Studies On Negative Pressure Sampling And Sample Preconcentration Techniques In Microchip Sieving Electrophoresis

Microchip electrophoresis (MCE) has been advanced remarkably by the applications of several separation modes and the integration with several chemical operations on a single planer substrate. Currently, negative pressure injection method has been widely used in microchip capillary electrophoresis for its rapid and bias-free sample introduction. The present work first proposed the negative pressure sampling method for chip-based sieving system analysis and its application in on-line sample preconcentration.In Chapter 1, pressure sample injection method and online sample preconcetnration techniques in microchip are reviewed, including positive and negative pressure sample injection method and focusing, electrophoresis, and static online sample preconcentration method.In Chapter 2, a rapid and variable-volume sample loading scheme for chip-based sieving electrophoresis was developed by negative pressure combined with electrokinetic force. This was achieved by using a low-cost microvacuum pump and a single potential supply at a constant voltage. To reduce the hydrodynamic resistance in the sampling channel, sieving material was only filled in the separation channel between the buffer waste reservoir (BW) to the edge of the crossed intersection. The injected sample plug length is in proportion with the loading time. It is easy to freely choose the sample plug volume in this method by simply changing the loading time. The sample consumption was about 1.7 X 102 nL at a loading time of 1 s for each cycle. Only 0.024 s was required to transport bias-free analyte to the injection point, and can inject high quality sample plug with non-distorted shape into the separation channel. The system has been proved to possess an exciting potential for improving throughput, repeatability, sensitivity and separation performance of chip-based sieving electrophoresis.In Chapter 3, we present a novel isotachophoresis - gel electrophoresis (ITP-GE) microchip system designed for rapid and efficient isotachophoretic preconcentration coupled with gel electrophoresis separation by using a negative pressure sampling technique. The overall ITP - GE procedure involves only three steps:sample loading, ITP preconcentration and GE separation. A sandwiched sample zone between a leading and a terminating electrolyte zonewas formed in the channel intersection in less than 1 s. The performance of present approach was evaluated by using DNA fragments as model analytes. Compared to conventional cross microchip GE using electrokinetic pinched injection, an average signal enhancement of 185-fold was obtained with satisfactory resolution. The results demonstrated the ITP - GE approach possessing an exciting potential of high sensitivity and short sampling time with significant simplification in operation and instrumentation.In chapter 4, we applied the variable-volume negative pressure sample loading scheme to field amplified sample injection-isotachophoresis-gel separation (FASI-ITP-GE) of proteins. A sandwiched sample zone between a leading and a terminating electrolyte zone was formed in injection procedure, and the injected sample plug length is in proportion with the loading time. Futhermore, FASI can achieved in sample injection procedure. Stacked sample plug was introduced into the ITP channel for ITP stacking, saving the effective ITP channel length. Compared FASI-ITP-GE to conventional cross microchip GE using electrokinetic pinched injection, exceeding 1000-fold signal enhancement of protein was obtained with satisfactory resolution.