380 



RADIOISOTOPES IN BIOLOGY AND AGRICULTURE 



Table 8-4. Summary of Chromatographic Separation of Inorganic Anions 



Detection 



Arsenate. . . . 



Arsenite 



Bromate. . . . 



Bromide 



Carbonate. . . 

 Chlorate. . . . 



Chloride 



lodate 



Iodide 



Nitrate 



Nitrite 



Phosphate. . . 



Sulfate 



Thiocyanate. 



Ammoniacal AgNOs 

 KI, HCl 

 AgNOs, H2S 



AgNOa, H2S 

 KI, HCl 



Fe(N03)3, HoOo; AgNOs, H.S 

 Universal indicator 

 KI, HCl 



Ammoniacal AgNOs 

 Universal indicator 

 Fe(N03)3, HoO.,; AgNOs, H2S 



* Solvent composition: A, pyridine-10 per cent H2O; B, butanol-pyridine-1.5 N 

 NHs; C, butanol-1.5 A^ NH4OH. 



(From Richard J. Block, Raymond Lestrange, and Gunter Zweig, "Paper Chroma- 

 tography, A Laboratory Manual," Academic Press, Inc., New York, 1952.) 



It was obvious that the C^^ had been incorporated into several different 

 compounds, but the identification of the compounds corresponding to the 

 darkened areas or spots required further investigation. It was first nec- 

 essary to prepare a "map" for the given solvent system using known 

 compounds detected by chemical reagents or known isotope-labeled com- 

 pounds. Comparison of the experimental chromatogram with the map 

 gives a clue as to the nature of the compound. Further evidence was 

 obtained by elution of the spot and chemical treatment to yield a sub- 

 stance that could be chromatographed again for comparison with a known 

 sample of the expected resultant compound. For example, a character- 

 istic spot was believed to be due to phosphoglyceric acid ; hydrolysis gave 

 a radioactive compound that was found to be identical with a sample of 

 authentic glyceric acid. As added identification, the unknown radioac- 

 tive material can be chromatographed together with an authentic sample 

 of the suspected substance. If the two compounds are identical, there 

 should be complete coincidence of the spots due to the two substances. 



Another example in plant metabolism is presented by the papers of 

 Steward and associates in connection with the estimation of the free amino 

 acids of plant cells. Dent, Stepka, and Steward (42) were able to isolate 

 from potato tuber 24 well-defined spots giving typical ninhydrin reactions. 



