224 



WALDO E. COHN 



cytidylic acid peak, deoxy-5-methylcytidylic acid can be separated [see 

 Sect. III.l.e(3)]. 



d. Use of Higher pH; the Separation of Inorganic Phosphate and Nonnucleo- 

 tide Phosphoric Acid Esters 



Elution of nucleotides by dilute acids involves both principles of elution 

 mentioned earlier, charge adjustment (pH dependent) and ionic strength 

 adjustment (chloride-ion dependent). With increase in pH it is also neces- 

 sary to increase the chloride ion in order to maintain a reasonable rate of 

 elution; thus at pH 5.6, a chloride concentration of about 0.02 M is neces- 

 sary to maintain rates of elution comparable to those indicated for 0.003 

 N HCl in Fig. 8. Under these conditions, cytidylic and uridylic acids ap- 

 pear together, as do guanylic and adenylic acids (see Fig. 7). Inorganic 

 phosphate, which appears in the adenylic acid region at pH 2-3, precedes 

 the pyrimidine nucleotides by a wide margin at pH 6. Creatine phosphate 

 and sugar phosphates behave similarly in the acid region but are separable 

 from inorganic phosphate in the neutral region by virtue of the lower sec- 



10 2.0 30 



Uten HCl (from pH odiuttment ) 



Fig. 8. Separation of ribonucleotides by anion exchange in a chloride system 

 showing conformity with calculated theoretical curves (see text).'' 

 Exchanger: Dowex-2-chloride, 2.5 cm. X 0.94 cm.*. 

 Solution: 0.003 A^ HCl, 0.8 ml./min. 

 Sorbed materials: purified nucleotides, in amounts shown. 



