Naphthyl methacrylate-phenylene diacrylate-based monolithic column for reversed-phase capillary electrochromatography via hydrophobic and π interactions

A neutral naphthyl methacrylate-phenylene diacrylate-based monolith (NPM) was introduced for RP-CEC of various neutral and charged solute probes via hydrophobic and π interactions. The NPM column was prepared by the in situ polymerization of naphthyl methacrylate as the functional monomer and 1,4-phenylene diacrylate (PDA) as the crosslinker in a ternary porogenic solvent containing cyclohexanol, dodecanol and water. The NPM column exhibited cathodal EOF despite the fact that it was devoid of any fixed charges. NPM exhibited stronger EOF than its counterpart naphthyl methacrylate monolith (NMM) made from the in situ polymerization of naphthyl methacrylate and trimethylolpropane trimethacrylate (TRIM). As for NMM, it is believed that the EOF arises from the adsorption of mobile phase ions onto the monolith surface. The higher EOF exhibited by NPM may be attributed to the acrylate nature of PDA as compared to the methacrylate nature of TRIM, and therefore PDA has a higher binding capacity for mobile phase ions due to its higher polarity than TRIM. The adsorption of mobile phase ions together with the additional π interactions offered by the aromatic rings of the NPM matrix modulated solute retention and separation selectivity. The applications of NPM were demonstrated by the separation of a wide range of small and large solutes including peptides, tryptic peptide maps and proteins.     

Novel β-cyclodextrin derivative functionalized polymethacrylate-based monolithic columns for enantioselective separation of ibuprofen and naproxen enantiomers in capillary electrochromatography

A novel enantioselective polymethacrylate-based monolithic column for capillary electrochromatography was prepared by ring-opening reaction of epoxy groups from poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith with a novel β-cyclodextrin derivative bearing 4-dimethylamino-1,8-naphthalimide functionalities. Conditions for the ring-opening reaction with respect to different reaction parameters were thoroughly optimized to obtain high electroosmotic flow, separation efficiency and enantioselectivity for the analytes. The nonaqueous mobile phase composition regarding acetonitrile–methanol ratio and the concentration of electrolyte were examined to manipulate the hydrophobic inclusion and anion-exchange interaction between the analytes and chiral stationary phase. It was observed that in addition to β-cyclodextrin cavity, the electrostatic interaction exhibited pronounced influence on the enantioseparation of acidic analytes. Acidic enantiomers (ibuprofen and naproxen) could be separated with separation factor (α) values up to 1.08 and a maximum separation efficiency of 86 000 plates/m could be achieved.     

Sulfated and sulfonated polysaccharide as chiral stationary phases for capillary electrochromatography and capillary electrochromatography–mass spectrometry

The applications of polysaccharide phenyl carbamate derivatives as chiral stationary phases (CSPs) for capillary electrochromatography (CEC) are often hindered by longer retention times, especially using a normal-phase (NP) eluent due to very low electroosmotic flow (EOF). Therefore, in this study, we propose an approach for the aforementioned problems by introducing two new types of negatively charged sulfate and sulfonated groups for polysaccharide CSPs. These CSPs were utilized to pack CEC columns for enantioseparation with a NP eluent. Compared to conventional cellulose tris(3,5-dimethylphenyl carbamate) or CDMPC CSPs, the sulfated CDMPC CSP (sulfur content 4.25%, w/w) shortened the analysis time up to 50% but with a significant loss of enantiomeric resolution (∼60%). On the other hand, the sulfonated CDMPC CSP (sulfur content 1.76%, w/w) not only provided fast throughput but also maintained excellent resolving power. In addition, its synthesis is much more straightforward than the sulfated one. Furthermore, we studied several stationary phase parameters (CSP loading and silica gel pore size) and mobile phase parameters (including type of mobile phase and its composition) to evaluate the throughput and enantioselectivity. Using the optimized conditions, a chiral pool containing 66 analytes was screened to evaluate the enantioselectivity under three different mobile phase modes (i.e., NP, polar organic phase (POP) and reversed-phase (RP) eluents). Among these mobile phase modes, the RP mode showed the highest success rate, whereas some degree of complementary enantioselectivity was observed with NP and POP. Finally, the feasibility of applying this CSP for CEC–MS enantioseparation using internal tapered column was evaluated with NP, POP and RP eluents. In particular, the NP-CEC–MS provided significantly enhanced sensitivity when methanol was replaced with isopropanol in the sheath liquid. Using aminoglutethimide as model chiral analyte, all three modes of CEC–MS demonstrated excellent durability as well as excellent reproducibility of retention time and enantioselectivity.     

Preparation of a zeolite imidazole skeleton–silica hybrid monolithic column for amino acid analysis via capillary electrochromatography

We developed a novel, convenient and low-cost one-pot strategy for preparing a zeolitic imidazolate framework-8 (ZIF-8)–silica hybrid monolithic column by adding ZIF-8 directly to a polymer solution of the silica matrix. The simulated stationary phase and monolithic column prepared under optimal conditions were characterized using X-ray diffraction, scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis nitrogen physisorption and zeta potential. The results obtained confirmed the successful introduction of ZIF-8 into the silica monolithic column, and the prepared monolithic column exhibited good permeability and physicochemical stability. A capillary electrochromatography method was developed based on a ZIF-8–silica hybrid monolithic column through which 15 mixed amino acids, 4 neutral compounds, 4 nipagin esters and 2 chlorinated fungicides were separated in 14, 5, 7 and 6 min, respectively, under optimal conditions. The relative standard deviations retention times and column efficiencies in run-to-run, day-to-day and column-to-column varied in the ranges of 1.90%–2.21%, 2.13%–2.51% and 3.08%–6.65%, respectively, which demonstrated that ZIF-8–silica hybrid monolithic column exhibited satisfactory reproducibility and stability. The incorporation of ZIF-8 into a silica monolithic column is a promising method for preparing novel monolithic columns composed of a metal–organic framework.     

Optimization of photo-polymerized sol–gel monolithic stationary phases prepared in polyacrylate-coated fused-silica capillaries for capillary electrochromatography

The preparation of photo-polymerized sol–gel monolithic stationary phases (MSP) within 100 μm internal diameter polyacrylate-coated fused-silica capillaries for use in capillary electrochromatography (CEC) was optimized. Eight mixtures containing different amounts of methacryloxypropyltrimethoxysilane (MPTMS) as the polymeric precursor, hydrochloric acid solution as the catalyst, toluene as the porogen and bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (Irgacure 819) as the photo-initiator were irradiated at 370 nm inside the capillaries in order to complete the MSP polymerization, according to a fractional factorial experimental design 2_IV^4-1. All the preparation procedure, from capillary pretreatment until the MSP is ready to use in CEC, were made in less than four hours in mild conditions. A high pressurization injection device (HPID) useful for micro-volume syringes was built in order to achieve practical, controlled and precise injections of sols, solvents and electrolytes in the capillaries. The eight MSP were equally washed, conditioned and submitted to CEC procedures via short-end injection, which showed higher efficiency and peak height taking shorter analysis time. Electrochromatographic behaviors of the MSP were corroborated with morphological characterizations by scanning electron microscopy. The optimum condition, which allowed the separation of standard mixture containing thiourea (marker compound), naphthalene, acenaphthene, fluorene, phenanthrene and anthracene in twelve minutes without external pressure assistance, showed efficiencies up to 51,460 N/m, relative standard deviation from 0.05 to 3.3% for migration/retention time and from 0.14 to 1.6% for relative area (considering thiourea as an internal standard) and also showed no statistical evidence that three MSP prepared at the same condition are different within 95% confidence interval.     

Preparation and evaluation of tandem stationary phases for μ-HPLC and capillary electrochromatography

Tandem separation beddings were prepared within the capillary tube with a photopolymerized monolith initially formed by sol-gel technology in combination with microspheric octadecylsilane material by slurry packing. The chromatographic performance of the tandem stationary phases was evaluated in detail by varying the flow rate and the composition of the mobile phase using a self-installed capillary HPLC system. For the tandem stationary phases with a forepart monolithic length of 11 cm and 1 cm the lowest theoretical plate height for the retained component was 26 µm and 34 µm, respectively. After evaluation by capillary electrochromatography, enhanced chromatographic performance was obtained using a column with 1 cm monolithic inlet frit with a theoretical plate height up to 7.20 µm. A scanning electron micrograph with different cross-sections of the column showed that a porous network formed in the center of the capillary and a homogenous slurry packing of C₁₈ was obtained at the back part.     

High-resolution monolithic columns—a new tool for effective and quick separation

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a “classic” monolithic column, the first generation (Onyx? monolithic C₁₈, 100 mm?×?4.6 mm, Phenomenex); a “narrow” monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm?×?3 mm, Merck); and a recently introduced “high-resolution” monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm?×?4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile–phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.

Novel preparation of monolithic imprinted columns for electrochromatographic separation by photopolymerization

A monolithic molecularly imprinted polymer with specific recognition ability for 4-hydroxybenzoic acid (4-HBA) was prepared by in situ photopolymerization, using methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EDMA) as a cross-linking agent, toluene and isooctane as porogenic solvents and Irgacure 1800 as an initiator. Baseline separation of isomers of hydroxybenzoic acid was achieved in less than 8 min on this monolithic column using 4-HBA as template, but not on the blank polymer. Furthermore, some neutral compounds could also be baseline-separated on the imprinted polymer column in the mode of pressure-driven capillary electrochromatography.     

Metal organic framework–organic polymer monolith stationary phases for capillary electrochromatography and nano-liquid chromatography

In this study, metal organic framework (MOF)–organic polymer monoliths prepared via a 5-min microwave-assisted polymerization of ethylene dimethacrylate (EDMA), butyl methacrylate (BMA), and 2-acrylamido-2-methylpropane sulfonic acid (AMPS) with the addition of various weight percentages (30–60%) of porous MOF (MIL-101(Cr)) were developed as stationary phases for capillary electrochromatography (CEC) and nano-liquid chromatography (nano-LC). Powder X-ray diffraction (PXRD) patterns and nitrogen adsorption/desorption isotherms of these MOF–organic polymer monoliths showed the presence of the inherent characteristic peaks and the nano-sized pores of MIL-101(Cr), which confirmed an unaltered crystalline MIL-101(Cr) skeleton after synthesis; while energy dispersive spectrometer (EDS) and micro-FT-IR spectra suggested homogenous distribution of MIL-101(Cr) in the MIL-101(Cr)–poly(BMA–EDMA) monoliths. This hybrid MOF–polymer column demonstrated high permeability, with almost 800-fold increase compared to MOF packed column, and efficient separation of various analytes (xylene, chlorotoluene, cymene, aromatic acids, polycyclic aromatic hydrocarbons and trypsin digested BSA peptides) either in CEC or nano-LC. This work demonstrated high potentials for MOF–organic polymer monolith as stationary phase in miniaturized chromatography for the first time.     

Chiral metal–organic framework used as stationary phases for capillary electrochromatography

Metal–organic frameworks (MOFs) have received great attention as novel media in separation sciences because of their fascinating structures and unusual properties. However, to the best of our knowledge, there has been no attempt to utilize chiral MOFs as stationary phases in capillary electrochromatography (CEC). In this study, a homochiral helical MOF [Zn₂(D-Cam)₂(4,4′-bpy)]_n (D-Cam = D-(+)-camphoric acid, 4,4′-bpy = 4,4′-bipyridine) was explored as the chiral stationary phase in open tubular capillary electrochromatography (OT-CEC) for separation of chiral compounds and isomers. The MOFs coated column has been developed using a simple procedure via MOFs post-coated on the sodium silicate layer. The baseline separations of flavanone and praziquantel were achieved on the MOFs coated column with high resolution of more than 2.10. The influences of pH, organic modifier content and buffer concentration on separation were investigated. Besides, the separations of isomers (nitrophenols and ionones) were evaluated. The relative standard deviations (RSDs) for the retention time of run-to-run, day-to-day and column-to-column were 1.04%, 2.16% and 3.07%, respectively. The results demonstrated that chiral MOFs are promising for enantioseparation in CEC.     

Immobilization of chitosan in sol–gel phases for chiral open-tubular capillary electrochromatography

Three different approaches for immobilizing cross-linked chitosan molecules (CS-s) in sol–gel phases to form chiral OT-CEC capillaries were comparatively investigated in this study. To synthesize column I, a bare capillary was first silanized with triethoxysilane (TEOS) and then reacted with the reaction product of 3-glycidyloxypropyltrimethoxysilane (GTS) and CS-s. Column II was prepared by the silanization of a bare capillary with a mixture of TEOS and GTS silanes followed by reaction with CS-s. To obtain column III, all the reagents, including TEOS, GTS, and CS-s were reacted together in a bare capillary. The SEM images showed that the column I phase consisted of two distinct layers, GTS and TEOS sol–gel films, while column II and III phases were homogeneous phases. By elemental analysis, the chitosan contents of the columns were found to decrease in the order column I > II > III, which corresponded to the order of the electroosmotic mobility values obtained from the measurements of the electroosmotic flow in the columns. The retention factor and the selectivity for the chiral separation of phenylglycine enantiomers in the optimized Tris running buffer (100 mM, pH 7.5) also followed this decreasing order. Besides the strength of the interaction with the immobilized functional chitosan, the hydrophobicity of the column affected the resolution of enantiomeric samples. The hydrophilic alanine sample could only be resolved by column III, but the hydrophobic tryptophan and catechin enantiomers were better separated by columns I and II. A reverse-phase mechanism has been found in the separations. Furthermore, the resolution and analysis time of column I and II phases were superior to the phase simply bonded with molecular chitosan.     

Enhancement of intramolecular excimer formation and photodimerization of anthrylmethyl α,ω-alkanedioates via hydrophobic interactions

The fluorescence spectra and photodimerization of anthrylmethyl a,w-alkanedioates (A-Mn-A) both in organic and in aqueous organic mixed solvents have been studied.In aqueous organic mixed solvents strong intramolecular excimer emission is detected and the quantum yield for the intramolecular photodimerization is significantly greater than those in organic solvents.These observations suggest that hydrophobic interactions force A-Mn-A molecule to self-coil.The ratio of the head-to-head to head-to-tail products in the intramolecular photodimers of A-Mn-A depends on the length of the linking chain.This work presents a successful example of application of hydrophobic interactions to enhancement of large-ring formation.     

Csaba Horváth's contribution to the theory and practice of capillary electrochromatography

This overview has been written as a tribute to a luminary of chromatography--Csaba Gyula Horváth, who passed away earlier this year. Since the scope of his work was enormous, the following pages focus only on one single aspect of his scientific activities, capillary electrochromatography. He was a visionary, recognizing the great potential of this method and devoted a large part of his efforts to studies of problems related to CEC, covering a huge variety of issues embracing the theoretical foundation, instrumentation, and column technology. During the period of time from 1996 to 2004, Csaba Horváth published almost 30 excellent papers concerning capillary electrochromatography, which are reviewed in this article.     

Open tubular capillary electrochromatography with block co-polymer coating for separation of β-lactam antibiotics

Unique block co-polymer P(MAn-St-NIPAm) has been successfully synthesized by reversible addition fragmentation chain transfer radical polymerization protocol. Based on the tunable hydrophobic/hydrophilic properties of the block co-polymer, a new open tubular capillary electrochromatography (OT-CEC) system has been constructed with the prepared block co-polymer as the coating and applied in analysis of β-lactam antibiotics in serum samples.     

Fast separations of chiral β-blockers on a cellulose tris(3,5-dimethylphenylcarbamate)-coated zirconia monolithic column by capillary electrochromatography

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) is an excellent chiral selector for enantioseparation of a wide variety of chiral compounds. The monolithic chiral columns are becoming popular in liquid chromatography and capillary electrochromatography. In this work, we present the fast separation of chiral β-blockers on a CDMPC-modified zirconia monolithic column by capillary electrochromatography (CEC). The porous zirconia monolithic capillary column was prepared by using the sol-gel technology and then zirconia surface modified with CDMPC. The enantioseparations were performed in reversed-phase (RP) eluents of a phosphate solution (pH 4.4) modified with acetonitrile or alcohol. The enantioseparations of a set of eight chiral β-blockers were achieved in less than one minute. Influences of the applied voltage, column temperature, concentration of acetonitrile and the type of alcohol as the organic modifier in the mobile phase, and sample injection time on enantioseparation were investigated. CEC separations at the applied voltage of 10 kV and 15 °C in the ACN-modified mobile phase provided the best resolutions for the analytes studied. Run-to-run and day-to-day repeatabilities of the column in the RP-CEC separation were less than 1 and 2%, respectively.     

Investigation on chiral capillary columns and their application in separation of enantiomers

Six chiral diamide stationary phases (CSPs), namely N-(3-carbobenzoxypropionyl)-L-Val-tert-butylamide (CSP-1), N-undecenoyl-L-Val-S-α-phenylethylamide (CSP-2), N-undecenoyl-L-Val-R-α-phenylethylamide (CSP-3), OV-225-L-Val-tert-butylamide (CSP-4), XE-60-L-Val-tert-butylamide (CSP-5) and polycyanoethyl vinyl siloxane-L-Val-tert-butylamide (CSP-6), were inves-tigated and CSP-6 was crosslinked within narrow bore (70 μm) fused silica capillary columns. Theseparation of amino acid enantiomers on this narrow bore column by gas chromatography (GC) isillustrated.     

In-column “click” preparation of hydrophobic organic monolithic stationary phases for protein separation

Two types of macroporous organic polymer monoliths based on glycidyl methacrylate (GMA), 4-vinylbenzyl chloride (VBC) and divinylbenzene (DVB) were prepared inside stainless-steel tubes. Azide functionalities were firstly introduced on the surfaces of poly(GMA-co-DVB) and poly(VBC-co-DVB) monoliths to provide reactive sites for click chemistry. With the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths was developed. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by the scanning electron microscopy, mercury intrusion porosimeter, Fourier transform infrared spectroscopy, and elemental analyses, respectively. The chromatographic performances of the “clicked” stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min.     

Porous layer open-tubular column with styrene and itaconic acid-copolymerized polymer as stationary phase for capillary electrochromatography–mass spectrometry

Enhancing the specific surface area of stationary phase is important in chromatographic science, especially in open-tubular column in which the coating only exists on the inner surface. In this work, a porous layer open-tubular (PLOT) column with stationary phase of styrene and itaconic acid-copolymerized polymer was developed. Thermal-initiated polymerization method with strategies like controlling the ratio of reaction reagents to solvents and reaction time, confinement by the narrow inner diameter of capillary were used for preparing the stationary phase with uniform structure and relatively thick layer. Due to the high separation efficiency and capacity, the PLOT column was used for capillary electrochromatography (CEC) separation of multiple groups of analytes like alkylbenzenes, phenyl amines, phenols, vanillins, and sulfonamides with theoretical plates (N) up to 1,54,845 N/m. In addition, due to high permeability of the CEC column and large electroosmotic flow mobility generated by abundant carboxyl groups in the coating material, the PLOT-CEC column was successfully coupled with mass spectrometry (MS) through a sheath flow interface. The developed PLOT-CEC-MS method was used for the analysis of antiseptics like parabens and herbicides like pyridines.     

Zeolitic imidazolate framework-67–modified open-tubular column with cyclodextrin for enantioseparation in capillary electrochromatography

The histidine-modified zeolitic imidazolate framework [His-ZIF-67] was prepared with the histidine, 2-methylimidazole, and Co²⁺ under ambient temperature. His-ZIF-67 was bonded via a glycidyl methacrylate copolymer to the internal surface of capillary and then functionalized with the NH₂-β-cyclodextrin (NH₂-β-CD). The materials were characterized by field emission scanning electron microscopy, high-resolution transmission electron microscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy, N₂ adsorption–desorption isotherm, X-ray diffraction, and X-ray photoelectron spectroscopy. In comparison with the NH₂-β-CD@capillary, the NH₂-β-CD@His-ZIF-67@capillary-coated column shows significantly enhanced resolution for chiral molecules. The NH₂-β-CD@His-ZIF-67@capillary column achieved the baseline separation of amlodipine and metoprolol (the resolution of amlodipine: 1.70; metoprolol: 1.50) and the partial separation of atenolol and propranolol (the resolution of atenolol: 1.03; propranolol: 0.60). These were attributed to the histidine modification and the features of ZIF-67, including an excellent surface area and the abundant porosity. The pH and proportion of organic modifier in the buffer were crucial for enantioseparation performance and were evaluated in detail. The fabricated NH₂-β-CD@His-ZIF-67@capillary-coated column showed good stability and repeatability (relative standard deviation <6.3%). The molecular modeling with AutoDock and grand canonical ensemble was carried out to evaluate the interactions between chiral stationary phase and racemic drugs.     

A novel coating method for CE capillary using carboxymethyl-β-cyclodextrin-modified magnetic microparticles as stationary for electrochromatography enantioseparation

Analytical and Bioanalytical Chemistry - Magnetic microparticles (MMPs) have been extensively studied and aroused considerable interest in separation science owing to their superior...