Study Of Capillary Coating Method And Apply To The Analysis Of Proteins

Capillary Electrophoresis(CE) is a micro separating and analytical technique of liquids, where the capillary is taken as the separation channel and high electrostatic voltage field as the driving force. The separation of samples is based on various characteristics, such as charge, molecular mass, isoelectric point, polarity, affinity and phase partition, etc. Compared with the traditional separation and analysis technology, CE has the advantages of fast separation, high separating efficiency, less sample consumption and high level of automation, which makes CE one very powerful tool for biomolecular separation of polypeptides, proteins, DNA and amino acids. In recent years, with the rapid development of coupling techniques of CE-MS, CE-LIF, etc, the capillary electrophoresis technology has shown more and more broad application prospects in life science, pharmaceutical analysis, food, chiral separation and clinical disease.However, the most used fused silica capillary has the inner wall of Si-OH, which dissociates into Si-O- when in buffer conditions with pH>3. The dissociation of Si-OH causes the instability of electroosmotic flow(EOF) and makes the capillary’s inner wall absorb basic proteins markedly. For this reason, problems arise, such as poor peak symmetry of protein, poor reproducibility of migration time, decreasing separation efficiency and low protein recovery. The coating modification to the capillary inner wall is the most effective and most commonly used solution to these problems.The coating modification mainly refers to that polymer materials bind to the capillary’s inner wall and form physical or chemical coating. With the nature of the capillary walls changed, EOF is controlled and the absorption of basic proteins is restrained. This thesis studied two coating methods combined with various factors affecting the effects of coating and established the systematical methods of detailed coating process and their effect-testing standard. Various factors affecting the effects of coating were studied and the differences of these factors were compared. Then the coated capillary were applied to different patterns of capillary electrophoresis for further quality testing and practical utilization. This paper mainly includes the following parts:(1) Firstly, capillary electrophoresis of different patterns and their basic principles were summarized. Undesired problems of the instability of electroosmotic flow and protein absorption under the common conditions of separation and analysis were introduced, as well as their solutions. In addition, the patterns and corresponding advantages of coating methods together with capillary’s types and prices in market were overviewed.(2) Both the purchased neutral coating liquid and the PB-DS-PB coating in Ref were explored and compared. Factors affecting coating effects included coating temperature, coating time, coating pressure and these factors were studied one by one and a whole set of systematic coating methods were established.(3) Based on the capillary zone electrophoresis(CZE), a set of methods for the coating evaluation were founded. The coated capillary in the optimizing process was used for sample detecting. The stability of EOF in varying pH, reproducibility, chemical stability and separating effects of basic proteins were adopted as reference parameters for the quality test of coated capillary.(4) The qualified coated capillary was applied to practical use. Different patterns of CE, such as affinity capillary electrophoresis, capillary isoelectric focusing, capillary electrophoresis with fluorescence detector were adopted for the separation and analysis of practical samples.