22 PHOTOCHEMICAL PRINCIPLES 



larger angle of 32 ± 2°, the peak position is shifted to 668 m/^. This 

 range of 20 m/x for the peak position shows the extreme differences 

 that may be given by various types of spectrophotometers. The impli- 

 cations of this experiment by Latimer are that small differences 

 reported from different laboratories for the absorption peaks of bi- 

 ological materials should be disregarded, and even large differences 

 should be interpreted with much caution. 



If we want to compare action spectra with absorption spectra of 

 the same material we are at once faced with the problem of how an 

 absorption spectrum really can be measured without any such distor- 

 tion being introduced by the peculiarities of a particular measuring 

 apparatus. For pigment identification or for comparing the absorption 

 spectra of living cells with action spectra it is usually not worth while 

 to worry about the reflection of light from the front surface of the 

 sample. The measurement of absorption for purposes of calculating 

 quantum yields, however, requires this further refinement. 



MEASUREMENT OF ABSORPTION SPECTRA OF LIVE CELLS 



Light entering a suspension of cells or a whole organ is to various 

 degrees reflected, transmitted, scattered, and absorbed. Since the light 

 from the sample comes out in various directions, it is difficult to 

 measure. Probably the soundest procedure theoretically, and in many 

 cases the simplest for practical problems, is to put the sample inside a 

 white sphere having a photocell in its wall. The photocell is supposed 

 to catch light coming out from the sample with equal effectiveness in 

 all directions. Such Ulbricht spheres have been used for years to 

 measure the total output of electric lamps. The spectrophotometer 

 designed by Hardy and made by the General Electric Company is 

 built around such a sphere. Some integrating sphere attachments are 

 available for other types of recording spectrophotometers. However, 

 it is quite simple to make a perfectly adequate laboratory setup from 

 a good monochromator and an appropriate photocell or photomulti- 

 plier. The home-made devices have the virtue of flexibility and adapt- 

 ability to specific problems that may be very difficult to attain with 

 well-made commercial instruments. Some of the theory of integrating 

 spheres has recently been worked out in detail and usable conclusions 



