48 GROWTH HORMONES IN PLANTS 



and ligroin, and the insoluble portion fractionated over sodium 

 bicarbonate. (5) The residue of the acid fraction was dissolved 

 in 60 per cent ethyl alcohol and shaken up with benzene. The 

 active substances were extracted from the benzene solutions with 

 50 per cent methanol and finally with water. After removal'of 

 the alcohol, the aqueous solution was extracted with ether, and 

 the residue again treated with petroleum ether and ligroin as 

 above. (6) The portion insoluble in ligroin was heated on a 

 water bath six to eight times, each time with 50 to 100 cc. xylene; 

 the growth substance went into solution almost entirely. (7) 

 After removal of the xylene, the residue was extracted about six 

 times on the water bath, each time with 50 to 80 cc. cyclohexane; 

 the active substance then remained in the insoluble residue. The 

 effectiveness after this step was (at most) 2,000,000 AE per 

 milhgram. (8) After treatment with cyclohexane, lead-salt 

 fractionation was carried out. The precipitate, in a weakly 

 acid medium, was discarded; determinations on the filtrate 

 showed it to have an effectiveness of about 3,000,000 to 6,000,000 

 AE per milligram. (9) The usual calcium-salt precipitation fol- 

 lowed, from which only the filtrate was treated further. (10) The 

 growth-substance concentration in the filtrate was only slightly 

 greater than in the previous step, so that the highly active product 

 (about 1.5 g.) was dissolved next in 18 cc. alcohol and mixed 

 with 180 cc. water; 5 g. barium acetate in saturated aqueous 

 solution was added. (11) Then normal potassium hydroxide 

 solution was added until no more precipitate formed. The 

 precipitate was entirely inactive; the filtrate, after decomposition 

 with acid, showed an effectiveness of about 15,000,000 AE per 

 milligram. Crystals formed in tliis residue after it had remained 

 in the refrigerator for 2 days. These crystals were filtered off 

 and washed with a little cold chloroform. The crude product 

 of about 135 mg. had a melting point of 148 to 156°C. (12) This 

 product was recrystallized from chloroform and yielded 111 mg. 

 The crystals were rhombic plates with a melting point of 161 

 to 162.5°C. and an effectiveness of 20,000,000 AE per milligram. 



These rhombic plates were three times recrystallized from 

 chloroform, giving 100, 91, and 82 mg., respectively. The melt- 

 ing point remained constant at 164°C. 



Another quantity of material was prepared in the same way 

 as the foregoing and eventually recrystallized four times from 



