Dissolve the residue from the benzene extract in a small quantity of chloroform,warming,if necessary,and completely transfer the solution,with the aid of a few mLof chloroform,to a 20-×150-mm test tube.Carefully evaporate the chloroform on a steam bath with the aid of a current of air.Add 100mg of trimethylacethydrazide ammonium chloride and 500µLof glacial acetic acid to the test tube,insert the stopper loosely,and heat in a boiling water bath for 5minutes.Cool the reaction mixture in an ice bath,dissolve in a small volume of cold water,and completely transfer,with the aid of a small volume of water,to a 125-mLseparator containing 25mLof cold water.Neutralize the solution to litmus with 1Nsodium hydroxide (approximately 6mL),and wash at once with three 15-mLportions of chloroform.Combine the chloroform washings in another separator,and wash them with 5mLof water.Discard the chloroform,and add the wash water to the first separator.Add 2mLof dilute sulfuric acid (1in 2),and allow to remain at room temperature for 2hours.Add 15mLof chloroform,shake vigorously for 1minute,and allow the layers to separate.Transfer the chloroform layer to another separator,and repeat the extraction of the water layer with three additional,successive 15-mLportions of chloroform.Wash the combined chloroform extracts with 5mLof water,filter through chloroform-washed cotton into a beaker,evaporate to a small volume,and transfer completely,with the aid of several small portions of chloroform,to a tared 25-mLbeaker.Evaporate on a steam bath with the aid of a current of air to dryness,and dry the residue of estrone in a vacuum desiccator to constant weight:the weight of the residue,corrected for the residue of a reagent blank similarly prepared,indicates the amount of C18H22O2in the volume of Injection taken.