PAPEK CHROMATOGRAPHY 371 



Mercuric nilrate-atnmotiium .sulfide reagent: Chn)iiiat()}j;i;iins nrc dippcti into a 

 0.25 M solution of mercuric nitrate in 0.5 A'^ nitric acid and then heated for 10 niin at 

 80°C. They are next washed by dipping in 0.5 N nitric acid and then in water. After 

 having dried at room temperature, tlie .sheets are (lii)jM'd in a sohition of one part 

 ammonium sulfide (reagent strength) and lour parts water. Purines and pj-rimidines 

 give black spots against a white to gray background. The minimum amount detect- 

 able is from 5 to 10 ^g- The spots are permanent. 



a-NaphthoI-hypochlorite reagent: Chromatograms are first sprayed with a 0. 1 per cent 

 solution of Qf-naphthol in 1 A' sodium hydroxide. The a-iuiphthol reagent may bo 

 used for as long as 1 to 2 months. After having been dried, the sheets are sprayed 

 with a solution consisting of one volume of Clorox diluted with one volume of water or 

 ethanol. Urea appears as a blue-green spot. Arginine appears as a red spot against a 

 white background but fades rapidly. Arginine is detected only at levels above 10 /ug. 



Naphthoresorcinol reagent: To 90 ml of 2 per cent trichloroacetic acid is added 0. 100 g 

 naphthoresorcinol dissolved in 10 ml of 95 per cent ethanol. The reagent must be 

 prepared immediately before use. The sheets, after having been sprayed, are heated 

 10 min at 85 to 90°C. Hexoses produce blue and brown colors. 



Naphthylethylenediamine reagent: Chromatograms are sprayed with a mixture of 

 equal volumes of 0.5 per cent sodium nitrite and 0.5 A^ sulfuric acid. After drying, 

 the sheets are sprayed with a 0.1 per cent solution of A'-l-naphthylethylenediamine 

 hydrochloride. This reagent is useful in detecting diazotizable amines. No such 

 compounds were observed in a large number of urine chromatograms. 



A^inhydrin reagent: This reagent consists of a 0.2 per cent solution of ninhydrin 

 (triketohydrindene hydrate) in water-saturated n-butanol. The reagent, if kept 

 tightly stoppered in a brown bottle, may be used for as long as 1 month. After 

 having been sprayed, the sheets are heated for 5 to 7 min at 90°C to promote color 

 development. IMost amino acids react to give purple spots, although there are 

 notable exceptions. Phenylalanine, tyrosine, and aspartic acid give blue colors; 

 tryptophan, olive-brown; asparagine, brown; proline, yellow. The reagent is not 

 specific for amino acids; certain amines, ethanolamine, peptides, ephedrine, etc., give 

 colors. As little as 0.2 ng of certain amino acids may be detected. The presence of 

 high salt concentrations may cause rapid fading of the colors and may even prevent 

 color development entirely. The presence of strong acids in the atmosphere or on the 

 chromatograms (as with the butanol-ethanol-hydrochloric acid solvent) causes redden- 

 ing of the usual purple color and promotes rapid fading. 



Orcinol reagent: To prepare the reagent, 0. 1 g orcinol in 10 ml of 95 per cent ethanol 

 is added to 90 ml of 2 A' hydrochloric acid containing 0.01 per cent ferric chloride. 

 The reagent should be prepared immediately before use. The sheets are sprayed and 

 heated 10 min at 85 to 95°C. Pentoses produce green spots. Heating must be care- 

 fully controlled to prevent destruction of the paper by the strong acid. 



Phenol-hypochlorite reagent: Chromatograms are first sprayed with 5 per cent phenol 

 in 95 per cent ethanol. After having been dried, the sheets are sprayed with 5.25 

 per cent sodium hypochlorite (Clorox). Colors develop immediately. The back- 

 ground darkens on standing. This reagent reveals a number of common urinary 

 constituents. Urea, at concentrations above about 5.0 /ug, appears as a yellow-green 

 spot. Chloride, at concentrations as low as 1.0 jug, produces a blue spot. Basic 

 cations produce yellow areas. Alost amino acids at high concentration (25 jug) pro- 

 duce blue or green colors. Arginine, however, yields orange; cystine and cysteine, 

 brown; and tryptophan, pink. 



If urea alone is to be measured, preliminary spraying of the resolved chromatograms 

 with 1 A' sodium hydroxide solution prevents the color develoi)ment of other urinar}' 

 constituents. For chromatograms resolved in a pliciiol solvent, the preliminary 



