648 ABSORPTION SPECTRA OF PIGMENTS IiY VITRO CHAP. 21 



number of such molecules thus converts the spectrum of "normal" chloro- 

 phyll into one of "tautomerized" chlorophyll. In pure polar solvent, on the 

 other hand, polar molecules surround the chlorophyll molecule from all 

 sides and thus cause a diffei-ent and more radical change in its absorption 

 spectrum. 



The absorption spectrum of the "activated" solution is affected by in- 

 creasing temperature, indicating a shift toward dissociation of the equilib- 

 rium 



(M+~ = polar molecule, tChl = tautomeric chlorophyll). The interpre- 

 tation of these interesting results will be discussed in chapter 23 (page 769) 

 after presentation of the corresponding fluorescence data. 



Evstigneev, (xavrilo^'a and Krasnovsky (1949") noted that polar mole- 

 cules have no effect on absorption spectrum and fluorescence of magnesium- 

 free compounds (pheophytin and phthalocyanine) and therefore ascribed 

 this effect to the binding of these molecules bj^ the residual valencies of 

 magnesium. 



A few words can be added here on the effect of dissolved gases on the absorption 

 spectrum of chlorophyll solutions. Padoa and Vita (1932) described changes in the ab- 

 sorption spectra of chlorophylls a and b (in benzene solutions) in contact with nitrogen, 

 oxygen, carbon monoxide and carbon dioxide. A strong effect was observed in the case 

 of carbon monoxide — a result taken as an indication of the existence of a chlorophyll- 

 carbon monoxide complex, similar to carboxyhemoglobin. However, the spectra re- 

 produced in the paper of Padoa and Vita are so different from the true spectrum of 

 chlorophyll, that they must have been obtained with some decomposition products 

 rather than with the intact pigment. Katz and Wassink (1939) found practically iden- 

 tical extinction curves for colloidal aqueous bacteriochlorophyll extracts in atmospheres 

 of oxygen, hydrogen sulfide, nitrogen, hydrogen and air. 



Evstigneev, Gavrilova and Krasnovsky (1949^) asserted that the pres- 

 ence of oxygen does have a certain effect on the spectrum of chlorophyll 

 (a + b, or pure h) in toluene. Upon evacuation, the absorption coefficient 

 decreased in both maxima, which were shifted slightly toward the red. In 

 chlorophyll b, a new maximum of absorption appeared, when air was re- 

 moved, at 670 mju. These changes were reversible ; but irreversible changes 

 were noted in the ultraviolet part of the spectrum. Similar changes were 

 observed in carbon tetrachloride and heptane, but not in pyridine, ethanol, 

 acetone or benzene. Addition of one drop of alcohol, pyridine or acetone 

 to 10 cc. toluene destroyed the effect of evacuation. Later (1949^) the 

 same investigators found that the effects they had ascribed to the admission 

 of air were actually caused by the admission of water vapor. 



These results obviously bear a relation to Livingston's conclusions that 

 chlorophyll is present, in nonpolar solvents, in a state different from that to 



I 



