The solution of the barium salts of fraction 2 was concentrated by evaporation, and to a portion excess of H2SO4 was added, the BaSO4 filtered off, and the filtrate carefully neutralised with ammonia and a portion added to a solution of neutral ferric chloride. It was coloured dark red, and a finely-divided precipitate of basic acetate of iron came down on boiling. A second portion of the concentrated solution was heated with sulphuric acid and gave off a strong smell of acetic acid. To a third portion silver nitrate was added, and a slight excess of acetic acid; a little silver was deposited in the form of a film, showing a trace of formic acid.

Having thus shown the presence of butyric, acetic, and formic acids in the drench, we proceeded to ascertain the quantities formed in a normal fermentation of bran without skins. For this purpose a drench was made in a clean vessel with 10 litres of distilled water and 200 grm. of bran mashed at a temperature of 38° C., and, after cooling to 33°, inoculated with bacteria from an actual drench which was fermenting vigorously; this was kept in the drench house so that the fermentation and general conditions might be exactly similar. In 48 hours all gases had ceased to be evolved, and the true fermentation was at an end. The liquid had an acid, not unpleasant smell, and was acid to litmus paper. The bran was strained off through muslin, washed with a little water, and well squeezed; the liquid measured 10 litres. Three litres of this were taken for the separation and estimation of the volatile acids, the remainder being set aside for examination and estimation of the non-volatile acids, etc. Two of the three litres were placed in a distilling flask with 5 grm. of pure CaCO3, and distilled down to one litre. The distillate was alkaline to litmus, and had a peculiar fishy smell; the remaining litre of drench was added, and the liquid taken down until the distillate ceased to be alkaline and only bad a faint smell. The alkaline distillate gave a yellow precipitate with Nessler’s solution, as well as the following reactions:—

AgNO3A faint brown precipitate.
HgCl2A yellowish-white curdy precipitate.
PbAA brownish-white precipitate.
CuSO4A dirty blue precipitate, which changed to a brownish turbidity on boiling.

HCl was added to the distillate in slight excess and the liquid evaporated to a small bulk; to a portion chloroform and alcoholic potash were added, and the liquid heated; no smell of isocyanides was given off; the body is, therefore, not a primary amine. Phosphomolybdic acid gives no precipitate, therefore the body is not an alkaloid. From the above tests and its characteristic smell, we conclude that the body is trimethylamine. The platinum salt was formed by evaporating the above concentrated solution of trimethylammonium hydrochloride with excess of platinum chloride, a precipitate of the platinum salt insoluble in alcohol being formed. There was not, however, a sufficient quantity from the three litres to ascertain the molecular weight.

Proceeding with the estimation of the volatile acids, 100 c.c. N HCl being required to completely neutralise the CaCO3 used, 50 c.c. were first added and the distillation continued; the distillate was only very faintly acid. Four fractions were now distilled off, using respectively 10, 10, 10, and 20 c.c. N HCl.[165]

Fraction 1 was boiled with excess of barium carbonate, filtered, the BaCO3 washed with hot water, and the filtrate evaporated to dryness, and the Ba salts dried at 130° C. till the weight was constant; the salts were then decomposed with strong sulphuric acid, ignited, and the barium sulphate weighed.

Fractions 2, 3, and 4 were treated in an exactly similar way, the barium salts obtained and the weight and percentage of barium sulphate being shown in the following table:—

Fraction

Weight of
Salts

Weight of
BaSO4

Per Cent
BaSO4

I.

0·55850·499189·365

II.

0·50600·461891·265

III.

0·316750·2941592·86

IV.

0·44750·425295·02

Calculating the fractions 1 and 2 as mixtures of barium butyrate and acetate, and fractions 3 and 4 as mixtures of barium acetate and formate, we may summarise the results thus:—

Fraction

Weight of
Salt

Ba
Acetate

Ba
Butyrate

Ba
Formate

I.

0·55850·49040·0681

..

II.

0·50600·50300·0030

..

III.

0·316750·2749

..

0·04185

IV.

0·44750·2629

..

0·18460
  Total 1·82875 1·53120·07110·22645