70 



HYDROGEN ION CONCENTRATION 



point was calculated at pH 6.09 and was found by the above de- 

 scribed method to be between pH 6.3 and 5,8, 



2, With difficultly soluble ampholytes the following method^" 

 may be used which is based upon the principle that the undissociated 

 portion of an ampholyte is less soluble than its ionized portion. 

 An equal ample amount of an ampholyte is added to each of a series 

 of hot acetate buffer solutions and the appearance of crystalliza- 

 tion is observed, A suitable ampholyte for this purpose is m- 

 aminobenzoic acid. In the following table the original pH of the 



TABLE 13 



Phenylalanine as base. 



3.75 

 4.10 

 4.27 



Isoelectric point calculated at pH 



4.58 



pH before 



addition of 



phenylalanine 



pH after 



addition of 



phenylalanine 



Phenylalanine as acid , 



4.66 

 5.07 

 5.26 

 5.45 

 5.73 



4.01 

 4.22 

 4.43 



4.48 

 4.57 



4.55 

 4.78 

 4.74 



4.72 



4.77 



Difference in 

 pH 



+0.28 

 +0.12 

 +0.16 



-0.01 



-0.11 

 -0.29 

 -0.52 

 -0.73 

 -0.96 



buffer solutions is given in the upper and the observed extent of 

 crystallization in the lower line: 



pH 5.4 5.1 4.8 4.5 4.2 3.9 3.6 3.3 3.1 



Crystallization: 0+ + + + + + + + + + + 000 



It appears then that the crystallization maximum is in the close 

 vicinity of pH 4.2, while the isoelectric point, as calculated from the 

 dissociation constants found in the literature, is at pH 4.1. The 

 crystallization optimum of p-aminobenzoic acid may be determined 

 with equal definiteness. The relative sharpness of definition of 

 such optima depends upon the value of the product ka X kb, just 

 as the sharpness of the maximum of the dissociation-residue curve 



" L. Michaelis and H. Davidsohn, Biochem. Zeitschr. 30, 140 (1910), 



