Investigation of natural auxins and growth inhibitors 



about equal parts of anhydrous acetonitrile and hexane or heptane. Under 

 these conditions, lAA and IAN remain in the acetonitrile phase, as well as 

 ethylindole acetate (lAE), although there might be, possibly, some losses of 

 the latter compound. 



3. Chromatographic purification — It was found that some of the coloured 

 impurities of plant extracts are strongly absorbed on paper, so that when a 

 chroraatogram is eluted with ether, some of the coloured material remains on 

 the paper. Two types of chromatographic purifications have been used : 



(a) The crude extract was first chromatographed in distilled HoO and 

 dried. The initial spot was then cut off and the rest of the paper eluted with 

 ether or absolute ethanol. 



(b) The crude extract was first chromatographed in ziopropanol (80) 

 -|- ammonia (10) -{-water (10) (v/v) and dried. Then the initial spot was cut 

 off and discarded, and the rest of the paper cut transversely in the middle. 

 The lower portion then contains mainly the acid auxins and the upper one 

 the neutral auxins which can be eluted and rechromatographed separately. 



During the purification procedure, there is always a possibility of loss of 

 active material and alteration of labile compounds. It has been possible, 

 however, to avoid any purification by using crude extracts of small 

 quantities of plant material and a very sensitive bio-assay test. The 

 procedure adopted will be described below. 



THE CHROMATOGRAPHIC PROCEDURE 



In order to obtain an initial spot as small as possible, the plant extract is 

 applied on the starting line of the paper strip by means of a tuberculin 

 syringe {Figure 4) in a stream of air to allow the rapid evaporation of the 



Figure 4- Tectinique used to obtain small 

 initial spots on cliromatograms. The extract 

 (£) was taken up in a tuberculin syringe (.S) 

 and deposited on the starting point of a paper 

 chromatogram (C) laid down on a perforated 

 paper disk (P). A stream of air, Jittered through 

 cotton, was blown through the glass tubing at 

 the right and was sucked into the suction flask 

 connected with a vacuum pump, thus activating 

 the evaporation of the extract on the paper strip 

 {cf> Nitsch and Nitsch, 1955). 



solvent (Nitsch and Nitsch, 1955). After complete drying, the paper strip is 

 equilibrated overnight over the solvent in a large glass tube {Figure 5), a 

 technique originally used by Stowe and Thimann ( 1 954) . All the experiments 

 reported were performed at room temperature in a dark cabinet. 



The finding of a suitable solvent is the key to successful chromatographic 

 separation of a mixture of compounds. More or less empirically, many 



