SEPARATION BY PAPER CHROMATOGRAPHY 245 



rated, the thick papers Whatman No. 3 (grained surface) and No. 3MM (smooth 

 surface) are useful. No. 3 is approximately 2.2 times as thick as No. 1, and more than 

 10 mg. of a substance can be separated from a mixture applied as a band across a 

 single sheet (183^" x 22''^"). S-Hydroxymethjlcytosine was isolated from bacterio- 

 phage DNA on this type of chromatogram in amounts adequate for crj'stallization 

 and analysis." 



Filter papers contain a certain amount of ultraviolet-absorbing material which 

 may be elated by a chromatographic solvent, especially if the latter is acid, and col- 

 lect in a band at or behind the solvent front. This can be removed by prior washing of 

 the paper, which is desirable in preparative work. In quantitative work, it may gen- 

 erally be allowed for by taking appropriate blanks. For successful chromatography 

 of phosphoric esters in many solvents, however, thorough washing of the paper is 

 essential, as otherwise the presence of metallic ions maj^ cause streaking or double 

 spots.'-' '^ 



With a given solvent system, precise Rf values^^ are influenced by (a) the 

 composition of the vapor phase in the chromatography vessel, which, 

 ideally, should be in equilibrium with the solvent mixture before a nni is 

 started, (b) the temperature, which affects partition coefficients, (c) the 

 direction (ascending or descending) and length of run, since the composition 

 of the solvent may change during its passage through the paper, ^^ and (d) 

 the paper, of which some variation is found even between batches of one 

 grade. If these conditions are adequately controlled Rp values can be ac- 

 curately reproduced. ^^ However, as has frequently been observed, a charac- 

 teristic pattern of the spots can be maintained despite considerable varia- 

 tion in absolute values of Rp , so that precise control is usually unnecessary. 

 The sensitivity to environmental conditions depends on the particular sol- 

 vent system. 



III. Detection of Purine and Pyrimidine Derivatives on Filter Paper 



1. Purine and Pyrimidine Bases 



In the earlier experiments, a variety of means were tried for determining 

 the positions of nucleic acid derivatives on filter paper. Hotchkiss- cut the 

 paper into narrow bands, each of which was eluted for measurement of its 

 ultraviolet extinction in the spectrophotometer. Vischer and Chargaff ^ ■ '^ 

 treated the paper with salts of mercury, and, after the excess had been 

 washed out, the mercury fixed by the purine and pyrimidine bases was 

 made visible by conversion to black mercuric sulfide. Another chemical 



" G. R. Wyatt and S. S. Cohen, Biochem. J., 55, 774 (1953). 

 '2 C. S. Hanes and F. A. Isherwood, Nature 164, 1107 (1949). 

 13 K. C. Smith and F. W. Allen, Federation Proc. 12, 269 (1953). 



movement of band 



" Defined' as Rp = 1 — 7 — ^ '■ ^ : — ^ ,. . ,. 



movement oi advancing front or liquid 



16 L. Horner, W. Emrich, and A. Kirshner, Z. Elektrochem. 56, 987 (1952). 



18 E. C. Bate-Smith, Biochem. Soc. Symposia {Cambridge, Engl.) No. 3, 62 (1949). 



" E. Vischer and E. Chargaff, J. Biol. Chem. 176, 703 (1948). 



