64 



FLUORESCENCE SPECTROPHOTOMETRY 



showed that the absorption maximum of protochlorophyll in barley was 

 at 650 m/A. The fluorescence spectra are normally at longer wavelengths 

 than the absorption maximum. This discrepancy is now satisfactorily 

 cleared up. It is due to an effect which is shown in Fig. 14, the 

 fluorescence spectrum of the inner seed coat of banana squash. There 

 is a fluorescence peak at about 635 and another one somewhat be- 

 yond 650 m/A while the major peak comes at about 700 m/x. Offhand, 



600 



700 



750m; 



6S0 



WAVELENGTH 



Fig. 13. Fluorescence spectrum of protochlorophyll in acetone and in a heated 

 water suspension of etiolated barley leaf macerate. (Smith, unpublished.) 



this looks like a beautiful composite curve for a mixture of three pig- 

 ments. The acetone extract, however, gives a fluorescence curve for 

 pure protochlorophyll. The residue, after acetone extraction, shows a 

 trace of protochlorophyll fluorescence and nothing else. The absorp- 

 tion curve of the extract agrees with that of protochlorophyll. Here 

 we have an excellent illustration of data which can be most mislead- 

 ing. The squash seed coat is a very highly colored material in which 

 intense reabsorption of the fluorescent light takes place. The 705-m/x 

 peak we believe to be merely the unreabsorbed minor long-wave- 

 length band of protochlorophyll. The other peaks are the major 



