26 : 3/ Absorption Spectrophotometry 491 



if A/// is small. From I oc (V) 8 , one may write 



/ + A/ _ (V + FA) 8 _ F + 8F 



/o ^o ^o 



Combining the last two relationships leads to 



AF 

 AD ^3-=- (11) 



V 



If only the intensity in a narrow wavelength band is measured, the 

 numerical coefficient in Equation 1 1 might come out closer to 4. 



If it is desired to measure optical densities or optical-density changes 

 as small as 0.001, then the electrical power source must be so constant 

 that the voltage changes are less than 3 parts in 10 4 . For sensitive 

 spectrophotometry, necessary to observe small changes in enzyme con- 

 centrations, it is sometimes essential to have an optical-density noise 

 level below 10 ~ 5 . This means the maximum noise voltage must be no 

 greater than 3 parts in 10 6 . 



B. Monochromators 



In order to make spectrophotometric measurements, as contrasted to 

 photometric measurements, it is necessary to have some method of 

 distinguishing light of different wavelengths. Our eyes do this in a 

 complex fashion, presenting the information as the sensation of color. 

 In a colorimeter, a series of colored glasses or filters is used for this 

 purpose. These separate rather broad wavelength bands of light. For 

 precision work, it is more convenient to produce narrow, sharp wave- 

 length bands with monochromators than with filters. Two general 

 types of monochromators are widely employed; these are the prism type 

 and the grating type. 



The action of a prism of dispersing a white light into a spectrum of 

 colored light was discovered by Isaac Newton (the "father of physics"). 

 It is illustrated in Figures 6, 7, and 8. The case shown in Figure 6 is 

 simplest to analyze. The angle y between the incoming and outgoing 

 rays is called the deviation. It is a minimum when the light rays in 

 the prism are parallel to the base. The more general case shown in 

 Figure 7 is harder to analyze. 



For all transparent media, n varies with the wavelength. Accord- 

 ingly, the deviation cp will also vary with the wavelength. Figure 8 is 

 a simplified diagram of a prism monochromator. The filament source 

 emits light which passes through slits S x . These act as a point source 

 for lens 1, which converts the light beam to parallel light. On passing 

 through the prism, the light is dispersed, light of each wavelength coming 

 out at an angle dependent on the index of refraction n. Lens 2 focuses 



