Distribution of natural hormones 



developed from that of Boysen-Jensen (1941) and similar to that of Bennet- 

 Clark and Kefford (1953) was used. The acidic components were taken out 

 from the ether extract by shaking repeatedly with 1 per cent sodium 

 bicarbonate solution. The combined aqueous layers were titrated to pH 4 

 with X sulphuric acid and re-extracted with ether. This ether extract was 

 dried over sodium sulphate in a refrigerator overnight and then concentrated 

 to small bulk in a water bath not exceeding 45°C. The concentrated acid 

 fraction so obtained was spotted on to the starting line of a Whatman No. 1 

 chromatogram paper, usually in 3 small spots about half an inch apart. 



Extract spotted - 

 here 



Storting line 



Mixture of authentic 

 lAAand IAN 

 spotted here 



10 



lAA 



reference 

 spot 



IAN 



reference 

 spot 



-Solvent 

 front 



0*- 



Ri 



r 



-f-O 



Figure. 1. Division of chromatogram for biological assay {see text). 



The chromatograms were developed in the descending manner in aqueous 

 butanol/ammonia solvent (200 ml «-butanol : 6 ml 0-880 ammonia : 36 ml 

 water) at a controlled temperature of 20°C for about 14 hours, by which time 

 the solvent front had travelled about 10 inches. The paper was then removed 

 from the tank and quickly dried by hanging in a fume cupboard. It was then 

 cut transversely into 10 strips of equal size, representing R^ values of 0-0-1, 

 0- 1-0-2, etc. These will be referred to as strip 1, strip 2, etc. (see Figure 1). 



The biological activity of these strips was determined by a special modifica- 

 tion of the wheat cyhnder test of Smith, Wain, and Wightman (1952). Each 

 chromatogram strip was immersed in 3 ml 1 per cent sucrose solution in a 

 small glass dish. Ten 5 mm segments from wheat coleoptiles, threaded on 

 glass capillaries, were floated on top of the solution and the dishes placed in a 

 water-saturated atmosphere at 25°C. The lengths of the coleoptile segments 

 were measured after incubation for 24 hours. 



In all experiments a mixture of known indole compounds was run 

 simultaneously as authentic spots and their positions were detected by 

 spraying with Ehrlich rej^gent. Under these conditions lAA has RfO-\7 and 

 runs in strip 2 of the chromatogram, and IAN has R^ 0-82 and runs mainly 

 in strips 8 and 9. 



EXPERIMENTAL RESULTS 



The results from an experiment in which a solution containing 1 mg lAA and 

 1 mg IAN was separated into acid and non-acid fractions, both of which were 

 assayed by the methods described above, are shown in Figure 2. All the lAA 

 activity is in strip 2 of the chromatogram. The IAN activity is divided between 

 the fractions, the larger part being in the non-acid fraction. Since equal 

 amounts were taken, it is clear that IAN is several times as active as lAA in 

 this particular test. 



33 



