26 : 2/ Absorption Spectrophotometry 



485 



20 



o 



x 





cS 



c 

 .g 



o 



Oxidized Form 

 Reduced Form 



Although absorption spectra are widely used, the units and symbols 

 with which the data are reported are very varied. In the language of 

 information theory, the spectrophotometrically obtained information is 

 coded in different ways. It is necessary 

 to know which code is being used. 

 This section describes some of these 

 codes and their basis in the physical 

 theory of absorption of electromag- 

 netic radiation. 



Suppose a beam of monochromatic 

 light is incident normally on a thin 

 sheet of material of thickness Ax, as 

 illustrated in Figure 5. The incident 

 intensity is represented by I x . In 

 passing through the material, some of 

 the light will be absorbed. The in- 

 tensity I 2 leaving the thin sheet will 

 be less than I x . If the sheet is suffi- 

 ciently thin, the change in intensity A/ 

 will be proportional to Ax. Experi- 

 mentally, it is found also that A/ is 

 proportional to I ± for monochromatic 

 light. Expressing these ideas symbol- 

 ically, one may write 



Figure 

 duced 



A/= h 



h 



lil x b,x (1) 



300 340 380 420 



Wavelength (mu,) 



2. Absorption spectra of re- 

 and oxidized diphosphopyri- 

 dine nucleotide. The role of DPN 

 in oxidative phosphorylation is dis- 

 cussed in Chapter 18. After J. B. 

 Nielands and P. K. Stumpf, Outlines 

 of Enzyme Chemistry (New York: John 

 Wiley and Sons, Inc., 1958). 



where jx is a proportionality constant 



depending on the material making up the thin sheet. 



If now one allows the thickness Ax to become infinitesimal, Equation 1 

 may be rewritten as 



dl 



dx = -^ 



(2) 



A sample of finite thickness x may be considered to be composed of 

 many such thin sheets. If I now is defined to mean the intensity 

 entering the sample at x = 0, and I x is the final intensity leaving the sample 

 at x, Equation 2 may be integrated to yield 



1 x — J e 



(3) 



This is known as Lambert's law 1 and /x is called the absorption coefficient. 



1 Also called Bouger^s law, in some texts. 



