The Fabrication And Application Of Covalent Organic Polymers-based Composites As Mixed-mode Stationary Phase For High Performance Liquid Chromatography

The mixed-mode chromatographic（MMC）stationary phase,which could afford multiple interactions between the stationary phase and analytes,has emerged as one of the most attractive candidates for HPLC separation and analysis.Compared to traditional single-mode stationary phases,the mixed-mode stationary phases possess the inherent virtues,including flexible separation selectivity,remarkably high loading capacity,excellent separation efficiency,and high biocompatibility,making them become good alternative stationary phases for the separation of small molecules or macromolecules in real samples with complicated matrices.Up to date,more and more advanced materials,such as graphene（GR）,carbon dots（CDs）,metal organic frameworks（MOFs）,and covalent organic frameworks（COFs）,have been developed and used as novel MMC stationary phases because of their glamorous properties.Even so,there are several inevitable problems,including the complicated experimental procedures,rigorous synthetic conditions,time-consuming period,nonuniform morphology,and broad distribution of particle size on the development of MMC stationary phases integrated with advanced materials.These undesirable defects cause inconvenience and obstacles in their further applications.Covalent organic polymers（COPs）,an emerging type of amorphous organic porous materials built from organic building blocks stitched via strong covalent bonds,have raised significant concerns due to their high specific surface area,excellent chemical/thermal stability,various functionalities,and versatile surface chemistry characteristics.More importantly,COPs own the vital merits of easy operation,cost-effective,time-saving,and easy modification,which endow them with promising potential in various fields.These attractive advantages also make COPs good candidates as novel separation media in HPLC.However,research on the COPs-based MMC stationary phase is still in its infancy.In order to exploit the application potential of the COPs-based MMC stationary phase in HPLC separation and analysis,several Si O₂@COPs core-shell composites with different structures and characteristics were designed and synthesized as novel MMC stationary phases for HPLC.Then,their retention mechanisms and separation performance were also detailedly investigated and evaluated.The packed MMC columns were further applied to separate diverse analytes in different real samples.These studies provide a robust pathway for the fabrication of novel MMC stationary phases for HPLC,and further reveal the great promising of COPs in HPLC separation and analysis.The research contents are listed as follows:（1）Facile fabrication of silica@covalent organic polymers core-shell compositesas the mixed-mode stationary phase for hydrophilic interaction/reversed-phase/ion-exchange chromatographyThe increasing demands for high-throughput analysis in various fields have promoted the development of efficient separation tools and techniques.As the most important means to improve the performance of chromatographic separation,the exploration of new MMC stationary phases with low back pressure,excellent separation capacity,and high column efficiency has attracted substantial attention.COPs are a promising class of cross-linked polymeric networks that attracted extensive attention in separation and analysis fields.Exploring facile and convenient strategy to prepare COPs-based mixed-mode stationary phases for HPLC has seriously lagged and has never been reported.In this chapter,we describe a facile in-situ grow strategy for fabrication of silica@COPs core-shell composites（Si O₂@Tp BD-（OH）₂）as a novel mixed-mode stationary phase for HPLC.Owing to the co-existing of abundant hydroxyl,carbonyl,imine,cyclohexyl groups,and benzene rings in the skeleton of COPs shell,the developed mixed-mode stationary phase exhibits HILIC/RPLC/IEC retention mechanisms.The content of acetonitrile,p H value,and salt concentration in the mobile phase were investigated on Si O₂@Tp BD-（OH）₂ packed column.In comparison to conventional single-mode columns,the Si O₂@Tp BD-（OH）₂ column showed flexible selectivity,enhanced separation performance,and superior resolution for benzene homologues,PAHs,nucleosides and bases,and acidic organic compounds.The column efficiency of p-nitrobenzoic acid was up to 54440 plates per meter.The packed column also possessed outstanding chromatographic repeatability for six nucleosides and bases with the RSDs of 0.07-0.23%,0.58-1.77%,and 0.31-1.23%for retention time,peak area,and peak height,respectively.Besides,the Si O₂@Tp BD-（OH）₂ column offered baseline separation of multiple organic pollutants in lake water,which verified its great potential in real sample analysis.Overall,the silica@COPs core-shell composites not only provide a new candidate of mixed-mode stationary phases,but also extend the potential application of COPs in separation science.（2）Core-shell hydroxyl-modified covalent organic polymers as the mixed-modestationary phase for high performance liquid chromatographyIntroduction of divers functional groups with muitiple interaction sites to the skeleton of stationary phase via proper synthesis methods is the key to fabricate novel MMC stationary phase.However,the synthesis of COPs-based MMC stationary phases by selecting the organic building blocks is very limited.In this work,we employed a facile layer-by-layer（LBL）method to synthesize Si O₂@COPs core-shell microspheres.Hydroxyl groups were then introduced to the Si O₂@COPs skeleton via a post-modification strategy to enhance hydrophilicity.The obtained Si O₂@COPs-OH core-shell microspheres were characterized and further employed as stationary phase for HPLC.Benefiting from the unique structure and multiple functional groups of the COPs-OH shell,the Si O₂@COPs-OH stationary phase presents hydrophilic interaction/reversed-phase/ion-exchange mixed-mode retention mechanisms and provides flexible selectivity,excellent separation performance,and favorable reproducibility for a wide range of hydrophobic（alkylbenzenes,polycyclic aromatic hydrocarbons,anilines,substituted benzenes）,hydrophilic（nucleosides and bases）,and ionic（benzoic acids）analytes.Remarkably,the column efficiency for uracil was up to36580 plates m^-1 and the RSD values of the retention time were lower than 1.4%after10 consecutive injections.Moreover,the Si O₂@COPs-OH column was successfully applied to separate endocrine-disrupting chemicals（EDCs）in the river water.In short,this work provides a robust synthesized pathway for the functional COPs as mixed-mode stationary phase and further reveals the potential applications of functional COPs in separation science.（3）Layer-by-layer strategy for the controllable fabrication of Si O₂@COPs-Tp BD core-shell microspheres as the mixed-mode stationary phase for high performance liquid chromatographyThe isolation of the composites from excessive COPs remains a formidable taskduring the traditional one-pot synthesis process.As a versatile and straightforward approach,the layer-by-layer（LBL）method effectively avoids the isolation obstacle since the substrate separately reacts with the organic building blocks.Furthermore,the density of the COPs shell,which has a significant effect on the separation performance of the Si O₂@COPs stationary phase,can be easily tailored by the LBL method.In this work,1,3,5-triformylphloroglucinol（Tp）and benzidine（BD）were selected as organic building blocks to construct the COPs shell on the silica surface through the LBL method.The density of the COPs shell was effectively regulated by controlling the reaction runs of the LBL.The obtained Si O₂@COPs-Tp BD core-shell microspheres combine the advantages of the excellent column packing property of the spherical matrices and the outstanding separation ability of the COPs-Tp BD,making these composites promising as HPLC column packing materials.A variety of alkylbenzenes,PAHs,substituted aromatics,nucleosides and bases,and alkaloid compounds were applied to evaluate the retention properties and separation performance of the Si O₂@COPs-Tp BD packed columns with different shell densities.The hydrophobic,hydrophilic,π-π,hydrogen bonding,and EDA interactions provided by the COPs-Tp BD shell dominated the retention and separation of various analytes.Under the optimized conditions,the column efficiency for ethylbenzene and thymine were up to25757 and 18687 plates m^-1,respectively.Besides,the RSD of the retention time,peak width at half-height,peak area,and peak height for six alkylbenzenes and four nucleoside and bases were 0.27-0.84%,0.51-3.76%,1.02-3.03%,and 1.38-3.14%.Four sulfonamides in the milk sample were also successfully separated on the Si O₂@COPs-Tp BD packed columns.All results imply the feasibility of controllable fabricating the Si O₂@COPs-Tp BD core-shell composites through the LBL method and further expand the potential application of COPs in pharmaceutical separation and analysis.