CHEMICAL ESTIMATION 



the fluorescence in the aqueous layer to estimate the cocarboxylase. 

 This method obviously suffers from severe limitations, and is only 

 applicable when the aqueous phase is free from coloured impurities 

 and other fluorescent substances. 



A simplified base-exchange method, not involving adsorption 

 columns, was used by E. Papageorge and M. V. Lamar *^ for the 

 estimation of aneurine in urine by three different thiochrome methods ; 

 agreement was generally good. The use of benzene sulphonyl chloride 

 to destroy aneurine is recommended for the estimation of non-thio- 

 chrome fluorescent impurities. 



A different principle was used for the estimation of aneurine by 

 H. Wachsmuth.*^ On adding potassiimi mercuric iodide solution to 

 a weakly acid solution of aneurine a crystalline precipitate of aneurine 

 iodomercurate was formed. The iodine in this precipitate or in the 

 excess of the reagent was titrated after oxidation to iodate by treat- 

 ment with bromine. 



Physico-chemical Method 



Although aneurine exhibits characteristic absorption bands in the 

 ultra-violet region of the spectrum, these can only be used for esti- 

 mating the vitamin when relatively pure solutions are available. The 

 only physical method of estimating aneurine that appears to be 

 promising is the polarographic method, although it does not seem to 

 have been generally adopted. J. J. Lingane and O. L. Davis ^^ dis- 

 covered that, with potassium chloride as base solution, aneurine gave 

 a step with a half -wave potential of — 1-25 volts vs. the saturated 

 calomel electrode, and that riboflavine and nicotinic acid gave steps 

 at other voltages, so that it was possible to estimate all three vitamins 

 in one and the same solution at the same time. 



When electrolysed in a very dilute solution containing ammonium 

 chloride, boric acid and potassium chloride or in a phosphate buffer 

 solution, aneurine gave a current-voltage curve with a prominent 

 maximum at 17 volts t;s."the saturated calomel electrode ; ^* this is 

 believed to arise from a catalytic effect of the aneurine. 



References to Section 9 



1. H. W. Kinnersley and R. A. Peters, Biochem. J., 1934. 28, 867. 



2. H. W. Kinnersley and R. A. Peters, ibid., 1938, 32, 15 16. 



3. H. J. Prebluda and E. V. McCollum, Science, 1936, 84, 488 ; /. 



Biol. Chem., 1939, 127, 495. 



4. D. Melnick and H. Field, /. Biol. Chem., 1939, 127, 505, 531. 



5. D. Melnick and H. Field, ibid., 1939, 130, 97- 



6. A. D. Emmett, G. Peacock and R. A. Brown, ibid., 1940, 135, 131. 



7. L. J. Harris and W. D. Raymond, Biochem. J., 1939. 33, 2037. 



41 



