Electrophoretic Conditions Research Articles

The use of experimental design for the development of a capillary zone electrophoresis method for the quantitation of captopril.

A capillary zone electrophoresis (CZE) method for the quantitation of captopril (CPT) using UV detection was developed. Influence of electrolyte concentration and system variables on electrophoretic separation was evaluated and a central composite design (CCD) was used to optimize the method. Variables investigated were pH, molarity, applied voltage and capillary length. The influence of sodium metabisulphite on the stability of test solutions was also investigated. The use of sodium metabisulphite prevented degradation of CPT over 24 hours. A fused uncoated silica capillary of 67.5cm total and 57.5 cm effective length was used for analysis. The applied voltage and capillary length affected the migration time of CPT significantly. A 20 mM phosphate buffer adjusted to pH 7.0 was used as running buffer and an applied voltage of 23.90 kV was suitable to effect a separation. The optimized electrophoretic conditions produced sharp, well-resolved peaks for CPT and sodium metabisulphite. Linear regression analysis of the response for CPT standards revealed the method was linear (R2 = 0.9995) over the range 5-70 μg/mL. The limits of quantitation and detection were 5 and 1.5 μg/mL. A simple, rapid and reliable CZE method has been developed and successfully applied to the analysis of commercially available CPT products.

Capillary Electrophoresis as Tool for Diastereomeric Separation in a Trichilia catigua Fraction.

The tree Trichilia catigua, popularly known as "catuaba", shows several biological activities and has emerged as a potential source of new drugs. Considering that more than 10 species are known under the same popular name, regulatory agencies require more rigorous quality control of this medicinal plant. To develop and validate a methodology using capillary electrophoresis (CE) with ultraviolet (UV) detection for analysing polyphenols in the ethyl-acetate fraction (EAF) of Trichilia catigua. Different electrophoretic conditions (such as wavelength of UV detection, voltage, buffer concentration and pH, cyclodextrin type and concentration) were investigated. After optimisation, borate buffer 80mmol/L at pH8.80 with 2-hydroxypropyl-β-cyclodextrin 10mmol/L was selected as background electrolyte. A voltage reduction was used to improve the separation of a diastereomeric pair of cinchonains. The method proved to be simple, sensitive, accurate, linear, precise and reproducible. For the first time in natural products analysis, a voltage reduction and hydroxypropyl-β-cyclodextrin were used to improve the separation of diastereomeric pairs. Until now, this is the only described methodology able to separate catechin, epicatechin, cinchonains Ia, Ib, IIa, and IIb from Trichilia catigua samples on the same run in less than 12min. When compared to the high performance liquid chromatography with photo-diode array detection (HPLC-PDA) method previously developed by our research group, the CE method was more efficient, faster, less expensive and less polluting. Our results demonstrate that this method could be employed in a quality-control laboratory for the quantification of polyphenols in EAF of Trichilia catigua. Copyright © 2016 John Wiley & Sons, Ltd.

A High Voltage Power Supply That Mitigates Current Reversals in Capillary Zone Electrophoresis-Electrospray Mass Spectrometry.

Capillary electrophoresis coupled with electrospray ionization typically employs two power supplies, one at each end of the capillary. One power supply is located at the proximal (injection) end of the capillary. The power supply located at the distal (detector) end of the capillary drives the electrospray. Electrophoresis is driven by the difference in potential between these power supplies. Separations that employ large capillary inner diameter, high conductivity background electrolyte, and high separation potentials generate higher current than that produced by the electrospray. Excess current flows through the electrospray power supply. Most power supplies are not designed to sink current, and the excess current will cause the electrospray voltage to deviate from its set point. We report a simple circuit to handle this excess current, allowing separations under a wide range of electrophoretic conditions. Graphical Abstract ᅟ.

Preparation of platinum-free tubular dye-sensitized solar cells by electrophoretic deposition

Tubular dye-sensitized solar cells (DSSCs) were developed by replacing expensive materials with those of lower cost as follows: (1) substituting conductive glass electrodes for titanium (Ti) wires, and, (2) use of a mixture of multi-walled carbon nanotubes, MWCNTs and poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate), and PEDOT-PSS in place of a platinum (Pt) catalyst. Platinized counter electrodes were used as the standard counter electrodes for comparison. The effects of the chemical treatment of a titanium wire substrate and electrophoretic deposition conditions on the efficiency of the resulting DSSCs were also investigated. Chemical treatment of titanium wires was done by soaking the wires in HF-HNO3 solutions at three different concentrations, 0.8, 1.6 and 2.4 M, for three different soaking durations, 5, 10 and 15 min. The optimal condition was found to be a HF-HNO3 concentration of 0.8 M and a soaking duration of 10 min. Film coating on working electrodes was performed using an electrophoretic technique at three different voltages, 5, 8 and 10 V, and four different coating durations, 1, 3, 5 and 7 min. Then, the optimal voltage of 5 V and deposition duration of 5 min was applied for film deposition on counter electrodes. The efficiency of the resulting DSSC with a CNTs/TiO2 counter electrode was 0.03%. The addition of PEDOT-PSS improved the efficiency of this DSSC to 0.08%.

Synthesis of Unsaturated Nonionic Poly(ester‐sulfones) via Acyclic Diene Metathesis (ADMET) Polymerization and Anode‐Selective Electrophoretic Deposition

Nonionic aliphatic polymers containing ester and sulfonyl moieties, [poly(ester‐sulfones)s] were found to undergo anode‐selective electrophoresis under electrophoretic deposition conditions. Herein are reported the syntheses of unsaturated nonionic polyesters containing sulfide linkages and double bonds on the polymer backbone via acyclic diene metathesis polymerization using a Grubbs' catalyst. Subsequent oxone oxidation was carried out, affording the corresponding poly(ester‐sulfone), followed by electrophoretic deposition of the unsaturated poly(ester‐sulfone) onto stainless steel. Subsequent UV‐irradiation cured the deposited film, improving the peeling strength of the coating. image

Effect of electrophoretic condition on the electromagnetic interference shielding performance of reduced graphene oxide-carbon fiber/epoxy resin composites

Abstract Reduced graphene oxide modified carbon fiber (rGO-CF) was synthesized by electrophoretic deposition and chemical reduction. The GO concentration, electrophoretic time and voltage were taken as variables. Epoxy based composites (rGO-CF/EP) were prepared through molding process and the effects of electrophoretic condition on electromagnetic interference (EMI) shielding performance of rGO-CF/EP were studied. Scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy showed that rGO nanosheets were successfully immobilized on CF. The shielding effectiveness (SE) of composites was tested, and the electrophoretic time and voltage have positive impact on SE value. When the voltage increased to 21 V for 60 min, the rGO-CF/EP composite obtained maximum EMI SE of 37.6 dB, which had a 47.6% improvement than that of CF/EP. Furthermore, a high calculated electrical conductivity of 7.20 S/m was achieved at the same condition. Therefore, controlling the electrophoretic condition is available to achieve lightweight high EMI shielding rGO-CF/EP.

SEM Analysis of Electrophoretically-Deposited Cobalt Ferrite Films

Cobalt ferrite nanoparticles were synthesized and electrophoretically deposited onto aluminum foil, graphite paper, and carbon felt. The resulting cobalt ferrite films were studied to determine their potential for use as an electrocatalyst for the oxidation of ammonium sulfite to ammonium sulfate for a proposed sulfur ammonia thermochemical cycle for hydrogen production. The effects of electrophoretic deposition conditions on deposit morphology and the effects of deposit morphology on electrochemical activity in 2M ammonium sulfite were investigated by SEM analysis.

Determination of HMG-CoA reductase inhibitors by micellar electrokinetic chromatography

Abstract Objective: In this study we report the development of a simple, rapid and efficient capillary electrophoresis method for the simultaneous determination of atorvastatin, fluvastatin, lovastatin and simvastin. Methods: Capillary zone electrophoresis proved to be efficient for the simultaneous separation of atorvastatin and fluvastatin, but could not resolve the determination of lovastatin and simvastatin. The simultaneous separation of all four statins was achieved by applying a micellar electrokinetic chromatographic method, after transforming lovastatin and simvastatin in β-hydroxyl acid forms through alkaline hydrolysis. The optimum electrophoretic conditions and analytical parameters were investigated and the analytical performances of the method were verified with regard to linearity, precision, accuracy, LOD and LOQ. Results: The optimum electrophoretic separation conditions were: 25 mM sodium tetraborate with 25 mM sodium dodecyl sulphate buffer electrolyte at pH 9.5, applied voltage + 25 kV, separation temperature 25 °C, injection pressure/time 50 mbar/1 minutes, UV detection at 230 nm. Using the optimized electrophoretic conditions we succeeded in the simultaneous determination of the four statins in approximately 3 minutes, the order of migration being: atorvastatin, fluvastatin, lovastatin, simvastatin. The proposed method has been applied to the determination of the analytes in pharmaceutical tablets formulations. Conclusions: The capillary electrophoretic method developed in the present work proved to be suitable for the routine analysis of statins and can be adopted as quality control protocol in pharmaceutical analysis.

SEM Analysis of Electrophoretically-Deposited Cobalt Ferrite Films

Cobalt ferrite nanoparticles (20 nm) were synthesized and electrophoretically deposited onto aluminum foil, graphite paper, and carbon felt (3 mm thickness) from a 100% ethanol bath. Linear sweep voltammetry was used to test the electrocatalytic activity of the deposits relative to blank substrates for the oxidation of ammonium sulfite to ammonium sulfate in a proposed sulfur ammonia (SA) water-splitting solar thermochemical cycle. Subsequently, scanning electron microscopy (SEM) was used to study the morphology of the cobalt ferrite films. The effects of electrophoretic deposition conditions on deposit morphology and the effects of deposit morphology on electrochemical activity in 2 M ammonium sulfite were investigated. EPD on aluminum and graphite paper was performed such that particles were deposited on only one side of the substrate. For the 3 mm thick carbon felt, particles were deposited on both sides of the substrate to improve deposit uniformity throughout the sample. Both deposition time (0-10 minutes) and deposition current (0-16 mA) were varied to analyze their effects on the resulting deposit films for all substrates. For the EPD conditions studied, a critical deposit thickness was found for graphite paper and carbon felt for maximum electrochemical current density, where additional deposited particles reduced the electrocatalytic activity of the deposits. This thickness was estimated to be 3 particle layers for the graphite paper substrate. At this thickness, the deposit had a 5.5 fold increase in activity relative to a blank graphite paper substrate. SEM micrographs showed that the deposit uniformly covered the substrate and had noticeable cracking. For the carbon felt, the critical deposit thickness was observed to be approximately 1-2 particle layers via SEM analysis. Moreover, SEM images showed that the deposit was a uniform film with no cracking. At this thickness, the deposit had a 4.3 fold increase in activity compared to a blank carbon felt substrate.

Screening neuraminidase inhibitors from glycosaminoglycan and natural extract by capillary electrophoresis

An electrophoretically mediated microanalysis (EMMA) method for screening neuraminidase inhibitors in depolymerized glycosaminoglycan and natural extracts is described. In the present method, enzyme and substrate were individually introduced into the capillary as distinct plugs, and then mixed for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at 214 nm represents the enzyme activity. The electrophoretic conditions for the enzyme reaction and separation of substrate and product were optimized in this study. Under the optimal conditions, the Michaelis–Menten constant and the inhibitive mechanism of zanamivir were studied, which agreed with the literature data. Furthermore, the inhibitory ratios of enzymatic activity of depolymerized glycosaminoglycan and traditional Chinese drugs were determined. The EMMA method has superiority over traditional assay methods, in not only minimizing the false-positive results but also in simplifying the experimental procedure. Therefore, it could be employed to screen inhibitors from natural sources.

Determination of antazoline and tetrahydrozoline in ophthalmic solutions by capillary electrophoresis and stability-indicating HPLC methods

Capillary electrophoretic (CE) and high performance liquid chromatographic (HPLC) methods were developed and optimized for the determination of antazoline (ANT) and tetrahydrozoline (TET) in ophthalmic formulations. Optimum electrophoretic conditions were achieved using a background electrolyte of 20mM phosphate buffer at pH 7.0, a capillary temperature of 25°C, a separation voltage of 22kV and a pressure injection of the sample at 50mbar for 17s. HPLC analysis was performed with Kinetex (150×4.6mm ID×5μm) (Phenomenex, USA) analytical column with 1mLmin−1 flow rate of mobile phase which consisted of 0.05% TFA in bidistilled water (pH adjusted to 3.0 with 5M NaOH) and acetonitrile/buffer in the ratio of 63:37 (v/v) at room temperature. Injection volume of the samples was 10μL and the wavelength of the detector was set at 215nm for monitoring both analytes.Calibration graphs showed a good linearity with a coefficient of determination (R2) of at least 0.998 for both methods. Intraday and interday precision (expressed as RSD%) were lower than 2.8% for CE and 0.92% for HPLC. The developed methods were demonstrated to be simple and rapid for the determination of ANT and TET in ophthalmic solutions providing recoveries in the range between 97.9 and 102.70% for CE and HPLC.

Image Pretreatment Tools II: Normalization Techniques for 2-DE and 2-D DIGE.

Gel electrophoresis is usually applied to identify different protein expression profiles in biological samples (e.g., control vs. pathological, control vs. treated). Information about the effect to be investigated (a pathology, a drug, a ripening effect, etc.) is however generally confounded with experimental variability that is quite large in 2-DE and may arise from small variations in the sample preparation, reagents, sample loading, electrophoretic conditions, staining and image acquisition. Obtaining valid quantitative estimates of protein abundances in each map, before the differential analysis, is therefore fundamental to provide robust candidate biomarkers. Normalization procedures are applied to reduce experimental noise and make the images comparable, improving the accuracy of differential analysis. Certainly, they may deeply influence the final results, and to this respect they have to be applied with care. Here, the most widespread normalization procedures are described both for what regards the applications to 2-DE and 2D Difference Gel-electrophoresis (2-D DIGE) maps.

HOST IDENTIFICATION OF ARTHROPOD BLOODMEALS BY AGAR ELECTROPHORESIS*)

A relatively easy and inexpensive method of rapid gel electrophoresis suitable for the host identification of arthropod bloodmeals was investigated. Electrophoretic conditions (preparation of agar and buffer, voltage, temperature, time, etc.) are discussed. The method of preparing and applying the arthropod bloodmeal to the slides is discussed in detail. Two minutes after the start of the electrophoretic run the suitability of the arthropod meal for testing was apparent. When there was visible migration of more than one Hb fraction towards the anode, digestion has proceeded too far for host identification. Bloodmeals of Aedes togoi, Aedes aegypti, Culex fatigans, Anopheles sp., Rhodnius prolixus, Triatoma infestans, Triatoma dimidiata, Cimex lectularius, Pediculus humanus, Ornithodorus moubata, Ornithodorus savignyi and Ornithodorus rudis from man, Cebus and rhesus monkey, dog, cat, rabbit, guinea pig, hamster, rat, mouse, chicken, pigeon, snake, toad and frog were studied and compared with the serum and hemolyzed blood of the host species. It was possible to identify the host from the pattern of the arthropod bloodmeals. A number of details on arthropod digestion processes are given.

Optimization of a nano-enzymatic reactor for on-line tryptic digestion of polypeptide conjugates by capillary electrophoresis.

This work aims at studying the optimization of an on-line capillary electrophoresis (CE)-based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on-line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.

Bead Based Proteome Enrichment Enhances Features of the Protein Elution Plate (PEP) for Functional Proteomic Profiling.

A novel functional proteomics technology called PEP(Protein Elution Plate) was developed to separate complex proteomes from natural sources and analyze protein functions systematically. The technology takes advantage of the powerful resolution of two-dimensional gel electrophoresis (2-D Gels). The modification of electrophoretic conditions in combination with a high-resolution protein elution plate supports the recovery of functionally active proteins. As 2DE(2-Dimensional Electrophoresis) resolution can be limited by protein load, we investigated the use of bead based enrichment technologies, called AlbuVoid™ and KinaSorb™ to determine their effect on the proteomic features which can be generated from the PEP platform. Using a variety of substrates and enzyme activity assays, we report on the benefits of combining bead based enrichment to improve the signal report and the features generated for Hexokinase, Protein Kinase, Protease, and Alkaline Phosphatase activities. As a result, the PEP technology allows systematic analysis of large enzyme families and can build a comprehensive picture of protein function from a complex proteome, providing biological insights that could otherwise not be observed if only protein abundances were analyzed.

Internal standard method for the measurement of doxorubicin and daunorubicin by capillary electrophoresis with in-column double optical-fiber LED-induced fluorescence detection

An internal standard method has been developed for the simultaneous determination of anthracycline antibiotics, doxorubicin (DOX) and daunorubicin (DAN), in rabbit plasma using capillary electrophoresis (CE) with in-column double optical-fiber LED-induced fluorescence detection (CE-ICDOF-LED-FLD). Rhodamine B (RhB) was selected as an internal standard because its emission wavelength is similar to that of the anthracycline antibiotics. Parameters including buffer pH, buffer concentration, organic solvents and separation voltage have been investigated to explore the sensitivity and separation efficiency of DOX and DAN. The optimal electrophoretic separation conditions were a borate buffer (15mM, pH 9.0) containing 50% acetonitrile (v/v), 10s hydrodynamic injection at a height of 20cm and a separation voltage of 15kV. The developed CE-ICDOF-LED-FLD method provides limits of detection of 18 and 13ng/mL for DOX and DAN in rabbit plasma samples, respectively. The recoveries ranging from 93.7 to 104.8% and the relative standard deviations at 1.1–1.7% were achieved for DOX and DAN in spiked rabbit plasma samples.

Quadruple Response Factorial Design Optimization of Capillary Zone Electrophoresis Assay Procedure for Metformin and Sitagliptin Combination

The metformin–sitagliptin combination has been recently approved in 2012 for its better efficacy and safety for diabetes than using single drugs. The current manuscript describes an assay method for this recent combination by capillary zone electrophoresis (CZE). The factorial design optimization of electrolyte composition for quadruple response of resolution, migration time, peak area, and peak symmetry was conducted. The univarite method was applied for optimizing other electrophoretic conditions. The optimum electrolyte composition was 10 mM borate at pH 6.4. Sufficient separation with a resolution of 6.7 and peak symmetry of 0.932 and 0.985 were obtained. Satisfactory repeatability of migration time, peak area, peak height, and peak symmetry (RSD 0.16–2.4%) was obtained. The validation parameters were acceptable in terms of recovery, linearity, inter-mediate precision, limits of detection, and limits of quantification. The use of CZE provides reagent-saving and fast method comparing with reported HPLC methods.

Advances in Microscale and Nanoscale Mechanisms of Electrophoretic Deposition in Aqueous Media

The processing of ceramic thick and thin films, nano- and micro-scaled ceramic structures as well as bulk ceramics of high quality and precise dimensions under electrophoretic boundary conditions requires a full understanding of the dynamics of relevant interfacial mechanisms and interactions of colloidal phases at the nano- and micro-scale. Recent findings and latest insights on the importance of electrokinetic and electrohydrodynamic interfacial processes for membrane electrophoretic depositon in aqueous media are summarised. In this context, the paper addresses the fundamental importance of surficial charge heterogeneities, electric double layer instabilities, electrokinetically induced micro-vortex dynamics, as well as lateral and medial effective electrical field gradients. These phenomena are evaluated in terms of reasonable correlations and mechanistic coincidences of general EPD deposition principles. The experimental results are based on potentiometry, in-situ videomicroscopy, high-resolution as well as secondary electron microscopy. A numerical method for the simulation of the electrophoretic deposition process is suggested based on a multiphysical Finite Element approach given by Nernst-Planck, Poisson- and Navier-Stokes equations. The results of the simulations provide adequate agreement with experimental findings.

SDS-PAGE as a Tool for Hydrodynamic Diameter-Dependent Separation of Quantum Dots

The purpose of this study was to test quantum dots (QDs) separation by native and Tris–Glycine SDS-PAGE according to the protocols commonly used for protein analyses. To study the electrophoretic behaviour of quantum dots, ten samples of previously synthesized CdTe QDs stabilized with mercaptosuccinic acid (MSA) were used. Prior to electrophoresis the hydrodynamic diameters of QDs and zeta potentials were determined, as well as the fluorescence properties and stability of QDs in the running buffers. After verification of QDs stability and separation in native polyacrylamide gel, SDS-PAGE in gradient 4–20 % polyacrylamide gel was performed. Under UV irradiation a colour-dependent separation of QDs was observed, which was consistent with their hydrodynamic diameter distribution. The electrophoretic conditions were further optimized with respect to achieving the optimal colour separation, fluorescence stability and to minimize the time of analysis. Based on the results obtained, for further work 15 % polyacrylamide gels with SDS were used and the times (30–60 min) and voltage (100–150 V) used for separation were optimized. Under the optimal separation conditions (30 min, 100 V) the addition of MSA in the concentration range 0–4 mM was used to improve visualization of QDs with diameters in the range from 7 ± 2 to 4 ± 2 nm.

Cyclodextrine screening for the chiral separation of amlodipine enantiomers by capillary electrophoresis.

Amlodipine is a long acting, dihydropyridine type calcium channel blocker frequently used in the treatment of hypertension and coronary insufficiency. The calcium channel blocking activity resides primarily in the S-amlodipine enantiomer, while R-amlodipine is a potent inhibitor of smooth muscle cell migration. In this study capillary electrophoresis was applied for the enantiomeric separation of amlodipine using different native and derivatized; neutral and charged cyclodextrines as chiral selectors. The effects of pH and composition of the background electrolyte, concentration and type of chiral selector, capillary temperature, running voltage and injection parameters have been investigated. Stereoselective interactions were observed when using α-CD, β-CD, HP-β-CD, RAMEB, CM-β-CD and SBE-β-CD. Optimized separation conditions consisted on a 50 mM phosphate buffer, pH - 3.0, 20 mM RAMEB as chiral selector, + 25 kV applied voltage, 15°C temperature and UV detection at 238 nm. Using the optimized electrophoretic conditions we succeeded the chiral separation of amlodipine enantiomers in approximately 6 minute, the order of migration being R-amlodipine followed by S-amlodipine. The method was successfully applied for the determination of amlodipine enantiomers from commercially available pharmaceuticals. The linearity range, limits of detection and quantification, precision and accuracy were determined and the results obtained confirmed that the method was suitable for this purpose. It can be concluded that the proposed capillary electrophoresis methods can be useful for routine pharmaceutical applications with benefits of its effectivity, simplicity, short analysis time and low consumption of analytes, solvents and chiral selectors.