THE OCCURRENCE OF SOME PREVIOUSLY UNREPORTED FATTY ACIDS IN PEANUT OIL

THE OCCURRENCE OF SOME PREVIOUSLY UNREPORTED FATTY ACIDS IN PEANUT OIL BY HELEN L. WIKOFF, JOSEPH M. KAPLAN, AND ALVIN L. BERMAN (From the Department of Physiological Chemistry, The Ohio State University, Columbus) (Received for publication, January 12, 1944) One of the early investigations of peanut oil was that made by Jamieson, Baughman, and Brauns (l), who examined two types of crude oil which they had expressed from Virginia and Spanish types of peanuts, respectively. They isolated lignoceric, arachidic, stearic, and pahnitic acids and identified these products by melting point determinations and elementary analyses. The presence of oleic and linoleic acids in the original oil was established by the bromine content of the respective bromine addition products of the two acids. Holde, Bleyberg, and Rabinowitsch (2), working at a later date, claimed to have isolated behenic acid from peanut oil and reported the presence of palmitic, stearic, arachidic, lignoceric, and carnaubic acids, as well. Longenecker (3), using peanut oil to test a fractionation equipment, reported the presence of myristic and pahnitoleic acid in peanut oil, together with the acids noted by the previous investigators. Longenecker based the identification of the compounds which he reported on the iodine numbers and saponification equivalents of the fractions separated. In order to determine the nature of spoilage which had occurred in a large consignment of peanut butter, a study was made of commercial peanut oil. For this purpose w-e purchased an edible grade of peanut oil having an iodine number of 90.7, a saponification number of 193.3, and a refractive index of 1.45, at 20. The methyl esters from 9.5 kilos of peanut oil were prepared by refluxing in 1.5 kilo portions on a water bath 14 to 15 hours with 4 liters of methyl alcohol containing gaseous hydrogen chloride. After the mixture of esters had been cooled and washed three times with 5 or 6 liters of water, the last traces of which were removed under reduced pressure, 8956 gm. of methyl esters remained and were fractionally distilled four times at 15 mm. pressure. In the first three distillations, fractionations were made at 20, lo, and 5 intervals, respectively; a fourth refractionation, also at 5 intervals was made to obtain a sharper separation of esters. During the first two fractionations, distillation progressed at a rate such that the drops could just be counted as they fell into the receiving flask. During the last two refractionations, the rate was about 100 drops a minute. The final distillation yielded thirty-eight ester fractions, the 227

228 FATTY ACIDS IN PEAKUT OIL first of which boiled below 85 at 15 mm. pressure, and the last had not distilled at 265 under 15 mm. pressure. Since we could find no report of any low boiling acids in the esters present in peanut oil, we decided to examine the material more thoroughly in order to determine whether such compounds had escaped the notice of previous workers. EXPERIMENTAL Iodine and saponification numbers obtained for each of the thirty-seven fractions distilled from the crude esters comprise the data in Table I. The weights of the fractions plotted against fraction numbers are the basis of Fig. 1. Because the amounts of material in Fractions 23 to 28 were so great, Curve 2, in which the scale of weights is one-fiftieth that of Curve 1, was substituted in that area. A series of peaks at the maximum concentrations of the esters of the various acids present occurred at appropriate places for the esters of CS, C~Z, CH, C18, GO, and Cn acids. To show the distribution of the unsaturated acids, the iodine numbers were plotted against fraction numbers (Curve 1, Fig. 2). In Curve 2, the iodine absorption values were plotted against the fraction numbers; Curve 2, including that portion of Curve 2 which would have gone off the scale, shows the iodine absorption figures reduced to one-five-hundredth of their true values. The large peak in Curve 1 occurring for Fraction 26 is very indicative of unsaturated Cl8 acids; the peak at the same point in Curve 3 strengthens this possibility. Bromination of portions of the various fractions in diethyl ether containing a small amount of glacial acetic acid did not result in the formation of any ether-insoluble material. Unsaturat,ed acids containing more than two double bonds are, therefore, not present in the oil. Since only very small amounts of material mere present in several of the lower fractions, an additional 15.5 kilos of peanut oil were esterified and fractionally distilled. The use of a constant pressure regulator resulted in a sharper separation of fractions, so that no material distilled between 100-115. Isolation and IdentiJcation of Fatty Acids Saturated Acids. Caprylic Acid-The presence of caprylic acid in the fractions distilling below 100 was established as follows: Those fractions prepared without the use of the constant pressure regulator were combined and the saturated acids separated by distillation after bromination in ether, followed by removal of excess bromine as well as the ether. The esters so obtained distilled at 83 under 15 mm. pressure. This is the boiling point for methyl caprylate as found by Lewko-

WIKOFF, KAPLAN, AND BERMAN 229 witsch (4) and Jamieson (5). Those fractions prepared with the constant pressure regulator were combined and subjected to the lead-soap-ether method for removing unsaturated material. A saturated acid thus iso- TABLE I Some Constants of Fractions Made at 6 Intervals (16 Mm. Pressure) of Methyl Esters from Peanut Oil - Fraction No. B.p. 15 mm. Weight I No. odineabsorptioc L n per fraction _ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 85 85-90 go- 95 95-100 100-105 105-110 110-115 115-120 E&125 125-130 130-135 135-140 140-145 145-150 X0-155 155-160 X0-165 165-170 170-175 175-180 HO-185 185-190 190-195 195-200 200-205 205-210 210-215 215-220 220-225 225-230 230-235 235-240 240-245 245-250 250-255 255-260 260-265 Em. 16.7 14.5 6.5 2.0 2.5 1.2 1.7 7.0 10.5 13.5 22.0 69.0 67.5 54.5 46.0 16.0 5.0 17.5 14.1 18.0 24.7 53.5 158.0 396.0 1657.5 4107.7 870.8 107.4 48.2 31.0 15.2 17.0 29.2 43.0 29.8 61.8 32.5 1.7 1.3 2.1 m. 0.2839 0.1885 0.1365 1.5 0.105 1.9 0.1995 2.5 0.3375 1.5 0.360 1.7 1.173 2.4 1.62 2.5 2.725 2.7 0.702 3.5 0.175 4.1 0.08 17.1 1.2425 23.0 3.243 29.1 5.238 37.0 9.139 35.8 20.153 43.4 68.572 61.1 241.956 83.2 1379.04 106.3 4366.4851 103.5 901.278 91.4 98.1636 80.5 38.801 70.7 21.917 56.0 8.512 43.6 7.412 31.5 9.198 22.6 9.71 11.9 3.55 6.2 3.83 4.9 1.59 141.3 147.9 150.1 176.6 180.5 185.4 193.1 200.8 w1.0 202.8 209.6 209.7 210.4 219.1 224.5 234.6 237.3 244.3 252.2 262.7 271.3 277.5 277.9 279.6 287.8 294.0 301.1 304.6 317.0 322.3 339.3 341.o 354.6 Not determined.

/ Y h/ I I \,Y \ \ I \ / \ / \ 123456789/O 12 /4 /6 18 20 22 24 2% 2% 30 32 34 3637 CO Go c/z G4 C/G Cl8 C?O G2 Fraction Num her FIG. 1. Curves derived from the weights of the several fractions I/.? I I I 1 I= Iodine Number,\ 2: Iodine Absorp. per fraction - Gram I \\ 96-3=Iodine AbJorp. per Frocf/on - Gram I I I I I- _ 5 i: ;a- - =A G.? C/4 G c/e GO Gz Fraction Number FIQ. 2. Curves derived from the iodine numbers of the several fractions 230

WIKOFF, KAPLAN, AND BERMAN 231 lated, after several recrystallizations from 95 per cent ethyl alcohol, possessed the following constants: melting point 15.7, melting point of caprylic acid 16.5 ; molecular weight 147.6, molecular weight of caprylic acid 144.12. La&c Acid-Although the absence of capric acid seemed probable from the appearance of the curve (Fig. l), nevertheless, the fractions boiling between 115-130 were investigated for the presence of this acid. The saturated esters were separated from unsaturated material by bromination of the combined fractions and distillation as in the case of the acids previously described. The saturated material thus separated possessed the characteristics shown in Table II. Inspection of the data for the material boiling below 125 reveals that the melting points are too low and the molecular weights too high for capric acid. The melting point and molecular weight of the material distilling between 125-130 agree closely with the values for lauric acid. It is, therefore, probable that the small amount of material originally Observed for acid isolated TABLE II Identijication of Laurie Acid Fraction boiling (15 mm. pressure) at Accepted values 113-120~ 120-125~ 125-130 Capric acid Laurie acid M.p., C.................... 16.0 26.5 43.4 31.3 45.6 Mol. wt............. 176.1 191.6 199.01 172.15 200.19 distilling between 100-125 at 15 mm. consisted of a mixture of CS and Cl2 saturated acids. The presence of lauric acid previously suspected in Fractions 12 to 15, because of the characteristic peak in the curve (Fig. l), was definitely established by isolating the free acid from the esters and recrystallizing several times from 95 per cent alcohol. The acid so prepared possessed the following constants: observed melting point 43.5, melting point of lauric acid 43.6 ; observed molecular weight 201.9, molecular weight of lauric acid 200.19; iodine number 0.8, iodine number of lauric acid 0.0. Myristic Acid-The presence of myristic acid in the fractions boiling between 155-175 was suspected from the appearance of the curve (Fig. l), and was established in the following manner. The saturated material was separated from any unsaturated compounds present by the lead-soapether method. The acid isolated from the ether-insoluble lead-soap possessed the following constants when recrystallized several times from 95 per cent alcohol: observed melting point of the lead salt 108.0, melting point of lead myristate 107.0 ; observed melting point of the acid 53.5,

232 FATTY ACIDS IN PEANUT OIL melting point of myristic acid 54.0 ; observed molecular weight of the acid 228.9, molecular weight of myristic acid 228.2; observed iodine number 1.5, iodine number of myristic acid 0.0. Palmitic Acid-Pahuitic acid was isolated from the combined methyl esters boiling between 175-190 as well as from those boiling between 190-195 and 195-200 by the lead-soap-ether separation. The fatty acid so obtained possessed the constants shown in Table III after several recrystallizations from 95 per cent alcohol. TABLE III Zdentijcation of Palmitic Acid M.p. of lead salt, C..... 114 acid, C... 62 Mol. wt. of acid... 257.2 I No.of acid... 1.89 TABLE IV Identification of Steak Acid M.p. of lead salt, C... 118 1 I acid, C... 65 Mol. wt. of acid... 279.9 I No.of acid... 1.6 Fraction boiling at Accepted values for 175-1909 1 190-195~ 1 195-200 pahitic acid 110-111 110 112 62.1 61.9 62.6-63 256.4 257.4 256.3 1.07 3.75 0.0 Fraction boiling (15 mm. pressure) at 200-205~ 205-210~ 210-215~ 67 62.1 284.4 292 0.45 4.2 Accepted values for stearic acid 115.6 69.6 284.3 0 Undoubtedly, the peak for the CM saturated acid occurs in the fraction boiling between 190-195, for it is in this fraction that the closest agreement occurs. The saturated acid from the fraction boiling between 195-200 probably contains small traces of stearic acid. Stearic Acid--Rear% acid was present in several of the fractions but the best preparation was isolated by the lead-soap-ether method from the methyl esters boiling between 205-210 at 15 mm. The fatty acids so obtained (or their derivatives) possessed the constants given in Table IV after several recrystallizations from 95 per cent alcohol. Arachidic Acid-The presence of arachidic acid was definitely established in the fraction distilling between 215-220 at 15 mm. after separation by the lead-soap-ether method and also after low temperature crystallization of the methyl esters from acetone. Although stearic acid was present in

WIKOFF, KAPLAN, AXD BERMAN 233 the same fraction, the arachidic acid crystallized first from ethyl alcohol. The best arachidic acid preparation was that obtained by redistilling, at 5 mm. pressure, portions of the material boiling above 230 at 15 mm. The fraction boiling at 205-210 at 5 mm. pressure, when subjected to low temperature crystallization, yielded arachidic acid of high purity. Constants for the acid obtained from these fractions are given in Table V. Behenic A&-&-Behenic acid was identified definitely in the methyl esters distilling between 245-250 and 250-255 at 15 mm. It is probable that the mixture distilling between 240-245 at 15 mm. contained a mixture of arachidic and behenic esters, since after repeated attempts to purify the material by distillation at 0.3 mm. pressure, a melting point of 77.5 and TABLE V Identification of Arachidic Acid M.p. of acid, C... 74 76-77 I methyl ester, C.... 47.5 46.5 Mol. wt. of acid... 301.8 309.3 I No. of acid... 5.21 1.9 TABLE VI Identification of Behenic Acid 240-245, 1.5 mm. T Fraction boil- Fraction boiling ~~2~220, ing at 205210, 5mm. Fraction boiling at 245-ZSO, 2s2550, 15 mm. 15 mm. Accepted values for arachidic acid 77 46.5-47.5 312 0 Accepted values for ZlO-2200, behenic acid smm. M.p. of acid, C.............. 77.5 79 79.5 79.1 80.2 I I ester, C............. 49.540 49.7 50 49.6-50 50 Mol. wt. of acid............ 328 336 339 339.2 340 I No. of acid............ 7.8 0.9 0.5 2.47 0.0 a molecular weight of 328 were obtained for the acid. The purest acid was prepared from the fraction distilling between 210-220 at 5 mm. (obtained by redistilling at 5 mm. pressure portions of the material boiling above 230 at 15 mm.). Constants for the acids obtained from these fractions are given in Table VI. L&noceric Acid-Lignoceric acid was probably present to a slight extent in several fractions. The best preparation was obtained from the esters distilling between 260-265, at 5 mm. pressure. The material was fractionally crystallized from 95 per cent alcohol and again recrystallized several times after the acid had been freed from the esters. The constants obtained for these acids are given in Table VII.

234 FATTY ACIDS IN PEANUT OIL Unsaturated Acids. 9-10 O&c Acid-9-10 oleic acid was identified in several of the fractions beginning with the methyl esters distilling between 190-195 at 15 mm. pressure. I %0 general methods were used for the isolation of oleic acid: (a) separation by the lead-soap-ether method, followed by further purification by the barium-soap-benzene process; (5) TABLE VII Identijication of Lignoceric Acid Fraction boiling at 260-265, 15 mm. Mol.wt... 365 M.p.of acid, C... 83.1 I methyl esters, C... 55 I No... 0.49 TABLE VIII T Fraction boiling at 220-230, 5mm. 367 82.9 54.5 1.95 Constants of Oleic Acid and Derivatives Isolated from Various Fractions Accepted values for lignoceric acid 368.4 84.2 50.7 0 Acid isolated from fraction boiling (15 mm. COrrF pressure) at %te romy 190-1950 195-200 200-205 205-210 210-215 -- Mol. wt. of acid............. 283.5 259.5 284.7 282.29 266.03 282.0 I No. of acid... 88.45 87.04 93.3 102.6 83.47 89.93 M.p. of dihydroxy derivative.. 128 128.6 128.8 130 Mol. wt. of dihydroxy derivative... 314.7 315.1 315.2 316.0 TABLE Constants of Linoleic Acid and Derivatives Isolated from Various Fractions IX Acid isolated from fraction boiling COrE- (15 mm. pressure) at spmding values for 200-205 205-2100 210-215 215-220 220-2250225-2300 limtic -- ---- Mol. wt. of acid.. 289.9 243 292.5 273.5 277 282.1 I No.of acid... 154.7 141.2 141.1 140.2 112.8 113.2 181 M.p. of tetrabromide, C.. 113 111 113.5 113.0 113.5 114 low temperature crystallization of the methyl esters from an 8 per cent solution in acetone. Identification was made by means of the molecular weight, iodine number, and preparation of the crystalline dihydroxystearic acid by oxidation with alkaline permanganate solution (Table VIII). Linoleic Acid-Linoleic acid was identified in several fractions beginning

WIKOFF, KAPLAN, AND BERMAN 235 with the methyl esters distilling between 200-205 at 15 mm. pressure. Two general methods were used for the isolation of the linoleic acid: (a) separation by the barium-soap-ether method; (b) low temperature crystallization of the methyl esters from an 8 per cent solution in acetone. Identification was made by preparation of the crystalline tetrabromide, since in no case was it possible to isolate a pure preparation of the linoleic acid (Table IX). Although slight peaks occurred in the iodine absorption curve (Fig. 2) at the ranges 145-150 and 245-250 at 15 mm., we were unable to isolate additional unsaturated fatty acids from this material. The fractions boiling between 175-200 at 15 mm. were carefully examined for the presence of pahnitoleic acid previously reported by Longenecker (3). However, a redistillation of this material at 0.3 mm. pressure yielded oleic acid as the only unsaturated material present. SUMMARY 1. Caprylic and lauric acids were isolated for the first time from peanut oil. 2. The presence of myristic, palmitic, stearic, arachidic, behenic, lignoceric, oleic, and linoleic acids, previously reported as occurring in peanut oil, was confirmed. 3. The oleic acid found was the 9-10 isomer. BIBLIOGRAPHY 1. Jamieson, G. S., Baughman, W. F., and Brauns, D. H., J. Am. Chem. Sot., 45, 1372 (1921). 2. Holde, D., Bleyberg, W., and Rabinowitsch, I., Ber. them. Ges., 62, 177 (1929). 3. Longenecker, H. E., J. Sot. Ckm. Id., 56. T 310 (1937). 4. Lewkowitsch, J., Chemical technology and analysis of oils, fate and waxes, London, 6th edition, 1, 157 (1921). 5. Jamieson, G. S., Vegetable fate and oils, New York, 287 (1932).

THE OCCURRENCE OF SOME PREVIOUSLY UNREPORTED FATTY ACIDS IN PEANUT OIL Helen L. Wikoff, Joseph M. Kaplan and Alvin L. Berman J. Biol. Chem. 1944, 153:227-235. Access the most updated version of this article at http://www.jbc.org/content/153/1/227.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/153/1/227.citation.full.h tml#ref-list-1