52 4 PRACTICAL ORGANIC AND BlO-CHEMISTRY 



Chlorophyll 



Preparation. 



Chlorophyll and the other pigments may be extracted from fresh or dried 

 leaves. It is advantageous to use dried leaves ; their bulk is smaller with a 

 consequent saving of solvent and they are more easily ground to a powder. 

 Leaves contain 70-80 per cent, of water and may be dried in a steam oven. 



Many leaves are spoilt by keeping, e.g. grass, others are spoilt by drying, 

 but several varieties can be dried without any change taking place in the 

 chlorophyll. Dried stinging nettle leaves are the most convenient source. 



Fresh leaves must be used for the analysis of the amount of chlorophyll. 

 Of the several methods used by Willstatter, that of rapid extraction on a Buchner 

 funnel is the most convenient. 



The dried material (^kilo. 1 ) is placed upon a large Buchner funnel or similar 

 contrivance in a layer 4-5 cm. thick. It is slowly treated with 1*5-2 litres 

 of 90 per cent, alcohol, or better 80 per cent, acetone, suction being started 

 after the powder has become moistened and the solvent being gradually added. 

 The material loses its green colour when it is extracted so that solvent need 

 no longer be added when the layer on sampling shows the absence of green 

 colour. About 900 c.c. of extract is obtained from 500 gm. of material. 



Fresh material (2 '5 kilos.) is ground up. It gives a sticky olive-brown paste, 

 It is partially dried and cleaned by shaking in a bottle with 500 c.c. of acetone. 

 The filtered liquid has a yellow-brown colour, but contains no chloro- 

 phyll. The residue is freed from acetone by pressure at 200 atmospheres in 

 a Buchner press ; it weighs about '8 kilo, and is ground up to a powder. The 

 powder is shaken in a bottle with 1500 c.c. of acetone tor 5 minutes, which 

 gives a concentration of 80 per cent, acetone, and i litre of 80 per cent, acetone 

 is added. The liquid is filtered off on a Buchner funnel and the solid washed 

 three times with 500 c.c. of 80 per cent, acetone. The time occupied in the 

 process is i-J hours and 47 gm. of chlorophyll are extracted. 



This method was useful in preparing the pigments from brown algae. 



The extract, if it be concentrated and if it deposits an oily layer, is 

 diluted with -2 5 --5 times its volume of 80 per cent, acetone. It is shaken 

 with finely powdered talc which takes up some of the pigment. The pigment 

 is completely thrown out by adding 300 c.c. of water. The precipitate is 

 filtered off on a layer of talc ; the liquid is yellow-green in colour and is dis- 

 carded. The pigment on the talc is washed with 500-600 c.c. of 65 per 

 cent, acetone, which removes yellow pigment, and with water until the final 

 washings do not smell of acetone. The talc is sucked dry and shaken 

 in a bottle with ether and sodium sulphate. The ethereal solution is filtered 

 through sodium sulphate and evaporated to a syrup. The syrup is gradually 

 mixed with 400-500 c.c, of petroleum ether. The precipitated chloro- 

 phyll is filtered off. The yield is about 3 gm. and contains 90-95 per cent. 

 of chlorophyll. Some chlorophyll, which is contained in the petrol ether 

 filtrate, is obtained by washing it with 80 per cent, methyl alcohol and pre- 

 cipitating by adding water. 



Purification. 



The pigment contained in an 80 per cent, acetone extract is transferred 

 into petrol ether by pouring it into about an equal volume of this solvent 

 contained in a separating funnel. About one-eighth of its volume of water 

 is added and the mixture carefully shaken. The lower layer of a yellowish- 

 green colour is removed and the upper layer is shaken twice with about 250 

 c.c. of 80 per cent, acetone. The acetone is removed by shaking four times 

 with 125 c.c. of water. To remove xanthophyll the petrol ether is shaken 

 with 500 c.c. of 80 per cent, methyl alcohol four or more times until it shows 

 only a faint-yellow colour. This extract contains about *2 gm. of xantho- 



P h yii (p- 530)- 



The petrol ether is freed from acetone and methyl alcohol by shaking 

 four times with 500 c.c. of water. It loses its fluorescence, becomes cloudy 

 1 One kilo, costs 65-80 shillings. 



