Chromatographic Investigations of Indole Compoimds 75 



in one experiment, this amount being evenly distributed among four 

 petri dishes. A tissue-in-water treatment and a solution of each com- 

 pound untreated with tissue were included as controls in each experi- 

 ment. Bacterial contamination was negligible in all treatments since 

 streptomycin at a concentration of 10-*M was included in all solu- 

 tions. After 48 hrs. the solutions together with tissue were frozen 

 overnight at — 10° C. On the following day the solutions were thawed 

 and the four identically treated solutions of each compound com- 

 bined. During this process the tissue was removed and rapidly ground 

 to a fine paste and then recombined with the residual solutions. Com- 

 pounds present in this homogenate were removed by acidifying the 

 system to pH 2.5 and extracting three times with 200-ml. quantities 

 of ethyl acetate. The combined extract was dried over anhydrous so- 

 dium sulfate and concentrated for analysis by paper chromatography. 



Chromatographic analysis of each extract was carried out by the 

 ascending method using Whatman No. 1 paper in all glass tanks. 

 The solvent used in most instances was a mixture of 77-butanol, am- 

 monia (0.880) , and water (100:3:18 v/v) , although occasionally isopro- 

 panol, ammonia (0.880), and water (10:1:1 v/v) was employed for 

 comparative purposes. Temperature was controlled at 20° C. and 

 each chromatogram was developed for 12 hrs. Chromatograms for 

 chromogenic analysis were spotted with amounts of ethyl acetate ex- 

 tract equivalent to 1,000 /xg. of the compound in the original solution. 

 After development, the papers were dried in air and in most cases 

 sprayed with Ehrlich's reagent applied as 1 per cent p-dimethyla- 

 minobenzaldehyde dissolved in 50 per cent alcoholic HCl. Other 

 sprays, such as the Salkowski reagent and nitrous acid reagent which 

 were prepared by conventional methods, were also used. 



When preparing chromatograms for biological examination, ethyl 

 acetate extract equivalent to 2,000 fxg. of the original compound was 

 evenly distributed over 20 spots on a 10-inch wide sheet of Whatman 

 No. 1 paper. After development, a longitudinal strip containing two 

 spots was removed from one side of the chromatogram and sprayed 

 with Ehrlich's reagent to establish the position of indole compounds. 

 The rest of the sheet was cut transversely into twenty strips of equal 

 size each corresponding to one-twentieth of the distance travelled by 

 the solvent front. Each strip was thus equal to half an Rf unit, i.e., 

 to 0.05, 0.05 to 0.1, 0.1 to 0.15, etc. The strips were placed in petri 

 dishes, one per dish, containing 10 ml. of distilled water, and the bio- 

 logical activity of any compound present in each strip was then de- 

 termined by the wheat cylinder test. The activity of a control strip 

 taken from above the starting line of the chromatogram was deter- 



