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Messrs. I. Jorgensen and F. Kidd. 



reprecipitated with petrol-ether, until- finally a blue-black macrocrystalline 

 powder is obtained, which is a mixture of the two chlorophylls a and b. 



As the purification of chlorophyll is somewhat difficult it is always, 

 advisable to test the purity of a chlorophyll preparation before use. We 

 therefore give below the criteria of purity of the pigment, more particularly 

 because some investigators, Ewart (2), for instance, state that Willstatter's 

 methods have been followed, although it is quite clear that the product 

 which they describe as chlorophyll cannot have been of even moderate 

 purity. 



Criteria of Purity of Clilorophyll. 



1. The substance should be free from yelloiv pigments and colourless 

 impurities. — Fortunately, in "Willstatter's method of purification, the yellow 

 pigments and colourless impurities are eliminated together, so that, 

 generally, chlorophyll free from the yellow pigments is also free from the 

 colourless impurities which are present along with the pigments in the 

 chloroplasts. It is sufficient, therefore, to test for the presence of the yellow 

 pigments. 



This test is based on the fact that, by strong alkalis, chlorophyll is 

 saponified to the water-soluble salts of the acids called chlorophyllins, while 

 the yellow pigments are not attacked by alkalis. A 30-per-cent. solution of 

 potassium hydroxide in methyl alcohol is therefore added to an ethereal 

 solution of chlorophyll, and water added after saponification is complete, 

 The green chlorophyllin salts are then found in the aqueous alkaline layer 

 and any yellow pigments present will immediately be detected by the colour 

 they give to the ethereal layer. 



Hence, if the chlorophyll is pure, the ethereal layer should remain 

 colourless after such a saponification test. 



2. The chlorophyll itself should remain unaltered during its extraction and 

 2mrification. — The changes in the composition of chlorophyll most likely to 

 occur during its preparation are — 



(a) The replacement of the magnesium by hydrogen, giving the 

 magnesium-free derivative, phseophytin. This takes place in an acid 

 medium. The presence of phaeophytin is easily discovered, as it gives in 

 solution a characteristic absorption spectrum containing two lines in the 

 green, one just before the Fraunhofer line E, and one between the lines E and 

 F. These lines are not found in the chlorophyll spectrum, nor in the 

 absorption spectrum of a freshly prepared leaf extract which contains no 

 phteophytin. 



Pure chlorophyll should therefore give in solution an absorption spectrum 

 similar to that from a freshly prepared leaf extract. 



