220 THE VITAMINS 



3.46 per cent, ammonia-nitrogen 1.9, and reducing sugars as dextrose 

 19 per cent of the total solids. "The final active material gave a faint 

 carbylamine test for amino nitrogen, reduced potassium permanganate 

 and ammoniacal silver nitrate quickly, and gave a faint coloration with 

 ferric chloride. Bromine water produced a slight cloudiness. The 

 phthalic anhydride and Liebermann reactions for phenols were too 

 faint to be considered positive. Further extraction of the dried acetone- 

 soluble material with absolute ethyl ether, in which the vitamin is 

 practically insoluble, reduced the total active solids to 0.28 milligram 

 per cubic centimeter of lemon juice." 



A slightly different method was described by Sipple and King 

 (1930). The lemon juice was decitrated with neutral lead acetate as 

 in the method of Grettie and King and was immediately adjusted to 

 pH 7.4 to 7.6 with diluted ammonium hydroxide. The yellow active 

 precipitate was centrifuged quickly, dissolved in dilute acetic acid and 

 the process repeated, beginning with the addition of dilute ammonium 

 hydroxide. An aqueous solution of the active precipitate was then 

 treated with two successive portions of n-h\ity\ alcohol as in the method 

 of Grettie and King, the filtrate concentrated, dissolved in 98 per cent 

 alcohol, and treated with one volume peroxide-free absolute ethyl ether 

 which removed more inactive material in the form of a white crystalline 

 precipitate. 



The active liquid was found to contain 0.56 milligram of total 

 solids per cubic centimeter of the equivalent lemon juice, with an 

 inappreciable amount of ash. Reducing substances calculated as glucose 

 amounted to 0.45 milligram per cubic centimeter; ammonium salts ex- 

 pressed as nitrogen, 0.045 milligram ; and total nitrogen as determined 

 by the micro-Kjeldahl method, 0.145 milligram of nitrogen per cubic 

 centimeter. The ferric chloride test for phenols was negative and the 

 carbylamine reaction mildly positive. Ammoniacal silver nitrate was 

 reduced slightly. 



Attempts to use barium acetate in place of lead acetate and sodium 

 hydroxide in place of ammonium hydroxide gave negative results. This 

 is interpreted as indicating that the precipitation of the vitamin by 

 lead at a pH of 7.4 to 7.6 is a more or less specific adsorption phe- 

 nomenon. 



The alcohol-ether preparation was finally purified still further by 

 evaporating to dryness in vacuo and extracting the residue with abso- 

 lute ether. This removed a significant amount of amorphous material, 

 leaving the total solids of the active fraction equivalent to 0.28 milli- 

 gram per cubic centimeter of lemon juice. 



