From the facts mentioned above it is quite probable that the clupanodonic acid previously described in the literature was not a pure compound, but rather a mixture of a number of acids. Accordingly a number of attempts were made to separate the fatty acids of menhaden oil, the most successful procedure being applied to cod and herring oils.

Separation By Fractional Distillation

The esters from 3 kg. of menhaden oil obtained in the previous experiment were fractionated 6 times. The pressure during each distillation was maintained at 15 mm. and on the last distillation the fractions were collected in such a way that certain ones would represent as far as possible only acids of the same carbon content. Although the intermediate fractions had such properties as to indicate that the principal fractions were not chemical individuals, the boiling point, index of refraction, molecular weight and iodine number indicated the predominance in each of these fractions of acids of a certain group.

Table IV. Results Of Analysis Of The Fractions From The Sixth Distillation Of The Methyl Esters From Three Kilograms Of Menhaden Oil

From the series of analyses just tabulated the following conclusions may be drawn: (1) It is possible by fractionation of the methyl esters to make a rough separation of acids according to molecular weight. (2) The important acids have a carbon content ranging from C14 to C22. Those of the lower molecular weights are largely saturated. (3) The iodine numbers and mean molecular weights of the fractions rise rapidly with the boiling points. The index of refraction of the esters was also found to increase regularly. When the index of refraction was plotted against iodine numbers practically a straight line was obtained, the main divergence being with the last point. The same general relationship is seen when the index of refraction is plotted against molecular weight, the breaks at the beginning and end of the series giving the graph the form of the sign of integration. (4) The polybromide number of the esters rises with the boiling point, while the bromine content of the derivatives also increases. Unsaturation undoubtedly increases with an increase in molecular weight.

Separation By Metallic Salts

It is apparent from the data previously given that a number of highly unsaturated acids occur in fish oils. It is furthermore probably true that no one has succeeded in isolating any of them in the free state. The clupanodonic acids of Tsujimoto and of Riedel, previously referred to, were undoubtedly complex mixtures. Various methods have been proposed for the separation of the saturated and unsaturated fatty acids. Most of the methods depend upon differences in the solubility of metallic salts of the unsaturated acids in organic solvents, those of the unsaturated acids being most soluble. Such separations are not quantitative, due to the tendency of the slightly unsaturated acids to follow the saturated acids in their behavior, and also to an apparent mutual solubility of the salts of the two types, one in the other.

The Lead Soap-Ether Method

Five hundred g. of refined menhaden oil (iodine number 183.9; saponification number 191.8) was treated according to the usual procedure for the lead soap-ether method 14,15 using only such modifications as were necessitated by the large size of the sample. The lead salts so separated were decomposed as usual with hydrochloric acid and the solid and liquid acids examined. The liquid acids weighed 300 g., showed an average molecular weight of 328.6 (the abnormally high value is probably due to the presence of a small amount of retained ether) and an iodine number of 237.9; whereas the solid acids weighed 150 g. and showed a molecular weight of 264.6 and an iodine number of 55.3. The liquid acids were then converted into their methyl esters and distilled under 15 mm. pressure.

Table V. The Distillation Of The Methyl Esters Of The Unsaturated Acids Of Menhaden Oil As Obtained By The Lead Soap-Ether Method

This distillation was twice repeated, when the fraction boiling at 235-250° had an iodine number of 334.1 and a molecular weight of 326.5. Since, however, the lower fractions gave evidence of the presence of saturated acids as impurities and since the method applied to large quantities was so tedious, further investigation by this procedure was discontinued.