Buffering Capacity of the Carrier Ampholytes

Everywhere in the pH-gradient there must be a sufficient buffering capacity to prevent the protein components of the sample specimen from affecting the pH. In other words, each carrier ampholyte must have sufficient buffering capacity at its isoelectric point. 

If the charge Q is plotted against pH, the result is identical with the titration curve of the ampholyte. The buffering capacity can thus be expressed  

The buffering capacity seems also to be a function of pi — pKf. Its numerical value is largest for small differences between pK). and the isoelectric point. As the difference between pl and pK). increases, the buffering capacity diminishes, linearly at first, and then exponentially. 

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Figure 5-4 Schematic of an lEF procedure, i, A homogeneous mixture of carrier ampholytes, pH range 3 to 10, to which proteins A, B, and C with pi 8, 6, and 4, respectively, were added. 91, Current is applied and the carrier ampholytes rapidly migrate to the pH zones where the net charge is zero (the pi value). Ill, The proteins A, B, and C migrate more slowly to their respective pi zones where migration ceases.The high buffering capacity of the carrier ampholyte creates stable pH zones in which each protein may reach its pi.

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