IV. DETERMINATION 93 



plants. According to the results of these workers, the stereoisomers do not 

 have full biological activity^^- " and should therefore be separated for 

 exact values. But in most cases the error arising from failure to separate 

 the stereoisomers is insignificant enough to be neglected. On the whole, it 

 is a recurring question for the analyst as to what degree of exactness is 

 required in each case. In case of clinical determinations, it is practically 

 always sufficient to determine the total content of carotene, especially 

 because the analytic method is not exact, since only very small quantities 

 of material are available. 



The preparation of vegetable material for analysis requires considerable 

 skill, and it varies to a certain extent for each plant. It is not possible to 

 transfer the results from one case to another (see also Zscheile and Whit- 

 more^^). The difficulties begin with the collection of the fresh vegetable 

 material. These samples are suitably and immediately stored in the cold, 

 if possible below 0°; otherwise a loss of carotene may occur. Conditioned in 

 this way the carotene content remains unchanged for a long time. Zscheile 

 and Whitmore*^ found that fresh alfalfa leaves stored for 66 days at — 18** 

 had the same carotene content as on the first day. Before extraction the 

 fresh vegetable samples should be treated A\dth boiling water for 5 to 10 

 minutes in order to destroy the enzymes. According to investigations of 

 H. Siillmann^^-®' a rapid enzymatic destruction takes place in the presence 

 of unsaturated fatty acids. To neutralize the vegetable acids Zscheile^^ adds 

 magnesium carbonate to the extractant. He uses 40 % acetone as solvent 

 for the extraction of fresh vegetable material. In other cases methanol, 

 ethanol, petroleum ether, and ether are suitable solvents. Dried material is 

 more difficult to extract than fresh plant tissues. An extended extraction 

 Avith boiling solvents often leads to a loss of carotene and also isomerization. 

 For the extraction of dried alfalfa Zscheile and Whitmore^^ recommended 

 a mixture of hexane and acetone (7:3). Investigation of the methods for 

 extracting carotene from dried vegetable material must be regarded as not 

 yet concluded. 



The analytic separation of the extract is done by chromatography. Here 

 the selection of the adsorption agent depends on the composition of the 

 extract. In use are aluminum oxide, ^^ calcium hydroxide, ^^ magnesium 



58 H. J. Deuel, Jr., C. Johnston, E. Sumner, A. Polgdr, and L. Zechmeister, Arch. 



Biochem. 5, 107 (1944); 6, 157 (1945); 7, 247 (1945). 

 "H.J. Deuel, Jr., C. Hendrick, E. Straub, A. Sandoval, J. II. Pinckard, and L. 



Zechmeister, Arch. Biochem. 14, 97 (1947). 

 " F. P. Zscheile and R. A. Whitmore, Anal. Chem. 19, 170 (1947). 

 " J. B. Sumner and A. L. Dounce, Enzymologia 7, 130 (1939). 

 «« H. Sullmann, Helv. Chim. Acta 24, 646 (1941). 

 8> E. V. Hove, Science 98, 433 (1943). 

 " G. Wendland, Vitamine u. Hormone 5, 46 (1944). 

 «3 A. Fujita, T. Narita, and M. Agisaka, Biochem. Z. 308, 420 (1941). 



