PROPERTIES OF GROWTH SUBSTANCES 45 



the acid solution extracted with ether. The amount of residue 

 fluctuated greatly, as did the content of physiologically active 

 substance. In several cases more than 80 per cent of the auxin 

 was present in the precipitate in the alkaline medium, so that 

 this was important in the further steps of preparation. When 

 only 40 to 60 per cent of the active substance pre\dously present 

 was in the precipitate, the filtrate was acidified with glacial 

 acetic acid, concentrated until removal of the alcohol, then 

 extracted with ether. The ether residue containing the remain- 

 ing amount of active substance was further treated, either 

 alone or together with the precipitate fraction (above mentioned). 

 In most cases the greater amount of the active substance remained 

 in the filtrate so that the precipitate could be disregarded. The 

 remaining portion of the active fraction weighed 3.17 g. (1.08 to 

 7.6 g.). At this point, usually about 60 per cent of the active 

 substance originally present in the urine was still present; it had 

 been concentrated eleven hundred times. 



7. Calcium-salt Precipitatiori. — -The most active residue from 

 the lead-salt fractionation, usually about 3 g., was dissolved in 

 30 cc. alcohol, and 300 cc. water were added; at tliis point, the 

 solution became slightly turbid. A concentrated aqueous solu- 

 tion of 6 g. calcium acetate was added to this solution, and then 

 a normal solution of potassium hydroxide was added during 

 frequent shakings until no further precipitation took place. The 

 precipitate was filtered off and repeatedly washed with aqueous 

 alcohol. The resulting filtrate was acidified with glacial acetic 

 acid and extracted with ether. The ether was evaporated, and 

 the ether residue weighed 2.25 g. The active substance was 

 sixteen hundred times more concentrated than in the original 

 material. 



8. Lactone Formation. — The clear, reddish-brown syrup was 

 boiled for one hour with 10 to 15 cc. 1.5 per cent methyl alcohol 

 in hydrochloric acid. After removal of the alcohol in a vacuum 

 the residue was taken up in ether, and the ether solution shaken 

 up twice with 2 per cent sodium bicarbonate solution and twice 

 with water. The auxin remained in the neutral fraction (the 

 ether solution). The ether solution was evaporated, and the 

 residue weighed 1.2 g. (0.47 to 2.45 g.). At this point, the active 

 substance was 2,500 times more concentrated than in the original 

 material. 



