762 FLUORESCENCE OF PIGMENTS IN VITRO CHAP. 23 



nonfluorescent either because of rapid internal conversion of excitation 

 energy, or because of the occurrence of internal oxidation-reductions (dis- 

 proportionations). It was already mentioned above that combination 

 of dimerization with resonance exchange of energy can lead to strong 

 quenching even when the number of dimeric molecules is very low. 



Whether self-quenching is due to pre-existing dimers (or polymers) or 

 to encounters between excited and nonexcited monomers (in which dimers 

 are formed) can often be deduced from observations of the absorption 

 spectra: In the first case the absorption spectrum of the dyestuff must 

 change with concentration (as observed, e. g., by Rabinowitch and Epstein 

 with thionine and methylene blue) ; in the second case. Beer's law must 

 be obeyed {i. e., the absorption spectrum of the dye must be independent 

 of concentration). Quenching by a chain of energy exchange reactions, 

 with a dimer as occasional link in the chain, suggested by Forster, also does 

 not require marked deviations from Beer's law, since the number of dimers 

 present can be very small. 



Which of the several possible quenching or self-quenching processes 

 actually limits the yield of fluorescence in a given solution is not easy to 

 say. If the pigment is stable in light, and its fluorescence is unchanged 

 after long illumination, "physical" quenching is the likely mechanism. 

 True, even when chemical quenching does occur, the dyestuff may be 

 photostable, if the quenching reaction is reversible; and the yield of 

 fluorescence may remain unchanged with time, even when quenching ini- 

 tiates an irreversible sensitized chemical reaction, if the products of this 

 reaction do not quench fluorescence stronger (or weaker) than the originally 

 present molecular species. Usually, however, chemical quenching is not 

 entirely reversible, but causes a more or less rapid chemical change of the 

 fluorescent pigment; and sensitized chemical reactions often do lead to 

 the formation of products whose presence changes the intensity of fluores- 

 cence. When this is the case, the fluorescence yield must change with time 

 — either because the original fluorescent compound is converted into a new 

 one, with different properties, or because the fluorescence yield of the 

 original species is changed by the accumulation of the products of the 

 sensitized reaction. Therefore, whenever the yield (or the spectrum) of 

 fluorescence changes with time, the indication is strong that chemical fac- 

 tors account for at least part of the quenching (for examples, see page 764). 



A systematic study of the effects of solvent, concentration, admixtures, 

 temperature and other factors on the yield of fluorescence is needed to 

 elucidate the quenching problem, which was discussed above on the basis 

 of general possibilities more than on the basis of actual observations with 

 chlorophyll. Studies of this kind would be' of particular interest for under- 

 standing the mechanism of sensitized photochemical reactions, such as 



