This was contained in a narrow-necked litre flask fitted with a rubber stopper, and narrow delivery tube dipping under mercury, and sterilised with all the usual precautions. The fermentation began on the second day, reached its height from the 6th–8th day, and continued for 39 days, when gas ceased to come off. The examination of the gases will be described later on. When the fermentation was over, the liquid was brought to boiling temperature. It was then examined for the volatile acids in exactly the same manner as we described in our previous paper. 140 c.c. normal HCl was added and distillation commenced; the distillate was acid. The distillation was continued until the distillate ceased to be acid, forming fraction I. Three more fractions were now distilled off using respectively 10, 20, and 17 c.c. N_{1}HCl. The fractions were boiled with excess of BaCO_{3} filtered, the BaCO_{3} washed with hot water, the filtrate evaporated to dryness, and the barium salts dried at 130° C. till the weight was constant.[177] The salts were then decomposed with strong H_{2}SO_{4}, ignited, and the barium sulphate weighed. The following is a tabulated statement of the results:-- [177] _Vide_ Note on the Estimation of Butyric Acid, W. H. Willcox, J. Chem. Soc., Nov. 21, 1895. ----------+---------+---------+------------- Fraction |Weight of|Weight of|Per Cent. of | Salts |BaSO_{4} | BaSO_{4} ----------+---------+---------+------------- I. | 1·2420 | 1·0915 | 87·88 II. | 0·9915 | 0·9170 | 92·49 III. | 1·2155 | 1·1980 | 98·56 IV. | 0·6350 | 0·6230 | 98·11 ----------+---------+---------+------------- Calculating fraction I. as a mixture of barium acetate and butyrate, and fractions II., III., and IV. as mixtures of barium acetate and formate,[178] we get:-- [178] _Vide_ J. Chem. Soc. (Trans.), lix., 94, App. II. ----------+---------+--------+-------+-------- Fraction |Weight of| Ba | Ba | Ba | Salts |Butyrate|Acetate|Formate ----------+---------+--------+-------+-------- I. | 1·2420 | 0·2630 |0·9790 | .. II. | 0·99151 | .. |0·89330| 0·0982 III. | 0·2155 | .. |0·44000| 0·7755 IV. | 0·6350 | .. |0·2552 | 0·3798 ----------+---------+--------+-------+-------- Totals | 4·0840 | 0·2630 |2·5675 | 1·2535 ----------+---------+--------+-------+-------- Calculating the barium salts into their respective acids we get:--