248 G. R. WYATT 



Nucleotides and other phosphoric acid esters on paper chromatograms can be de- 

 tected by sprajang with an acid molybdate solution, partial hydrolysis of the ester, 

 and reduction of the resulting phosphomolybdate complex to a blue-colored com- 

 pound.^* The necessary hydrolysis may be effected by heating the papers after spray- 

 ing, by ultraviolet irradiation,'^ or by previous spraying with a solution of phospha- 

 tase."- '* It is claimed that 0.1 ng. P can be detected. If the water in the reagent is 

 partially replaced by acetone^' the papers may be dipped in it instead of sprayed. 

 Nucleotides can also be detected by fixation of uranium,^" and phosphates, by fixa- 

 tion of ferric iron;" these reactions avoid the need for hydrolysis. 



IV. Solvent Systems 

 1. General and Theoretical Considerations 



The solvent systems with which successful separations were first ob- 

 tained on paper chromatograms consisted of organic fluids saturated with 

 water. Their effect was satisfactorily interpreted as resulting from the par- 

 tition of solutes between a water-poor mobile phase and a water-rich phase 

 held by the strongly hydrophilic cellulose fibers ; and for a number of amino 

 acids and carboxylic acids, partition coefficients calculated from Rp values 

 on the basis of this theory agree well with the coefficients directly meas- 

 ured. ''• ^2 A minor role may be played by adsorption and ion exchange, since 

 the cellulose fibers are electronegative in water, and carry a small number 

 of aldehyde and carboxyl groups.^ It was subsequently found that the sol- 

 vent need not be saturated with water, since the binding of water by the 

 cellulose results in a partition effect with miscible solvents just as with 

 water-saturated ones.^' In a further innovation, it was discovered that 

 separations may be obtained in the absence of any organic solvent, using 

 salt solutions, or even with water alone. Separations with water as the sol- 

 vent, apparently due to adsorption by the paper, may also be interpreted 

 as the result of partition between water and a water-cellulose complex. 

 [See below, Section IV.2.] 



The influence of the composition of the solvent system on the movement 



" Hanes and Isherwood'" spray the chromatograms at a rate of 1 ml. per 100 cm.^ 

 with a solution containing: 5 ml. 60% HCIO4, 10 ml. N HCl, 25 ml. 4% (NH4)2Mo04 , 

 and water to 100 ml.; then heat them to 85° for 7 min., and subsequently expose 

 them to H2S. Benson et al.*'' describe a similar reagent. 



3« R. S. Bandurski and B. Axelrod, /. Biol. Chem. 193, 405 (1951). 



" N. G. Doman and Z. S. Kagan, Biokhimiya 17, 719 (1952), seen only in abstract, 

 Chem. Abstracts 47, 4795 (1953). 



38 E. Fletcher and F. H. Malpress, Nature 171, 838 (1953). 



39 S. Burrows, F. S. M. Grylls, and J. S. Harrison, Nature 170, 800 (1952). 



*° B. Magasanik, E. Vischer, R. Doniger, D. Elson, and E. Chargaff, /. Biol. Chem. 



186,37 (1950). 

 " H. E. Wade and D. M. Morgan, Nature 171, 529 (1953) 

 <2 A. A. Benson, J. A. Bassham, M. Calvin, T. C. Goodale, V. A. Haas, and W. 



Stepka, J. Am. Chem. Soc. 72, 1710 (1950). 

 « H. R. Bentley and J. K. Whitehead, Biochem. J. 46, 341 (1950). 



