The mentioned immobilized PH gradients isoelectrofocusing is a electrophoresis technique developed in 80' s. An approximate linear pH gradient is genarated by titrating weak acidic and basic acrviamide derivaties which then covalent bound into the polyacrylamide matrix. The pH gradient is stable and independent of electric field fluctuation. The present method provides higher resolution and larger loading capacity comparing with conventional carrier ampholyte isoeletrofocusing. It can analyse and purify protein with only minor pI difference.