24 PHOTOSYNTHESIS 



of the chlorophyll a absorption band lies at a longer wave- 

 length than that of chlorophyll b, and conversely at the 

 violet end (Fig. 3.3). Hence by measurements of the absorp- 

 tion of light at two specified wavelengths in an acetone 

 extract of green leaves the quantities of both chlorophylls a 

 and h may be determined (Mackinney, 1941). If i^ees ^^^ 

 Z)645 are the optical densities at the two wavelengths 663 m^ 

 and 645 m/i then these are related to the concentrations of 

 chlorophyll a, Ca and chlorophyll b, Cf, by the following 

 equations: 



Z)g63=82-04Ca + 9-27^6 



A45=i675Ca + 45-6c5 



or C=Ca + Cft = 0-0202Z)g45+0-00802Z)gg3 



Both the extinction coefficient and the position of the band 

 maxima vary with the solvent used, and small amounts of 

 impurity in the solvent may appreciably alter the absorption 

 curves. 



2. Fluorescence. In organic solvents both chlorophylls 

 show a characteristic fluorescence spectrum consisting of a 

 strong band in the red and a weaker band in the far red. The 

 intensity of the fluorescence of a solution in acetone or ether 

 represents about 10% of the light energy absorbed. Just as 

 with the absorption curves the position of the bands and the 

 intensity of fluorescence are different in different solvents. 

 Thus Livingston found that solutions of ethyl chlorophyl- 

 lide in pure dry benzene showed very little fluorescence, the 

 intensity being one-twentieth that in methanol or ether. If 

 a little water, methanol, or ether is added to the benzene the 

 fluorescence is now even greater than that in pure methanol. 

 The second solvent need be added, however, only in an 

 amount equal on a molecular basis to the amount of chloro- 

 phyll present and thus it has been suggested that the 

 fluorescence of chlorophyll is due to the formation of a 

 chlorophyll complex formed with the added substance. The 

 fluorescence of solutions of chlorophyll can be 'quenched' 

 by the addition of such substances as quinone and by other 

 -oxidizing agents, and also by phenyl hydrazine (a very low 



