Abstract
A detailed study of our former procedure for the preparation of the ovarian hormone from liquor folliculi has shown that several time-consuming steps may be omitted. Tests have indicated that the acetone precipitation of phospholipins may be dispensed with, since the bicarbonate of the liquor folliculi furnishes sufficient alkali to saponify the lipins during the concentration of the alcoholic extract.
Another decided improvement has been effected by a study of the distribution ratios of the hormone and cholesterol between 70 per cent alcohol and petroleum ether. The cholesterol is twenty-nine times as soluble in petroleum ether as in 70 per cent alcohol. A few extractions of the alcoholic solution of the hormone with petroleum ether almost quantitatively remove the cholesterol but only a relatively insignificant fraction of the hormone.
The procedure now advocated is designed to separate the hormone from the soaps and cholesterol. It is the following: The alcoholic extract of fresh liquor folliculi is distilled to dryness and the residue is dissolved in a small amount of water which is transferred to a separatory funnel. The aqueous solution is extracted five times with an equal volume of ether; (in case emulsions form, the addition of a little alkali will aid the separation materially); the ether solutions are combined and washed with a little water, then with dilute acid and again with water. The ether is distilled off and the residue washed with 70 per cent alcohol and petroleum ether into a separatory funnel. If the volume of alcohol used is 100 cc. and the extract represents one liter of liquor folliculi, five washings with 25 cc. of petroleum ether are sufficient to reduce the quantity of cholesterol in the alcohol to less than .O1 mg., while the loss of hormone will amount to less than ten per cent.
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