24 PHOTOCHEMICAL PRINCIPLES 



ments are described by Shibata (1958). Barer (1955) has reduced 

 the scattering from cell walls by suspending cells in concentrated 

 protein solutions to measure their absorption spectra. Bateman and 

 Monk (1955) have used a glass flask packed in white powder as an 

 integrating sphere. The flask is filled with water for the incident 

 intensity reading, then with the highly diluted sample. 



We have seen how light scattering can greatly distort absorption 

 spectra of live cells and how some of this trouble can be avoided. If 

 the absorption of tissues or cells are correctly measured by these or 

 similar procedures, the absorption can be compared directly with 

 action spectra. 



ACTION SPECTRA 



One of the basic principles of photochemistry is that only light that 

 is absorbed has a chemical effect. For this reason, the effectiveness of 

 different wavelengths in causing chemical action is proportional to the 

 absorption by the active pigment concerned. An action spectrum, 

 when determined with appropriate precautions, thus gives the absorp- 

 tion spectrum and hence identifies the light-absorbing pigment. 



When no inactive pigments are present, the action spectrum may be 

 practically identical with the absorption spectrum of the whole system. 

 At the other extreme the action spectrum may have bands that are not 

 identifiable in the absorption spectrum and may show no effectiveness 

 whatever for some pigments the absorption of which is evident. The 

 basic requirement that should be met in determining precise action 

 spectra is to use a sufficiently thin layer of lightly pigmented material. 

 It is necessary to make sure in this way that all parts of the sample 

 receive equal intensities of light. In addition to this basic optical 

 requirement, the chemical requirement of not saturating the system 

 with light must be met. This means that intensities should be used 

 in the range where the effectiveness of the light is proportional to 

 its intensity. Even if these two requirements are satisfied, the result- 

 ing action spectra may be distorted if the system contains a high con- 

 centration of other pigments which are inactive but which may act as 

 an internal screen for some colors. Fortunately, internal scattering is 



