244 G. R. WYATT 



chromatography on ion-exchange columns and electrophoresis on filter 

 paper, is playing an important part in investigations into the specificity of 

 nucleases and the molecular structure of nucleic acids. 



The present article aims to provide a practical guide to paper chromato- 

 graphic methods for quantitative analysis of nucleic acids and for separa- 

 tion and estimation of their components and related substances. 



II. General Technique of Paper Chromatography 



The general procedures used in chromatography on filter paper are by 

 now well known and are covered by several reviews and books.*-^* Both 

 the original descending technique (in which the upper edge of the paper 

 dips into a trough containing the solvent^), and the ascending technique 

 (in which the paper, rolled into a cylinder, stands in a dish of the solvent*) 

 have been widely used, and give similar results. The ascending method is 

 convenient for small two-dimensional chromatograms but becomes im- 

 practical when the solvent is required to flow more than about 25-30 cm. ; 

 the descending method has the advantages that the solvent may be allowed 

 to flow an indefinite distance and that the solvent in the trough may be re- 

 newed while retaining the vapor in the tank. 



The troughs required for descending chromatography may be made of plastic or 

 stainless steel, but for chemical reasons glass is to be preferred, and it is perhaps 

 worth describing an especially simple method of making glass troughs.' Using a cut- 

 ting diamond mounted at the end of a steel rod,i" two longitudinal scratches are made 

 inside a length of glass tubing about 2.5 cm. in diameter, separated from one another 

 by about 90° around the circumference of the tube. The tube is then gently tapped on 

 the outside with a piece of metal following the lines of the scratches, and will crack 

 along them producing a trough whose ends can be sealed in a flame. 



The grade of filter paper most frequently used has been Whatman No. 1, which is 

 satisfactory for quantitative and qualitative work with nucleic acid derivatives. 

 Whatman No. 4 is a faster running paper; in some solvent systems, however, this 

 may result in poorer resolution. Schleicher and Schvill.No. 597 paper has also been 

 used with similar results, although different grades of paper may give different mobil- 

 ities with the same solvent system. When larger quantities of material are to be sepa- 



3 A. J. P. Martin, Ann. Rev. Biochem. 19, 517 (1950). 



* R. J. Block, R. Le Strange, and G. Zweig, "Paper Chromatography." Academic 

 Press, New York, 1952 



^ J. N. Balston and B. E. Talbot, "A Guide to Filter Paper and Cellulose Powder 

 Chromatography." Reeve Angel and Co., London, and W. and R. Balston Ltd., 

 Maidstone, 1952. 



* F. Cramer, "Papierchromatographie," 2nd ed. Verlag Chemie, Weinheim, 1953. 

 ^^ E. and M. Lederer, "Chromatography." Elsevier Publishing Co., Amsterdam, 



1953. 

 ^ R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 38, 224 (1944). 

 « R. J. Williams and H. Kirby, Science 107, 481 (1948). 

 ' This process was first demonstrated to the writer by Dr. R. Markham. 

 '" Glass-cutting tool no. 6686, obtainable from A. Gallenkamp and Co. , Ltd. , London. 



