ISOELECTRIC POINT OF PROTEIN SOLUTIONS 39 



water. The powdered gelatin was then put into a cylindrical 

 funnel and the acid allowed to drain off. The powdered gelatin 

 in the funnel was then perfused six or eight times, with constant 

 stirring, each time with 25 c.c. cold water i.e., water not above 

 5C. to remove the excess of acid and the salts. The water 

 must be cold to prevent the powdered granules from coalescing 

 since otherwise the washing would be incomplete. After the 

 liquid was drained off from the filter, the volume (i.e., the rela- 

 tive swelling of the gelatin) was measured; then the gelatin was 

 melted by heating to 45C. and enough water was added to bring 

 the volume in each case to 100 c.c. Then the conductivity, 

 osmotic pressure, and viscosity were measured in a way to be 

 described in a later chapter, and the pH was also determined, 

 either colorimetrically (which gives fairly accurate results with 

 gelatin but not with the other proteins) or preferably with the 

 hydrogen electrode. In the experiment represented in Fig. 3 the 

 pH was measured colorimetrically. A glance at the figure shows 

 that the ordinates of the curves representing the values for 

 osmotic pressure, conductivity, swelling, etc., drop very sharply 

 at pH 4.7, i.e., the isoelectric point of gelatin. By this method 

 the approximate location of the isoelectric point can be recognized 

 at a glance from the osmotic pressure measurements, the conduc- 

 tivity measurements, etc. The P.D. measurements would also 

 show a minimum at the isoelectric point. 



The lowest curve in Fig. 3 represents titration for Br. Gelatin 

 should exist in the form of gelatin bromide only on the acid side of 

 the isoelectric point and titration for Br should be negative 

 when the pH is above 4.7. The curve shows that no Br was 

 found when pH was equal or greater than 4.7; while it was found 

 on the acid side increasing in quantity the lower the pH. On the 

 alkaline side of the isoelectric point the gelatin existed still in 

 the state of Ca gelatinate. In this experiment the mass of the 

 gelatin was diminished by solution and washing to 0.8 gm. or 

 possibly a little less. 



We shall see later, that when powdered gelatin is put into an 

 acid solution, e.g., N/100 or N/1,000 HBr, the concentration of 

 the acid inside the gelatin granules is considerably lower than in 

 the outside solution. This is due to the establishment of a 

 Donnan equilibrium. 



