SEPARATION BY PAPER CHROMATOGRAPHY 247 



the positions of substances absorbing ultraviolet light of the wavelength used appear 

 as white areas on a dark background. These spots may then be traced on the chromat- 

 ogram itself. Slightly greater sensitivity is claimed for a technique identical in 

 principle but utilizing the 257-m/x and 275-miu emission lines from a cadmium arc.^^ 



It has been pointed out by Smith and Markham^^ that guanine and com- 

 pounds containing it fluoresce quite strongly in Hght of wavelengths 253.7 

 and 265 mju, and are thus easily differentiated from other nucleic acid de- 

 rivatives. Acid conditions are required, and may be created by exposing 

 the chromatogram to fumes of hydrochloric acid. Xanthine behaves simi- 

 larly .^^^ The effect may be recorded photographically by inserting between 

 the chromatogram and the photographic paper a sheet of cellulose nitrate, 

 which transmits only the fluorescent light. 8-Azaguanine and its compounds 

 fluoresce under both acid and basic conditions,-^'' and may be detected on 

 chromatograms by their fluorescence with greater sensitivity than by their 

 absorption of ultraviolet light.^" 



With the device described by Paladini and Leloir,'' in conjunction with the Beck- 

 man spectrophotometer, a continuous record may be obtained of the ultraviolet ab- 

 sorption of strip chromatograms. 



2. Nucleosides and Nucleotides 



The techniques using ultraviolet light described above are of course also 

 applicable to nucleosides and nucleotides. Some color reactions of the sugar 

 and phosphate portions of these compounds are also useful on occasion, as 

 in identification of unknowns or where it is necessary to use a chroma- 

 tographic solvent which itself absorbs in the ultraviolet range. 



Buchanan, Dekker, and Long'^ have developed means of detecting both ribo- and 

 deoxyribonucleosides on chromatograms by reactions of the sugars. The czs-glycol 

 structure present in ribosides may be oxidized either with periodate, the resulting 

 aldehydes being made visible with Schiff 's reagent, or with lead tetraacetate, in which 

 case white spots remain when the uncombined lead on the paper is converted to lead 

 dioxide. Other substances having this configuration, including adenosine-5'-phos- 

 phate, react, but riboside-2'- and -3'-phosphates and deoxyribosides do not. The 

 sensitivity of both methods is reported as about 20 /ug. of nucleoside. Deoxyribosides 

 on paper can be detected by adaptations of the Dische diphenylamine reaction or of 

 the Feulgen reaction, or of the reaction with cysteine.'' The last method is sensitive 

 to 10 iig. of deoxyriboside. [Cf. Chapters 9 and 17.] 



The positions of deoxyribosides on paper chromatograms have also been deter- 

 mined by virtue of their ability to promote growth of Labctobacillus leichmannii.^* 



28 J.-E. Edstrom, Nature 168, 876 (1951). 



" J. D. Smith and R. Markham, Biochem. J. 46, 509 (1950). 



"» J. Kream and E. Chargaff, J. A771. Chem. Soc. 74, 4274 (1952). 



'« R. E. F. Matthews, Nature 171, 1065 (1953). 



»i A. C. Paladini and L. F. Leloir, Anal. Chem. 24, 1024 (1952). 



32 J. G. Buchanan, C. A. Dekker, and A. G. Long, J. Chem. Soc. 1950, 3162. 



" J. G. Buchanan, Nature 168, 1091 (1951). 



3* V. Kocher, R. Karrer, and H. R. Muller, Intern. Z. Vitaminforsch. 21, 403 (1950). 



