26 : 1/ Absorption Spectrophotometry 483 



Any absorption or emission of electromagnetic energy which varies in 

 a characteristic fashion with wavelength can be used to study the nature 

 of, or measure the amounts of, certain biochemical substances. By and 

 large, one obtains different types of information from different regions 

 of the spectrum. The specific absorption bands of many biologically 

 important molecules lie in the visible and ultraviolet regions of the 

 spectrum. Measurements in these regions will be emphasized in this 

 chapter. 



A number of different terms are used to describe the equipment 

 employed to measure spectra in the visible, ultraviolet, and infrared 

 regions. An instrument which presents the spectrum in such a fashion 

 that it can be observed with the eye as a detector is called a spectroscope. 

 Apparatus arranged to measure the wavelengths at which bright 

 emission lines or dark absorption lines occur is referred to as a spectrometer. 

 If a detector other than the eye is usee}, the spectrometer is not restricted 

 to visible light. A colorimeter is made by placing spectrally selective 

 filters in the path of a white light; the colored light beam so produced 

 may be split in two and passed through two solutions. Any piece of 

 apparatus which compares the absorption of two solutions or emission 

 of two sources is called a photometer. A monochromator selects a narrow 

 wavelength band from the incident light. A monochromator combined 

 with a photometer is called a spectrophotometer. The latter is the most 

 widely used form of analyzing equipment in current biochemical and 

 biophysical research, although colorimeters are still used for some 

 clinical measurements. Spectrophotometry was markedly improved by 

 the development of electronic circuits. Although spectrophotometry 

 was used before the impact of electronics was felt in biology, the wide- 

 spread application of spectrophotometry has resulted from the availa- 

 bility of convenient electronic equipment. Today, every clinical 

 hospital laboratory, every biochemical research laboratory, and most 

 microbiology and physiology laboratories make use of absorption 

 spectrophotometry. 



A large number of basic physical concepts are necessary in order for 

 one to understand the origin and the nature of characteristic spectra of 

 biochemical molecules. Complex physical equipment is necessary to 

 make optimum use of the information available from these spectra. 

 Accordingly, absorption spectrophotometry is one of the important 

 fields of instrumentation within the general framework of biophysics. 



The present chapter is a description of some types of equipment used 

 in spectrophotometry. No attempt has been made to be complete or to 

 provide an instruction manual for the use of a specific spectrophotom- 

 eter. Rather, it is hoped that the reader will find an indication of the 

 types of measurements possible, as well as of certain modifications of 



