Chapter 21 

 ABSORPTION SPECTRA OF PIGMENTS IN VITRO 



A. Absorption Spectra of Chlorophyll 

 AND Its Derivatives* 



1. Absorption Spectra of Chlorophylls a and b 



The spectra of chlorophylls a and b have been studied in detail because 

 of their theoretical interest, as well as because of their usefulness for the 

 spectrophotometric determination of these pigments. Until recently, the 

 results of different authors did not agree ver}^ well, either in the exact posi- 

 tions of the band maxima, or in the values of the extinction coefficients. 

 Lately, improved chromatographic purification methods have enabled 

 Zscheile and co-workers (cf. Zscheile 1934, 1935; Zscheile and Comar 1941; 

 Comar and Zscheile 1941; Harris and Zscheile 1943) and Mackinney 

 (1938, 1940, 1941) to obtain preparations of chlorophylls a and b that could 

 meet high standards of spectroscopic purity and reproducibilitj'. 



Zscheile, Comar and Mackinney (1942) studied samples of chlorophyll prepared by 

 the first two investigators at Purdue and by the third one at Berkeley, measuring the 

 extinction coefficients by means of two different photoelectric spectrophotometers. 



For Zscheile and Comar's "wet" preparation of chlorophyll a (cf. page 60-1), the 

 two instruments gave practically identical extinction curves. (In the region between 430 

 and 660 m/x, all deviations were within 2%.) This shows how successfully large photo- 

 metric errors (which are common in visual and photographic determinations of absorption 

 curves) can be eliminated by the use of photoelectric devices. 



The spectra of the solutions of chlorophylls a and b prepared by Mackinney in the 

 dry state were, on the whole, similar to that of Zscheile's moist preparation; but dif- 

 ferences up to 10% in chlorophyll a and 15% in chlorophyll b did occur between the ex- 

 tinction curves determined in the two laboratories, as well as between these two curves 

 and that of Zscheile's preparation. The deviations varied irregularly with wave length, 

 indicating the probable presence, in Mackinney's preparation, of an admixture of colored 

 components. One may regret that no measurements were made below 430 m^, since 

 earlier experiments have shown a particularly strong variability of the absorption curve 

 in this spectral region [cf. page 607). 



The differences between the absorption curves of Zscheile and Mackinney appear 

 minor when compared with the discrepancies that existed between the curves published 

 by earlier investigators {cf. Table 2LI). These discrepancies must have been due to the 

 use of less reliable photometric devices, and to the inferior purity of the earlier chloro- 



* Bibliography, page 668. 



603 



