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Isoelectric focusing (IEF) separation has been regarded as a high-resolution separation technique for amphoteric analytes, such as proteins and peptides.To achieve the high throughput analysis, recently the minaturization of IEF on microchip has been paid more and more attention.In our recent work, an IEF microchip was fabricated on glass with the channel width of 100 μm and depth 40 μm.The EOF was eliminated by permanently coating the channel wall with polyacrylamide, and the focused analytes was driven to the detector by changing the catholyte with buffer.