228 CAROTINOIDS AND RELATED PIGMENTS 



xanthophyll a will be a yellow zone due to xanthophylls a' and 

 a", whose differentiation will be described in a moment; near the top 

 of the column will be a narrow yellow zone, due to xanthophyll (3. 

 If a stream of benzene is run through the column at this point, the 

 carotin and xanthophyll a will be quickly washed away and the yellow 

 zone containing xanthophylls a' and a" will slowly separate into two 

 zones. These can now be washed out of the column with petroleum 

 ether containing one per cent absolute alcohol, leaving xanthophyll (3 

 still adsorbed. This pigment can be removed, however, by petro- 

 leum ether containing 10 per cent absolute alcohol. 



While a chromatographic analysis of an unknown pigment solution 

 is instructive it does not necessarily provide a means of definite iden- 

 tification of any xanthophyll pigments which may be present. Any 

 pigments differentiated by this test must be submitted to further ex- 

 amination. When several pigments are shown by such an analysis, a 

 second chromatographic separation should be carried out on a solution 

 of each of the pigments for the purpose of purifying it as far as pos- 

 sible. Comparison can then be made with the known properties of 

 solutions of xanthophylls a, a', a" and ft, which are as follows: 



Xanthophyll a. This pigment is quantitatively removed by 80-90 

 per cent alcohol, preferably methyl alcohol, from its solution in petro- 

 leum ether. It is adsorbed by an excess of CaC0 3 from pure, abso- 

 lutely alcohol-free, low boiling petroleum ether. It is the least ad- 

 sorbed by CaC0 3 from CS 2 of any of the xanthophylls. Its carbon 

 disulfide solutions are orange to red orange. Its alcoholic solution is 

 bleached by addition of concentrated mineral acids, passing through 

 a green color before fading. Its spectroscopic absorption bands are 

 identical with those of crystalline xanthophyll. Plate 1 shows these 

 bands in alcohol and carbon disulfide in comparison with those of 

 carotin. The measurements of these bands, using a solution contain- 

 ing 5 mg. of pigment per liter, are stated by Willstatter and Stoll 

 to be as follows: 



Xanthophyll in Xanthophyll in carbon 



alcohol (HM.) disulfide (M.H) 



5mm. 10mm. 10mm. 20mm. 



Band I 484-472 488-471 515-501 516-501 



Band II 454-441 454-440 482-469 483-467 



Band III 419- 420- 447-441 



Xanthophylls of and a". These pigments are quantitatively ex- 

 tracted from petroleum ether by 80-90 per cent alcohol, preferably 



