604 ABSORPTION SPECTRA OF PIGMENTS IN VITRO CHAP. 21 



phyll preparations — the latter clearly indicated by the relatively high absorption in the 

 green {cf. last column in Table 21. IB). 



A spectroscopically important impurity likely to be present in many 

 chlorophyll preparations is the magnesium-free pheophytin, formed from 

 chlorophyll whenever the latter comes in contact with acids. Elimination 

 of magnesium from chlorophyll may take place even in living plants, e. g., 

 under the influence of acid fumes (cf. Stern 1935, 1938; and Tiegs 1938); 

 it can easily occur during extraction, when the pigments are exposed to the 

 action of acids contained in the cell sap. (Harris and Zscheile added mag- 

 nesium carbonate to the extracting solvent to neutralize these acids.) 

 This "primary" pheophytin is removed during chromatographic separation 

 (according to Zscheile 1941, a pheoph>'tin layer in the chromatogram was 

 responsible for his earlier belief that leaf extracts contain a "chlorophyll 

 c," cf. Vol. I, p. 402); but some pheophytin can again be formed afterward, 

 e. g., under the influence of atmospheric carbon dioxide. 



As shown in figures 21.18 and 21.19, the pheophorbides (and this ap- 

 plies to pheophytins as well) have rather strong absorption bands in the 

 green. Zscheile and Comar (1941) and Harris and Zscheile (1943) found 

 that the ratios of the extinction coefficients in the maxima of the red 

 chlorophyll bands (660 mn for component a in ethyl ether and 642.5 mfx 

 for component b in the same solvent) and of the green bands of pheophytin 

 (505 and 520 niyu, respectively) reach 52 in solutions of the purest prepara- 

 tions of chlorophyll a, and 19 in similar preparations of chlorophyll b, 

 but may drop to as low as 20 and 4.5, respectively, after these preparations 

 have been allowed to stand for as little as a single day in the dry state. 

 Zscheile and Comar (1941) recommended therefore that drying be avoided 

 altogether in the preparation of spectroscopically pure chlorophyll solu- 

 tions. More recently, Zscheile, Comar and Mackinney (1942) succeeded 

 in preparing dry chlorophyll a which could be stored and still showed, 

 upon dissolution, the high ratio of extinctions in the red and in the green 

 indicative of high purity; but no standard procedure for obtaining such 

 stable preparations could be given. 



Zscheile, Comar and Harris (1944) found that the spectra of ethereal 

 solutions of pure preparations of chlorophyll a show signs of deterioration 

 after about one week storage at 0-5° C. in darkness. Crude ether extracts 

 from leaves, on the other hand, proved to be comparatively stable — some 

 gave no evidence of spectroscopic change even after 14 weeks storage (at 

 — 20° C). Fresh corn leaves could be stored at —20° C, for a whole 

 month without deterioration of chlorophj'-ll. 



Another problem of chlorophyll purification is the elimination of traces 

 of chlorophyll a from chlorophyll b. According to Zscheile, supposedly 

 "pure" chlorophyll b, used by many earlier observers, did contain up to 



