116 AARON BENDICH 



manifested at values below pH 1, has not been measured.) The curves for 

 the cationic form, at pH 1.0, and for the neutral or nondissociated form, 

 at pH 7.2, cross at point a, indicating that at this wavelength (266 mju) 

 the two forms have the same extinction coefficient. This point is common 

 to the curves at all pH values from 1.0 to 7.2, and is called an isosbestic point 

 (point of equal extinction). ^^^'^27 Isosbestic point a delineates the equilibrium 

 (pK'ai) between the cationic and neutral forms; isosbestic points 6, c, and 

 d (pH 7 to 14) are concerned with the equilibrium between the neutral and 

 anionic forms. Since the neutral form is conmion to both equilibria, its 

 curve should (and does) pass through all the isosbestic points. The sharp- 

 ness of the isosbestic points is one measure of the purity of the absorbing 

 substance and of the precision in the recording of the spectra. "If curves at 

 a sufficient number of pH values are run, the plot of extinction coefficients 

 vs. pH at a given wavelength would give a titration curve. "^^^ From this 

 the apparent pKo can be calculated. For homogeneous substances, a pKa 

 value calculated from the extinctions at one wavelength should be the 

 same as that calculated from other wavelengths. 



From these considerations, it follows that the nature and even the po- 

 sition (s) of potentially tautomeric groups of an unknown pyrimidine (or 

 purine) derivative may be diagnosed from a study of its spectral behavior 

 as a function of pH. The molar concentration need not be known since the 

 spectral behavior is essentially independent of the concentrations usually 

 employed in ultraviolet spectrophotometry. 



Molecular extinction coefficients and absorption maxima are listed in 

 Table I for pyrimidines and purines often encountered in nucleic acid 

 studies. Whatever differences exist between these and other published 

 values are probably due to small variations in the instruments in use. 



2. Chemical Properties 



"As the simpler compounds are much less well known than the highly 

 hydroxylated or amino members, a rather distorted impression of pyrimi- 

 dine chemistry has grown up, much as if the behavior of benzene were 

 known only through the reactions of compounds like phloroglucinol."^^* 

 An analogous statement may be made in regard to purine. Pyrimidine and 

 purine are probably the most stable members of the series and can survive 

 treatment with strong oxidizing (but not reducing) agents, concentrated 

 sulfuric (100°) and nitric acids.^^'^^* The general stabihty of pyrimidine 

 may be attributed^^^ to the influence of the ring nitrogens which results in 



'25 A. Thiel, A. Dassler, and F. Wiilfken, Forischr. Chem. Physik u. physik. Chem. 18, 

 (3), 79 (1924). 



327 W. R. Erode, "Chemical Spectroscopy," 2nd ed. p. 249. Wiley, New York, 1943. 



328 B. Lythgoe, Quart. Revs. (London) 3, 181 (1949J. 



