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.     

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.     

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.     

Study of peak shape and efficiency in butyl acrylate-based monolithic columns for capillary electrochromatography

The study and modelling peak shape of in capillary electrochromatography (CEC), obtained using butyl acrylate (BA)-based monolithic columns, is described in this article. A modified-Gaussian model, which is a function of conventional experimental parameters: retention time (t_R), peak height (H₀) and standard deviation (σ₀) at the peak maximum, and left (A) and right (B) halfwidths, was used to describe the peaks of thiourea and several polyaromatic hydrocarbons compounds, which were eluted under several applied voltages. A mean relative error below 2% in the prediction of peak shape is obtained. Based on peak parameters, an easy and reliable estimation of global chromatographic performance, efficiency and peak capacity of BA-based monoliths was also considered. A comparison in terms of chromatographic performance of BA-based monoliths in CEC with CE mode and RPLC microparticulate columns was also performed.     

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。     

Fast separation and determination of coumarins in Fructus cnidii extracts by CEC using poly(butyl methacrylate‐co‐ethylene dimethacrylate‐co‐[2‐(methacryloyloxy)ethyl] trimethylammonium chloride) monolithic columns

A rapid CEC method with poly(butyl methacrylate‐co‐ethylene dimethacrylate‐co‐[2‐(methacryloyloxy)ethyl] trimethylammonium chloride) monolithic column has been developed for separation and determination of four coumarins (isopimpinelline, bergapten, imperatorin, and osthole) in Fructus cnidii extracts. The effect of polymerization condition including the monomers ratio and the porogens ratio were studied. The mobile‐phase composition, such as the composition of organic solvent, the concentration and pH of buffer, was also optimized. Under the same condition (50% ACN and 50% of a 10 mM sodium dihydrogen phosphate electrolyte at pH 4.95), in contrast to 25 min of analysis time in HPLC and 10 min of analysis time in pCEC, a fast separation of these analytes was achieved in less than 5 min in CEC. Method validation was developed in accordance with the analytical procedures. Intra‐ and interday precisions (RSD) for relative retention time and peak area were less than 1.69 and 4.63%, and LODs were lower than 0.5 μg/mL. Calibration curves of four compounds also showed good linearity (r²>0.995). The mean recoveries ranged between 93.91 and 98.65%. With this CEC system, the quality of F. cnidii extracts from various resources was evaluated by determining the contents of the four coumarins.     

Pressure‐assisted CEC versus CEC using methacrylate‐based monolithic columns: Influence of the polymerization‐mixture composition

Pressure‐assisted CEC (pCEC) can either be performed on a CE instrument by adding pressure at the column inlet, or by applying voltage on a capillary liquid chromatography system. This study investigates the pressure's added value in pCEC using an LC instrument as well as the influence of the polymerization‐mixture composition on monolithic columns in such experimental circumstances. Two factors of the polymerization mixture, which is used to prepare the monolithic capillary columns, were varied according to an experimental design approach: the pore‐forming solvent/total monomer ratio and the pore‐forming solvents composition. Initially, the effect of the resulting stationary phase on the elution behavior of mainly pharmaceutical compounds was studied. Four responses were used to evaluate the effects on the chromatography: retention time, retention factor, peak asymmetry and number of theoretical plates. After processing the results, the stationary phase composition with the best chromatographic behavior was determined and tested. The advantageous properties of this stationary phase compared with the design center‐point column were demonstrated. Secondly, the results of these pCEC experiments were compared with those generated in an identical experimental setup previously performed using CEC. Chromatographic conditions were chosen so that the center‐point column showed similar retention in CEC and pCEC. The expected advantage (faster analysis) and drawback (decreased efficiency) of pCEC in the analysis of pharmaceuticals was evaluated. Analysis time and efficiency were both found to depend greatly on the porosity of the column. The conclusion of this comparison is that pCEC did not have a significant added value to CEC. However, this was mainly due to the instrument's limitation of the pressure‐driven flow over the column. A clear benefit of the pCEC setup was apparatus‐related, i.e. the presence of a loop injection system on the pCEC instrument, which avoids the injection problems that were occasionally observed in CEC.     

Sodium desoxycholate‐assisted capillary electrochromatography with methacrylate ester‐based monolithic column on fast separation and determination of coumarin analogs in Angelica dahurica extract

A rapid and sensitive CEC method with methacrylate ester‐based monolithic column has been developed for separation and determination of five coumarins (byakangelicin, oxypeucedanin hydrate, xanthotoxol, 5‐hydroxy‐8‐methoxypsoralen and bergapten) in Angelica dahurica extract. Surfactant sodium desoxycholate (SDC) was introduced into the mobile phase as the pseudostationary to dynamically increase the selectivity of analytes instead of increasing the hydrophobicity of stationary phase. In addition, other factors, pH of phosphate buffer, ACN content and applied voltage, for instance, have also an obvious effect on the resolution but little on the retention time. Satisfactory separation of these five coumarins was achieved within 6 min under a 30:70 v/v ACN–buffer containing 20 mM sodium dihydrogen phosphate (NaH₂PO₄) and 0.25 mM SDC at pH 2.51. The RSDs of intraday and interday for relative peak areas were less than 3.0% and 4.7%, respectively; and the recoveries were between 87.5% and 95.0%. The LODs were lower than 0.15 μg/mL and the LOQs were lower than 0.30 μg/mL, respectively, while calibration curves showed a good linearity (r² > 0.9979). Finally, five target coumarins from the crude extracts of A. dahurica were separated, purified, and concentrated by D‐101 macroporous resin, and were successfully separated and quantitatively determined within 6 min.     

Naphthyl methacrylate‐based monolithic column for RP‐CEC via hydrophobic and π interactions

A neutral naphthyl methacrylate‐based monolith (NMM) was introduced for RP‐CEC of various aromatic compounds via hydrophobic and π interactions. It was characterized over a wide range of elution conditions to gain insight into its RP retention mechanism toward the various solute probes under investigation. First, the NMM column exhibited cathodal EOF at various mobile phase compositions and pH suggesting that although the NMM column is void of fixed charges, it acquires a negative zeta potential. It is believed that the negative zeta potential is imparted by the adsorption of mobile phase ions to the NMM surface. The NMM column exhibited π–π interactions in addition to hydrophobic interactions due to the aromatic and nonpolar nature of its naphthyl ligands. In all cases, the retention of the various aromatic test solutes including PAHs, benzene derivatives, toluene derivatives, anilines and toluidine, tolunitrile and nitrotoluene positional isomers on the NMM column were compared to their retention on an octadecyl acrylate‐based monolithic column. Not only were the values of the retention factors of the various solutes on the NMM column higher than those obtained on the octadecyl acrylate‐based monolithic column under otherwise the same CEC conditions, but the elution orders were also different on both columns with a superior and unique selectivity exhibited by the NMM column.     

Pressurized CEC of neutral and charged solutes using silica monolithic stationary phases functionalized with 3-(2-aminoethylamino)propyl ligands

A novel silica monolithic stationary phase functionalized with 3-(2-aminoethylamino)propyl ligands for pressurized CEC has been presented. The monolithic capillary columns were prepared by a sol-gel process in 75 microm id fused-silica capillaries and followed by a chemical modification. The diamino groups on the surface of the stationary phase are meant to generate the chromatographic surface and a substantial anodic EOF as well as to provide electrostatic interaction sites for charged solutes. The electrochromatographic characterization and column performance were evaluated by a variety of neutral and charged solutes. It was observed that the anodic EOF for the diamine-bonded monolith was greatly affected by the reaction time with 3-(2-aminoethylamino)propyltrimethoxysilane and the PEG amount in the sol-gel reaction mixture in addition to the mobile phase conditions. The monolithic stationary phase exhibited hydrophilic interaction chromatographic behavior toward neutral solutes. Good separations of various solutes including phenols, nucleic acid bases, nucleosides and nucleotides were achieved under different experimental conditions. Fast and efficient separations were obtained with high plate counts reaching more than 130,000 plates/m.     

A strong inorganic acid‐initiated methacrylate polymerization strategy for room temperature preparation of monolithic columns for capillary electrochromatography

A facile strong inorganic acid‐initiated methacrylate polymerization strategy was developed for fabricating monolithic columns at room temperature. The prepared monoliths were characterized by FTIR spectrometry, mercury intrusion porosimeter and SEM, while their performance was evaluated by CEC for the separation of various types of compounds including alkyl benzenes, polycyclic aromatic hydrocarbons, nonsteroidal anti‐inflammatory drugs, anilines, and nitrophenol isomers. The column‐to‐column and batch‐to‐batch reproducibility for the prepared monoliths in terms of the RSD of EOF flow velocity, retention factor, and the minimum plate height of naphthalene ranged from 3.4 to 12.4%. The fabricated monoliths gave excellent performance for the separation of the test neutral compounds with the theoretical plates of 170 000–232 000 plates per meter for thiourea, and 77 400–112 300 plates per meter for naphthalene. The proposed strong inorganic acid‐initiated methacrylate polymerization strategy is a promising alternative for fabricating organic polymer‐based monoliths.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

Photografted methacrylate‐based monolithic columns coated with cellulose tris(3,5‐dimethylphenylcarbamate) for chiral separation in CEC

A chiral capillary monolithic column for enantiomer separation in capillary electrochromatography was prepared by coating cellulose tris(3,5‐dimethylphenylcarbamate) on porous glycidyl methacrylate‐co‐ethylene dimethacrylate monolith in capillary format grafted with chains of [2(methacryloyloxy)ethyl] trimethylammonium chloride. The surface modification of the monolith by the photografting of [2(methacryloyloxy)ethyl] trimethylammonium chloride monomer as well as the coating conditions of cellulose tris(3,5‐dimethylphenylcarbamate) onto the grafted monolithic scaffold were optimized to obtain a stable and reproducible chiral stationary phase for capillary electrochromatography. The effect of organic modifier (acetonitrile) in aqueous mobile phase for the enantiomer separation by capillary electrochromatography was also investigated. Several pairs of enantiomers including acidic, neutral, and basic analytes were tested and most of them were partially or completely resolved under aqueous mobile phases. The prepared monolithic chiral stationary phases exhibited a good stability, repeatability, and column‐to‐column reproducibility, with relative standard deviations below 11% in the studied electrochromatographic parameters.     

Determination of tocopherols in vegetable oils by CEC using methacrylate ester-based monolithic columns

The separation and determination of tocopherols (Ts) in vegetable oils by CEC using methacrylate ester-based monolithic columns has been developed. The effects of pore size of the monolithic columns were studied, and the composition of mobile phase was optimized. The optimal pore size of the monolith was obtained with 12 wt% 1,4-butanediol in the polymerization mixture. Excellent resolution between tocopherols was achieved within 10 min analysis time with a 99:1 v/v MeOH-aqueous buffer containing 5 mM tris(hydroxymethyl)aminomethane at pH 8.0. The LODs were lower than 2.3 microg/mL, and interday and column-to-column reproducibilities at 25 microg/mL were better than 5.6%. Using a 93:7 v/v MeOH-aqueous buffer, both tocopherols and tocotrienols (T(3)s) of grapeseed and palm oils were resolved. Application to the detection of olive oil adulteration with low-cost edible oils was demonstrated.     

Dual‐templates molecularly imprinted monolithic columns for the evaluation of serotonin and histamine in CEC

To extend the application of molecularly imprinted polymers, the dual‐templates molecularly imprinted monolithic columns were developed in a capillary format. Two templates serotonin and histamine were simultaneously imprinted using two different functional monomers such as methacrylic acid (MAA) and methylenesuccinic acid (MSA) in a mixture of ethylene glycol dimethacrylate (EDMA) as a cross‐linker and AIBN as polymerization initiator dissolved in DMF as porogen. The resulting molecular imprinted polymers (MIPs) were characterized based on their performance in the CEC separation of two imprinted templates. The optimization parameters such as pH, ACN composition, and concentration of the eluent were varied to achieve best resolution and efficiency for CEC separation of templates with each MIP column. It was found that the MIP monolith column fabricated using MSA offered better resolution and separation efficiency compared to column fabricated with MAA. This work utilized the dual‐templates imprinting approach successfully and broadens the scope of multi‐templates imprinting capabilities in capillary format in CEC application.     

Preparation and evaluation of lauryl methacrylate monoliths with embedded silver nanoparticles for capillary electrochromatography

In this article, capillary columns constituted by lauryl methacrylate monoliths with embedded silver nanoparticles (AgNPs) were developed and tested. Two incorporation approaches of AgNPs in monoliths were explored. The AgNPs were either photogenerated in situ during polymerization of the monolith by UV irradiation, or incorporated to the polymerization mixture (ex situ). The influence of the AgNP concentration on the morphological and chromatographic properties of the polymer matrix was investigated, and both the in situ and ex situ approaches were comparatively discussed. The morphology of the monoliths was characterized by electron microscopic techniques, and their electrochromatographic performance was also evaluated with test mixtures of neutral compounds (sterols, fatty acid methyl esters, tocopherols, and polyaromatic hydrocarbons).     

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.     

Comparison on photo-initiators for the preparation of methacrylate monolithic columns for capillary electrochromatography

The synthesis of lauryl methacrylate monoliths for capillary electrochromatography by UV polymerization using several free-radical initiators (α,α′-azobisisobutyronitrile, 2,2-dimethoxy-2-phenylacetophenone, dibenzoyl peroxide (BPO) and lauroyl peroxide (LPO)) has been investigated. Using a 1,4-butanediol/1-propanol mixture as porogenic solvent, the influence of each initiator and its content on the morphological and electrochromatographical properties of beds was evaluated. Under their respective optimum content, satisfactory separations of a test mixture of PAHs with similar efficiencies (minimum plate heights of 8.0–12.7 μm obtained from Van Deemter plots) were achieved for the four investigated photo-initiators. The columns photo-polymerized with LPO provided the best compromise between chromatographic performance and analysis time. Moreover, this initiator showed a fine control in the column retention properties. The resulting monolithic columns exhibited a good run-to-run repeatability in the tested chromatographic parameters (RSD < 2.4%) for all initiators investigated; and satisfactory column-to-column repeatability (RSD < 6.0%), except for beds photo-polymerized with BPO (RSD < 10.8%).     

Peak spreading in linear gradient elution chromatography with a thin monolithic disk

The peak spreading of DNAs of various sizes [12-mer, 20-mer, 50-mer and 95-mer poly(T)] in linear gradient elution (LGE) chromatography with a thin monolithic disk was investigated by using our method developed for determining HETP in LGE. Electrostatic interaction-based chromatography mode (ion-exchange chromatography, IEC) was used. Polymer-based monolithic disks of two different sizes (12 mm diameter, 3mm thickness and 0.34 mL; 5.2 mm diameter, 4.95 mm thickness and 0.105 mL) having anion-exchange groups were employed. For comparison, a 15-μm porous bead IEC column (Resource Q, 6.4mm diameter, 30 mm height and 0.97 mL) was also used. The peak width did not change with the flow velocity for the monolithic disks where as it became wider with increasing velocity. For the monolithic disks the peak width normalized with the column bed volume was well-correlated with the distribution coefficient at the peak position K(R). HETP values were constant (ca. 0.003-0.005 cm) when K(R)>5. Much higher HETP values which are flow-rate dependent were obtained for the porous bead chromatography. It is possible to obtain 50-100 plates for the 3mm monolithic disk. This results in very sharp elution peaks (standard deviation/bed volume=0.15) even for stepwise elution chromatography, where the peak width is similar to that for LGE of a very steep gradient slope.     

Separation of parabens in capillary electrochromatography using poly(styrene-divinylbenzene-methacrylic acid) monolithic column

In this study, a series of poly(styrene-divinylbenzene-methacrylic acid) monolithic capillaries was used as the separation column of CEC for the analyses of parabens in commercial pharmaceutical and cosmetic products. The results showed that the chromatographic characteristics of these analytes were strongly affected by the preparation condition of the monolithic column including monomer content, porogenic solvent composition, and polymerization time. Baseline separations were markedly sped up by lowering the polymerization time without any obvious loss of resolution. Furthermore, mobile-phase composition (pH, ACN, and electrolyte concentration) was also able to effectively improve the separation behavior. Similar to the influence of lowering the polymerization time, retention times for all analytes were significantly shortened in the CEC method by decreasing the electrolyte concentration in the mobile phase.