Electrochromatographic Separation Research Articles

Butyl methacrylate based monoliths with different cross‐linking agents using DMF‐aqueous buffer as porogen

A simple porogen containing only DMF and aqueous buffer was used for synthesis of monolithic stationary media for CEC). Butyl methacrylate (BMA)-based capillary monoliths were obtained using proposed porogen together with acrylic/methacrylic cross-linking agents with different alkyl chain lengths. For this purpose, ethylene glycol dimethacrylate, butanediol dimethacrylate and hexanediol diacrylate (HDDA) were used. The monoliths with better electrochromatographic separation performance were obtained when the acrylic cross-linking agent with the longest alkyl chain length (i.e. HDDA) was used with the proposed porogen. The electrochromatographic separation of alkylbenzenes, phenols and benzoic acids were sucessfully performed in CEC particularly using poly(BMA-co-HDDA) monolithic stationary phase with the column efficiency up to 270 000 plates/m.

Preparation and Evaluation of C18 Modified Capillary Open-Tubular Column Based on Thiol-ene Click Chemistry for Capillary Electrochromatography

Abstract A novel C 18 open-tubular column (C 18 capillary) for capillary electrochromatography was prepared based on thiol-ene click chemistry between n -octadecanethiol and vinyl-functionalized capillary (vinyl capillary). The inner wall of capillary was chemically modified with vinyltrimethoxysilane to provide ene-functionality. Then n -octadecanethiol was chemically bonded on the surface of vinyl capillary via thiol-ene click chemistry. The surface features of the prepared C 18 capillary was investigated using scanning electron microscopy. The effects of buffer pH on the electroosmotic flow of C 18 capillary, vinyl capillary was compared with bare capillary. The experiment results indicated that the lowest EOF profile was obtained on the C 18 capillary under the same experimental conditions compared with vinyl capillary and bare capillary. To evaluate the column performance, test mixture of three polycyclic aromatic hydrocarbons (PAHs) were electrochromatographic separation on C 18 capillary, vinyl capillary and bare capillary. The electrochromatographic retention behavior of the selected model PAHs on C 18 capillary was also investigated. The experiment results showed that C 18 capillary exhibits typical reversed-phase electrochromatographic behavior toward PAHs. Compared with vinyl capillary, the relatively good peak shape and no peak tailing was observed for the separation of basic model analytes on C 18 capillary, which may be attributed to embedded polar sulphur groups on the surface of the C 18 capillary that effectively shield the adsorption of basic analytes onto the residual silanol groups.

Open Tubular Microreactor with Enzyme Functionalized Microfluidic Channel for Amperometric Detection of Glucose

AbstractA simple and efficient method using enzyme immobilized microfluidic channel as open tubular microreactor was designed for amperometric detection of glucose. The microreactor was composed of a polydimethylsilicone/glass hybrid device with three reservoirs, a cooling cave and a 6 cm capillary with a sampling fracture as microchannel. The microchannel was further modified by thermal polymerization, followed by covalently attaching with glucose oxidase. Through fracture sampling and electrochromatography separation, the production via enzymatic reaction was determinated by Pt electrode at the end of capillary. The linear range for the detection of glucose was 0.05–7.5 mmol·L−1 with detection limit of 23 µmol·L−1. The inter‐ and intra‐chip reproducibilities for determination of 2.5 mmol·L−1 glucose were 98.5% (n=5) and 96.0% (n=5), respectively. With the advantage of flexible assembly, rapid efficiency, good stability and low‐cost, this microreactor provided a potential platform for establishing a portable enzyme‐based chemical detection system in practical application.

Diamino moiety functionalized silica nanoparticles as pseudostationary phase in capillary electrochromatography separation of plant auxins

A novel and simple method for the preparation of silica nanoparticles having surface-functionalized diamino moiety (dASNPs) was reported in our paper and characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectrometry, and thermogravimetry techniques. To test this method practically, in this contribution we describe the enhanced separation of four plant auxins - indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (dCPAA), and 2-(1-naphthyl) acetic acid (NAA) - by capillary electrochromatography using diamino moiety functionalized silica nanoparticles as pseudostationary phase (PSP) in the running buffer. The effect of pH, buffer concentration, and diamino moiety functionalized silica nanoparticles concentration on the selectivity of separation was investigated. A combination of the nanoparticles and running buffer reversed the electroosmotic direction making possible the rapid and efficient separation of the auxins from the auxins migrated in the same direction with the EOF under optimum experimental conditions. A good resolution of four auxins was obtained within 5.5 min under optimum experimental conditions. The precision (RSD, n = 5) was in the range of 0.72-0.91% and 1.89-2.23% for migration time and peak area response, respectively. The detection limits were 0.48, 0.44, 0.46, and 0.42 μM for NAA, IBA, IAA, and dCPAA, respectively. Furthermore, the method was successfully tested for the determination of IAA in the grapes.

Effect of ion adsorption on CEC separation of small molecules using hypercrosslinked porous polymer monolithic capillary columns

Both poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) and poly(4-methylstyrene-co-vinylbenzyl chloride-co-divinylbenzene) monolithic columns have been hypercrosslinked and for the first time used to achieve capillary electrochromatographic separations. Although these columns do not contain ionizable functionalities, electroosmotic flow was observed due to adsorption of ions from a buffer solution contained in the mobile phase on the surface of the hydrophobic polymer. An increase of more than one order of magnitude was observed with the use of both monolithic polymers. The hypercrosslinking reaction creates a large surface area thus enabling adsorption of a much larger number of ions. Alkylbenzenes were successfully separated using the hypercrosslinked monolithic columns.

New “one-step” method for the simultaneous synthesis and anchoring of organic monolith inside COC microchip channels

A new method for monolith synthesis and anchoring inside cyclic olefin copolymer (COC) microchannels in a single step is proposed. It is shown that type I photoinitiators, typically used in a polymerization mixture to generate free radicals during monolith synthesis, can simultaneously act as type II photoinitiators and react with the plastic surface through hydrogen abstraction. This mechanism is used to "photograft" poly(ethylene glycol) methacrylate (PEGMA) on COC surfaces. Contact angle measurements were used to observe the changes in surface hydrophilicity when increasing initiator concentration and irradiation duration. The ability of type I photoinitiators to synthesize and anchor a monolith inside COC microchannels in a single step was proved through SEM observations. Different concentrations of photoinitiators were tried. Finally, electrochromatographic separations of polycyclic aromatic hydrocarbons were realized to illustrate the beneficial effect of anchoring on chromatographic performances. The versatility of the method was demonstrated with two widely used photoinitiators: benzoin methyl ether (BME) and azobisisobutyronitrile (AIBN).

Acrylate ester-based monolithic columns for capillary electrochromatography separation of triacylglycerols in vegetable oils

A simple and reliable method for the evaluation of triacylglycerols (TAGs) in vegetable oils by capillary electrochromatography (CEC) with UV–Vis detection, using octadecyl acrylate (ODA) ester-based monolithic columns, has been developed. The percentages of the porogenic solvents in the polymerization mixture, and the mobile phase composition, were optimized. The optimum monolith was obtained at the following ratios: 40:60% (wt/wt) monomers/porogens, 60:40% (wt/wt) ODA/1,3-butanediol diacrylate and 23:77% (wt/wt) 1,4-butanediol/1-propanol (14 wt% 1,4-butanediol in the polymerization mixture). A satisfactory resolution between TAGs was achieved in less than 12 min with a 65:35 (v/v) acetonitrile/2-propanol mixture containing 5 mM ammonium acetate. The method was applied to the analysis of TAGs of vegetable oil samples. Using linear discriminant analysis of the CEC TAG profiles, the vegetable oils belonging to six different botanical origins (corn, extra virgin olive, hazelnut, peanut, soybean and sunflower) were correctly classified with an excellent resolution among all the categories.

Capillary electrochromatographic testing of monolithic silica columns synthesized according to an experimental design approach

Monolithic silica capillary columns synthesized following a three-level design were evaluated for the electrochromatographic separation of acidic and neutral compounds. The influences of four factors in the sol-gel synthesis, i.e. the concentrations of tetramethylorthosilicate (TMOS) and PEG in the starting mixture, the gelation temperature and the silanization modifying time, on the electrochromatographic performance of the resulting C18-silica capillary monoliths were studied. The considered responses were retention factor, resolution, symmetry factor, column efficiency, electrokinetic porosity and the equivalent length of the monolith. The four factors were varied to change the pore structure and the surface coverage with octadecyl moieties, resulting in nine stationary phases. The retentive properties of the columns were initially characterized with alkylbenzenes. Next, the separation for acetylsalicylic acid (ASA) and its related compounds was optimized and used to evaluate the performance of the nine stationary phases considering six responses. A compromise between the different responses was found around higher concentrations of tetramethylorthosilicate and PEG with a lower gelation temperature and a modifying time of 2 h. Column efficiencies up to 96,000 plates/m and resolutions above 1.9 were obtained for the acetylsalicylic acid separation, with a sufficient EOF to yield rapid analysis, which showed improvements over the center-point stationary phase.

Development of a new open-tubular capillary electrochromatography method for in vitro monitoring of toxic aromatic amines distribution in rat blood

Exposure to aromatic amines from different industrial and agricultural activities entails a substantial risk of deleterious somatic effects, genetic damage and cancer development. Thus, a new and simple method for separation and analysis of aromatic amines has been developed by open-tubular capillary electrochromatography with a novel amphipathic block copolymer (poly(tert-butyl acrylate)(127)-block-poly(glycidyl methacrylate)(86)) coating based on its self-assembled property. Key factors affecting the separation efficiency of the test analytes, such as pH, buffer concentration and selective solvent, were studied in detail. Meanwhile, method validation was well evaluated by linearity (≥0.998), detection limit and recovery. Application of this developed protocol on in vitro monitoring of the target aromatic amines distribution in rat blood demonstrated its potential usage for separation and determination of aromatic amines in biological samples. Additionally, for assimilating more polymeric materials into analysis of aromatic amines, the effect of morphology changes of the amphipathic block copolymer coating on open-tubular capillary electrochromatography separation was also studied, and the result revealed that the block copolymer coating could play the same role as surfactant.

Preparation of silica-based nanoparticle having surface-bound octanoyl-aminopropyl moieties and its applications for the capillary electrochromatography separation of charged and neutral compounds

A kind of novel amphiphilic silica-based nanoparticle having surface-bound octanoyl-aminopropyl moieties (OA-NP) with the diameter of ~250 nm was successfully prepared and characterized by elemental analysis, Fourier transform infrared spectrometry. The potential use of OA-NP as pseudostationary phase in capillary electrochromatography for the separation of aromatic acids, basic, and neutral compounds was investigated. Five aromatic acids were separated rapidly with high column efficiency as they migrate in the same direction with the EOF under optimum experimental conditions. Under a running buffer with the composition of 40% methanol, 10 mM phosphate buffer (pH 5.5) with 1.0 mg/mL OA-NPs added, basic compounds investigated were baseline resolved with relatively symmetrical peaks. Due to the existence of polar acyl amide group on the surface of OA-NPs, "silanol effect" that occurs between the positively charged basic analytes and the silanols of the capillary column was greatly suppressed. Furthermore, the newly synthesized OA-NPs were also tried for the separation of some neutral analytes, and satisfactory separations were obtained.

Two-dimensional strong cation-exchange liquid chromatography/reversed-phase pressurized capillary electrochromatography for separation of complex samples

A 2-D separation platform was constructed using micro strong cation-exchange liquid chromatography (μ-SCXLC) and reversed-phase pressurized capillary electrochromatography (RP-pCEC) for the analysis of complex samples. Samples were fractionated by the first-dimension μ-SCXLC with a linear solvent gradient and then injected into the second-dimension RP-pCEC for further separation. The μ-SCXLC/RP-pCEC 2-D system with three separation mechanisms, namely strong cation-exchange, reversed-phase chromatography and electrophoresis, provided high selectivity, high resolution and high peak capacity compared to one-dimensional chromatographic approaches. Separation effectiveness of this 2-D system was demonstrated by the analysis of different kinds of complex samples, such as traditional Chinese medicine Cortex Phellodendri, bovine serum albumin (BSA) tryptic digest and real serum tryptic digest. A theoretical peak capacity of approximately 1200 was achieved, which proves its promising potential for the separation and analysis of complex samples.

Zirconia nanoparticles-coated column for the capillary electrochromatographic separation of iron-binding- and phosphorylated-proteins

A ZrO(2) nanoparticles (ZrO(2)NPs)-coated column was prepared through a sol-gel process using zirconium(iv) oxychloride, which reacted with silanol groups of the fused-silica capillary. The condensation reaction was carried out at 350 °C for 8 h. Electroosmotic flow (EOF) measurements and scanning electron microscopy (SEM) images were used to characterize the ZrO(2)NPs fabricated on the inner wall of the capillary. Below the pI value (pH 5-6), cathodic EOF elucidated that the phosphate buffer adsorbs tightly on the zirconia surface, resulting in a negatively charged surface. In this work, iron-binding proteins, phosphorylated proteins and glycoproteins were selected as the model compounds. The effects of pH, concentration, buffer type and the organic modifier were studied to optimize the separation efficiency. Iron-binding proteins exhibited a retention time for myoglobin (Mb) < hemoglobin (Hb), which corresponded to the binding constants for ZrO(2)NPs. The α- and β-subunit of Hb could be separated in borate buffer (20 mM, pH 9.0) with MeOH (20%, v/v). Greater affinity of α-casein and bovine serum albumin (BSA) for the stationary phase as the pH decreased was found by comparison with that of conalbumin (ConA) and transferrin (Tf). Interestingly, 14 peaks for glycoisoforms of ovalbumin (OVA) were observed using borate buffer (40 mM, pH 9.0). The established method was also applied to the determination of analytes in the egg whites of chicken and duck eggs.

Carbon nanotube based separation columns for high electrical field strengths in microchip electrochromatography

Electrically insulated carbon nanotube (CNT) based separation columns have been fabricated that can withstand an electrical field strength of more than 2.0 kV cm(-1) without bubble formation from electrolysis. The carbon nanotubes were grown in a pillar array defined by photolithographic patterning of the catalyst layer used for synthesis of the nanotubes. Multiwall carbon nanotubes are inherently electrically conductive and cannot be used as a continuous layer in the microfluidic channels, without short circuiting the electrical field in the separation column, when the field strength is more than a couple of 100 V cm(-1). Here, the carbon nanotubes are grown in an array of hexagonal pillars, where the nanotubes in the individual pillars are not in direct electrical contact with the nanotubes of the adjacent pillars. This makes it possible to increase the electrical field strength from around 100 V cm(-1) to more than 2.0 kV cm(-1) and thereby to use the CNT columns for electrokinetic separations with the high electrical field strengths that are typically used in this application. An electrochromatographic separation of two Coumarin dyes was demonstrated on the CNT column with an acetonitrile content of 90%.

Preparation and evaluation of a monolithic molecularly imprinted polymer for the chiral separation of neurotransmitters and their analogues by capillary electrochromatography

A monolithic molecularly imprinted polymer (MIP) column was prepared as the stationary phase for the capillary electrochromatographic (CEC) separation of a group of structurally related compounds including dopamine (DA), (±)-epinephrine (EP), (−)-isoproterenol (ISO), (±)-norepinephrine (NE), (±)-octopamine (OCT), and (±)-synephrine (SYN). Here, (−)-NE was used as the template. Either methacrylic acid (MAA) or itaconic acid (IA) together with a mixture of ethylene glycol dimethacrylate (EDMA) and α,α′-azobis(isobutyronitrile) (AIBN) in N, N-dimethylformamide (DMF) was introduced into a pre-treated, silanised, fused-silica capillary by a thermal non-covalent polymerisation procedure. Optimised conditions for the polymerisation reaction were assessed by the separation efficiency of the template. Both the template/monomer/cross linker molar ratio and the compositions of the functional monomer, cross-linker, and porogen affected polymerisation. The optimum in situ polymerisation reaction was performed at 65 °C for 17 min. By varying CEC parameters like eluent composition and pH, we observed that the addition of SDS to the eluent clearly improved the CEC separations. With a mobile phase of citrate buffer (10 mM, pH 3)/SDS (40 mM)/acetonitrile (2/2/1, v/v/v) solution and an applied voltage of 10 kV, the six related structures of the template and their enantiomeric mixtures were satisfactorily separated at 30 °C.

Electrochromatography in cyclic olefin copolymer microchips: A step towards field portable analysis

In order to develop a portable and disposable instrument for on-site analysis of neutral compounds, a lauryl methacrylate monolith has been synthesized into a cyclic olefin copolymer microdevice for reversed-phase electrochromatography purposes. This monolith was tested in capillary to evaluate electrochromatographic performances in terms of electroosmotic flow (EOF) mobility, retention and efficiency prior to its transfer into the microfluidic device. The produced monolithic bed exhibited a good run-to-run repeatability, column-to-column reproducibility and batch-to-batch reproducibility, with relative standard deviation (RSD) values below 9% for EOF mobility, retention factors and heights of theoretical plate. The electrochromatographic performances of the monolith were optimized by reducing irradiation time. Photopolymerization time of 10 min was found to be the best process in order to obtain a robust, retentive and efficient system. The on-chip performances of this monolith were evaluated in detail for the reversed-phase electrochromatographic separation of polycyclic aromatic hydrocarbons, with plate heights reaching down to 15 μm when working at optimal velocity. Aiming at the maximum simplification of instrumental fabrication and operation, a direct injection from a 2 μL droplet was compared with more conventional dynamic injection process.

Polymer Monoliths with Exchangeable Chemistries: Use of Gold Nanoparticles As Intermediate Ligands for Capillary Columns with Varying Surface Functionalities

Newly developed porous polymer monolithic capillary columns modified with gold nanoparticles coated with exchangeable functionalities allow easy switching of separation modes by a simple ligand exchange process. These columns are prepared from a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith through reaction of its epoxide moieties with cysteamine to afford a monolith rich in surface thiol groups. Gold nanoparticles prepared via in situ reduction of chloroauric acid within the column become attached to the surface of the pores of the monolith. Alternatively, a solution of colloidal gold nanoparticles can be pumped through the thiol modified column to achieve their attachment. While the first approach is faster, it affords a lower coverage of nanoparticles than the second method, while both methods preserve the excellent hydrodynamic properties that are typical of the monolithic columns. Functionalization of the surface of the bound gold nanoparticles is then carried out using low molecular weight thiol-containing surface ligands. The dynamic nature of the bond between gold and these surface ligands enables the replacement of one surface ligand by another through a simple solution exchange process. This novel approach expands the application range of monoliths as a single column can now be used in different separations modes. Applications of the columns with exchangeable chemistries are demonstrated with the capillary electrochromatographic separation of peptides and the nano-high-pressure liquid chromatography (HPLC) separation of proteins in both reversed phase and ion exchange modes.

Electrochromatographic characterization of methacrylate ester-based monolith and capillary electrochromatography separation of flavonoids

A porous polymethacrylate ester-based monolithic column for capillary electrochromatography (CEC) was designed by mean of in situ co-polymerizing lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a ternary porogenic solvent including cyclohexanol, 1,4-butanediol and water. After investigating the influence factors of the CEC monolithic columns, four flavonoids (i.e., Rutin, Quercetin, Kaempferol, and Quercitrin) were separated and assayed to evaluate this monolithic column with CEC method. Under optimum conditions, the CEC method exhibited high separation efficiency, with rapid separation time of 3–4 min, for the four flavonoid samples using 10 mM phosphate buffer containing 70% acetonitrile (pH 9.0). Importantly, the proposed method could provide a promising approach for rapid separation and detection in biomedicine.

Preparation of monolithic polylaurylmethacrylate column and its application in capillary electrochromatographic separation of myoglobin digests

The monolithic polylaurylmethacrylate column was prepared in a single step using the monomer solution containing lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA), and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), with the ternary porogenic solvent consisting of 1-propanol, 1,4-butanediol and H2O. The effects of AMPS content and the ratio of monomer solution to porogenic solvent were investigated. The optimal mass ratio of monomer solution to porogenic solvent was 35:65, the monomer solution was composed of 59.5% (w/w) LMA, 40% (w/w) EDMA and 0.5% (w/w) AMPS, and the porogenic solution was composed of 60% (w/w) 1-propanol, 30% (w/w) 1,4-butanediol and 10% (w/w) H2O. The prepared monolithic column was successfully applied in the capillary electrochromatographic (CEC) separation of myoglobin digests under the optimized mobile phase.

Microchip electrochromatography: the latest developments and applications

This review summarizes recent developments and applications of microchip electrochromatography (microCEC) mainly in the past five years between 2005 and 2009 with a focus on column technologies. In addition, some new improvements in the chip design and fabrication, sample preconcentration, electroosmotic flow control as well as mechanisms that govern electrochromatographic separation are described and reviewed. The features and limitations of several practical aspects of their applications are highlighted.

The synthesis of chloropropyl-functionalized silica hybrid monolithic column with modification of N,N-dimethyl-N-dodecylamine for capillary electrochromatography separation

A chloropropyl-functionalized silica (CP-silica) hybrid monolithic column was synthesized within the confines of a capillary via the sol–gel process using tetramethoxysilane (TMOS) and (3-chloropropyl)-trimethoxysilane (CPTMS) as the precursors. The resulting CP-silica hybrid monolith inside the capillary showed homogeneous macroporous morphology and was well attached to the inner wall of the capillary. The obtained CP-silica hybrid monolithic capillary column demonstrated the inherent hydrophobic property and could be applied as a reversed-phase stationary phase in CEC directly. Due to the great chemical reactivity of the incorporated chloro groups on the hybrid silica monolithic matrix, the chloropropyl moieties on the surface of the hybrid silica monolith matrix could be conveniently further modified by a tertiary amine of N,N-dimethyl-N-dodecylamine (DMDA) via the nucleophilic substitution reaction at 70 °C to introduce a dodecyl groups (C12) onto the CP-silica hybrid monolithic matrix. The resulting C12-silica hybrid monolithic column not only demonstrated the significantly enhanced hydrophobic property in the separation of alkylbenzenes in reversed-phase capillary electrochromatography (RP-CEC), but also the strong electroosmotic flow (EOF) in a wide pH range. Five alkylbenzenes could be baseline separated in 3 min with column efficiency ranging from 189 700 to 221 000 N/m with a 70% ACN running buffer in CEC.