372 RADIOISOTOPES IN BIOLOGY AND AGRICULTURE 



spraying with phenol may be ignored. In this case the amount of phenol remaining 

 on the chromatogram must be controlled by rather careful partial drying for 8 min 

 ( ± 30 sec) at 90°C. The sheets must be sprayed immediately after this drying. 



Picric acid reagent: Chromatograms are sprayed with 0.5 N sulfuric acid and heated 

 for 1 hr at 100°C. They are then sprayed with a 1.3 per cent solution of picric acid in 

 95 per cent ethanol, which is combined immediately before use with one-fifth its 

 volume of 10 per cent sodium hydroxide. Creatine and creatinine appear as orange 

 spots against a yellow background. Allantoin gives a faint orange spot. A niunber 

 of other urinary constituents cause a slight fading of the yellow background color. 

 The colors developed for creatinine and creatine are permanent. As little as 0.2 /zg is 

 detectable. If creatinine alone is to be determined, the preliminary hydrolytic treat- 

 ment with sulfuric acid may be omitted. 



Silver nitrate reagent {a)nmoniacal): Equal volumes of 0. 1 A'^ silver nitrate and 5 A^ 

 ammonium hydroxide are mixed immediately before use. Chromatograms may be 

 sprayed with or dipped into the reagent. It is advisable that the sprayer be washed 

 immediately after use several times with 5 N ammonium hydroxide, followed by 

 washing with water to prevent clogging by deposition of silver and insoluble silver 

 salts. Uric acid develops almost immediately as a black spot again.st a white back- 

 ground. For further development the sheet should be heated for 10 min at 100°C, 

 thus darkening the background to light brown. Carbohydrates then appear as dark 

 brown spots. Chloride and phosphate produce bleached areas on the paper. The 

 weak-acid alkaline area produces a spot slightly darker brown than the background, 

 but at high concentrations (50 to 100 fxg sodium acetate) this may also appear as a 

 bleached area. Most of the amino acids cause a slow bleaching of the background 

 color to white or yellow. The bleaching effect of the amino acids appears to be 

 different from the immediate bleaching of inorganic ions. The latter seems to be 

 due to simple interference with the contact of the reagent with the paper. As little 

 as 0.2 fig uric acid and 0.5 /ug glucose can be detected using this reagent. The amino 

 acids can be seen only at concentrations of 30 to 50 /ig; chloride and phosphate are 

 detectable at 5 to 10 ^g. The chromatogram becomes very dark after standing for 

 several days, and the originally darkened areas can no longer be detected. This 

 reagent cannot be used for the color development of chromatograms that have been 

 developed in solvents containing phenol unless the phenol is previously removed by 

 drying the sheet for at least 48 hr at room temperature. 



Sulfa nilic acid reagent (diazotized): Sulfanilic acid (4.5 g) is dissolved in 45 ml of 

 12 A' hydrochloric acid with warming, and the solution is diluted to 500 ml with water. 

 A 100-ml aliquot of this dilute solution is chilled in an ice bath, and 100 ml of a 4.5 

 per cent solution of sodium nitrite is added. This mixture is allowed to stand for 

 15 min in the ice bath. This reagent may be kept in the refrigerator without deterio- 

 ration for 1 to 3 days. Immediately before use an aliquot of this solution is mixed 

 with an equal volume of a 10 per cent solution of sodium carbonate. This reagent is 

 particularly useful in the determination of histidine and a number of unidentified 

 substances present in urine. Pink, red, orange, and purple spots are developed against 

 a light yellow background. The colors are stable, but contact with vapors of phenol, 

 collidine, or lutidine causes darkening of the background. The minimum concentra- 

 tion of histidine detectable is about 0.2 ^g- This is a general reagent for phenols and 

 may be used to detect thyroxine and its analogues. 



Zinc uranyl acetate reagent: Uranyl acetate (5.0 g) is mixed with 3 ml of warm 30 

 per cent acetic acid, and the mixture diluted to 25 ml. A second solution is prepared, 

 consisting of 15 g zinc acetate mixed with 3 ml of 30 per cent acetic acid and diluted 

 to 25 ml. The two solutions are mixed and warmed gently. Sodium chloride (0. 1 g) 



