alcohol was run through the cellulose very slowly and then as much as possible was removed with suction. The cellulose was stored as a n alcoholdamp powder in closed containers. Air drying made it lumpy and reduced its capacity, but i t could be restored by washing with water and alcohol. Soybean oil and cottonseed oil were randomized with 0.5% sodium methoxide (6) by stirring under a nitrogen atmosphere at 60' C. for at least 4 hours. The catalyst was washed out and the dry oils were analyzed as usual. F a t t y acid analyses were made by converting the oils to methyl esters by transesterification. The methyl esters were analyzed by gas-liquid partition chromatography on a 7.5-foot Craig polyester succinate column (17) at 185' C. using a Chromacon Model No. 9410. Saponification values mere determined b y a microprocedure (4). Iodine values were determined by a micromodification of the Hunter and Hyde procedure ( l a ) .
846
ANALYTICAL CHEMISTRY
Analysis of Various Fats by Mercaptoacetic Acid Method
%J
I.V. Residue I.V. 63.0 0.31 44.6 1.16 18.5 27.3 1.98 34.6 33.3 3.78 20.0 25.9 48 64 18.8 1.10 10.8 44 4.01 8.7 19.6 16.8 1.2 1.5 74.4 2.2 80.6 24.2 19.9 11.6 6.2 30.1 3.6 10.6 32.3 4.0
% Residue 1.00 1.26 2.75 1.14 3.05
M.P. of % ResiI.V. Residue I.V. due 44 52 18.0 24.9 48 50 18.8 50 55 7.6 31 54 3.4 2.72 2.4 60 48 1.0 26 1.5 26 21.8 1.78 13.6 1.1 27.5 0 . 5 40 1.8 40
S.V. of
Residue 204 177 203 189 75 139 244 200 205 203 270 266
yo GS, Corr. 0.0
0.4 0.1 0.9 0.0
0.0
0.6 0.0
2.5 4.1 72.2 76.2 0.9 249 27.2 260 28.0 0 0.0 On subsequent cycles a small column was used with all fats except cocoa butter 74.8 80.2 4.36 28.9 30.0
RESULTS
The analyses of several commercial fats and oils are shown in Table I. All the residues had iodine values indicating that they were contaminated with unreacted fat. Lengthening the time of reaction and increasing the amount of mercaptoacetic acid above the amounts indicated in the procedure did not decrease the amount of unreacted fat. However, if the reaction mixture was extracted and the reaction products were removed, the residue would react with more mercaptoacetic acid to give a lower iodine value and decreased amount of residue. At least a 200% excess of mercaptoacetic acid is used in the reaction. Less than this gave residues contaminated with greater amounts of unreacted fat. A homogeneous reaction mixture decreased the amount of unreacted fat in the product. The reaction mixture is nonhomogeneous a t first, but as the reaction proceeds and mercaptoacetic-glycerides are formed, the reaction mixture becomes homogeneous if sufficient unsaturated glycerides are present, and if the ratio of fat to mercaptoacetic acid is properly balanced. The reaction is sensitive to the cleanliness of the glassware in which it is carried out. This has been noted by other workers ( 7 ) . Reaction vessels cleaned in nitric-sulfuric acid inhibited the reaction. The yield of residue was not affected by the addition of selenate, ferric, or cupric ions, unless the level was so high that considerable oxidation of the mercaptoacetic acid occurred. If this happened, more unreacted fat was found in the residue due to a decrease in the excess mercaptoacetic acid. Ultraviolet light did not affect the yield of residue. Exclusion of air from the reaction
% GSS*
Reported n.d. (6)
0.0-0.1 ( 6 ) >O.O (6) n.d. 161 n.d. (6j Low (6) n.d. (6) 2 . 2 (8)
84-86 (6) 84-86 (6) 2 . 5 (6) 18-41 (8) 15-41 (8) RThich required
mixture resulted in very little reaction of the fat with the mercaptoacetic acid. Oxygen appears to be necessary to initiate the free radicals involved in the reaction. Increasing the temperature of the reaction misture above 50" C. resulted in a less complete reaction. A temperature of 50" C. was chosen, as most fats are liquid at this temperature, and lower temperatures did not lead to appreciably loner yields of residue. I n the isolation of the trisaturated glycerides from the mercaptoaceticglycerides formed in the reaction, the fats with the highest amounts of longchain saturated fatty acid gave the most trouble. This is presumably because they are rich in disaturated triglycerides. Hilditch (8) has pointed out the difficulty of removing the disaturated monoazelaoglycerides from the trisaturated glycerides in the permanganate procedure. I n fats which are rich in polyunsaturated glycerides, the mercaptoacetic-glycerides are removed almost completely by the extraction procedure alone. Fats that are rich in disaturated triglycerides are still heavily contaminated by the mercaptoaceticglycerides after extraction, as shown by their high acid titer. I n the separation of ion exchange cellulose, those fats richest in long-chain saturated fatty acids required the most cellulose to remove the mercaptoacetic-glycerides. The residues obtained by this procedure have appreciable iodine values even after being recycled several times. Although the percentage of residue gives a reliable upper limit to the value of the trisaturated glyceride which may be valuable for many purposes, the estimate of the trisaturated glyceride should be improved by applying a correction factor. I n making this correction i t mas assumed that the contaminating substance was a n unreacted disaturated
mono-olein. This assumption is based on the folloning consideration: Because only one double bond of an unsaturated glyceride must react to be separated from the trisaturated glyceride, the glycerides having the fea est double bonds should accumulate in the residue. Thus the trisaturated glyceride will be contaminated mainly with monounsaturated glyceride, and oleic acid is the major monounsaturated fatty acid in the fats tested. The residues that were obtained from the highly unsaturated vegetable oils and which are very lon- in trisaturated glyceride, have iodine values indicating that glycerides with more than one double bond are present. I n these cases a more accurate correction might be made b y making a more detailed analysis of the fatty acids present rather than by using the iodine alone. The n eights of the residues agreed within a relative error of 3% n-hen several determinations were made by the procedure given above. J17hen the amount of residue was very small the relative error was increased somewhat because of weighing error. The accuracy of the trisaturated glyceride determination is influenced b y the accuracy and magnitude of the iodine value of the residue. This in turn is influenced by the number of cycles run with some fats. The absolute error in the trisaturated glyceride content of coconut oil and butterfat is estimated to be no more than +17c. The error in the trisaturated glyceride content of lard is estimated to be no more than 1 0 . 5 % . K h e n the trisaturated glyceride content is less than 1Ycqthe error in the determination is probably no more than +0.27,. To show that no rearrangement and interesterification of the fat occurred during the reaction, trilaurin n as mixed n i t h peanut oil a t the 10, 25, 50, and i 5 % level. The reaction was run through one cycle and the residue recovered. The residue \\as corrected for the amount recovered from peanut oil alone, which yielded trilaurin recoveries of 98 to 1067,. If there had been interesterification the recoveries would have been lon-. The saponification values of the rcsidues were in the range to be expected for each of the facts examined, except in some cases where there n-as very little residue. Theqe low saponification values may be due to the presence of uneaponifiable material or to contamination during the procedure. Likewise the melting points of the residues were in the range expected for the fats examined except where there 1%as very little residue. I n these cases the melting points were usually rather high because no trisaturated glyceride seemed to be present. These high melting points may be due to the presence of
unsaponifiables, the formation of trans double bonds during the reaction, or contamination during the procedure. Because most unsaponifiables are unsaturated, most of them are probably removed during the procedure. Table I shows t h a t most of the unsaturated vegetable oils gave residues of less than 1%. Also the residues t h a t were left had rather high iodine values. The residues were semisolid at room temperature, and did not have the appearance and consistency of longchain trisaturated glycerides. It was concluded that the trisaturated glyceride content of most of these oils must be less than 0.1%. This is particularly interesting in the case of soybean oil which is said t o follow the theory of random distribution @ I ) , and which should have more than 0.1% trisaturated glycerides. T o check this, a sample of the soybean oil was randomized with sodium methoxide. This randomized oil was analyzed and yielded a larger amount of residue which was estimated t o contain 0.4% trisaturated glycerides. The fatty acid analysis of the soybean oil indicated it had the following molar composition: 11.7% palmitic, 5.6y0 stearic, 28.4y0 oleic, 5o.1yO linoleic, and 4.2oj, linolenic. Thus it should contain about 0.5yo b y weight of trisaturated glycerides, if it were randomly distributed. Cottonseed oil also exhibited no detectable trisaturated glyceride. Its fatty acid composition n-as 1.1% myristic, 29.0% palmitic, 2.2% stearic, li.8y0 oleic, and 49.9y0 linoleic. By random distribution theory it should contain 3.470 trisaturated glyceride. Randomization of the cottonseed oil produced a detectable amount of trisaturated glyceride, but the value was still below that predicted by random theory. This is presumably because the randomization reaction was not carried to completion. Contrary to other highly unsaturated vegetable oils, peanut oil appeared t o have about 0.6% trisaturated glycerides. However, the high saponification value and low melting point indicate t h a t there is little of the long-chain trisaturated glyceride that would be expected in peanut oil. Additional experiments have indicated that the sample of peanut oil TT-hose analysis is given in Table I \vas adulterated with small amounts of coconut oil. The trisaturated glycerides from this peanut oil when converted to methyl esters and analyzed by gas chromatography gave a pattern typical of coconut oil. Other samples of peanut oil have given no detectable trisaturated glyceride. The results show that the mercaptoacetic acid method is capable of giving a reasonably accurate estimate of the trisaturated glyceride present in natural fats. Besides providing a method for
determining trisaturated glyceride, this method also gives a method for the quantitative isolation of trisaturated glyceride. However, the amounts of fat that may be handled conveniently are rather small. Also it is difficult to remove the last traces of unsaturated constituents from the trisaturated glyceride. Presumably the same method that has been applied to triglycerides could also be applied to methyl esters t o yield a measure of the saturated fatty acids present in a fat. illso it might be possible to get more information about the glyceride structure of fats by resolving the mercaptoacetic-glycerides into fractions based on the number of acid groups per molecule. LITERATURE CITED
(1) Axberg, G., Holmberg, B., Ber. deut. chem. Ges. 66B, 1193 (1933). (2) Bailey, A. E., “lfelting and Solidification of Fats,” p. 153, Interscience, New York, 1950. (3) Cama, J. S., Chakrabarty, hI., Hilditch, T. P., Meara, M.L., J . Sci. Food Agr. 4,321 (1953). (4) Chandlee, P., Eshelman, L. R.,
Pauls, J. F., Hammond, E. G., Bird, E. W., unpublished method. (5) Ec$y, E. R7., “Vegetable Fats and Oils, p. 146, Reinhold, Yew York, 1954. (6) Eshelman, L. R., Hammond, E. G., J . Am. Oil Chemists’ SOC. 35,230 (1958). ( 7 ) Fore, S. P., O’Connor, R. T., Goldblatt, L. A., Ibid., 35, 226 (1958). (8) Hilditch, T. P., “The Chemical Constitution of Katural Fats,” 1st ed., p. 405, Wiley, New York, 1940. (9) Ibid., p. 412. (10) Hilditch, T. P., Lea, C. H., J . Chem. SOC.1927, 3106. (11) ,Hoog, H., Eichwald, E., Xec. trav. chzm. 58, 481 (1939). (12) Hunter, L., IIyde, F., Analyst 58, 523 (1933). (131 Kartha. il. R. S.. J . Am. Oil Chemists’ ’ doc. 30, 280 (1953): (14) Knight, H. B., Jordan, E. F., Roe, E. T., “Biochemical Preparations,” Vol. 2, p. 100, Wiley, New York, 1952. (15) Koenig, N. H., Swern, D., J . Am. Chem. SOC.79, 362 (1957). (16) Lakshminarayana, G., Rebello, D., American Oil Chemists’ Society, October 1958. (17) Murty, K. L., Craig, B. AI., American Oil Chemists’ Society, October 1958. (18) Peterson, E. 9., Sober, H. A., J . Am. Chem. SOC.7 8 , 751 (1956). (19) Ralston, -4.W.,“Fatty Acids and Their Derivatives,” p. 551, Wiley,’New York, 1948. (20) Reiser, R., Dieckert, J. IT’., J . Am. 021 Chemists’ SOC.31, 625 (1954). (21) S_cholfield,C. R., Hicks, 31.-1., Zbid., 34, ( 7 (1957). (22) Ting, I., h’ew Zealand J . Sci. Technol. 29A, 240 (1948). (23) Wheeler, D. R., Riemenschneider, R. W.,Sando, C. E., J . Biol. Chem. 132, 687 (1940). RECEIVEDfor review August 17, 1959. Accepted March 1, 1960. Journal paper No. 5-3714 of The Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project 1128. Suppofted in part by a grant from The American Dairy Association. VOL. 32, NO. 7, JUNE 1960
0
847
