ION-EXCHANGE CHROMATOGRAPHY 219 



by cation exchange, utilizing ionic-strength adjustment without any charge 

 adjustment (5-methylcytosine follows cytosine by a very slight margin, 

 whereas cytidine precedes it by a factor of 3.4'^). Uridine and cytidine have 

 been separated from pyridine or enzymic digests by sorption of the latter 

 on a sulfonic acid resin, followed by displacement with ammonia or pyri- 

 dine.2^'2* The possibilities of a separation of the bases by charge adjust- 

 ment (e.g., by increasing pH in a sodium or ammonium cycle, as Stein and 

 Moore^ have done for amino acids) has not been tried on the bases, but 

 Reichard and Estborn^'^ have utihzed this principle for the preparation of 

 four (N'^-labeled) deoxynucleosides in an ammonium cycle. Since adenosine 

 was not present in their mixture (the enzyme preparation having converted 

 it to deoxyinosine) , no information is at hand as to how well adenosine and 

 guanosme might separate in such a system. Extensive hydrolysis at the 

 acid-sensitive glycosidic linkage of the purines has accompanied other ex- 

 periments along this line.^" Such hydrolysis has also been observed with the 

 purine ribonucleotides in the hydrogen cycle. '^' ^' 



3. Anion Exchange 



a. General 



The anionic properties of the bases reside chiefly in the keto groups, 

 which can enolize and thus develop ionizable hydrogens. While the hydroxyl 

 groups of ribose are also weak acids, it has seldom been necessary to resort 

 to the high pH required to ionize them. The attachment of ribose to the 

 purine and pyrimidine bases does not interfere with the acidic group (ex- 

 cept in the case of adenine), so that the behavior of bases and ribosides is 

 again very similar. The absence of glycosidic lability in alkaline regions 

 make the anion-exchange method preferable to cation exchange. In addi- 

 tion, it is possible to exploit the method of elation bj' charge adjustment, 

 thus removing the substances by very dilute (e.g., 0.01 M) solutions buf- 

 fered in the region of the pK's involved instead of the concentrated acid 

 solutions employed in cation-exchange separations. 



h. Bases 



The five bases normally considered as constituents of nucleic acid have 

 enolic pK's in the descending order cytosine, adenine, thymine, guanine, 

 and uracil. The order of elution by NH4OH-NH4CI buffers (Fig. 3) shows 

 two aberrations from this sequence: adenine follows guanine and uracil 

 precedes thymine. In the latter case, the additional methyl group may be 

 responsible for the higher distribution coefficient. 



" D. T. Elmore, Nature 161, 931 (1948); J. Chem. Soc. 1950, 2084. 



" R. J. C. Harris and J. F. Thomas, Nature 161, 931 (1948); /. Chem. Soc. 1948, 1936. 



" P. Reichard and B. Estborn, Acta Chem. Scand. 4, 1047 (1950). 



