Carbon Assimilation. 
289 
extractions of chlorophyll. Now it has already been pointed out in an 
earlier section of this chapter that the efficiency of a solvent for 
extraction is not necessarily conditioned by the solubility of extracted 
chlorophyll in it. Thus pure extracted chlorophyll is easily soluble 
in benzene, in petrol ether and in water-free acetone, as well as in 
alcohol, ether, and carbon-disulphide, yet chlorophyll is only very 
slowly extracted from dried leaf powder by pure alcohol, ether and 
acetone, and not at all by benzene, petrol ether and carbon-disulphide. 
On the other hand it is immediately extracted by methyl alcohol. 
The foundation then of Willstatter’s method of extraction is 
the use of solvents containing a moderate content of water. This 
latter forms a salt solution with some of the cell contents, and this 
salt solution effects an alteration in the condition of the chlorophyll 
which thus becomes easily soluble in the organic solvent. If the 
solvent contains the correct water-content the pigment is almost 
entirely extracted. The pure solvents are effective in the order, 
methyl alcohol, acetone, ethyl alcohol, ether. When 1% water is 
added, acetone, ethyl alcohol and methyl alcohol are all equally 
effective as solvents. With a higher water-content acetone is 
better than any of the others. The best solvent is acetone contain¬ 
ing 15% (by volume) of water. Willstatter however, uses 80% 
acetone, because with this somewhat higher water-content, a 
quantity of accompanying substances are not extracted and the 
separation of the pigments becomes easier. If alcohol is used for 
chlorophyll extraction the most satisfactory solvent is one contain¬ 
ing 10% (by volume) of water. 
The earlier extractions of chlorophyll were always made with 
hot or at least warm solutions. All Willstatter’s extractions have 
been made in the cold, i.e., at ordinary laboratory temperature, 
thus preventing any alteration in the pigments which might take 
place with rise of temperature. 
(4) The Method of Extraction of the Pigments. The nettle 
leaves having been collected, their stalks are removed and the leaves 
dried at air temperature. They are then powdered as finely as 
possible, and the resulting powder then dried at a temperature of 
30°C. to 40 n C. A quantity of this powder, say 500 grams, 1 is then 
put on a filter paper in a Buchner funnel 24 cms. in diameter and 
sucked to it by means of a strong water pump, or better, by a 
vacuum pump. Half a litre of solvent is now allowed to permeate 
the powder on the filter paper for five minutes without the use of 
1 The quantities of material and reagents and the dimensions of apparatus 
quoted in this and succeeding sections are those given by Willstatter and 
Stoll (1913). 
