ION-EXCHANGE CHROMATOGRAPHY 



225 



ondary pK of phosphoric acid esters. The data" on the separation of these 

 substances are summarized in Fig. 10. 



e. Isomeric Nucleotides 



(1) Choice of Anion. Other acids and their salts may be used in place of 

 chloride. In general, those anions \nth a greater affinity than chloride for 

 the exchanger (e.g., Br~, SO4 ) vaW effect a given rate of elution with 

 lower ionic strength or lower hydrogen-ion concentration or both; those 

 with a lesser affinity (e.g., formate, acetate) \\\\\ require a lower pH or a 

 higher anion concentration. The effectiveness of a given anion in replacing 

 a specific solute will depend upon its affinity for the exchanger, which is a 

 function of pK, and upon intrinsic properties. Formate has received much 

 attention,"' ^^ partly because of the ease of obtaining solutions of stable pH 

 in the region of 3-4 and partly because of the ease with which it and its 



500 1000 1500 2000 Z500 



m. FROM pH EQUILIBRATION 



Fig. 9. Separation of deoxyribonucleotides by anion exchange." 

 Exchanger: Dowex-1-chloride, 200-400 mesh, 8 cm. X 0.72 cm.^. 

 Eluting solution: HCl as shown, 1 ml./min. 



Sorbed material: mixed deoxynucleotides isolated by preliminary ion exchange 

 from 150 mg. DNA digested with deoxyribonuclease followed by intestinal phos- 

 phatase plus arsenate (method of Klein and Thannhauser used by Volkin ei alP 

 and Sinsheimer and Koerner^*). 



