304 RIBOFLAVIN 



all tissues is present in the form of the dinucleotide> Spleen contains an 

 enzyme that rapidly degrades the phosphate-bound forms of riboflavin to 

 the free vitamin.^ 



It has been shown that riboflavin phosphoric acid is able to form loose, 

 non-dialyzable complexes, for instance with a solution of pseudoglobulin 

 or albumin from horse serum. The separation of the flavin component and 

 the protein in this case can be achieved by precipitation of the latter with 

 ammonium sulfate.^ 



In order to liberate riboflavin from its natural protein-bound forms, it 

 is necessary to treat the mashed tissues with suitable solvents at room tem- 

 perature or at the boiling point of the solvent. Methanol, ethanol, acetone, 

 undiluted or diluted with water, and aqueous acid solutions have been used 

 for extraction of the vitamin. For instance, riboflavin from fresh or dried 

 plants has been extracted in good yields by boiling the material with 70 % 

 methanol for 45 minutes.^ 



For the isolation of riboflavin from the extracts, it sometimes is useful 

 first to remove lipids by extraction wdth ether, in which the vitamin is 

 insoluble. Salts and glycogen in some cases can be eliminated from ribo- 

 flavin concentrates by fractionate precipitation with alcohol or acetone. 

 Impurities from fermentation liquors may be precipitated by means of 

 acetone, and crude riboflavin can be recovered from the concentrated filtrate 

 by the addition of more acetone.'^ The vitamin can be extracted Avith butanol 

 and then precipitated from the extract by addition of petroleum ether. ^ 

 In the isolation of riboflavin from whey, the accompanying creatinine has 

 been removed by picric acid precipitation. 



Precipitation of riboflavin occurs with lead acetate and with silver nitrate 

 in neutral solution, or with phosphotungstic acid in N II2SO4; from the 

 latter precipitate the phosphotungstic acid can be extracted with amyl alco- 

 hol. Silver nitrate or mercuric sulfate in acid solution leaves the vitamin in 

 solution but precipitates some accompanying substances. 



Good adsorbents for riboflavin are fuller's earth in acid solution, Florisil, 

 Floridin XXF, and Frankonit in neutral solution. One of the best eluents 

 is pyridine, diluted with aqueous methanol or ethanol i^'* ammonia, tri- 

 ethanolamine, 0.1 iV NaOH in 60% ethanol, boiling 60% ethanol, 80% 



4 O. A. Bessey, O. H. Lowry, and R. H. Love, /. Biol. Chein. 180, 755 (1949). 



^ J. 1 J. Cvammer, Nature 161, 349 (1948); R. S. Comline and F. R. Whatley, /7>/r/. 



161, 350 (1948). 

 « T. Wagner-Jauresg .'hkI II. Arnold, Biochem. Z. 299, 280 (1938). 

 ' Merck and Co., British Pat. ()21 ,401 (Aug. 15, 194G) [C.A. 43, 71S9 (1949)]. 

 « Merclv and Co., U. S. Pat. 2,355,220 (Aug. 8, 1944) [CI. 38, 6488 (1944)1. 

 ^* P. Gyorgy, R. Kuhn, and T. Wagner-Jauregg (to I. (!. Farl)enindustne A. G.), 



(icrnian Pat. 607,512 (Nov. 22, 1932). 



