The Extraction of Pure Pigments from the Leaf. 2 3 



(6) The Separation of the two Chlorophyll Components from one 

 another. Although no doubt for many plant physiological purposes 

 it will be sufficient to extract a mixture of the pure chlorophyll 

 pigments, yet in other cases it will doubtless be of the first import- 

 ance to obtain the two chlorophyll components isolated from one 

 another. In order to make this review as complete as possible we 

 have therefore thought it worth while to give Willstatter's method 

 of separation of chlorophyll a and chlorophyll b in spite of its 

 laboriousness. 



The principle involved in the separation is that of the distribu- 

 tion of the two components in petrol ether and methyl alcohol. In 

 a mixture of these two solvents the a component goes to the petrol 

 ether, the b to the methyl alcohol. 



Eight grams' of chlorophyll isolated according to the method 

 described in section 5 are dissolved in 150 to 200 c.c. of ether, and 

 filtered into a 7-litre separating funnel containing 4 litres of petrol 

 ether (S.G. '64 to -66). The chlorophyll begins to precipitate out, 

 and 50 to 100 c.c. of methyl alcohol are added to clear it again. 



Before separating the components by fractionation the ether is 

 first removed by washing with 2 litres of 80% methyl alcohol once 

 or twice. 



The chlorophyll b is now separated by repeated extractions (14 

 of them) with 2 litres of 85% or 90% methyl alcohol. The compo- 

 nent a remains in the petrol ether. The methyl alcohol must first 

 be saturated with petrol ether (5-5% and 10% respectively is required 

 for this) and immediately before use it must be acidified with '01 

 gram oxalic acid per litre. 



(7) Purification of Chlorophyll b. The first methyl alcohol extract 

 is brought to a concentration of about 90% by the addition of a 

 litre of methyl alcohol. It is washed with a litre of petrol ether, 

 separated from it, added to 2 litres of ether and mixed with much 

 water, by which means the chlorophyll b is brought into ethereal 

 solution. 



The seeond methyl alcohol extract is similarly mixed with a 

 litre of methyl alcohol. It is shaken with the washed petrol ether 

 of the first extract to which has been added another ^-litre of petrol 

 ether. The solution containing component b is separated from the 

 petrol ether and added to the ether solution of the first extract to 

 which another ^-litre of ether is added. 



Each of the petrol ether portions used in washing is freed by 

 ■ See note on page 20. 



