352 MISSOURI AGRICULTURAL EXP. STA., RESEARCH BULLETIN NO. IO 



Experiment 2. 



Fifteen grams of the fat was treated as in Experiment i. In ad- 

 dition the carbon bisulphide solution of the unsaponifiable ether extract 

 was filtered through a column of CaCO 3 in a manner identical with the 

 chromotographic experiment with alfalfa hay. As far as could be de- 

 tected with the eye all the pigment passed quite rapidly through as an 

 unadsorbed rose colored zone, which spread out considerably in its 

 passage through the column but showed no differentiation into zones. 

 The absorption bands of the filtered pigment were identical with the 

 bands of carotin. 



After the carbon bisulphide had washed out all the pigment and 

 was passing through colorless, a stream of petroleum ether contain- 

 ing ten per cent alcohol was run through the column. As it passed 

 through it gathered a zone of yellow color, leaving the column pure 

 white. This pigment was collected at the mouth of the tube, its solu- 

 tion evaporated, and the residue dissolved in carbon bisulphide. The 

 light-orange colored solution showed two strong absorption bands and 

 a third fainter one. 



The carbon bisulphide solution of the main pigment was now evap- 

 orated to dryness and the residue dissolved in ninety-five per cent 

 alcohol. The alcohol was diluted with water to an eighty to ninety per 

 cent solution and extracted with petroleum ether (b. p. 30-50 C). The 

 bulk of the pigment went into the petroleum ether and a second 

 extraction with fresh petroleum ether took out still more pigment. A 

 third extraction with fresh petroleum ether, however, left the alcohol 

 layer considerably more colored than the petroleum ether layer. The 

 alcohol layer was now evaporated and the residue dissolved in 

 carbon bisulphide, giving an orange yellow solution which showed 

 three strong absorption bands. There seemed to be three or four 

 times as much of this pigment as of the xanthophyll which had been 

 adsorbed by the CaCO 3 in the chromotogramm, and together they 

 probably amounted to ten per cent of the total pigment. 



All the xanthophyll pigments were now combined (they were 

 all in carbon bisulphide solution) and the resulting solution analyzed by 

 means of a chromotogramm. As the orange-yellow solution was washed 

 through the column by a stream of carbon bisulphide it took on 

 the appearance as shown in Figure III. Zones two and three were 

 collected together, and showed three absorption bands. (See Table 4.) 

 Their solution was evaporated and the residue dissolved in petroleum 



