26 : 4/ Absorption Spectrophotometry 495 



recording is necessary. This is not difficult with appropriate electronic 

 circuits. 



The essential role of electronics in all of the natural sciences cannot 

 be overemphasized. This has been especially true in biophysics, where 

 almost all measurements are made with electronic equipment. The 

 material in every chapter in this text depends for its validity on measure- 

 ments made with electronic tools. Spectrophotometry is no exception to 

 this rule. 



4. Flow Systems 



To study rapid reactions, it is necessary to mix the reactants and start 

 observing the reaction before it has progressed too far. Often this is 

 not possible in cuvettes, where the time to mix the reactants is at least 

 1 sec. To avoid this difficulty, flow' systems are used in which the 

 mixing occurs just before the solution enters the path of the light beam. 

 For reactions which are not too rapid, the flow is stopped after mixing is 

 completed. Then the changes in optical density are recorded. This is 

 called the stopped-flow method. It can be used for reactions whose half- 

 times are greater than 30 msec. 



Some reactions take place so rapidly that the stopped-flow method is 

 too slow to be useful, so that it is necessary to observe the reaction 

 during flow. If the liquid flows at a constant velocity, a constant 

 optical density will be detected. This can be repeated at varying 

 velocities to obtain the optical density at varying times after mixing. 

 If, instead, the velocity is varied during flow, it is possible to observe the 

 reactions at various times after mixing. In principle, one could also 

 vary the distance, d, from the mixer to the observation point but this is 

 mechanically more cumbersome. 



A suitable flow system is diagrammed in Figure 10. The two react- 

 ants are stored in tanks 7\ and T 2 respectively. When the stopcocks 

 C 1 and C 2 are set in an appropriate fashion, liquid may be pulled from 

 the storage tanks into the syringes by raising the connecting bar. With 

 the stopcocks turned as shown in the diagram, liquid may be pushed 

 from the syringes, mixing at M, before flowing through the path of the 

 light beam d centimeters downstream. If the linear flow velocity is v, 

 the time t, after mixing, is given by 



d 



t = - 



V 



When flow starts, the old reactants are in the observation tube and 

 mixing chamber. Some of the mixed reactants may also have diffused 

 back up from M toward the stopcocks. The initial changes then have 



