Automation of electrophoretic microchips for sequential analysis of different samples is demonstrated. This system used an autosampler, which was on-line connected to the microchip and the whole process including sample loading and injection, analysis and data acquisition as well as washing were all automated. Rhodamin B at different concentrations was first loaded into a hydrodynamic flow stream by an autosampler, delivered to the microchip, and then sequentially injected into the electrophoretic microchannel for analysis and detection. Automation was achieved by running two independent programs, one for sample loading by an autosampler and the other one for electrophoretic injection by voltage switching, on the same computer. Using this sampling chip, each loaded volume (0.2-1 microL) can be injected for dozens of electrophoretic analyses (1-10 nL for each injection). The variances caused by the external connections, which did not affect the electrophoretic analysis but would cause band broadening of the loaded sample in the hydrodynamic flow stream, were theoretically deduced. Results indicate that the dead volume (approximately 300 nL) due to the connection fitting on the chip could lead to dilution of the loaded sample by a factor of one when 0.2 microL of sample was loaded. Such a design allows sequential analysis of a series of samples while the running buffer is continuously pumped into the connection capillary as well as microchannels for washing between two loaded samples to minimize cross contamination without human intervention. Using this sampling chip, the required sample amount and handling time can be greatly reduced compared to the manual method.