Natural auxins 



chromatography in the latter solvent. As far as the identity of the growth 

 peak found in the lAA region is concerned, it is most probably lAA since 

 the characteristic colour reaction of lAA was obtained on the paper in the 

 lAA position with both the Salkowski and the Ehrlich reagents. 



Figure 3. Histograms showing the growth- 

 promoting activity of 1 cm sections of chromato- 

 grams run in isobutanol-methanol-water (80 : 

 5; 15). Lyophilized immature bean seeds 

 {as in Figure 2, but 30 mg dry weight) were 

 first extracted at C for 1-5 hours with 

 10 + 5+5 c.c. of cold absolute methanol. 

 This extract gave the upper histogram. The 

 same bean material was then extracted for 

 another 1-5 hours at C with 10 + 5 + 5 c.c. 

 of cold ethyl acetate and chromatographed in 

 the same solvent {lower histogram) . 



THE PURIFICATION OF THE EXTRACTS 



To obtain a clean chromatogram with well-separated spots, one has generally 

 to purify the crude plant extract. Such a purification can be achieved in 

 several ways, among which we have used the following ones : 



1. Bicarbonate purification — This well-known technique, already used by 

 Boysen-Jensen (1941), purifies the acid auxins, especially lAA, very well. 

 They enter the aqueous phase as salts, whereas most of the coloured impurities 

 remain in the ether phase. Upon acidification of the aqueous fraction with 

 dilute HCl (Larsen, 1955), it is possible to re-extract the acid auxins with 

 fresh ether. Clean chromatograms of lAA which can be used for colour 

 reactions are obtained in this way. 



2. Acetonitrile purification — The bicarbonate technique, unfortunately, does 

 not work for neutral auxins. Adapting a technique devised originally for 

 insecticides by Jones and Riddick (1952), it was possible to eliminate at least 

 the fatty substances and some of the carotenoids from the extract (Nitsch, 

 1955). The original extract is evaporated down to dryness, then shaken with 



8 



