Variations in Quantity of Leaf Pigments. 43 



The phytochlorin is now separated by shaking 4 or 5 times 

 with 3% hydrochloric acid, 400 c.c. being used altogether, and then 

 several times with 5% acid until this is only feebly green. The 

 extracts with the stronger acid require to be fractioned. This is 

 effected by neutralising and extracting with 30 c.c. of ether, and then 

 repeatedly extracting the ether with 3% hydrochloric acid until the 

 volume of all the 3% hydrochloric acid extracts is brought up to 

 500 c.c. This constitutes the phytochlorin solution. The remaining 

 liquid contains the phytorhodin which is extracted 4 or 5 times 

 with 12% hydrochloric acid until the ether remains only slightly 

 reddish. 



3. Tlie Pi'eparatioH of the XatUhopliyll and Carotin Solutions. 

 The ether extract from 20 grams of fresh leaves is saponified with 

 2 c.c. of concentrated potash solution in methyl alcohol, the mixture 

 being strongly shaken, first by hand and then for half-an-hour on a 

 mechanical shaker. The liquid is then allowed to stand for some 

 time and if it still fluoresces red it is skaken again and, if necessary, 

 more potash is added. When the saponification is complete the 

 solution is transferred to a small separating funnel and shaken 

 with ether. A further 30 c.c. of ether is then added to the syrupy 

 chlorophyllin salts. After shaking with water the upper ethereal 

 layer contains the yellow pigments, the water the chlorophyllin salts. 

 (Cf. Section E, experiment 6). After separation of the two layers, 

 the watery layer may be again shaken with ether in order to 

 determine whether the whole of the yellow pigments have been 

 extracted. 



The two yellow pigments are then separated according to the 

 principle used in experiment 7. The ether solution is washed with 

 water and methyl alcoholic potash to remove traces of chlorophyllins 

 and small quantities of brown, acid, organic substances, and then 

 twice more with distilled water. The ether is then evaporated at 

 ordinary temperature under reduced pressure to a few c.c, and 

 then transferred to 80 c.c. of petrol ether in a separating funnel. 



From this the xanthophyll is extracted by repeated extractions 

 with methyl alcohol as follows. (1) 100 c.c. 85%, (2) 100 c.c. 90%, 

 (3) two extractions with 92%. If the last extraction is not 

 colourless, further additions of 92% methyl alcohol are made. 



In this way the xanthophyll in the methyl alcohol is separated 

 from the carotin which remains in the petrol ether. 



The xanthophyll is transferred to 130 c.c. of ether, by adding the 

 latter to the methyl alcohol followed by slow addition of water and 



