On-line Preconcentration And Separation Of Biological Macromolecules By Capillary Electrophoresis

Nucleic acids and proteins as most important bio-macromolecules in life science have been the focus of researchers from different fields. Capillary electrophoresis (CE) is an important separation means for these molecules due to its high separation efficiency, high speed, and less consumption of samples.The principle of CE, separation methods and research advance of capillary coating techniques and on-line concentration techniques are reviewed.A simple device and a new protocol for the preparation of cross-linked polyacrylamide (CPA) coating column were developed in the light of proteins adsorption issue. The electroosmotic flow was suppressed effectively and the adsorption of basic proteins was obvious alleviated. Separation efficiency up to 105 plants/m was achieved with RSD less than 5% both for intra-day assay and for inter-day assay. Non-gel sieving capillary electrophoresis of protein-SDS complexes was well demonstrated successfully with the CPA coated column.Injection preconcentration of real sample, plasma, was carried out for the first time with a porous conductive joint prepared by HF etching of a fused silica capillary. Preconcentration and separation of plasma proteins was realized with an uncoated capillary column, and working conditions were explored. About 120 times concentration was achieved by 300 seconds preconcentration as estimated by peak area.Electro-injection preconcentration and non-gel capillary electrophoresis separation of DNA was also demonstrated for the first time with a bare capillary with the porous joint. Low concentration of DNA was achieved after condition optimization. DNA detection limit as low as 0.05 ng/μL was demonstrated by a UV 254 nm photometric detector with 300 sec preconcentration, corresponding 10 to 60 times preconcentration as estimated by peak area.The porous membrane could not only be used for preconcentration of bio-macromolecules, but also for analysis of real sample of complex matrix. Together with conventional sample preparation methods, capillary electrophoresis with online porous joint could be developed into a powerful analytical means for low abundance protein and DNA analysis with its capability of online preconcentration, desalting, separation and online detection.