Variations In Electroosmotic Flow Research Articles

Variations in Electroosmotic Flow Outside Glass Nanopores with Species of Monovalent Cation

Nanopores are an important technology in molecular sensing systems. Their behaviour is strongly affected by flow characteristics that are not well understood. Utilising a novel 3 dimensional combination optical tweezers and capillary nanopore measurement system, we have measured flow behaviour outside conical glass nanopores as a function of species of group 1 cation. We present a full 3 dimensional flow field about the nanopore. We use this 3 dimensional measurement system to find the flow force as a function of distance from the pore, allowing us to determine the momentum flux for each salt condition. We find strong salt dependence on the strength of momentum flux from the nanopore. Additionally we find previously reported voltage dependent flow reversal to be dictated by salt species. For a subset of species we observe EOF in the expected direction as predicted by classical electrokinetic theory, while for others the direction of the flow is reversed. We present a model explaining the observed behaviour. These data and corresponding model are key to understanding ion transport, fluid flow and translocation dynamics in voltage driven nanopore systems.

Continuous Redirection and Separation of Microbeads by Faradaic Ion Concentration Polarization

AbstractHerein we report that faradaic ion concentration polarization (fICP) can be used to sort and separate micron‐scale objects within a bifurcated microelectrochemical device. In fICP, a bipolar electrode (BPE) is activated by a driving voltage, which results in formation of an ion depletion zone (IDZ) and an ion enrichment zone (IEZ). By judicious control of the placement of the BPE and control of the driving voltage, the IDZ and IEZ can be manipulated in a way that directs charged microbeads into a particular channel of the microelectrochemical device. The key findings are threefold. First, ∼100 % microbead redirection and separation may be achieved by fICP. Second, the tracking of microbeads as they traverse the channel length permits visualization of local variations in electroosmotic flow (EOF). Third, variations in solution conductivity in different regions of the channel lead to differences in EOF that exert a significant influence over microbead trajectory.

Range of potential and electrolyte concentration for increase in electroosmotic flow contribution with channel diameter/width in nanochannels

The variation of electroosmotic flow (EOF) contribution to total current with applied potential and electrolyte concentration has been investigated for different values of nanochannel diameter/width. The EOF contribution generally decreases with channel diameter/width and becomes negligible for wide channels. However, there is a range of applied potential and electrolyte concentrations in which EOF contribution increases with channel diameter/width. The nanochannel depletion with applied potential across the nanochannel leads to decrease in the ion drift current as well as EOF current, however, decrease in former is higher than the decrease in latter for a range of applied potential and electrolyte concentration, hence, the EOF contribution increases with channel diameter/width for a specific range of applied potential and electrolyte concentration.

Capillary isoelectric focusing with pH 9.7 cathode for the analysis of gastric biopsies.

Capillary isoelectric focusing tends to suffer from poor reproducibility, particularly for the analysis of complex protein samples from cellular or tissue homogenates. This poor reproducibility appears to be associated with erratic variations in electroosmotic flow. One cause of electroosmotic flow variation is degradation of the capillary coating caused by the extremely basic solution commonly used during mobilization and focusing; this degradation of the capillary coating can be reduced by employing a CAPS mobilization buffer at pH 9. Another cause of variation is protein adsorption to the capillary wall, which causes an increase in electroosmotic flow. The effects of protein adsorption can be reduced by use of surfactants in the buffer and by employing an extremely low sample loading. We report the use of CAPS mobilization buffer in combination with an ultrasensitive laser-induced fluorescence detector for the reproducible analysis of ∼2 ng of protein from a Barrett's esophagus biopsy.

Intrinsic charge ladders of a monoclonal antibody in hydroxypropylcellulose-coated capillaries

Capillary zone electrophoresis (CZE) has been used to resolve the charge heterogeneity of an intact ( approximately 150 kDa) monoclonal IgG antibody (mAb). Although this microheterogeneity can also be observed by isoelectric focusing, CZE allows the net charge of each variant to be measured as a function of pH and other solution conditions. Separation was achieved in both borate and Tris run buffers using capillaries that had been statically coated with hydroxypropylcellulose (HPC). The HPC coating makes inadvertent chromatographic retention of the mAb undetectably small and decreases electroosmotic flow (EOF) to approximately 10(-5) cm(2) V(-1) s(-1), with reasonable stability over dozens of runs under the conditions tested (pH 8.5 and 9.0 for each buffer). We also describe a novel means of measuring small, positive EOF coefficients and larger, negative net mobilities in the same run. This allows determination of accurate electrophoretic mobilities despite variations in EOF. The resolved mAb charge variants (which most likely result from deamidation or partial truncation) constitute what we call an "intrinsic" charge ladder. As with conventional charge ladders formed by deliberate modification of a homogeneous protein, net charge is obtained by extrapolating a plot of electrophoretic mobility versus (assumed) incremental charge difference. At a given pH, the mAb is more negatively charged in borate than in Tris, reflecting specific binding of the B(OH)(4)(-) anion. We also report hydrodynamic radii calculated from the slopes of these plots.

Analysis of neutral surfactants by non-aqueous capillary electrophoresis using an electroosmotic flow reversal

The separation of KM 20, that is in fact a mixture of non-ionic surfactants, was carried out by non-aqueous capillary electrophoresis. This complex mixture resulting from the condensation of ethylene oxide with fatty alcohols does not have chromophoric moieties. So, we analysed it after derivatization by means of 3,5-dinitrobenzoyl chloride. The proposed approach is based both on the formation of complexes with alkaline or ammonium cations in methanol and on the utilisation of a positively charged capillary. From a comparative study on the capillary treatment procedure, we used hexadimethrine bromide as electroosmotic flow reverser in order to obtain both repeatable analyses and good resolutions of the largest KM 20 oligomers. Then, among the five cations used to form complexes with KM 20, we pointed out that ammonium cation led to the best resolutions. Moreover, we evidenced that the counter-ion of this cation had a great influence on resolution because it modified the magnitude of electroosmotic flow. Ion pair formation that is more or less strong between ammonium and its counter-ion was involved in this variation of electroosmotic flow. So, we calculated the association constants for various ammonium salts in methanol. Then, using ammonium chloride as background electrolyte, we optimised the concentration of this salt, in methanol, in order to reach the optimal separation of KM 20 oligomers. Thus, a baseline separation was obtained by using 6 × 10 −2 mol/L NH 4Cl as running electrolyte. In these conditions, we separated, in about 30 min, more than 30 oligomers of KM 20. The distribution of these oligomers that was determined from the optimal separation, appeared consistent with that obtained from HPLC analyses. Indeed, we determined that the mean ethoxylation number was equal to 18 while its real value is equal to 20.

Pherogram normalization in capillary electrophoresis and micellar electrokinetic chromatography analyses in cases of sample matrix-induced migration time shifts

When analyzing bio-matrix samples using capillary electrophoresis (CE) or micellar electrokinetic chromatography (MEKC), unwanted shifts in the time axis are often observed, both between samples and standards and between samples, thus hampering identification. These shifts are caused by either or both of two sample matrix-induced effects: variations in stacking conditions (effective field strength or migration length) and variations in electroosmotic flow. Based on elementary CE principles and provided that any two peaks in the pherograms can be linked, these variations can be separately accounted and quantitatively corrected for, so that perfectly overlapping pherograms of standards and samples can be obtained after normalization. The method was validated using samples of a DNA ladder, separated in a sieving polymer. In addition, a number of data files from CE and MEKC analyses (steroids, opioids, β-blockers, amines, and inorganic anions) previously published by other authors were successfully normalized. A freeware computer programme, CEqualizer, for normalizing ASCII files of detector signals using the method described, is available to the CE community from http://www.ceyork.f2s.com.

Use of mobility ratios to estimate binding constants of ligands to proteins in affinity capillary electrophoresis

This work evaluates the use of mobility ratios ( M) to estimate binding constants of proteins to ligands using affinity capillary electrophoresis (ACE). This concept is demonstrated using two model systems: vancomycin (Van) from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). A plot of change in M (Δ M) over the concentration of ligand [L] versus Δ M yields a more useful representation of the Scatchard plot in capillary electrophoresis (CE) than traditional plots of the change in mobility Δ μ over [L] versus Δ μ in a wide set of circumstances, especially when comparing electropherograms obtained in the presence of substantial variations in electroosmotic flow. Altering the voltage and/or capillary length of the CE system produced only small variations in M, but much larger changes in the more standard measures of migration used by the μ form of analysis. The use of M in the Scatchard analysis offers a new approach to estimating binding constants of ligands to proteins using ACE.

Capillary zone electrophoresis in normal or reverse polarity separation modes for the analysis of hydroxy acid oligomers in neutral phosphate buffer

Capillary zone electrophoresis (CZE) with neutral phosphate buffer as the background electrolyte was used to analyse water-soluble oligomers obtained by polycondensation of racemic lactic acid. Two CZE separation modes were tested. The first mode was based on normal separation (injection at the anodic side) using a fused-silica capillary. Eight peaks were observed within a 60-min migration time range. They were ascribed to dimer and higher water-soluble oligomers. Peaks from dimer to tetramer were split due to sensitivity for the fine structures at the level of the distribution of chiral lactic acid moieties in oligomer chains. The second mode was based on reverse separation (injection at the cathodic side) using a fused-silica capillary modified by adsorption of a polycation on its inner wall. Under these conditions, oligomers were rapidly separated without peak splitting. Considering the forces which are involved in CZE, data were plotted as a function of 1/t scale, according to the equation [signal]=f((−1)k/t) where k=0 and k=1 for normal and reverse separation modes, respectively. Such a plot allowed direct comparison between the various runs after a simple translation along the 1/t axis, regardless of the separation mode and the variation of electroosmotic flow. The second separation mode allowed separation of 3-hydroxybutyric acid and 6-hydroxyhexanoic acid oligomers. For the former series of oligomers, a side reaction generating crotyl bonds was observed due to the high sensitivity of CZE. It was shown that separation was governed by the ratio charge/mass of the oligoesters whatever their structure.

Theoretical aspects on the regulation of electroosmotic flow in capillary electrophoresis by adding charged amphiphiles

The regulation of electroosmotic flow (EOF) in capillary electrophoresis is of great importance in many analytical applications. In this paper, the variation of EOF with the concentration of amphiphile is studied by adding a charged amphiphile to the background electrolyte (BGE) when using a C 8-coated capillary. A theory relating the change in concentration of amphiphile in the BGE to the variation in observed EOF is presented. The basis of the theory is that the adsorbed amphiphile creates an electrostatic surface potential that is calculated from the linearised Poisson-Boltzmann equation. The adsorption isotherm of the amphiphile to the capillary surface is described by an electrostatic surface potential modified linear isotherm. By assuming that the ζ-potential in the Smoluchowski equation is proportional to the electrostatic surface potential, an equation describing the EOF as a function of amphiphile concentration is obtained. The proposed theory is experimentally tested by adding octane sulfonate, tetrabutylammonium or tetrapentylammonium ion to the BGE. It is shown that, in these cases, the proposed theory describes the EOF as a function of amphiphile concentration well and that, as expected, the proportionality constant between the electrostatic surface potential and the ζ-potential is less than one. Some practical aspects and problems that occur with this type of measurement are also discussed.

Determination of binding constants of ligands to proteins by affinity capillary electrophoresis: compensation for electroosmotic flow.

This paper describes the estimation of binding constants (Kb) between carbonic anhydrase B (CAB, EC 4.2.1.1, from bovine erythrocytes) and charged benzenesulfonamides by affinity capillary electrophoresis (ACE) under conditions in which the migration time is affected by changes in electroosmotic flow and by nonspecific interactions accompanying changes in the concentration of ligand. Comparisons of values of migration times of the protein of interest, and of "noninteracting" marker proteins, with those of a neutral internal standard provide the basis for corrections for variable electroosmotic flow; these corrections make possible the estimation of Kb and its uncertainty even in the presence of substantial variations in electroosmotic flow.

Improvement of reproducibility in capillary zone electrophoresis by sequential application of high voltage and pressure

In capillary zone electrophoresis (CZE) problems arise concerning the reproducibility of the measurements. This is due to variations in electroosmotic flow and analyte velocities. The discontinuous use of high voltage for the separation of analytes and the pressure for the transport to the detector offers the possibility to suppress the main causes for these phenomena. The procedure is demonstrated for the separation of 4 phenol derivatives. An increase in reproducibility is found for standard solutions from about 3% to 1%.

Evaluation of surface‐bonded polyethylene glycol and polyethylene imine in capillary electrophoresis

AbstractA procedure for bonding polyethylene glycol (PEG) and polyethylene imine (PEI) onto the capillary surface through a highly crosslinked polysiloxane layer containing epoxy groups has been developed. Systematic variation of the coatings can be used to (a) allow separations over a pH range of 3 to 9, (b) provide for adjustment of electro‐osmotic flow, and (c) reduce the adsorption of peptides and proteins. The variation of electroosmotic flow on these coated columns at different operational conditions can be explained using electrical double layer theory. Separations of substituted aromatic compounds and peptides on these coated columns were much improved over similar separations on untreated columns. The results indicate that PEI coated columns are suitable for the separation of basic proteins and PEG coated columns are suitable for acidic proteins. Separations of simple oligonucleotides which differ by only one base were also achieved.