24 Carbon Assimilation. 



means of water from its methyl alcohol, whereon the pigment in it 

 is precipitated. 



The third and fourth and fifth alcohol extracts are similarly 

 treated. The content of component h is now considerably reduced. 



The sixth methyl alcohol extract is treated with 900 c.c. methyl 

 alcohol, and each successive extract with 100 c.c. less, so that to 

 the 14th extract only 100 c.c. methyl alcohol is added. 



These extracts are cleaned in pairs with 1 litre of petrol ether, 

 for the second of each pair a further J-litre of petrol ether is added. 



All extracts thus cleaned are added to the same ether solution 

 which is increased by continual additions of ether, beginning with 

 1 litre, and decreasing in amount to about J-litre with the 10th 

 extract.' 



A 15th and 16th extraction with methyl alcohol is made in 

 order to free chlorophyll a from the last traces of chlorophyll b. 



The chlorophyll b solution is now freed from methyl alcohol by 

 washing with water, it is dried with sodium sulphate and evaporated 

 to 500 c.c, and then to 30 or 40 c.c. in vacuo. 



The chlorophyll b is then precipitated by the addition of 

 300 c.c. petrol ether of B.P. 30" to ZQ^C. and filtered on talc. The 

 filtrate contains much chlorophyll a. It is purified by solution in 

 ether and precipitation with petrol ether, which is repeated several 

 times. It is finally filtered and dried in a vacuum desiccator. 



(8) Purification of Chlorophyll a. The petrol ether solution, 

 from which the last traces of chlorophyll b have been removed as 

 indicated in the preceding section, is further purified by shaking it 

 three times with 2 litres of 90% methyl alcohol. 



The methyl alcohol is removed and the petrol ether solution 

 of chlorophyll a is washed with water until the chlorophyll is 

 precipitated in quantity. Talc is added to the extent of from 30 to 

 100 grams, and the whole filtered on a layer of talc on a Buchner 

 funnel. The petrol ether should then run off colourless. 



The talc is washed with petrol ether of low B.P. and sucked 



dry with the pump till all petrol ether small has disappeared. It 



is then transferred to a bottle and shaken with as little ether as 



possible. On filtration on a small Buchner funnel the beautiful 



deep blue ether solution of chlorophyll a runs through. The 



chlorophyll and talc are completely freed from one another by 



further filtration. 



' The large quantities of ether are necessary because the watery methyl 

 alcohol dissolves much ether and the petrol ether which separates out on dilution 

 makes it difficult to carry the chlorophyll over from methyl alcohol to ether. 



