378 



RADIOISOTOPES IN BIOLOGY AND AGRICULTURE 



Table 8-2. Summary of Chromatographic Separation of Organic Substances 



{Continued) 



contains ample testimony to its ever-increasing use. It is the intention 

 here to illustrate how the combined techniques of paper chromatography 

 and radiotracers are mutually advantageous. 



The use of radioisotopes extends the sensitivity of an ordinary chro- 

 matographic procedure by facilitating the detection and estimation of the 

 separated components. There are three general methods for association 

 of the radioisotope with the resolved constituents of the chromatogram, 

 as discussed by Winteringham et al. (25) : Labeling of initial mixture: This 

 is usually accomplished by incorporation of the radioisotope into the 

 metaboHzing system, e.g., growing of plants with C^"* and S^^ and later 

 separating the amino acids; or by administering P^^ to animals and later 

 separating the thyroid hormones and/or protein complex. It is also pos- 

 sible to treat the mixture with a labeled reagent before the chromato- 

 graphic separation. Labeling of chromatogram: It is possible that labeled 

 reagents could be applied to the completed chromatogram so as to 

 react selectively with the separated constituents. Neutron activation of 

 the chromatogram: It is also possible that the completed chromatogram 

 could be irradiated in the pile to produce measurable radioactivity in the 

 separated constituents. The success of this method would be dependent 

 upon producing a significantly higher radioactivity in the elements of the 

 separated constituents than in the filter paper. 



A classical illustration of the use of paper chromatography to resolve 

 mixtures is the research of Calvin and coworkers (40, 41) in studies on 

 photosynthesis (see Chap. 1). In a typical study, Chlorella were exposed 

 to C^^ for a 60-sec period of photosynthesis and then killed and extracted 

 with alcohol. The extracts were concentrated to a small volume, and an 

 aliquot placed in the corner of a square sheet of filter paper and dried with 

 an air stream. A two-dimensional chromatogram was prepared using 

 phenol-water as the first solvent and butanol-propionic acid-water as the 

 second solvent. The chromatogram was exposed on X-ray film to pro- 

 duce the results shown in Fig. 8-7. In this chromatogram the origin was 

 near the lower right corner, and the phenol-water was used to develop 

 running from right to left, whereas the second solvent was employed 

 running from bottom to top. 



