Capillary Electrophoretic System Research Articles

Electric-field induced phase transitions in capillary electrophoretic systems

The movement of particles in a capillary electrophoretic system under electroosmotic flow was modeled using Monte Carlo simulation with the Metropolis algorithm. Two different cases with repulsive and attractive interactions between molecules were taken into consideration. Simulation was done using a spin-like system, where the interactions between the nearest and second closest neighbors were considered in two separate steps of the modeling study. A total of 20 different cases with different rates of interactions for both repulsive and attractive interactions were modeled. The movement of the particles through the capillary is defined as current. At a low interaction level between molecules, a regular electroosmotic flow is obtained; on the other hand, with increasing interactions between molecules, the current shows a phase transition behavior. The results also show that a modular electroosmotic flow can be obtained for separations by tuning the ratio between molecular interactions and electric field strength.

Implementation of a ultraviolet area imaging detector for analysis of polyvinyl alcohol microbubbles by capillary electrophoresis

Contrast agents are widely used to enhance the image quality in clinical imaging using e.g. ultrasound. The contrast agents used for ultrasound imaging are mainly microbubbles (MBs) with a soft or hard shell encapsulating a core of gas. In the present study, MBs with a hard shell of polyvinyl alcohol (PVA), and a core of air were analysed in a capillary electrophoretic system using a UV area imaging detector. The detector was operating at 3 wavelengths; 214 nm, 255 nm and 525 nm, and the highest absorbance for individual PVA-MBs were obtained at 214 nm. Two detection windows and a vertical loop capillary position enabled tracking of the PVA-MBs both in an upward and a downward flow direction, where PVA-MBs had different flow distributions and slightly higher average flow velocity upwards, attributed to temperature differences in the capillary that was part within the instrument and part outside. The tracking also allowed counting and quantification of the PVA-MBs. Separation of PVA-MBs from proteins present in human blood plasma was achieved, with multi-wavelength imaging showing best contrast at 525 nm. The PVA-MBs absolute values of negative zeta potential and anionic mobility when injected from plasma in the pH 12 background electrolyte are higher than those obtained for MBs injected from buffer, consistent with their increased negative charge due to a protein corona coating of the PVA-MBs.

A pressure-driven capillary electrophoretic system with injection valve sampling.

To improve repeatability and efficiency and to simplify the operation procedure of capillary electrophoresis (CE), a pressurized CE system (p-CE) with injection valve sampling was developed. It consisted of one high-pressure pump, a six-port injection valve, a PEEK cross, a separation and back pressure capillary, an ultraviolet-visible detector and a high voltage power supply. The pressure-driven flow ranging from 4.5 nL min(-1) to 0.81 μL min(-1) in the separation capillary was produced by splitting to the flow from the high-pressure pumps (0.005-0.4 mL min(-1)). Nano-volume sample injection (<10 nL) was conducted by a micro-volume rotary injector (0.5-5 μL) with flow splitting. In the p-CE system, the new commercial capillary could be directly used without any wash, and the capillary-flush process between runs was also eliminated. In this case, the analytes were driven toward the outlet of the separation capillary by the pressurized flow, the electric field force and minute electroosmotic flow, and they were separated owing to the electrophoretic mobility. The p-CE system allows for the independent variation of the pressurized flow rate and electrical field and electrophoretic separation of good repeatability (below 3%) under high electrical fields (500-1000 V cm(-1)) and flow rate gradient modes. The feasibility of the p-CE system in real analysis was demonstrated by iodate quantification in iodized table salts. The separation of iodide and iodate was realized within 0.3 min, proving its high analytical speed.

Fingerprinting postblast explosive residues by portable capillary electrophoresis with contactless conductivity detection

A portable capillary electrophoretic system with contactless conductivity detection was used for fingerprint analysis of postblast explosive residues from commercial organic and improvised inorganic explosives on various surfaces (sand, concrete, metal witness plates). Simple extraction methods were developed for each of the surfaces for subsequent simultaneous capillary electrophoretic analysis of anions and cations. Dual-opposite end injection principle was used for fast (<4 min) separation of 10 common anions and cations from postblast residues using an optimized separation electrolyte composed of 20 mM MES, 20 mM l-histidine, 30 μM CTAB and 2 mM 18-crown-6. The concentrations of all ions obtained from the electropherograms were subjected to principal component analysis to classify the tested explosives on all tested surfaces, resulting in distinct cluster formations that could be used to verify (each) type of the explosive.

Detection and quantification of 8‐hydroxy‐2′‐deoxyguanosine in Alzheimer's transgenic mouse urine using capillary electrophoresis

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the major forms of oxidative DNA damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8-OHdG by using CE-LIF detection. The method involved the use of specific antibody to detect the DNA lesion (8-OHdG) and consecutive fluorescence labeling. Next, urinary 8-OHdG fluorescently labeled along with other constituents were resolved by capillary electrophoretic system and the lesion of interest was detected using a fluorescence detector. The limit of detection was 0.18 fmol, which proved sufficient sensitivity for detection and quantification of 8-OHdG in untreated urine samples. The relative standard deviation was found to be 11.32% for migration time and 5.52% for peak area. To demonstrate the utility of this method, the urinary concentration of 8-OHdG in an Alzheimer's transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages, such as high separation efficiency, good selectivity, low limit of detection, simplicity and low cost of analysis.

PCR Multiplex fluorescente para detecção de bactérias em sêmen bovino

Este estudo teve como objetivo avaliar o limiar de detecção da técnica de PCR multiplex fluorescente aliada a eletroforese capilar na detecção de agentes infecciosos em amostras de sêmen experimentalmente contaminadas com concentrações decrescentes das bactérias Brucella abortus, Leptospira interrogans sorovar pomona, Campylobacter fetus e Haemophilus somnus. Amostras de sêmen bovino foram experimentalmente contaminadas com concentrações decrescentes de bactérias obtidas através de diluições seriadas na base 10 de modo a obter-se amostras contendo desde 1 vez até 10-7 bactérias/mL a partir da concentração inicial de Leptospira pomona, Brucella abortus, Campylobacter fetus e Haemophilus somnus. As diluições foram efetuadas individualmente para cada bactéria, bem como nas diferentes concentrações necessárias para a padronização do teste de multiplex PCR. As extrações de DNA de todas as soluções contendo espermatozóides e bactérias analisadas no presente estudo foram realizadas segundo protocolo descrito por Heinemann et al. (2000). Os produtos de PCR multiplex foram avaliados por eletroforese em gel de poliacrilamida 8% e separação eletroforética por sistema capilar em equipamento automático de análise de fragmentos de DNA MegaBace. Observou-se a amplificação de fragmentos de 193pb, 330pb, 400pb e 415pb a partir do DNA de B. abortus, L. pomona, H. somnus, C. fetus, respectivamente. Na análise por eletroforese capilar de produtos da PCR multiplex do DNA para detecção simultânea dos quatro patógenos observou-se a sinal de positividade até a diluição de 10-3 bactérias/mL vezes da concentração inicial da solução estoque de cada bactéria. A técnica de PCR multiplex aliada à eletroforese capilar foi usada pela primeira vez para o diagnóstico direto de quatro bactérias patogênicas no sêmen, demonstrando ser um método rápido na detecção de bactérias causadoras de doenças reprodutivas.

Erratum: A capillary electrophoretic system based on a novel microemulsion for the analysis of coenzyme Q10 in human plasma by electrokinetic chromatography

Erratum: A capillary electrophoretic system based on a novel microemulsion for the analysis of coenzyme Q10 in human plasma by electrokinetic chromatography

In situ determination of nerve agents in various matrices by portable capillary electropherograph with contactless conductivity detection

Rapid, efficient and robust methods for sampling and extracting genuine nerve agents sarin, soman and VX were developed for analyzing these compounds on various solid matrices, such as concrete, tile, soil and vegetation. A portable capillary electrophoretic (CE) system with contactless conductometric detection was used for the in situ analysis of the extracted samples. A 7.5 mM MES/HIS-based separation electrolyte accomplished the analysis of target analytes in less than 5 min. The overall duration of the process including instrument start-up, sample extraction and analysis was less than 10 min, which is the fastest screening of nerve agents achieved with liquid phase separation methods to date. The procedure can easily be performed by a person in a protective suit and is therefore suitable for real-life applications. The CE results were validated by an independent GC–MS method and a satisfactory correlation was obtained. The use of a proper sampling strategy with two internal standards and “smart” data-processing software can overcome the low reproducibility of CE. This has a significant impact on the potential acceptance of portable CE instrumentation for the detection and analysis of genuine chemical warfare agents (CWA).

The use of novel ionic liquid‐in‐water microemulsion without the addition of organic solvents in a capillary electrophoretic system

In this work, a new ionic liquid-in-water (IL/W) microemulsion without requiring toxic organic solvents was investigated as a pseudostationary phase (PSP) in CE. As observed during the IL/W microemulsion system, a fast and an efficient separation of eight phenolic acids was achieved using 1-butyl-3-methylimidazolivmhexa fluorophosphate (bmimPF(6)) as oil drops, Tween 20 as the surfactant, and borate as the BGE. The effects of oil phase, surfactant, buffer and pH on the separation were explored in detail to evaluate the novel PSP. In contrast, the detection efficiency of these same analytes was markedly decreased using oil-in-water (O/W) MEEKC. We have also validated the practicality of the IL/W microemulsion method by quantitative determination of acidic compounds in pharmaceutical injection. The results obtained indicated that an additional association between the IL cations and analytes tested seemed to play a prominent role in the separation mechanism exhibited by this novel PSP compared with the conventional O/W MEEKC.

Determination of synthetic pharmaceuticals in phytotherapeutics by capillary zone electrophoresis with contactless conductivity detection (CZE-C 4D)

In this work, a method for simultaneous determination of amfepramone, fenproporex, sibutramine and fluoxetine was developed by capillary zone electrophoresis with capacitively coupled contactless conductivity detection (C 4D) using a homemade capillary electrophoretic system. The optimized conditions for the separation of the pharmaceuticals by CZE were as follows: 50 mmol L − 1 phosphate buffer (pH 5.0) in 50/50 (v/v) mixture of water/acetonitrile as the working electrolyte, 15 kV separation voltage, 25 °C separation temperature, hydrodynamic injection by gravity using 20 cm injection height and 60 s injection time. The detection by C 4D was carried out by using a homemade detector, which employs a sinusoidal wave generator operating at 600 kHz frequency and 2 V pp wave amplitude. The optimized and validated CZE-C 4D method was applied for the determination of the studied pharmaceuticals as adulterants in phytotherapeutic formulations commercialized in Brazil for slimming purposes.

A capillary electrophoretic system based on a novel microemulsion for the analysis of coenzyme Q10 in human plasma by electrokinetic chromatography

A new analytical method for determination of coenzyme Q10 (2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone, CoQ10) in human plasma was developed based on CE using a double tensioactive microemulsion. CoQ10 was quantitatively extracted into 1-propanol/hexane and quantified by MEEKC. The microemulsion was prepared by mixing 1.4% w/w sodium bis(2-ethylhexyl) sulfosuccinate, 4% w/w cholic acid, 1% w/w octane, 8.5% w/w butanol, 0.1% w/w PVA and 85% w/w 10 mM Tris buffer at pH 9.0. The optimized electrophoretic conditions included the use of an uncoated silica capillary of 60 cm total length and 75 mum id, an applied voltage of 20 kV, room temperature and 214 nm ultraviolet detection. Selectivity, linearity, LOD, LOQ, precision and accuracy were evaluated as the parameters of validation. Owing to its simplicity and reliability, the proposed method can be an advantageous alternative to the traditional methodology for the quantitation of CoQ10 in human plasma with good accuracy and precision.

Application of a fluorescence stereomicroscope as an in-line detection unit for electrophoretic separation of bacteria

Electrophoretic separation of bacteria as well as potential applications of electrophoresis in microbiological medical analysis has gained the interest of several research groups. The undesired effects such as aggregation and/or adhesion of the cells have a strong effect on the separation process. For a better understanding of the phenomena we decided to apply a fluorescence stereomicroscope as an in-line detection unit for observation of electrophoresis of microorganisms. The microscope was connected to the capillary electrophoretic system which allowed observing the moving bacteria through a 10 cm wide window created in the capillary that was kept in a special holder. The experiments showed that the stained bacteria adhered to the inner capillary surface thus creating a green fluorescent layer. The short (2 mm) polymeric monolithic bed, the pore size of which disabled bacteria cells to pass through, was also photosynthesized in the centre of the window and served to preconcentrate the bacterial suspension.

Dissociation Kinetics and Complexation Equilibrium Studies of a ZnII Complex with 1,5-Bis(2-hydroxy-5-sulfophenyl)-3-cyanoformazan during Capillary Electrophoretic Separation Process

Abstract The dissociation kinetics and the complexation equilibrium of a ZnII complex with 1,5-bis(2-hydroxy-5-sulfophenyl)-3-cyanoformazan (hscf: H5L) during capillary electrophoretic (CE) separation process were studied. The solvolytic dissociation reaction rate constants of the ZnII–hscf complex ([ZnL]3−) (kd) in the pH range of 6.4 to 8.0 were determined with a CE-based dissociation kinetic analysis method. Small kd values on the order of 10−5 s−1 showed the remarkable kinetic inertness of [ZnL]3−, which was detectable in the CE system. The stability constant of [ZnL]3− (KZnL) at 293 K and I = 0.10 was estimated to be [ZnL3−]/{[Zn2+][L5−]} = 1024.2±0.3 through the spectrophotometric titration. The thermodynamic stability of [ZnL]3− in the pre-capillary complexation stage was thus demonstrated. In contrast, it was shown that the CE separation environment is thermodynamically unsuitable for the metal complexes based on the conditional stability constant of [ZnL]3−, which was determined under CE separation conditions, and the concentration of free HSCF in the complex band in the capillary. Thus, the “stability” of metal complex in the CE system strongly depends on kinetic inertness rather than thermodynamic stability, though their thermodynamic stability is necessary for the pre-capillary derivatization step.

Diagnosis of transient electrokinetic flow in microfluidic channels

Measuring the electro-osmotic velocity distributions in microchannels is usually performed for steady-state electrokinetic flows. Characterizing time-dependent electrokinetic flows is of importance to the development of microfluidic devices such as rapid capillary electrophoretic separation system, ac pumps, novel micromixers, etc. In this paper, we use a micron-resolution particle image velocimetry (micro-PIV) based phase locking technique with an ordinary PIV charge coupled device (CCD) camera to carry out an experimental study of the transient electrokinetic flow in microchannels by synchronizing different trigger signals for the laser, CCD camera, and in-house designed high-voltage switch. With the transient micro-PIV technique, we further propose a method to decouple the particle electrophoretic velocity from the micro-PIV measured velocity and to determine the zeta potential of the channel wall. The time evolution of the full-field, electro-osmotic velocity distributions in both open- and closed-end rectangular microchannels is obtained. Using the slip velocity approach and the measured channel zeta potential, the theoretical predictions of the transient electro-osmotic flow in the open- and closed-end microchannels are obtained, and they are found to be in good agreement with the experimental results.

Direct sample injection for capillary electrophoretic determination of organic acids in cerebrospinal fluid

Organic acids in cerebrospinal fluid (CSF) are potential diagnostic markers for neurological diseases and metabolic disorders. A capillary electrophoretic (CE) method for the direct analysis, i.e., without any sample preparation, of six organic acids in CSF was developed. A capillary coating consisting of a triple layer of charged polymers (polybrene-dextran sulfate-polybrene) was used in combination with a negative separation voltage, providing fast and efficient analysis of acidic compounds. Separation conditions, such as background electrolyte (BGE) concentration and pH were optimized, and the influence of albumin and sodium chloride was systematically studied using a set of test compounds. With injection volumes of ca. 44 nL, plate numbers of up to ca. 150,000 were obtained with a BGE of 200 mM sodium phosphate (pH 6.0). It appeared that high sodium chloride concentrations in the sample hardly affected the peak width and shape of the organic acids, most probably due to transient isotachophoresis effects occurring in the sample zone. Adverse effects of CSF proteins, which frequently compromise the CE performance, could be effectively minimized by the triple layer coating in combination with rinses of 0.1 M hydrochloric acid. Overall, the developed CE system allowed direct injections of CSF samples, yielding good separation efficiencies and stable migration times (RSDs<2%) for organic acids. Validation of the method with artificial and real CSF samples showed good linear responses (r>0.99), and LODs for the organic acids were in the range of 2-8 microg/mL when applying UV detection. RSDs for migration times and peak areas were <2% and <7%, respectively. The applicability of the CE system is shown for the determination of organic acids in CSF samples.

Water-Vapor Plasma-Based Surface Activation for Trichlorosilane Modification of PMMA

Separation rates and resolutions within capillary electrophoretic (CE) systems can be enhanced when surface zeta potentials are uniform with minimum deviations from ideal pluglike flow. Microfluidic CE devices based on poly(methyl methacrylate) (PMMA) are being developed due to the optical clarity, availability, stability, and reproducible electroosmotic flow (EOF) rates displayed by this polymer. Control of EOF in polymer-based CE systems can be achieved by surface zeta potential alteration through chemical modification. Herein, a method will be presented for the surface functionalization of PMMA with chemistry analogous to formation of trichlorosilane self-assembled monolayers on SiO2. The current method involves two separate steps. First, surface activation with water-vapor plasma introduces surface hydroxylation. Second, treatment of the plasma-treated PMMA with a substituted trichlorosilane solution forms the functional surface layer. The modified surfaces were characterized using several analytical techniques, including water contact angle, X-ray photoelectron spectroscopy, Fourier transform infrared-attenuated total reflection, secondary ion mass spectroscopy, and measurement of EOF velocities within PMMA microchannels.

Observation of the complex formation between Cu(II) and protein by capillary electrophoretic system incorporating an UV/CL dual detector

We investigated the complex formation between Cu(II) and human serum albumin (HSA) through a biuret reaction by use of capillary electrophoretic system incorporating an ultra-violet absorption (UV) and chemiluminescence (CL) dual detector. Cu(II)-tartrate complex and Cu(II)-human serum albumin complex were detected by UV detection (282 nm) with on-capillary, followed by CL detection (luminol-hydrogen peroxide CL reaction) with end-capillary. We examined the effects of the reaction time and temperature on the UV and CL responses. On the basis of the obtained results we considered the Cu(II)-human serum albumin complex formation processes and its catalytic activity for the CL reaction. The system easily, rapidly, and simultaneously produced useful information concerning the complex formation of Cu(II) and human serum albumin due to the presence of the both detectors.

Flow Counterbalanced Capillary Electrophoresis Using Packed Capillary Columns: Resolution of Enantiomers and Isotopomers

A method with the ability to increase greatly both the resolution and efficiency of a given capillary electrophoretic system is described. This method differs from traditional capillary electrophoresis (CE) in that a counterflow is induced in the direction opposite to the electrokinetic migration of the analyte. This has the effect of extending not only the time the analytes migrate in the electric field but also the effective length and the effective applied voltage of the system. Previous work in our group with flow counterbalanced capillary electrophoresis has utilized an open tube of small inner diameter to reduce peak broadening caused by hydrodynamic flow. Narrow-diameter capillaries (5-10 microm) restricted analysis to fluorescent analytes and laser-induced fluorescence detection. The method described here uses a capillary of much larger inner diameter (75 microm) that has been packed with nonporous silica particles. The packing material reduces the amount of band broadening caused by pressure-induced flow relative to that experienced in an open tube. A larger diameter capillary allows the detection of analytes by UV absorption, not only eliminating the need to tag analytes with fluorescent tags but also allowing for the detection of a much broader range of analytes. The system was evaluated by studying the separations of several enantiomers using only beta-cyclodextrin as the chiral selector. The system was also used to resolve the two naturally occurring isotopes of bromine and to resolve phenylalanine from phenylalanine-d8. Relative to traditional CE, large improvements in resolution and separation efficiency have been achieved with this method.

Capillary electrophoretic system incorporating an UV/CL dual detector

We developed a capillary electrophoretic system incorporating an ultra-violet absorption (UV)/chemiluminescence (CL) dual detector, taking advantage of the CL reaction of luminol-hydrogen peroxide and the batch-type CL detection cell. UV detection was carried out using the on-capillary method while CL detection was performed using the end-capillary method. Examination of isoluminol isothiocyanate (ILITC) as a model sample revealed two main peaks with UV detection and one main peak with CL detection. The first peak in the UV detection data corresponded to the main peak in the CL detection data. We then determined that the ILITC sample included natural ILITC as well as an impurity that had absorption behavior but did not have CL properties and labeling ability. Furthermore, the components of a mixture containing glycine, glycylglycine and glycylglycylglycine, all labeled with ILITC, were well separated and detected using the present system. The present system easily, rapidly, and simultaneously produces useful information due to the presence of both UV and CL detectors.

Separation of low-molecular mass peptides by capillary electrophoresis with the use of alkylamines as dynamic coating agents at low pH

Modification of the silanophilic activity of the inner surface of the capillary wall was studied in a capillary electrophoretic system using alkylamines containing background electrolytes at acid pH. The effect of the following amine additives was investigated: (1) alkyl-α,ω-diamines (1,2-diaminoethane, 1,4-diaminobutane, 1,7-diaminoheptane, spermine), (2) polymeric amines (polyethylenimine, polybrene), (3) cationic amine surfactants (cetrimide, hexamethonium bromide). A seven membered test mixture of peptides (Gly–Pro–Ala, Pro–hPro, Gly–Pro–Arg, Gly–Pro–Gln, Lys–Pro–Gly, Asn–Pro–Gly, His–Pro–Gly) possessing one or more amino groups was used for selectivity evaluation. Under optimised concentration of the amine modifiers the selectivity was always improved (except for polybrene), particularly with the fast moving analytes. The best results were obtained with 1,2-diaminoethane and 1,7-diaminoheptane. On the other hand with slowly moving peaks the best separations were obtained with 1,7-diaminoheptane, hexadecyltrimethylammonium bromide and hexamethonium bromide, i.e. with modifiers possessing large aliphatic domains which are likely to be hydrophobically bonded with the separated solutes. The selectivity improvement with fast moving members of the test mixture can be ascribed to the decrease of the electroosmotic flow, while the improved separation with slowly moving peaks appears to reflect the altered interaction with the hydrophobized capillary wall. As expected the endoosmotic flow was in all cases decreased. The practical applicability of using amine based dynamic modifiers of the capillary wall was demonstrated on a natural peptide mixture (bacterial collagenase hydrolysate of collagen types I and III).