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 to that in the symmetrical injection. Furthermore, using heterogeneous surface patches is a very effective way to enhance the concentration gradient generated in the chamber. Different patches for certain chambers are investigated. 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 heterogeneous patch.