14 II. METHODS OF INVESTIGATION 



cients of sharp maxima being depressed, those of sharp minima increased, 

 with respect to those which would be found with strictly monochromatic 

 light. 



When more than one absorbing substance is present in solution and 

 Beer's law still applies, each substance operates independently on 

 the light beam. Hence the resultant density is the sum of the den- 

 sities due to the individual components. 



3.1.2. The Absorption Curve. A plot of one of the photometric 

 constants against the wavelength of the incident light constitutes a 

 spectrophotometric curve. For the purposes of biochemistry, the 

 most convenient constants are the specific and molar (or millimolar) 

 extinction coefficients, so that the curve is an absorption curve. 

 Some workers, however, use transmittance curves, and certain com- 

 mercial spectrophotometers are calibrated in terms of transmittance. 



The absorption bands visible in a simple spectroscope are represented on 

 an absorption curve by peaks, the clear portions of tlie spectrum by troughs. 

 Some spectra however, appear to have distinct bands visually, but their 

 corresponding absorption curves show only regions of changing curvature, 

 with no minima separating them. Such spectra are frequently found in mix- 

 tures of substances which separately have sharp bands, but whose bands 

 overlap on superposition. They may, however, also occur in pure substances. 



A pure substance, under identical conditions, has a characteristic 

 absorption curve, and the molar extinction coefficient at any wave- 

 length is a constant. The wavelengths usually recorded are those of 

 the maxima of absorption, particularly that of the most intense band, 

 where several exist. 



When the absorption curves of two substances in equal concentra- 

 tion are superposed, they will in general cross at one or more points, 

 called isosbestic points. The absorption curves of equilibrium mix- 

 tures of two interconvertible substances of constant total concen- 

 tration will likewise pass through the isosbestic points, and the 

 experimental determination of this condition is a criterion of an 

 equilibrium mixture. 



3.1.3. Determination of Concentration by Spectrophotom- 

 etry. When a single absorbing substance is present in solution. 

 Beer's law has been shown to apply, and the specific or the molar 

 extinction coefficient at a suitable wavelength has been measured, 

 the concentration of the substance in such a solution can be calcu- 

 lated from its extinction. The most suitable wavelength is usually 



