CHLOROPHYLL 327 



alcohol or 80 per cent acetone, yielding a green solution with 

 a strong red fluorescence. 



3. Phase Test. — Prepare an ethereal solution of chlorophyll 

 as follows : About 15 c.c. of an 80 per cent acetone extract of 

 dried leaves are poured into 30 c.c. of ether contained in a tap 

 funnel and mixed with 50 c.c. water. The ethereal solution 

 rises to the surface. It should be washed four times with 

 50 c.c. of water each time by carefully allowing the water to 

 run down the side of the funnel without shaking. If a 30 per 

 cent methyl alcoholic solution of potash is now run under the 

 ether layer a brown colour is produced at the junction of the 

 two liquids. The colour gradually changes to olive-green and 

 finally back to the original green. The reaction, which is 

 known as the " Phase Test," is due to the saponification of the 

 chlorophyll with formation of the potassium salt of chloro- 

 phyllin. Consequently on dilution with water the green colour 

 remains in the aqueous layer and is no longer soluble in ether. 



4. Separation of Chlorophylls from Carotinoids. — Shake 

 vigorously 5 c.c. of an ethereal solution of chlorophyll (pre- 

 pared as above) with 2 c.c. of concentrated methyl alcoholic 

 potash. When the green colour has returned, dilute with 

 10 c.c. water, added in portions, and add a little more ether. 

 On shaking, two layers separate ; the lower aqueous alkaline 

 layer contains the chlorophyllin, while the ether contains 

 carotinoids. 



5. Separation of Xanthophyll from Carotin. — Wash the 

 ethereal solution of these two substances obtained from 

 previous experiment with a little water and evaporate to i c.c. 

 Dilute with 10 c.c. of light petroleum, and shake up two or 

 three times with 10 c.c. of 90 per cent methyl alcohol until 

 the latter is no longer coloured. The methyl alcohol will 

 contain the xanthophyll, while the carotin will be in the light 

 petroleum. 



6. Action of Acid on Chlorophyll. — Shake 2 c.c. of an 

 ethereal solution prepared as above with a little 20 per cent 

 hydrochloric acid and a few drops of water ; run off the lower 

 aqueous layer and evaporate the ether solution over a water 

 bath. Dissolve the residue in 5 c.c. of alcohol ; note the 



