THE PIGMENTS OF LEAVES 525 



and the chlorophyll is precipitated. The suspension in the petrol ether is 

 shaken with sodium sulphate and about 35 gm. of talc and filtered through 

 a layer of 1 5 gm. of talc, which is stirred up to facilitate the filtration. The 

 filtrate contains carotin which may be isolated from it (p. 530). 



The chlorophyll on the talc is washed with petrol ether and finally with 

 petroleum ether of boiling-point 30-50. It is dissolved in 250 c.c. of 

 ether, the ethereal solution is filtered through sodium sulphate, evaporated 

 to 25 c.c., again filtered and evaporated to 5 c.c. The chlorophyll is pre- 

 cipitated by adding 200 c.c. of petroleum ether. It is filtered off on talc, 

 dissolved in ether and the solution evaporated in a desiccator. The yield is 

 about 3 gm. from 500 gm. and represents three-fourths of the total pigment. 



Properties. 



Chlorophyll (a + b) forms a blue-black solid with metallic lustre having 

 a crystalline appearance under the microscope. On powdering it becomes 

 a dull-green or a blue-black powder. It shows no sharp melting-point ; de- 

 pending on the rate of heating ; it forms oily drops at 93-96 and 103-105. 



It is easily soluble in absolute alcohol, ether, benzene, chloroform, 

 carbon disulphide, most easily soluble in pyridine. The alcoholic solution 

 has a blue-green colour. The ethereal solution is a beautiful blue-green 

 with a strong fluorescence and a bluer tone than the alcoholic. In carbon 

 disulphide its solution is green in colour. It is not so easily soluble in 95 

 per cent, ethyl or methyl alcohol and it is soluble with difficulty in 90 per 

 cent, alcohol. It is soluble with difficulty in cold petrol ether, more easily 

 in the hot solvent. It dissolves easily in petroleum ether containing methyl 

 or ethyl alcohol. 



In anhydrous alcoholic solution chlorophyll undergoes a remarkable 

 change termed allomerisation which makes its isolation very difficult. This 

 change is hindered by adding a trace of acid, '01 gm. of oxalic acid in 1000 

 c.c. of alcohol. Its spectrum shows a marked absorption band in the red 

 region and a fainter one towards the orange. The violet end is completely 

 absorbed. 



Tests for the Purity of Chorophyll. 



(1) It must leave an ash consisting of pure magnesium oxide correspond- 

 ing to 4*5 per cent, of its weight. If phaeophytin be present, the spectrum 

 shows absorption bands near E and between E and F. 



(2) Phase Test. On saponification with methyl alcoholic potash in ethereal 

 solution, the colour becomes brown changing in a few minutes back to the 

 original green. 



This does not occur with the allomerised material. 



(3) It must contain no yellow pigments. The ethereal solution in the 

 above phase test must be colourless. On slowly adding water to the alkaline 

 solution, the ether should become yellow. 



(4) Hydrolysis Test. A few mgm. of the substance are boiled with 3-4 c.c. 

 of methyl alcoholic potash for several minutes avoiding too great a concentra- 

 tion. The solution is acidified and extracted with 30 c.c. of ether. The 

 ethereal solution is extracted with (a) 4 per cent, hydrochloric acid, (b) 9 per 

 cent, hydrochloric acid to remove phytochlorin e and phytorhodin g. It is 

 subsequently shaken with 1 2 per cent, hydrochloric acid when the ether be- 

 comes quite colourless. On neutralising and shaking with ether, it shows the 

 red colour of phytorhodin. The colour is not red if allomerised chlorophyll 

 be present. 



(5) Phytol should form one-third of the molecule. This is most con- 

 veniently tested by extracting the saponified ethereal solution with 22 per cent, 

 hydrochloric acid which dissolves the chlorophyllides. 



Separation of Chlorophyll into its Components a and 6. 



The separation of the mixture of the a and b chlorophylls depends upon 

 the partition of the constituents between the solvents, petroleum ether which 

 dissolves chlorophyll a and methyl alcohol which dissolves chlorophyll b. 



