CHEMICAL ESTIMATION 



20. K. C. Hamner, W. S. Stewart and G. Matrone, Food Res., 1943, 



8, 444. 



21. A. P. Meiklejohn, Biochem. J., 1943, 37, 340. 



22. H. P. Sarett and V. H. Cheldelin, /. Biol. Chem., 1944, 155, i53- 



23. P. M. West and P. W. Wilson, Science, 1938, 88, 334. 



24. L. Emerique-Blum and A. LwofE, Bull. Soc. Chim. bioL, 1940, 22, 179. 



25. C. F. Niven and K. L. Smiley, /. Biol. Chem.. 1943, 150, i. 



26. W. B. Emery, N. McLeod and F. A. Robinson, Biochem. J., 1946, 



40, 426. 



27. E. E. Fitzgerald and E. B. Hughes, Analyst, 1949, 74, 340. 



28. A. L. Bacharach and W. F. J. Cuthbertson, ibid., 1948, 73, 334. 



29. A. Jones and S. Morris, ibid., 1949, 74, 333. 



9. CHEMICAL ESTIMATION OF ANEURINE 

 Azo Method 



The first chemical test proposed for vitamin B^ was the so-called 

 formaldehyde-azo test of H. W. Kinnersley and R. A. Peters,^ who 

 found that vitamin B^ concentrates gave a pink colour with diazo- 

 tised sulphanilic acid and formaldehyde solution and that the colour 

 increased slowly in intensity for thirty to sixty minutes, thereafter 

 remaining constant for a considerable time. The method was later 

 modified by them ^ to make it quantitative ; the error was stated to 

 be not greater than 5 %. H. J. Prebluda and E. V. McCollum ^ and 

 D. Melnick and H. Field * used diazotised ^-aminoacetophenone (with- 

 out formaldehyde) for estimating aneurine. The latter authors ex- 

 tracted the coloured product with xylene to make the test more 

 specific, and thereby reduced the error to 2 %. By hydrolysing 

 cocarboxylase preparations with yeast phosphatase the test was 

 made applicable to the estimation of the coenz3mie. In a later paper, 

 Melnick and Field ^ introduced adsorption on Permutit, followed by 

 elution with potassium chloride solution, to effect purification. 



The azo method is now one of the standard methods of estimating 

 aneurine, and has been modified by various workers to eliminate 

 interference from other compounds present in the material to be 

 assayed (cf. Emmett et al.^). Diazotised ;/)-aminoacetophenone was 

 used by L. J. Harris and W. D. Raymond,^ by E. F. Yang and B. S. 

 Piatt, 8 and by Y. Sakurai et al.^ The last-named group of workers 

 purified the sample by adsorption of the aneurine on " acid clay " and 

 elution of the adsorbate with alcoholic phenol. H. Willstaedt ^^ used 

 diazotised 2 : 4-dichloroaniline, which gives a yellowish-red colour, as 

 the reagent. E. R. Kirch and O. Bergeim ^^ used _^-carbethoxybenzene 

 trichloroacetate and extracted the colour with isoamyl alcohol ; both 

 vitamin A and vitamin C interfere, however, and must be removed, 

 the former by extraction with isoamyl alcohol before addition of the 



37 



