Study Of The Thermal Expansion Pump For Capillary HPLC And New Methodologies For Protein Micro-separation

Miniaturization is a trend of modern science and technology, especially in analytical chemistry. Separation and analysis in micro-scale has been an important task for the development of modern chromatography. In the miniaturized system, the sample consumption and analysis costs can be greatly reduced, high detection sensitivity and fast analysis speed can be obtained, environmental pollution can be minimized, and miniaturized analytical instruments would be possible. In recent years, capillary HPLC, capillary electrophoresis and micro-fluidic chip have attracted extensive research interest and developed rapidly in instruments and methods. It has been recognized that micro-flow pump is a key component of the miniaturized system, micro-fluidic driving and controlling is its basic technology, and protein separation and analysis in micro-scale would lead to a better understanding of life. A pump that can produce high pressure and constant flow at micro-flow rate range is always desired for capillary HPLC and miniaturization of HPLC. The separation and analysis of trace, low concentration and complex protein is concerned by experts. In this study, we focused on developing a novel thermal expansion pump that can produce continuous flow for capillary HPLC, inventing a solid-support florescent derivatization method for protein in low concentration and establishing a new 2D-nano-HPLC separation method employing a biphasic column for trace protein analysis. The research work in this thesis is divided into five chapters.In Chapter 1, micro-flow pumps and technologies of micro-fluidic driving were summarized, requirements and current technologies of micro-flow pumps for capillary HPLC were included, separation methods and corresponding detection techniques commonly used for protein separation and analysis in micro-scale were introduced. The research background was demonstrated.In Chapter 2, the basic research of thermal expansion pump (TEP, for short) was done. The TEP has almost no moving part and no mechanical consumptive hardware to change frequently. It is running in silence. Its overall costs of manufacture and routine running are very low. It is also easy to fabricate and assembly without complicated technologies. TEP utilizes the volume expansion of liquid in a heating chamber for fluid delivery, which is mainly depends on temperature elevation. The mechanism of TEP was investigated and described more clearly. Three main factors to influence the accuracy of fluidic output, including volume thermal expansion, liquid compression, and liquid mass loss in the heating chamber during output, were analyzed in detail and a(T, P)was discussed for special. Theoretical equation for controlling fluidic output of this pump by accurate temperature control had been established and pump initial temperature was taken into account for practical application. According to the theory, water was selected as the liquid medium used for TEP and chamber volume of TEP was estimated to match capillary HPLC. Copper and stainless steel were chosen as the pump material. The heating control system was set up based on PID algorithm and relevant software was completed. The prototype was fabricated and optimized in heating control and pump structure. The novel pump is capable of generating a stable and continuous flow at high pressure (above 10MPa) from nano-liters to micro-liters per minute without splitting and demonstrated by a series of experiments. It is expected to apply in the UPLC system. We also explored its miniature potential.In Chapter 3, we developed, for the first time, a TEPs system that is capable of generating continuous flow for capillary HPLC, and applied it in nano-HPLC system coupled with laser induced fluorescence detector (LIF). The TEPs system employing two groups of thermal expansion pumps (TEPs) working by turns were fabricated, and a controlling strategy for the pump system to maintain a continuously delivery without pressure fluctuation even at switching point was also developed. Both isocratic and gradients of binary solvent delivery by the TEPs were performed. Reproducibility and standard deviation at different flow rates were determined. A novel nano-HPLC system employing the TEPs system was set up for the first time. A result of RSD=4% for flow and RSD=2% for retention times at 500 nL/min was achieved. We have successfully established a nano-HPLC-LIF system, consisting of the TEPs system as the main innovation.In Chapter 4, a method based on solid-support reaction was initially developed to realize fluorescent derivatization of picomoles of protein at concentrations as low as 10-8 M, and the method has been successfully applied to real samples. A simple, low-cost homemade capillary C18 cartrideges was fabricated as the solid-support reactor with Sol-gel and slurry packing methods. FITC was selected as fluorescent reagent and effects of reaction conditions on solid-support has been evaluated and optimized. Fluorescent derivatiozation of protein in 10-8 M with FITC on solid-support were realized for the first time. Compared with solution derivatization, lower detection limit of protein can be obtained by this method. And the use of the capillary solid-support reactor allows easy handling of protein with small volume. We also developed a method based on capillary-size exclusion chromatography (cSEC) for fluorescent labeled protein purification withμL volume, effectively reducing the interference of fluorescent intruders in analysis. In Chapter 5, a novel nano-HPLC system for two-dimensional separation was first reported, which coupling weak anion exchange chromatography (WAX) and reverse phase chromatography (RPLC) by employing a biphasic column. Combining with high sensitive laser-induced fluorescence (LIF) detection method, the system was applied for trace complex protein analysis. We packed WAX resin and RP C18 particles in a fused silica capillary column (100μm i.d.) in sequence. There are many advantage by using this biphasic column:1. the flow rate was decreased, thus the sensitivity of system was increased; 2. WAX separated by charge and RP separated by hydrophobicity, the two dimensions of chromatography were orthogonal, which improved peak capacity of system effectively; 3. the unique biphasic column effectively eliminated the valves and dead volume commonly associated with complex chromatography. In our initial research, we described the structure and separation process of this system, optimized and demonstrated its detection sensitive and separation capability roughly for trace complex peptides and proteins analysis.In summary, this thesis focuses on the key problems of separation and analysis in micro-scale. We initially developed a novel thermal expansion pump for capillary HPLC, which is capable of generating continuous flow, and utilized it in fabrication of nano-HPLC-LIF system. This work will bring more breakthroughs both in scientific research and technical applications. We also developed a method of solid-support fluorescent derivatization for protein in low concentration, and established a nano-HPLC-LIF system employing WAX-RPLC biphasic column for trace complex protein analysis. Both of them provide an important foundation to realize the single-cell analysis by liquid chromatography in the future.