398 RADIOISOTOPES IN BIOLOGY AND AGRICULTURE 



(Dowex 50) in the sodium form, with stepwise elution accomplished by- 

 sodium citrate buffers of increasing pH (10, 10a, 10b). 



Ions of Different Size. Williams and Johnson (11) noted that inor- 

 ganic salts interfered in an otherwise satisfactory method for the estima- 

 tion of pectin by electrodeposition. Interfering electrolytes were readily 

 removed by passing the solution through a sulfonic acid resin (H+ form) 

 and then through a weakly basic anion exchanger (free-base form). The 

 large size of the anion prevented the pectic acid from being retained by the 

 anion exchanger. This principle has also been used for the characteriza- 

 tion of pectins, the determination of the degree of substitution of cellulose 

 xanthate, and the purification of direct cotton dyes (2). 



Conversion of Nonelectrolytes to Ions by Complex Formation. The 

 isolation of radiochemically pure compounds has been a major difficulty 

 in biosynthetic procedures. This is particularly true for the sugars. 

 Advantage has been taken of the fact that sugars and certain other poly- 

 hydroxy compounds react with borate ions to form negatively charged 

 complexes that can then be resolved by ion-exchange chromatography 

 (12 to 16). This method has been most fruitful both for (luantitative 

 analysis of sugars found in biological materials and for isolation of labeled 

 sugars prepared biosynthetically. Advantages in isolation are that car- 

 rier is not needed, thus allowing the sugars to be obtained with a high 

 specific activity, and also that the method can handle large amounts of 

 sugar. 



The details of procedure can be noted from Table 9-3, which presents 

 a summary of some of the separations that have been accomplished. The 

 concentration of sugar in the eluate can be measured by the radioactivity 

 present, usually C^'', when radioisotopes are used. The colorimetric 

 anthrone method has been used for hexoses and polysaccharides, and the 

 orcinol reaction has been used for the pentoses. These procedures are 

 not specific for any one carbohydrate, but if a single sugar is present, as 

 in the eluates from the resin column, then the method can be made quan- 

 titative for that sugar. The borate ions were not found to interfere with 

 the colorimetric determinations. 



With similar techniques, phosphogiyceric acid, fructose diphosphate, 

 and the adenosine polyphosphates were separated from sugar monophos- 

 phates and from each other. A sequence of eluting solutions of varying 

 borate concentration, chloride concentration, and pH was used (17). 



TYPES OF APPLICATIONS 



Illustrative applications described in this section have been grouped to 

 point up the various experimental objectives that can be and have been 

 achieved. 



