QUANTITATIVE KSTIMATIOX OI< CAROTlttOlDS 259 



Certain animal (issues whose carotinoid content may he desired may 

 contain pigments soluble in alcohol or ether whose presence would in- 

 terfere with the direct analysis of the extracts. Tissues such as liver, 

 spleen, kidney, heart, etc. fall in this class. In most cases the foreign 

 pigments can be removed by saponih'cation, but this must be conducted 

 with great care to avoid the production of other foreign pigments which 

 may be extracted from the soap by ether and interfere, not only with 

 the analysis, but also with the true demonstration of the presence of 

 carotinoids. Bile pigments, if present, can usually be removed by 

 treating the fresh tissues with lime water, previous to the extraction 

 with ether, in order to form ether-insoluble calcium salts. It must be 

 admitted, however, that the quantitative analysis of tissues of this 

 character for carotinoids requires considerable study before it can be 

 concluded that the method proposed is entirely free from error. 



The final colorimetric comparison with the standard hardly needs 

 further comment. It is obvious that the 0.2 per cent K,Cr 2 O 7 solution 

 is the most convenient to use. For animal tissues, and perhaps some 

 plant tissues, the amount of pigment present may be so low that a con- 

 venient quantity of tissue will not yield sufficient pigment to match the 

 dichromate standard at any of the equivalent carotinoid depths given 

 by Willstatter and Stoll. It is convenient in these cases to set the 

 unknown solution at a given depth of say 50 mm. or 100 mm. and 

 match its color with the standard dichromate. The question then arises 

 as to the carotin or xanthophyll equivalent of the dichromate depth 

 found in such an analysis. For this purpose the writer has constructed 

 the curves shown in Chart 1. These curves are based on the somewhat 

 meager data given by Willstatter and Stoll for the comparative color of 

 5 x 10~ 5 molar carotin and xanthophyll solutions with the standard 

 dichromate solution and also the comparative color of the two pig- 

 ment solutions. 



The method of using these curves involves no difficulties, but for the 

 sake of clearness one or two examples may be given. 



1-A'ample 1. A 25 mm. layer of melted butter fat from cows on fresh 

 pasture grass was found to require 36.9 mm. of 0.2 per cent K 2 Cr 2 7 in 

 the Kober colorimeter. Referring to the carotin curve in Chart 1 it 

 is seen that 36.9 mm. of standard dichromate equals 45.2 mm. of 

 0.00268 per cent carotin solution. 



Therefore, 0.00268 : x --- 25 : 45.2 



x -- 0.00485 per cent carotin in the butter fat 

 (ignoring the sp. g. of the fat). 



