The weak sulphuric solution, thoroughly freed from the inactive ingredients and from the lead, obtained according to the above procedure may be concentrated in vacuo and a sulphuric acid solution of mercury oxide added until no further precipitate is produced. The precipitate is filtered off, washed and treated in an aqueous supension with hydrogen sulphide. After the liquid has been separated by filtering from mercury sulphide, it is freed from hydrogen sulphide by evaporation in a vacuum or by introducing an excess of carbon-dioxide and evaporated to dryness in vacuo. The remaining residue is a yellow-brown mass, easily soluble in water, said to contain the active substances of the parent material in a concentrated and very pure form.

The solution freed from inactive ingredients and from lead, obtained according to the first example, is acidified with oxalic acid and a solution of phosphotungstic acid is added until no further precipitate is produced. The precipitate, separated by filtering and washed with water containing oxalic acid, is suspended in water and the remaining oxalic acid is precipitated with calcium carbonate. The precipitate is filtered off and the filtrate, freed from calcium compounds, is evaporated to dryness in vacuo. There is obtained a yellow-brown crystalline powder, easily soluble in water, which is said to contain the vitamin of the parent material in a highly concentrated, very pure and permanent form, and which is suitable for purposes of injection.

13 Gams, British Patent 103,294, Jan. 4, 1917; Ghem. Abs. 11, 1522, 1917; Gams and Schrciber, U. S. Patent 1,235,196, July 31, 1917.

The use of "activated " fuller's earth was introduced by Seidell.14 Washed and pressed brewers' yeast, or other source of vitamin is digested for 36 hours at 37.5° C, and the clear liquid from the resulting mass is mixed with about 60 grams per litre of finely divided fuller's earth. The mixture is shaken and treated with about 1 per cent of normal hydrochloric acid to assist settling. The sediment is washed with dilute acid and with ethyl alcohol and then dried in vacuo over sulphuric acid. The vitamins contained in the yeast extract are almost completely absorbed by the fuller's earth. The solid extract thus obtained can be taken either in liquid suspension or as capsules, about 5 grams per day being an adult dose.

The particular grade of fuller's earth found by Seidell to be most useful is a kind obtained from Surrey, England. By the autolysis of fresh yeast followed by filtration a clear reddish brown filtrate is obtained containing over 20 per cent of solids. This is very rich in vitamin. If fuller's earth is added in the proportion of 50 grams per litre and kept in intimate contact with the liquid for about half an hour and then removed by filtration, the yeast liquor is found to contain practically the same amount of solids originally present, but all of the vitamin is now firmly attached to the fuller's earth and repeated washing does not remove any appreciable amount of vitamin from it.

Physiological experiments have shown that no deterioration occurred in samples of the "vitamin-activated fuller's earth" kept over two years. Large amounts of it can be readily accumulated and after being uniformly mixed, it can be standardised by physiological tests for its vitamin content. Such material forms a particularly satisfactory starting point for the comparative study of various methods for the isolation of vitamins.

Although vitamin B is destroyed by heating to 120* C. for two hours, in the dry state in combination with fuller's earth it can be heated to at least 2009 C. without appreciable deterioration. Various attempts have been made to remove the vitamin, in its pure form, from its combination with fuller's earth, principally by treatment with dilute alkali solutions, but all attempts have so far been unsuccessful.14a.

Eddy and Roper15 applied this method to the separation of the vitamin from pancreatic extract. The minced glands were extracted with 95 per cent alcohol, containing enough hydrochloric acid to make it about 0.8 per cent acid. The filtered extract was evaporated to dryness in a current of air at 25° C. The residue was extracted with water, filtered and concentrated in an air current so as to make 1 cc. correspond to 2.7 grams of pancreas. Fifty grams of Lloyd's reagent16 were added per liter of extract, and the mixture shaken in a mechanical shaker. After standing over night the sediment was removed with the suction funnel, dried in a current of air, re-powdered, and finally dried in a vacuum desiccator. When the activated Lloyd powder is dried it retains its power as a vitamin carrier, and when fed to rats exercises the same power as water solutions of vitamins.

14 Seidell, U. S. Patent 1,173,317; J. S. C. I. 35, 663, 1916; U. S. Pub. Health Reports, 31, 364, 1916.

14a Seidell, Jour. Ind. Eng. Chem. 1921, 73. Details of experiments on the isolation of the antineuritic vitamin by silver nitrate precipitation are given by Seidell (J. Ind. Eng. Chem. 1921, 1111).

15 Eddy and Roper, Proc. Soc. Exp. Biol. Med. 14, 62, 1916; Am. J. Dis. Child. 14, 189, 1917.

Frankel and Schwarz17 prepared an active fraction from yeast by extracting the yeast with 80 per cent alcohol, removing the fats, and treating with basic lead acetate. The lead in the active filtrate is removed with hydrogen sulphide, and the filtrate further precipitated with mercuric chloride. The precipitate is then decomposed with hydrogen sulphide, freed from hydrochloric acid, and concentrated in a vacuum. The concentrated syrup is precipitated with picrolonic acid, which removes an inactive picrolonate, and the active substance is finally precipitated with phosphotungstic acid. The precipitate is decomposed with baryta and sulphuric acid, and concentrated in a vacuum. The active base thus obtained is said to be twenty-two times more potent than the original alcoholic extract when tested by its power to accelerate fermentation.