SEPARATION BY PAPER CHROMATOGRAPHY 261 



hours, a suitable volume is about 10 fx\., and using less does not result in 

 significantly smaller final spots. 



A substance may be eflficiently eluted in a small volume by cutting out 

 a pointed band of paper including the spot, and allowing water to diffuse 

 through it toward the point. The solute is carried with the water, which 

 may be collected from the point of the paper in a small vessel,^^ in a pipet^^ 

 or directly on another sheet of filter paper for re -chromatography.^^ This 

 technique has been used to concentrate minor components such as 5-methyl- 

 cytosine from nucleic acids for estimation.*^ 



For quantitative estimation, spots are cut out and eluted by soaking in a 

 volume of liquid appropriate for the cells in which the extinctions are to be 

 read (4-5 ml. for the 1-cm. cells of the Beckman or similar spectrophotom- 

 eter). 0.1 iV HCl is an eluent'^ in which the nucleic acid bases are suffi- 

 ciently soluble, and in which some of them have higher extinction coeffi- 

 cients than in neutral or alkaline solutions. Standing overnight at room 

 temperature, with shaking before and after, effects quantitative elution. 

 To allow for ultraviolet-absorbing substances in the paper, blanks are cut 

 equal in area to the spots and at equal distances from the starting line, and 

 are eluted and read at the same wavelengths as the corresponding spots. 

 As a further precaution against error due to ultraviolet-absorbing contami- 

 nants, Vischer and Chargaff^^ read the extinctions of all eluates both at the 

 absorption peak of the substance being estimated and at another reference 

 wavelength where its absorption is low, basing their calculations on the 

 difference. The same procedure was adopted by Crosbie et al.^^ in analyzing 

 HCl and HCIO4 hydrolysates, and it is evident that it will generally reduce 

 the error from contaminants, although their absorption at the two wave- 

 lengths will rarely be precisely equal. Spectral data for use in estimating 

 nucleic acid derivatives by the absorption at their maxima and by the dif- 

 ference method are given in Tables III and IV, respectively. [Cf. Beaven, 

 Holiday and Johnson, Chapter 14.] Hotchkiss^ gives tables of the absorp- 

 tion of nucleic acid derivatives at 5-mM wavelength intervals, which may 

 be used to compute the composition of binary mixtures from density read- 

 ings at two or more wavelengths. 



The methods which have been described are suitable for estimation of purine and 

 pyrimidine derivatives in the range of 5 to 100 Mg- per spot. Quantitative analyses in 

 duplicate have been conveniently carried out on samples of 0.3 to 1.0 mg. of nucleic 

 acid by making up hydrolysates to a volume of 25 m1-, from which two 8-^1. portions 

 are taken for chromatography and two 2-^1. portions for phosphorus estimation."' «' 

 In an ultra-micro adaptation of paper chromatography recently described by Ed- 

 strom," only one hundredth of this amount of material is required. The hydrolysate 



" C. E. Dent, Biochem. J. 41, 240 (1947). 



92 R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 41, 590 (1947). 



" A. M. Moore and J. B. Boylen, Science 118, 19 (1953). 



