Neutral Analytes Research Articles

Novel chiral stationary phases based on 3,5-dimethyl phenylcarbamoylated β-cyclodextrin combining cinchona alkaloid moiety.

Novel chiral selectors based on 3,5-dimethyl phenylcarbamoylated β-cyclodextrin connecting quinine (QN) or quinidine (QD) moiety were synthesized and immobilized on silica gel. Their chromatographic performances were investigated by comparing to the 3,5-dimethyl phenylcarbamoylated β-cyclodextrin (β-CD) chiral stationary phase (CSP) and 9-O-(tert-butylcarbamoyl)-QN-based CSP (QN-AX). Fmoc-protected amino acids, chiral drug cloprostenol (which has been successfully employed in veterinary medicine), and neutral chiral analytes were evaluated on CSPs, and the results showed that the novel CSPs characterized as both enantioseparation capabilities of CD-based CSP and QN/QD-based CSPs have broader application range than β-CD-based CSP or QN/QD-based CSPs. It was found that QN/QD moieties play a dominant role in the overall enantioseparation process of Fmoc-amino acids accompanied by the synergistic effect of β-CD moiety, which lead to the different enantioseparation of β-CD-QN-based CSP and β-CD-QD-based CSP. Furthermore, new CSPs retain extraordinary enantioseparation of cyclodextrin-based CSP for some neutral analytes on normal phase and even exhibit better enantioseparation than the corresponding β-CD-based CSP for certain samples.

Simultaneous separation and concentration of neutral analytes by cyclodextrin assisted sweeping-micellar electrokinetic chromatography.

An on-line cyclodextrin assisted sweeping-micellar electrokinetic chromatography (CD assisted sweeping-MEKC) was developed for the simultaneous separation and concentration of four neutral analytes (erianin, dendrophenol, naringenin and scoparone) in Dendrobium officinale Kimura et Migo (D.officinale). The D.officinale was directly determined by this on-line stacking method after simple extraction and dilution. The optimized background solution (BGS) was 50mM phosphoric acid (PA) containing 100mM SDS and 30% (v/v) methanol. The best separation and concentration performance of analytes dissolved in 90mM CD and 100mMPA was achieved in a short analysis time when injected at 50mbar for 100s. Compared with conventional sweeping-MEKC and MEKC method, significant improvement in enrichment efficiency was achieved by using this proposed method. A series of validation studies of the present method was performed under the optimal conditions. Good linearities were obtained with the correlation coefficients in the range of 0.994-0.999, the detection limits were ranged from 13 to 40ng/mL. Sensitivity enhancement factors (SEFs) were in the range of 28.5-46.8 compared with traditional injection (injection time 3s). Therefore, the proposed method was successfully applied for the separation and concentration of neutral analytes in real samples.

A sensitive two-step stacking by coupling field-enhanced sample injection and micelle to cyclodextrin stacking for the determination of neutral analytes

To promote the detection sensitivity of neutral analytes, a sensitive two-step stacking strategy by coupling field-enhanced sample injection and micelle to cyclodextrin stacking in reverse migrating micelles MEKC has been established by selecting jujuboside A and B as models. The stacking mechanism, affecting factors as well as analytical performances of the proposed method were investigated. Compared with typical injection, the sensitivity enhancement factors of this method were up to 140- and 152-fold for jujuboside A and B, respectively. The enhancements were 8–10 times better than only single stacking strategy by MCDS. The LODs were 0.2–0.3 μg mL−1, interday and intraday%RSD were both lower than 4.3%, and a good linearity (r>0.999) was obtained. Feasibility in real samples analysis was evaluated by using Semen Ziziphi Spinosae dispensing granule and rat's urine. The results suggested that the strategy was simple, reliable, sensitive and promising for the analysis of neutral analytes in complex sample matrix.

Open-tubular Capillary Electrochromatography with Janus Structured Au-Fe3O4 Nanoparticles Coating as Stationary Phase.

A novel Au-Fe3O4 nanoparticles-based capillary column was fabricated by magnetic approach for open-tubular capillary electrochromatography (OT-CEC). The bifunctional dumbbell-like Janus Au-Fe3O4 nanoparticles (Au-Fe3O4 NPs) were prepared through a hydrothermal synthesis strategy, and the morphology was characterized by transmission electron microscopy (TEM). Multilayers Au-Fe3O4 NPs were easily coated onto the inner surface of silica capillary by an external magnetic field to generate an Au-Fe3O4 NPs-based column. Compared with a bare capillary, the modified surface exhibited more stable and suppressed electroosmotic mobility. The column showed good separation efficiency for neutral analytes in the OT-CEC separation mode, with theoretical plate numbers of up to 79705 per meter for naphthalene. The successful separation of dihydroxy benzene isomers and proteins demonstrated that the column exhibits a reasonable separation performance. The reproducibility of the Au-Fe3O4 NPs capillary was studied, with relative standard deviations (RSD) for day-to-day and column-to-column less than 1 and 1.75%, respectively.

Cover feature: Analyte focusing by micelle collapse for liquid extraction surface analysis coupled with capillary electrophoresis of neutral analytes on a solid surface

Cover feature: Analyte focusing by micelle collapse for liquid extraction surface analysis coupled with capillary electrophoresis of neutral analytes on a solid surface

Mass Spectrometry Based Approach for Organic Synthesis Monitoring.

Current mass spectrometry-based methodologies for synthetic organic reaction monitoring largely use electrospray ionization (ESI), or other related atmospheric pressure ionization-based approaches. Monitoring of complex, heterogeneous systems may be problematic because of sampling hardware limitations, and many relevant analytes (neutrals) exhibit poor ESI performance. An alternative monitoring strategy addressing this significant impasse is condensed phase membrane introduction mass spectrometry using liquid electron ionization (CP-MIMS-LEI). In CP-MIMS, a semipermeable silicone membrane selects hydrophobic neutral analytes, rejecting particulates and charged chemical components. Analytes partition through the membrane, and are then transported to the LEI interface for sequential nebulization, vaporization, and ionization. CP-MIMS and LEI are both ideal for continuous monitoring applications of hydrophobic neutral molecules. We demonstrate quantitative reaction monitoring of harsh, complex reaction mixtures (alkaline, acidic, heterogeneous) in protic and aprotic organic solvents. Also presented are solvent-membrane compatibility investigations and, in situ quantitative monitoring of catalytic oxidation and alkylation reactions.

Fast super/subcritical fluid chromatographic enantioseparations on superficially porous particles bonded with broad selectivity chiral selectors relative to fully porous particles

Superficially porous particles (SPPs) have shown advantages in enantiomeric separations in HPLC by conserving selectivity while providing higher efficiency separations with significantly reduced analysis times. The question arises as to whether the same advantages can be found to the same extent in super/subcritical fluid chromatography. In this work, the low viscosity advantage of carbon dioxide/MeOH mixtures is coupled with high-efficiency 2.7 μm superficially porous particles for enantiomeric separations. Given the fact that the viscosity of the mobile phase is typically ten times lower than liquid mobile phases it is possible to use flow rates as high as 14 mL/min on 5 cm packed columns. Superficially porous particles (SPPs) were grafted with teicoplanin (TeicoShell), a chemically modified macrocyclic glycopeptide (NicoShell), vancomycin (VancoShell), and isopropyl derivatized cyclofructan-6 (LarihcShell-P). One hundred chiral analytes were separated in a very short time frame, as little as 0.2 min (13 s). Even shorter separations can be obtained with advances in SFC instrumentation. The LarihcShell-P is the only chiral crown ether-based selector which showed high selectivity for primary amines. The Teicoshell column offered unique separations for acidic and neutral analytes. The NicoShell and the VancoShell were useful in separating amine (secondary and tertiary) containing pharmaceutical drugs and controlled substances. By chemically modifying a macrocyclic glycopeptide (NicoShell) we report the first enantiomeric separation of nicotine under SFC conditions within 3 min with a resolution of >3. Additionally, van Deemter plots are constructed comparing the fully porous particles and superficially porous particles bonded with the same chiral selectors. In toto the SPP advantages also were found for SFC. However instrumental shortcomings involving extra column effects and pressure limitations need to be addressed by instrument manufacturers to realize the full advantages of SPPs and other smaller particle supports.

One-pot preparation of zwitterionic sulfoalkylbetaine monolith for rapid and efficient separation of lysozyme from egg white

A porous zwitterionic monolithic column was prepared to rapidly and efficiently separate lysozyme from egg white. The monolith was synthesized in a stainless steel HPLC column (5 cm × 4.6 mm i.d.) by in-situ thermal initiated co-polymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (MSA) and ethylene dimethacrylate (EDMA). Due to the combination of quaternary ammonium and sulfonic groups on the monolithic matrix in one-pot process, the hydrophobic carbon chain and hydrophilic radical were obtained, which provided multiple driving forces for neutral, basic and acidic analytes, thus mix-mode chromatography mechanism contributed to the retention of different charged proteins. Properties such as composition, morphology and stability of the MSA-co-EDMA monolithic column were characterized by various analytical methods and the results showed that the monolith has large through-pores, good hydrophilicity and permeability. The effects of mobile phase pH and ionic strength on proteins were investigated, drawing the conclusion that the main adsorption and elution mechanism of lysozyme on MSA-co-EDMA monolith was electrostatic interaction, while those of other proteins included hydrophobic, hydrophilic and electrostatic interactions. Therefore, efficient separation of lysozyme and other proteins could be successfully achieved by switching the pH of mobile phase. Lysozyme can be adsorbed using 20 mmol/L phosphate buffer (pH 7.0) and eluted with 20 mmol/L phosphate buffer (pH 2.0). To prove the practicality of the monolithic column, it was also applied in the separation of lysozyme in egg white, which means the work has the potential for further development in proteome analysis of real biological samples.

Chirality Discrimination at the Single Molecule Level by Using a Cationic Supermolecule Quasi-Gated Organic Field Effect Transistor.

Achieving rapid and highly sensitive small molecule chiral discrimination is a great challenge in modern-day analytical sciences. Herein, an organic field effect transistors (OFET) is developed by employing an imidazolium 3,5-dimethylphenylcabamoylated-β-cyclodextrin (Im+-Ph-β-CD) as both the recognition unit and a quasi gate, which induces a secondary accumulation channel of electrons in the n-type transistor to achieve the signal transduction and amplification via field effect. The charge of the imidazolium group is partially shielded due to its self-inclusion in the CD cavity, and this shielding effect is reduced at varying degrees in the existence of isomers due to the competitive inclusion. Consequently, the different weak intermolecular interactions related to the target-induced CD-enantiomer complexation with different geometry and stabilization energy for each isomer can be transformed to electronic signals based on the variety of Im+-Ph-β-CD's effective charge rather than the intrinsic charge of analytes, hence leading to chiral differentiation, and the hydrogen-bonding network of Im+-Ph-β-CD membrane further magnifies the signal. This working strategy even allows chiral discrimination of electrically neutral analytes. The as-prepared sensor affords rapid and real-time discrimination to small molecule enantiomers at single molecule level with a limit of detection of 8.1 × 10-19 M in a 200 μL volume (about 100 small molecules). Moreover, we prove the great potential of the chiral organic field effect transistor in quantitative analysis of commercial medicines.

Analyte focusing by micelle collapse for liquid extraction surface analysis coupled with capillary electrophoresis of neutral analytes on a solid surface.

Liquid extraction surface analysis (LESA) has an advantage of directly sampling analytes on a surface, thus avoiding unnecessary dilution by homogenization of the bulk sample commonly practiced in solid sample analysis. By combining LESA with CE, the additional advantage of separating analytes before detection can be accomplished. For neutral molecules, MEKC needs to be used. Since the detection sensitivity of CE in general suffers from the small capillary dimension, analyte focusing by micelle collapse was employed for enhanced extraction in LESA and sample preconcentration for MEKC. In addition, using a commercial CE instrument, the LESA process was performed much faster and more reliably compared to our first demonstration of LESA-CE using a homemade CE setup. Three neutral water-insoluble pesticides sprayed on an apple skin were directly extracted, preconcentrated, and analyzed by the automated LESA-analyte focusing by micelle collapse-MEKC with high sensitivity in 10min. The relative standard deviations of the migration times and peak heights were 0.8-2.1 and 1.2-3.0%, respectively when ametryn was used as an internal standard. The limits of detection obtained with UV absorbance at 200nm were 1.8-6.4 ppb.

Admicelles in open-tube capillaries for chromatography and electrochromatography

Surfactant bilayers or admicelles at the solid surface-liquid interface inside 50–200 μm inner diameter (i.d.) open-tube fused-silica capillaries were developed as ‘soft’ stationary pseudophases for the liquid chromatographic (LC) separations of neutral and charged analytes. Admicelles were formed in-situ from buffered aqueous mobile phases with cetytrimethylammonium bromide at concentrations between the critical surface aggregation concentration and critical micelle concentration, which were determined by electroosmotic flow measurements using capillary electrophoresis. There were no micelles in the mobile phase solution. Also, there was no solid phase that is classically required in LC. Pressure and voltage driven modes or open-tubular admicellar liquid chromatography (OT-AMLC) and electrochromatography, respectively were proposed based on the separation of neutral analytes. The parameters (i.e., pH, concentration of surfactant, salt, and methanol in the mobile phase and capillary i.d.) that affected the surprising chromatographic effect of admicelles at the interface were investigated. The analytical performance of OT-AMLC for small molecules were found acceptable. Applications to environmental water and biological (HepG cell line metabolism media) samples analysis with appropriate sample preparation procedures were also conducted. The use of pseudophases at the solid surface-liquid interface could be a viable solution to problems associated with the use of solid stationary or support materials in nano- and micro-liquid chromatography and electrochromatography.

Dual enantioselective LC–MS/MS method to analyse chiral drugs in surface water: Monitoring in Douro River estuary

This work presents the development of an enantioselective method to quantify chiral drugs (CDs) in surface water and its application in the Douro River estuary monitoring. Different classes of CDs were targeted, including 23 compounds, namely beta-blockers, antidepressants, one beta2-adrenergic agonist, non-steroidal anti-inflammatory drugs, stimulants, and some illicit drugs as cocaine (COC) and its metabolites, and amphetamines. The analytical method was based on an innovative application of solid phase extraction (SPE), followed by liquid chromatography-tandem mass spectrometry (LC–MS/MS) using a triple quadrupole analyzer. The ground-breaking approach of SPE consists in the use of Oasis® MCX cartridges to pre-concentrate 500 mL of water samples, allowing the simultaneous extraction of acidic, basic and neutral analytes, rather than the conventional recovery of basic compounds only. Two chiral columns were used for enantiomeric separation in reverse elution mode, a Chirobiotic™V and a Pirkle type Whelk-O®1, for basic and acidic compounds, respectively. The method validation demonstrated good linearity (r2 > 0.99), selectivity and sensitivity, with method detection limits between 0.01 and 2.66 ng L−1 and method quantification limits between 0.02 and 5.71 ng L−1. The developed method was successfully applied to monitor daily variations along one week in surface waters collected in 5 locations of the Douro River estuary. Tramadol (TRM) and its metabolite N-desmethyltramadol (NDT), presented high concentrations near the affluent of a tributary river, while the second eluted enantiomer of O-desmethyltramadol (ODT) was found at high concentrations at the mouth of the Douro River. The metabolite NDT was quantified at higher concentrations than TRM. Venlafaxine (VNF) was found at high concentrations near the affluent of the same tributary river, but its metabolite, O-desmethylvenlafaxine (ODV), was found at concentrations 3 times higher. COC was found every day at all sampling points along the estuary, with slight variations.

Preparation of a monolithic column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction for capillary liquid chromatography

A monolithic capillary column with a mixed-mode stationary phase of reversed-phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in-situ copolymerization of a homemade monomer N,N-dimethyl-N-acryloxyundecyl-N-(3-sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed-mode stationary phase. It was demonstrated that the mixed-mode stationary phase displayed typic dual retention mechanisms of reversed-phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.

Separition of steroid hormones by microemulsion electrokinetic chromatography involving ionic liquids

Microemulsion electrokinetic chromatography (MEEKC) is a promising method for separating ionic and neutral analytes in the complex matrix, where a microemulsion is used as the background electrolyte. Current work identified the possibility of using hydrophilic ionic liquids (ILs) based on imidazole (C 12 MImCl, C 16 MImCl) as modifiers and surfactants, as well as hydrophobic ILs (C 6 MImBF 4 ) as an “oil” in the microemulsion for the separation of steroid hormones by MEEKC. The factors such as ILs concentration, nature and pH of the background electrolyte, and the ratio of the components of the microemulsion, that influence the efficiency and separation selectivity of analytes were found. Suitable conditions for the complete separation of a model mixture of steroid hormones: cortisol, cortisone, 11-deoxycortisol, 11-deoxycorticosterone, corticosterone were obtained. The analytical characteristics of methods (optimized conditions) for the determination of steroids by micellar and microemulsion electrokinetic chromatography were compared. It was investigated that the addition of 15 mM 2-hydroxypropyl-β-cyclodextrin increases the separation selectivity and reduces the analysis time. The application of the on-line concentration methods made it possible to reduce the detection limits of analytes to 50 ng / ml. The mode for electrophoretic determination of steroid hormones in biological fluids (urine, blood serum) by MEEKC with the use of ionic liquids has been proposed using the previously obtained results. Keywords : imidazolium ionic liquids, steroid hormones, microemulsion electrokinetic chromatography, microemulsion DOI: http://dx.doi.org/10.15826/analitika.2019.23.2.001 (Russian) L.A. Kartsova, E.A. Bessonova, D.O. Moskvichev Saint-Petersburg State University, Institute of Chemistry, 26 Universitetskii prospect, St. Petersburg, Petergof, 198504, Russian Federation

Enrichment and Separation of Cationic, Neutral, and Chiral Analytes by Micelle to Cyclodextrin Stacking-Micellar Electrokinetic Chromatography.

Analyte focusing by micelle to cyclodextrin stacking (MCDS) in micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) and fused silica capillaries is demonstrated for neutral, cationic, and chiral analytes. The stacking was at a dynamic boundary formed between the injected charged SDS micelles and neutral γ-cyclodextrin (γ-CD) zones, where the analytes bound inside micelles were released due to the formation of stable SDS-CD inclusion complexes. The complex formation reduced or eliminated the affinity of the analytes to the micellar phase. There was reversal (for charged) or nulling (for neutrals) of the analyte's effective electrophoretic mobility that caused the analytes to accumulate at the boundary. Under the conditions where the SDS micelles velocity is faster than the electroosmotic flow (using acidic buffer), MCDS was conducted by injection of a long plug of sample in a micellar diluent after injection of a CD solution plug into a capillary that was filled with MEKC background solution. By simply extending the length of the CD plug, chiral separations of chlorpheniramine and phenoxyacid herbicides were achieved without optimizing the MEKC conditions. The analytical figures of merit including linearity and repeatability for the tested compounds were found acceptable, and the sensitivity enhancement factors were up to 171. The stacking strategy in MEKC was applied to metabolic stability studies of small molecules with HepG2 cell line, where the samples were only treated with acetonitrile and then diluted with the micellar diluent (demonstrating the reduction of tedious sample preparation requirements for biological samples prior to chemical analysis).

Micellar Electrokinetic Chromatography.

Micellar electrokinetic chromatography (MEKC) is a mode of capillary electrophoresis that allows for the separation of neutral molecules in an electric field. Typically, neutral molecules move with electroosmotic flow (EOF) or bulk flow during electrophoretic separations resulting in no temporal resolution between mixtures of neutral analytes. Inclusion of surfactant micelles in the separation buffer allows for the separation of neutral analytes from one another through association with the micelle. Here we outline the implementation of MEKC for the separation of neutral molecules using a mixture of nitroaromatic explosives and their degradation products serving as a test analyte mixture.

Effect of nature of electrolytes on retention and selectivity in hydrophilic interaction liquid chromatography

This work investigates the selectivity and retention changes on four classes of HILIC columns upon addition of 1–20 mM electrolyte. The effect of four electrolytes (Na+Cl−, Na+ClO4−, Na+PF6−, and Na+CF3CO2−) were tested on neutral, cationic, anionic, and zwitterionic analytes under HILIC conditions (70–90% ACN). These electrolytes alter the retention and selectivity on silica, zwitterionic and diol columns through ion exchange and ionic screening mechanisms. Neutral analytes were unaffected by addition of 1–20 mM electrolyte, indicating minimal change to the retentivity of the water layer. Cationic and anionic analytes increase and decrease in retention, respectively (Na+PF6− ≈ Na+ClO4− > Na+CF3CO2− > Na+Cl−). Chaotropic electrolytes accumulate at the mobile phase/water layer interface, enhancing the ion exchange and ionic screening effects of the mobile phase electrolyte. Altering the buffer cation (Li+, Na+, K+) caused small but statistically significant changes in retention.

Covalent organic framework TpPa-1 as stationary phase for capillary electrochromatographic separation of drugs and food additives.

Because of unique properties like permanent porosity, low density and large surface area, covalent organic frameworks are attractive microporous materials as stationary phase for CEC. Among them, COF-TpPa-1 possesses extraordinary chemical stability and common features of covalent organic frameworks. So, COF-TpPa-1 could be a promising material as stationary phase to enhance separation selectivity and tolerate the acid or alkaline electrochromatographic separation conditions. Here, we report the preparation of COF-TpPa-1 modified capillary column by in situ growth for electrochromatographic separation. The immobilization of COF-TpPa-1 on the inner wall of capillary column was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and X-ray diffraction. The fabricated COF-TpPa-1 modified capillary column indicated good resolution for the separation of neutral analytes, nonsteroidal anti-inflammatory drugs and food additives in open-tubular CEC mode. Compared with bare capillary column, the COF-TpPa-1 modified capillary column improved separation selectivity remarkably for tested analytes, including hydrophobic, π-π and hydrogen bond interactions. Besides, the prepared capillary column showed excellent repeatability and stability. The intraday, interday and column-to-column relative standard deviations of migration time for neutral analytes were less than 3.54%. This work shows enamine-linked covalent organic frameworks have great potential as stationary phases in CEC.

A Critical Review on Colorimetric and Fluorescent Probes for the Sensing of Analytes via Relay Recognition from the year 2012–17

AbstractDesigning an effective sensor molecule for the selective detection of analyte with dual sensitivity is an innovative approach and is expected to offer huge biological and environmental benefits. Contrary to other sensing process, the reversible recognition of two different analytes while varying the binding sites and signaling subunits is entirely a new concept as the metal ion present in the ensemble can be easily displaced in the presence of ionic or neutral analytes. A reversible strategy can also be employed in the simultaneous recognition and identification of two different analytes with the aid of highly selective and sensitive chemosensors. This review gives an overview of the recent inventions (from 2012 to 2017) in the field of colorimetric and fluorescent receptors towards the detection of two analytes via a relay recognition process. A special emphasis is placed on different mechanisms associated with the sensing process assisted by fluorescence induced receptors which will help to design an effective sensor system for the selective detection of anions or amino acids or metal ions.

Poly(2-carboxyethyl acrylate-co-ethylene glycol dimethacrylate) monolithic precursor. Part II. Carbodiimide assisted post-polymerization modification with tris and d-Glucamine for use in hydrophilic interaction capillary liquid chromatography

A carboxylated precursor monolith (carboxy monolith), namely poly(2-carboxyethyl acrylate-co-ethylene glycol dimethacrylate), was prepared by the in situ co-polymerization of 2-carboxyethyl acrylate (CEA) with ethylene glycol dimethacrylate (EDMA), see the preceding article in this issue. This carboxy monolith was subjected to post-polymerization modification (PPM) with Tris and d-Glucamine in the presence of N-(3-dimethylamineopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC), a water soluble carbodiimide. EDAC with its zero-length carboxyl-to-amine coupling ability works by activating the surface carboxyl groups of the monolith for direct reaction with the primary amines of Tris and d-Glucamine via amide bond formation. The Tris-amide (TA) and d-Glucamide ligands imparted the poly(CEA-co-EDMA) monolith a polar character for use in hydrophilic interaction liquid chromatography (HILIC). The TA- and d-Glucamide-poly(CEA-co-EDMA) monolithic capillary columns were evaluated using neutral and charged polar analytes, such as phenolic acids, nucleic acid bases and nucleosides. The difference in polarity between these monolithic capillary columns is noticeable in terms of the numbers of hydroxyl group per ligands, and therefore, the d-Glucamide-poly(CEA-co-EDMA) monolith bearing five hydroxyl groups per ligand could separate more polar species compared to the TA-poly(CEA-co-EDMA) monolith which has three hydroxyl groups per ligand. Solute’ retention on both types of HILIC columns were reproducible from run-to-run, day-to-day and column-to-column.