PAPER CHROMATOGRAPHY 369 



as yellow ininicdiately ; the background fades to j'ellow on standing. All these 

 substances can be detected in amounts as low as 5 Mg- 



Aniline acid phthalate reagent: To 100 ml of water-saturated H-butanol are added 

 l.(')6 g phthalic acid and 0.03 g aniline. The reagent is stal)le for 2 to 3 months at 

 room temperature. On sheets sprayed with this reagent and heated 5 min at 100°C, 

 aldopentoses appear as red spots and hexoses as green to brown spots. 



Azide-iodine reagent: Fifty milliliters of a 0.1 A' iodine solution (aqueous solution 

 prepared using potassium iodide to effect solution) is added to 50 ml of 95 per cent 

 ethanol, and 1.5 g sodium azide is dissolved in this solution. If kept tightly stoppered 

 in brown bottles, this reagent is stable indefinitely. This reagent is particularly useful 

 for the detection of sulfur-containing amino acids. Methionine, cj-stine, and cysteine 

 decolorize the reagent immediately, appearing as white areas against a brown back- 

 ground. After an hour these spots show considerable fluorescence under ultraviolet 

 light. These amino acids may be detected in concentrations of about 5 )ug or more. 

 Spots must be marked soon after development, because within a few hours the back- 

 ground fades to give a uniformly colored sheet. Rose and yellow spots due to uniden- 

 tified constituents appear on urine chromatograms. 



Bromine reagent: Liquid bromine (0.5 ml) is added to 50 ml glacial acetic acid and 

 50 ml water. This reagent is stable for 2 or 3 weeks if kept in the refrigerator. After 

 the chromatograms are sprayed with this mixture, they are heated in an oven at 

 90°C for 3 to 5 min. Histidine may be detected by this procedure in amounts of 

 25 Mg or more. On exposing the damp sheet to ammonia vapor, the brown histidine 

 spot becomes purple, and the sensitivity is increased so that 10 ^g becomes visible. 

 The histidine spot does not fade. In addition to histidine, a number of unidentified 

 urinary constituents show up with this reagent as pink, brown, and green spots. Some 

 of these spots show a bright yellow fluorescence under ultraviolet light as well. Their 

 ability to fluoresce decreases after several daj's. 



Bromocresol green indicator reagent: This reagent is a tj^pical acid-base indicator 

 and, as such, can be used to detect acidic or basic substances on the chromatogram. 

 A 0.04 per cent solution in 95 per cent ethanol is emploj-ed. Before using the solution, 

 the color should be adjusted to blue-green with dilute sodium hydroxide solution. 

 The indicator solution is stable indefinitely. Chromatograms developed in acidic or 

 basic solvents must, of course, be thoroughly dried before spraying. Acids give 

 yellow spots against a blue-green background, and bases show intensification of the 

 blue basic color. Acidic areas on urine chromatograms correspond to chloride, sulfate, 

 phosphate, citrate, lactate, and hippuric acid. Urea appears as a slightl}' basic 

 spot, and the weak acid alkali gives a strong basic spot. Some metal ions, e.g., lead 

 and copper, give a bright pink color with this indicator. The colors developed are 

 affected bj' acidic or basic substances in the atmosphere but are otherwise quite stable. 



2,6-DichIorophenolindophenol reagent: A 0.4 per cent solution of 2,()-dichlorophenol- 

 indophenol in ethanol is sprayed on the chromatograms. The reagent is stable 

 indefinitely. This reagent acts as an acid-base indicator as well as an oxidation- 

 reduction indicator. Initially the background is blue, with acidic areas appearing as 

 pink spots. The most conspicuous spot in urine is chloride (pink). The weak-acid 

 alkali spot appears as a darker blue area. Although amino acids do not appear as 

 acidic or basic substances, they become apparent as completely bleached areas in 

 1 hr to several days, depending on the concentration present. Ascorbic aci<l causes 

 almost immediate bleaching. Creatinine and creatine appear as bleached areas in 

 about 45 min. Lactic and hippuric acids, as well as chloride at fairly high concentra- 

 tions, cause slow bleaching. Urea may appear as a blue area at high concentrations. 

 The bleached areas remain unchanged indefinitely, although the background changes 

 from blue to pink in 48 to 72 hr, necessitating the marking of the acid areas before this 



