Field-amplified sample injection combined with water removal by electroosmotic flow pump in acidic buffer for analysis of phenoxy acid herbicides by capillary electrophoresis

A procedure that combines two common stacking techniques, field-amplified sample injection and water removal, with an electroosmotic flow pump, is used to separate phenoxy acid herbicides by capillary zone electrophoresis. Before sample loading, a long plug of water was hydrodynamically injected into the capillary both to serve as the medium to permit a high electric field strength and to contain sample anions. Because of this long length of water, the number of ions injected into the capillary was greatly increased. Electrokinetic injection at reversed voltage was then used for introducing negatively charged ions from the diluted sample into the column. The water was removed from the capillary using the electroosmotic flow (EOF) pump when the EOF of the background electrolyte was suppressed. This method afforded a sensitivity enhancement of greater than 3,000 times. Combined with solid-phase extraction, detection limits for the phenoxy acid herbicides as low as 0.01 ng/mL could be achieved.     

Electroosmotic and pressure-driven flow in open and packed capillaries: velocity distributions and fluid dispersion

The flow field dynamics in open and packed segments of capillary columns has been studied by a direct motion encoding of the fluid molecules using pulsed magnetic field gradient nuclear magnetic resonance. This noninvasive method operates within a time window that allows a quantitative discrimination of electroosmotic against pressure-driven flow behavior. The inherent axial fluid flow field dispersion and characteristic length scales of either transport mode are addressed, and the results demonstrate a significant performance advantage of an electrokinetically driven mobile phase in both open-tubular and packed-bed geometries. In contrast to the parabolic velocity profile and its impact on axial dispersion characterizing laminar flow through an open cylindrical capillary, a pluglike velocity distribution of the electroosmotic flow field is revealed in capillary electrophoresis. Here, the variance of the radially averaged, axial displacement probability distributions is quantitatively explained by longitudinal molecular diffusion at the actual buffer temperature, while for Poiseuille flow, the preasymptotic regime to Taylor-Aris dispersion can be shown. Compared to creeping laminar flow through a packed bed, the increased efficiency observed in capillary electrochromatography is related to the superior characteristics of the electroosmotic flow profile over any length scale in the interstitial pore space and to the origin, spatial dimension, and hydrodynamics of the stagnant fluid on the support particles' external surface. Using the Knox equation to analyze the axial plate height data, an eddy dispersion term smaller by a factor of almost 2.5 than in capillary high-performance liquid chromatography is revealed for the electroosmotic flow field in the same column.     

Electrokinetic injection for stacking neutral analytes in capillary and microchip electrophoresis

An on-column mechanism for electrokinetically injecting long sample plugs with simultaneous stacking of neutral analytes in capillary electrokinetic chromatography is presented. On-column stacking methods allow for the direct injection of long sample plugs into the capillary, with narrowing of the analyte peak width to allow for an increase in the detected signal. Low-pressure injections (approximately 50 mbar) are commonly used to introduce sample plugs containing neutral analytes. We demonstrate that injection can be accomplished by applying an electric field from the sample vial directly into the capillary, with neutral analytes injected by electroosmotic flow at up to 1 order of magnitude faster than the corresponding pressure injections. Since stacking occurs simultaneously with electrokinetic injection, stacking is initiated at the capillary inlet, resulting in an increased length of capillary remaining for separation. Reproducibility obtained for peak height and peak area with electroosmotic flow injection is comparable to that obtained with the pressure injection mode, while reproducibility of analysis time is markedly improved. Electrokinetic stacking of neutral analytes utilizing electroosmotic flow is demonstrated with discontinuous (high conductivity, high mobility) as well as continuous (equal conductivity, equal mobility) sample electrolytes. Injecting neutral analytes by electroosmotic flow affords a 10-fold or greater decrease in analysis times when capillaries of 50-microm i.d. or smaller are used. This stacking method should be exportable to dynamic pH junction stacking and electrokinetic chromatography with capillary arrays. Equations describing this electrokinetic injection mode are introduced and stacking of a neutral analyte on a microchip by electrokinetic injection using a simple cross-T channel configuration is demonstrated.     

Effect of direct control of electroosmosis on peptide and protein separations in capillary electrophoresis.

The separations of peptide and protein mixtures in capillary zone electrophoresis (CZE) at various solution conditions were studied with the direct control of electroosmosis. The zeta potential at the aqueous/capillary interface and the resulted electroosmosis in the presence of an electric field were directly controlled by using an additional electric field applied from outside of the capillary. The controlled electroosmotic flow affected the migration time and zone resolution of peptide and protein mixtures. The changes in the magnitude and polarity of the zeta potential caused the various degrees of peptide and protein adsorption onto the capillary through the electrostatic interactions. The separation efficiencies of peptide and protein mixtures were enhanced due to the reduction in peptide and protein adsorption at the capillary wall. The direct manipulations of the separation efficiency and resolution of peptide and protein mixtures in CZE were demonstrated by simply controlling the zeta potential and the electroosmotic flow with the application of an external electric field.     

Induced pressure pumping in polymer microchannels via field-effect flow control

Microfluidic field-effect flow control (FEFC) modifies the zeta potential of electroosmotic flow using a transverse electric field applied through the microchannel wall. Previously demonstrated in silicon-based and glass microsystems, FEFC is presented here as an elegant method for flow control in polymer-based microfluidics with a simple and low-cost fabrication process. In addition to direct FEFC flow modulation, independent transverse electric fields in connected microchannels are demonstrated to produce a differential pumping rate between the microchannels. The different electroosmotic pumping rates formed by local zeta potential control induce an internal pressure at the microchannel intersection, resulting in hydrodynamic pumping through an interconnecting field-free microchannel. Modulation of the voltages applied to the gate electrodes adjusts the magnitude and direction of the bidirectional pressure pumping, with fine resolution volume flow rates from -2 to 2 nL/min in the field-free microchannel demonstrated.     

Electroosmotic capillary flow with nonuniform zeta potential

The present work is an analytical and experimental study of electroosmotic flow (EOF) in cylindrical capillaries with nonuniform wall surface charge (zeta-potential) distributions. In particular, this study investigates perturbations of electroosmotic flow in open capillaries that are due to induced pressure gradients resulting from axial variations in the wall zeta-potential. The experimental inquiry focuses on electroosmotic flow under a uniform applied field in capillaries with an EOF-suppressing polymer adsorbed onto various fractions of the total capillary length. This fractional EOF suppression was achieved by coupling capillaries with substantially different zeta-potentials. The resulting flow fields were imaged with a nonintrusive, caged-fluorescence imaging technique. Simple analytical models for the velocity field and rate of sample dispersion in capillaries with axial zeta-potential variations are presented. The resulting induced pressure gradients and the associated band-broadening effects are of particular importance to the performance of chemical and biochemical analysis systems such as capillary electrokinetic chromatography and capillary zone electrophoresis.     

Theoretical description of the influence of external radial fields on the electroosmotic flow in capillary electrophoresis

The effect of applying a radial voltage on the electroosmotic flow in capillary electrophoresis has been studied from a theoretical point of view. Based on Stern's model for the electric double layer on the surface of a fused silica capillary and on the Gouy-Chapman theory for the diffuse layer, equations describing the relation between the electroosmotic mobility and the radial electric field were derived. The thickness of the stagnant solution layer on the surface of the capillary, an important parameter in the calculations, was estimated from the electroosmotic mobility found in high-pH solutions. The theory developed predicts the experimental findings that the effect of the radial field levels off at high applied voltages and that it is smaller when the electroosmotic mobility without radial field is already high. The theoretical results were compared with experimental data taken from the literature. A good quantitative agreement was found.     

Preparation and characterization of monolithic porous capillary columns loaded with chromatographic particles

Using sol-gel technology, a porous glass matrix (xerogel) is formed in a capillary column and acts as a support for a stationary phase of chromatographic particles used in capillary electrochromatography. Preparation of the sol-gel matrix and immobilization of the octadecylsilica (ODS) stationary phase occur in a single step. The presence of the particles in the column greatly reduces matrix cracking caused by internal pressure differentials within the pores of the sol-gel matrix. Good electroosmotic flow is achieved in part because of the inherent negative charge of both the particles and the sol-gel matrix. The performance of these sol-gel/ODS capillary columns was evaluated with a mixture of aromatic and nonaromatic organic compounds. Efficiencies of up to 80?000 plates/m were observed in columns with immobilized 3-μm ODS particles. The efficiency and resolution are enhanced when 3-μm ODS particles are used in place of the 5-μm particles.     

Fabrication and characterization of a fritless microfabricated electroosmotic pump with reduced pH dependence

A fritless electroosmotic pump with reduced pH dependence has been fabricated on a glass microchip and its performance characterized. The chip design consists of two 500-microm channels, one packed with anion exchange beads and the other packed with cation exchange beads, which produce convergent electroosmotic flow streams. The electroosmotically pumped solution flows away from the intersection of the two pumping channels through a field-free channel. This simple design allows for the production of a fritless electroosmotic pump and easy replacement of the ion exchange beads whose charged surfaces generate the flow. The pump was found to produce volumetric flow rates of up to 2 microL/min for an applied voltage of 3 kV at a pH of 6.8. Moreover, the electroosmotic pump can generate high flow rates over an extended pH range of at least 2-12, a significant advantage over previously fabricated electroosmotic pumps, which typically have a more limited range in which they can achieve high flow rates.     

Fast, accurate mobility determination method for capillary electrophoresis

A new method for accurately determining effective mobilities and electroosmotic flow rates for capillary electrophoresis is described. The proposed method can be performed using most commercial capillary electrophoresis instruments. Problems inherent to the conventional mobility determination method such as a variable electroosmotic flow during the run and migration through unthermostated regions of the capillary are eliminated with the use of the proposed method. In addition, very low effective mobilities and electroosmotic flow rates can be measured quickly and reproducibly. Also, cation mobilities and anion mobilities can be measured in a single run regardless of the magnitude or direction of the electroosmotic flow.     

Dynamic aspects of electroosmotic flow in a cylindrical microcapillary

Dynamic aspects of the electroosmotic flow (EOF) in a cylindrical capillary are analysed. An analytical solution for electrostatic potential of the double layer has been derived by solving the complete Poisson–Boltzmann equation for arbitrary zeta-potentials under an analytical scheme. Transient EOF field in response to the application of time dependent electric field is obtained analytically by using Green’s function method. Specifically, sinusoidally alternating (AC) electric fields are used to study the effect of frequency-dependent oscillation on the EOF. Limiting cases of zero frequency and pulsed electric field are also discussed.     

Speciation of mercury by hydrostatically modified electroosmotic flow capillary electrophoresis coupled with volatile species generation atomic fluorescence spectrometry

A novel method for speciation analysis of mercury was developed by on-line hyphenating capillary electrophoresis (CE) with atomic fluorescence spectrometry (AFS). The four mercury species of inorganic mercury Hg(II), methymercury MeHg(I), ethylmercury EtHg(I), and phenylmercury PhHg(I) were separated as mercury-cysteine complexes by CE in a 50-cm x 100-microm-i.d. fused-silica capillary at 15 kV and using a mixture of 100 mmol L(-1) of boric acid and 12% v/v methanol (pH 9.1) as electrolyte. A novel technique, hydrostatically modified electroosmotic flow (HSMEOF) in which the electroosmotic flow (EOF) was modified by applying hydrostatical pressure opposite to the direction of EOF was used to improve resolution. A volatile species generation technique was used to convert the mercury species into their respective volatile species. A newly developed CE-AFS interface was employed to provide an electrical connection for stable electrophoretic separations and to allow on-line volatile species formation. The generated volatile species were on-line detected with AFS. The precisions (RSD, n = 5) were in the range of 1.9-2.5% for migration time, 1.8-6.3% for peak area response, and 2.3-6.1% for peak height response for the four mercury species. The detection limits ranged from 6.8 to 16.5 microg L(-1) (as Hg). The recoveries of the four mercury species in the water samples were in the range of 86.6-111%. The developed technique was successfully applied to speciation analysis of mercury in a certified reference material (DORM-2, dogfish muscle).     

On-line concentration of proteins and peptides in capillary zone electrophoresis with an etched porous joint

A novel approach for on-line concentration of proteins and peptides in capillary electrophoresis (CE) is presented. A short section (approximately 0.5-1 cm) along the capillary was etched with HF. The etched section became a porous membrane that allowed electrical conductivity but prevented passage of the analyte ions. The capillary was isolated into two parts by the etched section. Thus, we were able to use three buffer vials to perform CE experiments in the capillary by applying high voltages independently. Concentration and separation were performed at the two respective regions. When high voltage was applied to the concentration capillary (between the inlet end and the etched section), proteins and peptides were concentrated at the etched portion, because the porous capillary wall allowed only small buffer ions to pass through and there was no electric field gradient beyond that point. After focusing, the narrow sample zone was introduced into the separation capillary (between the etched section and the outlet end) by hydrodynamic flow or by electroosmotic flow. Finally, conventional CE was carried out for separation of the analytes. Several different concentration schemes for proteins and peptides were successfully demonstrated by using this new approach.     

Determination of accurate electroosmotic mobility and analyte effective mobility values in the presence of charged interacting agents in capillary electrophoresis

A method for determining the accurate effective mobility value of an analyte in the presence of a charged interacting agent, such as a charged cyclodextrin, a micellar agent, a protein, or a DNA fragment that binds the traditional electroosmotic flow markers, is presented. Part of the capillary is filled with the charged interacting agent-containing background electrolyte; the other part is filled with the charged interacting agent-free background electrolyte. The analyte band is placed in the charged interacting agent-containing background electrolyte zone, while a neutral marker (electroosmotic flow marker) is placed in the adjacent charged interacting agent-free background electrolyte zone. The initial, preelectrophoresis distance between the analyte band and the neutral marker band is determined by pressure mobilizing the bands past the detector and recording the detector trace. Subsequently, by applying reverse pressure, the bands are moved back into the first portion of the capillary and a brief electrophoretic separation is carried out. Then, the bands are pressure mobilized again past the detector to obtain their final, postelectrophoresis distance. If (i) the neutral marker does not come into contact with the charged interacting agent and (ii) the analyte does not migrate out of the homogeneous portion of the charged interacting agent zone, the accurate effective electrophoretic migration distance of the analyte, corrected for bulk flow transport, can be determined. The actual electric field strengths in the different zones of the heterogeneously filled capillary can be calculated from the integral of the electrophoretic current and the conductivity of the charged interacting agent-containing background electrolyte measured in a separate experiment. Once the effective mobility of an analyte in the charged resolving agent-containing background electrolyte is determined by this method, the analyte becomes a mobility reference probe for that background electrolyte and can be used to calculate the bulk flow mobility in subsequent conventional CE separations utilizing the same charged interacting agent. The new method can also be used to probe the interactions of the charged interacting agents and the wall of the capillary.     

Separation of tissue proteins of human lung carcinomas by partial-filling capillary electrophoresis

Tissue proteins from human squamous cell lung carcinomas (SQCLC) and small cell lung carcinomas (SCLC) were separated in 0.01% hydroxypylmethyl cellulose (HPMC) linear polymer sieving solutions in the inlet portion of the capillary and next to the outlet of the capillary, followed by capillary zone electrophoresis (CZE) in 40 mM phosphate buffer, pH 2.5. A proper HPMC concentration could cause a molecular sieving effect through the formation of an entangled polymer network. The migration time of the analyte in this matrix depended on the size and electrophoretic mobility of the analyte, the mesh size, and the electric field strength. In the CZE separation, the electroosmotic flow and the charge-to-size ratio of the analyte were important parameters. HPMC concentration and zone length were examined to optimize the separation. Applying this partial-filling technique to the separation of water-soluble proteins from human lung tissues, we found a greatly improved resolution and increased peak intensity. The capillary electrophoresis patterns of normal, SQCLC, and SCLC were obtained and compared for their molecular classifications.     

Monitoring electroosmotic flow by periodic photobleaching of a dilute, neutral fluorophore

Electroosmotic flow has been monitored in a capillary using a method based on periodic photobleaching of a neutral, fluorescent buffer additive. Rhodamine B was determined to be neutral between pH 6.0 and 10.8 and was added to the running buffer at a concentration of 400 nM. Rhodamine B was photobleached by opening a shutter under computer control for 250 ms every 5.00 s, to expose the dye to a laser beam and create a photobleached zone. The time was measured for the photobleached zone to migrate 6.13 mm to a downstream laser-induced fluorescence detector, to determine the rate of electroosmotic flow in the entire capillary. The flow rate was sampled every 5.00 s, and the precision of the flow measurements was 0.7% or better. Three fluorescent compounds were separated and detected by capillary electrophoresis with laser-induced fluorescence detection, while simultaneously monitoring the electroosmotic flow rate.     

Laser modification of preformed polymer microchannels: application to reduce band broadening around turns subject to electrokinetic flow

A pulsed UV excimer laser (KrF, 248 nm) was used to modify the surface charge on the side wall of hot-embossed microchannels fabricated in a poly(methyl methacrylate) substrate. Subablation level fluences, less than 2,385 mJ/cm2, were used to prevent any changes in the physical morphology of the surface. It is shown that the electroosmotic mobility, induced by an electric field applied along the length of the channel, increases by an average of 4% in the regions that have been exposed to UV laser pulses compared to nonexposed regions. Furthermore, application of UV modification to electroosmotic flow around a 90 degrees turn results in a decrease in band broadening, as measured by the average decrease in the plate height of 40% compared to flow around a nonmodified turn. The ability to modify the surface charge on specific surfaces within a preformed plastic microchannel allows for fine control, adjustment, and modulation of the electroosmotic flow without using wall coatings or changing the geometry of the channel to achieve the desired flow profile.     

Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions

A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field.     

Open tubular capillary electrochromatography: technique for oxidation and interaction studies on human low-density lipoproteins

A novel, open tubular capillary electrochromatographic method was developed for the in vitro oxidation of low-density lipoprotein (LDL) particles. Low-density lipoprotein particles with molar mass of approximately 2.5 MDa yielded a stable stationary phase at temperatures 25 and 37 degrees C and at pH values from 3.2 to 7.4. The quality of the coatings was not influenced by variations in the LDL concentration in the coating solutions (within the range of 2-0.015 mg/mL) with the coating procedure used in the study. Radiolabeled LDL stationary phases and scanning electron microscopy, employed to shed light on the location and coating density of LDL particles on the inner surface of the capillary wall, confirmed the presence of an LDL monolayer and almost 100% coating efficiency (99 +/- 8%). In addition, the radioactivity measurements allowed estimation of the amount of LDL present in a single capillary coating. Capillaries coated with human LDL particles were submitted to different oxidative conditions by changing the concentration of the oxidant (CuSO4), oxidation time, pH value, and temperature. The oxidation procedure was followed with electroosmotic flow mobility, which served as an indicator of the increase in total negative charges of LDL coatings, and by asymmetrical field flow fractionation, which measured the changes in size of the lipoprotein particles. The results indicated that oxidation of LDL was progressing with increasing time, temperature, and concentration of the oxidant as expected. The oxidation process was faster around neutral pH values (pH 6.5-7.4) and inhibited at acidic pH values (pH 5.5 and lower).     

Approaches for the simultaneous extraction of tetrabromobisphenol A, tetrachlorobisphenol A, and related phenolic compounds from sewage sludge and sediment samples based on matrix solid-phase dispersion

A procedure based on matrix solid-phase dispersion (MSPD) for sample preparation in the analysis of some bromophenols and halogenated bisphenols in sediments and sludges has been developed. For the first time ever, MSPD was applied for the extraction of organic contaminants from sediment and sewage sludge samples. The influence of experimental conditions on the yield of the extraction process and on the efficiency of the built-in cleanup step was thoroughly evaluated. Analysis of the extracts was performed by nonaqueous capillary electrophoresis coupled with photodiode array ultraviolet detection, using large-volume sample stacking injection based on the electroosmotic flow pump as an on-column preconcentration technique. The method was applied to the analysis of real sludges from urban sewage treatment plants, as well as river and marine sediment samples.