250 G. R. WYATT 



exclusively with two similar ring systems, but does account for such facts as 

 the better separation of 5-methylcytosine from cytosine with increasing 

 chain length of the alcohol used.** The nucleic acid derivatives have num- 

 erous possibilities for hydrogen bonding, which undoubtedly contributes 

 to the different order of movement of the nucleotides given by solvents 

 active in forming hydrogen bonds, such as phenol and butyric acid, than 

 by aliphatic alcohols. 



d. Salt Content 



Salt will generally decrease the mutual solubility of water and an organic 

 solvent, and will thus alter the partition of a chromatographic solvent sys- 

 tem, and for this reason the local presence of salt may cause distorted spots. 

 Addition of salt to a system selectively slows the movement of solutes, and 

 relatively strong salt solutions containing little or no organic solvent may 

 be used to obtain chromatographic separations on filter paper. Hagdahl and 

 Tiselius*^ have separated amino acids using 3 M phosphate buffer as the 

 solvent; they term this "salting-out chromatography" and attribute the 

 effect to reductions in solubility due to the presence of salt, with consequent 

 increase in apparent adsorption. With similar "salting-out solvents" (5% 

 ammonium citrate or sodium or potassium phosphate overlayered with 

 isoamyl alcohol, which is slightly soluble in water and has the effect of pro- 

 ducing more compact spots), Carter^^ has obtained separations of nucleic 

 acid components. The principle has been further applied by Markham and 

 Smith, ^"^ who used 0.8 saturated ammonium sulfate containing 2 % isopropa- 

 nol. It is notable that these systems separate substances in a different order 

 from the more usual organic systems, and that they are capable of resolving 

 nucleoside-2'- and -3 '-phosphates, a separation not accomplished in any 

 system of the more usual type. 



Two practical considerations to be borne in mind when preparing solvent 

 systems are: (a) it is preferable to select volatile substances (ammonium 

 sulfate might with advantage be replaced by ammonium carbonate) and 

 (b) when working with nucleic acid derivatives it is of course particularly 

 desirable to avoid substances absorbing in the ultraviolet range. 



2. Separation of Purine and Pyrimidine Bases and Nucleosides 



At the time of writing, over eighty solvent systems have been described 

 for separation of nucleic acid components on paper chromatograms. For- 

 tunately for the reviewer and for those using this technique, relatively few 

 of these show real advantages over others, and some of the simplest mix- 



« G. R. Wyatt, Biochem. J. 48, 581 (1951). 



" L. Hagdahl and A. Tiselius, Nature 170, 799 (1952). 



6» R. Markham and J. D. Smith, Biochem. J. 49, 401 (1951). 



