Negative pressure pinched sample injection for microchip-based electrophoresis.

A simple method for injecting well-defined non-biased sample plugs into the separation channel of a microfluidic chip-based capillary electrophoresis system was developed by a combination of flows generated by negative pressure, electrokinetic and hydrostatic forces. This was achieved by using only a single syringe pump and a single voltage supply at constant voltage. In the loading step, a partial vacuum in the headspace of a sealed sample waste reservoir was produced using a syringe pump equipped with a 3-way valve. Almost instantaneously, sample was drawn from the sample reservoir across the injection intersection to the sample waste reservoir by negative pressure. Simultaneously, buffer flow from the remaining two buffer reservoirs pinched the sample flow to form a well-defined sample plug at the channel intersection. In the subsequent separation stage, the vacuum in headspace of the sample waste reservoir was released to terminate all flows generated by negative pressure, and the sample plug at the channel intersection was electrokinetically injected into the separation channel under the potential applied along the separation channel. The liquid levels of the four reservoirs were optimized to prevent sample leakage during the separation stage. The approach considerably simplified the operations and equipment for pinched injection in chip-based CE, and improved the throughput. Migration time precisions of 3.3 and 1.5% RSD for rhodamine123 (Rh123) and fluorescein sodium (Flu) in the separation of a mixture of Flu and Rh123 were obtained for 56 consecutive determinations with peak height precisions of 6.2% and 4.4% RSD for Rh123 and Flu, respectively.