How pH gradient is generated?

It is technically difficult to generate a stable pH gradient by buffer system. A stable pH gradient is generated using Ampholytes. Ampholytes are synthetic, low molecular mass heteropolymer of oligoamino and oligocarboxylic acids (containing both acidic and basic groups). Using various combination of amino and carboxylic acid group, ampholytes with different isolectric points are synthesized. ampholytes have another property which makes them very useful. They have very high buffering capacity at their isolectric point. Mixture of ampholytes with different isoelectric point ranges are commercially available (pH 7-12; pH 4.0-7.0 etc). General structure of ampholytes are shown in Fig. 2.

Figure 2: General structure of ampholytes.

• -Ampholytes of a given range (for example pH 2.0-12.0) is mixed with arcylamide/bis acrylamide while being polymerized (no SDS). If this polymerization is done at pH 8.8 and electrophoresis is performed, what happens?
• Ampholyte molecules with maximum isolectric point (above pH 8.8) will have maximum positive charge and move fastest and reach closest towards negative electrode. The ampholyte with second maximum isolectric point will not move further because the space taken by ampholyte with higher isolectric point. As ampholytes have high buffering capacity at isolectric point a higher pH is maintained. As soon as it moves forward, it will experience a pH higher than its isoelectric point. Ampholytes will get negative charge and move back (Fig. 3).