20 Carbon Assimilation. 



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, i n petrol o th ei' 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 

 SO'C. to 40»C. A quantity of this powder, say 500 grams,' 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 



' The quantities of material and reagents and the dimensions of apparatus 

 quoted in this and succeeding sections are those given bv Willstatter and 

 Stoll (1913). ^ " 



