642 ABSORPTION SPECTRA OF PIGMENTS IN VITRO CHAP. 21 



for chlorophyll a — the former being the more po'ar of the two compounds. 

 For example, according to Harris and Zscheile, the red band of chlorophyll 

 a lies at 660 mn in ether and 664 m^t in methanol (AX = 4 m^), while the 

 corresponding values for chlorophyll b are 642.5 and 651 m^t, respectively 

 (AX = 8.5 m/x). Hydrogen bonding, too, may have to be taken into con- 

 sideration. 



If one compares the effects of varying refractivity of the solvents on the 

 spectra of different homologous solutes, one may expect the solute with the 

 stronger polarizability to exhibit the strongest shift. Polarizability in- 

 creases with the intensity and wave length of the first absorption band. 

 Thus, the spectra of dyestuffs should be more sensitive to solvent changes 

 than the spectra of noncolored substances, and the sensitivity of dyes of dif- 

 ferent color should increase with the shift of the main absorption band to- 

 ward longer waves. This is confirmed by the finding of Pruckner (1940) 

 that the solvent effect increases strongly from porphins through dihydro- 

 porphins (chlorins and phorbins) to tetrahydroporphins (bacteriochloro- 

 phyll). The first absorption band of chlorophyll is situated further toward 

 the red and is more intense than the first absorption band of the porphyrins ; 

 and the same is true of the first absorption band of bacteriochlorophyll 

 compared to that of chlorophyll. The two (or four) additional hydrogen 

 atoms contained in these compounds contribute electrons that are easily 

 excitable by light (thus giving rise to long-wave absorption bands) and 

 easily displaceable in electric fields (thus producing strong polarizability). 



Figure 21.25 shows the shifts of the band maxima of bacteriochlorophyll 

 and ordinary chlorophyll in the same solvents. This figure indicates that 

 on the w^ave length scale the solvent effect is about twice as strong for the 

 first band of bacteriochlorophyll as for the first band of ordinary chloro- 

 phyll. 



Katz and Wassink (1939) extrapolated the curves in figure 21.25 to 

 vacuum (refractive index 1) and predicted that the absorption peak of free 

 chlorophyll molecules — if it can ever be determined — will be found at 648 

 ± 5 m/i, and that of free bacteriochlorophyll molecvdes at about 740 m/x. 



In piperidine solution, the absorption peak of chlorophyll lies at 642 niyu, i. e., be- 

 yond the extrapolated Hmit for the free molecule. This demonstrates the existence of 

 exceptions to Kundt's rule, probably caused by specific chemical interactions between 

 solvent and solute. Other (less striking) exceptions from Kundt's rule have been dis- 

 cussed by Mackinney (1938, 1940) and Egle (1939). 



Theoretically, it would be more appropriate to plot, in figures 21.24 

 and 21.25, wave numbers (or frequencies) since these are proportional to 

 energies and therefore bear direct relationship to the terms in equation 

 (21.4). In a narrow spectral range, such as is used here for a given band of 

 a single pigment, linear extrapolation on a wave number scale would give 

 results not significantly different from those obtained by linear extrapola- 



