370 RADIOISOTOPES IN BIOLOGY AND AGRICULTURE 



change occurs. Most of the substances mentioned above are detectable in amounts 

 above about 5 ^g. 



2,G-Dichloroquinonechloroimide reagent: The reagent is prepared as a 1 per cent 

 sokition of 2,6-dichloroquinonechloroimide in 95 per cent ethanol. When stored in 

 the refrigerator, the reagent is stable for 2 to 3 weeks. On sheets sprayed with this 

 reagent, alkaline areas appear as blue spots, uric acid as a yellow spot, and creatinine 

 as a brown spot. On standing several hours, certain imidentified substances in urine 

 appear as brown, pink, and red spots on the chromatogram. Contact with phenolic 

 substances must be strictly avoided, and sheets that have been sprayed with DCC 

 should be kept apart from those developed with other reagents because of interaction 

 between this reagent and others, especially sulfanilic acid, the ferricyanide-nitro- 

 prusside reagent, and p-dimethylaminobenzaldehyde. 



p-Dimethylaminobenzaldehyde reagent: p-Dimethylaminobenzaldehyde (2.0 g) is 

 dissolved in 100 ml of 1.2 A^ hydrochloric acid. Storage in a refrigerator will keep 

 this reagent 3 to 5 days without deterioration. Urea and allantoin give bright 

 yellow spots against a white background. The colors do not fade on drying or stand- 

 ing. The minimum concentration of urea that is detectable is 5 to 10 yug. 



Ferric chloride reagent: A 1 per cent aqueous solution of ferric chloride is employed. 

 The reagent may be kept for 7 to 10 days in a refrigerator. Sheets must be sprayed 

 lightly to avoid streaking. Spots appear most clearly after the sheet has dried except 

 in the case of tartaric acid, which is visible as a yellow spot only on the wet sheet. 

 Phosphate and sulfate produce white areas against a light yellow background. 

 Amounts of phosphate and sulfate greater than 10 to 20 yug can be detected. Urea 

 appears as a pale area slightly lighter than the background and can be detected only 

 in fairly high concentrations (30 Mg)- Basic areas are light brown, presumably on 

 account of precipitation of ferric hydroxide. Phenol derivatives produce green to 

 purple spots. Aspirin appears in urine chromatograms as an intense purple spot. 

 As little as 0.5 ^g salicylic acid or acetyl salicylic acid can be detected. 



Ferricyanide-nitroprusside reagent {alkaline): Equal volumes of 10 per cent sodium 

 hydroxide, 10 per cent sodium nitroprusside, and 10 per cent potassium ferricyanide 

 solutions are mixed. The mixture is diluted with three volumes of water. After 

 standing for about 20 min, the initial dark color changes to a pale yellow, and the 

 reagent is ready for use. This reagent is quite unstable at room temperature but 

 may be kept for 2 to 3 weeks in the refrigerator without deterioration. Creatinine, 

 guanidine, glycocyamine, and arginine give rise to orange colors against a light yellow 

 background. Creatinine gives a blue color. Certain other nitrogenous compounds 

 also respond to the reagent. The colors are stable over long periods, provided precau- 

 tions are taken to exclude phenol vapors during development and contact with 

 phenolic substances afterward, since these cause marked color changes on the 

 chromatograms. 



Hydrolytic reagent: Following the procedure outlined for the picric acid reagent as 

 described below but omitting the picric acid spray, a pink color is developed with 

 indole and a gray color with tryptamine. In addition, several unknown urinary 

 constituents give pink, blue, and green colors. 



Lead cohaltinitrite reagent: Lead nitrate (11.5 g) and 15 g sodium nitrite are dissolved 

 in a small amount of water. When these are completely dissolved, 10 g cobalt nitrate 

 is added, and the solution diluted to 100 ml with water. This solution is allowed to 

 stand for at least 1 hr before filtering to remove the orange-brown precipitate. A 1: 2 

 dilution of the filtrate is prepared immediately before spraying. The reagent is 

 stable for 1 to 2 days at room temperature. Contact with the vapor of the reagent 

 while spraying should be avoided. Potassium and ammonium ions produce brown 

 spots against a light yellow background. 



