2 2 Carbon Assimilation. 



is then removed by adding 4 successive ^-litres of water, with 

 gentle rotation of the liquid and running off the lower layer each 

 time. (The first time 0-6 litre of acetone is removed and in 

 successive removals 0-5, 0-4 and 0-2 litre). In the acetone thus 

 removed are many of the impurities accompanying chlorophyll in 

 the crude extract. 



From the solution remaining, the xanthophyll is first separated 

 by shaking the solution with 3 successive additions of 2 litres of 

 80% methyl alcohol. After each addition and shaking, the methyl 

 alcoholic layer is removed, and if the last extract is still considerably 

 yellow, one or two further additions of methyl alcohol are made. 

 From these methyl alcohol extracts xanthophyll is prepared. 



From the petrol ether solution, which should now have a 

 volume of 3'6 litres, the last traces of acetone and methyl alcohol 

 are removed by washing with water four times, each time using 

 2 litres of water. With the disappearance of the last parts of the 

 acetone and methyl alcohol, the chlorophyll is precipitated as a 

 suspension from tiie petrol ether, which thus loses its fluorescence. 



This suspension \n petrol ether is shaken with some fused 

 sodium sulphate and about 150 gms. of talc, and then filtered 

 through a layer of talc on a Buchner funnel. From the filtrate 

 carotin can be isolated as described later. 



The talc and chlorophyll on the Buchner funnel are washed 

 with ordinary petrol ether until this runs off yellow in colour, and 

 then the washing is completed with 300 c.c. petrol ether of B.P. 30° 

 to 50°C. The talc is then sucked completely dry with the pump, 

 and the chlorophyll in it dissolved in pure ether. The ether 

 solution of chlorophyll so obtained is filtered through fused sodium 

 sulphate, concentrated to 100 c.c, filtered twice more, and evaporated 

 to 25 c.c. 



From this solution the chlorophyll is precipitated by the slow 

 addition of 800 c.c. of low B.P. petrol ether. The precipitate so 

 obtained may be a blue black powder easily filtered, or it may be so 

 fine that it has to be filtered on talc. 



The precipitate is again dissolved in ether, and the solution 

 concentrated to 20 c.c. and dried in a dish in a desiccator. 



The pure chlorophyll so obtained consists of about 13 grams 

 (i.e., 6-5 grams per kilo of dried leaves of mixed chlorophyll a and 

 chlorophyll b) forming a thin shining steel blue crust. The yield is 

 about 75% of the total chlorophyll content of the leaves. 



