1782 CHEMISTRY OF CHLOROPLAST PIGMENTS CHAP. 37B 



nitrogen, and the same factor also determines the tendency of the amine to 

 attach itself to the electrophilic carbon atom (9). 



According to Holt (1954) the brown "phase test intermediate" can be 

 obtained instantaneously by the action of sodium methanolate on 

 chlorophyll or other solvents (and is then comparatively long-lived at 

 room temperature). 



Weigl and Livingston (1952) could observe no isotopic exchange of hydrogen and 

 deuterium between watei' and chlorophyll a (or pheophylin a) in neutral organic solvents 

 (ether, dioxane, acetone, benzene); .similar negative results, ohtaincil by Xorris, Ruben 

 and Allen with tritium, were described on p. .557. 



5. Crystallization and Stability of Chlorophyll 

 and Bacteriochlorophyll 



(Addendum to Chapter 16, section A5) 



Despite occasional reporting of microcrystalline chlorophyll prepara- 

 tions, it seems that all pre\dously recommended methods lead to amor- 

 phous, more or less wax-like products. In particular, the procedure by 

 which Willstatter and StoU had obtained a product described as "micro- 

 crystalline," was found by Hanson (cf. p. 448) to yield a preparation with- 

 out a sharp x-ray diffraction pattern (Fig. 37B.7) — which is decisive proof 

 of the absence of a regular molecular arrangement. 



Jacobs, Vatter and Holt (1953, 1954) were able to obtain crystalline 

 chlorophylls a and b, as well as crystalline bacteriochlorophyll, by using 

 as guide the shift of the absorption band upon crystallization, first noted 

 with alkyl chlorophyllides (cf. Chapter 37C, section 3). The several meth- 

 ods they found to produce crystalline chlorophyll preparations had in com- 

 mon the presence of water during the precipitation, although this presence 

 alone is not sufficient for the purpose. Thus, chlorophyll precipitated 

 from acetonic solution by dilution with water remains amorphous unless 

 a small amount (> 100 p. p.m.) of Ca + + ions is present. (Calcium is not 

 incorporated in the precipitate; its effect must be an electric one, destroy- 

 ing the stability of the amorphous colloid.) Other and better methods to 

 prepare crystalline chlorophyll are: (1) adding water to a chlorophyll 

 solution in ether, and slowly evaporating the ether in vacuum; (2) adding 

 pentane to water-saturated ethereal solution of chlorophyll and removing 

 the ether by repeated washing with water; and (3) adding hexane to the 

 ether- water solution and removing ether by evaporation. In contrast to 

 the chlorophylls (and bacteriochlorophyll), the pheophytins can be crys- 

 tallized by evaporation of their ethereal solutions also in apparently com- 

 plete absence of water. The difficulty of crystallizing chlorophyll is thus 

 caused by the presence, in the same molecule, of the long hydrophobic 

 phytol chain, and the polar magnesium atom. 



Fig. 37B.6 is an electron micrograph of crystalline chlorophyll a. It 

 shows that the crystallization extends mainly in two dimensions; the ma- 



