Use of the sol-gel technique to prepare capillary columns coated with a macrocyclic dioxopolyamine for open-tubular capillary electrochromatography

Summary Columns for open-tubular (OT) capillary electrochromatography (CEC), coated with 1,4,7,10-tetraazacyclotridecane-11,13-dione (dioxo[13]aneN₄) by use of the sol-gel technique, have been investigated for the first time. Dioxol[13]aneN₄ was reacted with 3-(2-cyclooxypropoxy)propyltrimethoxysilane and the product was then mixed with tetraethoxysilane (TEOS) and water to form a glass matrix as a network in which the stationary phase (dioxo[13]aneN₄) was incorporated. In comparison with OTCEC columns prepared by the sol-gel process with TEOS only, the sol-gel-derived macrocyclic dioxopolyamine columns enabled better separations of a mixture of isomeric nitrophenols and benzenediols, a mixture of isomeric aminophenols and diaminobenzenes, and a mixture of four biogenic monoamine neurotransmitters. High efficiencies (60 000—340 000 plates m⁻¹) were achieved for isomeric benzenediols, aminophenols, nitrophenols, and diaminobenzenes. Migration time and theoretical plate number reproducibility was satisfactory;RSD was <2% for one column and <8.5% from column to column.     

Photopolymerized sol-gel frits for packed columns in capillary electrochromatography

Porous sol-gel frits are fabricated in a capillary column by filling it with a solution of 3-(trimethoxysilyl)propyl methacrylate, hydrochloric acid, water, toluene (porogen), and a photoinitiator (Irgacure 1800) and exposing it to UV light at 365 nm for 5 min. The separation column (30 cm x 75 microm I.D.) contains between the inlet and outlet frits a 15-cm packed segment filled with 5-microm silica particles modified with the chiral compound (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine. A detection window (1 mm long) is placed immediately after the outlet frit. To demonstrate the performance of this chiral separation column, mixtures of 16 different amino acids (three of which are not naturally occurring) derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole were separated by capillary chromatography. The enantiomeric separation of the column results in a resolution ranging from 1.21 to 8.29, and a plate height ranging from 8.7 to 39 microm.     

A novel sol-gel calix[4]arene-modified capillary column for open-tubular capillary electrochromatography

A novel open-tubular CEC (OT-CEC) column was prepared by immobilized 5,11,17,23-tetra-tert-butyl-25,27-diethoxy-26,28-dihydroxy-calix[4]arene (Calix[4]) on fused-silica capillary column with sol-gel technology. Calix[4] was initially reacted with gamma-glycidoxypropyltrimethoxysilane (KH-560) to form a new sol-gel precursor (calix[4]-KH-560), and then mixed with another precursor, namely tetraethoxysilane (TEOS). After hydrolysis and condensation, a sol-gel matrix was obtained, in which calix[4] was incorporated. Successful calix[4]-modified sol-gel coating was suggested by infrared (IR) spectra and greatly decreased EOF. In comparison with OT columns prepared by the sol-gel method with TEOS and KH-560 only, the calix[4]-modified sol-gel column showed greatly improved separation of isomeric toluidines, nitrophenols, picolines and neurotransmitters with structural similarity. Migration time and theoretical plate number reproducibility were satisfactory with RSDs less than 1 and 6% each for within column runs and not more than 3 and 7% each for column-to-column measurements, using toluidine and nitrophenol isomers as test solutes on this column. High separation efficiencies (96,000-300,000 plates/m) for basic toluidine isomers were obtained. This column was also successfully employed to combine extraction procedure for the determination of dopamine (DA) and norepinephrine (NE) in Portulaca oleracea L. The recoveries of DA and NE were 93.3 and 94.2%, respectively.     

Preparation of stationary phases for open-tubular capillary electrochromatography using the sol-gel method

Capillary electrochromatography requires the deposition of a stationary phase inside the capillary. In this paper the sol-gel method is proposed for this purpose. The gels were prepared externally and injected into a fused-silica capillary, where anchorage to the capillary wall was possible through condensation reactions between the silanol groups of the capillary wall and the residual silanol groups the gel. Contrary to a commonly used practice, alkaline pretreatment of the inner capillary wall prior to the introduction and anchoring of the gel was found to be only marginally effective in improving the mechanical stability of the column. The influence of various parameters, such as the pH, the water content, the presence of alcohol (ethanol) on the formation of tetraethoxysilane (TEOS)-n-octyltriethoxysilane (C8-TEOS) hybrid gels of varied composition is discussed. The pH and the amount of water present were found to be the determining factors in the preparation of a stable gel with the desired mechanical and chromatographic properties. By carrying out the gel formation at 80 degrees C, capillary columns could be produced in 2.5 h. While an acidic pH was required during (external) gel formation, subsequent treatment of the gel inside the capillary with an alkaline solution ('aging') was found to improve separation and stationary phase capacity significantly. The capillary columns were subsequently used to separate a mixture of polycyclic aromatic hydrocarbons in less than 3 min.     

Analytical separations in open-tubular capillary electrochromatography

This review represents a summary of recent progress in open-tubular capillary electrochromatography (OT-CEC) for chiral and achiral separations. The OT-CEC approach is an alternative to packed-CEC that could eliminate the problems associated with retaining frits and silica particles. In OT-CEC, the stationary phase is immobilized on the inner walls of the capillary. Preparation of the stationary phase is critical for OT-CEC. The preparation methods for capillary columns include (i) adsorption, (ii) covalent bonding and/or cross-linking, (iii) porous layers, (iv) chemical bonding after etching, (v) sol-gel, and (vi) molecular imprinting. Major developments, potential applications, technical difficulties and advantages associated with these wall coatings in OT-CEC are presented. In addition, the coupling of OT-CEC with mass spectrometry (MS) is briefly reviewed. Several applications of this hyphenated technique for analytical separations are also summarized.     

Metallomesogenic stationary phase for open-tubular capillary electrochromatography

A synthetic coppermesogenic polymer is prepared and then covalently bonded to the siloxane-based deactivated column as the stationary phases of open-tubular CEC with essentially high phase ratio. The EOF generated from the modified phase is surveyed through conventional aqueous buffers and hydroorganic mobile phases. Zeta potentials, which are computed from the EOF data and the ratio of dielectric constant to viscosity, are plotted as a function of pH, ionic molarity, and compositional range. These plots responsible for the electroosmotic characteristic of the bonded phases are found to be like those of bare fused-silica or deactivated columns through decreasing or increasing the ACN content in the mobile phase, respectively. This two-phase characteristic is basically derived from the polymeric configuration with carboxylato ligands attached onto the polysiloxane backbone. Phthalates and amino acids are suitable probes to examine the two phenomena, more-polar and less-polar mediums, respectively, and to judge whether the chromatographic retention is the major source of separation mechanism. With the mixing modes of Lewis acid-base interaction, dispersive force, and shape discrimination, the chromatographic partition adequately accomplishes the uneasily resolved separations by only CZE mode, although the electrophoretic migration is truly somewhat involved.     

Recent trends in open-tubular capillary electrochromatography

Capillary electrochromatography (CEC), which combines the advantages of the high efficiency of capillary electrophoresis (CE) and the high selectivity of liquid chromatography (LC), has recently received considerable attention. Most CEC experiments have been performed with capillary columns packed with small LC packing materials (1.5–5 μm particle diameter). However, problems such as difficulties in packing the small LC packing materials and fabricating the frits still exist in preparing the CEC column. The use of open-tubular columns in CEC is therefore an alternative approach that can eliminate the problems encountered in packed-column CEC. So far, several types of open-tubular columns have been proposed for CEC separations and in this article recent progress in this area is reviewed.     

Polymer-brush stationary phases for open-tubular capillary electrochromatography

Synthesis of poly(2-hydroxyethyl methacrylate) (PHEMA) brushes from the inside of silica capillaries by surface-initiated atom transfer radical polymerization (ATRP) yields unique stationary phases for open-tubular capillary electrochromatography (OT-CEC). Although PHEMA brushes have only a small effect on the separation of a set of phenols and anilines, derivatization of PHEMA with ethylenediamine (en) allows baseline resolution of several anilines that co-elute from bare silica capillaries. Derivatization of PHEMA with octanoyl chloride (C8-PHEMA films) affords even better resolution in the separation of a series of phenols and anilines. Increasing the thickness of C8-PHEMA coatings by a factor of 2 enhances resolution for several solute pairs, presumably because of an increase in the effective stationary phase to mobile phase volume ratio. Thus, this work demonstrates that thick polymer brushes provide a tunable stationary phase with a much larger phase ratio than is available from monolayer wall coatings. Through appropriate choice of derivatizing reagents, these polymer brushes should allow separation of a wide range of neutral molecules as well as compounds with similar electrophoretic mobilities.     

Alkylthiol gold nanoparticles in open-tubular capillary electrochromatography

Open-tubular columns for capillary electrochromatography (CEC) were formed by immobilising dodecanethiol gold nanoparticles on prederivatised 3-aminopropyl-trimethoxysilane (APTMS) or 3-mercaptopropyl-trimethoxysilane (MPTMS) fused-silica capillaries. The initial stage of this research involved the synthesis and characterisation of dodecanethiol gold nanoparticles, with tunnelling electron microscopy analysis of the dispersed phase of the gold nanoparticles dispersion in CHCl3, revealing spherical particles. The surface features of an Au-MPTMS coated capillary column were determined using scanning electron microscopy. The electroosmotic flow characteristics of Au-APTMS and Au-MPTMS capillary columns were then determined, by varying the pH and the voltage. The electrochromatographic properties of the gold nanoparticles CEC capillaries were investigated using a "reversed-phase" test mixture of thiourea, benzophenone and biphenyl and selected pyrethroid pesticides. Efficient separations of benzophenone and biphenyl solutes on Au-MPTMS and Au-APTMS capillary columns were obtained, as were linear plots of logarithm capacity factor versus % MeOH. A study of the reproducibility of retention for these solutes on Au-APTMS, Au-MPTMS and on a loosely coated capillary demonstrated the necessity of a coupling agent to prevent the gold nanoparticles from washing-off. These dodecanethiol gold capillary columns are easier to produce and operate than packed capillary columns. The research work confirms the use of gold nanoparticles as a novel phase for open-tubular CEC, demonstrating reproducible retention and characteristic reversed-phase behaviour.     

Packing columns for capillary electrochromatography

Considering the current interest in capillary electrochromatography (CEC), performed in packed columns, we present the different methods used to pack capillary columns for use in CEC. General considerations on column packing are given and the column fabrication process is discussed in sufficient detail to allow instruction to those who are not experienced in the field. Five different packing methods are discussed to deliver packing material into the capillary column from a practical view point: slurry pressure packing, packing with supercritical CO2, electrokinetic packing, using centripetal forces, and packing by gravity. Entrapment of particulate material by sintering and sol-gel technology is also mentioned. Although slurry pressure packing procedures are most common, higher separation efficiencies are obtained using other packing approaches. Electrokinetic packing seems to be the simplest technique to deliver the packing material into the capillary columns. Nevertheless, as with the other packing techniques, skill and experience are required to complete all the steps involved in the fabrication of packed columns for CEC.     

A new type of capillary column for open-tubular electrochromatography

A new type of open-tubular C(18) ester-bonded electrochromatographic column was prepared with sol-gel technology, followed by an on-column octadecyl silylation reaction. Glycidoxypropyltrimethoxysilane, a widely used and important silane agent, was used as the sol-gel precursor to form a thin coating layer on the wall of the fused-silica capillary. The C(18) groups were introduced into the coating layer by on-column esterification reaction with stearic acid. The electrochromatography behavior of the column was evaluated in terms of the separation of peptides. A high efficiency of 4.8x10(5) plates/m was achieved for a basic pentapeptide using the C(18 )ester-bonded column. In comparison with bare capillaries and glycidoxypropyltrimethoxysilane sol-gel-coated capillaries, the C(18) ester-bonded column showed the best separation of a mixture of seven pentapeptides under identical conditions of buffer, pH, and applied voltage.     

Comparison of separation behavior of benzodiazepines in packed capillary electrochromatography and open-tubular capillary electrochromatography

Packed column capillary electrochromatography (CEC), open-tubular CEC and microcolum liquid chromatography (LC) using a cholesteryl silica bonded phase have been studied to compare the retention behavior for benzodiazepines. It has been found that packed column CEC gives better resolution, faster analysis time than microcolumn LC for benzodiazepines maintaining similar selectivity except for some solutes which are charged species under the separation conditions. However, open-tubular CEC gave different selectivities to a larger extent for charged benzodiazepines from that which should be produced by the chromatographic properties of the cholesteryl silica phase. Charged species migration times are mainly influenced by electrophoretic mobility rather than the chromatographic interactions.     

Recent highlights in stationary phase design for open-tubular capillary electrochromatography

This review examines the most recent innovations made to achieve high performance in open-tubular capillary electrochromatography (OT-CEC) separations, focusing on the ingenious chemical and physical solutions made to increase the surface area and equip the stationary phase with exploitable selectivity. Among the approaches taken are chemically bonded ligands, etching with chemical bonding, sol-gels, molecularly imprinted polymers, porous layers, physically attached or adsorbed phases, and nanoparticle coatings. Particularly noteworthy are modern developments with macrocyclic receptor ligands, nanoparticles and open channel electrochromatography on-chip.     

Multi-walled carbon nanotube composites with polyacrylate prepared for open-tubular capillary electrochromatography

A new phase containing immobilized carbon nanotubes (CNTs) was synthesized by in situ polymerization of acid-treated multi-walled CNTs using butylmethacrylate (BMA) as the monomer and ethylene dimethacrylate as the crosslinker on a silanized capillary, forming a porous-layered open-tubular column for CEC. Incorporation of CNT nanomaterials into a polymer matrix could increase the phase ratio and take advantage of the easy preparation of an OT-CEC column. The completed BMA-CNT column was characterized by SEM, ATR-IR, and EOF measurements, varying the pH and the added volume organic modifier. In the multi-walled CNTs structure, carboxylate groups were the major ionizable ligands on the phase surface exerting the EOF having electroosmotic mobility, 4.0 × 10(4) cm2 V(-1)1 S(-1)1, in the phosphate buffer at pH 2.8 and RSD values (n=5), 3.2, 4.1, and 4.3%, for three replicate capillaries at pH 7.6. Application of the BMA-CNT column in CEC separations of various samples, including nucleobases, nucleosides, flavonoids, and phenolic acids, proved satisfactory upon optimization of the running buffers. Their optima were found in the borate buffers at pH 9.0/50 mM, pH 9.5/10 mM/50% v/v ACN, and pH 9.5/30 mM/10% v/v methanol, respectively. The separations could also be used to assess the relative contributions of electrophoresis and chromatography to the CEC mechanism by calculating the corresponding velocity and retention factors. Discussions about interactions between the probe solutes and the bonded phase included the π-π interactions, electrostatic repulsion, and hydrogen bonding. Furthermore, a reversed-phase mode was discovered to be involved in the chromatographic retention.     

Multi-wall carbon nanotubes bonding on silica-hydride surfaces for open-tubular capillary electrochromatography

Prepared multi-wall carbon nanotube (MWNT) materials, including untreated MWNT, HNO₃-treated MWNT and HNO₃-HCl-treated MWNT were covalently attached onto a silica-hydride-modified capillary by hydrosilation, using the abundant double bonds between the pentagon carbons in the MWNT structure. These MWNT-incorporated capillaries were characterized by SEM, ATR-IR and electroosmotic flow (EOF) measurements in phosphate buffers with a pH range of 3.7–9.3 and in the mixtures of acetonitrile modifier. The untreated capillary was assumed to carry some carboxylate groups formed on the non-acid-treated MWNTs, as it had higher EOF values than the hydride capillary. As the MWNTs were treated with HNO₃ and HCl solutions, the capillaries had increasingly higher EOF values. To examine the existence of an electrochromatography mechanism in the modified capillaries, a mixture of nucleosides and thymine was probed to check the velocity factor and retention factor. In addition to the π–π interaction between the probe solutes and the MWNT immobilized stationary phases; a reversed-phase mechanism could contribute to the chromatographic retention. For acidic tetracyclines, increasing the loadability of MWNTs resulted in a high retention factor and improved the separation resolution.     

Advances in capillary electrochromatography and micro-high performance liquid chromatography monolithic columns for separation science

Over the last decade, monoliths or continuous beds have emerged as an alternative to traditional packed-bed columns for use in capillary electrochromatography (CEC) and micro-high performance liquid chromatography (micro-HPLC). Monolithic columns can be divided into two categories: silica-based monolithic columns and rigid organic polymer-based monolithic columns resulting from the polymerization of acrylamide, styrene, acrylate or methacrylate monomers. In this paper, the chemistry and most recent applications of these various types of monoliths in both CEC and micro-HPLC are presented.     

Gold nanoparticle-coated capillaries for protein and peptide analysis on open-tubular capillary electrochromatography

We report a new method of immobilization of gold nanoparticles (AuNPs) on a fused-silica capillary through covalent binding. The resulting modified capillary was applied to electrophoretic systems to improve the efficiency of separation and the selectivity of selected solutes. The immobilization of AuNPs on the capillary wall was performed in a very simple and fast way without requiring heating. The surface features of an AuNP-coated capillary column were determined using the scanning electron microscopy. The chromatographic properties of AuNP-coated capillaries were investigated through variation of the buffer pH and separation voltage. Effective separations of synthetic peptides mixture were obtained on the AuNP-coated capillaries. The method shows a remarkable stability since it was reused about 900 times. The capacity factor was duplicated. Therefore, this modification is stable and can be applied to different separation purposes. A complex mixture of tryptic peptide fragments of HSA was analyzed in both the bare- and the AuNP-coated capillaries. Better electrophoretic peptide profile was observed when using the AuNP-coated capillary.     

开管毛细管电色谱分析3种解热镇痛药物

针对生物体液样品开展药物的绿色高效毛细管电泳分离分析具有重要的研究意义。该研究以3种解热镇痛药(4-氨基安替比林、氨基比林及非那西汀)为研究对象,以嵌段聚合物为涂层,建立了药物的开管毛细管电色谱(OT-CEC)分析新策略。首先,采用活性/可控自由基可逆加成-断裂链转移聚合方法,合成制备得到了两亲性嵌段聚合物-聚(苯乙烯-甲基丙烯酸缩水甘油酯)(P(St-GMA)),并将其涂覆到毛细管内壁;其次,通过考察影响OT-CEC分离效率的关键因素,包括嵌段聚合物的聚合时间、涂覆毛细管嵌段聚合物的浓度、电泳运行缓冲液的种类和pH值、有机溶剂添加剂等,优化了3种解热镇痛药物的OT-CEC分离条件;最终发现,不需添加任何有机溶剂及表面活性剂,仅采用50.0 mmol/L乙酸钠-乙酸(pH 5.7)作为OT-CEC的缓冲溶液,就能实现3种解热镇痛药物的基线分离。在8.0～2.5×10³μmol/L范围内,分析物峰面积与其对应的浓度呈现良好的线性关系,相关系数(R²)均大于0.995,检出限为1.0～2.5μmol/L。结果表明:P(St-GMA)在溶液中自组装...     

Silicate entrapped columns--new columns designed for capillary electrochromatography

Designed especially for capillary electrochromatography (CEC), silicate-entrapped columns are made by trapping particles of chromatographic packing material in a network of silica. Once entrapped, the capillary no longer requires frits. This renders a more homogeneous and stable packed bed. Accidental breakage of the fragile frits is not an issue with these robust columns. Columns packed with reverse-phase material subjected to silicate entrapment demonstrated faster separations of retained analytes and increased efficiencies compared with nonentrapped columns. The method was also used to prepare chiral CEC columns by entrapping a molecular imprinted polymeric (MIP) packing having minimal surface charge density, thus being unable alone to support sufficient electroosmotic flow for CEC.