Extraction and Measurement Techniques 53 



in most of the organic solvents. Of a large number of solvents tried 

 pure water was fovmd to be the most suitable for such neutral com- 

 pounds. In water the Rj values of most of the compounds which travel 

 fast with the organic solvents are reduced while the aliphatic acids of 

 indole travel faster. Thus water paired with any of the suitable solvents 

 mentioned above may be utilized with good success in two dimensional 

 chromatography of these indole compounds. 



After chromatographic separation of growth substances of the 

 indole group, perhaps the simplest means of detecting and identify- 

 ing the spots is by their fluorescence characteristics in ultra-violet 

 light. This method was first used by Pacheco (1951) and by Jerchel 

 and Midler (1951) for detecting and identifying indoleacetic acid. 

 The method has subsequently been extended for use with many indole 

 derivatives, as shown in table 2. 



The indole derivatives can also be detected on the paper by spray- 

 ing with a solution of 50 parts of 5% HCIO4 and 1 part of 0.5 M FeClg 

 (Pacheco, 1951; Bennet-Clark et al, 1952). Most of the indole com- 

 pounds give pink, orange, brown or blue colorations with this reagent 

 on heating (table 2). 



A 2 per cent solution of p-dimethylaminobenzaldehyde in 1.2 N 

 HCl (Berry et al, 1951; Pacheco, 1951) is also a very good indicator, 

 and the indole compounds show diverse colors after 24 hours. The 

 paper, however, becomes brittle after spraying with the HCl. 



Another color test for detecting indole compounds on paper is 

 the nitrous acid test. The paper is sprayed with a solution of KNO2 

 (1 g.), HNO3 (20 ml.) and 95^0 ethanol (80 ml.), which gives a red 

 color with indoleacetic acid and a yellow color with indolealdehyde 

 (von Denffer and Fischer, 1952). 



Instead of using color tests, bioassays for growth-promoting and 

 for growth-inhibiting substances can be used. A variety of growth tests 

 have been used for this purpose, including the cress root test (Bennet- 

 Clark et al, 1952), the Avena straight-growth test (Hancock and Bar- 

 low, 1953) and the pea root test (Audus and Thresh, 1953). The plant 

 material may be placed directly on the moistened chromatogram or the 

 spots may be eluted into a small volume of water and the growth test 

 carried out in the solution. 



Indoleacetic acid may be estimated by eluting the appropriate 

 part of the chromatogram with water and measuring the absorption 

 spectrum of the eluate at 280 m^. (Jerchel and Miiller, 1951) or at 

 other suitable Avavelengths depending on the color produced by the 

 reagent sprayed. A rough quantitative estimation of a substance can 



