Boronate Affinity Monolithic Column Research Articles

Selective recognition of nucleosides by boronate affinity organic-inorganic hybrid monolithic column

Boronic acids are important ligands used to selectively recognize and enrich compounds containing cis-diol groups such as nucleosides. In the present study, organic-inorganic hybrid [POSS-MAH-BPA] monolithic column was prepared for the first time in the literature as a new boronate affinity system for the recognition of nucleosides. The selectivity of the [POSS-MAH-PBA] boronate affinity monolithic column for the recognition of cis-diol containing adenosine nucleoside from its analogue molecule of deoxyadenosine triphosphate, dATP, non-cis-diol containing compound was investigated both by UV and HPLC studies. When the relative selectivity coefficients are compared, the [POSS-MAH-PBA] boronate affinity monolithic column is 4.25 times more selective for adenosine than [POSS-MAH] monolithic column. Besides, to determine endogenous nucleosides in biological fluids, which may serve as non-invasive cancer biomarkers, nucleosides were spiked into the urine solutions and passed through the [POSS-MAH-PBA] boronate affinity monolithic column, and the nucleosides were confirmed by HPLC. The adenosine recognition capability of the [POSS-MAH-PBA] boronate affinity monolithic column with an average enrichment factor of 48.9-fold was apparently superior to that of the [POSS-MAH] monolithic column.Methacryl Polyhedral Oligomeric Silsesquioxanes (POSS-MA) with nano-sized stable 3-dimensional architectures provided the advantage of being used as an adsorbent for the monolithic structure by providing high surface area, 507.60 m2/g, and enabling vinyl groups to function with amino acid-based MAH monomers capable of providing electrons to coordinate PBA. Recovery results of more than 90% for adenosine showed that the [POSS-MAH-PBA] boronate affinity monolithic column could be a promising adsorbent for selective adsorption of cis-diol containing compounds such as nucleosides.

Preparation of a boronate-affinity monolithic column for adsorption of nucleosides.

Chitosan is a considerably versatile and promising biomaterial and can easily form a 3D hierarchical porous scaffold. In this work, a novel boronate-affinity monolithic column modified with a boronic acid-chitosan complex was prepared and characterized by different methods such as Fourier transform infrared spectroscopy, scanning electron microscopy, thermal gravimetric analysis, specific surface area analysis and pore size distribution analysis. The synthesized monolithic column was used for polymer monolithic microextraction combined with high performance liquid chromatography for the simultaneous determination of cytidine, uridine, inosine, and guanosine in milk powder samples. Several parameters affecting extraction efficiency, including the eluent proportion, eluent flow rate, sample flow rate, sample volume, and sample pH were investigated. The boronate-affinity monolithic column showed high enrichment ability due to the selective formation of cyclic borate esters between nucleosides and boronic acid groups at high pH and the release of cyclic borates at low pH values. Under the optimum operating conditions, the linear range was 0.1-50 μg mL-1, and the correlation coefficients were in the range of 0.9993-0.9994. The LOD and LOQ were in the range of 0.0027-0.0034 and 0.0090-0.011 μg mL-1, respectively. In addition, the results of recovery and relative standard deviation were satisfactory.

Development of a new in-line coupling of a miniaturized boronate affinity monolithic column with reversed-phase silica monolithic capillary column for analysis of cis-diol-containing nucleoside compounds

In-line coupling of capillary columns is an effective means for achieving miniaturized and automated separation methods. The use of multimodal column designed to allow the direct integration of a sample preparation step to the separation column is one example. Herein we propose a novel in-line coupling at the capillary scale between a boronate affinity capillary column (μBAMC unit) and a reversed-phase separation column. This has been made possible due to the elaboration of a new and efficient μBAMC unit. A thiol-activated silica monolithic capillary column was functionalized through thiol-ene photoclick reaction. This simple and fast reaction allows to prepare stable μBAMC units having grafting densities of 1.93 ± 0.17 nmol cm−1. This grafting strategy increases the surface density by a factor 4 compared to our previous strategies and opens the frame to in-line coupling with reversed-phase capillary column. Proof of concept of the in-line coupling was done by coupling a 1-cm length μBAMC unit to a 7-cm length reversed phase capillary column. The conditions of loading, elution and separation were optimized for cis-diol nucleosides analysis (uridine, cytidine, adenosine, guanosine). A loading volume (at pH 8.5) of up to 21 hold volume (i.e 1 μl) of the μBAMC unit can be loaded without sample breakthrough. For the least retained nucleoside (uridine) a limit of detection of 50 ng mL−1 was estimated. Elution and full separation of the four nucleosides was triggered by flushing the multimodal column with an acetic acid (50 mM) / methanol (98/2, v/v) mobile phase.

Synthesis of boronate-functionalized organic-inorganic hybrid monolithic column for the separation of cis-diol containing compounds at low pH.

In this work, an organic-inorganic hybrid boronate affinity monolithic column was prepared via "one-pot" process using 4-vinylphenylboronic acid as organic monomer and divinylbenzene as cross-linker. The effects of reaction temperature, solvents and composition of organic monomers on the column properties (e.g. morphology, permeability, and mechanical stability) were investigated. A series of test compounds including small neutral molecules, aromatic amines, and cis-diol compounds were used to evaluate the retention behaviors of the prepared hybrid monolithic column. The results demonstrated that the prepared hybrid monolith exhibited mixed-interactions including hydrophilicity, cation exchange, and boronate affinity interaction. The run-to-run, day-to-day and batch-to-batch reproducibilities of the prepared hybrid monolith for thiourea's retention time were satisfactory with the relative standard deviations (RSDs) less than 0.09, 1.45 and 4.05% (n=3), respectively, indicating the effectiveness and practicability of the proposed method.

Development and application of a new in-line coupling of a miniaturized boronate affinity monolithic column with capillary zone electrophoresis for the selective enrichment and analysis of cis-diol-containing compounds

An integrated, miniaturized and fully automated system was developed for the analysis (preconcentration/purification, separation and detection) of cis-diol containing molecules in complex matrices. This innovative in-line coupling system was achieved via the in-situ and localized synthesis of a short segment of silica-based monolith at the inlet of a 75-μm inner diameter fused silica capillary. The monolithic segment was locally functionalized with an acrylamide derivative of phenylboronic acid by free radical photopolymerization within 10min of irradiation time. Efficiency of the photopolymerization reaction was followed by frontal affinity chromatography of 1,2-dihydroxybenzene (catechol) as cis-diol model solute. An active-site amount of 0.43nmolcm−1 (9.8nmolμL−1) of phenylboronic acid moieties was obtained, with a Kd value of about 290μM close to reported value for the phenyl boronate-catechol complex. The optimal conditions of use of the miniaturized boronate affinity monolithic column (μBAMC) were determined and adapted to the in-line coupling with capillary electrophoresis. Catechol was specifically preconcentrated in a pH 8.5 phosphate buffer/MeOH (80/20, v/v) mixture. A volume up to 20 times the monolith volume can be percolated with a quantitative recovery yield. Three catecholamines were purified, preconcentrated and in-line separated. Elution from the μBAMC was performed with a small plug of acidic solution, allowing field amplified sample stacking of solutes within the plug before their in-line electrophoretic separation at pH 8.75. This unique in-line coupling was successfully used for the fully automated analysis of catecholamines neurotransmitters in urine samples, highlighting the purification efficiency of the μBAMC and the potential of such a fully integrated approach. In addition to the low sample volume required (less than 2μL), the limits of detection (LOD) accomplished with this coupling were estimated at 9.0, 9.5 and 4.8ngmL−1 for dopamine, adrenaline and noradrenaline respectively, which improves the LOD of theses solutes compared to other CE methods.

A hybrid monolithic column based on boronate-functionalized graphene oxide nanosheets for online specific enrichment of glycoproteins

A hybrid monolithic column based on aminophenylboronic acid (APBA)-functionalized graphene oxide (GO) has been developed and used for selective enrichment of glycoproteins. The APBA/GO composites were homogeneously incorporated into a polymer monolithic column with the help of oligomer matrix and followed by in situ polymerization. The effect of dispersion of APBA/GO composites in the polymerization mixture on the performance of the monolithic column was explored in detail. The presence of graphene oxide not only enlarged the BET surface area from 6.3m2/g to 169.4m2/g, but also provided abundant boronic acid moieties for glycoprotein extraction, which improved the enrichment selectivity and efficiency for glycoproteins. The APBA/GO hybrid monolithic column was incorporated into a sequential injection system, which facilitated online extraction of proteins. Combining the superior properties of extraordinary surface area of GO and the affinity interaction of APBA to glycoproteins, the APBA/GO hybrid monolithic column showed higher enrichment factors for glycoproteins than other proteins without cis-diol-containing groups. Also, under comparable or even shorter processing time and without the addition of any organic solvent, it showed higher binding capacity toward glycoproteins compared with the conventional boronate affinity monolithic column. The practical applicability of this system was demonstrated by processing of egg white samples for extraction of ovalbumin and ovotransferrin, and satisfactory results were obtained by assay with SDS-PAGE.

Preparation of phenylboronate affinity rigid monolith with macromolecular porogen

Boronate-affinity monolithic column was first prepared via polystyrene (PS) as porogen in this work. The monolithic polymer was synthetized using 4-vinylphenylboronic acid (4-VPBA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinker monomer, and a mixture of PS solution in tetrahydrofuran, the linear macromolecular porogen, and toluene as porogen. Isoquercitrin (ISO) and hyperoside (HYP), isomer diol flavonoid glycosides, can be baseline separated on the poly(VPBA-co-EDMA) monolith. The effect of polymerization variables on the selectivity factor, e.g., the ratio of monomer to crosslinker (M/C), the amount of PS and the molecular weight of macromolecular porogen was investigated. The surface properties of the monolithic polymer were characterized by scanning electron microscopy and nitrogen adsorption. The best polymerization condition was the M/C ratio of 7:3, and the PS concentration of 40mg/ml. The poly(VPBA-co-EDMA) polymer was also applied to extract cis-diol flavonoid glycosides from the crude extraction of cotton flower. After treated by poly(VPBA-co-EDMA) for solid phase extraction, high purity ISO and HYP (>99.96%) can be obtained with recovery of 83.7% and 78.6%, respectively.

Preparation and characterization of fluorophenylboronic acid-functionalized affinity monolithic columns for the selective enrichment of cis-diol-containing biomolecules.

Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity monolithic column applying 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) as a ligand. The DFFPBA-functionalized monolithic column not only exhibited an ultrahigh boronate affinity toward cis-diol-containing biomolecules, but also showed great potential for the selective enrichment of cis-diol-containing biomolecules in real samples.

Online micro-solid-phase extraction based on boronate affinity monolithic column coupled with high-performance liquid chromatography for the determination of monoamine neurotransmitters in human urine

Quantification of monoamine neurotransmitters is very important in diagnosing and monitoring of patients with neurological disorders. We developed an online analytical method to selectively determine urinary monoamine neurotransmitters, which coupled the boronate affinity monolithic column micro-solid-phase extraction with high-performance liquid chromatography (HPLC). The boronate affinity monolithic column was prepared by in situ polymerization of vinylphenylboronic acid (VPBA) and N,N′-methylenebisacrylamide (MBAA) in a stainless capillary column. The prepared monolithic column showed good permeability, high extraction selectivity and capacity. The column-to-column reproducibility was satisfactory and the enrichment factors were 17–243 for four monoamine neurotransmitters. Parameters that influence the online extraction efficiency, including pH of sample solution, flow rate of extraction and desorption, extraction volume and desorption volume were investigated. Under the optimized conditions, the developed method exhibited low limit of detection (0.06–0.80μg/L), good linearity (with R2 between 0.9979 and 0.9993). The recoveries in urine samples were 81.0–105.5% for four monoamine neurotransmitters with intra- and inter-day RSDs of 2.1–8.2% and 3.7–10.6%, respectively. The online analytical method was sensitive, accurate, selective, reliable and applicable to analysis of trace monoamine neurotransmitters in human urine sample.

Synthesis of a SiO2/TiO2 hybrid boronate affinity monolithic column for specific capture of glycoproteins under neutral conditions

A unique boronate-functionalized SiO2/TiO2 hybrid monolithic column was synthesized by a facile approach. Although a conventional boronic acid, 4-vinylphenylboronic acid, was used as the affinity ligand, the prepared monolithic column exhibited specific capacity to capture glycoproteins including antibodies in aqueous solution at neutral pH. With the incorporation of titania, the monolith was highly hydrophilic, and the procedure of affinity chromatography could be performed under aqueous organic-solvent-free conditions.

One-step synthesis of an organic–inorganic hybrid boronate affinity monolithic column with synergistic co-monomers

An organic-inorganic hybrid and synergistic boronate affinity (BA) monolithic column was synthesized by a novel "one-step" approach. We report on the synthesis of a unique organic-inorganic hybrid affinity monolith by using TEOS, the organic silane, N-(β-aminoethyl)-γ-aminopropyltriethoxysilane (AEAPTES) and γ-MAPS as the co-precursors, 4-vinylphenylboronic acid as the functionalized monomer, and azobisisobutyronitrile (AIBN) as the initiator. Due to the presence of amino groups in the AEAPTES molecule, the prepared monolith exhibits several attractive properties. The monolith will be more hydrophilic and can bind with cis-diol compounds at neutral pH by a mechanism of intermolecular B-N coordination. The resulting hybrid boronate affinity monoliths have potential applications in specific recognition and enrichment of cis-diol biomolecules under neutral conditions.

Preparation of boronate affinity monolithic column with high specific surface using highly structurally symmetrical copolymerizing reagents

Boronate affinity monolithic column is an important chromatographic medium for the selective isolation of the cis-diol containing compounds. However, it is difficult to control the micro-structure of the monolithic capillary during its preparation process. In this article, using melamine and tris (2,3-epoxypropyl) isocyanurate (TEPIC), both are highly symmetrical in molecular structure, as the copolymerizing reagents, a boronate affinity monolithic capillary with high specific surface was prepared by in-situ polymerization according to the "teamed boronate affinity" principle. The pH for binding with cis-diol containing compounds of this monolithic capillary was decreased to neutral range due to the teamed bonorate affinity. The specific surface area was dramatically increased due to the use of the highly symmetrical copolymerizing reagents, which was 80.3 m2/g and apparently higher than those for other boronate affinity monolithic capillaries reported in references.

Enrichment of cis -diols active components in traditional Chinese medicines by boronate affinity monolithic column

A novel poly( 4-vinylphenylboronic acid (VPBA) -co-pentaerythritol triacrylate (PETA)) monolithic column was prepared by thermal-initiated copolymerization of VPBA and PETA in a binary porogen system comprising diethylene glycol and ethylene glycol. The fabricated monolithic column demonstrated good boronate affinity property, which can specifically capture cis-diols containing compounds. We developed such a monolithic column and applied it to enrich active components in Herba Taraxaci and Euonymus acanthocarpus. The extracts of the two traditional Chinese medicines were separated and enriched on microHPLC by the prepared monolith, and the fractions were collected and examined by HPLC. The results showed that the responses of the corresponding peaks were greatly improved, indicating that the poly( VPBA-co-PETA) monolithic column can be efficiently used to enrich active components in traditional Chinese medicines containing cis-diols group.

Preparation of organic-silica hybrid boronate affinity monolithic column for the specific capture and separation of cis-diol containing compounds

A new boronate-silica hybrid monolithic column was prepared using a one-pot approach with 3-acrylamidophenylboronic acid (AAPBA) as the boronate affinity ligand. The AAPBA-silica monolith exhibited several attractive advantages. First, it is highly hydrophilic, providing excellent specificity and avoiding the presence of organic solvent in the mobile phase. Second, due to its large surface area, it exhibited a high binding capacity, 49.5μmol/mL, the highest among the boronate affinity monolithic columns appeared in the literature. Third, the monolith can bind with cis-diol containing compounds at pH as low as 6.5, which not only avoids the use of basic pH conditions at which the silica monolith may hydrolysis but also facilitates the applications to wider sample range. Finally, the hybrid monolithic column exhibited apparent secondary separation capability, which allows for two-dimensional (2D) separation of cis-diol compounds in a single column. Due to these merits, the AAPBA-silica hybrid monolithic column can be a promising separation medium for the analysis of cis-diol containing compounds.

Synthesis and application of a macroporous boronate affinity monolithic column using a metal-organic gel as a porogenic template for the specific capture of glycoproteins

A macroporous boronate affinity monolithic column was prepared and applied to specifically capture glycoproteins using metal-organic gels (MOGs) as a porogenic template. This newly explored application of MOGs has proven to be a more convenient method for the formation of macropores in contrast to traditional porogenic methods. The poly (3-acrylamidophenylboronic acid-co-ethylene dimethacrylate) monolithic columns were synthesized in stainless columns by in situ polymerization. To fabricate the macroporous formation with a uniformed open-channel network, the preparation conditions, such as reaction temperature, the concentration of the MOGs and the ratio of monomers were systematically investigated. The prepared macroporous monoliths were characterized by scanning electron microscope (SEM) and mercury intrusion porosimetry. Furthermore, horseradish peroxidase (HRP) and transferrin (TF) were chosen as test glycoproteins, and the chromatographic analysis demonstrated that the macroporous boronate affinity monoliths exhibited a higher selectivity and better dynamic binding capacity toward glycoproteins compared with non-glycoproteins. The resulted affinity monolithic column was successfully employed to specifically capture TF from a bovine serum sample.