340 PHOTOSYNTHESIS 



smaller volume and weight of leaf material can be used, there is an economy 

 in solvents, because these do not become diluted by the water in the leaves 

 (this is 70-80 per cent of the fresh weight), the extraction is not dependent 

 upon the season of the year or the location of the plants, and very con- 

 siderable quantities of the pigments can be prepared. It should also be 

 stated, however, that in the drying of the plant material there are some 

 disadvantages. The leaves must be dried very carefully in order to avoid 

 destruction of the chlorophyll; some leaves, as, for example, elder and 

 the needles of conifers, lose considerable chlorophyll during drying. Some 

 plant material, also, cannot be stored without loss of chlorophyll unless 

 the powdered material is kept in vacuum desiccators over sulfuric acid. 



Precautions must also be taken in the extraction of these pigments 

 from the dry material. Chlorophyll is easily soluble in benzol and dry 

 acetone, but the pigment cannot be extracted by these solvents. The ease 

 of extraction is dependent not only upon the solubility in a given solvent. 

 The dry leaf material is extracted only very slowly by absolute alcohol, 

 ether, chloroform and acetone and not at all by benzol, petroleum ether 

 and carbon bisulfide. On the other hand, extraction can be accomplished 

 rapidly with methyl alcohol. Most of the solvents just mentioned ex- 

 tract chlorophyll quite readily if there is a certain amount of water pres- 

 ent ; the exception is methyl alcohol which extracts more easily in the 

 anhydrous condition. The cause for this seems to lie in the colloidal 

 state of the chlorophyll in the leaf. From colloidal aqueous solutions of 

 chlorophyll no pigment can be extracted by shaking with pure ether or 

 benzol, according to Willstatter and Stoll. If a small quantity of salt, 

 e.g., calcium chloride, is added to the colloidal chlorophyll solution, prac- 

 tically all the chlorophyll passes rapidly into the organic solvents. Willstat- 

 ter and Stoll have made determinations of the amount of chlorophyll ex- 

 tracted by different organic solvents containing varying quantities of 



water. 



For most purposes 80 per cent acetone by volume is most useful. 

 Willstatter and Stoll's method was to place the finely powdered leaf ma- 

 terial in a large Nutsch filter and the acetone is poured into this. By 

 alternate suction and percolation and adding the solvent in several por- 

 tions, the leaf material is extracted to a straw yellow color. The leaf 

 material must not form too thick a mat on the filter. In small quantities, 

 e.g., 500 grams of material the chlorophyll can be extracted with 1500 

 to 1600 cc. of solvent and in half an hour yield 900 cc. of extract con- 

 taining 4.5 grams of chlorophyll. 



For extracting the pigment from fresh material, this is first ground 

 very thoroughly, which forms a thick olive brown paste. Thus, for ex- 

 ample, starting with 2.5 kilos of fresh nettle leaves, the paste is shaken 

 with 1500 cc. of acetone and filtered with suction. The filtrate contains 

 no chlorophyll. The residue is subjected to a pressure of 200 atmos- 

 pheres in a Buchner press, and ground again. It is then shaken for five 

 minutes with 1500 cc. of acetone which, with the water remaining in the 



