Pump-free Negative Pressure Sampling Device For Rapid Sample Loading And Nonaqueous Electrophoresis-chemiluminescence On A Microchip

Microchip electrophoresis(MCE) has emerged as an important research branch of separation unit in the field of miniaturized tatal analysis systems (μTAS) and integrated with several detection techniques. Currently, the two main injection methods of MCE, bias electrokinetically pinched injection and non-biased pressure injection, both need complex energy system and external equipments, which can not meet the requirement of miniaturization. Chemiluminescence(CL) has been proven to be one of the most ideal detector in MCE and has the advantages including simple instrumental setup, low background noise, and low costs for operation and maintainance. Peroxyoxalate chemiluminescence(POCL) is currently accepted as the most efficient enzyme-free reaction, which must react in nonaqueous environment. Nonaqueous microchip electrophoresis(NAMCE) under organic solvent and in replacement of water is an effective technique of detecting hydrophobic fluorescent substance. The present work proposed a pump-free negative pressure sampling device for MCE and the negative pressure was produced by a pipet bulb. Meanwhile, research on NAMCE-POCL system was completed.In chapter 1, pressure injection methods for MCE in 2002-2009(including positive pressure injection and negative pressure injection), the mechanism of POCL and its integration with some analytical techniques(including flow injection analysis(FIA), high-performance liquid chromatography(HPLC), capillary electrophoresis(CE), MCE) are reviewed.In chapter 2, a pump-free, simple, and low-cost negative pressure sampling device for rapid bias-free sampling loading in MCE was developed. It composed of a pipet bulb, a 3-way electromagnetic valve and a single voltage supply at a constant voltage. Experimental results and the theoretical predications proved that the present device worked well in a wide vacuum degree ranged from -250 to -30 mbar with a satisfactory analytical precision and wide range of applications. Each injection, A pinched sample plug was formed at the channel intersection in less than 0.5 s and the sample consumption was calculated to be 51～12 nL. Combined with the on-chip integrated detector, it is anticipated that a portable analytical device would be developed in the near future. In chapter 3, NAMCE-POCL system was established and applied in separation of rhodamines. A simple and compact subatmospheric pressure fluid-driven device was developed for manipulating the whole analytical process, including sample loading, and POCL reagents transportation. A single voltage was supplied on buffer reservoir and buffer waste reservoir at a constant voltage without voltage switching operations. A porous polymer plug was created in the separation channel of the microchip as a select valve, which prevented the pressure-driven POCL reagents flowing back into the separation channel but allowed electrophoretic migration along the separation channel and realized variable-volume sample loading. By improving the reaction efficiency using a flow-type and spiral detection cell, not only greatly enhanced the sensitivity, but also improved the reproducibility since the waste produced by the POCL reaction would not accumulated in the detection. NAMCE using solvent reagent as running buffer media avoided the interface problem between water and organic phase because aqueous solution was used as running buffer media in previous reports while POCL reaction must react in organic environment. Optimized the POCL reaction condition of the model molecular rhodamine 6G(Rh6G) in NAMCE-POCL system. Under the optimized conditions, the detection limit of Rh6G was 2.1 nmol/L. The optimized condition was further applied in separation of rhodamine 123(Rh123) and Rh6G and determination of 1-aminopyrene. The detection limit of 1-aminopyrene was 35 nmol/L. The reproducibility of NAMCE-POCL was also evaluated, The RSDs for the migration time were 0.29～1.1%, the peak height 1.3～3.1%, and the peak area 2.5～6.9%. The results showed the proposed NAMCE-POCL system offered a number of benefits including good stability, good reproducibility, high sensitivity and wide range of applications.