Titration of Bacteriological Media 



31 



should end at a hydrogen ion concentration within the zone of 

 neutralization of the methylene derivative. To determine the 

 location of these zones we have plotted the titration curves of a 

 number of representative amino acids and ammonium salts be- 

 fore and after the addition of formalin 7 . The following pure 

 substances were titrated : glycine, alanine, phenylalanine, tryosine, 

 asparagine, aspartic acid, glutamic acid, ammonium chloride, 

 ammonium lactate, ammonium phosphate, ammonium carbonate. 

 Excluding ammonium phosphate and ammonium carbonate which 

 present a special problem it is found that the isoelectric zone of 

 each of these substances extends beyond P H 6.0 and as far as 

 P H 7.0, some as far as P H 8.0 8 . After the addition of formalin 

 the zone of neutralization for each substance was found to begin 

 on the acid side at about the hydrogen ion concentration here 

 listed; glycine P H 6.8, alanine P H 8.0, phenylalanine P H 7.6, 

 tryosine P H 7,6, asparagine P H 6.0, aspartic acid P H 8.0, glutamic 

 acid P H 8.0, ammonium chloride P H 6.0, ammonium lactate P H 

 6.0, ammonium phosphate P H 8.0, ammonium carbonate P H 7.0. 

 For the formol titration of most amino acids or of mixtures of 

 amino acids, therefore, their solutions should be reduced to a hy- 

 drogen ion concentration of not less than P H 7.0 and after the ad- 

 dition of formalin the titration should end at a hydrogen ion con- 

 centration not greater than P H 8.3 9 : Theoretically P H 8.4 is a bet- 

 ter end point than P H 8.0 but we have found the buffer effect of 

 the formalin so great at P H 8.4 that the end point of the titration 

 judged colorimetrically is very poor, and have obtained more 

 accurate results at P H 8.0. 



Sorensen recognized phosphates and carbonates as disturbing 

 elements in the formol titration of urine. In fact any substance 

 which exerts a buffer effect between P H 7.0 and P H 8.0 is a serious 

 source of error. To overcome this difficulty Henriques 10 (1909) 

 and Henriques and Sorensen (1909) described methods for the 



7 A correction was made for the buffer effect of the formalin itself. 



8 Of the substances titrated, tyrosine showed the greatest degree of disso- 

 ciation at P H 7.0 and was about 3.8 per cent, dissociated at this hydrogen ion 

 concentration. Glycine, leucine and alanine are less than 2 per cent, disso- 

 ciated at P R 8.0. 



9 Henriques and Sorensen (1909, 1910) recommend titration from P R 6.8 

 to about P H 8.4 or beyond. Northrop (1921) started his titration at P R 7.0. 

 io Henriques, V., Z. f. physiol. Chem., 1909, lx. 1. 



