272 



J. D. SMITH 



T 



10 20 30 



Distance moved (cm.) 



Fig. 2. Graph of mobility observed (in cm./2 hr./20 v. /cm.) against the mobility 

 (relative to uridylic acid) calculated from the charge and frictional resistance for 

 19 mono- and polynucleotides'" (two of the points are on top of others). 



While 3.5 is one of the most useful pH values, the four nucleotides having 

 the greatest differences in net charge at pH 3.5, it is not suitable for all 

 separations. Another useful pH is 5 where all mono-, di-, and trinucleotides 

 have net negative charges of approximately 1,2, and 3, respectively, and 

 so separate with relative mobilities of 1:1.4:1.5. It is important to note 

 that at pH 7-8, where the secondary phosphate is fully dissociated, the 

 mono-, di-, and trinucleotides have relative charges of 2, 3, and 4 and their 

 relative mobilities are 1, 1.05, and 1 so that in this pH range their separation 

 is not possible. 



3. Purines, Pyrimidines, and Nucleosides 



The ionizing groups on the bases and nucleosides are the amino and enol 

 groups (the basic dissociations of the purine rings have pK values between 

 and V^ and may be neglected). The dissociation constants of these are 

 close to, although not identical with, those of the corresponding nucleotides 

 (Table I), so their electrophoretic separation is similar to that of the cor- 

 responding nucleotides. At pH 3.5, for example, uridine is uncharged, 

 while cytidine, adenosine, and guanosine migrate towards the cathode in 



13 J. K Wood, J. Chem. Soc. 89, 1839 (1906). 



