44 GROWTH HORMONES IN PLANTS 



3. Fractionation with Sodium Bicarbonate Solution. — The ether 

 residue was dissolved again in the smallest possible amount of 

 pure ether (1 to 3 liters) and shaken up eight times, each time 

 with 500 cc. saturated bicarbonate solution. Dilute hydro- 

 chloric acid was added to the combined bicarbonate extracts, and 

 the acid solution was extracted with ether six to eight times ; 3 to 

 4 Uters of ether were necessary for this step. The ether was dried 

 over anhydrous sodium sulphate and was evaporated down. A 

 residue weighing 45 g. was obtained. The active substance at 

 this point had been concentrated seventy-eight times. 



4. Extraction with Petroleum Ether and Ligroin to Remove More 

 Inert Material. — The ether residue was heated for i^ hour on a 

 water bath with 400 cc. petroleum ether (b.p. 40 to 60°C.). 

 After cooling, the solution was carefully poured off, and the 

 extraction was repeated twice in the same way with fresh petro- 

 leum ether; the physiologically ineffective substances went into 

 solution in the petroleum ether and were removed in this way. 

 The auxin remaining in the syrup-like material was insoluble 

 in petroleum ether. This syrup was extracted three times, each 

 time by heating the residue with 400 cc. Hgroin (b.p. 100 to 

 120°C.). The residue weighed 19.7 g., and the active substance 

 was 238 times more concentrated than in the original material. 



5. Extraction with Benzene. — The syrup thus far purified was 

 dissolved in 300 cc. 60 per cent ethyl alcohol and shaken ten 

 successive times, each time with a fresh 100 cc. portion of benzene. 

 The combined benzene solutions were extracted three times with 

 water, using 300 cc. each time, then with 50 per cent methyl 

 alcohol three times, using 300 cc. each time. The methyl alco- 

 holic extracts were evaporated to dryness; the residues from the 

 methyl alcoholic and the aqueous extracts were combined, and 

 this was shaken several times with ether. After the ether was 

 dried and evaporated, the residue weighed 5.5 g. The active 

 substance was then six hundred times concentrated. 



6. Lead-salt Precipitation. — The ether residue was dissolved 

 in 125 to 150 cc. 96 per cent alcohol and mixed with a concen- 

 trated aqueous solution of 5 g. neutral lead acetate. The result- 

 ing precipitate contained almost no auxin. To the filtrate was 

 added drop by drop a 30 per cent sodium hydroxide solution 

 until a weak alkahne reaction was obtained. The precipitate 

 thus formed was filtered off, dissolved in dilute acetic acid, and 



