368 



RADIOISOTOPES IN BIOLOGY AND AGRICULTURE 



nium hydroxide is employed as above to maintain an alkaline atmosphere. After 

 24 hr a pink discoloration is detectable in the solvent mixture, and the solution must 

 be discarded. This solvent is useful in separating basic substances. 



Butanol-ethanol-hydrochloric acid solvent: 80 ml n-butanol, 20 ml of 95 per cent 

 ethanol, and 40 ml of 2 A^ hydrochloric acid. This solvent is especially useful in the 

 determination of cystine and histidine. 



Ethanol-hydrochloric acid solvent: 80 ml of 95 per cent ethanol and 20 ml of 0.1 A^ 

 hydrochloric acid. This solvent has been used exclusively in the determination of 

 sodium and potassium. 



Isohutyric acid-acetic acid solvent: 80 ml isobutyric acid, 20 ml glacial acetic acid, 

 and 20 ml water. This solvent has been used for the resolution of certain unidentified 

 urinary constituents. 



Detection of Spots. If radioactive materials are being studied, then 

 the spots may be readily detected by counting procedures or autoradio- 



grams. Descriptions have been pre- 

 sented of various automatic scanning 

 devices based on measurements of radio- 

 activity (13, 25 to 27) . These devices 

 are based upon a method of moving the 

 paper strip slowly past a Geiger tube, 

 with the counts being automatically 

 recorded on a chart which can be 

 matched against the chromatogram. 

 Figure 8-5 shows such an arrangement, 

 as developed by Frierson and Jones 

 (13) ; this type of assembly is also avail- 

 able commercially. Some substances 

 may be detected by virtue of their color 

 or fluorescence. However, in general, 

 it is necessary to use a reagent that 

 reacts with the substances being sep- 

 arated to produce a visible color. If 

 possible, the reagent should be apphed 

 in alcoholic solution, since this permits 

 rapid drying and minimizes the spread- 

 ing of spots. Usually the reagent is 

 apphed by hght spraying. Following 

 is a description of some of the com- 

 monly used color-developing reagents, 

 as employed by Williams (9), particularly in the analysis of urine: 



Alizarin-ammonia reagent: Chromatograms are sprayed with a 0.1 per cent solution 

 of alizarin in 95 per cent ethanol and then exposed to ammonia vapor. The reagent, 

 tightly stoppered, is stable indefinitely. Calcium and sodium salts of weak acids 

 appear as blue to purple spots against a lavender background. Acid areas appear 



Fig. 8-5. Assembl}^ for scanning of 

 paper strips. The strip is attached to 

 the chart of a recording Brown poten- 

 tiometer and is fed past a colli- 

 mated Geiger tube. The pulses are 

 fed to a count-rate meter which op- 

 erates the recorder. [From W. Joe 

 Frierson and John W. Jones, Radio- 

 active Tracers in Paper Partition 

 Chromatography of Inorganic Ions, 

 Anal. Chem.., 23: 1447-1452 (1951).] 



