Monolithic Column Research Articles

Development of an organic polymer monolith column for the nano liquid chromatography fast analysis of monoclonal antibody in infusion bags prepared in a hospital pharmacy.

Poly(butyl methacrylate-co-ethylene dimethacrylate) monolith was in situ prepared in a liquid chromatography capillary column with a 75 μm internal diameter. This monolith offered high permeability (5.3 ± 10-14m2) and good peak capacity (140 for a 15 cm column length at 300 nl/min with a 20 min gradient time). This is exemplified by its separation ability in reversed mode for subunit analysis of monoclonal antibodies after IdeS digestion (middle-up analysis). The potential of this column was also illustrated for the fast analytical control of therapeutic monoclonal antibodies in standardized infusion bags prepared in advance in a pharmacy department. Linearity analysis revealed the column's capability for accurate quantification analysis of the different dose bandings (in mg) of monoclonal antibodies in <2min. In addition, lifetime analysis data indicated that the column can be highly reproducible and has a long lifetime with stable and low back pressure. The variations observed on the peak shape and area between unstressed (intact) and stressed monoclonal antibodies indicated that our nano liquid chromatographic method was stability indicating. In addition, using a gradient elution mode, the presence of minor components in the infusion bags was visualized.

Construction of adamantane-based monolithic column with three-dimensionally porous structure for small molecules separation and biosample analysis

BackgroundThe fabrication technique of capillary column is the key to the development and application of capillary liquid chromatography (cLC) to improve separation efficiency for analytes. The capillary monolithic column possessed three-dimensionally connected porous or channel structures. Unique porous structure endows excellent permeability and high performance in diverse fields, especially in separation. Thereinto, organic monolithic columns have attracted widespread attention due to their advantages of simple preparation and excellent biocompatibility. However, their separation selectivity needs to be further developed and regulated to apply the separation of more diverse samples. ResultsA novel polymeric monolithic column was prepared via thermally initiated in situ copolymerization of 2-methyladamantan-2-yl acrylate (MADA) with ditrimethylolpropane tetraacrylate (DTTA) in fused silica. The prepared poly(MADA-co-DTTA) monolith showed adjustable permeability, developed porous structure and high thermal stability. Consequently, it exhibited excellent separation capability of small molecules (alkylbenzenes and polycyclic aromatic hydrocarbons). Especially, when acetonitrile/water (60/40, v/v) was used as the mobile phase, the theoretical plate numbers reached 84,000 plates m−1 for butylbenzene at a linear velocity of 0.5 mm s−1. Most importantly, the hydrophobicity of the poly(MADA-co-DTTA) monolithic column was regulated via host-guest interaction between adamantyl group and cucurbit [7]uril (CB[7]). Additionally, the poly(MADA-co-DTTA) monolith was further adopted for the analysis of the tryptic digest of proteins from HeLa by cLC-MS/MS. The 33,783 unique peptides and 5,299 proteins were identified on the monolith, which exhibited great separation ability for complex samples. Significance and noveltyDue to abundant pore structure and good chemical properties, the poly(MADA-co-DTTA) monolithic column exhibited high performance for the separations of small molecules and biological sample. Meanwhile, owing to the existence of adamantyl-group, CB[7] was immobilized on the poly(MADA-co-DTTA) monolithic column to fabricate poly(MADA-co-DTTA)-CB[7] by host-guest interaction. It is possible to adjust the surface chemistry of the monolithic materials to accommodate more complex analytes.

Quantitative analysis of lipids and nucleic acids in lipid nanoparticles using monolithic column

Quantitative analysis of lipids and nucleic acids in lipid nanoparticles using monolithic column

Development of an immobilized laccases - CDI CIMmultus® monolithic reactor for ecofriendly producing gallic acid-dextran conjugate

Enzymatic modification of polysaccharides is an efficient and environmentally friendly method to enhance their bioactivities. Immobilized enzymes represent a technology that enables the reuse of enzymes and increase their stability. In this study, laccases from Trametes versicolor were covalently immobilized on CIMmultus® CDI monolithic column, which was employed to graft gallic acid (GA) onto the backbone of T40 dextran. 1.43 mg laccases were successfully immobilized, yielding a mass conversion rate of 32.17 %. The kinetic parameters of free and immobilized laccases were respectively 0.0056 and 0.1770 U for Vmax and 0.0121 and 0.7510 mmol·l−1 for Km. Remarkably, 91.29 % of the enzymatic activity of immobilized laccases was preserved after catalyzing 1200 ml of reactant. A central composite design was carried out to investigate the influence of T40 dextran concentration and flow rate on the phenolization. Response surface methodology was used for predicting the optimal conditions, showed that a 2.07 % mass ratio of phenolization could be achieved with 2 % dextran and a flow rate of 0.89 ml·min−1. The GA-dextran conjugate, produced under the best conditions, revealed a DPPH radical inhibition activity of 63.97 % and an IC50 of 73.39 µg·ml−1. This work describes a new IMER for ecofriendly producing phenolized polysaccharides.

A Robust Chromatographic Method for Drug Release profiling of liposomal doxorubicin HCl

Liposomes are excellent drug delivery vehicles for chemotherapeutics as they may change the pharmacokinetics of therapeutic compounds, resulting in altered tissues distribution, and in some cases, reduced cytotoxicity and enhanced distribution and efficacy of the active pharmaceutical ingredient (API) at target tissues. Drug release profiles of liposomal formulations are crucial to support equivalence evaluation and quality control in pre- and post-approval stages. We developed an automated chromatographic method for quantifying the drug release profile of liposomal formulations containing doxorubicin to overcome the shortcomings of currently available methods. The newly developed method employs nanoparticle exclusion chromatography (nPEC), using a monolithic silica column coated with polyvinylpyrrolidone to separate the released drug from liposomal encapsulated drug. We evaluated the effects of pH, temperature, and ammonium formate concentration on the drug release rate. The optimized release buffer consisting of 5 % sucrose, 20 mM l-histidine, and 200 mM ammonium formate was selected for the drug release profiling of five liposomal formulations at 47 °C. The drug release profiles of five liposomal doxorubicin formulations were similar. Our automated method requires very small amounts of the sample and provides release profiles with high sensitivity and accuracy. In addition, this method can be applied to other liposomal products to allow for simple, fast, and accurate analysis of in vitro drug release profiling.

Seismic performance of an assembled monolithic concrete column

A prefabricated concrete column-to-column structure connected by high-strength bolts is proposed. To explore the seismic performance and failure form of the structure, the finite element analysis software is used to simulate the low-cycle repeated load test. The hysteretic curve and skeleton curve of the structure are obtained, and the stiffness degradation law, ductility and energy dissipation capacity of the structure are quantitatively analyzed to evaluate the seismic performance of the structure. The results show that the ductility coefficient of the structure is basically between 2.19 and 4.97, and the ductility coefficient of most specimens is greater than 3.0, indicating that the structure has good ductility and deformation capacity. The equivalent viscous damping coefficients of the specimens are in the range of 0.21–0.29, indicating that the structure has good energy dissipation capacity. In a certain range, increasing the thickness of the end plate has no significant impact on the energy consumption capacity of the structure. The compressive strength of the concrete in the core area is greatly improved under the constraint of the end plate bolts, and end plate bolt connection can well realize the transmission of force and give full play to the performance of materials.

Polymeric monolithic columns based on natural wood for rapid purification of targeted protein

With multiscale hierarchical structure, wood is suitable for a range of high-value applications, especially as a chromatographic matrix. Here, we have aimed to provide a weak anion-exchange polymeric monolithic column based on natural wood with high permeability and stability for effectively separating the targeted protein. The wood-polymeric monolithic column was synthesized by in situ polymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in wood, and coupled with diethylaminoethyl hydrochloride. The wood-polymeric monolithic column can be integrated with fast-protein liquid chromatography for large-scale protein purification. According to the results, the wood-polymeric monolithic column showed high hydrophilicity, permeability and stability. Separation experiments verified that the wood-polymeric monolithic column could purify the targeted protein (spike protein of SARS-COV-2 and ovalbumin) from the mixed proteins by ion exchange, and the static adsorption capacity was 33.04 mg mL−1 and the dynamic adsorption capacity was 24.51 mg mL−1. In addition, the wood-polymerized monolithic column had good stability, and a negligible decrease in the dynamic adsorption capacity after 20 cycles. This wood-polymerized monolithic column can provide a novel, efficient, and green matrix for monolithic chromatographic columns.

Significant Waste Reduction in Liquid Chromatography by a Reusable Large-Volume Monolithic Silica Column

Significant Waste Reduction in Liquid Chromatography by a Reusable Large-Volume Monolithic Silica Column

On the modelling of the effective longitudinal diffusion in bi-continuous chromatographic beds

We report on the possibility to extend to bi-continuous packings the two models for the effective longitudinal diffusion Deff, or B-term band broadening, recently proposed for discontinuous chromatographic beds. In bi-continuous packings, like monolithic columns, solutes experience a connected end-to-end pathway in both the mobile and stationary zones, as opposed to discontinuous packings, wherein the stationary adsorptive zone is distributed over a set of isolated elements. Since it is unclear whether a densely packed bed of spherical particles should be treated as a continuous or a bi-continuous medium, this extension is also crucial to fully understand the behaviour of packed particle beds. The proposed models for the effective longitudinal diffusion Deff originate from the adoption of the Two Zone Moment Analysis (TZMA) method by which Deff can be expressed as a linear combination of two essential quantities γm and γs, referred to as effective zone-diffusion factors. In the present work we propose two analytical models for γm and γs that now cover both the discontinuous and the bi-continuous case. To validate the theory, several bi-continuous packings are investigated, including the tetrahedral skeleton model (TSM), six different Triple Periodic Minimal Surface (TPMS) monoliths and randomly packed beds of spheres. For all of these, the models provide highly accurate results for Deff over a wide range of porosities and zone retention factors k″. The comparison with literature experimental data for both monolithic silica columns and columns packed with fully porous and porous-shell particles is also presented.

Selective hydrophobic interaction chromatography for high purity of supercoiled DNA plasmids.

High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material.

Determination of ω-end functionalities in tailored poly(2-alkyl-2-oxazoline)s by liquid chromatography and mass spectrometry.

The in-depth analytical characterization of polymers, in particular regarding intended biomedical applications, is becoming increasingly important to elucidate their structure-property relationships. Specifically, end group analysis of e.g. polymers featuring a 'stealth effect' towards the immune system is of particular importance because of their use in coupling reactions to bioactive compounds. Herein, we established a liquid chromatography (LC) protocol to analyse bicyclo[6.1.0]nonyne-functionalized poly(2-alkyl-2-oxazoline)s (POx)s as promising functional polymers that can be applied in strain-promoted click reactions. This work involved the synthesis of poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) by living cationic ring-opening polymerization (CROP) with different molar masses ranging from 2 up to 17.5 kDa and, to our knowledge, the first liquid chromatographic analysis of PMeOx. The developed analytical protocol enables the quantitative determination of post-polymerization reaction sequences with respect to the conversion of the ω-end groups. All synthesized polymers were straightforwardly analysed on a C18-derivatized silica monolithic column under reversed-phase chromatographic conditions with a binary mobile phase gradient comprising a mixture of acetonitrile and water. Subsequent mass spectrometry of collected elution fractions enabled the confirmation of the desired ω-end group functionalities and the identification of synthetic by-products.

Improved mRNA affinity chromatography binding capacity and throughput using an oligo-dT immobilized electrospun polymer nanofiber adsorbent

Increased demand for mRNA-based therapeutics and improved in vitro transcription (IVT) yields have challenged the mRNA purification platform. Hybridization-affinity chromatography with an immobilized oligo-deoxythymidilic acid (oligodT) ligand is often used to capture mRNA through base pairing with the polyadenylated tail. Commercially available oligodT matrices include perfusive cross-linked poly(styrene-divinylbenzene) 50 µm POROS™ chromatography resin beads and convective polymethacrylate CIMmultus® monolithic columns consisting of 2 µm interconnected channels. POROS™ columns may be limited by poor mass transfer for larger mRNAs and slow flowrates, while monoliths can operate at higher flowrates but are limited by modest binding capacity. To enable both high flowrates and binding capacity for mRNA of all lengths, prototype chromatography media was developed by Cytiva using oligodT immobilized electrospun cellulose nanofibers (Fibro™) with a 0.3–0.4 µm pore size. In this work, four polyadenylated mRNAs ranging from ∼1900–4300 nucleotides were used to compare the dynamic binding capacity (DBC) of Fibro™, POROS® and CIMmultus® columns as a function of residence time and binding buffer compositions. Fibro™ improved the DBC ∼2–4-fold higher than CIMmultus® and ∼2–13-fold higher than POROS™ across all residence times, mRNA length, and binding matrix compositions tested. CIMmultus® DBC was least dependent on residence time and mRNA size, while both Fibro™ and POROS™ DBC increased at slower flowrates and with shorter mRNA. Surprisingly, inverse size exclusion (ISE) experiments showed that POROS™ was not limited by diffusion and POROS™ along with CIMmultus® demonstrate higher mRNA permeation however the Fibro™ prototype is not in the final configuration. Lastly, IVT reaction products were subjected to purification and oligodT elution product yield, quality, and purity were consistent across the three matrices investigated. These results highlight the benefits of high DBC and equivalent product profiles offered by the oligodT Fibro™ prototype compared to commercially available oligodT media.

Direct Analysis of Whole Blood by a Disposable Monolithic Column Mass Spectrometry Analysis Kit†

Comprehensive SummaryA monolithic column‐based mass spectrometry (MS) analysis kit was prepared for whole blood analysis with MS. The kit is disposable and can be used for purification, storage, transportation and direct analysis of whole blood. The kit mainly consists of a capillary for quantitative microsampling, a cation exchange monolithic column for purification and storage, and a syringe for loading sample. This kit is very friendly to various users that one can easily siphon the blood in the kit followed by rapid clean‐up. We established a quantitative method using the kit with a limit detection as low as 0.33 nmol/L, and achieved more than five orders of magnitude enhancement in sensitivity compared to direct nanoelectrospray ionization MS analysis. The column can avoid analyte exposure to environment, which helps the storage of the sample for laboratory analysis. The relative standard deviation of immediate blood analysis and storage blood analysis within 10 d was less than 10%. This method has been successfully applied to the quantitative analysis of procainamide hydrochloride in 2 μL rat blood. These results indicate that this disposable kit does have the potential to achieve highly sensitive quantitative MS analysis in biological samples, which is expected to become a cost‐effective and powerful tool for in vitro diagnostics.

A novel chiral monolithic nano-column with 50 µm i.d. for the enantioseparation of chiral drugs by nano-liquid chromatography

A novel chiral monolithic nano-column was developed for the enantioseparation of chiral drugs by nano-liquid chromatography (nano-LC). The monolithic capillary with 50 µm i.d. was first prepared by an in-situ polymerization using butyl methacrylate (BuMA), ethylene dimethacrylate (EDMA) and NFMoc-Lysine modified GO nanoparticles. The final monolithic stationary phase was characterized by using FT-IR spectra of synthesized structures, scanning electron microscopy (SEM) and chromatographic analyses. After characterization, the prepared chiral monolithic column was investigated for the chiral separation of pharmaceutical racemates, including anti-inflammatory drugs (e.g., ketoprofen, ibuprofen, naproxen, etodolac and flurbiprofen) and β-blockers (e.g. propranolol, metoprolol and atenolol). It was demonstrated that NFMoc-Lysine modified GO nanoparticles incorporated chiral monolith could be successfully developed while this chiral monolith allowed sensitive chiral separation of chiral drugs. The chiral separation could be mostly achieved via the formation of π-π interactions as well as hydrophilic interactions between chiral stationary phase and enantiomers

Rapid preparation of a glycan oxazoline and a homogeneously glycosylated antibody with an enzyme-immobilized monolithic column

Chemo-enzymatic glycan engineering is considered to be one of the most promising strategies to enhance efficiency in pharmaceutical research. However, it is assumed that this technology has limited industrial application for the production of biological therapeutics because of the high cost of the process. In this study, we developed a scheme for rapidly preparing a glycan oxazoline and a homogeneously glycosylated antibody. The enzyme-immobilized monolith and the flow chemistry-based approach enabled a glycan oxazoline and a homogeneously glycosylated antibody to be obtained at the gram scale from starting materials (sialylglycopeptide and heterogeneously glycosylated protein) within 2.5 h. This cost-effective scheme for obtaining a large amount of glycan donors and homogeneously glycosylated proteins in a short time will be helpful to implement glycan engineering technology for industrial purposes such as pharmaceutical production.

Self-Centering of RC Columns with Prestressed Fe-SMA Reinforcement

Severe seismic events often lead to detrimental residual displacements in traditional monolithic bridge columns, resulting in serviceability issues and substantial repair costs. Conversely, post-tensioned segmental columns display beneficial self-centering behavior and minimal residual drifts owing to the post-tensioning force and rocking behavior at segment joints. Despite these advantages, such structures exhibit limited energy dissipation capacities and require heavy mechanical equipment for prestressing through conventional tendons. This paper discusses the feasibility of using prestressed Fe-SMA reinforcement for self-centering of existing and new bridge columns. Firstly, the results of an experimental study on the self-centering of existing monolithic columns are presented, followed by a numerical study on the feasibility of using Fe-SMA for the self-centering of segmental columns. A 3D finite element (FE) model, developed in ABAQUS, was validated against experimental results to evaluate the seismic performance of the proposed column system. The validated model was then utilized for a focused investigation into the influence of the ratio of energy dissipating (ED) bars to Fe-SMA bars, bond condition of Fe-SMA bars, and concrete confinement of segments on the self-centering and energy dissipation behavior of prestressed segmental columns. The results highlight the ability of Fe-SMA bars to provide effective self-centering and energy dissipation for existing and new bridge columns due to their self-prestressing and high ductility.

Chiral metal-organic frameworks grown in situ for monolithic capillary electrochromatographic enantioseparation.

Chiral metal-organic frameworks (CMOFs) with chiral selectivity are one of the high-quality stationary phases for capillary electrochromatography (CEC). However, there is a problem of unsatisfactory enantioseparation performance of capillary columns due to insufficient loading. In this work, a lamellar CMOF (Cu-TC) was grown in situ on the surface of the monolith in a capillary monolithic column to obtain a Cu-TC@monolithic column. The CEC system constructed based on the Cu-TC@monolithic column shows a satisfactory chiral separation performance. Compared with the Cu-TC-based coated column (Cu-TC@coated column), the enantioseparation performance of the CEC system based on the Cu-TC@monolithic column was greatly improved, and the resolutions (Rs) of the model analytes were increased by 80-500%. In addition, the effects of experimental conditions such as the number of cycles of Cu-TC in situ growth, buffer concentration, buffer pH, organic solvent addition and applied voltage on the performance of CEC were also investigated. Finally, the chiral selection mechanism of the stationary phase was explored by selective adsorption experiments. The present work provides a new idea for the development of capillary stationary phases, which has great potential considering the diversity of CMOFs.

A green liquid chromatographic method using ethanol in mobile phase for the determination of nimesulide and naproxen in gel formulations

Dеtеrmination of nonsteroidal anti-inflammatory drugs (NSAIDs) in pharmacеutical formulations hеlps to еnsurе that thеy arе manufacturеd to thе corrеct spеcifications. Consequently, simple, reliable, and environmentally friendly analytical methods are needed for the quality control of NSAID formulations. In this study, a novel and green liquid chromatographic method was developed for the determination of nimesulide (NIM) and naproxen (NAP) in gel formulations by employing ethanol as a green alternative to hazardous methanol and acetonitrile. The high viscosity of ethanol was compensated by employing a Chromolith HighResolution RP18e monolithic column (100 × 4.6 mm) with low flow resistance. Box-Behnken design with desirability function was employed for the optimization of selected significant parameters: pH (2.8-4), ethanol ratio in the mobile phase (35-55%), and flow rate (0.7-1.5 mL/min). Under optimum conditions, satisfactory separation of analytes was achieved within 5 min. Calibration curves for both analytes were linear between 1-50 μg/mL. Accuracies of intra- and inter-day experiments at low-, middle-, and high-quality control levels ranged from 99.0-101.5% with relative standard deviation values lower than 2.3%. The limits of detection were 0.27 and 0.62 µg/mL for NIM and NAP, respectively. The applicability of the method was demonstrated by analyzing gel formulations. The results of this study indicated that monolithic columns in combination with ethanol as a mobile phase component could be considered a desirable and green alternative for the routine analysis of NSAIDs in quality control laboratories.

Ductility of Precast Concrete Column Beams Using Dowels

Precast concrete structures are considered as monolithic structures, flexural reinforcement and beam column joint shear can be planned based on design capacity. This research is an experimental study. The objective is to assess the ductility of precast concrete column beams utilizing dowels. The test specimens comprise three configurations: monolithic beam column (BN), 2 post precast beam column (BG 1), and 4 post precast beam column (BG 2), with respective displacement values of 37.4 mm, 42.7 mm, and 47 mm. This research was carried out in the stages of calculating displacement ductility using displacement parameters, and curvature ductility using parameters of cross-sectional curvature or curvature values and load values due to cyclic loads. The research results show that the displacement ductility values for test specimens BN, BG 1 and BG 2 are 2.34, 2.21 and 2.06 respectively. The curvature ductility value for test specimens BN, BG 1 and BG 2 is more than the value required in ASCE 41-17, namely 0.01 in the LS condition and in the CP condition it is 0.015. Additionally, these values surpass the stipulated requirements. This leads to the conclusion that beam column connections, whether monolithic or precast, exhibit exceptional performance in withstanding earthquake loads under both moderate seismic conditions (moderate earthquakes) and severe seismic conditions (strong earthquakes)

Itaconic Acid-Based Organic-Polymer Monolithic Column for Hydrophilic Capillary Electrochromatography and Its Application in Pharmaceutical Analysis.

Itaconic acid is an excellent hydrophilic monomer owing to the dicarboxylic group possessing strong polarity. This study reports on the preparation of a new organic-polymer monolithic column poly(itaconic acid-co-3-(acryloyloxy)-2-hydroxypropyl methacrylate) (poly(IA-co-AHM)) featuring excellent hydrophilic chromatography ability and its application in pharmaceutical analysis. The monolithic column was successfully synthesized by using the monomer itaconic acid and the cross-linker AHM through an in situ copolymerization method. Optical microscopy, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) were employed for the characterization of the poly(IA-co-AHM) monolithic column, and all of these demonstrated that the prepared itaconic acid-based monolithic column exhibited satisfactory permeability and a homogeneous porous structure. Owing to the carboxylic groups of itaconic acid, a cathodic electroosmotic flow (EOF) was generated on the itaconic acid-based monolithic column among the pH ranges of the mobile phase from 4.0 to 9.0. Depending on the powerful hydrophilic interactions, different kinds of polar substances, including thioureas, nucleoside drugs, sulfonamides, and polypeptides, were separated efficiently by the itaconic acid-based monoliths poly(IA-co-AHM). The separations of polar compounds were successfully realized, even at a lower level of 50% acetonitrile content on this monolithic column. The highest column efficiencies corresponding to N,N'-dimethylthiourea and idoxuridine were 102 720 and 124 267 N/m, respectively. The poly(IA-co-AHM) monolithic column displayed excellent repeatability, whose relative standard deviations (RSDs) of the retention time and peak area were both lower than 5.0%. All experimental results demonstrated that the new itaconic acid-functionalized monolithic column was greatly appropriate to separate the polar compounds under the HILIC mode.