or as free acids:—
Fraction | Acetic | Butyric | Formic | |
I. | 0·2308 | 0·0385 | .. | |
II. | 0·2367 | 0·0017 | .. | |
III. | 0·1293 | .. | 0·0170 | |
IV. | 0·1237 | .. | 0·0748 | |
| Total | 0·7205 | 0·0402 | 0·0918 | |
Proceeding now to (3) non-volatile bodies, we find these to consist of (1) acids, and (2) soluble carbohydrates. For examination, the remaining 4 litres of experimental drench were evaporated down to 1 litre and filtered; the residue (consisting of starchy matter) was well washed and the washings added to the filtrate, the whole placed in a large distilling-flask and distilled with continued addition of distilled water until the distillate was no longer acid (this required the addition and distillation of 4 litres of distilled water). A further deposit of solid matter rendering boiling dangerous, the liquid was again filtered and the residue washed free from acid. This residue was found to consist of nitrogenous organic matter and calcium phosphate, together with a trace of calcium oxalate, both being derived from the bran.
The clear liquid containing the non-volatile acids and other bodies was further concentrated and made up to 500 c.c.; it was dark brown in colour and was very acid to litmus; owing to a trace of flocculent matter, it was again filtered.
The presence of lactic acid was shown in the following manner: 10 c.c. of the liquid were placed in a small distilling-flask along with 2 c.c. strong H2SO4 and about 0·5 grm. potassium chromate in a little water. This was distilled and the vapours received in a test-tube surrounded by cold water; on adding magenta solution, decolorised by SO2, to the liquid in the test-tube, a red colour was produced by the aldehyde formed from the lactic acid; aldehyde was also recognised by its smell. We find this an exceedingly delicate test for lactic acid, and as far as we know it is quite new in this form.
For 10 c.c. of liquid to be examined, we find 2 c.c. strong H2SO4 and 1 grm. of potassium chromate to be the best proportions. Formic, acetic, propionic, butyric, valerianic, succinic, malic, tartaric, and citric acids do not give the reaction.
The liquid was now tested for succinic and malic acids. 25 c.c. was taken and decolorised by treatment with pure animal charcoal for half an hour; the liquid was then filtered, the charcoal washed free from acid, and the filtrate concentrated; ammonia was added in slight excess, the precipitate of calcium phosphate filtered off. To the filtrate CaCl2 was added in slight excess to remove the remainder of phosphates, the liquid filtered, and the filtrate cautiously neutralised with HCl; the addition of neutral ferric chloride to a portion gave no precipitate, showing the absence of succinic acid. To the remainder an equal volume of absolute alcohol was added and the liquid boiled; there was no precipitate. Twice the volume of absolute alcohol was then added and gave no precipitate. On the addition of four times the volume of absolute alcohol, a slight white precipitate came down, which evidently consisted of dextrin, from the manner in which it settled round the sides of the tube, and from its insolubility on adding HCl. We therefore conclude that there is no malic acid present, and that the only non-volatile acid produced is lactic acid.
The acidity was first determined by titrating 10 c.c. of the solution with N/10 sodium hydrate, using glazed litmus paper to determine the point of neutralisation. The 10 c.c. required 6·66 c.c. N/10 NaOH, equivalent to 0·666 of N lactic acid = 0·7481 grm. per litre of original drench.
In order to separate the lactic acid from the colouring matter and other bodies (dextrin and soluble starch) present, 100 c.c. of the concentrated liquid was decolorised with 10 grm. of animal charcoal, the mixture filtered and the charcoal well washed, the filtrate evaporated to dryness, and 15 drops of N/10 H2SO4 added to decompose any salts of lactic acid present; this was now extracted with ether, the ethereal extract of lactic acid placed in a distilling-flask, the ether distilled off, and the residue boiled with distilled water and pure calcium carbonate. The small amount of calcium sulphate present was removed by boiling the filtrate from the CaCO3 with barium carbonate, and filtering. The filtered liquid now consisted of a solution of calcium lactate; it was evaporated to dryness in a platinum dish, and the residue dried at 110° C.; it was then washed first with ether, then absolute alcohol.[166]