COLORIMETRY-SPECTROPHOTOMETRY 1 27 



Another common use of the spectrophotometer is in the determination 

 of reaction rates. In this case we allow the chemical reaction to proceed 

 directly in the cuvettes. In the reaction 



A + B > C + D 



suppose component C has a maximum absorption at 365 m/^ and none 

 of the other components in the mixture, A, B, or D, has any appreciable 

 absorption at this wavelength. If the starting materials A and B are 

 placed in the cuvette in the spectrophotometer and the wavelength is set 

 at 365 mfJi, the measured increase in optical density will be a measure 

 of the appearance of material C. By carefully timing the reaction we have 

 an adequate and convenient measure of the rate of the reaction. 



Fluorescence measurements 



Fluorescence is the ability of certain molecules to re-emit absorbed 

 light. Since the molecule remains in the excited state for a short time, 

 the emitted light is of a longer wavelength than that absorbed. With 

 many kinds of compounds, especially at low concentrations, the fraction 

 of the absorbed quanta which appear as fluorescence is constant, or 

 nearly so. This means that solutions of greater concentration will absorb 

 more light and therefore will fluoresce more intensively. In certain situ- 

 ations it is advantageous to measure the fluorescence rather than the 

 absorption. For example, riboflavin absorbs light at the blue end of the 

 spectrum and shows a fairly strong yellow-orange fluorescence. The con- 

 centration of a solution could be determined by measuring either absorp- 

 tion or fluorescence. It happens, however, that several nonfluorescent 

 materials commonly present in solutions of this vitamin also absorb ap- 

 preciably at the same wavelengths. In such a mixed solution it is difficult 

 to ascribe any particular portion of the total absorption to the riboflavin. 

 Since it is the only component with marked fluorescence, measurement 

 of the fluorescence gives a more convenient measure of the concentra- 

 tion. 



Several fluorimeters are available commercially. Generally the light 

 beams are handled according to some modification of the diagram in 

 Fig. 9-11. The exciting beam enters the container from one side. The 

 fluorescent light passes through a filter, which cuts out the exciting light. 

 It then is measured by a photocell placed at right angles to the exciting 

 beam. Several of the commercially available instruments also have photo- 



