Studies Of Novel Capillary Electro-kinetic Microseparation Materials And Methods

Capillary electro-kinetic microseparation method includes capillary electrophoresis (CE) and capillary electrochromatography (CEC). The thesis focuses on solving the problems meet in the separations of biological molecules such as proteins, amino acids and so on with CE and CEC.A novel method for the fabrication of poly(vinyl alcohol) coated capillary column was developed to avoid the irreversible adsorption between proteins and capillary inner wall. The prepared column can promise high protein separation efficiency (generally higher than 600,000 plates/m), and excellent reproducibility. For example, relative standard deviations for the migration time of three basic proteins were smaller than 1.0% within 100 injections.A"both-ends-sample-injections"method was designed to simultaneous separation of both negatively and positively charged proteins with the running buffer of a neutral pH. Besides, electroosmotic pump-assisted protein CE was also developed for the separation of cationic, anionic and neutral proteins in a single run.To solve the problem (low phase ratio and low ligand capacity resulting in poor resolution) in open tubular CEC (OTCEC), tentacle-type polymer was designed as the OTCEC stationary phase. A tentacle-type metal-chelating polymer coated capillary column was introduced and used for the separation of amino acids and purine derivatives in this thesis. Immobilized metal capacity of the prepared tentacle-type column was nearly 900 times higher than that of mono-layer metal-chelating group modified one. Besides, the tentacle-type capillary can obviously increase the retention factor and resolution of analytes.To prepare a tentacle-type polymer coating easily functionalized with different ligand, a new method for grafting glycidyl methacrylate to the capillary inner wall was thus developed. The prepared polymer coated column could be easily modified with different chromatographic ligand by a ring-opening reaction of the epoxy groups. In this study, phenylalanine was selected as the ligand. Benezene derivatives, amino acids as well as protein mixtures were then separated, respectively. To improve the detection sensitivity in capillary electrokinetic microseparation methods, a novel negatively charged tentacle-type polymer-coated capillary column was fabricated and applied for on-line extraction and preconcentration of proteins. The prepared tentacle-type polymer coating can efficiently increase the protein adsorption capacity, thereby increasing the sensitivity enhancement factor. In this work, over 1500-fold sensitivity enhancement was realized for myoglobin. The developed method also promised for enrichment and separation of protein mixture.