The Preparation And Application Of Functionalized Metal-organic Framework-based High-performance Capillary Electrochromatography

Objective:（1）Nanoscale hierarchically porous Metal-organic frameworks（NHP-MOFs）,with lower mass transfer resistance and increasing effective interaction sites compared with microporous MOFs,which were introduced into capillary electrochromatographic（CEC）and explored as novel stationary phases with excellent mass transfer performance and abundant effective interaction sites for high-efficiency CEC separation of various analytes,to explore the influence of MOFs pore structure on separation performance,and enhance the application prospect of MOFs in the field of CEC stationary phases.（2）MOFs have exhibited increasingly wide utilization in the field of chromatographic separation.However,the previous studies on MOF-based stationary phases focused only on the synthesis and chromatographic performance of a certain kind of supported MOF coatings as stationary phases using the complicated,and time-consuming modification methods,which impeded the widespread application of MOFs in separation science.Therefore,it was necessary to explore and develop a new method for the preparation of MOFs stationary phase,providing a new idea for the design and preparation of novel functionalized MOFs chromatographic stationary phases.（3）Enzyme analysis was of great significance for disease diagnosis and development of new drugs.The technique of capillary electrophoresis（CE）integrated with immobilized enzyme reactor（IMER）usually suffers from the problems of lower enzyme loading and catalytic activity,as well as poor reusability of enzymes.A novel method for IMER-CE enzyme analysis was proposed to be constructed using novel functionalized metal-organic frameworks materials with excellent performance and strong enzyme loading capacity.Methods:（1）Based on the classical microporous Ui O-66,NHP-Ui O-66crystals were synthesized by a modulator-induced strategy.The two MOFs were adhered to the inner wall of the capillary by the adhesion of sodium silicate,and then solidified at high temperature under N₂ condition.The crystal morphology of MOFs was characterized by transmission electron microscopy（TEM）and scanning electron microscopy（SEM）;the crystal structure of MOFs was characterized by X-ray diffraction（XRD）;the specific surface area and pore size distribution of NHP-Ui O-66 crystals were analyzed by nitrogen adsorption-desorption analysis technology.The mass transfer ability of Ui O-66 and NHP-Ui O-66 was measured by adsorption kinetic experiments on isatin and coomassie brilliant blue G250.The chromatographic performance of fabricated NHP-Ui O-66 modifying OT-CEC was evaluated by the separation performance of neutral compounds[i.e.,chlorobenzenes,substituted benzenes and polycyclic aromatic hydrocarbons（PAHs）],charged compounds（i.e.,nucleosides）,medium molecular-weight compounds（i.e.,polypeptides）,and high molecular-weights analytes（i.e.,basic proteins）.The column capacity of the coating capillary was evaluated by investigating the effect of sample concentration on the separation efficiency.The stability and repeatability of NHP-Ui O-66 modified OT-CEC column were verified by the repeated separation of alkylbenzene compounds.（2）Due to the poor heterogeneous nucleation ability of MOFs,the coating preparation method was complicated and time-consuming.Cysteine（Cys）could effectively induce and accelerate the nucleation of MOFs crystals,so an efficient and general MOFs-coated stationary phase preparation strategy（immobilized Cys-triggered in situ growth,ICISG）can be explored and developed based on the induction of Cys.The distribution of Cys bound to the inner wall of the capillary through glutaraldehyde and 3-aminopropyltriethoxysilane（APTES）was characterized by energy-dispersive X-ray spectroscopy（EDS）spectra.Taking four MOFs consisted of different metal ions and organic ligands（ZIF-8,Ui O-66,HKUST-1,MIL-100（Fe））as an example,different MOFs Coated capillary were prepared by in situ growth on the inner wall of the Cys-modified column.The morphology and uniformity of ZIF-8 coatings induced by different amino acids were characterized by SEM and AFM.The crystal structure,morphology and distribution of the four high-quality MOFs crystalline coating columns were characterized by XRD,SEM,EDS and other tests.The chromatographic performance of the four fabricated MOFs modifying columns was evaluated by neutral compounds（i.e.,chlorobenzenes、substituted benzenes and halogenated benzenes）,acidic compounds（i.e.,diphenol isomers）,basic compounds（i.e.,diamine isomers）.Several factors affecting the performance of MOFs chromatographic stationary phase were initially revealed,and a comparative study was carried out with the unmodified capillary column and the Ala-induced ZIF-8 coated column.The column capacity of the four prepared MOFs-coated columns was evaluated and compared by testing the effect of sample injection concentration on the separation efficiency.The stability and repeatability of four MOFs-modified OT-CEC columns prepared by this strategy were verified by the repeated separation of alkylbenzene compounds.（3）Ui O-66-NH₂ with excellent stability was selected as the MOFs model to prepare modified capillaries by ICISG strategy,and acetylcholinesterase（ACh E）was adsorbed at one end of the capillary to construct a novel MOFs-IMERs-CEC.The crystal structure of the MOFs coating was characterized by XRD,and the surface morphology of the inner wall of the capillary was characterized by SEM;the chemical composition of the coating surface was characterized by attenuated total reflection Fourier transform infrared（ATR-FT-IR）.The separation performance of Ui O-66-NH₂ modified capillary was evaluated by CEC separation of neutral,acidic and basic compounds.The immobilization capacity of ACh E on GA and Ui O-66-NH₂ modified silica spheres was determined respectively to evaluate the enzyme loading capacity of Ui O-66-NH₂ coating.Using acetylthiocholine（ATCh）as substrate to investigate the Michaelis-Menten constant,enzyme inhibition kinetics,stability and reproducibility of the prepared Ui O-66-NH₂-IMER,and compared with CE-integrated IMERs prepared by traditional covalent bonding method.In addition,the method was applied to the detection of organophosphorus pesticides in leguminous vegetable samples,proving the application potential of this method in practical sample analysis.Results:（1）The XRD patterns of the synthesized Ui O-66 and NHP-Ui O-66 were in accordance with the corresponding simulated one in the prominent diffraction signals,indicating the successful preparation of Ui O-66 and NHP-Ui O-66 crystals.SEM and TEM characterization results showed that Ui O-66 and NHP-Ui O-66 were nanoscale crystals with particle sizes of about 100 nm and 50nm,respectively.The hierarchical pore distribution（0.6-20 nm）and high specific surface area(881.7 m²·g^-1)of NHP-Ui O-66 were confirmed by N₂ adsorption-desorption.In the dye adsorption experiment,the adsorption rate and adsorption capacity of NHP-Ui O-66 nanoscale crystals for isatin and G250 were higher than those of Ui O-66 crystal,indicating that the as-prepared NHP-Ui O-66nanocrystals were very suitable for the rapid diffusion of the guest molecules,and had excellent mass transfer performance.The SEM characterization of the capillary column found that the inner wall of the two kinds of MOFs-coated capillaries became rough with a large number of crystalline materials,indicating the successful preparation of the two kinds of MOFs-coated columns.The separation performance of HP-Ui O-66 coated column was compared with unmodified column and microporous Ui O-66 modified column under the same CEC conditions.In contrast,the NHP-Ui O-66 coated column has significantly improved resolution,separation efficiency and column capacity for the six types of analytes.In addition,the retention time of substituted benzenes and the separation performance of the NHP-Ui O-66 modified column did not change significantly after 120 consecutive runs.Therefore,the prepared NHP-Ui O-66coated column had good repeatability and stability.（2）The characterization of Cys-modified capillary by EDS found that C,N,O,S elements were uniformly distributed on the inner wall of Cys-modified capillary,indicating that Cys was uniformly grafted on the inner wall of the capillary.The characterization by SEM and AFM showed that both Cys and Ala could induce the in-situ growth of ZIF-8,but the MOFs coating induced by Cys was more uniform and denser.The SEM images of the remaining MOFs coatings also showed the corresponding crystal morphologies,and the crystal structures of the four MOFs coatings were further confirmed by XRD.Under the same chromatographic conditions,the separation performance of the ZIF-8-coated column induced by Cys was better than that of Ala,which was consistent with the characterization results of SEM.By optimizing the CE conditions,the analytes achieved high-efficient baseline separation on the four MOFs-coated columns prepared by the ICISG strategy.Compared with the MOFs modified column prepared by other method,the separation performance of the MOFs modified column obtained by the ICISG strategy was significantly improved.At the same time,the chromatographic properties such as separation efficiency and column capacity of the four MOFs modified columns were also quite different,especially the MIL-100（Fe）modified column with larger pore size has the best separation efficiency and column capacity.Finally,after 200 consecutive runs,the four MOFs-modified capillary columns maintained good separation performance for substituted benzene with no obvious changes in migration time and peak width.（3）After repeated in situ growth of Ui O-NH₂-66,SEM characterization showed that a dense crystalline film could be formed on the inner wall of the capillary,and there’s no obvious change after immobilizing ACh E.The formation of Ui O-66-NH₂ and the successful immobilization of ACh E on Ui O-66-NH₂ were further confirmed by XRD and ATR-FT-IR,and the immobilization of ACh E had no obvious effect on the crystal structure of Ui O-66-NH₂.By optimizing the chromatographic conditions,the selected analytes achieved high-efficiency baseline separation on the prepared Ui O-66-NH₂-coated column.Compared with the uncoated capillary column,the resolution and separation efficiency of the Ui O-66-NH₂-coated column for the three types of analytes were significantly improved under the same CE conditions.The loading capacity of Ui O-66-NH₂ modified silica spheres for ACh E was much higher than that of GA.The Michaelis constant of Ui O-66-NH₂-IMER measured by nonlinear regression curve was 0.77±0.06 m M,which was lower than free enzyme（1.25±0.06 m M）.The enzyme inhibition kinetics study showed that the half maximal inhibitory concentration(IC₅₀)value of TAC on Ui O-66-NH₂-IMER was 0.28μM,which was close to the value of 0.25μM reported in previous study.After 40consecutive runs,the enzymatic activity of Ui O-66-NH₂-IMER remained at93.5%,showing its excellent long-term stability.The standard addition experiment found that malathion exhibited a good linear relationship（R²=0.990）in the concentration range of 0.1～3.0μg/m L,and the limit of detection（LOD）was 40 ng/m L.The spiked recoveries of malathion in the three soybean samples ranged from 96.4%to 104.5%with relative standard deviations（RSD,n=5）ranged from 2.3%to 4.1%.Conclusion:（1）NHP-Ui O-66 were successfully synthesized and corresponding modified capillary columns were prepared.As a stationary phase,NHP-Ui O-66 exhibits superior performance in CEC separation,which can effectively combine the high specific surface area of microporous Ui O-66,as well as the low mass transfer resistance and effective action sites of NHP materials.NHP-MOFs stationary phase materials were not only expected to be widely used in the field of CEC separation,but also contribute to the development of other types of chromatographic stationary phases.（2）An efficient and versatile MOFs-coated stationary phase preparation strategy（ICISG）was developed,which can efficiently prepare various types of MOFs-coated capillary stationary phases for high-efficiency electrochromatographic separation,and four MOFs Coated capillary column with excellent separation performance were successfully applied.This strategy provided a new idea for the design and preparation of novel functionalized MOFs chromatographic stationary phases,and also provided guidance for the construction of MOFs-IMER.（3）A Ui O-66-NH₂-IMERs-CEC microanalysis system with excellent enzyme immobilization and electrochromatographic separation performance was successfully constructed for high-efficiency online enzyme assay.The system effectively combined the advantages of MOFs as the stationary phase for electrochromatography and the support matrix for enzyme immobilization,with greatly improved separation ability,excellent stability and repeatability,higher catalytic efficiency and larger enzyme loadability.The detection of organophosphorus pesticides in actual samples hados good accuracy and practicability,thus possessing an enormous prospect for high-efficiency and robust CEC-integrated enzyme analysis.