The following conclusions may be drawn from the examination of the ester fractions of the various oils in the preceding table.

The highly unsaturated acids would seem to contain 16, 18, 20 and 22 carbon atoms, the molecular weights of the tetra-unsaturated acids being 248, 276, 304 and 332, respectively. The lowest mean molecular weight determined was 266.8 (see below), which is 9 points lower than that for clupanodonic acid. The highest value found was 324.7, which is only 7 units below that for the C22 acid.

From the examination of the iodine numbers it will be seen that up to the last two fractions the unsaturation increases with the boiling point and molecular weight. The calculated iodine numbers of the methyl esters of the acids which may be present are: hexadecatrienoate, 288.5; clupano-donate, 350.1; arachidonate, 319.3; eicosapentenoate, 401.7; docosatetren-oate, 293.5; docosapentenoate, 369.0; docosahexenoate, 445.2.. Fraction 2 when slowly distilled into 3 sub-fractions gave a fraction having an iodine number of 316.5 and a molecular weight for its acids of 266.8, which data indicate a mixture composed of 2/3 methyl clupanodonate and l/3 methyl hexadecatrienoate. The data for Fraction 3 are very close to the theoretical values for methyl clupanodonate. The data for Fractions 6 and 7 indicate mixtures of methyl arachidonate and eicosapentenoate, while those for Fraction 9 indicate methyl docosatetrenoate, docosapentenoate and docosahexenoate.

The percentages of bromine in the polybromides of the various fractions show that the degree of unsaturation increases with an increase in the boiling point of the fraction and, therefore, with the molecular weight. One of the sub-fractions obtained from Fraction 2 and also Fraction 3 yielded bromides with 68.59 and 68.6% of bromine which are almost in exact agreement with the calculated value for methyl octobromostearate. This compound has not been previously prepared. It is a white amorphous solid which melts at 240°, uncorr.

The following percentage values for bromine content have been calculated: octobromostearate, 68.79; octobromo-arachidate, 66.78; decabromo-arachidate, 71.66; octobromobehenate, 64.90; decabromobehenate, 69.90; dodecabromobehenate, 73.72. Fractions 6 and 7 would seem from this also to consist of methyl arachidonate and eicosapentenoate, while Fractions 9 and 10 are probably chiefly methyl docosapentenoate. Since Fraction 10 yields a bromide which contains more bromine than Fraction 9, the former undoubtedly contains some of the methyl docosahexenoate. This is the principal evidence which we have of the presence of this acid.

The polybromide numbers of the fractions furnish little evidence of the nature of the fatty acids. The very important fact which should be observed here is that they are only about 1/3 of the theoretical value, which is always in the vicinity of 300.

On the basis of the analyses of Fraction 3, it is believed that this fraction is almost pure methyl clupanodonate. It is further believed that this represents the first time that a derivative of clupanodonic acid has been prepared in a reasonably pure state. The constants of methyl clupanodonate based on this sample are, iodine number (Wijs) 348.8; b. p. (15 mm.) 215°; n20, 1.4860. The octobromide is described above.

The data in Table VII for cod and herring oils offer no evidence to contradict our conclusions based on the more complete distillation of the esters of menhaden oil, but rather confirm most of these data. The poly-bromide numbers of the mixed esters of the 3 oils are 105.3, 125.8 and 113.3, values which are about 1/3 of those calculated.

The Nature Of The Bromination Reaction

A low yield of polybromides upon bromination of the pure, highly unsaturated esters of linolenic and linolic acids has been reported by Rollett17 and Erdmann.18 The most satisfactory explanation of the liquid bromides is that perhaps they are isomeric soluble bromides with correspondingly different properties. To furnish further light on this problem the following experiment was carried out. Starting with menhaden oil, the methyl esters were prepared and these reduced to the esters of the unsaturated acids. Then, under quantitative conditions, the supposedly pure unsaturated acids were prepared by saponification and brominated, and both the liquid and solid bromides weighed, reduced and again brominated. Suitable analyses were made at each step. The diagram, Table VIII.

Table VIII Quantitative Bromination And Reduction Of Menhaden Oil Esters

1900 g. of menhaden oil → 1337 g. of methyl esters → 500 g. of polybromides (68.62% of bromine) → 47 g. of methyl esters → 40 g. of fatty acids. Iodine number, 360.5. Molecular weight, 304.2

The acid obtained here represents that from an additional 7 g. of solid bromides from the same point in a parallel experiment shows the result of this experiment. Although no final conclusion may be drawn from these data, the following suggestions may be made.

The liquid bromides seem to contain in general considerably less bromine than the solid. When reduced, the former give a product with an iodine number considerably less than the original acid, while the molecular weight is only slightly less. These in turn yield only a small amount of solid bromides on bromination. The low bromine content of the liquid bromides and the decrease in iodine number suggest a change in the degree of unsaturation.

17 Rollett, Z. physiol. Chem., 62, 410, 421 (1909).

18 Erdmann and Bedford, Ber., 42, 1324 (1909).

There seem to be four possibilities of change here. (1) One double bond may have been saturated during the reduction. (2) There may have been a rearrangement leaving one of the unsaturations in a position where it cannot be brominated, since it is known that an α-β unsaturation causes a reduction in the iodine number of an acid. (3) Oxidation may have occurred. (4) A double bond may have disappeared by ring formation as from a 1-5 di-ene grouping. The second and fourth possibilities are the * most reasonable. In support of the second it has been noticed repeatedly in this Laboratory that at 0° bromine is not added to the unsaturated acids with the same ease as iodine monochloride or monobromide in attempts at the determination of the bromine absorption values of drying vegetable and fish oils. In support of the possibility of ring formation we may recall the fact that when the highly unsaturated esters of cod oil were accidentally overheated, there was a rapid lowering of the iodine and polybromide numbers with no change in molecular weight. Bromination might catalyze a similar change.

Whatever may be the answer to the question of the liquid bromides, apparently 1/3 or less of the ester brominated goes to form the normal ether-insoluble bromides, while the other 2/3 forms a product with 4 to 8% less bromine, which is soluble in ether. The liquid bromides on debromination yield a product from which a smaller proportion of poly-bromides may be obtained on rebromination in ether solution.

Presumably the material soluble in ether after bromination consists of two portions: the solid bromides which are dissolved in the ether solution of the liquid bromides, and the true liquid bromides, either isomeric with the others or formed from altered unsaturated acids.