786 FLUORESCENCE OF PIGMENTS IN VITRO CHAP. 23 



values obtained with nitrophenol and chlorophyll a, in a series of alcohols 

 (from methanol to octanol), were not inversely proportional to the viscosi- 

 ties of these solvents. This may mean that, although ki is a measure of 

 the probability of quenching by kinetic encounters, the frequency of such 

 encounters is not the simple function of viscosity assumed in eq. 23.16D. 



Livingston and Ke found that the absorption spectrum of chlorophyll a 

 was not markedly affected by the presence of even a large amount of the 

 strongest quenchers listed in Table 23.IIIC (e. g., 0.3 mole/1, of C6H5NO2), 

 thus indicating that quenching was not due to a preliminary dark chemical 

 reaction — such as complex formation — between chlorophyll a and the 

 quencher. 



The considerable effect, which the comparatively weak quencher, 

 phenylhydrazine, has on the absorption spectrum of chlorophyll h, was 

 noted before (chapter 21, page 649); no similar effect was observed with 

 chlorophyll a. 



Reversibility of quenching could be conclusively proved only for the 

 gaseous quencher, oxygen, since it could be easily removed. (It will be 

 recalled in this connection that the fluorescence of allomerized chlorophyll a 

 in methanol is only about one-half as intense as that of the original solu- 

 tion; in other words, a slow irreversible change is superimposed on the 

 reversible quenching by oxygen.) Dilution experiments with one weak 

 organic quencher, phenylhydrazine, indicated that quenching probably is 

 reversible in this case, too. 



Franck and Livingston (1941) had discussed the possible existence of a residual 

 fluorescence which could not be further reduced by quenchers. (Such a "nonquench- 

 able" fluorescence is to be expected if the electronically excited molecule requires a cer- 

 tain time to assume the configuration suitable for chemical quenching; the fraction of 

 total fluorescence, emitted between excitation and the attainment of this configuration, 

 would then be "unquenchable".) In the system (chlorophyll a in CH3OH + nitro- 

 benzene), a weak fluorescence appeared even in a 4 M solution of the quencher; however 

 it was too weak {F = O.OOSFo) to consider it as a definite confii'mation of this prediction. 



Whatever the interpretation of the quadratic term in the denominator 

 of equation (23.16C), the large values of the linear term found with oxidiz- 

 ing molecules and the low values found with reducing molecules (including 

 the substrates whose autoxidation is sensitized by chlorophyll, such as 

 allylthiourea) undoubtedly are significant. Livingston and Ke stressed 

 a detailed similarity of the hst of strong quenchers in table 23.IIIC with 

 the hst of substances known to inhibit certain polymerization processes; 

 but rather than looking for a causative relation between the two effects, one 

 should perhaps consider both as conse(iuences of the same oxidative proper- 

 ties of the quenchers (or inhibitors). 



Quenching of chlorophyll fluorescence by kinetic encounters with oxi- 



