[HARDING] THE "NINHYDRIN" REACTION 35 



of amino acid a-nitrogen in minute amounts, which will estimate 

 from 0-05 mgs. to 0-005 mgs. of a-nitrogen in amino acids with an 

 accuracy of about one per cent. Herzfeld (6) also devised a method of 

 estimating amino acids by a quantitative application of the "ninhydrin" 

 reaction ; but Harding and MacLean found the results untrustworthy 

 and a criticism of this has appeared in their paper. The colorimetric 

 method of Harding and MacLean uses as its standard the blue color- 

 ation developed by heating 1 c.c. of alanine solution containing • 05 

 mgs. of a-nitrogen with 1 c.c. of one per cent "ninhydrin" and 1 

 c.c. of ten per cent pyridine solution in a rapidly boiling water-bath 

 for twenty minutes. This amount of color and its method of prepara- 

 tion has been used as a standard in the general investigation into the 

 "ninhydrin" reaction which has been in progress in this laboratory 

 during the past eighteen months and the results of which are announced 

 in a preliminary way in this communication. It will be evident that 

 the ability to work in a quantitative manner has enabled us to draw 

 conclusions which otherwise would have been impossible. 



THE CHARACTERISATION OF THE AMMONIUM SALT OF DIKETOHYDRIN- 

 DYLIDENE-DIKETOHYDRINDAMINE IN SOLUTION. 



From the brief account of the position of the "ninhydrin" reaction 

 which has just been given, it is evident that some method of definitely 

 characterising the blue coloring matter produced by amino acids and 

 triketohydrindene hydrate is necessary, when only small amounts 

 are present in solution. This we have been able to do in three ways. 



(1) The coloring matter gives a broad absorption band in the 

 visible spectrum when viewed in dilute solution. This band extends 

 from the red into the green part of the spectrum, blocking out almost 

 entirely the whole of the yellow. ' 



(2) The blue color changes to a purple when viewed in artificial 

 light, resembling very much the color of dilute potassium perman- 

 ganate when viewed in daylight. 



(3) The color is resistant to mild oxidation. Thus the passage 

 of a rapid current of air for five minutes through the standard color 

 has no effect on the quantity present. 



We have also prepared the ammonium salt diketohydrindylidene- 

 diketohydrindamine from hydrindantin by the action of ammonium 

 carbonate and ammonium acetate according to the directions of Ruhe- 

 mann and have been unable to detect any difference between it and 

 the coloration given by amino acids with triketohydrindene hydrate. 

 This further evidence only emphasises Ruhemann's view of the reac- 



