SEPARATION BY PAPER CHROMATOGRAPHY 255 



obtained if the acid solvent is used first ; the rate of flow of the second solvent 

 is then more rapid than in untreated paper. The spacing of spots with such 

 a system is shown in Figure 1 . 



Carter's dibasic sodium phosphate system is also included in Table I 

 (solvent i). Pyrimidines are well separated from purines, but within each 

 class there is little resolution. The use of water, or 0.01 M phosphate buffer, 

 to bring about rapid separation of pyrimidines from purines was first de- 

 scribed by Zamenhof et al.^^ Slightly greater dispersion oi Rp values is 

 given by water adjusted to pH 10, according to Levenbook,^^ whose data 

 on this system are included in Table I (solvent j). The chief virtue of water 

 as a solvent is its rapid flow, so that purine bases can be completely sepa- 

 rated from pyrimidines in 3 hours. 



3. Separation of Nucleotides 



Because of the strong polarity of their phosphoryl groups, nucleotides 

 do not move at appreciable rates in relatively nonpolar solvent systems 

 such as water-saturated n-butanol. Their movement may be accelerated by 

 increasing the content of water or other polar components in the system, 

 or by suppressing phosphoryl dissociation by addition of acid. These con- 

 ditions prevent full advantage being taken of the ionic differences in the 

 constituent purine and pyrimidine bases, and separation of the nucleotides 

 by paper chromatography has proven somewhat difficult. Effort has been 

 expended on the problem in a number of laboratories, and some of the more 

 satisfactory solvents which have been developed are shown in Table II. 

 None, however, is entirely satisfactory for separation of the nucleotides of 

 the four bases from PNA in a single run, although these may be resolved by 

 two-dimensional chromatography or by electrophoresis on filter paper. 

 [Cf. Smith, Chapter 8.] 



The two solvent systems containing hydrochloric acid (Table II, solvents a and 

 b) both separate the pyrimidine ribonucleotides and the purine bases excellently from 

 one another. In solvent a cytosine and thymine deoxyriboside diphosphates are also 

 resolved from the pyrimidine ribonucleotides.*^ The purine nucleotides are not satis- 

 factorily resolved in either solvent. The isopropanol mixture is the faster running of 

 the two. A mixture of n-butanol, ethanol, and 5 N HCl (3:2:2 by vol.) resolves pur- 

 ines and pyrimidine nucleosides and nucleotides, with the following Rp values;" gua- 

 nine, 0.24; adenine, 0.35; cytidine, 0.43; cytidylic acid, 0.54; uridine, 0.63; uridylic 

 acid, 0.78. 



Solvent c is one of several mixtures of acetone with carboxylic acids tested by 

 Burrows et al.^^ It affords good separation of guanylic, cytidylic, and uridylic acids; 



^* S. Zamenhof, G. Brawerman, and E. Chargaff, Biochim. et Biophys. Acta 9, 402 



(1952). 

 *^ L. Levenbook, personal communication, 1953. 



66 L. L. Weed and D. W. Wilson, J. Biol. Chem. 202, 745 (1953). 



67 J.-E. Edstrom, Biochim. et Biophys. Acta 9, 528 (1952). 



