Prediction of the location of stationary steady-state zone positions in counterflow isotachophoresis performed under constant voltage in a vortex-stabilized annular column

A theoretical model is presented and an analytical expression derived to predict the locations of stationary steady-state zone positions in ITP as a function of current for a straight channel under a constant applied voltage. Stationary zones may form in the presence of a countercurrent flow whose average velocity falls between that of a pure leader zone and of a pure trailer zone. A comparison of model predictions with experimental data from an anionic system shows that the model is able to predict the location of protein zones with reasonable accuracy once the ITP stack has formed. This result implies that an ITP stack can be precisely directed by the operator to specific positions in a channel whence portions of the stack can be removed or redirected for further processing or analysis.     

Separation of proteins by zone electrophoresis on-line coupled with isotachophoresis on a column-coupling chip with conductivity detection

This feasibility study deals with the separations of proteins by an on-line combination of zone electrophoresis (ZE) with isotachophoresis (ITP) on a poly(methylmethacrylate) column-coupling (CC) chip with integrated conductivity detection. ITP and ZE provided specific analytical functions while performing the cationic mode of the separation. ITP served, mainly, for concentrations of proteins and its concentrating power was beneficial in reaching a low dispersion transfer (injection) of the proteinous constituents, loaded on the CC chip in a 960 nL volume, into the ZE separation stage. This was complemented by an electrophoretically driven removal of the sample constituents migrating in front of the focused proteins from the separation system before the ZE separation. On the other hand, ZE served as a final separation (destacking) method and it was used under the separating conditions providing the resolutions and sensitive conductivity detections of the test proteins. In this way, ITP and ZE cooperatively contributed to low- or sub-microg/mL concentration detectabilities of proteins and their quantitations at 1-5 microg/mL concentrations. However, a full benefit in concentration detectabilities of proteins, expected from the use of the ITP-ZE combination, was not reached in this work. Small adsorption losses of proteins and detection disturbances in the ZE stage of separation, very likely due to trace constituents concentrated by ITP, appear to set limits in the detection of proteins in our experiments. The ITP-ZE separations were carried out in a hydrodynamically closed separation compartment of the chip with suppressed hydrodynamic and electroosmotic flows of the electrolyte solutions. Such transport conditions, minimizing fluctuations of the migration velocities of the separated constituents, undoubtedly contributed to highly reproducible migrations of the separated proteins (fluctuations of the migration time of a particular protein were typically 0.5% RSD in repeated ITP-ZE runs).     

10,000-fold concentration increase in proteins in a cascade microchip using anionic ITP by a 3-D numerical simulation with experimental results

This paper describes both the experimental application and 3-D numerical simulation of isotachophoresis (ITP) in a 3.2 cm long "cascade" poly(methyl methacrylate) (PMMA) microfluidic chip. The microchip includes 10 × reductions in both the width and depth of the microchannel, which decreases the overall cross-sectional area by a factor of 100 between the inlet (cathode) and outlet (anode). A 3-D numerical simulation of ITP is outlined and is a first example of an ITP simulation in three dimensions. The 3-D numerical simulation uses COMSOL Multiphysics v4.0a to concentrate two generic proteins and monitor protein migration through the microchannel. In performing an ITP simulation on this microchip platform, we observe an increase in concentration by over a factor of more than 10,000 due to the combination of ITP stacking and the reduction in cross-sectional area. Two fluorescent proteins, green fluorescent protein and R-phycoerythrin, were used to experimentally visualize ITP through the fabricated microfluidic chip. The initial concentration of each protein in the sample was 1.995 μg/mL and, after preconcentration by ITP, the final concentrations of the two fluorescent proteins were 32.57 ± 3.63 and 22.81 ± 4.61 mg/mL, respectively. Thus, experimentally the two fluorescent proteins were concentrated by over a factor of 10,000 and show good qualitative agreement with our simulation results.     

Direct determination of valproate in serum by zone electrophoresis–isotachophoresis on a column‐coupling chip

A feasibility study was performed using zone electrophoresis (ZE) coupled on‐line with isotachophoresis (ITP) sample pretreatment on a poly(methyl methacrylate) column‐coupling chip with integrated conductivity detection for direct determination of drugs in serum. Valproic acid (an antiepileptic drug), having a therapeutic range of 0.35–0.69 mmol/L (50–100 mg/L), was a test analyte while reference serum samples served as proteinaceous matrices. ITP provided in the ITP‐ZE combination a multitask sample pretreatment: (1) separation of the analyte from the serum matrix and its concentration into a narrow ITP band, (2) removal of the matrix constituents migrating in the ITP stack from the separation compartment of the chip, (3) ITP stacking of the drug released on a continuous electrophoretic decomposition of the drug‐protein complex. A high sample loadability, closely linked with the use of ITP in the first separation stage, made it possible to inject diluted serum samples with the aid of a 0.95 μL sample channel of the chip. Consequently, a 1–2 μmol/L concentration limit of quantitation for valproate from the response of the conductivity detector in the ZE stage of the combination was reached. The drug could be reliably determined in less than 10 minutes also in instances when its concentration in serum was below the lower value of the therapeutic range. 90–94% recoveries of valproate from serum samples were obtained in its direct ITP‐ZE determination when the filtration of the diluted serum (a 0.45 μm pore size filter) was the only pre‐column sample handling operation. No disturbances attributable to the precipitation of proteins from the loaded samples in the chip channels were detected.     

Simulation of electrophoretic separations by the flux-corrected transport method

Electrophoretic separations at typical experimental electric field strengths have been simulated by applying the flux-corrected transport (FCT) finite difference method to the transient, one-dimensional electrophoresis model. The performance of FCT on simulations of zone electrophoresis (ZE), isotachophoresis (ITP), and isoelectric focusing (IEF) has been evaluated. An FCT algorithm, with a three-point, central spatial discretization, yields numerical solutions without numerical oscillations or spurious peaks, which have plagued previously-published second-order solutions to benchmark ZE and ITP problems. Moreover, the FCT technique captures sharp zone boundaries and IEF peaks more accurately than previously-published, first-order upwind schemes.     

Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments

Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection.     

High-resolution modeling of isotachophoresis and zone electrophoresis

The space-time conservation element and solution element (CESE) method is applied to simulate the ITP and zone electrophoresis (ZE) separation phenomena. The CESE method expresses the governing equation in the integral form of the conservation law, and has a second-order accuracy in both space and time. The current results show that the CESE solutions for the ITP and ZE phenomena are more accurate than those obtained using conventional numerical schemes, which are characterized by serious numerical diffusion and oscillation. Furthermore, the CESE method suppresses the numerical oscillations or peaks observed in the results obtained using traditional second-order finite difference schemes. Finally, the results reveal that the CESE method accurately models the sharp boundaries between adjacent ITP samples under steady-state conditions. Overall, the results presented in this study demonstrate the numerical accuracy of the CESE method and confirm its applicability to the modeling of a range of electrophoretic phenomena.     

Isotachophoresis with counterflow in an open capillary: Computer simulation and experimental validation

The purpose of applying a countercurrent flow to isotachophoretic migration is to increase the effective separation channel length during ITP. However, severe dispersion induced by applying a counterflow can be detrimental to ITP. This paper uses numerical simulations in a 2D axisymmetric domain to investigate the dispersion caused by a parabolic counterflow in open‐capillary ITP. Counterflow in these simulations was generated by applying a back pressure to stop the isotachophoretic stack, i.e., forming stationary ITP zones. It is found that dispersion is strongly related to analyte molecular diffusivity: R‐phycoerythrin, due to its small diffusivity, showed ～20‐fold increase in zone width in stationary counterflow ITP, compared to ITP in the absence of counterflow, while fluorescein only had ～10% increase in zone width under similar operating conditions. Applying the Taylor–Aris dispersion formula in counterflow ITP simulations provided only a rough estimate of the dispersion, e.g., overestimation of analyte zone widths. Experiments on counterflow ITP were conducted in a silica capillary that was covalently and dynamically coated to exclude electroosmosis effect. The counterflow was generated by adjusting the relative height of the fluids in the two reservoirs at the capillary ends. Good qualitative agreement between simulations and experiments was found.     

Sample pre-concentration by isotachophoresis in microfluidic devices

We have designed microfluidic devices with the aim of coupling isotachophoresis (ITP) with zone electrophoresis (ZE) as a method to increase the concentration limit of detection in microfluidic devices. We used plastic multi-channel chips, designed with long sample injection channel segments, to increase the sample loading. The chip was designed to allow stacking of the sample into a narrow band by discontinuous ITP buffers and subsequent separation in the ZE mode. In the ITP-ZE mode, with a 2-cm long sample injection plug, sensitivity was increased by 400-fold over chip ZE and we found that the separation performance after the ITP stacking was comparable to that of regular chip ZE. We report sub-picomolar limits of detection of fluorescently labeled ACLARA eTag reporter molecules electrokinetically injected from cell lysate sample matrixes containing moderate salt concentrations. We evaluated sample injections from buffers with varied ionic strengths and found that efficient stacking and separations were obtained in both low and high conductivity buffers, including physiological buffer with at least 140 mM salt. We applied ITP-ZE to the analysis of a cell surface protease (ADAM 17) which used live intact cells in physiological buffers with detection limits below 10 cells/assay.     

Integrated isotachophoretic preconcentration with zone electrophoresis separation on a quartz microchip for UV detection of flavonoids

A quartz microchip integrated isotachophoretic (ITP) preconcentration with zone electrophoresis (ZE) separation was fabricated using a novel multi-point pressure method featured in normal temperature and lower pressure during bonding process. ITP followed by subsequential ZE of two flavonoids, quercetin and isorhamnetin on the microchip was performed consecutively on the homemade microfluidic workstation with UV detection, resulting in a decreased detectable concentration of 32-fold, compared to the ZE mode only, and their detection limits decreased down to 0.2 microg/mL and 1.2 microg/mL, respectively.     

Determination of free sulfite in wine by zone electrophoresis with isotachophoresis sample pretreatment on a column-coupling chip

This work deals with the determination of free sulfite in wine by zone electrophoresis (ZE) with on-line isotachophoresis (ITP) sample pretreatment on a column-coupling (CC) chip with conductivity detection. A rapid pre-column conversion of sulfite to hydroxymethanesulfonate (HMS), to minimize oxidation losses of the analyte, was included into the developed analytical procedure, while ITP and ZE were responsible for specific analytical tasks in the separations performed on the CC chip. ITP, for example, eliminated the sample matrix from the separation compartment and, at the same time, provided a selective concentration of HMS before its transfer to the ZE stage of the separation. On the other hand, ZE served as a final separation (destacking) method and it was used under the separating conditions favoring a sensitive conductivity detection of HMS. In this way, ITP and ZE cooperatively contributed to a 900 microg/l concentration detectability for sulfite as attained for a 60 nl load of wine (a 15-fold wine dilution and the use of a 0.9 microl sample injection channel of the chip) and, consequently, to the determination of free sulfite when this was present in wine at the concentrations as low as 3 mg/l. The separations were carried out in a closed separation compartment of the chip with suppressed hydrodynamic and electroosmotic flows. Such transport conditions, minimizing fluctuations of the migration velocities of the separated constituents, made a frame for precise migration and quantitation data as achieved for HMS in both the model and wine samples. Ninety percent recoveries, as typically obtained for free sulfite in wine samples, indicate promising potentialities of the present method as far as the accuracies of the provided analytical results are concerned.     

Electrophoretic separations on chips with hydrodynamically closed separation systems

This review focuses on capillary electrophoretic separations performed on capillary electrophoresis chips (CE chips) with hydrodynamically closed separation systems in a context with transport processes (electroosmotic flow (EOF)) and hydrodynamic flow (HDF)) that may accompany the separations in these devices. It also reflects some relevant works dealing with conventional CE operating under such hydrodynamic conditions. The use of zone electrophoresis (ZE), isotachophoresis (ITP) and their on-line combination (ITP-ZE) on the single-column and column-coupling CE chips with the closed separation systems and related problems are key topics of the review. Some attention is paid to sample pretreatment in the separations performed on the CE chips. Here, mainly potentialities of the ITP-ZE combination in trace analysis applications of the miniaturized systems are discussed in a broader extent. Links between the ZE separation and detection provide a frame for the discussion of current status of the detection on the CE chips. Analytical applications illustrate potentialities of the CE chips operating with the closed separation systems (suppressed HDF and EOF) to the determination of small ions present in various matrices by ZE, ITP and ITP-ZE.     

Characterization of voltage degradation in dynamic field gradient focusing

Dynamic field gradient focusing (DFGF) is an equilibrium gradient method that utilizes an electric field gradient to simultaneously separate and concentrate charged analytes based on their individual electrophoretic mobilities. This work describes the use of a 2-D nonlinear, numerical simulation to examine the impact of voltage loss from the electrodes to the separation channel, termed voltage degradation, and distortions in the electric field on the performance of DFGF. One of the design parameters that has a large impact on the degree of voltage degradation is the placement of the electrodes in relation to the separation channel. The simulation shows that a distance of about 3 mm from the electrodes to the separation channel gives the electric field profile with least amount of voltage degradation. The simulation was also used to describe the elution of focused protein peaks. The simulation shows that elution under constant electric field gradient gives better performance than elution through shallowing of the electric field. Qualitative agreement between the numerical simulation and experimental results is shown. The simulation also illustrates that the presence of a defocusing region at the cathodic end of the separation channel causes peak dispersion during elution. The numerical model is then used to design a system that does not suffer from a defocusing region. Peaks eluted under this design experienced no band broadening in our simulations. Preliminary experimental results using the redesigned chamber are shown.     

Electrokinetic analyte transport assay for alpha-fetoprotein immunoassay integrates mixing, reaction and separation on-chip

A rapid and highly sensitive CE immunoassay method integrating mixing, reaction, separation, and detection on-chip is described for the measurement of alpha-fetoprotein (AFP), a liver cancer marker in blood. Antibody-binding reagents, consisting of 245-bp DNA coupled anti-AFP WA1 antibody (DNA-WA1) and HiLyte dye-labeled anti-AFP WA2 antibody (HiLyte-WA2), and AFP-containing sample were filled into adjacent zones of a chip channel defined by the laminar flow lines of the microfluidic device using pressure-driven flow. The channel geometry was thus used to quantitatively aliquot the reagents and sample into the chip. DNA-WA1 was electrokinetically concentrated in the channel and sequentially transported through the AFP-sample zone and HiLyte-WA2 zone by ITP in such a manner that the AFP sandwich immune complex formation took place in the sample and HiLyte-WA2 zones. The sandwich AFP immune complex was then detected by LIF after CGE in a separation channel that was arranged downstream of the reaction channel. AFP was detected within 136 s with a detection sensitivity of 5 pM. The on-chip immunoassay described here, applying ITP concentration, in-channel reaction, and CGE separation, has the potential of providing a rapid and sensitive method for both clinical and research applications.     

A Theoretical Study on the Relationships between Kohlrausch' Regulating Function and Moving Chemical Reaction Boundary

In 1897, Kohlrausch formulated an electrophoretic mathematics model. By using the model conjoined with divergence theorem, electroneutrality condition and some assumptions, for instance constant relative mobility and no diffusion and convection, He derived the so-called beharrliche function,viz.,Kohlrausch' regulating function1. The function shows that, for a steady-state boundary, the sums of the ionic concentrations divided by their mobilities on either side homogeneity solutions of a boundary are the same values and instead constant as a function of time and location. The regulating function is of university importance. It is found the function is of validity for moving boundary electrophoresis, isotachophoresis (ITP), zone electrophoresis(ZE) and isoelectric focusing (IEF),including capillary electrophoresis(CE).     

Inverse cationic ITP for separation of methadone enantiomers with sulfated β‐cyclodextrin as chiral selector

Chiral ITP of the weak base methadone using inverse cationic configurations with H⁺ as leading component and multiple isomer sulfated β‐CD (S‐β‐CD) as leading electrolyte (LE) additive, has been studied utilizing dynamic computer simulation, a calculation model based on steady‐state values of the ITP zones, and capillary ITP. By varying the amount of acidic S‐β‐CD in the LE composed of 3‐morpholino‐2‐hydroxypropanesulfonic acid and the chiral selector, and employing glycylglycine as terminating electrolyte (TE), inverse cationic ITP provides systems in which either both enantiomers, only the enantiomer with weaker complexation, or none of the two enantiomers form cationic ITP zones. For the configuration studied, the data reveal that only S‐methadone migrates isotachophoretically when the S‐β‐CD concentration in the LE is between about 0.484 and 1.113 mM. Under these conditions, R‐methadone migrates zone electrophoretically in the TE. An S‐β‐CD concentration between about 0.070 and 0.484 mM results in both S‐ and R‐methadone forming ITP zones. With >1.113 mM and < about 0.050 mM of S‐β‐CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S‐β‐CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

A method to determine quasi-steady state in constant voltage mode isotachophoresis

Identification of the steady state is very challenging in isotachophoresis (ITP); especially in complex microgeometries, such as dog-leg channels or cross-channel junctions. In this work, an elastic matching method is applied to determine the quasi-steady state in microscale ITP. In the elastic matching method, the similarity between two profiles is calculated by comparing intensity distribution of two images or profiles. To demonstrate this similarity-based analysis technique for ITP, a constant voltage mode ITP model is developed and applied to a five-component ITP system. Hydrochloric acid and caproic acid are used as the leader and terminator, respectively, while histidine is used as the counter-ion. Two sample components, acetic acid and benzoic acid, are separated under the action of an applied electric field in both straight and dog-leg microchannels. This analysis shows that conductivity profiles provide a better measure to determine the quasi-steady state in an ITP process. For a straight microchannel, the quasi-steady state is achieved in less than a minute with a total potential drop of 100?V in a 2?cm long channel. In a straight channel, a true steady state can be achieved for ITP with appropriate countercurrent flow where stationary zones are formed, but the time it takes to reach the steady state is much longer than the without counter flow case. The numerical results indicate that a steady state cannot be reached in a dog-leg microchannel because of sample dispersion and refocusing at and near the intersections and at the branch channels. However, the elastic matching method can be used to determine the quasi-steady state in a dog-leg microchannel.     

Analysis of sub-ppb levels of Fe(II), Co(II), and Ni(II) by electrokinetic supercharging preconcentration, CZE separation, and in-capillary derivatization

The analysis of sub-ppb levels of Fe(II), Co(II), and Ni(II) in heat exchanger fluids of nuclear power plants is needed to monitor corrosion. A method involving preconcentration with electrokinetic supercharging (electrokinetic injection with transient ITP), CZE separation, and in-capillary derivatization with ortho-phenanthroline (o-Phe) for direct UV detection was thus developed. First, a multizone BGE was loaded into the capillary by successive hydrodynamic introduction of zones of (i) o-Phe-containing BGE, (ii) BGE for the zonal separation, and (iii) ammonium-based leading electrolyte. Metal cations were electrokinetically injected and stacked at the capillary inlet behind this last leading zone. Finally, a terminating electrolyte zone was hydrodynamically introduced. When a constant voltage was applied, metal ions kept on concentrating isotachophoretically, then separated in CZE mode, were complexed by migrating through an o-Phe zone, and finally detected by direct absorbance. To detect extremely thin peaks, it was attempted for the first time to focus the derivatization reagent by inducing a second transient ITP, before labeling analytes, already separated in CZE mode. With this arrangement, LODs were about 30 ppt in pure water. In heat exchanger fluid matrices containing 1000 ppm bore and 2 ppm lithium, only Fe(II) cation was detected among the three cations of interest at the 1 ppb level using the present method, and its LOD was about ten times higher, due to the lower loading rate during electrokinetic injection.     

Isotachophoresis of beta-blockers in a capillary and on a poly(methyl methacrylate) chip

Analysis of the beta-blockers oxprenolol, atenolol, timolol, propranolol, metoprolol, and acebutolol in human urine by a combination of isotachophoresis (ITP) and zone electrophoresis (ZE) was investigated. Methods were developed with a conventional capillary electrophoresis (CE) apparatus and a poly(methyl methacrylate) (PMMA) microchip system. With CE the separation of oxprenolol, atenolol, timolol, and acebutolol from a standard solution containing 5 microg/mL of each compound was accomplished by performing ZE with transient ITP. The electrolyte system consisted of 10 mM sodium morpholinoethane sulfonate (pH 5.5) and 0.1% methylhydroxyethylcellulose as the leading electrolyte and 30 mM ortho-phosphoric acid (pH 2.0) as both the terminating and the ZE background electrolyte. With the microchip system the separation of oxprenolol and acebutolol from a standard solution containing 10 microg/mL of each compound was accomplished by a coupled-channel ITP-ZE device using the same leading electrolyte solution as the CE system but 5 mM glutamic acid (pH 3.4) as terminating and background electrolytes. The systems were used for analyses of patient urine samples. Water-soluble hydrophilic matrix compounds were removed from the urine samples by solid-phase extraction (SPE). Limits of quantification below 5 microg/mL could be achieved. The PMMA ITP-ZE chip has not earlier been used for analyses of any drugs from urine samples.