On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens

An improved technique, pressurized capillary electrochromatography (pCEC) coupling with end-column amperometric detection (AD), was developed and used for the separation and determination of estrogens. The effects of pH value, composition of mobile phase, concentration of the surfactant sodium dodecyl sulfate (SDS) and applied voltage on separation were investigated. The electrochemical oxidation of diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX) could be reliably monitored with a carbon electrode at 0.9 V (vs. Ag/AgCl). The pCEC analyses were performed on a capillary separation column packed with 3 microm C18 particles with an acetonitrile/water (31%: 69%) mobile phase containing Tris buffer (5 mmol/L, pH 4.5) and 4 mmol/L SDS. High voltage up to 12 kV reduced the retention time dramatically and still provided a baseline resolution. In addition, supplementary pressure prevented bubble formation and provided reliability and reproducibility of the pCEC performance. The detection limits for the three estrogens ranged from 1.2 to 2.2x10(-7) mol/L, about 10 20-fold lower than those obtained with pCEC-UV detection. To evaluate the feasibility and reliability of this system, the proposed pCEC-AD method was further demonstrated with fish muscle samples spiked with estrogens.     

Direct determination of amino acids by pressurized capillary electrochromatography with chemiluminescence detection

A pressurized CEC (pCEC) coupled with on-column chemiluminescence (CL) detection was developed for direct determination of amino acids, which was based on the principle of an enhanced effect of Cu(II)-amino acid complexes on the CL reaction between luminol and hydrogen peroxide in alkaline solution. The effects of some important factors on pCEC separation and CL intensity were systemically investigated. Baseline separation of amino acids including L-histidine (L-His), L-threonine (L-Thr), and L-tyrosine (L-Tyr) was achieved by using a monolithic column with a mobile phase of 5.0x10(-3) mol/L phosphate buffer at pH 8.0 that contained 25% v/v methanol and 5.0x10(-4) mol/L luminol and 1.0x10(-5) mol/L Cu(II) at an applied voltage of -5 kV. The calibration curves of the analytes by plotting the peak height against corresponding concentration were linear over the range of 3.2x10(-6)-3.2x10(-4) mol/L for L-His, 4.1x10(-6)-4.1x10(-4) mol/L for L-Thr, and 6.0x10(-7)-3.0x10(-4) mol/L for L-Tyr. The LODs for L-His, L-Thr, and L-Tyr were 6.4x10(-7), 8.4x10(-7), and 3.0x10(-7) mol/L (S/N = 2), respectively. The proposed method was applied to the analysis of amino acid injection sample with satisfactory results. Mean recoveries for three amino acids were from 84.3 to 89.6%.     

Separation and determination of seven fluoroquinolones by pressurized capillary electrochromatography

In this paper, pressurized CEC was used for the separation and determination of seven fluoroquinolones (FQs). The effect of different experimental conditions, such as the concentration and pH of the buffer, the organic modifier concentration, the surfactant and ion-paring agents added to the electrolyte, and applied voltage were studied. All the seven FQs were baseline separated using mobile phase containing 27% v/v ACN, 5 mmol/L Na2HPO4 buffer (pH 4.0 adjusted using citric acid), 11 mmol/L SDS, and 0.01% TEA v/v at detection wavelength of 287 nm and at an applied voltage of -10 kV. The calibration curves were linear (r>0.9991) over a concentration range of 1.0-50.0 mg/L for norfloxacin (NFLX); 2.5-50.0 mg/L for fleroxacin (FLX), ciprofloxacin (CPFX), and lomefloxacin (LMX); and 5.0-50.0 mg/L for enoxacin (ENX), ofloxacin (OFLX), and gatifloxacin (GFLX). The detection limits (S/N = 3) for ENX, OFLX, FLX, NFLX, CPFX, LMX, and GFLX were 0.5, 0.8, 0.4, 0.2, 0.4, 0.5, and 1.0 mg/L, respectively. The method is simple, rapid, and reproducible. It was successfully applied to the analysis of fish muscle samples spiked with FQs. Mean recoveries ranged from 81.6 to 97.6%.     

Determination of pyrethroid pesticide residues in vegetables by pressurized capillary electrochromatography

A simple and rapid pressurized isocratic capillary electrochromatography (pCEC) method has been developed to separate six pyrethroid pesticides. The effects of pH of buffer, organic solvent content, buffer concentrations and applied voltage on the separation of six pyrethroids were investigated. Under the optimized conditions, the pCEC method developed allows baseline separation of a complex mixture of six pyrethroids in <20 min. The method is applied to the analysis of these pesticide residues in Chinese cabbage. The limits of quantification (LOQ) ranged from 0.5 to 0.8 μg/ml (corresponding to 0.05 and 0.08 mg/kg in the vegetable sample), with relative standard deviations (R.S.D.) <5.0%. Mean values of recoveries for six pyrethroids ranged from 89.6 to 96.3%, respectively.     

Splitless on-line coupling of capillary high-performance liquid chromatography,capillary electrochromatography and pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy

A mixture of unsaturated fatty acid methyl esters was separated with a new splitless capillary set-up. With the employed apparatus configuration different capillary separation techniques such as capillary high-performance liquid chromatography (cHPLC), capillary electrochromatography (CEC) and pressurized capillary electrochromatography (pCEC) could be applied. The detection and identification of the sample compounds were accomplished by hyphenating these capillary separation techniques with nuclear magnetic resonance (NMR) spectroscopy using a novel configuration of the detection capillary set-up. Using modified electrokinetically driven separation techniques, the electric field was applied solely across the separation column. With this improved interface for capillary liquid chromatography-NMR on-line coupling, the stereochemical assignment of the cis and trans configuration of unsaturated fatty acids could be easily accomplished. Finally, the results of cHPLC-NMR, CEC-NMR and pCEC-NMR coupling experiments were compared.Dedicated to Professor Günter Häfelinger on the occasion of his 65th birthday     

Preparation and evaluation of C18-bonded 1-microm silica particles for pressurized capillary electrochromatography

Nonporous silica spheres (1 microm) were synthesized and bonded with octadecylsilane functionality. These stationary phase particles were packed electrokinetically into fused-silica capillaries with 100 microm id for a length of 20 cm, which was evaluated by using pressurized CEC (pCEC). The efficiency of the C18 RP column was characterized through the theoretical plates of thiourea, benzyl alcohol, toluene, styrene, and naphthalene. The effects of experimental parameters such as the applied voltage, sample size, pump flow rate, pH value and the concentration of the buffer solution, and the content of methanol in the mobile phase, on-column efficiency were evaluated. Column efficiency as high as 200 000 theoretical plates per meter for naphthalene was obtained with the optimal condition of 70% v/v methanol and 30% v/v of 10 mmol/L phosphate buffer (pH 7.8) at an applied voltage of 10 kV and a supplementary pressure of 500 psi.     

Quantification of domoic acid in shellfish tissues by pressurized capillary electrochromatography

A method was developed to quantify domoic acid (DA), the chemical responsible for amnesic shellfish poisoning (ASP), by pressurized CEC (pCEC). The effect of different experimental conditions on the separation of DA and matrix solutes, such as the content of ACN in mobile phase, pH and concentration of buffer, supplementary pressure and applied voltage, were investigated. Under the optimal conditions, the pCEC method separated DA from shellfish matrices within 6 min. By using supplementary pressure, bubble formation in the capillary column was completely suppressed. The method was repeatable, sufficient accurate and sensitive for rapid screening of DA in shell seafood.     

Separation of rhubarb anthraquinones by capillary electrochromatography

Summary A rapid, simple method for packing capillary electrochromatography (CEC) columns with HPLC stationary phases is described. The basis of the method is the use of a vacuum to suck a slurry of stationary phase into the fused-silica tubing, a procedure which takes approximately ten seconds only, then compression of the stationary phase by means of an HPLC pump. These packed CEC columns have been investigated for the separation of five anthraquinones from rhubarb. Separation of the anthraquinones inRheum palmatum L. under optimized conditions is presented.     

Epinephrine‐modified methacrylate monolithic column with fumed silica nanoparticle for pressurized capillary electrochromatography

Via the ring‐opening reaction of epoxy groups with epinephrine, a novel epinephrine functionalized polymethacrylate monolith with fumed silica nanoparticles has been fabricated for pressurized capillary electrochromatography. The preparation of epinephrine‐modified monoliths has been optimized. In addition, morphology, electroosmotic flow, separation mechanism and column performance have been studied. The internal structure of the monolithic stationary phase was more uniform and the column efficiency increased after the incorporation of nanoparticles. With this column, satisfactory separation capability of aromatic compounds and alkaloids has been achieved and the column efficiency for naphthalene reached 138 696 plates/m. As for the real sample, 3 alkaloids were separated in Huanglian Shangqing capsules, a Chinese traditional medicine.     

Preparation and evaluation of a neutral methacrylate-based monolithic column for hydrophilic interaction stationary phase by pressurized capillary electrochromatography

A polar and neutral polymethacrylate-based monolithic column was evaluated as a hydrophilic interaction capillary electrochromatography (HI-CEC) stationary phase with small polar–neutral or charged solutes. The polar sites on the surface of the monolithic solid phase responsible for hydrophilic interactions were provided from the hydroxy and ester groups on the surface of the monolithic stationary phase. These polar functionalities also attract ions from the mobile phase and impart the monolithic solid phase with a given zeta potential to generate electro-osmotic flow (EOF). The monolith was prepared by in situ copolymerization of a neutral monomer 2-hydroxyethyl methacrylate (HEMA) and a polar cross-linker with hydroxy group, pentaerythritol triacrylate (PETA), in the presence of a binary porogenic solvent consisting cyclohexanol and dodecanol. A typical HI-CEC mechanism was observed on the neutral polar stationary phase for both neutral and charged analytes. The composition of the polymerization mixture was systematically altered and optimized by altering the amount of HEMA in the polymerization solution as well as the composition of the porogenic solvent. The monoliths were tested in the pCEC mode. The resulting monoliths had different characteristics of hydrophilicity, column permeability, and efficiency. The effects of pH, salt concentration, and organic solvent content on the EOF velocity and the separation of nucleic acids and nucleosides on the optimized monolithic column were investigated. The optimized monolithic column resulted in good separation and with greater than 140,000 theoretical plates/m for pCEC.     

Rapid separation and determination of carbamate insecticides using isocratic elution pressurized capillary electrochromatography

An isocratic elution pressurized CEC (pCEC) system was used to separate and determine ten carbamate insecticides. It was found that introduction of the electrical field, supplementary pressure, and SDS in the proposed method greatly improved the speed, column efficiency, selectivity, and repeatability for separation and determination of carbamates. On a capillary column of 75 microm ID packed with 3 microm octadecyl silica, baseline separation and detection of ten analytes was performed by using a mobile phase consisting of 30% v/v ACN and 70% v/v of 5 mmol/L ammonium acetate (pH 6.5) containing 1 mmol/L SDS and 0.01% triethylamine (TEA). Under the optimum conditions ten carbamate insecticides could be completely separated within 20 min. For the real vegetable samples, an SPE procedure for the cleanup of matrices was carried out prior to pCEC analysis. The detection limits of 0.05-1.6 mg/kg for ten carbamates and mean recoveries of 51.3-109.2% for eight kinds of vegetable samples at different concentrations of carbamates with RSD less than 11.4% were obtained, respectively. The proposed method has been proved to be effective in the rapid analysis of carbamate residues in vegetables.     

Molecularly imprinted polymers as tools for the screening of felodipine from dihydropyridine calcium antagonists by pressurized capillary electrochromatography

A group of structurally similar dihydropyridine calcium antagonists (DHPs) and related compounds were used to simulate a combinatorial library. A molecularly imprinted polymer (MIP) comprising felodipine (FLD) was synthesized in situ inside the capillary for use in the separation of FLD from other DHPs by pressurized electrochromatography (pCEC). To evaluate the feasibility of using the MIP columns for the separation of FLD, parameters including pH, the applied voltages, and the effect of organic modifier were studied. The results indicated that the MIP columns demonstrated better recognition properties over a pH range of 4–6. The efficiency (plates/m) at pH 5.0 for the non-imprinted analytes was 117,000 for thiourea, 18,700 for nicarpidine, 17,300 for nisoldipine, and 14,600 for nifedipine; however, the efficiency for the imprinted analyte FLD was low, as evidenced by the broad peak, yielding only 5,100 plates/m. The column efficiency was also investigated under both micro-HPLC and pCEC conditions.     

毛细管电色谱和加压毛细管电色谱的进展与应用

毛细管电色谱(CEC)以内含色谱固定相的毛细管为分离柱,以电渗流为驱动力,既可以分离带电物质也可以分离中性物质。它结合了毛细管电泳和高效液相色谱两者的优点,兼具高柱效、高分辨率、高选择性和高峰容量的特点,同时具有色谱和电泳的双重分离机理。然而,“纯粹”的电色谱在实际应用中有着天然的弱点,即: 在电流通过毛细管柱中的流动相时容易产生气泡(焦耳热作用),从而使电流中断和电渗流停止,毛细管柱必须被重新用流动相润湿后方能再次使用。加压毛细管电色谱(pCEC)将液相色谱中的压力流引入CEC系统中,不仅解决了气泡、干柱等问题,而且实现了定量阀进样和二元梯度洗脱。CEC和pCEC作为微分离领域的两种前沿技术,满足了当前复杂样品分析和分析仪器微型化的需求,近年来获得了广泛的关注。本文综述了这两种技术近来的发展,包括仪器、色谱固定相的发展,总结了其在生命科学、药物分析、食品安全以及环保样品分析等方面的应用进展,评述了各方法的特点,并展望了CEC和pCEC今后的发展和应用前景。     

Separation of enantiomers by capillary electrochromatography

The current popularity of capillary electrochromatography (CEC) has led to an increasing number of studies on the development and evaluation of enantioselective CEC systems. These studies clearly demonstrate that the most prominent advantage of electrically driven separation methods, the vastly increased column efficiency as compared to pressure-driven chromatography, can also be experimentally achieved for the separations of enantiomers. In analogy to high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), several approaches have been used. The addition of a chiral selector to the mobile phase is the simplest method. Less erroneous and more elegant approaches are those that use open-tubular, conventional packed, and monolithic columns containing chiral stationary phases that stereoselectively interact with enantiomers. This review evaluates the new techniques and compares them to enantioselective HPLC and CE. Further, it describes the various concepts of enantioselective CEC and focuses on the current ‘state-of-the-art' column technology.     

Development of a quality evaluation method for Fructus schisandrae by pressurized capillary electrochromatography

A pressurized CEC (pCEC) method with postcolumn detection cell had been developed for quantifying the lignans from Fructus schisandrae extracts. The effects of different experimental conditions, such as the ACN content of the mobile phase, the concentration and pH of the buffer, the applied voltage, and the supplementary pressure were studied. Five lignans (schisandrin, gomisin A, schisantherin C, deoxyschizandrin, schisandrin B) were baseline separated using a mobile phase of ACN-phosphate buffer (pH 5.4; 5 mM) (40:60 v/v) under -4 kV applied voltage. The method showed the satisfactory retention time and peak area repeatability. The calibration curves were linear in the range 50.0-1000.0 microg/mL for schisandrin, 20.0-500.0 microg/mL for gomisin A, 10.0-200.0 microg/mL for schisantherin C, 20.0-500.0 microg/mL for deoxyschizandrin, and 20.0-500.0 microg/mL for schisandrin B. The correlation coefficients were between 0.9978 and 0.9989. With this pCEC system, fingerprints of F. schisandrae were preliminarily established to distinguish two members S. chinensis (Turcz.) Baill. and S. sphenanthera Rehd. Et Wils. of F. schisandrae by characteristic peaks, and evaluate the quality of various sources of raw materials by determining the contents of the five lignans.     

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.     

Investigation of enantiomer recognition of molecularly imprinted polymeric monoliths in pressurized capillary electrochromatography screening the amino acids and their derivatives

Molecularly imprinted monolithic columns were prepared for chiral separation of tyrosine and its amino acid derivatives by in situ therm-initiated copolymerization of methacrylic acid, 4-vinylpyridine and ethylene glycol dimethacrylate. The enantiomers were rapidly separated on monolithic columns in less than 10 min by pressurized capillary electrochromatography (pCEC). The influences of several parameters such as the content of cross-linking monomer on the composition of the pre-polymerization mixture were systematically investigated. The influence of the pCEC conditions including the composition of the mobile phase was also optimized to obtain the good enantioseparation. It was found that in addition to molecularly imprinted recognition, chromatographic retention and electrophoretic migration play important roles in the retention and chiral recognition of molecularly imprinted polymer (MIP) columns. The cross-selectivity for similar amino acids and its derivatives were systematical investigated for understanding the recognition mechanism on the MIP monolithic columns. The results indicated that molecularly imprinted polymer recognizes the template molecule by its molecular shape defined binding cavity.     

Monolithic silica columns with mixed mode of hydrophilic interaction and weak anion-exchange stationary phase for pressurized capillary electrochromatography

A silica-based monolithic column as polar stationary phase is proposed for pressurized CEC (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by 3-aminopropyltrimethoxysilane to produce a column for hydrophilic interaction applications. The amino groups on the surface of the polar stationary phase generated anodic EOF under acidic conditions and served at the same time as a weak anion-exchanger. The anion solutes such as nucleotides were separated by the mixed mode mechanism, which comprised hydrophilic interaction, weak anion-exchange, and electrophoresis. The influences of buffer concentration and organic modifier content on the separation of nucleotides by pCEC have been investigated. In addition, the monolithic silica columns were also able to separate various polar compounds such as phenols, nucleic acid bases, and nucleosides in the hydrophilic interaction CEC mode.     

Separation and determination of six catechins in tea by pressurized capillary electrochromatography

An efficient pressurized capillary electrochromatography (pCEC) method has been successfully developed for the determination of six catechins in tea. The separation was performed on a reversed-phase EP-100-20/45-3-C18 capillary column (total length of 45?cm, effective length of 20?cm, diameter of 100?μm, ODS packing inside for 3?μm). The mobile phase ratio of organic phase, the concentration of phosphate buffer and sodium heptanesulfonate, separation voltage, and other experimental conditions were investigated and optimized. The mobile phase was 15?mM NaH₂PO₄ and 12?mM sodium heptanesulfonate (pH 3.0)/methanol (64:36) at a flow rate of 0.04?mL/min. Under optimal conditions including applied voltage of ?4?kV and a UV detection wavelength of 230?nm, the six catechins in the tea were well separated. The calibration curves for the analytes had good linearity in the range of 8.02?μg/mL–202.13?μg/mL with a correlation coefficient of 0.9928–0.9997. The limits of detection (LOD) for the six catechins were 4.62?μg/mL–11.63?μg/mL (S/N?=?3). The recoveries of the six catechins were 96.2%–108.4% with a relative standard deviation (RSD) between 0.78% and 4.51%. The method has been used for the determination of six catechins in tea samples with good results.     

Rapid separation and determination of microcystins using monolithic columns in isocratic elution mode by pressurized capillary electrochromatography

As a new wave of technology, polymethacrylate-based monolithic column was prepared and its application in the separation of three kinds of microcystins (MCs) in pressurized capillary electrochromatography with ultraviolet detection was studied. The key factors affecting the separation performance, such as monolithic column, pressure of the pump, component and concentration of mobile phase and the voltage, were investigated and optimized in detail. A baseline separation could be achieved in less than 6 min using a 5 mM borate buffer with a pH of 9.6 and 10% acetonitrile as the mobile phases in isocratic elution, under a voltage of +13 kV and a supplementary pressure of 7.5 MPa. The calibration curves were linear with correlation coefficient r>0.998 over a range of 0.10-25.00 mg/L. The LODs for the three MCs were in the range of 0.03-0.09 mg/L. This method was successfully applied to separate MCs from other compounds in spiked tap water after solid-phase extraction. The lower LODs for MC-LR, MC-YR and MC-RR were obtained to be 0.10, 0.13, 0.16 microg/L, respectively. These results make it clear that this proposed system is accurate and robust enough to be used as a fast separation tool for routine monitoring of MCs in real water samples.