Various methods have been devised for obtaining preparations of vitamins in concentrated form for use where the fresh foodstuffs are not available or where it is desirable to eliminate every variable in nutritive factors except the one under consideration.

The first concentrate of A was the "butter oil"1 prepared by Osborne and Mendel,2 but this was found to deteriorate on keeping, even under the best conditions, so that within a year its efficacy was almost entirely lost. A more satisfactory preparation was obtained from dried plant tissues by extracting with ether. The oily residue obtained when the ether was distilled amounted to about three per cent of the weight of the dried plant. Most of these oils were potent in stimulating growth. A striking exception was found in the extract of dried tomato which was not effective, although the dried tomato itself gave the best growth obtained in the whole series of experiments carried out at that time. The great variation in the ease with which different plant tissues yield their vitamin to solvents has already been noted,4 and apparently the grasses, clover, alfalfa, and timothy, and spin&ch are particularly favorable sources for this type of preparation.

Zilva5prepared alcoholic extracts of A from fresh cabbage and carrots by allowing the finely triturated foodstuff to stand for 12 to 18 hours with alcohol, in the proportion of 100 g. of foodstuff to 500 c. c. of alcohol. This was then filtered and the filtrate evaporated in vacuo at 35° C. The residue is said to be effective in promoting growth. Steenbock and Boutwell6 regard alcohol as the most satisfactory solvent for the extraction of A from plant products, having found it considerably more effective than ether in this respect. Benzene also proved fairly satisfactory although inferior to alcohol.

Since A is not destroyed by drying at moderate temperatures, dried plant products may be used as a convenient source of supply. Osborne and Mendel7 found 0.1 gram of dried alfalfa, clover, timothy, or spinach as effective as an equal quantity of butter-fat, while dried tomato was still better. The products used were prepared from the plant materials by heating in a large drier, through which a current of air circulated at 60° C. or less, and grinding the dry residues to a powder.

1 Osborne and Mendel, J. Biol. Chem. 41, 649, 1020.

2 See p. 00.

4 See pp. Ill, 112.

5 Zilva, Bioch. J. 14, 404, 1020.

6 Steenbock and Boutwell, J. Biol. Chem. 42, 131, 1020.

In making an extract of A, McClendon7a uses green leaves or fruit skins. These are dried, preferably in the absence of oxygen, and then ground to a powder. The powder is moistened with alcohol by being thrown into a container half filled with boiling 95 per cent alcohol and allowed to remain without application of heat for twenty-four hours. The mass is then placed in a strong canvas bag and subjected to a pressure of 5000 lbs. to the square inch. The press-cake may be ground in a mill and re-extracted if desired in the same manner. If, however, it is desired to recover the solvent, a preliminary concentration may be carried out in a vacuum pan. The product is a sticky powder when absolutely dry, but it absorbs water from air and becomes pasty. It contains resinous or fatty substances. When made from spinach a hygroscopic substance of waxy constituency is obtained. This preparation of A contains sufficient fat-soluble vitamin to afford normal growth in a rat, when 0.05 to 0.1 gram is added daily to a ration free from this factor.

The methods which have been used for preparing concentrates of vitamin B fall into three classes: (1) those in which an extract of the food substance is evaporated to dryness at low temperature; (2) those in which the vitamin is precipitated from an extract containing it, the precipitate after purification is decomposed, and the resulting solution evaporated in vacuo; and (3) those in which the vitamin is absorbed by fuller's earth or some similar absorptive agent and the "activated " product thus obtained used without further treatment.

The first method of procedure was used by Bosshard and Hefti,8 who treat substances rich in vitamins, particularly yeast or rice bran, with dilute mineral acids at 80° C. until the biuret reaction gives a negative result. The product is filtered, freed from mineral acid, and then evaporated to dryness at the lowest possible temperature, either without further treatment, or after the conversion of the amino acids present into their calcium or sodium salts. By avoiding too high a temperature or acid concentration, the vitamins are said to be brought unchanged into solution.

7 Osborne and Mendel, J. Biol. Chem. 41, 549, 1920. 8 McClendon, J. Biol. Chem. 1921, 411.

9 Bosshard and Hefti, J. 8. C. I., 1920,675A; German Patent 320,785, March 19, 1916.

In a process recommended by the Soc. anon, pour Find. chim. A, Bale,9 vitamin preparations are made by comminuting vegetable substances so as to retain the juice, adding a small proportion of acid, if necessary, so as just to neutralize any alkali that may be present, and drying in vacuo at a temperature below 100° C. Preparations of this sort can be baked into loaves by adding flour and a distending agent such as sodium bicarbonate or yeast.9*

Voegtlin10 gives the following method for the extraction of vitamin B from animal tissues:

Extract From Ox Liver

Fresh ox liver was minced in a meat chopper and dried, first on glass plates at about 40* C. in a current of air from an electric fan, and then in a vacuum desiccator. The dried material was then ground to a fine powder and extracted with 05 per cent alcohol for several days at 37* C. About two liters of alcohol were used for each kilo of dried material. One half of the alcohol was used for the first extraction, and the remainder for the second extraction. During the extraction the bottles containing the material were occasionally shaken. The alcohol was then filtered off and evaporated in vacuo at 35 to 40° C, until the residue in the flask showed a tendency to form viscous bubbles which rise in the flask. The viscous residue was then poured into a separatory funnel and extracted with about twice its volume of ether. Three distinct layere formed, upon allowing the contents of the funnel to stand for three hours. The upper layer consisted of an ethereal solution of fats and lipoids, the middle layer of a white or yellowish insoluble residue, and the lower layer of a yellowish or brown colored oil-like substance. The material contained in the lower layer will hereafter, for the sake of brevity, be designated as the "vitamin" fraction.

The "vitamin" fraction was then run off into another separatory funnel, containing ether, and thoroughly agitated. After it had settled out, the oily material was run off into a dish and the ether evaporated off in vacuo. The yellowish oil had a pleasant, nutty odor, and was soluble in water. Cooper states that this material is soluble in absolute alcohol, but it was found that its solubility was very slight in absolute alcohol; whereas 50 per cent alcohol dissolves it quite readily. In case a sharp separation of the various layers was not produced upon extraction with ether, as much oily material as possible was run off, the mixture of oil and solid material was run into a dish, freed from ether and the mixture extracted with a small amount of 50 per cent alcohol. After filtering, the alcoholic solution was again poured into ether and the lower layer was treated as above. The various vitamin fractions were then united and the ether evaporated in vacuo.