Pathways of Decomposition of Indole Derivatives 185 



MATERIAL AND METHODS 



Chemicals 



Some of the indole derivatives were purchased, others were gen- 

 erously supplied by Drs. J. A. Bentley, D. Von Denffer, A. W. Gal- 

 ston, R. A. Gray, Q. Mingoia, G. F. Smith, and K. V. Thimann. N- 

 hydroxyindole-3-acetic acid was synthesized according to Houft et al. 

 (14). 



Decomposition of aqueous solutions at a concentration of 0.1 per 

 cent was induced by aging (minutes, hours, days, or years, according 

 to the substance), heating, ultraviolet radiation (exposure for several 

 minutes to a Hanovia Utility ultraviolet lamp) or by oxidants like 

 ferric chloride. The decomposed solution was either directly applied 

 to chromatograms and ionograms or extracted first in n-butyl alcohol. 

 In some cases ether fractionation of the neutral and acid decomposi- 

 tion products was performed. 



Chromatography and Electrophoresis 



Uni- and bi-dimensional ascending chromatography was per- 

 formed with Whatman No. 1 paper and the following solvents: (a) 

 water, (b) acetone and water (8:2), (c) 16 per cent NaCl and 2 per 

 cent acetic acid (1:1), (d) 25 per cent acetic acid, and (e) isopropanol, 

 28 per cent ammonia and water (8:1:1). In the case of (a) and (b), 

 the chromatogram was usually run in an atmosphere saturated with 

 vapors of acetic acid. Addition of a few ml. of acetone on the walls 

 of the tank improved some of the separations in the case of (a). In 



Fig. 3. A, lonochromatogram of ultraviolet decomposition products of indole-3- 

 acetic acid. The limits of the fluorescent zones (a, c, e) and of the ultraviolet- 

 absorbing zone f (lAA) in the ionogram were traced with a pencil (cf. Figure 4). 

 The substances which remained at the origin (O) formed a reddish brown double 

 line (zone b) due to chromatographing during the repeated application of the 

 decomposed solution on the ionogram. The ionogram was next machine-sewed 

 onto two filter paper strips. The sewing line on the upper strip is shown as a 

 horizontal interrupted line on top of the ionogram at the bottom of the figure. 

 Ascending chromatography with acetone and water 8:2. Spots II, III, and XII 

 and its tail are double as they originate from the double line of zone b. Their 

 slight displacement to the right of that line is jDrobably due to the holes of the 

 sewing line which diverted the flow of the solvent during chromatography. B, 

 Upper part of the chromatogram after exposure to ultraviolet radiation. Spots 

 XIII and XIV faded considerably and new fluorescent spots (VIII, XV, XVI) ap- 

 peared. After treatment of the chromatogram with A^ HCl, spots IX, X, XII and 

 its tails, XIII, XV, and XVI exhibited different shades of red. VIII was slightly 

 yellowish. Conventional hatching indicating approximate fluorescence colors as 

 in Figure 7. 



