454 NIACIN 



1. Isolation of Nicotinic Acid 



Nicotinic acid is easily isolated from most natural materials, the exact 

 technique to be adopted depending on the nature of the material. In yeast, 

 nicotinic acid may be extracted directly without preliminary hydrolysis.^ 

 With most materials, acid or alkaline hydrolysis is necessary to liberate 

 the free acid. Removal of fat with suitable solvents is a desirable preliminary 

 to hydrolysis, especially when dealing with animal tissue. Nicotinic acid 

 may be extracted directly from the acidified hydrolyzed material using 

 organic solvents (see p. 460). The free acid may be separated from this 

 extract as such, or in the form of an ester, or as the copper salt. The free 

 acid can be obtained from the copper salt using hydrogen sulfide. Nicotinic 

 acid can be purified by recrystallization from water or alcohol or by subli- 

 mation. 



Nye et al}° isolated nicotinic acid from Neurospora mycelium in the fol- 

 lowing fashion. The mycelium was extracted with acetone. The acetone 

 extract was dried, redissolved in water, filtered, and treated with Ba(0H)2 

 and heat. After neutralization with H2SO4 filtration and further adjustment 

 of the pH to 4.2 with HCl, the nicotinic acid was adsorbed on charcoal 

 (Norit A). The active material was eluted with hot 4% aniline in water, 

 the aniline removed with ether, and the watery eluate taken to dryness. 

 The dry residue was dissolved in hot absolute alcohol, the solution filtered, 

 again taken to dryness, and dissolved in water. Nicotinic acid was crystal- 

 lized from the concentrated aqueous solution and was recrystallized from a 

 1:4 mixture of acetic acid and benzene. In a later publication^^ this proce- 

 dure was modified. A paper strip chromatogram developed with n-butanol 

 saturated with 0.2 A'' ammonium hydroxide was used to separate nicotinic 

 acid (labeled with C") from unwanted materials. Nicotinic acid could be 

 eluted from the appropriate part of the paper strip and crystalhzed with 

 added "carrier" nicotinic acid. The very small amount of nicotinic acid 

 (300 to 900 7) which was present in the extracts studied by these workers 

 made special techniques necessary. Yanofsky and Bonner^- also have de- 

 scribed procedures for isolating small quantities of nicotinic acid using 

 paper strip chromatograms. 



2. Isolation of Nicotinamide 



This substance can be isolated usually by water extraction, followed by 

 partial hydrolysis with 0.1 N sulfuric acid to liberate free nicotinamide. 



20 J. F. Nye, H. K. Mitchell, E. Leifer, and W. H. Langham, J. Biol. Chem. 179, 783 

 (1949). 



21 E. Leifer, W. H. Langham, J. F. Nye, andH. K. Mitchell, J. Biol. Chem. 184, 589 

 (1950). 



22 C. Yanofsky and D. M. Bonner, J. Biol. Chem. 190, 211 (1951). 



