CHLOROrilVLL AXD THE CHLOROPLASTS 343 



hoi extraction is then added to the ether containing the first extract and 

 another liter of ether is added. The petroleum ether, used for wash- 

 ing, is freed from methyl alcohol by a stream of water, whereby some 

 dissolved pigments are precipitated. 



The third and fourth methyl alcohol extracts are treated in the same 

 manner, but the amount of the h component becomes less in each ex- 

 traction. To the sixth methyl alcohol extract, before washing with 

 petroleum ether, only 900 cc. of methyl alcohol are added, to the seventh 

 800 cc, the eighth 700 cc. and to the fourteenth only 100 cc. They are 

 all washed with the same petroleum ether used in the first and second 

 extractions and are added to the same ether solution, always with the 

 addition of more ether, at first 1000 cc. and after the tenth extraction 

 500 cc. 



When there is a great deal of chlorophyll h in the original chlorophyll, 

 the sixth and seventh extraction are also brought to 90 per cent by the 

 addition of methyl alcohol ; to the following extractions less of the methyl 

 alcohol is added. 



The sixteenth and seventeenth extractions have the purpose of free- 

 ing the chlorophyll a from the last traces of chlorophyll h. These methyl 

 alcohol extractions are rich in chlorophyll a and are therefore well washed 

 with petroleum ether. 



The petroleum ether solution, thus freed from chlorophyll h, contains 

 the chlorophyll a component. The solution is washed with water until 

 the chlorophyll has been precipitated quantitatively. This precipitate is 

 adsorbed with 30-100 grams of talc and the whole is filtered carefully 

 with suction through a layer of talc, and washed with low-boiling petroleum 

 ether. The suction is continued until there is no odor of petroleum ether. 

 The chlorophyll a is then extracted from the talc with pure ether and 

 filtered several times. Most of the ether is evaporated and the last por- 

 tion of the ether is permitted to evaporate in a vacuum desiccator. The 

 chlorophyll a remains as a beautifully shining, blue-black mass. 



The chlorophyll h component, which has been extracted by means 

 of methyl alcohol, is now in an ether-petroleum ether solution. This is 

 washed with water to free it from methyl alcohol and, after drying with 

 sodium sulfate, is concentrated to about 500 cc. and finally at reduced 

 pressure, to 30 to 40 cc. From this solution the main portion of the 

 chlorophyll h is precipitated by the addition of 300 cc. petroleum ether 

 (30-50°). This precipitate is filtered immediately onto talc. The filtrate 

 contains considerable quantities of chlorophyll a. The precipitate also 

 contains some chlorophyll a, and has to be dissolved again in ether and 

 reprecipitated with low-boiling petroleum ether. This dissolving and re- 

 precipitation process has to be repeated two to three times if there are 

 relatively large quantities of chlorophyll a present. Chlorophyll h is more 

 easily filtered than the a component; the former can therefore be col- 

 lected on a hard filter and finally dried in a desiccator. 



