128 QUANTITATIVE STUDIES 



Adherence to the Beer-Lambert Law 



When an adequate optical system has been used, and losses 

 of light by scattering and refraction minimised, the difference of 

 the intensities of the light incident upon the specimen and that 

 transmitted is due to absorption of photons. The question then 

 arises: what is the relationship between the amount of light 

 absorbed and the number of absorbing molecules. It is com- 

 monly assumed that this relationship is given by the Beer-Lam- 

 bert law, i.e.: 



E = log I /I = ecd 



where E = extinction, I = intensity of light entering specimen, 

 I = intensity of light leaving specimen, e = extinction coefficient, 

 c = concentration of absorbing substance, and d = thickness of 

 absorbing layer. This postulated linearity between the extinc- 

 tion and concentration is by no means an unbreakable rule, and, 

 unfortunately, in biological systems, as Caspersson has noted, it 

 is difficult to obtain adequate evidence of the extent to which the 

 rule is valid. The conditions prevailing in fixed specimens are 

 difficult to evaluate from the point of view of the relationship be- 

 tween E and c. Errors due to fluorescence may also be of im- 

 portance: these can be detected by use of a fluorescence micro- 

 scope. 



Commoner has made valuable contributions to this field. In 

 an elegant paper he showed that the Beer-Lambert law was valid 

 for the pigments in Coleus hair cells. And in an interesting paper 

 written with Lipkin he pointed out that, where the specimen 

 under examination contains optically anisotropic molecules, 

 marked deviations from the Beer-Lambert law occur if the aniso- 

 tropic molecules are markedly oriented. In the case of an army 

 of molecules which are completely oriented, with unpolarised 

 light the extinction is proportional to the number of molecules 

 only at relatively low extinction values (less than 0.15). As the 

 concentration rises E deviates increasingly from the linear rela- 

 tionship, and as E approaches 0.3 the extinction becomes inde- 

 pendent of the number of absorbing molecules: the proportion of 

 light absorbed may never exceed 50 percent. With such a sys- 

 tem, containing oriented anisotropic molecules, it is possible to 



