The application of the barium soap-benzene method16 was attempted. This is based on the solubility of the barium soaps of fatty acids with more than one double bond in cold benzene containing about 5% of 95% alcohol, added to bring a trace of moisture into the benzene, while the barium salts of the acids of the oleic and the saturated series will dissolve in the hot benzene-alcohol but crystallize on cooling.

14 Gusserow, Ann., 27, 153 (1828).

15 Varrentrapp, ibid., 35, 197 (1840).

16 Farnsteiner, Z Nahr. Genussm., [2] 1, 390 (1898).

The sodium soaps of menhaden oil were neutralized with acetic acid and precipitated in the cold with an excess of barium chloride solution. The soaps so precipitated were filtered on a Biichner funnel, and when transferred to a large flask and heated on a steam-bath for a few minutes so agglutinated and shrunk that practically all of the occluded water could be decanted. The soaps were then treated with the benzene-alcohol mixture and refluxed for 30 minutes on a steam-bath. The solvent was poured off and the residual soaps were treated with a fresh portion of the solvent. This treatment was repeated until the soaps were completely disintegrated and practically all dissolved. The combined solutions were then allowed to cool overnight, and filtered from the crystalline precipitate. About 8 liters of solvent was required for 500 g. of the oil. The acids from both filtrate and precipitate were recovered by decomposing the soaps with hydrochloric acid and distilling the benzene. The last traces of benzene were removed by heating in a vacuum. The results obtained in the first trial distillation of 500 g. were so satisfactory that 3 additional distillations were made, the products being combined for analysis.

One kg. of these liquid acids was converted into their methyl esters and distilled thrice under 15 mm. pressure. A special Claisen flask was made of Pyrex glass for this distillation by Mr. Paul Anders. The flask had an especially wide neck and side tube, to prevent the liquid from bumping over, and the side arm was inserted a short distance into the upright side tube in order to prevent esters condensing on the stopper from running into the receiver. The side arm was also especially long so that it might be surrounded by a small Pyrex condenser while it conducted the distillate directly into a Raikow receiver which permitted five fractions to be taken without breaking the vacuum. The rubber stoppers were protected from the hot ester vapors by pinning a thin sheet of cork over their lower surfaces.

Table VI. Analytical Constants Of The Methyl Esters Of The Liquid Acids Of Menhaden Oil Obtained By The Barium Soap Separation, After Three Distillations

Fractions 8-12, boiling over 5° ranges from 235-260°, had mean molecular weights ranging from 304 to 336 and iodine numbers for the esters ranging from 316 to 348. Fraction 8 gave values corresponding to the theoretical values for the methyl ester of arachidonic acid. Probably the higher-boiling fractions contain docosapentenoic and docosahexenoic acids.

The polybromide numbers of Fractions 8 and 11 were determined by the following procedure.

Place 1-2 g. of the ester in a weighed 50cc. centrifuge tube and add 35 cc. of anhydrous ether. Place the tube in a cooling bath kept below 0° and add bromine, with vigorous stirring to a distinct excess or until the solution is colored red. Allow the tube and contents to stand in an icebox overnight in the dark, centrifuge, decant the ether, and wash the precipitate 4 times by centrifuging with 40cc. portions of ether. Dry the tube and contents at 60° for 2 hours and weigh. The weight of bromides, divided by the weight of ester and multiplied by 100, gives the "polybromide number." In order to determine the nature of the residual acids in the ether filtrates, they were shaken with sodium thiosulfate solution to remove the excess of bromine, then dried with calcium chloride, the ether was allowed to evaporate and the residue finally dried in a vacuum oven at 60°.

Fraction 8 showed a polybromide number of 97.90, giving 69.61% of bromine in the bromides, and 56.56% of bromine in the ether-soluble portions, while for Fraction 11 the polybromide number was 101.13 and the bromine percentages 70.07 and 56.05 respectively.

Fraction 8, above, could not be pure methyl arachidonate since this would yield a bromine derivative with 66.78% of bromine, whereas the derivative formed actually contained 69.61%. If this fraction contained a considerable quantity of methyl docosapentenoate, which gives a polybromide with 71.66% of bromine, such results might be obtained. However, the substitution of this acid in part or entirely would give iodine numbers which would be much higher than those obtained. It is more probable that the fraction is a mixture of methyl arachidonate and methyl docosapentenoate with small quantities of the esters of less unsaturated acids.

An examination of the ether-soluble bromides leads to one of two conclusions: either the liquid bromides are not completely saturated with bromine, because of some sort of steric hindrance, or they are isomeric liquid bromides mixed with esters of the saturated acids or with bromides of less unsaturated acids.