SEPARATION BY PAPER CHROMATOGRAPHY 249 



of substances has been discussed in terms of partition theory by Martin.^- ^^ 

 The following factors are among the most important which may be utilized 

 in preparing solvents to effect desired separations and in using paper 

 chromatography in the identification of unknown substances. 



a. Water content 



By using a miscible organic solvent, the water content may be varied 

 over a wide range. As water is added to the moving phase, the rates of migra- 

 tion of solutes will increase in proportion to their polarity. For example, by 

 altering the water content of propanol-water mixtures, the relative posi- 

 tions of adenine and adenylic acid-^ or 5-methylcytosine and 5-hydroxy- 

 methylcytosine^^ may be reversed. 



h.pH 



Since ionization will alter the partition of a solute in favor of the aqueous 

 phase, one can regulate the relative rates of movement of ionizable sub- 

 stances by control of pH, having regard to their dissociation constants. 

 [Cf. Cohn, Chapter 6, and Jordan, Chapter 13.] Thus, in neutral aqueous 

 n-butanol uracil migrates more rapidly than cytosine, but if the solvent is 

 made basic by the presence of sufficient ammonia the movement of uracil 

 and thymine may be slowed until the former has an Rp less than that of 

 cytosine. This may be explained by ionization of enolic hydroxyls (pKi = 

 9.5 and 9.9 for uracil and thymine, respectively*^), the hydroxyl of cyto- 

 sine having too high a pK (12.2) to be more than slightly affected by am- 

 monia. The relative mobilities are of course sensitive to the precise con- 

 centration of ammonia (cf. Table I, solvents a, h, and c). Addition of strong 

 acid or base to a chromatographic solvent may have further effects on par- 

 tition coefficients; for example, hydrochloric acid added to aqueous alco- 

 hols decreases the Rp's of all the purine and pyrimidine bases, but the 

 effect is differential and at about 1 A^ HCl the relative positions of adenine 

 and cytosine are interchanged.'*^ 



c. Nature of the Organic Components 



The importance of van der Waals' forces and of hydrogen bonding in 

 determining the partition of a system has been stressed by jNIartin.*^ When 

 solvent and solute are similar in structure, for example both aromatic or 

 both aliphatic, closer fit between their molecules, resulting in greater van 

 der Waals' forces and higher Rp values, may be expected. This principle is 

 of limited utility in separating nucleic acid bases, where we are concerned 



** A. J. P. Martin, Biochem. Soc. Symposia iCa77ibndgc, Engl), No. 3, 4 (1949). 



« G. R. Wyatt and S. S. Cohen, Nature 170, 1072 (1952). 



« D. Shugar and J. J. Fox, Biochim. et Bwphys. Acta 9, 199 (1952). 



" G. R. Wyatt, Biochem. J. 48, 584 (1951). 



