A kind of PH gradient microfluidic chips through soft-lithography microfabrication for isoelectric focusing (IEF) and high performance liquid chromatography (HPLC) is introduced here. These pH gradient chips have the advantages such as easy fabrication, controllable pH range and precision, isoelectric focusing and separation at the same time, low voltage for isoelectric focusing, and time stable pH gradient. This method has potential application to sample preparation, separation and analysis of microfluidic chips.