CHLOROPHYLL AND THE CHLOROPLASTS 341 



plant material, becomes diluted to about 80 per cent by volume, and to 

 this another liter of 80 per cent acetone is added. It is filtered and 

 washed three times with 80 per cent acetone. The extract contains the 

 chlorophyll. This method is very serviceable for the extraction of plant 

 material which is dried with difficulty, e.g., mucilaginous plants. 



For the separation of the 80 per cent acetone extract into the dififer- 

 ent pigments Willstatter and StoU recommend the following procedure. 

 The acetone extract (from 2 kilograms of leaves) is poured into a separa- 

 tory funnel containing 4000 cc. of i>etroleum ether (0.64-0.66) and 500 cc. 

 of water are added, shaking carefully. The water is drawn off, and the 

 petroleum ether layer is washed twice, each time with a liter of 80 per 

 cent acetone ; this removes colorless impurities. The acetone which has 

 been dissolved in the petroleum ether layer is removed by washing four 

 times with 500 cc. each of water. The petroleum ether is now shaken 

 with three lots of 2000 cc. each of 80 per cent methyl alcohol. If the 

 last extract is still appreciably colored, the petroleum ether is shaken 

 once or twice more with methyl alcohol. The methyl alcohol extracts 

 contain xanthophyll and a small quantity of chlorophyll h. The xantho- 

 phyll may be purified according to the method described below. 



The isolation of cliloropiiyll from the petroleum ether solution is accom- 

 plished by first washing this with water, four times, each with 2000 cc, 

 in order to remove the methyl alcohol and acetone. Thereby the petroleum 

 ether solution loses its fluorescence, becomes turbid and the chlorophyll 

 begins to precipitate. The suspension in petroleum ether is shaken with 

 anhydrous sodium sulfate and talc and is filtered through a thin layer 

 of talc with suction. The filtrate contains besides a little chlorophyll, 

 considerable carotin. The talc holding the chlorophyll is washed with 

 petroleum ether until the filtrate is colored but slightly yellow and then 

 with petroleum ether of boiling point 30-50°. This is sucked ofif com- 

 pletely and the chlorophyll is dissolved immediately from the talc in 

 ether. The ether solution is filtered through anhydrous sodium sulfate 

 and concentrated to 100 cc, filtered again and concentrated to 25 cc 

 The chlorophyll is precipitated by the addition of 800 cc. low-boiling 

 petroleum ether. A blue-black powder is formed or sometimes a sus- 

 pension of fine particles which can be filtered only on talc. This is 

 again dissolved in 20 cc. of ether and permitted to evaporate in a desic- 

 cator. There are thus obtained 6.5 grams of chlorophyll from a kilo of 

 leaf material. 



Certain tests can be applied to the chlorophyll preparation, obtained 

 in the manner described, in order to determine its purity. The ash 

 should amount to 4.5 per cent and consist of pure magnesium oxide. The 

 ash of pure chlorophyll contains no phosphorus nor iron; the presence 

 of both of these elements has been a matter of dispute for some time. 

 The chlorophyll preparation should contain no yellow pigments. The 

 presence of the yellow pigments becomes evident in the following test. 

 The ether solution is shaken with methyl alcoholic potassium hydroxide. 



