1 1 2 COLORIMETRY-SPECTROPHOTOMETRY 



lines wherever light is absorbed; the pattern of these dark lines is char- 

 acteristic of the material being examined. 



The absorption of light depends upon the displacement of electrons, 

 and the phenomenon should also occur in reverse. If a molecule is 

 heated to a sufficiently high temperature, electrons can be displaced. As 

 they fall back to their normal levels they emit the excess energy as light. 

 If we examine this light with a spectroscope we find that the bright 

 lines, or emission lines, correspond at various regions in the spectrum 

 with the dark absorption lines in our previous spectrum. 



The molecule in the excited state is unstable, and the energy must be 

 dissipated in a small fraction of a second. There are several possible 

 fates for this excitation energy. Far more common than any of the others 

 is the conversion of this energy into heat, which is transferred to other 

 atoms and molecules in the vicinity. The displaced electron falls back 

 to its normal energy level. In some cases, the energy may be used to drive 

 a chemical reaction. The whole field of photochemistry is founded upon 

 this ability. The absorbing molecule itself may be changed in the photo- 

 chemical reaction or it may serve as a sensitizer for some other reaction. 

 A third possible fate for the quantum of absorbed energy is its re-emis- 

 sion as light in the process of fluorescence. Since, during the lifetime of 

 the excited state, a small fraction of a second, a portion of the energy 

 will be dissipated as heat, the quantum re-emitted must be smaller and 

 therefore of a longer wavelength than the quantum absorbed. In certain 

 kinds of molecules a fourth fate is possible. The excited molecule may 

 change from the unstable excited state into a longer-lived excited state. 

 After a period of seconds, or even minutes or hours, the quantum is 

 re-emitted as light, this time of a much longer wavelength. This phenom- 

 enon, known as phosphorescence, probably occurs only rarely in biologi- 

 cal materials. 



Analytical instruments 



The simplest kind of colorimetric instrument uses the human eye as 

 a light detector. The technique consists of comparing the color of the 

 "unknown" solution with the color of a set of solutions of known con- 

 centration. The human eye is an amazingly sensitive instrument, and 

 quite small differences in concentration can be detected. In the ordinary 

 practice the "unknown" is held between two of the "known" solutions, 

 and by trial and error we find that pair of standard solutions between 



