Capillary Electrophoresis Equipment Research Articles

Monoalkyl carbonates in carbonated alcoholic beverages

The presence of monoethyl carbonate (MEC) in beer and sparkling wine is demonstrated for the first time, as well as the formation of this species in drinks prepared with a distilled beverage and a carbonated soft drink. A capillary electrophoresis (CE) equipment with two capacitively coupled contactless conductivity detector (C4D) was used to identify and quantify this species. The concentrations of MEC in samples of lager beer and rum and cola drink were, respectively, 1.2 and 4.1mmol/l, which agree with the levels of ethanol and CO2 available in these products. Previous results about the kinetics of the reaction suggest that only a small amount of MEC should be formed after the ingredients of a drink are mixed. However, in all three cases (whisky and club soda; rum with cola; gin and tonic water), MEC was quickly formed, which was attributed to the low pH of the drinks.

Human complement factor 3 polymorphism determination by capillary electrophoresis of serum

Variability of complement factor 3 (C3) mobility in serum protein electrophoresis was investigated. We found that the migration time of C3 can be reproducibly determined (beween-run CV=0.76%) using clinical capillary electrophoresis (CE) equipment (the Capillarys™ 2 system, Sebia). Moreover, we found a significant difference (p<0.001) in migration times of the major C3 phenotypes FF (fast-fast), FS (fast-slow) and SS (slow-slow). Glycosylation did not significantly affect test results. This is the first report on the migration time of C3 phenotypes on a clinical CE instrument. The presented method allows faster data than agarose-electrophoresis or genotyping. Moreover, reference ranges for serum C3 concentration depend on C3 phenotype, which allows a better tailored clinical interpretation of C3 concentrations.

Implementation of a field programmable gate array for wireless control of a lab-on-a-robot

We report the design of a digital system to wirelessly control microchip capillary electrophoresis (CE) equipment and a mobile unit for chemical analysis. The digital system consists of an embedded processor designed for digital control, decoding and applying of wirelessly-transmitted test parameters, data acquisition, and mobility control. The design is implemented on a field programmable gate array (FPGA) and its development board interfaces with four digital-to-analog converters on a newly-designed 3-channel high voltage power supply, electrochemical detector, wireless modems for communications with a base unit, mobile platform motor controllers, GPS sensor, and an air micropump. The FPGA allows for all the interfacing hardware to perform CE and transmission of the data acquired from the interfacing electrochemical detector. The work described herein extends the utilization of microchip capillary electrophoresis to include remotely controlled field applications.

Otimização da utilização de marcadores moleculares microssatélites e sua aplicação em estudos com plantas daninhas

Apesar de existirem marcadores moleculares mais específicos, os marcadores microssatélites apresentam grande potencialidade de utilização na área de plantas daninhas devido à sua crescente disponibilização em outras espécies e à qualidade das informações proporcionadas. O uso convencional dos marcadores moleculares microssatélites demanda grande quantidade de trabalho e recursos financeiros. O objetivo deste trabalho foi descrever a técnica da cauda fluorescente como forma de otimização da utilização de marcadores moleculares microssatélites, utilizando como exemplo um estudo de identificação de híbridos entre arroz-vermelho e cultivado. Foram utilizadas como modelo plantas de arroz cultivado, arroz-vermelho e o híbrido originado do cruzamento artificial dessas plantas. A técnica da cauda fluorescente consiste na síntese do iniciador forward com a sequência desejada e a adição da sequência de um iniciador universal, que corresponde à chamada cauda. A detecção da amplificação é realizada em equipamento de eletroforese capilar automatizada, através da utilização de um iniciador universal sintetizado com fluoróforo. O sistema desenvolvido foi eficiente na identificação da hibridização entre arroz cultivado e vermelho e apresenta viabilidade de utilização, por exemplo, em estudos de fluxo gênico da resistência a herbicidas e de caracteres relacionados à adaptação diferencial entre essas plantas. A técnica da cauda fluorescente possibilitou o uso de diversos marcadores moleculares a partir de um único marcador fluorescente e viabilizou a realização das análises em multiplex. O aumento da disponibilidade e do conhecimento de técnicas moleculares pode proporcionar melhor elucidação em vários estudos relacionados a espécies de plantas daninhas que possuem pouca disponibilidade de marcadores moleculares específicos.

Reliable low-cost capillary electrophoresis device for drug quality control and counterfeit medicines

The proportion of counterfeit medicines is dramatically increasing these last few years. According to numerous official sources, in some pharmaceutical wholesalers in African countries, the proportion has reached 80%. Unfortunately, this situation is far to be improved due to lack of suitable analytical equipment allowing rapid actions of the Regulatory Agencies based on scientific consideration, at affordable cost and all over the drug supply chain. For that purpose, a network group considered that mater by building a low-cost original capillary electrophoresis (CE) equipment equipped with a new deep UV detector based on LED technology. The generic conditions for analysis were investigated: capillary zone electrophoresis (CZE) performed at acidic pH for basic drug molecules (i.e., quinine, highly used as the last antimalarial rampart), basic pH for compounds such as furosemide (a common diuretic drug) and at neutral pH for a well known antibiotic combination, trimethoprim/sulfamethoxazol. To evaluate the ability of the CE equipment for quantification, a full validation and a method comparison study were carried out for the CZE method dedicated to quinine determination. The validation involved the use of accuracy profile and total error concept to monitor the adequacy of the results obtained by the new prototype. The method comparison was based on the Bland and Altman approach by comparing results obtained by the low-cost CE and a conventional set-up. Subsequent validation studies were realized with neutral and acidic drug molecules, each focusing on a single concentration level calibration curve in order to maintain as low as possible the expenses due to reagents and thus the cost of analysis, as important advantages of CE for drug quality control.

Ionic mobility of the solvated proton and acid–base titration in a four-compartment capillary electrophoresis system

Although H+ and OH− are the most common ions in aqueous media, they are not usually observable in capillary electrophoresis (CE) experiments, because of the extensive use of buffer solutions as the background electrolyte. In the present work, we introduce CE equipment designed to allow the determination of such ions in a similar fashion as any other ion. Basically, it consists of a four-compartment piece of equipment for electrolysis-separated experiments (D. P. de Jesus et al., Anal. Chem., 2005, 77, 607). In such a system, the ends of the capillary are placed in two reservoirs, which are connected to two other reservoirs through electrolyte-filled tubes. The electrodes of the high-voltage power source are positioned in these reservoirs. Thus, the electrolysis products are kept away from the inputs of the capillary. The detection was provided by two capacitively coupled contactless conductivity detectors (C4D), each one positioned about 11 cm from the end of the capillary. Two applications were demonstrated: titration-like procedures for nanolitre samples and mobility measurements. Strong and weak acids (pKa < 5), pure or mixtures, could be titrated. The analytical curve is linear from 50 μM up to 10 mM of total dissociable hydrogen (r = 0.99899 for n = 10) in 10-nL samples. By including D2O in the running electrolyte, we could demonstrate how to measure the mixed proton/deuteron mobility. When H2O/D2O (9 : 1 v/v) was used as the solvent, the mobility was 289.6 ± 0.5 × 10−5 cm2 V−1 s−1. Due to the fast conversion of the species, this value is related to the overall behaviour of all isotopologues and isotopomers of the Zundel and Eigen structures, as well as the Stokesian mobility of proton and deuteron. The effect of neutral (o-phenanthroline) and negatively charged (chloroacetate) bases and aprotic solvent (DMSO) over the H+ mobility was also demonstrated.

Quantification of 4-aminopyridine in plasma by capillary electrophoresis with electrokinetic injection

A rapid and sensitive CE method for the determination of 4-aminopyridine in human plasma using 3,4-diaminopyridine as an internal standard was developed and validated. The analytes were extracted from 0.5-mL aliquots of human plasma by liquid-liquid extraction, using 8 mL of ethyl ether, and injected electrokinetically into capillary electrophoresis equipment. The instrumental conditions were obtained and optimized by Design of Experiments (DOE--factorial and response surface model), having as factors: separation voltage, ionic strength (buffer concentration), pH and temperature. The response variables were migration time, resolution, tailing factor and drug peak area. After obtaining mathematically predicted values for the response variables with best factors combinations, these were reproduced experimentally in good agreement with predicted values. In addition to optimal separation conditions obtained by Design of Experiments, sensitivity was improved using electrokinetic injection at 10 kV for 10 s, and a capillary with 50 cm effective length and 100 microm I.D. The final instrumental conditions were voltage at 19 kV, capillary temperature at 15 degrees C, wavelength at 254 nm, and phosphate buffer 100 mM, pH 2.5 as the background electrolyte. This assay was linear over a concentration range of 2.5-80 ng/mL with a lower limit of quantification of 2.5 ng/mL. The relative standard deviation for the assay precision was <7% and the accuracy was >95%. This method was successfully applied to the quantification of 4-aminopyridine (4-AP) in plasma samples from patients with spinal cord injury.

Analytical Assessment of a Home Made Capillary Electrophoresis Equipment with Linear Charge Coupled Device for Visible Light Absorption Detection in the Determination of Food Dyes

The performance of modular home made capillary electrophoresis equipment with spectrophotometric detection, at a visible region by means of a miniaturized linear charge coupled device, was evaluated for the determination of four food dyes. This system presents a simple but efficient home made cell detection scheme. A computer program that converts the spectral data after each run into the electropherograms was developed to evaluate the analytical parameters. The dyes selected for analytical evaluation of the system were Brilliant Blue FCF, Fast Green FCF, Sunset Yellow FCF, and Amaranth. Separation was carried out in a 29 cm length and 75 µm I.D fused silica capillary, using 10 mmol L−1 borate buffer at pH 9, with separation voltage of 7.5 kV. The detection limits for the dyes were between 0.3 and 1.5 mg L−1 and the method presented adequate linearity over the ranges studied, with correlation coefficients greater than 0.99. The method was applied for determination and quantification of these dyes in fruit juices and candies.

PH programming in capillary electrophoresis by means of temperature programming

When using a background electrolyte with a buffer having strong temperature dependence of p K, different operating temperatures result in different operating pH values, using the same background electrolyte (BGE). It has been shown that this can be used to fine-tune selectivities of sample mixtures of weak analytes. Capillary electrophoresis (CE) equipment is designed to operate under isothermal conditions, but by proper programming, a very reproducible temperature and thus BGE pH program during the analysis can be realized. This was experimentally verified and illustrated by computer simulations. Equipment characteristics have been determined, and possibilities and restrictions to make use of this feature are presented.

Determination of gaseous and particulate carbonyls in air by gradient‐elution micellar electrokinetic capillary chromatography

A new continuous-flow gradient-elution micellar electrokinetic capillary chromatography method is developed for the determination of airborne carbonyls after derivatization with 2,4-dinitrophenylhydrazine. A total of 16 carbonyls can be determined with detection limits ranging from 0.94 to 8.50 mg/L, working range from 4.72 to 346 mg/L, and repeatabilities (relative standard deviation, n=5) from 1.23 to 4.6% or 3.93 to 7.6% for migration time and peak area, respectively. Coupling with denuder-filter sampling, a preliminary survey has been conducted to determine gaseous and particulate carbonyls from air sampled at a roadside station. The method is shown to have sufficient sensitivity for 1-h sampling of ambient carbonyls with detection limits ranging from 0.045 to 1.2 microg/m3 and working range from 0.11 to 43.3 microg/m3 at a flow rate of 10 Lpm. The method requires minimal modification of commercially available capillary electrophoresis equipment and can differentiate gaseous and particulate carbonyls to provide essential information and objective data for adopting effective measures to combat the discharge of carbonyl compounds to the atmosphere.

Capillary electrophoresis for short chain organic acids in faeces: Reference values in a Mediterranean elderly population

There is increasing evidence that gut microflora and fermentation processes in the large intestine are important for health, and that health-promoting effects are mediated by fermentation products. Usually analytical methods for these compounds are tedious. A simple and rapid procedure of aqueous extraction from the stools has been optimized. After extraction, an aliquot of the aqueous layer was directly injected into the capillary electrophoresis equipment. Oxalic, formic, fumaric, 2-ketoglutaric, succinic, citric, acetic, propionic, 2-ketoisovaleryc, butyric, isovaleric lactic, glyceric 2-hydroxybutyric, and valeric acids were separated and identified. Electrophoretic conditions were: phosphate buffer 234 mM pH 6.10 with 12% (v/v) methanol with a coated capillary at −10 kV of applied potential. The method was validated for a representative group of compounds: acetic, propionic butyric, 2-hydroxybutiric, isovaleric, and oxalic acids, including the comparison of results with ionic chromatography. Finally 136 samples from healthy humans aged 60–80, both male and female living in Spain, were measured. They could be used as reference values for further studies.

IGentifier: indexing and large-scale profiling of unknown transcriptomes

Development and refinement of methods to analyse differential gene expression has been essential in the progress of molecular biology. A novel approach called iGentifier is presented for profiling known and unknown transcriptomes, thus bypassing a major limitation in microarray analysis. The iGentifier technology combines elements of fragment display (e.g. Differential Display or RMDD) and tag sequencing (e.g. SAGE, MPSS) and allows for analysis of samples in high throughput using current capillary electrophoresis equipment. Application to epidermal tissue of wild-type and mlo5 barley (Hordeum vulgare) plants, infected with powdery mildew [Blumeria graminis (DC.) E.O. Speer f.sp.hordei], led to the identification of several 100 genes induced or repressed upon infection with many well known for their response to fungal pathogens or other stressors. Ten of these genes are suggested to be classified as marker genes for durable resistance mediated by the mlo5 resistance gene.

Direct determination of chlorophenols present in liquid samples by using a supported liquid membrane coupled in-line with capillary electrophoresis equipment

Actually there is a great trend on the development of effective analytical methods for monitoring trace levels of various phenols which can indicate, among others compounds, the water quality. A simple, inexpensive supported liquid membrane (SLM) device was used in combination with commercially available capillary electrophoresis (CE) equipment for the direct determination of chlorophenols in surface water samples. The manifold was used simultaneously to extract and preconcentrate the analytes from liquid samples. In the extraction set-up, the donor phase (4 mL) was placed in the CE vial, where a micro-membrane extraction unit (MMEU) accommodating the acceptor phase (100 μL) in its lumen was immersed. The supported liquid membrane was constructed by impregnating a porous Fluoropore Teflon (PTFE) membrane with a water-immiscible organic solvent (dihexyl ether). The extraction process was optimized with regard to the pH of the donor and acceptor phases, membrane liquid, extraction time and voltage applied to the inlet or outlet vial during extraction. The chlorinated phenols pentachlorophenol (PCP), 2,3,6 trichlorophenol (TCP) and 2,6 dichlorophenol (DCP) were thus efficiently separated by CE, using tris(hydroxymethyl)aminomethane (Tris) and an NaH 2PO 4 solution containing 1% (v/v) methanol at pH 10.5 as running buffer.

New supported liquid membrane‐capillary electrophoresis in‐line arrangement for direct selective analysis of complex samples

An in-line coupling of a micro-membrane extraction unit, based on supported liquid membrane, with commercially available capillary electrophoresis equipment is described. A main characteristic of this micro-membrane device, made from a simple Eppendorf tube, is that it permits the application of voltage in the acceptor solution to be applied during the extraction process. This has been shown as an alternative to enhance sensitivity, as the analytical signal achieved by applying 10 kV for 20 min was similar to that obtained without the application of voltage and with extraction time of 60 min. In addition, the design has been made permitting both in-line hydrodynamic and electrokinetic sample introduction into the electrophoretic capillary. The analytical potential of the proposed system has been demonstrated by the direct determination of nitroimidazoles from pig liver tissue. The high efficiency of the proposed system allowed the extraction and the determination of the analytes to be performed from a simple tissue homogenate obtained in water. The precision of the analysis of spiked samples, expressed in terms of relative standard deviation, was better than 4.8%.

On-line conversion and determination of artemisinin and its kinetic parameters using orthogonal design by coupling of flow injection with capillary electrophoresis

A rapid, accurate and improved capillary electrophoresis (CE) coupled with flow injection (FI) method with 6.5 cm long separation capillary for the determination of artemisinin and its relevant kinetic parameters by on-line treatment with alkali has been developed. Orthogonal experimental design has been utilized to optimize experimental factors that affect the analysis of artemisinin. The buffer pH value (pH), the concentration of the buffer ( C N a 3 P O 4 ), the applied voltage ( V a), the concentration of alkali ( C NaOH), the heating time ( t h) and the reaction temperature ( T r) are selected as the investigating variables. The different effects on the peak area are studied by the orthogonal design method. As a result, the optimum experimental factors are 10.5 for pH, 10 mmol/L for C N a 3 P O 4 , 2.1 kV for V a, 0.4 mol/L for C NaOH, 2 min for t h, and 40 °C for T r. Analysis is carried out in less than 2 min after conversion of artemisinin in the FI system that is coupled to CE equipment via a split–flow interface cell. The method has been used to determine not only the content of artemisinin in medicine plant Artemisia annua L., but also the activation energy and the chemical respond order of the conversion from artemisinin to Q292.

Disposable twin gold electrodes for amperometric detection in capillary electrophoresis.

We describe a simple and easy way to construct gold microelectrodes for amperometric detection in capillary electrophoresis (CE). The gold microelectrodes, in single or twin sets, were obtained from recordable compact discs (gold-sputtered type), which present highly reproducible surface characteristics. The performance of these electrodes was evaluated by using a home-made CE equipment. The basic steps for the electrode construction are: drawing on a microcomputer; laser printing of the design on wax paper; heat-transfer of the toner onto the gold surface of a peeled recordable compact disc (CD-R); etching of the gold layer from unprinted regions; removal of the toner with a solvent; sealing of unused electrode areas with varnish. One electrode at a time was connected to a potentiostat (or two, to a bipotentiostat) and operated in a wall jet configuration relative to the CE capillary outlet. The amperometric signals were integrated for quantification purposes. Repetitive injections (n = 10) of a mixture containing iodide, ascorbic acid, dipyrone, and acetaminophen (20, 200, 500, and 100 microM), presented relative standard deviations of 2.9, 4.5, 6.1, and 4.0%, respectively. For these analytes, the detection limits (S/N = 3, 30 s of 100 mm hydrodynamic injection) were 0.1, 0.5, 3.1, and 1.1 microM, respectively.

Capillary electrophoresis of neurotransmitters with amperometric detection at melanin-type polymer-modified carbon electrodes

This paper reports on the advantages of combining the separation capability of capillary electrophoresis using a novel home-made electrochemical cell adapted to commercially available capillary electrophoresis equipment with the permselective properties of melanin-type polymer-modified carbon electrodes as amperometric detectors. The excellent permselective properties of the melanic polymer have allowed a large improvement on the quantification of dopamine, epinephrine and norepinephrine. A linear response was obtained in the range 8–200 pg of dopamine with sensitivities between 3- and 5-times higher than those at the bare carbon electrodes and detection limits at submicromolar level (41 fmol in the volume injected). The effect of the carbon electrodes size, and capillary electrophoresis parameters on the analytical performance is also discussed.

Multímetro interfaceado de baixo custo para aquisição de dados

A digital multimeter (~U$ 240.00 on the national market) connected to a microcomputer by a RS-232 serial interface is proposed for data acquisition in equipment with analog output. Data are measured at the rate of 2 points per second and stored in text files by the software that accompanies the device, running in a Windows environment. The performance of the multimeter was verified by monitoring the transient signals generated in flow injection systems associated with fluorimetric, spectrophotometric and flame photometric detection. In addition, the performance of the proposed device was similar to that attained by employing an interface card with a 12-bit analog-to-digital converter for acquisition of the signals generated by a capillary electrophoresis equipment with oscillometric detection.

Enhancing sensitivity in capillary electrophoresis

Capillary electrophoresis (CE) provides an efficient separation methodology for a wide variety of chemical components in diverse matrices. However, the concentration-sensitivity of CE is usually much lower than that of liquid chromatography (LC) and inadequate for many practical applications. In recent years, this shortcoming has been addressed by developing various approaches to enhance the sensitivity of CE, and these are the subject of this article. They involve either the use of improved detection systems or the development of automatic pre-concentration methodologies for both on-capillary and on-line coupled systems. This article discusses recently reported detection modes based on the use on non-absorbance detectors and chemiluminescence and derivatization reactions. Also, it reviews sensitivity enhancements based on on-capillary and on-line coupled pre-concentration techniques. By virtue of such valuable features as low cost, flexibility, miniaturization and ease of use, continuous-flow systems coupled to CE equipment using a UV/Vis detector are shown to be especially suitable for routine use.

Determination of myo-inositol phosphates in food samples by flow injection-capillary zone electrophoresis.

A capillary electrophoresis (CE) method with indirect photometric detection was developed to identify and quantify myo-inositol phosphates in food samples. A flow-injection (FI) system including a micro-column containing anionic exchange resin was used for the solid-phase extraction of the myo-inositol phosphates with a view to their preconcentration. The FI system was automatically coupled to CE equipment via a mechanical interface. The overall analysis time was shortened by incorporating an FI system for myo-inositol hexakisphosphate monitoring. The limit of detection for myo-inositol phosphates as determined by FI-CE ranged from 11 to 26 micromol/L and the coefficient of variation from 3.9 to 5.0%. On the other hand, the limit of detection and coefficient of variation for myo-inositol hexakisphosphate as monitored by the FI system were 75 micromol/L and 2.9%, respectively. The proposed method was successfully applied to a variety of food samples with recoveries ranging from 96.0 to 107.7% and the precision from 3.9 to 7.9%. Based on the results, the content of myo-inositol hexakisphosphate in nuts was two or three times higher than that in legumes.