Capillary Electrochromatographic Separation Research Articles

Chiral-induced covalent organic framework as novel chiral stationary phase for chiral separation using open-tubular capillary electrochromatography

As a novel class of chiral stationary phase (CPS) material, chiral covalent organic frameworks (CCOFs) have already shown great promise in open-tubular capillary electrochromatography (OT-CEC) for chiral separation. The synthesis methods of CCOFs used in OT-CEC mainly include bottom-up, post modification and chiral induction. The CCOFs synthesized by bottom-up and post modification strategies already have lots of applications in capillary electrochromatography, however, the chiral-induced synthesized via an asymmetric catalytic strategy has not yet been reported for using as the chiral stationary phase (CPS) in OT-CEC or even in chromatographic separation. Herein, the chiral-induced COF (Λ)-TpPa-1 was synthesized by asymmetric catalytic synthesis and coated on the inner surface of a capillary by an in-situ growth strategy as the CPS for chiral drug separation. The baseline separation of six enantiomers was achieved within 14 min, with a high-resolution (Rs) range from 1.85 to 6.75. Moreover, the resolution and migration time of the capillary keep stable within 160 runs, showing its superior stability and repeatability. This research provides a new idea for the development and application of novel CPS materials in the field of capillary electrochromatography separation, also shows the new application of chiral induced COFs. Furthermore, the chiral-induced CCOFs can be easily applied to other chromatographic separation fields, exhibiting its extensive application value in chiral analysis separation.

Construction of chiral crown ethers in robust covalent organic frameworks for electrochromatographic enantioseparation

ABSTRACT Capillary electrochromatography (CEC) is a rapidly emerging separation technique that merges the high separation efficiency of capillary electrophoresis with the exceptional selectivity of liquid chromatography. However, it remains a synthetic challenge to design functional chiral stationary phases (CSPs) with high chemical stability against acid and base in CEC enantioseparation. Here we demonstrate that incorporating chiral crown ethers into stable covalent organic frameworks (COFs) enables efficient and stable separations of racemates by CEC. This facilitates the crafting of two three-dimensional (3D) chiral COFs by polycondensation of a chiral 1,1'-binaphyl-20-crown-6-derived dialdehyde and tetraamines with diisopropyl substituents. Both feature an 11-fold interpenetrated diamond framework, characterized by tubular open channels decorated with chiral crown ethers serving as enantioselective recognition and binding sites. These frameworks demonstrate excellent stability in water, acid and base, thanks to the presence of bulky isopropyl groups that shield the dynamic imine linkages. Moreover, the precisely defined COF channels enhanced the accessibility of the enclosed crown ethers to the analytes while providing strong protection against harsh environments, rendering them suitable for CSPs in CEC separations. They can effectively separate some important enantiomers, including ketones, epoxides and alkaline substances, when utilized as coatings on chiral columns, particularly facilitating the chiral separation of drugs. This study advances the application of COFs in electrochromatographic separations, expanding the scope of porous materials design and engineering to create COFs with targeted enantioselective properties.

Multilayer coatings based on gold nanoparticles and polymers with bovine serum albumin as a functional layer for the chiral separation in capillary electrochromatography.

In this study, we developed physically adsorbed multi-layer coatings using poly-l-lysine or poly(diallyldimethylammonium chloride) and gold nanoparticles, which were functionalized with bovine serum albumin for the chiral separation in electrochromatography. The approach involves sequentially depositing positively charged polymers and negatively charged citrate-stabilized gold nanoparticles. By repeating this modification cycle, we created two- and four-layer coatings, which were sequentially functionalized with albumin forming three- and five-layer coatings that were finally applied for the separation of enantiomers of dl-tryptophan. The formed coatings exhibit stability across a pH range of 2-10 and feature a dense, uniform surface, as confirmed by scanning electron microscope images. The number of layers impacted nanoparticle deposition density, with five-layer coatings being denser than three-layer ones. Five-layer coatings enable baseline separation of dl-tryptophan enantiomers, whereas three-layer coatings require the presence of albumin in the background electrolyte for separation. Therefore, increasing the number of layers and gold nanoparticles density enhances albumin active center concentration on capillary walls, improving the separation of dl-tryptophan enantiomers. The five-layer coatings can be easily fabricated and possess good repeatability of analytes migration time.

In situ growth of imine-based covalent organic framework as stationary phase for open-tubular capillary electrochromatographic separation.

Designing advanced stationary phases to improve separation efficiency is essential in capillary electrochromatography. Due to their outstanding performance, covalent organic frameworks have recently demonstrated considerable promise in the field of separation science. Herein, an open-tubular capillary electrochromatography method was reported using porous imine-based covalent organic framework with sufficiently available interaction sites as stationary phase. The imine-based covalent organic framework coated capillary was easily prepared via an in situ growth method at room temperature, and its separation performance was evaluated, indicating the high separation efficiency for three types of analytes, including herbicides, polybrominated dibenzofurans, and bisphenols. Moreover, the imine-based covalent organic framework coated capillary showed good reproducibility and stability, with intraday (n=3), interday (n=3), and column-to-column (n=3) relative standard deviations of retention time and peak areas of less than 5%. The separation efficiency of the coated capillary remained unchanged even after 200 runs and the maximum theoretical plates reached up to 85595 N/m for 4,4'-ethylidenebisphenol. It was predicted that the imine-based covalent organic framework stationary phase would be a strong contender for chromatographic separation with high efficiency.

Sulfonic acid functionalized monolithic column for high selectivity capillary electrochromatography separation.

A new nano-scale spherical vinyl-functionalized covalent organic polymer (TAPT-DVA-COP) with uniform sizes around 300nm was initially constructed using 2,5-divinyl-1,4-benzaldehyde (DVA) and 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT) as monomers. Then, a sulfonic acid (-SO3H) modified COP termed COP-SO3H was developed based on post-sythesis method employing TAPT-DVA-COP as precursor. Capillary electrochromatography (CEC) monolithic columns were fabricated using the physical doping technique to exhibit the application potential of TAPT-DVA-COP and COP-SO3H. Compared to the TAPT-DVA-COP monolithic column, the COP-SO3H monolithic column achieved a highly selective separation between analytes with different properties, including monosubstituted benzenes, alkylbenzenes, hydroxybenzoates, nucleoside bases, and biogenic amines. Non-covalent interaction (NCI) analysis and experimental data show that the synergism of the sulfonic acid group and aromatic moieties on COP-SO3H endows the new stationary phase with diverse interactions, including ion exchange, hydrophobic, π-π and hydrogen bonding. In addition, the COP-SO3H monolithic column exhibited good reproducibility and excellent potential for the determination of hydroxybenzoates in compact powders and alkylbenzenes in effluent samples.

A monolithic azacalix[4]pyridine column for high-resolution and high-efficiency pressurized capillary electrochromatographic separation

Herein, a highly stable (NAllyl)4-bridged alyzacalix[4]pyridine monolithic column (nAC4P@capillary) was fabricated by copolymerizing nAC4P with GMA and EDMA into a stationary phase through a simple olefin addition reaction. The physicochemical properties of the prepared materials were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), energy dispersive X-ray (EDS), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR) and elemental analysis (EA). A wide range of analytes with various properties was well separated and demonstrated the synergistic effect of the various interaction sites. The maximum column efficiencies (Nmax) for all the analytes reached 62,368 plates/m, and the relative standard deviations (RSDs) of the daily retention times of alkylbenzenes, measured over six months were all lower than 5.63%. Non-covalent interaction analysis reveals that nAC4P@capillary can effectively combine the advantages of electronic configurations, π-π interactions, CH-π interactions, O–H…N hydrogen bonds and lone pair-π interactions, providing excellent prospects for high-performance chromatographic separation.

Homomesoporous Metal-Organic Framework for High-Performance Electrochromatographic Separation.

Metal-organic frameworks (MOFs) have exhibited tremendous potential in the area of separation science. However, most of the developed MOF-based stationary phases contained only microporous structures and suffer from limited separation performance. 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. As a proof of concept, noninterpenetrated mesoMOF-1 with uniform mesopore sizes (22.5 × 26.1 Å) and good stability was facilely grown on the inner surface of capillaries and applied as a homomesoporous MOF coating-based stationary phase for high-efficiency electrochromatographic separation. Seven types of analytes with different molecular dimensions were all baseline separated on a mesoMOF-1 coated column with high theoretical plate numbers and excellent repeatability, 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. These results indicate that the homomesoporous MOF-based stationary phase can effectively balance the kinetic diffusion (mass transfer capability) and thermodynamic interactions (the strength of adsorption interaction), having great potential for high-performance chromatographic separation.

Synthesis of spherical three-dimensional covalent organic frameworks and in-situ preparation of capillaries coated with them for capillary electrochromatographic separation

Covalent organic frameworks (COFs) are divided into two-dimensional (2D) and three-dimensional (3D) structures according to the connection dimension of covalent bonds. 3D COFs have smaller pore size and larger surface area, which would facilitate the separation of small organic molecules with similar structures and properties in capillary electrochromatographic (CEC) separation. However, the application of 3D COFs in CEC is still in its early stages. Thus, 3D COFs (3D-IL-COF-1 and 3D-IL-COF-2) were synthesis by a new synthesis method in this work. The related characterization results confirmed that spherical 3D-IL-COFs formed by the accumulation of small polyhedral particles were successfully synthesized. Then, 3D-IL-COFs were introduced into the inner wall of capillary by in-situ growth method to prepare the 3D COFs coated capillaries for the first time. The electrochromatographic separation performance of the 3D-IL-COFs coated capillaries was investigated using the 3D-IL-COF-1 coated capillary as the model. The results indicate that the 3D-IL-COF-1 coated capillary with uniform coating exhibits a broad-spectrum separation capacity not only for acidic, basic and neutral analytes but also for neutral isomers, which shows that 3D-IL-COFs becomes an attractive potential stationary phase for electrochromatographic separation.

Study of the separation ability differences of three covalent organic frameworks as coated materials in capillary electrochromatography

Covalent organic frameworks (COFs) have great potential applications in chromatographic separation. So it is crucial to understand the relationship between the separation ability of COFs and their structures. Here we report a strategy to evaluate the separation ability of three 2D COFs and explore the relationship between separation ability and their molecular structures. The three 2D COFs (COF-LZU1, COF-42 and COF-LZU8) have one same building unit 1,3,5-triformylbenzene, while varied from the conjugated linking units and functional side-chains. They were used to construct coated capillary column for capillary electrochromatographic separation of same groups of phthalates. They exhibited different separation efficiencies. COF-42 and COF-LZU8 coated capillary columns provided good signal resolutions and high column efficiencies with high theoretical plate numbers. It is demonstrated that COFs with hydrazone unit and longer side-chains provided higher selectivity and resolutions for the phthalates separation. Molecular simulations and DFT calculations were further proceeded to explore the deep reason why the three COFs coated CEC displayed different separation ability based on the host−guest interactions on molecular level. This work highlights a new opportunity to select or design functional COFs and improve their efficiency in chromatographic separation based on host−guest chemistry.

In situ synthesis of a spherical covalent organic framework as a stationary phase for capillary electrochromatography

Covalent organic frameworks (COFs) are a novel type of crystalline porous organic polymer materials recently developed. It has several advantages in chromatographic separation field, such as high thermal stability, porosity, structural regularity, and large specific surface area. Here, a novel spherical COF 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 2,5-bis(2-propyn-1-yloxy)-1,4-benzenedicarboxaldehyde (BPTA) was developed as an electrochromatographic stationary phase for capillary electrochromatography separation. The COF TAPB-BPTA modified capillary column was fabricated via a facile in situ growth method at room temperature. The characterization results of scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD) confirmed that COF TAPB-BPTA were successfully modified onto the capillary inner surface. The electrochromatography separation performance of the COF TAPB-BPTA modified capillary was investigated. The prepared column demonstrated outstanding separation performance toward alkylbenzenes, phenols, and chlorobenzenes compounds. Furthermore, the baseline separations of non-steroidal anti-inflammatory drugs (NSAIDs) and parabens with good efficiency and high resolution were achieved. Also, the prepared column possessed satisfactory precision of the intra-day runs (n = 5), inter-day runs (n = 3), and parallel columns (n = 3), and the relative standard deviations (RSDs) of the retention times of tested alkylbenzenes were all less than 2.58%. Thus, this new COF-based stationary phase shows tremendous application potential in chromatographic separation field.

Metal-Organic Frameworks-Based Immobilized Enzyme Microreactors Integrated with Capillary Electrochromatography for High-Efficiency Enzyme Assay.

Enzyme assays are important for studying enzyme-mediated biochemical reactions and for clinical diagnosis and drug development. The technique of an immobilized enzyme microreactor (IMER) integrated with capillary electrophoresis (CE) has been frequently utilized in online enzyme assays. However, the traditional approaches for IMER-CE enzyme analysis have some defects such as low loading capacity and poor stability. Herein, metal-organic frameworks (MOFs), which have enormous potential in the fields of enzyme immobilization and capillary electrochromatographic (CEC) separation, were first explored as novel support materials with good enzyme immobilization performance and stationary phases with excellent separation abilities to construct an integrated MOFs-IMER-CEC microanalysis system for a high-efficiency online enzyme assay. As a proof-of-concept demonstration, acetylcholinesterase (AChE) was immobilized on a densely packed UiO-66-NH2 nanocrystal coating on a capillary inner surface with abundant intercrystalline mesoporosity and was employed as a highly effective and robust IMER for CEC-integrated online enzyme analysis. The excellent separation performance of the UiO-66-NH2-modified capillary was verified by high-efficiency separation of three types of neutral, acidic, and basic compounds. The Michaelis-Menten constant and enzyme inhibition kinetics of UiO-66-NH2-IMER were systematically assessed, exhibiting distinct advantages such as remarkably increased enzyme loadability, superior affinity for substrates, and greatly improved stability and repeatability compared to CE-integrated IMERs prepared by the traditional covalent bonding method. Furthermore, the developed method was successfully utilized for detecting organophosphorus pesticides in leguminous vegetable samples, demonstrating its strong practicality. The study not only proposed a novel support material and construction strategy for a high-performance microchannel-based IMER but also can be widely used in bioanalysis and biosensing research.

Application of Sol-Gels Modified with Natural Plants Extracts as Stationary Phases in Open-Tubular Capillary Electrochromatography.

Ethanol extracts of three widely growing plants were added to silica sol–gel solutions, which were subsequently applied as wall surface modifiers in inner quartz capillaries. Modified capillaries were used for open-tubular capillary electrochromatographic separation of nucleotides and amino groups containing biological compounds (neurotransmitters, amino acids and oligopeptides). The experiments were performed at physiological pH 7.40, and eventual changes of effective mobilities were calculated. Specific compounds characteristic for each plant were tested as sol–gel additives as well, and thus-modified capillaries were used for the separations of the same analytes under identical conditions. The aim of this study was to find out possible interactions between physiological compounds and extracts of freely available plants anchorded in the sol-gel stationary phase in the flowing system. Even though the amount of the modifier in each capillary is very small, basic statistical evaluation showed some not negligible changes in effective mobility of tested analytes. These changes were bigger than ±5% for separations of nucleotides in capillaries with curcuma, Moringa or the mixture of synthetic additives as the sol-gel aditive, and for separations of amino compounds where these changes varying by additive, analyte by analyte.

Metal-Organic Framework-Based Enzyme Composites as Chiral Stationary Phase for High-Efficiency Capillary Electrochromatographic Enantioselective Separation

Metal-Organic Framework-Based Enzyme Composites as Chiral Stationary Phase for High-Efficiency Capillary Electrochromatographic Enantioselective Separation

Enzyme Metal-Organic Framework Composites as Chiral Stationary Phase for High-Efficiency Capillary Electrochromatographic Enantioselective Separation

Enzyme Metal-Organic Framework Composites as Chiral Stationary Phase for High-Efficiency Capillary Electrochromatographic Enantioselective Separation

A Monolithic Azacalix[4]Pyridine Column for High-Resolution and High-Efficiency Capillary Electrochromatographic Separation

A Monolithic Azacalix[4]Pyridine Column for High-Resolution and High-Efficiency Capillary Electrochromatographic Separation

Rapid polymerization of polyhedral oligomeric siloxane-based zwitterionic sulfoalkylbetaine monolithic column in ionic liquid for hydrophilic interaction capillary electrochromatography

A novel polyhedral oligomeric siloxane (POSS)-based zwitterionic monolithic capillary column was prepared via one-pot polymerization in ionic liquid porogen, using N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine (DMMSA) and methacrylic ethyl trimethylammonium chloride (META) as binary functional monomers, and methacryl substituted POSS as cross-linker. The pore structure, permeability and homogeneity were well tuned by optimizing the polymerization conditions. The resultant monolith was characterized by scanning electron microscopy, nitrogen adsorption/desorption isotherm and Fourier transform infrared spectroscopy. The incorporation of zwitterionic ligand (DMMSA), quaternary amine group (META) and rigid POSS skeleton endows the hybrid organic-silica monolith with high hydrophilicity, electrostatic interaction and good mechanical stability, as well as a tunable electroosmotic flow over wide pH range. A close investigation of capillary electrochromatographic separations of different types of polar compounds such as bases, nucleosides and benzoic acids on such stationary phase exhibited a retention independent column efficiency up to 118,000 plates/m (thiourea), as well as a mixed-mode hydrophilic interaction chromatography (HILIC) retention mechanism including weak electrostatic interaction, hydrophobic interaction and anion exchange.

A lipase-based chiral stationary phase for direct chiral separation in capillary electrochromatography

Candida antarctica lipase B (CALB) is a natural biocatalyst with an intrinsically strong chiral environment and a high degree of enantio-selectivity, which is widely used in the separation of racemates. Here, a facile and efficient covalent immobilization approach was utilized to immobilize CALB onto the capillary inner wall as a novel chiral stationary phase to explore and broaden its application in the direct chiral separation by electrochromatography. The obtained CALB immobilized capillary column was characterized by scanning electron microscopy (SEM), fluorescence imaging and Fourier transform infrared spectroscopy (FT-IR). The enantioseparation property of the CALB immobilized capillary column was confirmed by direct chiral separation of several pairs of monoamine neurotransmitter enantiomers in OT-CEC mode. Outstanding enantioseparation performance for three types of monoamine neurotransmitter enantiomers including epinephrine, norepinephrine and phenylephrine was obtained by the CALB immobilized column. Thanks to the effectiveness of covalent bonding method and the intrinsic stability of CALB, the prepared CALB immobilized capillary columns were quite steady and reproducible. The relative standard deviations for retention times of the enantiomers were as follows: for intra-day (n = 5) runs (≤0.25%), inter-day (n = 3) runs (≤0.72%) and between-columns (n = 3) (≤2.42%). After 90 consecutive runs in CEC mode, the CALB immobilized column still exhibited desirable enantionseparation performance.

In situ room-temperature preparation of a covalent organic framework as stationary phase for high-efficiency capillary electrochromatographic separation

Covalent organic frameworks (COFs), considered as a series of newly emerging porous organic materials, have been widely utilized in separation fields. Herein, a novel COF (TFPB-BD) was first employed as stationary phase for high-efficiency capillary electrochromatographic separation. Benzidine (BD) and 1,3,5-Tris-(4-formylphenyl)benzene (TFPB) were selected as organic linkers and then introduced into the aldehyde group modified capillary for the in situ growth of TFPB-BD onto the capillary inner wall at room temperature. The morphology and formation of TFPB coated capillary column were confirmed by a variety of tools including Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). It's interesting that the TFPB globular crystals with nanoscale were uniformly and densely modified on the capillary inner surface. Hence, the prepared column exhibited prominent separation performance for the test analytes including alkylbenzenes, chlorobenzenes and phenolic compounds with high efficiency and high resolution. The maximum column efficiency can reach about 1.8 × 105 plates•m−1. Additionally, the high resolutions of anilines, amino acids and parabens were also achieved on the TFPB modified capillary. The precisions (RSDs) of the retention times of alkylbenzenes of intra-day runs (n = 3), inter-day runs (n = 3) and parallel columns (n = 3) were all less than 2.83%. This innovative COF-based stationary phase gives great promise for the chromatographic separation field.

Dendritic Mesoporous Silica Nanospheres: Toward the Ultimate Minimum Particle Size for Ultraefficient Liquid Chromatographic Separation.

Use of smaller particle size of packing materials in liquid chromatography leads to faster separation and higher efficiency. This basic law has driven the evolution of packing materials for several generations. However, the use of nanoscale packing materials has been severely hampered by extremely high back pressure. Here, we report a new possibility of solving this issue via introducing novel nanomaterials with highly favorable structures. n-Octyl-modified monodispersed dendritic mesoporous silica nanospheres (DMSNs) with an unprecedentedly small diameter (ca. 170 nm) and appropriate pore size (5.6 nm) were controllably synthesized and demonstrated to be a practically applicable packing material offering ultrahigh efficiency. The center-radial centrosymmetric mesopore channels significantly improved the permeability of packed capillaries, enabling column packing and capillary electrochromatographic separation on regular instruments. Due to the unique morphology, very tiny particle size, and highly uniform packing, the packed column exhibited ultrahigh efficiency up to 3 500 000 plates/m. Powerful separation capability was demonstrated with glycan profiling of cancerous and normal cells, which revealed that cancerous cells exhibited characteristic N-glycans. Because DMSNs with tunable particle size and mesopores can be controllably prepared, DMSNs hold great potential to be a new record toward the ultimate generation of packing materials for ultraefficient liquid chromatographic separation.

Open tubular capillary column immobilized with sulfobutylether-β-cyclodextrin for chiral separation in capillary electrochromatography.

A novel chiral open tubular capillary column was fabricated with sulfobutylether-β-cyclodextrin and glycidyl methacrylate for enantioseparation in capillary electrochromatography. First, the pretreated silica-fused capillary was treated with 3-trimethoxysilyl propyl methacrylate to attach double bond ligand onto the surface. A copolymer layer was formed on the surface of capillary using glycidyl methacrylate and ethylene dimethacrylate by in situ one-pot polymerization. Sulfobutylether-β-cyclodextrin was encapsulated inside the copolymerized layer. The morphology of the developed column was characterized by field emission scanning electron microscopy. The effect of organic percentage and pH value of the mobile phase on electroosmotic flow and resolution was also investigated. The performance of the fabricated column was validated by separation of amlodipine besilate, 2,3-diphenylpropionic acid, tropic acid, and pantoprazole enantiomers with good resolutions of 3.67, 4.82, 3.34, and 2.61, respectively. The repeatabilities of column-to-column and day-to-day through relative standard deviation were found better than 4%, exhibiting satisfactory repeatability of the developed column. The results reveal that open tubular capillary columns modified with β-cyclodextrin show a great prospect for enantioseparation of chiral drugs in capillary electrochromatography.