Principle of Carrier Ampholyte Generated pH Gradients

Carrier ampholytes are mixtures of amphoteric buffers and possess the following properties:

• high buffering capacity and solubility at the pI,
• good and regular conductivity at the pI,
• absence of biological effects,
• low molecular weight.

The pH gradient is produced by the electric field. For example, in a focusing gel with the usual concentration of 2 to 2.5% (w/v) carrier ampholyte (e.g. for gradients from pH 3 to 10) the gel has a uniform average pH value. Almost all the carrier ampholytes are charged: those with the higher pI positively, those with the lower pI negatively.

When an electric field is applied, the negatively charged carrier ampholytes migrate towards the anode, the positively charged ones to the cathode and their velocity depends on the magnitude of their net charge.

The anodal end of the gel becomes more acidic and the cathodal side more basic.

The carrier ampholyte molecules with the lowest pI migrate towards the anode and those with the highest pI towards the cathode. The other carrier ampholytes align themselves in between according to their pI and will determine the pH of their environment. A stable, gradually increasing pH gradient from pH 3 to 10 results.

These pH gradients are employed for Isoelectric Focusing and Titration Curve Analysis.