In two-dimensional microchip electrophoresis (2D-MCE) which is performed by the on-line connection of different separation modes on a single microchip, there are great advantages such as high resolution and low consumption of samples.However, there remains some drawbacks, including the low concentration sensitivity and requirement of troublesome procedure to fill a second matrix only in a specific region of channels.To overcome these drawbacks, a novel technique for online coupling of sample preconcentration by large-volume sample stacking with an electroosmotic flow pump (LVSEP) with 2D-MCE was developed for a highly sensitive and high peak-capacity analysis of biomolecules.In experimental, a 1 ×TAE buffer (pH 8.3) and 2% (w/v) hydroxylpropyl methyl cellulose (HPMC) were used as a background solution (BGS) and sieving matrix, respectively.By using the difference in the backpressure due to the channel configuration (Fig.1), a low-ionic strength (Ⅰ) sample and polymer solutions were easily introduced into the first-and second-dimension channels (Fig.2a), respectively.