HYDROLYSIS — ESTIMATION OF BASES IN PNA 201 



two wavelengths, \i andX2, is the sum of the densities due to each component at each 

 wavelength.^' ^-^ ^^ Thus from the Beer-Lambert law two simultaneous equations 

 can be written in which the concentrations of A and B are expressed as a function of 

 their molecular extinction coefficients and the optical densities at the respective 

 wavelengths as follows :^^ 



CaEaXi + CbEbxi = jDxi 

 and 



CaEa\2 + CbEb\2 = D\2 



Ca and Cb are the respective concentrations of A and B expressed as moles per liter; 

 Ea\i, Eb\i, EA\2f s-rid Eb\2 are the respective molecular extinction coefficients of A and 

 B at Xi and X2; and Dx^ and Dx2 are the optical densities at the two wavelengths. 

 Solution of the equations for Ca and Cb gives the following: 



^ £^sXiJDx2 — EbmDx, 



EamEbXi — EaXiEbm 



and 



Cb = 



EaXuDxi — EaXjDx^ 



EaXiEbXi — EaXxEbX". 



The pairs of wavelengths selected for the estimation of adenine and guanine or of 

 cytidine and uridine are somewhat arbitrary, but, in order to achieve as high a sensi- 

 tivity as possible for the detection of either component, it is desirable to use wave- 

 lengths where relatively large differences in the molecular extinctions of the two 

 compounds occur, but at which both show appreciable absorption. If one of the 

 wavelength pairs is selected near either end of the ultraviolet spectrum as well, a 

 farther check is obtained as to whether or not ultraviolet-absorbing impurities are 

 present which absorb appreciably at the shorter or longer wavelengths. Similarly, by 

 using wavelengths at which intersection points of the respective absorption curves 

 occur, estimates of the total concentration of both components can be made, or, by 

 using a wavelength at which one component fails to absorb, the other can be esti- 

 mated independentl3^ 



In the analysis of PNA performed in the author's laboratory* • 2- t\vo 

 wavelength pairs, 262 and 280 m/x, and 262 and 240 m/i, were used for esti- 

 mation of adenine and gvianine and both intersection points at 252 m^z and 

 276 m/i for calculation of total purine bases. The respective equations using 

 molecular extinction values for highly purified samples of adenine and 

 guanine are as follows: 

 Adenine 



^ 6.66D262 - 7.65Do8o ^ 9.40Dj62 - 7.65D.40 



262-280 mti C = ■ 262-240 m^, C = — 



5.U X 10^ 8.25 X 10^ 



'* If the optical density ratios of the pure substances at the wave lengths chosen are 

 appreciably-d-ifferent, the relative amounts of the two components present can be 

 determined most simply by extrapolation of the optical density ratio. 



