THE PIGMENTS OF LEAVES 52; 



The Chlorophyllides. 



Preparation. 



Chlorophyll is partially hydrolysed by chlorophyllase with separation of 

 phytol; the carboxyl group is esterified with methyl or ethyl alcohol with 

 the formation of methyl or ethyl chlorophyllide. Chlorophyllide may also be 

 obtained. 



Leaves rich in chlorophyllase are used, e.g. those of Galleopsis tetrahit. 



(1) Ethyl Chlorophyllide. Crystalline Chlorophyll. 



1 kilo, of powdered fresh leaves is mixed with 2 litres of 90 per cent. 

 alcohol and allowed to stand 12-24 hours. The solid is filtered off on a 

 Buchner funnel and washed with 2*5 litres of acetone in portions of 500 c.c. 

 The combined alcohol and acetone solutions are mixed with i5ogm. of coarse 

 talc and diluted by stirring with 4-5 litres of water in the course of i hour. 

 The chlorophyllide separates and is filtered off on talc. The talc is rapidly 

 washed with (i) 250 c.c. of 55 per cent, acetone and (2) 250 c.c. of 55 per 

 cent, alcohol, which are removed by suction, (3) 1500 c.c. of petrol ether, (4) 

 500 c.c. of ether. These solutions contain all the. xanthophyll and carotin 

 (p. 530). Though the ether has a green colour it contains very little chloro- 

 phyllide. The talc is treated with 250 c.c. of absolute alcohol containing 

 005 gm. of oxalic acid to prevent allomerisation and the deep green filtrate 

 is mixed with 3-4 litres of ether. The ether is shaken with 2 litres of water 

 to remove alcohol, dried with sodium sulphate and evaporated to 1500 c.c. 

 It is filtered and evaporated to 20 c.c. Ethyl chlorophyllide crystallises out 

 during the evaporation and on standing. The yield is 4*5-5 gm. or 'i per 

 cent, of fresh leaves. 



(2) Methyl Chlorophyllide. 



2 kilos, of dried leaves are treated with a mixture of 3*2 litres of acetone 

 and o'8 litre of 80 per cent, methyl alcohol for about 40 hours. The mass is 

 filtered through muslin on a Buchner funnel and washed with 4-5 litres of 

 acetone in portions, allowing each portion to soak into the material before 

 sucking it off. The filtrate occupying a volume of 6-7 litres is mixed with 

 300 gm. of coarse talc, stirred well and diluted slowly (in i hours) with 7 

 litres of water. Methyl chlorophyllide crystallises out, is filtered off on a layer 

 of talc and washed with (i) 500 c.c. of 50 per cent, acetone, (2) 500 c.c. of 

 50 per cent, alcohol. The talc is sucked dry, shaken with 2 litres of petrol 

 ether, filtered off and washed with (i) petrol ether, (2) ether. The talc is 

 treated with 800 c.c. of absolute alcohol containing -02 gm. of oxalic acid and 

 the methyl chlorophyllide obtained in the same way as ethyl chlorophyllide. 

 A yield of *5 gm. per kilo, is obtained. 



(3) Chlorophyllide. 



24 kilos, of fresh leaves are placed in 29 litres of pure acetone in 3 large 

 vessels. After 3-4 hours the chlorophyllide is present in the solution, which 

 is separated by centrifugalising, mixed with 500-600 gm. of talc and diluted 

 slowly with twice the volume of water. The liquid is decanted and the talc 

 with the crystals of chlorophyllide filtered off on a Buchner funnel. It is 

 divided into 4 portions. It is washed on the funnel with (i) ether until the 

 solvent becomes pale green, (2) 300 c.c. of acetone. The chlorophyllide 

 dissolves in the acetone. It is transferred to ethereal solution (2 litres for 

 each portion) by adding water. The ether is fieed from acetone by washing 

 with water and dried with sodium sulphate. The ethereal solution is rapidly 

 evaporated nearly to dryness. The chlorophyllide crystallises out in a yield 

 of 12 gm. or 0*05 per cent, of the fresh leaves. 



Separation of the a and b Chlorophyllides. 



The separation of these constituents is effected in a similar way to that of 

 the a and b chlorophylls by partition between solvents. 



The b components of methyl chlorophyllide dissolve in 50-60 per cent, 

 methyl alcohol, of chlorophyllide in 40-50 per cent, methyl alcohol, the a 

 components in an ether-petrol ether mixture. 



