Preparation Of Electrophoretic Microcolumn Packed With Quartz Microncry Stals And Its Electrophoretic Separation

Capillary electrophoresis(CE) has been employed widely owing to its high separation efficiency,fast analytical velocity,low sample consumption and multiform separation modes.On the other hand,CE has its shortcomings too,such as low sample loadability and dissatisfactory detection sensitivity,resulting from its small inner diameter and short light path of the separation capillary.Thus,under the precondition of reduced thermal effect.it is an effective means to enhance sample capacity and detection sensitivity by using large electrophoresis channel.In this dissertation,quartz microncrystals of uniform size and regular shape are prepared by hydrothermal synthesis.The electrophoresis microcolumn is prepared by packing a 2 mm I.D.fused-silica tube with quartz microncrystals.By the on-line coupling of electrophoretic microcolumn,electrokinetic flow analysis(EFA) system and Ultraviolet-visible(UV-vis) spectrophotometer,a total analysis system of EFA-microcolumn electrophoresis-UV detection is developed and used in the separation and determination of amino acids(AAs).1.Quartz microncrystals of uniform size and regular shape are prepared by hydrothermal synthesis.The effects of different hydrothermal reaction conditions, including reaction temperature and time,the concentration of KOH solution and the mass of silica gel,on the crystalline phase and size of the product are investigated. The products of different crystalline phase and size are prepared under different reaction conditions.The optimal conditions are as followed:350℃,2 h,27 mL 0.46 mol/L KOH and 2.0 g silica gel.The average dimension of the quartz microncrystals is 9μm×3.8μm,the uniform size can satisfy the packing requirement of the electrophoretic microcolumn.2.The laboratory-made electrophoretic microcolumn unit was composed by one 100 mm×2 mm I.D.fused-silica tube packed with the quartz microncrystals (separation column),one jointed 20 mm×1.5 mm I.D.fused-silica tube(detection tube) by fritting,and one on-column detection optical unit assembled with two quartz lenses of 4 mm focus and their brass holders fixed to the separation column.By packing with the quartz microncrystals,the thermal effect in the electrophoretic microcolumn is limited effectively and the precondition of electrophoresis separation in large channel is created.And the improved on-column detection optical unit not only can reduce the sample diffusion,but also increase the light path,so the separation efficiency and detection sensitivity are enhanced finally.3.Different electrophoretic microcolumns are prepared by packing the 9μm length quartz microncrystals prepared by hydrothermal synthesis and the purchased industrial quartz microparticles from 45μm to 360μm.The thermal effect in electrophoretic microcolumn can be limited by packing with the quartz microparticles. The influence of thermal effect on separation is explored by using tryptophan and tyrosine as target analytes.Firstly.the influence of different size of packed quartz microparticles on the porosity,thermal effect,whirling flow and the final separation efficiency are investigated.The results indicate that by the reduce the size of packed quartz microparticles,the porosity is reduced from 52%to 40%,the thermal effect and whirling flow are also reduced,so the final separation efficiency is enhanced from 3.0×10~3 to 4.0×10~4 plates/m.Secondly,by improving shape uniformity of packed quartz microparticles,the electrophoretic microcolumn is packed more regularly,and then the whirling flow is reduced more effectively.Finally,the 9μm length quartz microncrystals prepared by hydrothermai synthesis have smaller size and more uniform shape compared with the purchased quartz microparticles.Therefore,the electrophoretic microcolumn packed with the microncrystals has the low porosity, thermal effect and whirling flow,so the high separation efficiency of 4.0×10~4 plates/m is obtained.Furthermore,the influence of different separation buffer solution on thermal effect and separation efficiency are also explored.4.The electrokinetic flow analysis(EFA) system is proposed and bases on sequential injection analysis(SIA) system,multicommutation analysis(MA) system and electroosmotic pump.The EFA system possesses the advantages of simpleness, convenience,automaticity,multifunction and controllability by replacing peristaltic pump and mechanical pump in traditional flow analysis by electroosmotic pump as the driving equipment of pump carrier,and changing syringe valve and selection valve to solenoid valve.A total analysis system of EFA-microcolumn electrophoresis-UV detection is composed by coupling the electrophoretic microcolumn packed with quartz microncrystals,EFA transportation system and UV-vis spectrophotometer.The total analysis system has the advantages of simpleness, convenience,modularization,multifunction and controllability.If a fiber spectrophotometer is used as its detector,a portable total analysis system can be employed for in-situ and real time analysis and has its wide application. 5.The laboratory-designed and assembled total analysis system is used in the on-line separation and determination of standard mixture of underivatized tryptophan, phenylalanine and tyrosine,and the separation conditions are optimized.The results indicate that the thermal effect in electrophoretic microcolumn is limited by packing with the quartz microncrystals.With the separation buffer solution of 1.5 mmol/L disodium phosphate(pH 11.5) containing 25%(v/v) methanol and 10%(v/v) acetonitrile,the separation efficiency of three amino acids are 4.5×10~4.4.0×10~4 and 3.7×10~4 plates/m,respectively.The sample capacity of the electrophoretic microcolumn achieves 35μL,the limits of detection are 0.035,0.22 and 0.20μmol/L, respectively,and linear calibration ranges are more than two orders of magnitude. With the same column,the relative standard deviations(RSD) of the peak height and migration time are less than 4.8%and 0.35%(n=5),respectively.Between columns, the RSD of the peak height and migration time are less than 7.1%and 2.15%(n=10), respectively.Moreover,the underivatized tryptophan,phenylalanine and tyrosine in compound AAs injection samples are analyzed by the total analysis system.The analyte recoveries are obtained in the range of 91.3%-105.8%.The analyte concentrations found in four compound amino acid injection samples are close to the labelled concentrations.With high separation efficiency,large sample capacity, enhanced detection sensitivity and satisfactory analysis precision,the proposed microcolumn electrophoresis can be used for the sample analysis of low concentration and large volume.It can also be developed as preparative electrophoresis.