Investigation of natural auxins and growth inhibitors 



considered, nor their R/s (see also Figure 6(a)); secondly, the removal of 

 uopropanol changes the R/s but does not impair the separation of the 

 compounds — in fact, it improves it; thirdly, the removal of water definitely 

 ruins the value of the solvent. Thus, from all components of the mixture only 

 one is really indispensable, namely water. This is true even in the case of 

 water-insoluble compounds such as hexane, as demonstrated by Nitsch and 

 Nitsch (1955). It is probable that water, at least in the vapour state, has to 

 impregnate the paper fibres in order to give a good chromatogram. These 

 investigations give support to the use of water alone previously reported by 

 Bitancourt, Schwartz, and Dierberger (1954) and by Sen and Leopold (1954). 

 These authors, however, did not equilibrate their paper strips over the water 



(^) 



I/^ 



^lAE o 



IAN 



05 1 2 3315 678 3 10 



Normal ify of NH^OH -* p H — 



Figure 6. (a) R/s of I A A, lAE, and IAN in water to which various concentrations of ammonia have 

 been added, {b) The effect of pH on the R/s oflAA, lAE, and IAN in water. pH 3-0 has been obtained 

 both with citrate-phosphate buffer (about 0-03 M) and HCl (0-001 M) ; pH'5 4-0-7-0 with citrate- 

 phosphate buffers [about 0-03 M) ; pH 8-0 with citrate-phosphate buffer and with borate-KC\ buffer 

 {about 0-05 M) ; pHV 9-0- 10-0 with borate-KCl buffers {about 0-05 M) {cf. Nitsch and Nitsch, 1955). 



solvent before chromatography. It has been shown (Nitsch and Nitsch, 1955) 

 that a preliminary equilibration improves markedly the quality of the 

 chromatography in water by giving compact spots. 



The use of water alone in the chromatographic separation of auxins has its 

 limitations. First of all, it does not separate the acid auxins one from another 

 (for example, lAA and indole-3-butyric acid (IBA) practically run together, 

 as shown in Table 2). In the second place, when fatty material is present in 

 the plant extract, the auxins, more soluble in fats than in water, may not 

 move readily out of the initial spot, and streaking may occur. For these 

 reasons, it was necessary to improve the water solvent. Changing the pH 

 did not help at all {Figure 6(b)), but the addition of an organic solvent such as 

 acetonitrile, or an alcohol, allowed a good separation of several acid auxins 

 (Figure 7) , whereas the neutral ones, such as IAN and I AE, ran together. It was 

 then concluded that no one solvent could be ideal for compounds as widely 

 different in their physico-chemical properties as acids, esters, and nitriles, 

 and that it was better to look (a) for a solvent separating the acid compounds, 

 (b) for another one separating the neutral substances. 



For acid auxins there exists already an excellent solvent, the one developed 

 by Stowe and Thimann (1954). The need for a different mixture was not 

 felt until it was discovered, in the course of the search for a solvent applicable 



11 



