Development And Application Of Key Components Of Quantitative Capillary Electrophoresis And Capillary Electrochromatographic Open Tubular Columns

In recent years,with the development of science and technology,the complexity and trace analysis of samples become particularly prominent.Conventional analytical tools are unable to meet the requirment of high efficiency and rapid analysis.Especially for the biological macromolecules,such as proteins,because of their complex structure and large molecular weight,the separation and purification from their complex biological matrices are difficult task that requires application of sophisticated and reliable technology.Microscale electrokinetic separation techniques,including capillary electrophoresis（CE）and open tubular capillary electrochromatography（OT-CEC）,have attracted extensive research interests and have proved to be powerful analytical tools for a variety of applications,especially in the separation and analysis of biological macromolecules,with the advantages of high separation efficiency,high resolution,high selectivity and fast speed.However,traditional CE instrument can not be widely utilized for quantitative analysis of samples due to the defects of the injection design.In OT-CEC,the disadvantage is the low separation capability that arises from the low phase ratio on the limited surface area.Therefore,there is an urgent need to improve the accuracy and precision of the CE apparatus and develop a new open tubular column to solve the above problems.Automated,quantitative capillary electrophoresis（qCE）and open tubular column were developed and studied in this dissertation.We aim to establish and develop a novel,quantitative,efficient technology to meet the demand of complex samples separation.The contents of this dissertation are divided into seven chapters.The first chapter provided introduction on conventional CE and OT-CEC,focusing mainly on the characteristics,theory,related instruments and application of various coating techniques in protein separation.In second chapter,we focused on the design of automated,quantitative capillary electrophoresis apparatus and study the key components,such as auto-sampler,electric decoupler,micro-fluidic syringe pump,high voltage power supply and so on.The investigation on the performance of qCE instrument,such as baseline noise and drift of the detector,detection limit,repeatability,etc.The relative standard deviation（RSD）of migration time for thiourea was 0.8%,and RSD of peak area was0.7%.For the four nucleosides,the RSD of migration time was 0.6%,the RSD of peak area was in the rang from 1.1%to 1.8%,and day-to-day RSD2.1%to 3.3%.The results show that qCE has good repeatability,stability,and is expected to be more widely used in the separation and analysis of biochemical substances.Third chapter described the preparation of P-fSiO₂@C18 open tubular column to solve the problem of low surface area,low phase ratio,low column capacity and absorb problem.We developed a method for column preparation,which is simple,fast,stable and has good reproducibility.In OT-CEC mode,separation and analysis of neutral substance,standard proteins and real biological samples were realized,respectively.Based on P-fSiO₂@C18 OT-CEC,the method is expected to be further applied to the separation and analysis of more complex compounds.Fourth chapter is the establishment of a novel separation mode that combined automatic qCE instrument with OT-CEC for protein separation.Fifth chapter is about the investigation on the fabrication and application of P-fSiO₂ open tubular column.Experimental parameters such as mobile phase composition,ionic strength of the buffer,separation voltage,pH were optimized for the separation of 8 sulfonamides in OT-CEC mode,and the column efficiency of sulfaquinoxaline was as high as228,000 theoretical plates/m.The run-to-run RSD of the analytes was in the range of 2.4%to 4.3%,and the day-to-day RSD was 2.3%to 4.9%,which showed the excellent stability.The OT-CEC model based on P-fSiO₂open tubular column is expected to be a new method for separation and analysis of sulfonamides.Sixth chapter is the investigation of Au-Fe₃O₄ nanoparticles based open tubular column.The Janus structured Au-Fe₃O₄ nanopartices was sythsized by hydrothermal method.Using the properties of magnetic adsorption,open tubular column with Au-Fe₃O₄ coating was prepared.The capillary column exhibits quite reproducible migration times for the test solutes,with values of RSD of 0.3%,2.9%,and 2.7%for run-to-run,day-to-day and column-to-column,respectively.The method was used for the separation of neutral substance,phenolic isomers and protein samples.Through the investigation of the separation ability of neutral substances such as thiourea,naphthalene and biphenyl,the retention mechanism of Au-Fe₃O₄ stationary phase has been investigated.The study of the separation ability of the phenol isomers indicate that gold nanoparticles have certain retention ability to the phenolic hydroxyl groups.The new type of open tubular column can also be used for isolation of ovalbumin,transferrin and ovomucin in egg protein samples.The last chapter provided the summary and prospect.