CHEMISTRY OF PURINES AND PYRIMIDINES 111 



pounds are characterized either by a very high point or by infusibility (the 

 latter is especially true of the hydroxylated purines). ^°^ This traditional 

 approach (including mixed melting points) can, therefore, only be of limited 

 value in the assessment of purity and identity, and in some instances (see 

 discussion on orotic acid, above) it may even be misleading. However, the 

 number of physical criteria of purity that are available (-when taken to- 

 gether) is sufficient to satisfy the most fastidious worker. 



When the amount of a specimen to be tested is small, paper chromatog- 

 raphy (Chapter 7) is the method of choice. Microgram quantities are used, 

 and the sample, after elution and spectrographic evaluation, is subjected 

 to reanalysis on paper. The specimen, to be judged "pure," must show a 

 single "spot" in more than one solvent system and constant spectral 

 characteristics (at more than one pH) after each chromatographic run. 

 For radioactive materials, coincidence with radioactivity must be estab- 

 lished. The same considerations apply to column chromatography (Chapter 

 6) in that a single band must be demonstrated and the variables are the 

 adsorbents (anion- or cation-exchange resins, starch, etc.) as well as the 

 solvent systems. It is highly unlikely that a mixture of pyrimidines or 

 purines would behave as a homogeneous substance when subjected to all 

 these tests.^'" 



When an adequate supply of a preparation is available, and a standard 

 specimen of unquestioned purity is desired, the above techniques as well 

 as the follo\ving are used. The substance is subjected to repeated fractiona- 

 tion by crystallization (salt as well as neutral forms), passage through a 

 column, countercurrent distribution, etc., until constancy is achieved with 

 respect to properties such as those hsted in Tables I and II, and a cor- 

 responding constancy with respect to isotope content if the compound is 

 so labeled. The determination of elementary composition presents no special 

 problem with pyrimidines and purines. 



Additional criteria are the reproducibility of ultraviolet data, dissocia- 



'•" The eutectic mixtures of a number of pyrimidine and purine derivatives with di- 

 cyandiamide exhibit well-defined melting points which are much lower than those 

 of the original derivatives (K. Dimroth and H.-G. Meyer-Brunot, Biochem. Z.-323, 

 343 (1952)). The determinations are easily carried out on tiny quantities. 



"" It is possil)le, for example, that the growth response of a bacterial mutant may be 

 due to a small impuritj' in a purine or pyrimidine preparation. The importance of 

 using homogeneous compounds in biological studies, therefore, cannot be stressed 

 too strongly. Yet, despite the ease and simplicity of paper chromatography, the label 

 on a bottle has often been accepted too literally. As examples of extreme inhomo- 

 geneity, the author has examined by paper chromatography a number of commer- 

 cially available "hypo.xanthine" preparations only to find (with one exception) 

 these to contain, on the average, about 20% of adenine. One sample of "guanine" 

 was found to show some four to five "spots, " and only one small "spot" corresponded 

 in Rf value to guanine. 



