4.4.5 Capillary Isoelectric Focusing (CIEF)

Capillary isoelectric focusing separates peptides and proteins based on their isoelectric point (pI)--the point at which they carry no net charge. A pH gradient is formed within the capillary using ampholytes—zwitterionic molecules with both an acidic and basic moiety.

The capillary is filled with a mixture of solutes and ampholytes, and a pH gradient is formed with a basic solution at the cathode and an acidic solution at the anode. When an electric field is applied, ampholytes and solutes migrate through the pH gradient until they reach the pH that corresponds to their isoelectric point, where they become uncharged. The solute zones will maintain a narrow breadth as any solute which migrates into the zone of a different pH becomes charged and migrates back into the pH of its isoelectric point. After this equilibrium occurs, a steady state is reached and hydraulic pressure is used to pass the zones through to the detector.

EOF must be minimized to prevent flushing ampholytes through the capillary.