iv] CARBON ASSIMILATION 37 



The petrol ether extract is then washed with 80 / acetone to remove 

 colourless impurities, and with 80 /o methyl alcohol to remove xantho- 

 phyll. Finally all traces of acetone and methyl alcohol are removed by 

 washing with water. This renders the chlorophyll insoluble in petrol 

 ether, since it is only soluble in this solvent if traces of other solvents 

 are present. Hence the pigment is precipitated out as a fine suspension, 

 leaving the carotin in solution. The chlorophyll is filtered off through 

 powdered talc, taken up in ether, reprecipitated by petrol ether and 

 finally obtained as a blue-black micro-crystalline substance. The col- 

 loidal solution or sol is made by dissolving 04 gm. of pure chlorophyll in 

 3 c.c. of absolute alcohol and pouring into 300 c.c. of distilled water. 



The advantage of using such a solution is that the experimental 

 conditions, in all probability, approach more nearly to the conditions in 

 the plant, and reactions with other substances take place more readily 

 than when the chlorophyll is used as a film. The use of pure, instead of 

 crude, chlorophyll is also important as by this means it is possible to 

 determine the changes taking place in chlorophyll itself without complica- 

 tions arising from the accompanying impurities. The discordant results 

 of various workers on this subject are doubtless due to the employment 

 of crude chlorophyll. Ethyl alcohol is the best solvent for preparing 

 the sol since it does not produce formaldehyde when exposed to light 

 under ordinary circumstances in glass vessels. Methyl alcohol and 

 acetone should be avoided as they themselves either contain or give rise 

 to formaldehyde. 



The chlorophyll sol is electro-negative. It is stabilized by weak 

 alkalies, but precipitated by weak acids. 



Working with such a colloidal solution the results may be summarized 

 as follows. 



When a chlorophyll sol is exposed to light in an atmosphere of 

 nitrogen in a sealed tube, no apparent change takes place in the chloro- 

 phyll, and no formaldehyde is produced. 



When exposed in an atmosphere of carbon dioxide in a sealed tube, 

 the chlorophyll rapidly turns yellow- or brown-green. In the case of 

 sols of high concentration, the colour-change is preceded by precipitation 

 of the pigment. The same change takes place in the dark, only more 

 slowly. No formaldehyde is produced, and no absorption of carbon 

 dioxide could be detected. The yellow product has been shown to be the 

 magnesium-free derivative, phaeophytin, which is produced from the 

 pigment by the action of acids. The changes observed are explained by 

 the fact that the carbon dioxide, acting as a weak acid, first precipitates 



