PREPARATION OF CORTICAL HORMONE 237 



filtered to free it from the separated lipids. The clear filtrate 

 is distilled in vacuo at 35 to 40° until all the acetone is removed. 

 The completeness of this removal may be determined from the 

 specific gravity of the residual aqueous fluid, which should be 

 over 0.99 at room temperature. After being chilled in the ice 

 chest, the solution is again filtered to remove the separated 

 lipids. The filtrate is carefully neutralized with an aqueous 

 solution of NaOH until neutral (pH 7.0) and shaken at inter- 

 vals with activated charcoal (norit or decolorizing carbon). 

 One gram of charcoal is used for every 200 grams of glandular 

 material originally extracted. After some hours (6 or more, 

 depending on the efficiency of the shaking) the carbon is col- 

 lected on a Buchner funnel. The filtrate contains only a trace 

 of hormone, but this can be recovered by treatment again with 

 charcoal. 



Before utilizing the charcoal-hormone combination prepared, 

 as described above, it is necessary to remove most of the epi- 

 nephrine and other impurities which contaminate it. To re- 

 move these impurities the charcoal-hormone combination is sus- 

 pended in about 3 times its volume of 10 per cent hydrochloric 

 acid (1 part of 33 per cent HC1 to 10 parts of water). After 

 thorough agitation, the suspension is filtered on a Buchner 

 funnel. The charcoal is then suspended in about 3 times its 

 volume of a 0.1 n sodium hydroxide solution, agitated, and 

 again filtered. It is then again washed with 3 volumes of 2 

 per cent hydrochloric acid and finally washed with a little 

 distilled water and filtered. It is now ready for use. The 

 charcoal-hormone combination may also be effectively freed of 

 adsorbed impurities by washing on the Buchner funnel with 

 dilute ammonia, water, and ethyl alcohol, respectively. These 

 reagents elute an inappreciable amount of the hormone. 



The amounts of acid and alkali used for washing the char- 

 coal are relatively small, and hence do not remove an appre- 

 ciable quantity of the hormone. The trace removed can be 

 reclaimed by neutralizing the washings and readsorbing on a 



