HYDROGEN ION CONCENTRATION AND PROTEOLYSIS. 153 



Schiff studied further to apply tliis reaction to the quantitative deter- 

 mination of amino acids, polypeptides and other similar compounds. 

 His attempt was not wholly successful because he neglected to consider 

 aU factors involved in the process. For instance, the amount of potassium 

 hydroxide, water and other substances which are used in the titration 

 should be considered in order to obtain accurate results. This was shown 

 by Sorensen, who pointed out the reversibility of such a reaction as is 

 indicated below : — 



CHa CH3 



I I 



CH.NH2+HCHO -> CHN.CH2+H2O 



I I 



COOH +KOH <- COOK +H2O 



He also found that the equilibrium of the system depends upon the 

 quantity of each of the chemicals present. For example, from the above 

 reaction it can be clearly seen that an increase of potassium hydroxide, 

 or, in other words, of hydroxyl ions, in the system would result in an action 

 similar to an increase of the formaldehyde. In order to control these 

 sources of error, Sorensen chose an indicator which turns with such a 

 high concentration of hydroxyl ions that the process could be finished 

 from left to right. He used phenolphthalein and titrated to a strong red 

 color v,it\i n/5 KOH. Thus he studied the method very carefully, and 

 succeeded in conducting it with such precision that he had only 0-5 per 

 cent, error when he observed certain conditions which will be enumerated 

 in an example to be cited soon. 



Further, Sorensen extended the formol titration method to proteolysis, 

 and found that it can be depended upon for accurate quantitative deter- 

 minations. He based his principle upon the fact that proteolysis is mainly 

 the hydrolytic splitting of protein, and takes place by the addition of a 

 water molecule. It is a differential method as illustrated below: — 



X— Y =Z, where X is amount of amino acids present after proteolysis takes place; 

 Y, the same, before; Z the same produced by the proteolysis. 



In order to carry out the determination, all other acids in the liquid 

 should be eliminated; that is, the liquid must contain as many H ions as 

 OH ions at the beginning. If all these precautions are observed the amount 

 of alkali used in the titration after the addition of neutral formaldehyde 

 should be equal to the amount of the amino acid group present. After 

 finding X and Y, Z, or the index of degree of proteolysis, can be determined. 



Example. 

 Preparation of Solutions and Reagents, 

 (a) 125 c. c. 4 per cent. Witte's peptone solution ] 



75 c. c. 0.2 per cent, pancreatin solution / total volume, 250 c. c. 

 50 c. c. distilled water J 



(6) Standard acid solution: n/5 HCl is prepared by standardizing against sodium 



oxalate which is obtained from the Bureau of Standards. 

 (c) Standard alkali solution: n/5 KOH is prepared by titrating against the above 

 standard acid. 



