Concentration gradient in a chamber appended to a microchannel is important to cell-movement control and to the concentration-gradient based assays on Lab-on-a-Chip devises. In this paper, the effects on the concentration field of the asymmetrical injection, the Peclet number, the mobility ratio of electrophoresis to electroosmosis, the chamber's downstream position, and the chamber's geometry parameters are investigated. The most sensitive parameter is the asymmetrical injection, which can increase the concentration gradient twice as large as that in the symmetrical injection. Additionally, use of heterogeneous surface patches is a very effective way to enhance the concentration gradient generated in the chamber. Furthermore, the influence of a fixed cell-sized particle on the concentration gradient in the chamber and around the particle is examined as an example. The existence of the particle decreases the concentration difference between the front and the back of the particle. Finally, experimental visualization of the concentration fields was conducted, and good agreements were found between the numerical simulation results and the experimental results of the concentration fields generated in a micro-chamber with/without a particle and with/without a heterogeneous patch.