ION-EXCHANGE CHROMATOGRAPHY 



221 



.BEST CHROMATOGRAPHIC 

 RANGE 



80 160 240 J20 



RELATIVE DISTRIBUTION COEFFICIENT (ml per ml rRA-flOO TO C/C . 5 1 



Fig. 5. Relative distribution coefficients (ml. effluent per ml. exchanger to C/Co = 

 0.5) of bases and ribosides as a function of pH in 0.01 M Cl~, as derived from break- 

 through experiments. '^ 



Exchanger: 1 ml. IRA-400. 



(Circled letters T, C, A, U, and G are taken from Fig. 20, to show the influence of 



borate ion on thymidine (no effect), cytidine, adenosine, uridine, and guanosine. 



respectively.) 



c. Ribosides 



The similarity in the behavior of the ribosides is shown in Fig. 4, in 

 which a mixture of bases and ribosides has been chromatographed. This 

 method has been appUed to the large-scale separation of the deoxynucleo- 

 sides^" for preparative purposes. 



A summary of breakthrough sorption experiments on bases and nucleo- 

 sides, for the purpose of determining relative distribution coefficients, is 

 given in Fig. 5. 



III. Separation of Nucleotides 

 1. Mononucleotides 



a. Ionic Properties 



The nucleotides differ markedly in ion-exchange characteristics from the 

 bases and ribosides by virtue of the presence of the strongly acidic phos- 

 phoryl groups. Phosphate esters are stronger acids than inorganic ortho- 

 phosphate itself, with pK values of about 1 and 6 for the first and second 

 dissociation, respectively, as against 2 and 7 for orthosphophate. The dif- 

 ference in secondary ionization constants makes possible the easy separation 

 of inorganic phosphate from phosphoric acid esters in the region of pH 6 

 (see Sect. Ill.l.rf). 



b. Distribution Coefficients 



Among the nucleotides, anionic behavior is predominantly a function of 

 the phosphate group, but this is modified by the ionic and nonpolar proper- 

 ties of the nucleoside residues. An assessment of the total charge due to the 



