94 VITAMINS A AND CAKOTENES 



oxide,^ magnesium hydroxide, ^^ magnesium carbonate,®^ calcium phos- 

 phate,^^ silicic acid,^^ zinc carbonate," and bone powder. ^^ 



It is necessary to test in each case whether the extracted pigments can be 

 directly separated by chromatography or whether a preliminary purification 

 is necessary. Such a purification can be achieved by saponification whereby 

 the chlorophyll is separated and carotenoid esters, (e.g., xanthophyll esters) 

 are saponified. After the unsaponifiable fraction has been isolated it can be 

 suitably partitioned between 95 % methanol and petroleum ether, the chro- 

 matographic analysis being carried out with the epiphase (petroleum ether 

 phase) . Of the more important carotenoids, carotene and lycopene are to be 

 found in the epiphase (see table in Karrer and Jucker, p. 29 in ref. 12). It 

 should be noticed that cryptoxanthin and rubixanthin will also become 

 epiphasic with 90 % methanol. Instead of methanol, 94 % aqueous diacetone 

 alcohol can be used to ad vantage. ^^ According to Haagen-Smit^^ the separa- 

 tion of xanthophyll can be performed by extraction with 85 % phosphoric 

 acid. As solvent for the chromatographic adsorption n-hexane (b.p. 65 to 

 67°) or pure petroleum ether (optically pure) (b.p. 40 to 60°) is used almost 

 exclusively. Technical details about these procedures are given by Zech- 

 meister^^ and Zscheile^^ (see also Karrer and Jucker^^ and Hesse"). How- 

 ever, the quantitative exactness of the chromatographic adsorption analysis 

 is limited. Losses through oxidation as a consequence of an increased re- 

 activity while being adsorbed, through the action of light, and through 

 isomerization and persistent adhesion of the pigment to the adsorbent 

 cannot be completely excluded. These losses increase, the smaller the 

 quantity of the substances being determined. In general, they range from 

 2 to 5 %. With quantities of 150 to 30 7 they can range from 20 to 50 % 

 (see Gstirner, p. 39 in ref. 22). Hence, for exact values, it is necessary to 

 determine the average losses with known quantities of the carotene and to 

 correct the analytic figures accordingly. 



The identification of the isomeric and stereoisomeric carotenes isolated 

 from the chromatogram is done spectroscopically (for the absorption curves 



64 A. R. Kemmerer and G. S. Fraps, Ind. Eng. Chem. Anal. Ed. 15, 714 (1943). 

 " A. R. Kemmerer, /. Assoc. Offic. Agr. Chemists 27, 542 (1944) ; 28, 563 (1945) ; 29, 18 

 (1946). 



68 L. A. Moore, Ind. Eng. Chem. Anal. Ed. 12, 726 (1940) ; 13, 600 (1941) ; 14, 707 (1942). 

 6^ G. Hesse, Die Adsorptionsmethoden im chemischen Laboratorium. W. de Gruyter, 



Berlin, 1943. 

 6» T. B. Mann, Analyst 69, 34 (1944) . , 



69 D. M. Hegsted, J. W. Porter, and W. H. Peterson, Ind. Eng. Chem. Anal. Ed. 11, 

 256 (1939). 



"> A. J. Haagen-Smit, C. E. P. Jeffreys, and J. G. Kirchner, Ind. Eng. Chem. Anal. 



Ed. 15, 179 (1943). 

 " L. Zechmeister, Chem. Revs. 34, 267 (1944). 

 " F. P. Zscheile, Arch. Biochem. 5, 77, 211 (1944). 



