766 



FLUORESCENCE OF PIGMENTS IN VITRO 



CHAP. 23 



This conclusion follows from some remarkable observations described 

 by Livingston, Watson and McArdle (1949). Contrary to what was gener- 

 ally assumed before, they found that chlorophyll solutions in nonpolar 

 organic solvents do not fluoresce at all (or only very weakly), but that traces 

 of polar admixtures, such as water or methanol, are sufficient to "activate" 

 their fluorescence. 



In this study, Livingston and co-workers used a mercury arc for excita- 

 tion (mainly the lines 436 and 405 m/x) . The fluorescent light was filtered 

 through a deep-red filter, so that only the second fluorescent band of chloro- 

 phyll a (720 m/i) was measured. This eliminated self-absorption. (It was 

 also stated that the position of the second band is less strongly affected by 

 the solvent than that of the first band, which can be shifted by as much 

 as 7.6 m^u, cf. table 23. IC; but this seems strange, since one would not 



Fig. 23.5. 



UJ 



o 



z 



UJ 



o 

 (/) 



UJ 



cr 

 o 



_I 

 U. 



u. 

 o 



z 



UJ 



0.2 0.4 0.6 0.8 1.0 



SQUARE ROOT OF MOLE FRACTION OF ALCOHOL 



Intensity of fluorescence of chlorophyll a in the system octanol-benzene 

 (after Livingston 1948). 



anticipate substantial differences in solvent effects on two emission bands 

 originating, presumably, in the same excited state and leading to two ad- 

 joining vibrational levels of the same lower state.) 



Chlorophyll solutions in dry hydrocarbons were obtained by evaporat- 

 ing in vacuum a solution of chlorophyll a or h, dissolving the residue in dry 

 hydrocarbon, evaporating again, and repeating this operation until all 

 water (which may have been present in chlorophyll from its preparation) 

 had been removed. (Disappearance of fluorescence was used as criterion 

 of dryness.) Various polar "activators" were then added, dissolved in the 

 same dry hydrocarbon. 



In purest dry benzene, the intensity of fluorescence (F) was <3% of that 

 ordinarily observed in the same solvent (Fo) ; even this weak fluorescence 



