254 



G. R. WYATT 



fff in Isoproponol-HCI 



Fig. 1. Diagram of the positions of nucleic acid derivatives on a two-dimensional 

 chromatogram run on Whatman No. 1 paper by the descending technique first in 

 solvent/ (Table I), then in solvent c (Table I; however, in the large chromatogram 

 tank used here, the effective NH3 concentration is reduced). A, adenine; AA, yeast 

 adenylic acid; AR, adenosine; C, cytosine; CA, cytidylic acid; CR, cytidine; G, 

 guanine; GA, guanylic acid; GR, guanosine; HMC, 5-hydroxymethylcytosine; HX, 

 hypoxanthine; HXR, inosine; MC, 5-methylcytosine; T, thymine; TDR, thymidine; 

 U, uracil; UA, uridylic acid; UR, uridine; X, xanthine. 



This solvent resolves up to 75 ng. per spot of each of the DNA bases in 35 

 cm. movement of the front. Uracil is also resolved; 5-hydroxymethylcyto- 

 sine, however, runs together with cytosine. Ribosides run at similar rates 

 to their bases, and deoxyribosides rather faster; purine deoxyribosides are 

 decomposed by the acid. 



When using solvents containing a high proportion of hydrochloric acid, the acid 

 must be thoroughly removed from the paper at the conclusion of the run by evapora- 

 tion at not too high a temperature (to avoid charring). Residual acid may damage 

 photographic paper used for printing the chromatograms and, according to Schramm 

 and Kerekjarto,^' on exposure to ultraviolet light may liberate chlorine which de- 

 stroys cytosine by oxidation. 



For mixtures too complex to be resolved in one dimension, a useful two- 

 dimensional system is the isopropanol-HCl solvent in combination with 

 n-butanol-NHj or with isopropanol-NHs. Better movement of guanine is 



" G. Schramm and B. von Kerekjdrt6, Z. Naturforsch. 7b, 589 (1952). 



