Constant Electroosmotic Flow Research Articles

Nanoparticle gel electrophoresis: Bare charged spheres in polyelectrolyte hydrogels

Nanoparticle gel electrophoresis has recently emerged as an attractive means of separating and characterizing nanoparticles. Consequently, a theory that accounts for electroosmotic flow in the gel, and coupling of the nanoparticle and hydrogel electrostatics and hydrodynamics, is required, particularly for gels in which the mesh size is comparable to or smaller than the particle radii. Here, we present an electrokinetic model for charged, spherical colloidal particles undergoing electrophoresis in charged (polyelectrolyte) hydrogels: the gel-electrophoresis analogue of Henry’s theory for electrophoresis in Newtonian electrolytes. We compare numerically exact solutions of the model with several independent asymptotic approximations, identifying regions in the parameter space where these approximations are accurate or break down. As previously assumed in the literature, Henry’s formula, modified by the addition of a constant electroosmotic flow mobility, is accurate only for nanoparticles that are small compared to the hydrogel mesh size. We derived an exact analytical solution of the full model by judiciously modifying the theory of Allison et al. [15] for uncharged gels, drawing on the superposition methodology of Doane et al. [14] to account for hydrogel charge. This furnishes accurate and economical mobility predictions for the entire parameter space. The present model suggests that nanoparticle size separations (with diameters ≲40nm) are optimal at low ionic strength, with a gel mesh size that is selected according to the particle charging mechanism. For weakly charged particles, optimal size separation is achieved when the Brinkman screening length is matched to the mean particle size.

Capillary Coated with Graphene and Graphene Oxide Sheets as Stationary Phase for Capillary Electrochromatography and Capillary Liquid Chromatography

Graphene oxide (GO) nanosheets were immobilized onto the capillary wall using 3-aminopropyldiethoxymethyl silane as coupling agent. Graphene coated column (G@column) was fabricated by hydrazine reduction of GO modified column. Scanning electron microscopy (SEM) images provided visible evidence of the GO grafted on the capillary wall. Energy dispersive X-ray spectrometry (EDS) indicated the high coverage of the GO on the capillary wall. The G@column exhibited a pH-dependent electroosmotic flow (EOF) from anode to cathode in the pH range of 3-9 while the graphene oxide coated column (GO@column) showed a constant EOF. Both GO@column and G@column were evaluated for open-tubular capillary electrochromatography (OT-CEC). The GO@column was also evaluated for open-tubular capillary liquid chromatography (OT-CLC). Good separation of the tested neutral analytes on the GO@column was achieved on the basis of a typical reversed-phase behavior. On the contrary, G@column showed poor separation performance because of the strong π-π stacking and hydrophobic interactions between graphene and polyaromatic hydrocarbons. The high coverage of GO improved the column phase ratio which makes the GO@column promising for OT-CLC separation. Five of the major known proteins including three glycoisoforms of ovalbumin in chicken egg white were identified in a single run on the GO@column with phosphate buffer (5 mM, pH 7.0) and an applied voltage of 20 kV. The run-to-run, day-to-day, and column-to-column reproducibilities are evaluated by calculating the relative standard deviations (RSDs) of the EOF in OT-CEC and retention time of naphthalene in OT-CLC, respectively. These RSD values were found to be less than 3%.

Linear voltage profiles and flow homogeneity in pressurized planar electrochromatography

Planar electrochromatography (PEC) is an emerging technique for thin-layer chromatography (TLC) where electroosmosis is the driving force for the solvent, not capillary action. This allows for much faster and constant flow rates in turn yielding increased zone capacities and efficiencies. Instrumental designs have changed greatly over the last few years solving many of the initial instrumentation challenges. We have previously shown that low applied pressure (or no applied pressure) PEC instruments do not give linear voltage drops across the separation path length of a TLC plate, which in turn results in non-stable electroosmotic flow (EOF). By the use of our unique reader electrode grid we have the ability to monitor the potential at eight discrete positions throughout the 10-cm separation path length. We now show that high-pressure PEC instruments, most commonly referred to as pressurized planar electrochromatography (PPEC) do show a linear voltage drop and constant EOF. We compare plate equilibration times of PPEC and low-pressure PEC, use of increased field strengths, as well as sample application designs. In addition, we discuss the use of rhodamine B as a visual marker for reproducible migration and calculation of theoretical plates.

Stable Permanently Hydrophilic Protein-Resistant Thin-Film Coatings on Poly(dimethylsiloxane) Substrates by Electrostatic Self-Assembly and Chemical Cross-Linking

Poly(dimethylsiloxane) (PDMS) is a biomaterial that presents serious surface instability characterized by hydrophobicity recovery. Permanently hydrophilic PDMS surfaces were created using electrostatic self-assembly of polyethyleneimine and poly(acrylic acid) on top of a hydrolyzed poly(styrene-alt-maleic anhydride) base layer adsorbed on PDMS. Cross-linking of the polyelectrolyte multilayers (PEMS) by carbodiimide coupling and covalent attachment of poly(ethylene glycol) (PEG) chains to the PEMS produced stable, hydrophilic, protein-resistant coatings, which resisted hydrophobicity recovery in air. Attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy revealed that the thin films had excellent chemical stability and resisted hydrophobicity recovery in air over 77 days of measurement. The spectra also showed a dense coverage for PEG dialdehyde and excellent resistance to protein adsorption from undiluted rat serum. Atomic force microscopy revealed dense coverage with PEG dialdehyde and PEG diamine. Contact angle measurements showed that all films were hydrophilic and that the PEG dialdehyde-topped thin film had a virtually constant contact angle (approximately 20 degrees ) over the five months of the study. Electrokinetic analysis of the coatings in microchannels always exposed to air also gave good protein separation and constant electroosmotic flow during the five months that the measurements were done. We expect that the stable, hydrophilic, protein-resistant thin-film coatings will be useful for many applications that require long-term surface stability.

Microchannel protein separation by electric field gradient focusing

A microchannel device is presented which separates and focuses charged proteins based on electric field gradient focusing. Separation is achieved by setting a constant electroosmotic flow velocity against step changes in electrophoretic velocity. Where these two velocities are balanced for a given analyte, the analyte focuses at that point because it is driven to it from all points within the channel. We demonstrate the separation and focusing of a binary mixture of bovine serum albumin and phycoerythrin. The device is constructed of intersecting microchannels in poly(dimethylsiloxane)(PDMS) inlaid with hollow dialysis fibers. The device uses no exotic chemicals such as antibodies or synthetic ampholytes, but operates instead by purely physical means involving the independent manipulation of electrophoretic and electroosmotic velocities. One important difference between this apparatus and most other devices designed for field-gradient focusing is the injection of current at discrete intersections in the channel rather than continuously along the length of a membrane-bound separation channel.

Application of hydrophobic anion-exchange phases in capillary electrochromatography

Capillary electrochromatography (CEC) requires stationary phases that enable appropriate electroosmotic propel under various conditions. Analyte retention can be controlled through hydrophobic or electrostatic interaction with the packing material. The development and characterization of new strong anion-exchange materials with additional hydrophobic moieties (SAX/C 18 mixed-mode phases) is described. The synthesis was based on polymer encapsulation of porous silica. The phases were systematically characterized by means of elemental analyses, HPLC frontal analyses and CEC experiments. The studies focused on the influence of various parameters (e.g., pH, kind of buffer, capillary wall) on the electroosmotic flow (EOF). Phases with high anion-exchange capacity generated a fast and constant EOF over a wide pH range. Long-time stability of EOF and hydrophobic retention under CEC conditions were demonstrated within the course of 100 consecutive injections. The applicability of the SAX/C 18 phases in appropriate buffer systems is demonstrated for neutral, acidic and basic compounds.

Use of a mixed-mode packing and voltage tuning for peptide mixture separation in pressurized capillary electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer detector.

A mixed-mode (reversed-phase/anion-exchange) stationary phase has been used as the capillary column packing for investigation of the separation of peptide mixtures in pressurized capillary electrochromatography (pCEC). This stationary phase contains both octadecylsilanes and dialkylamines. The amine groups of the stationary phase determine the charge density on the surface of the packing and can produce a strong and constant electroosmotic flow (EOF) at low pH. A comparison was made in terms of the capability of separating tryptic digests between the mixed-mode phase and C18 reversed phase. In addition, the constant EOF enabled the tuning of the retention and the selectivity of the separation by adjusting the mobile phase pH from 2 to 5. Furthermore, the magnitude and the polarity of the electric voltage were demonstrated to greatly influence the elution profiles of the peptides in pCEC. An ion trap storage/reflectron time-of-flight mass spectrometer was used as an on-line detector in these experiments due to its ability to provide rapid and accurate mass detection of the sample components eluting from the separation column.

Separation of weak organic acids and bases by capillary zone electrophoresis in a sulfonated-polymer wall-modified open tubular fused-silica capillary

A sulfonated-polymer wall-modified open tubular fused-silica capillary has a nearly constant electroosmotic flow (EOF) over the pH range studied (pH 2–9) in capillary zone electrophoretic (CZE) applications. The EOF is less than that obtained with a bare fused-silica capillary above pH of ∼6.5 but is greater at lower pH values. Altering buffer pH influences weak acid and base analyte migration time according to analyte p K a value while maintaining a nearly constant EOF. Very weak organic bases can be separated as cations in an acidic buffer prior to neutral analyte migration while weak acid analyte migration order can be manipulated by buffer pH according to the p K a values for the weak acid analytes. EOF can also be reduced by increasing mono- and divalent cation concentration in the buffer. Divalent cations reduce EOF to a much greater extent while hexadecyltrimethylammonium salt causes an EOF reversal that is independent of pH throughout the pH range studied. Migration order and resolution in the CZE separations of organic acids, phenols, pyridines, pyrimidines, purine bases, and nucleosides can be improved by using Mg 2+ as a buffer additive and adjusting buffer pH depending on p K a values of the analytes.

Separation and analysis of cyclodextrins by capillary zone electrophoresis

α-, β-, and γ-Cyclodextrins were separated by capillary zone electrophoresis using indirect UV photometric detection. The cyclodextrins are separated and detected after complexation with benzoic acid or benzoic acid derivatives using a constant electroosmotic flow. Enhanced resolution of mixtures of cyclodextrins was obtained by adjusting the pH of the electrolyte and the concentration of benzoic acid. High pH conditions resulted in short analysis times with a low selectivity, whereas high benzoic acid concentrations resulted in long analysis times with a high selectivity. Detection limits were found at 50 μM for α-, β-, and γ-cyclodextrins, respectively. The method allows the separation and analysis of cyclodextrins in complex samples such as fermentation broth, urine, plasma, and pharmaceutical formulations with minimal sample pretreatment.

Biomedical Applications of On-Line Preconcentration-Capillary Electrophoresis Using an Analyte Concentrator: Investigation of Design Options

Abstract A method to perform on-line sample preconcentration of serum immunoglobulin E by affinity capture is described. Purified anti-IgE antibodies were covalently bound to an analyte concentrator-reaction chamber or cartridge. The immunoglobulins (IgE) were bound to and eluted from the cartridge by the optimum dissociating buffer system, and the eluent(s) were then subjected to capillary electrophoresis. The first design used was a 5 mm solid-phase cartridge fabricated by assembling a bundle of multiple microcapillaries in which a monoclonal antibody directed against IgE was covalently bound to the surface of every microcapillary. The whole assembly was connected, through sleeve connectors, to the capillary column for affinity capillary electrophoresis. The second design used consisted of an analyte concentrator-reaction chamber that was fabricated from a solid rod of glass. Several small diameter passages or through holes containing a similar surface area was tested for the same experiments and performance as described above. A major advantage of these designs, over previously described designs, is the absence of frits and beads. The previously reported designs consisted of derivatized beads confined to the concentrator cartridge by a frit at each end. After limited usage of the cartridge, the beads tends to pack at the outer frit. This lead to restricted flow through the concentrator chamber and ultimately clogging of the system. The designs reported here allows for a constant electroosmotic flow, superior reproducibility of the electropherograms, a reduced possibility of blocking the microcapillaries, and increase number of usages of the cartridge. The use of this novel analyte concentrator design for the determination of immunoglobulins in biological fluids is demonstrated by capillary electrophoresis of IgE in serum. The general utility of this technique for a variety of biomedical applications is discussed.

On the measurements of electrophoretic mobilities by means of capillary isotachophoresis at a constant voltage.

A method for measuring electrophoretic mobilities by means of isotachophoresis (ITP) at a constant voltage as described by H. Carchon and E. Eggermont (Electrophoresis, 1982, 3, 263-274) is analyzed. An error made in this work, disregarding the pH shift arising at the initial discontinuity on the leader-terminator boundary, has been corrected. This method has been carefully studied and generalized for the presence of constant electroosmotic flow in a capillary. The limits of its applicability and the diffusionless ITP theory in general are discussed. A detailed study of the evolution of initial discontinuity (stationary boundary) showed some anomalies not reported previously, particularly non-monotonic concentration profiles in the vicinity of stationary boundaries. Moreover, in some cases, diffusion effects and the contribution of H+ ions can also strongly influence the behavior of moving boundaries. Computer modelling (confirmed by experimental data) showed that these effects could lead to the decay of the ITP train, despite the fact that the steady state diffusionless ITP theory predicts its stability.

Self-regulating dynamic control of electroosmotic flow in capillary electrophoresis

Capillary electrophoresis (CE), primarily because of the impressive separation power and the compatibility with large biomolecules, has established itself as an important separation tool over the past several years. One of the major barriers limiting the broad, routine application of the technique is the validation of the reliability of migration times and the quantitative accuracy when dealing with real samples. In particular, sample matrices can alter the nature of the surface of the inner wall of the capillary, leading to irreproducible results. Even when the temperature is constant and when adsorption of the analytes onto the capillary wall can be neglected, which is the case of pure zone electrophoresis (CZE), the [zeta] potential of the wall can still be altered by the injected ions. In this report, the authors demonstrate the utility of a concept for maintaining a constant electroosmotic flow rate for sample ions in CZE, independent of the changing sample matrix composition. Such an interactive control of electroosmotic flow has the advantage that the instantaneous flow rate need not be monitored. Once the stationary front is established, no further external intervention is needed. 23 refs., 2 tabs.

Capillary zone electrophoresis of biological substances with fused silica capillaries having zero or constant electroosmotic flow.

A series of capillary surface modifications entailing multilayered coatings were introduced and evaluated in capillary zone electrophoresis of biological substances, e.g., proteins, peptides, oligosaccharides and nucleotides. In one set of surface modifications, large molecular weight hydroxypropyl cellulose afforded "zero" flow capillaries, which were used as precursors for developing anodal flow capillaries. When "zero" flow capillaries were further functionalized with a charge polyethyleneimine layer to which a top polyether layer was covalently attached, the resulting anodal electroosmotic flow was relatively weak due to the high viscosity of the coated wall imparted by the hydroxypropyl cellulose layer. Capillaries with relatively strong and constant anodal electroosmotic flow were best achieved when the inner capillary surface was first chemically derivatized with methylated (i.e., quaternarized) polyethyleneimine hydroxyethylated. This hydroxylated and permanently charged polymeric coating yielded constant anodal flow regardless of electrolyte pH. The hydroxyl groups of the charged polymeric coating permitted the covalent attachment of polyether chains, which minimized electrostatic interaction between the positive charges of the polymeric layer and oppositely charged biopolymers. Under these conditions, rapid transport of acidic biopolymers past the detection point in the separation capillary could be achieved, and relatively high plate counts were obtained.