774 FLUORESCENCE OF PIGMENTS IN VITRO CHAP. 23 



together in an "encounter") rather than by the frequency of collisions. The distinction 

 between eiicounters (or "co-ordinations") and collisions, and its importance for reaction 

 kinetics of condensed systems was discussed by Rabinowitch (1937). 



The unusual magnitude of the constant (23.12) is illustrated by the 

 fact that for nine other dyes, investigated by the same authors, the self- 

 quenching constants were at least 2000 times smaller (and thus of the 

 order of magnitude to be expected from diffusion constants) . 



It seemed thus that the conclusions of Weiss and Weil-Malherbe were 

 in need of confirmation. Their results might have been vitiated by self- 

 absorption (which the authors dismissed as unimportant "because there 

 was no evidence of surface fluorescence"). In their set-up, the fluores- 

 cent beam was in line with the (ultraviolet) excitmg beam — an arrangement 

 that favors self-absorption. Less mutual overlapping of the fluorescence 

 band and absorption band may explain why self-absorption did not affect 

 equally strongly the results obtained with the other nine dyes. 



That the observation of Weiss and Weil-Malherbe was due to self- 

 absorption was later confirmed by Livingston and co-workers (1948), who 

 showed experimentally that, if care is taken to avoid self-absorption, no 

 concentration quenching of chlorophyll fluorescence can be noted up to 

 1.5 X 10~^ mole/1. Weiss (1948) agreed with this, but stated that con- 

 centration quenching does occur at still higher concentrations. Livingston 

 and co-workers (1948), using a cell 1 mm. thick, extended their measure- 

 ments up to 0.10 mole/1, of chlorophyll in butyl ether and found in fact a 

 strong concentration quenching above 2 X 10~^ mole/1, (fig. 23.7); at 

 7 X 10 ~^ mole/1. , the fluorescence yield was only 7% of that in dilute solu- 

 tion. The insert in fig. 23.7 shows the sigmoid shape of the curve. 



The absorption spectrum of chlorophyll a in butyl ether, measured at 

 2 X 10 ~^ mole/1, (a concentration at which self-quenching reduces the 

 fluorescence to about 40% of the maximum), appeared not to differ sig- 

 nificantly from the spectrum of dilute solution. 



A further pertinent observation was that the partly quenched fluores- 

 cence of a concentrated chlorophyll solution was more strongly depressed 

 by an increase in temperature than the fluorescence of a dilute solution. 



These observations — the unchanged absorption spectrum, the sigmoid 

 quenching curve and the enhancing effect of temperature on concentration 

 quenching — can all be fitted into the picture of quenching as the result of 

 dissipation of excitation energy in a "weak link" in the energy exchange 

 chain, such as a dimeric molecule (Forster) or a "hot" molecule of the 

 monomer (Franck and Livingston). The concentration at which the 

 quenching becomes noticeable (2 X 10"^ mole/1.) is of the same order of 

 magnitude as that calculated by FSrster for the onset of the energy ex- 

 change (of. chapter 32). 



