The Rational Construction And Application Of High-performance MOFs Electrochromatographic Stationary Phases

Capillary electrochromatography（CEC）is an important class of chromatographic separation technique for trace separations and is widely used in various fields such as life sciences,pharmaceutical analysis,clinical diagnostics and environmental monitoring.The core part of CEC separation is the chromatographic column,so the establishment of novel chromatographic stationary phases is the trend of CEC development.As a novel class of organic-inorganic hybrid crystalline porous materials,metal-organic frameworks（MOFs）,have huge application potential in the field of CEC stationary phase.Up to now,a growing amount of research has focused on the MOFs in chromatographic stationary phases.However,since most of the previously reported MOF-based stationary phases only have microporous structures,they usually suffer from critical issues such as high mass transfer resistance,low separation selectivity,and limited column capacity,which place severe limitations on the electrochromatographic separation performance and application range of MOFs stationary phase.To address the problem,homomesoporous MOFs and HP-MOFs were explored as the novel stationary phase for high-performance capillary electrochromatographic separation.Based on the advantages of HP-MOFs,an integrated HP-MOFs-IMER-CEC was constructed for a high-efficiency online enzyme assay.In addition,it is also very important to develop functional stationary phase materials to improve the selectivity of CEC.In this study,chiral functional polydopamine was used as a chiral medium,and a variety of amino acid enantiomers were successfully separated in a short time.The details are as follows:Objective:（1）The non-interpenetrated mesoMOF-1 with uniform mesopore sizes（22.5×26.1（?））and good stability was facilely grown on the inner surface of capillaries and utilized as a homomesoporous MOF coating-based stationary phase for OT-CEC separation of various analytes with different molecular weight.In addition,the intrinsic link between the structure of MOF-based stationary phase and the interaction and retention mechanisms of different molecular-weight analytes were systematically explored and disclosed in detail.（2）HPU-3 was grown on the inner wall of capillaries by the ICISG using template-free synthesis as a HP-MOF coating-based stationary phase for OT-CEC separation of various analytes with different molecular weight and high-efficiency online enzyme assay.（3）There are striking differences in vital movement between D-AAs and L-AAs.And the question of how to obtain highly optically pure amino acid enantiomers has long been of great significance,especially for the food and pharmaceutical industries.A new chiral ligand exchange capillary electrochromatography（CLE-CEC）method was developed for the high-performance enantioseparation of D,L-amino acids（D,L-AAs）.The potential mechanism of the greatly enhanced enantioseparation performance obtained by PDA/L-Arg@capillary was also explored.Moreover,the proposed method was further utilized for studying the enzyme kinetics of L-glutamic dehydrogenase.Methods:（1）The mesoMOF-1 was facilely and efficiently grown on the inner-wall of capillaries via our previously established immobilized cysteine-triggered in situ growth（ICISG）method.After being pre-modified with Cys,the obtained capillaries were used for in situ growth of HKUST-1 nanocrystals（the isostructural analogue of mesoMOF-1 with interconnected 3D square pores of 9×9（?））through an ICISG strategy.The crystal morphology and structure of MOFs on the inner walls of capillaries was characterized by scanning electron microscopy（FESEM）,attenuated total reflectance Fourier transform infrared spectroscopy（ATR-FTIR）and X-ray diffraction（XRD）.The separation performance of mesoMOF-1@capillary was systematically evaluated by the separation of various types of analytes with different molecular dimensions,including the compounds with a small size（incl.substituted benzenes,halobenzenes,purine alkaloids,nonsteroidal anti-inflammatory drugs（NSAIDs）,and steroid hormones）,the analytes with a medium size（i.e.,quinolones）,and the compounds with a larger size（i.e.,polypeptides）.The mass transfer performance of mesoMOF-1 and HKUST-1 was systematically investigated through a series of adsorption kinetic experiments were carried out on three pairs of compounds of different sizes.The column capacities of HKUST-1@capillary and mesoMOF-1@capillary were evaluated using fluorobenzene,androstenedione,and lomefloxacin as model compounds,respectively.The repeatability of mesoMOF-1@capillary was investigated by determining the relative standard deviations（RSDs）of the migration times of substituted benzenes under the optimal CE conditions.（2）HPU-3（pore size,13 nm）was grown on the inner wall of capillaries by the ICISG using template-free synthesis.The crystal morphology and structure of MOFs on the inner walls of capillaries was characterized by scanning electron microscopy（FESEM）,attenuated total reflectance Fourier transform infrared spectroscopy（ATR-FTIR）,X-ray diffraction（XRD）.The separation performance of mesoMOF-1@capillary was systematically evaluated by the separation of various types of analytes with different molecular dimensions,including the compounds with a small size（incl.halobenzenes,chlorobenzene）,the analytes with a medium size（i.e.,purine alkaloids,steroid hormones）,and the compounds with a larger size（i.e.,quinolones）.An integrated HP-MOFs-IMER-CEC microanalysis system was constructed using xanthine oxidase（XOD）as the model enzyme,adsorbed on HPU-3 crystals coating on a capillary inner surface.Successful growth of HPU-3 on the inner walls of capillaries was confirmed by FESEM.The crystal structure and chemical composition of MOFs coating on the inner walls of capillaries was characterized by XRD and ATR-FTIR.The incubation conditions（incubation time,p H and concentration of incubation buffer,etc.）were also optimised,and then the optimized incubation conditions were used to investigate the Michaelis-Menten constant（K_m）,maximum reaction rate(V_max),stability and reproducibility of HPU-3-IMER-XOD with xanthine as the substrate,and compare with Ui O-66-IMER（pore size,0.6 nm）.（3）A novel L-Arg modified PDA coated capillary（PDA/L-Arg@capillary）was firstly prepared through the basic amino acid-induced PDA co-deposition strategy and employed to constitute a new CLE-CEC system for the chiral separation of D,L-AAs with L-Arg as the immobilized ligand coordinating with Zn（II）in running buffer.The inner surface images and properties of PDA/L-Arg@capillary were characterized by FTIR,ATR-FTIR,FESEM and EOF.The preparation parameters and CLE-CEC operation conditions of PDA/L-Arg@capillary were optimized based on the separation performance of Dns-D,L-Ala.And it was further used for the enantioseparation of several different Dns-D,L-AAs under the optimal conditions.The repeatability and stability of PDA/L-Arg@capillary was evaluated based on the RSDs of retention time of Dns-D,L-Asp,Dns-D,L-Ser and Dns-D,L-Ala.The potential mechanism of the greatly enhanced enantioseparation performance was also explored by comparing with bare column and PDA coated column.Moreover,the proposed method was further utilized for studying K_mand V_maxof L-GLDH.Results:（1）FESEM suggested the successful fabrication of HKUST-1 and mesoMOF-1 coatings inside the capillaries and the thickness of the mesoMOF-1layer on the capillary inner wall was about 90 nm,very close to that of HKUST-1（approximately 80 nm）.All results of ATR-FTIR,XRD and the water contact angle measurements confirmed the successful formation of HKUST-1 and mesoMOF-1 layers.At the respective optimal CE separation conditions,baseline separation of the compounds with a small size(incl.substituted benzenes,halobenzenes,purine alkaloids,nonsteroidal anti-inflammatory drugs（NSAIDs）,and steroid hormones,the analytes with a medium size（i.e.,quinolones）,and the compounds with a larger size（i.e.,polypeptides）with high column efficiency could be achieved on mesoMOF-1@capillary,exhibiting significantly improved separation selectivity and column efficiency in comparison to a microporous HKUST-1 coated column.The maximum column efficiencies of the mesoMOF-1 coated column for substituted benzenes and halobenzenes reached up to 1.4×10⁵ plates/m,and its mass loadability was also much higher than that of the HKUST-1 coated column.In addition,based on the analysis of adsorption kinetics and chromatographic retention behaviors,the interaction and retention mechanisms of different molecular-weight analytes on mesoMOF-1 coated stationary phases were systematically explored and disclosed in detail.（2）FESEM demonstrated that both HPU-3 and Ui O-66 were successfully grown on the inner walls of the capillary,with smaller crystal size and more mesoporous structure between the nanocrystals in HPU-3@capillary.It was confirmed by XRD that the useage of acetic acid and ultrapure water as efficient modulator did not affect the crystal structure.The formation of HPU-3 and the successful immobilization of XOD on HPU-3 were confirmed by ATR-FTIR.At the respective optimal CE separation conditions,baseline separation of the compounds with a small size（incl.halobenzenes,chlorobenzene）the analytes with a medium size（i.e.,purine alkaloids,steroid hormones）,and the compounds with a larger size（i.e.,quinolones）with high column efficiency could be achieved on HPU-3@capillary,exhibiting significantly improved separation selectivity and column efficiency in comparison to a microporous Ui O-66 coated column.The maximum column efficiencies of the HPU-3 coated column for chlorobenzene reached up to 1.9×10⁵ plates/m.Meanwhile,based on the changes of substrate peak area,the V_max values for Ui O-66-IMER was 64.85m M/min and the K_m value was 1.03 m M,while the V_max value for HPU-3-IMER was 309.42 m M/min and the K_m value was 1.91 m M.The V_maxvalue of HPU-3-IMER is much higher than that of Ui O-66-IMER,significantly enhancing the catalytic efficiency of XOD after 60 consecutive runs,the enzymatic activity of HPU-3-IMER remained at approximately 80.91%.In addition,after at least 5runs per day for 10 days,HPU-3-IMER maintained at 81.28%,demonstrating its excellent long-term stability and storage stability.（3）Assisted by hydrothermal treatment,the robust immobilization of L-Arg on capillary inner wall could be facilely achieved within 1 h.As for FESEM,a rough inner surface with some visible small aggregates could be observed,which demonstrated the successful fabrication of L-Arg modified PDA coating on the capillary inner wall by FTIR and ATR-FTIR.Eight D,L-AAs were completely separated and three pairs were partially separated under the optimal conditions.The prepared PDA/L-Arg@capillary showed good repeatability and stability.The RSDs of intra-day,inter-day and column-to-column were all below 5.8%.Besides,PDA/L-Arg@capillary could be performed for 80 consecutive injections with-out significant signs of degradation in the retention times and the enantioseparation performance.Based on the changes of substrate peak area,the values of V_max and K_m were calculated to be 414.94 m M/min and 5.41 m M.The data were similar as previously reported values,confirming the reliability of the developed CLE-CEC method in enzyme kinetics study.Conclusion:As a novel class of crystalline porous materials,MOFs have obvious advantages in CEC chromatographic separation compared with traditional inorganic porous materials,so they are more and more widely used.However,the commonly used microporous MOFs-based stationary phases for CEC have many shortcomings which greatly limit their separation efficiency and resolution,including high mass transfer resistance and low utilization of interaction sites.In this work,homomesoporous and hierarchically porous MOFs excellent mass transfer capability were explored as the novel stationary phase for high-performance capillary electrochromatographic separation in the field of bioanalysis.The presented novel MOFs-based stationary with excellent mass transfer capability for CEC analysis are not only helpful to overcome the shortcomings of traditional CEC stationary phases,but also to extend the application potential of MOFs in CE and CEC.The primary conclusion are as follows:（1）Herein,homomesoporous MOFs with excellent mass transfer capability and strong thermodynamic interactions are first explored as the novel stationary phase for high-performance capillary electrochromatographic separations.The homomesoporous MOF-based stationary phase can effectively balance the size exclusion,adsorption interaction and mass transfer capability,having great potential for high-performance chromatographic separation.This study not only discloses the strong correlation between the pore size,ligand composition,and the electrochromatographic retention behaviors of MOF-based stationary phases but also provides a new route to rationally designing high-performance MOF-based chromatographic stationary phases.（2）HPU-3 was grown on the inner wall of capillaries by the ICISG using template-free synthesis as a HP-MOF coating-based stationary phase for OT-CEC separation of various analytes with different molecular weight and high-efficiency online enzyme assay.This study not only reported for the first time an efficient and convenient method for the preparation of HP-MOFs coated stationary phase,but also proposed a strategy for the construction of HP-MOFs-IMER-CEC microanalysis system.（3）A novel CLE-CEC method was firstly developed with L-Arg as the immobilized chiral ligand for D,L-AAs enantioseparation.The hydrothermal-assisted in-situ PDA-based co-deposition strategy for immobilizing L-Arg chiral ligands could effectively avoid the time-consuming and sophisticated fabrication processes of the previously reported modification method.Benefitting from the strong synergistic effect between the immobilized L-Arg in the PDA/L-Arg coating and the free ligands in the buffer solutions,complete separation of eight Dns-D,L-AAs was successfully achieved on the constructed CLE-CEC system.Moreover,the presented method could further be utilized for studying the enzyme kinetics of L-GLDH,exhibiting its promising prospects in enzyme assays and other related applications.