The present invention provides a mobility electrophoresis separation device, its operating method, and an interface between liquid chromatography and mass spectrometry. The mobility electrophoresis separation device comprises a separation capillary, a syringe pump for injecting a buffer solution, a syringe for injecting a sample solution, and two electrodes disposed apart from each other on either side of the separation capillary. A sample solution is injected by a syringe at a position of the capillary channel, and a buffer solution is injected into the capillary channel upstream the first position, and carries the sample solution to flow downstream. While the mixed liquid flows through the capillary, an electric field is applied in the direction of the flow. Different ions in the sample are thus separated in the flow due to their different velocities traveling in the flow.
