Fast determination of prominent carotenoids in tomato fruits by CEC using methacrylate ester-based monolithic columns

In this study, the major carotenoids (beta-carotene and lycopene) present in tomato fruits were analyzed by CEC with a methacrylate ester-based monolithic column. The effects of the porogenic solvent ratio, and the hydrophobicity of bulk monomer employed were examined on carotenoids separations. A fast separation of these analytes was achieved in less than 5.0 min in a mobile phase containing 35% THF, 30% ACN, 30% methanol, and 5% of a 5 mM Tris aqueous buffer, pH 8, with lauryl methacrylate-based monoliths. The CEC method was evaluated in terms of detection limit and reproducibility (retention time, area, and column preparation) with values below 1.6 microg/mL and 7.2%, respectively. The proposed procedure was successfully applied to the determination of both carotenoids in fruits of several tomato-related species and its usefulness to analyze large series of samples for nutritional quality screening trials in tomato breeding programs is demonstrated. To our knowledge, this is the first work that exploits the powerful and user-friendly monolithic technology for quality breeding and germplasm evaluation program purposes.     

Rapid determination of sterols in vegetable oils by CEC using methacrylate ester‐based monolithic columns

A method for the determination of sterols in vegetable oils by CEC with UV–Vis detection, using methacrylate ester‐based monolithic columns, has been developed. To prepare the columns, polymerization mixtures containing monomers of different hydrophobicities were tried. The influence of composition of polymerization mixture was optimized in terms of porogenic solvent, monomers/porogens and monomer/crosslinker ratios. The composition of the mobile phase was also studied. The optimum monolith was obtained with lauryl methacrylate monomer at 60:40% (wt:wt) lauryl methacrylate/ethylene dimethacrylate ratio and 60 wt% porogens with 20 wt% of 1,4‐butanediol (12 wt% 1,4‐butanediol in the polymerization mixture). Excellent resolution between sterols was achieved in less than 7 min with an 85:10:5 v/v/v ACN–2‐propanol–water buffer containing 5 mM Tris at pH 8.0. The limits of detection were lower than 0.04 mM, and inter‐day and column‐to‐column reproducibilities at 0.75 mM were better than 6.2%. The method was applied to the determination of sterols in vegetable oils with different botanical origins and to detect olive oil adulteration with sunflower and soybean oils.     

Analyses of benzophenones by capillary electrochromatography using methacrylate ester-based monolithic columns

In this study, eight benzophenones, which are commonly used as UV filters in various cosmetics and plastics, were analyzed by capillary electrochromatography with a methacrylate ester-based monolithic column. The effects of the composition and pH of mobile phase, porogenic solvent ratio, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) content on benzophenone separations were examined. For all benzophenones, separation performances were markedly improved in monolithic columns with larger 1-propanol ratio and higher AMPS content. Furthermore, a twofold increase in AMPS content almost reduced the separation time in half when a monolithic column had an adequately high surface area, i.e. monolithic column was produced in a higher ratio of 1-propanol. As well, the retention behaviors of these analytes in the monolithic column were strongly influenced by the level of acetonitrile in the mobile phase, and the pH of the mobile phase also had an apparent influence on separation resolution.     

Analyses of preservatives by capillary electrochromatography using methacrylate ester-based monolithic columns

Five common food preservatives were analyzed by capillary electrochromatography, utilizing a methacrylate ester-based monolithic capillary as separation column. In order to optimize the separation of these preservatives, the effects of the pore size of the polymeric stationary phase, the pH and composition of the mobile phase on separation were examined. For all analytes, it was found that an increase in pore size caused a reduction in retention time. However, separation performances were greatly improved in monolithic columns with smaller pore sizes. The pH of the mobile phase had little influence on separation resolution, but a dramatic effect on the amount of sample that was needed to be electrokinetically injected into the monolithic column. In addition, the retention behaviors of these analytes were strongly influenced by the level of acetonitrile in the mobile phase. An optimal separation of the five preservatives was obtained within 7.0 min with a pH 3.0 mobile phase composed of phosphate buffer and acetonitrile 35:65 v/v. Finally, preservatives in real commercial products, including cold syrup, lotion, wine, and soy sauces, were successfully determined by the methacrylate ester-based polymeric monolithic column under this optimized condition.     

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.     

CEC separation of heterocyclic amines using methacrylate monolithic columns

Two methacrylate-based monolithic columns, one with a negatively charged group (sulfonic group) and another with a new monomer N,N-dimethylamino ethyl acrylate (DMAEA), were prepared and tested for the separation of basic compounds by CEC. This new monolithic stationary phase was prepared by the in situ polymerization of DMAEA with butyl methacrylate and ethylene dimethacrylate, using a ternary porogenic solvent consisting of water, 1-propanol and 1,4-butanediol. The performance of this column was evaluated by means of the analysis of a family of heterocyclic amines. Separation conditions such as pH, amount of organic modifier, ionic strength and elution mode (normal or counterdirectional flow) were studied. At the optimal running electrolyte composition, and using the counterdirectional mode, symmetrical electrochromatographic peaks were obtained, with the number of theoretical plates up to 30,000 and a good resolution between closely related peaks. The 2-acrylamido-2-methyl-1-propane-sulfonic acid column was used for CEC-MS, taking advantage of the compatibility of its elution mode (normal flow) with the MS coupling.     

A model of flow-through pore formation in methacrylate ester-based monolithic columns

The main factors affecting the porosity of methacrylate-ester based monolithic columns were investigated. We prepared 23 monolithic capillary columns with porosity controlled by varying the proportions of butyl methacrylate and ethylene dimethacrylate monomers and of 1,4-butanediol and 1-propanol as the porogen solvent in the polymerization mixtures by thermally initiated in-situ polymerization in fused-silica capillaries. Using mixture design software, we systematically varied the composition of the polymerisation mixtures to find significant factors affecting flow-through pore formation. Multivariate analysis of the experimental data obtained for the fabricated columns yielded a model for prediction of the flow-through porosity in monolithic beds as a function of the composition of the polymerization mixture used to prepare polymethacrylate monolithic capillary columns. The mean error of prediction was lower than 8% for eight columns prepared independently of the original set of 15 columns used to derive the flow-through model. The flow-through porosity increases with increasing concentration of the binary porogen solvent mixture, the concentration of 1,4-butanediol being the main factor enhancing flow-through pore formation. On the other hand, increasing concentrations of the hydrophobic monomer butyl methacrylate and increasing concentrations of 1-propanol have a negative effect on flow-through pore formation. The capillary columns prepared with a high proportion of flow-through pores and a minimum amount of mesopores can be used for fast gradient separations of both low-molecular weight compounds and biopolymers.     

Study of elution behaviour with gradient voltage in CEC using methacrylate monolithic columns

A theoretical study on the retention behaviour and chromatographic performance of neutral solutes using a lauryl methacrylate‐based monolithic column under voltage gradient mode in CEC was carried out. Through a flexible mathematical function based on a modified Gaussian model, the peak shape of compounds was firstly fitted under constant and gradient voltage. Using the peak shape parameters and retention time, the estimation of global chromatographic performance, efficiency and peak capacity under several voltage conditions was performed. The influence of voltage gradient on the separation efficiency is discussed and simple equations are presented to calculate retention and peak widths under voltage gradient conditions. A comparison in terms of chromatographic performance of a test mixture of neutral solutes under constant and gradient voltage modes was also carried out. The experiments carried out under gradient voltage showed better efficiencies (172 000 plates/m) and lower peak widths than those obtained under constant voltage (52 000 plates/m).     

Preparation and characterization of hexyl methacrylate monolithic columns for CEC

The preparation of hexyl methacrylate (HMA) monolithic columns for CEC separations has been investigated with two initiation systems: (i) ammonium peroxodisulphate and TEMED to activate the polymerization reaction, and (ii) by thermal initiation with AIBN. For both initiators, the influence of composition of porogenic solvent on morphological and chromatographic properties of monoliths was investigated. Two porogenic solvent systems, aqueous and non-aqueous media, were also studied for monolithic beds polymerized with AIBN. Under optimal conditions, low minimum plate heights (9.6 mum for peroxodisulphate, 8.4 and 10.0 mum for AIBN in aqueous and non-aqueous porogenic solvents, respectively) were obtained. A comparison in terms of chromatographic performance of HMA monoliths with butyl methacrylate columns polymerized with both initiators was also performed. The resulting HMA-based stationary phases also exhibited a good repeatability and column-to-column reproducibility, with RSD values below 5.6% in the studied electrochromatographic parameters. The potential of HMA-based columns was demonstrated by the analysis of complex mixtures of polyaromatic hydrocarbons and anabolic steroids.     

甲基丙烯酸酯整体柱毛细管电色谱法快速分析白芷中的主要活性成分

以甲基丙烯酸酯整体柱为分离柱,建立了一种快速分离、分析白芷药材提取物中的主要活性成分欧前胡素(imperatorin)、异欧前胡素(isoimperatorin)、珊瑚菜内酯(phelloptorin)和发卡二醇(falcarindiol)的毛细管电色谱(CEC)方法。在整体柱制备实验中对三元致孔剂组成成分的比例进行了系统的考察。在分离实验中对流动相的组成(包括有机相组成、缓冲液浓度和缓冲液的pH值)进行了优化。最终的优化条件为: 流动相为乙腈-20 mmol/L NaH2PO4(pH 4.95)(50:50, v/v),分离电压为－25 kV。结果表明,所制备的甲基丙烯酸酯毛细管整体柱具有良好的渗透性和重现性;4种分析物的标准曲线线性关系良好(r2＞0.997),检出限均小于0.34 mg/L,加样回收率为95.18%～98.44%。该方法快速、简便、可靠。应用该方法对18个不同产地的白芷样品进行了测定,并对其药材质量进行了评价。     

Lauroyl peroxide as thermal initiator of lauryl methacrylate monolithic columns for CEC

The preparation of lauryl methacrylate (LMA)-based monolithic columns for CEC using lauroyl peroxide (LPO) as thermal initiator of polymerization has been investigated. The influence of initiator amount and composition of porogenic solvent on the physical and electrochromatographic properties of the resulting LMA-based monoliths was evaluated. A comparison with LMA-based columns thermally polymerized with AIBN was performed. At a given porogenic solvent composition, LMA stationary phases initiated with LPO showed higher permeabilities and better efficiency values than those prepared using AIBN as initiator. The optimum polymerization mixture found for LPO initiator provided a minimum plate height of 9.5 mum in a polycyclic aromatic hydrocarbon mixture. The produced monolithic beds also exhibited a good run-to-run repeatability and column-to-column and mixture-to-mixture reproducibility, with RSD values below 5.3% for the retention factors, areas and plate heights.     

Photo‐polymerized lauryl methacrylate monolithic columns for CEC using lauroyl peroxide as initiator

Lauryl methacrylate (LMA)‐ester based monolithic columns photo‐polymerized using lauroyl peroxide (LPO) as initiator were prepared, and their morphological and CEC properties were studied. The composition of the polymerization mixture (i.e. ratios of monomers/porogenic solvents, 1,4‐butanediol/1‐propanol and LMA/crosslinker) was optimized. The morphological and chromatographic properties of LMA columns were evaluated by means of SEM pictures and van Deemter plots of PAHs, respectively. The polymerization mixture selected as optimal provided a fast separation of a mixture of PAHs with excellent efficiencies (minimum plate heights of 8.9–11.1 μm). Satisfactory column‐to‐column (RSD<4.5%) and batch‐to‐batch reproducibilities (RSD<6.3%) were achieved. The LMA columns photo‐polymerized with LPO were compared with those prepared with AIBN. Using PAHs, alkylbenzenes and basic compounds for testing, the columns obtained with LPO gave the best compromise between efficiency, resolution and analysis time.     

Comparison of thermal‐ and photo‐polymerization of lauryl methacrylate monolithic columns for CEC

Lauryl methacrylate‐based (LMA) monolithic columns for CEC, prepared using either thermal initiation or by UV‐irradiation in the presence of AIBN have been compared. Thermal polymerization was carried out at 70°C for 20 h. For UV initiation, the effects of the time exposure to UV light and irradiation energy were investigated. For each initiation process, the influence of composition of porogenic solvent (1,4‐butanediol/1‐propanol ratio) on the physical and electrochromatographic properties of the resulting monoliths was also evaluated. Photochemically lauryl methacrylate stationary phases initiated showed higher permeabilities and better efficiencies than those prepared by thermal initiation. After optimization of polymerization mixture, photopolymerized columns provided a permeability of 4.25×10^?13 m² and a minimum plate height of 13.4 μm for a mixture of polycyclic aromatic hydrocarbons. Similar column‐to‐column and batch‐to‐batch reproducibilities, with RSD values below 11.6 and 11.0 % for the thermal‐ and UV‐initiated columns, respectively, were obtained.     

Simultaneous analysis of cationic, anionic, and neutral compounds using monolithic CEC columns

A new capillary electrochromatography (CEC) column for the simultaneous analysis of cationic, neutral, and anionic compounds using CEC-ESI-MS is described. Three different silica monolith columns were prepared by changing the poly(ethylene glycol) (PEG) contents for comparison of the separation property of these columns. Different separation programs were used for the simultaneous separation of different charged compounds under the same conditions. The column prepared with 80 mg of PEG separated typical compounds within 15 min using 1 M formic acid as the electrolyte. The analytes migrated in the order of cationic, neutral, and anionic compounds, which means that the migration order was mainly determined by the electrophoresis. The hydrodynamic flow by pressure from the inlet side was significant for a stable analysis to be achieved. The effect of the composition of the sheath liquid was also examined. All analytes (14 amino acids, thiourea, urea, citric acid, and ATP) were detectable when 1% acetic acid in 50% (v/v) methanol was used as the sheath liquid.     

Determination of tocopherols and sterols in vegetable oils by solid-phase extraction and subsequent capillary gas chromatographic analysis.

In general, analyses of tocopherols and sterols are performed separately in vegetable oils. By applying solid-phase extraction (SPE) prior to capillary gas chromatography, a simple and reliable procedure for the quantification of both tocopherols and sterols in a single analytical run has been developed. SPE was used as sample clean up procedure for the separation of these minor components from the triacylglycerol matrix, replacing time consuming saponification or on-line LC-GC. The analysis of tocopherols and free sterols in five different vegetable oils illustrates robustness and reliability of this method outlined. Quantification of the analytes was performed by external calibration with reference substances and internal standardization. The recovery of the procedure as well as the repeatability of the quantitative results have been evaluated.     

电感耦合等离子体发射光谱法测定植物油中的磷

用电感耦合等离子体发射光谱法(ICP-AES)测定了植物油中的磷.采用多谱线拟合技术(MSF)校正了铜对P213.617 nm和P214.914 nm光谱干扰.比较了活性炭炭化灰化法和微波消解法两种样品前处理方法对分析结果的影响.结果表明这两种前处理方法所得结果都能与国标磷钼蓝分光光度法的分析结果吻合,其中活性炭炭化灰化法的方法检出限(0.053 mg/kg)较微波消解法的方法检出限(0.42 mg/kg)更低,所以对低含量的磷的检测结果其相对误差及精密度更好.该法应用于植物油中磷的测定.     

Determination of DNA entrapment into liposomes using short monolithic columns

A high-performance liquid chromatography (HPLC) method for the determination of DNA entrapment efficiency in liposomes has been developed. Plasmid DNA was encapsulated into positively charged liposomes. Non-entrapped DNA was separated by ultracentrifugation from liposomes and supernatant was chromatographed on Convective Interaction Media (CIM) DEAE disk. The elution of DNA was monitored by the absorbance at 260 nm and the quantity of DNA in the tested sample was calculated from the integrated peak areas using the appropriate standard curve. This method is fast, simple, precise and does not require any kind of DNA labelling in contrast with mostly used methods for determination of DNA entrapment efficiency.     

Efficiency of methacrylate monolithic columns in reversed-phase liquid chromatographic separations

Butyl-methacrylate-based porous monoliths were prepared inside fused-silica capillaries as reversed-phase separation media for liquid chromatography (LC) and capillary electrochromatography (CEC). During our previous research on methacrylate-based monoliths for reversed-phase separations, we noticed that a separation efficiency of up to 300,000 plates/m can easily be obtained in the CEC mode for unretained compounds. However, the efficiencies for retained compounds were much lower in reversed-phase systems, especially in pressure-driven LC. In this work methacrylate-based columns were prepared and characterized in terms of efficiency and retention in reversed-phase (pressure-driven) LC and in CEC. Much attention has been paid to the mass-transfer mechanism in the stationary phase. Factors that affect the plate heights for specific compounds have been investigated. A possible explanation for the relatively low separation efficiency of retained compounds and suggestions to improve molecular mass transfer are provided.     

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.     

Preparation and evaluation of butyl acrylate-based monolithic columns for CEC using ammonium peroxodisulfate as a chemical initiator

Acrylate-ester-based monoliths for CEC using peroxodisulfate as a chemical initiator were prepared. The influence of two ternary porogenic solvents on the physical and chromatographic properties of butyl acrylate monolithic stationary phases was investigated. The composition and the ratio of porogenic solvent were adjusted to obtain highly permeable rigid monoliths with adequate column efficiency. Among the prepared butyl acrylate monoliths, those polymerized from a ternary porogenic solvent of acetonitrile/ethanol/water exhibited the most promising performance with a minimum plate height for naphthalene of 10.5 microm and a bed permeability of 7.3 x 10(-14) m(2). A comparison in terms of efficiency and permeability with thermal and UV initiation using alpha,alpha'-AIBN was also performed. The resulting monolithic stationary phases were evaluated in terms of reproducibility, giving RSD values below 5.1% in the electrochromatographic properties studied.