292 Ingvar Jorgensen and Walter Stiles. 
(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 1 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 second 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 
1 See note on page 289. 
