ION-EXCHANGE CHROMATOGRAPHY 



223 



none). Since uridylic acid has no cationic group, we express the data in 

 terms of iiridyhc acid ehition (the parallelism of ionosinic acid should be 

 noted). The relative positions of cytidylic and of adenylic acids are main- 

 tained at all pH values; those of uridylic and guanylic acids become re- 

 versed at low pH due to the development of a charge upon the latter. The 

 same is true of adenylic acid and its derivative, inosinic acid. 



c. Elution with Dilute HCl 



A separation of four purified ribose nucleotides by dilute HCl at pH 2.5 

 is shown in Fig. 8.^^ The mixture was sorbed from dilute ammoniacal solu- 

 tion, with total chloride concentration less than 0.01 J\I, followed by 0.01 

 M NH4CI until the effluent pH fell to 7. (This NH4CI wash removes any 



Fig. 7. Observed relative distribution coefficients (positions of elution peaks) of 

 ribonucleotides as functions of pH, derived from column chromatography (see Fig. 8). 



bicarbonate present and thus prevents CO2 formation when the acid is 

 started, as well as eliminating a long delay until all amphol^'tes on the 

 resin are neutralized; it is generally advisable when proceeding from alka- 

 line to acid solutions.) The order predicted from Fig. 6 is not observed with 

 respect to the uridylic-guanylic acid sequence, nor with respect to the dis- 

 tance between cytidylic and adenylic acids; the aberrations are accounted 

 for by the factor discussed and are set forth in Fig. 7. Fig. 8 also illustrates 

 the use of specific ultraviolet absorption characteristics to follow the course 

 of a separation and the extent to which the sj^stem follows the Mayer 

 and Tompkins eciuation,* derived upon the assumption that equilibrium 

 between exchanger and solute is achieved throughout the elution sequence. 

 The separation of the deoxynucleotides (Fig. 9-^"-^) follows a similar pat- 

 tern with thymidylic taking the place of uridylic acid. From the deoxy- 



2' E. Volkin, J. X. Khym, and W. E. Cohn, J. Am. Chem. Soc. 73, 1533 (1951). 



28 R. L. Sinsheimer and J. F. Koerner, Science 114, 42 (1951). 



" R. O. Hurst, J. A. Little, and G. C. Butler, J. Biol. Chem. 188, 705 (1951). 



