II. CHEMISTRY 455 



This solution can then be extracted with n-butanol or chloroform,-^- ^^ in 

 which nicotinamide is soluble. The chloroform solution may be subjected 

 to fractional distillation, nicotinamide distilling at 150 to 160° at 5 X 10~* 

 mm. Hg.^ The distillate may be recrystallized from organic solvents such 

 as chloroform, benzene, or ethylene glycol. 



Warburg and Christian^ have described the procedure they used to ob- 

 tain nicotinamide from red blood cells. Coenzyme I (DPN) and coenzyme 

 II (TPN) were first isolated in a purified state (see p. 482 for method). The 

 coenzyme (DPN) could then be hydrolyzed with dilute sulfuric acid, lib- 

 erating free nicotinamide. Adenine could be precipitated as the silver salt. 

 The mother liquor and washings from this step were cleared of silver with 

 hydrogen sulfide and the nicotinamide extracted with amyl alcohol. After 

 filtration and concentration, nicotinamide was precipitated from the amyl 

 alcohol solution with picrolonic acid. The picrolonic acid salt could be 

 recrystallized from hot water. Treatment with hydrochloric acid yielded 

 the free nicotinamide hydrochloride,^ which was in turn recrystallized from 

 hot ethanol. 



The procedure used by the Wisconsin workers in isolating nicotinamide 

 from liver was somewhat involved but essentially as follows.^"- ^^' ^^^ -® 

 Liver paste was suspended and extracted with a 40 % ethanol, 50 % diethyl 

 ether, and 10 % water mixture. The concentrate from this step was ex- 

 tracted with acetone. Several successive concentrations and extractions 

 with amyl alcohol, 95 % ethanol, acetone, and finally water were used. The 

 nicotinamide was adsorbed from the water extract using charcoal (Norit A) 

 and eluted with a mixture of 4 parts methanol and 1 part pyridine. Metha- 

 nol and pyridine were removed by evaporation. The dry residue was ex- 

 tracted with acetone and the extract evaporated to dryness. This dry resi- 

 due was placed in a molecular still and held at 160 to 165° and 1 X 10~* mm. 

 pressure for 3 hours. The distillate was dissolved in alcohol and treated 

 with alcoholic mercuric chloride to yield a precipitate which was dissolved 

 in dilute HCl and decomposed with H2S. Crystalline nicotinamide appeared 

 in the concentrated filtrate. 



Bovarnick-^ has described a procedure by which she isolated pure nico- 

 tinamide from heated asparagine-glutamic acid mixtures. Presumably, a 

 paper strip chromatogram technique similar to that described for nicotinic 

 acid could be used in the isolation of small quantities of nicotinamide, 

 although this has not yet been reported. It is known, however, that nico- 



" H. von Euler, F. Schlenk, L. Melzer, and B. Hogberg, Hoppe-Seyler's Z. physiol. 



Chem. 258, 212 (1939). 

 " P. Karrer and H. Keller, Helv. Chim. Acta 22, 1292 (1939). 

 " C. A. Elvehjem and C. J. Koehn, Jr., /. Biol. Chem. 108, 709 (1935). 

 2« C. J. Koehn, Jr., and C. A. Elvehjem, J. Biol. Chem. 118, 693 (1937). 

 " M. R. Bovarnick, J. Biol. Chem. 153, 1 (1944). 



