530 BIOTIN 



in which the proportion of free vitamin H was increased by high-pressure 

 hydrolysis. ^^ After precipitation with alcohol and acetone the filtrate con- 

 taining the active principle was treated with phosphotungstic acid, and 

 the active precipitate decomposed with barium hydroxide.^'' This crude 

 material, containing approximately 0.1 % biotin (vitamin H) was subjected 

 to esterification conditions with methanol and HCl, and the ethyl acetate- 

 soluble material from this procedure was dissolved in chloroform and passed 

 through a column of activated aluminum oxide. -^ The column was then 

 eluted in succession with chloroform, acetone, and then repeatedly with a 

 mixture of 90 % acetone and 10 % methanol. By this procedure a fortyfold 

 concentration of the active principle was accomplished. A repetition of 

 the same chromatographic procedure achieved a further threefold con- 

 centration. The active material from this second elution was dissolved in 

 chloroform, and the solution was extracted with dilute hydrochloric acid. 

 After second esterification of this acid extract, crystals of the methyl ester 

 of biotin were obtained after concentration of the ethyl acetate solution 

 of the esterified active substance. The crystals were purified by two crys- 

 tallizations from methanol-ether mixtures, and then by sublimation in a 

 high vacuum (10~^ mm.). The sublimate was crystallized from methanol 

 and ether in long, fine needles, m.p. 166 to 167° (uncorrected). The ester 

 was subjected to several recrystallizations and sublimations in vacuo without 

 changing its biological activity and melting point.^^- ""* Expressed in terms 

 of vitamin H units, the crystalline ester had a potency of 27,000 vitamin H 

 units per milligram. The same order of activity was also found for a crystal- 

 line biotin preparation obtained by Kogl,^ as further proof for the identity 

 of biotin and vitamin H.^^ 



With a modification of the procedure used for the isolation of biotin 

 from liver, Melville et al}'-' have succeeded in obtaining pure biotin from a 

 crude milk concentrate which is a more readily available natural source of 

 biotin. After the usual esterification procedure the biotin ester was ad- 

 sorbed on Decalso from chloroform solution and eluted with a 5 % methanol- 

 acetone mixture. The eluate was passed through a column of commercial 

 activated alumina. The column was treated with a 10% methanol-acetone 

 mixture from which, after removal of the solvent, the crystalline methyl 

 ester was obtained. Further purification consisted in washing with ethyl 

 acetate, followed by sublimation in vacuo and crystallization from a meth- 



23 V. (lu Vigneaud, K. Hofmann, D. B. Melville, and P. Gyorgy, /. Biol. Chcm. 140, 



643 (1941). 

 ^* P. Gyorgy, C. S. Rose, K. Ilofmann, D. B. Melville, and V. du Vigneaud, Science 



92, 609 (1940). 

 25 D. B. Melville, K. TTofmann, E. Hague, and V. du Vigneaud, J. Riol. Chcm. 142, 



615 (1942). 



