498 Absorption Spectrophotometry /26 : 5 



the test sample. The spectrum obtained in this fashion is a difference 

 spectrum. 



In some cases, a suspension of cells or cell fragments will undergo 

 specific changes in absorption during the reaction as well as more general 

 changes due to such factors as settling and swelling. This can be taken 

 into account by measuring the optical density at two neighboring wave- 

 lengths, only one of which is altered specifically by the reaction being 

 studied. If the original light beam is passed alternately through two 

 monochromators, and these two beams are then recombined to pass 

 through a common sample, the nonspecific changes in optical density 

 can be subtracted out by the associated electronic equipment. The 

 device used for this operation is called a dual-beam spectrophotometer. It 

 is less sensitive to light-level fluctuations than is a single, unsplit-beam 

 spectrophotometer. 



Both the split-beam and dual-beam spectrophotometers employ some 

 form of light chopper. All light choppers have an inherent noise which 

 cannot be eliminated. Accordingly, one can measure smaller changes 

 in optical density by regulating the light source than one can through 

 the use of split- or dual-beam spectrophotometers. The extremes of 

 light regulation necessary to do this with a single beam make split- and 

 dual-beam spectrophotometers more convenient for most purposes. 



Instead of presenting a detailed description of split- and dual-beam 

 spectrophotometers, the action of one particular split-beam spectro- 

 photometer is outlined below. It is shown in block-diagram form in 

 Figure 12. 



Light proceeds through the monochromator and is then reflected 

 alternatively by the sector mirror R, so that it passes through sample 

 holder C 1} and by the fixed mirror M, so that it passes through sample 

 holder C 2 . Thereafter, the light is reflected by two fixed mirrors M' 

 and M" so that it converges on the photomultiplier cathode. To detect 

 low signal intensities, the load resistor must be large. A d-c amplifier 

 with feedback acts as a current amplifier, presenting sufficient current to 

 operate the following circuits. The potential from the d-c amplifier is 

 proportional to the intensity, I 1} of light coming through C ± for half a 

 cycle and then to I 2 coming through C 2 for the next half cycle. This 

 potential is passed through the logarithmic attenuator; the signals 

 produced are proportional to log I x and to log I 2 respectively, each for 

 half a cycle. The output of the logarithmic attenuator is fed into an 

 a-c amplifier. 



The a-c amplifier responds only to the differences from the average of 

 the two signals. Denoting its rms output by e, one may write 



e = A [log I x - log 7 2 ] = A log f 



