BIOTIN 



2. ISOLATION OF BIOTIN 

 a-Biotin 



The procedure used by F. Kogl and B. Tonnis ^ to isolate a-biotin 

 methyl ester from egg-yolk was as follows : The yolks of looo fresh 

 eggs were treated with acetone and the filtrate was concentrated and 

 treated with four volumes of alcohol. The active precipitate was 

 dissolved in water and impurities were removed by precipitation with 

 lead acetate. The filtrate was freed from lead, and phosphotungstic 

 acid was added to precipitate the active principle. The precipitate 

 was decomposed with baryta and the solution was shaken with charcoal. 

 After washing the adsorbate with 50 % alcohol, an active fraction 

 was obtained by elution with 60 % acetone containing 2-5 % of 

 ammonia. A second precipitation with phosphotungstic acid and 

 decomposition with baryta gave an active fraction soluble in alcohol. 

 This was freed from impurities by precipitation with mercuric chloride 

 and then esterified with methanolic hydrogen chloride. Further 

 impurities were removed by precipitation with picrolonic acid and 

 then with rufianic acid. Finally, a very potent preparation was 

 obtained by decomposition of the reineckate, the yield being 4 mg., 

 or 8 % of the material originally present. From a second preparation, 

 starting with a large quantity of dried egg-yolk, the methyl ester was 

 obtained in crystalline form by high vacuum distillation and crystal- 

 lisation from a mixture of chloroform and light petroleiun. The 

 substance had a m.p. of 146 to 147° C. and was active on Saccharomyces 

 in a dilution of i in 10,000,000,000. 



An improved method of isolating biotin from egg-yolk was subse- 

 quently described by F. Kogl and L. Pons,^ who used molecular 

 distillation to purify the crude product ; by means of this modification, 

 the yield was increased to 10 or 20 %. The biotin methyl ester was 

 crystallised from mesityl oxide which gave a purer product, m.p. 

 161 to 165° C. 



P -Biotin 



Gyorgy et al} used the following method for the preparation of a 

 " vitamin H " concentrate from liver : a residue obtained in the pre- 

 paration of " Campolon " was digested with papain or autoclaved 

 with acid, and the active factor was concentrated by a variety of 

 operations, including adsorption on charcoal, elution with a mixture 

 of pyridine, methanol and water, precipitation with phosphotungstic 

 acid, and precipitation with gold chloride. Yeast autolysates were 

 also concentrated by similar methods, but the procedure was more 

 difficult than with liver. 



V. du Vigneaud et al} used as the starting-material a liver concen- 



406 



