VII. ESTIMATION 247 



probably due to the lad tliat \\\o color reaction is carrictl out at a pll of 

 7.0 to 7.3 iiustoad ot at the hi.i;hor pll of 10 to II, which is rc(iuii('(l lor tlic 

 coupling reaction witli iliazotized sult'aiiilic acid. Solutions containing 0.5 

 to 2.0 7 of pyridoxine per milliliter can 1)(> measured by this procedure. 

 The use of tliazotized ^^-aminoacetophenone instead of sulfanilic acid in 

 the color development permits a simpler and more efficient purification 

 process. A synthetic resin, Amberlite No. IR-4, is used instead of phospho- 

 tungstic acid in the purification process prior to the color development. 

 This resin adsorbs soluble interfering sul)stances, thus preventing their 

 simultaneous adsorption on Superfiltrol with pyridoxine; the vitamin is 

 eluted from the Superfiltrol with alkaline ethyl alcohol. 



7. DiAZOTIZED p-NlTROANILINE TeST 



Swaminathan^' used this phenol reagent to assaj^ for pyridoxine in food- 

 stuffs, but it has not been used by many other investigators. Nassi^^ modi- 

 fied the method somewhat by introducing a blank determination. He ob- 

 served that pjaido.xine, irradiated with ultraviolet light, lost the capacity 

 to produce an orange color with diazotized p-nitroaniline. He suggested, 

 therefore, that colorimetric tests be conducted on non-irradiated and ir- 

 radiated aliquots of the sample; the difference between the two values 

 would more closely approximate the true pyridoxine content of the sample. 

 However, considerable caution in interpretation must be exercised in view 

 of the presence of pyridoxal and pyridoxamine in natural materials. 



8. Indophenol Test 



Gibbs-" observed manj- years ago that phenols which had unsubstituted 

 para positions formed, under the proper conditions, blue indophenol salts. 

 Pyridoxine gives a positive Gibbs test when reacted with 2,6-dichloro(iui- 

 nonechlorimide.^ Scudi and his coworkers-''-^ developed an analytical pro- 

 cedure for the estimation of pyridoxine based upon this indophenol reaction, 

 which has become the most widely used and accepted of all colorimetric 

 tests. As little as 0.5 7 pyridoxine in 1 ml. of solution can be analyzed by 

 this method. However, even this chlorimide reagent, which is more specific 

 than other reagents, reacts with compounds not possessing Bo activity. 

 Most of the chemical procedures that have utilized this indophenol reac- 



'9 L. Xassi, Boll. soc. Hal. biol. sper. 16, 690 (1941). 



2" H. D. Gibbs, /. Biol. Chem. 72, 649 (1927). 



" J. V. Scudi, H. F. Koones, and J. C. Keresztesty, Proc. Soc. Exptl. Biol. Med. 43, 



118 (1940). 

 " J. V. Scudi, K. Uuna, and W. Antopol, J. Biol. Chem. 135, 371 (1940). 

 " J. V. Scudi, W. A. Bastedo, and T. J. Webb, J. Biol. Chem. 136, 399 (1940). 

 2^ J. V. Scudi, J. Biol. Chem. 139, 707 (1941). 



