Determination of metal cations on miniaturised planar polymeric separation devices using isotachophoresis with integrated conductivity detection

The feasibility of using miniaturised planar polymeric separation devices for the isotachophoretic separation of metal cations was demonstrated. Devices were produced in silicone rubber using a cast moulding fabrication technique. Detection was performed using an integrated single electrode conductivity detector, a design which offers simple fabrication and high resolution. The electrical characteristics of the devices were found to be suitable for performing electroseparations with a power dissipation of up to 1.5 W m-1 being achieved. The separation of a sample containing a mixture of the four metal ions lithium, lanthanum, dysprosium and ytterbium was reproducibly achieved using miniaturised devices. A comparison with a capillary scale separation of the same mixture was made. The miniaturised separations were achieved in under 600 s, which is less than half the time taken for the capillary scale separations.     

Rapid chloride analysis using miniaturised isotachophoresis

A new design of miniaturised separation device for performing isotachophoresis (ITP) has been produced. The device contains a simple arrangement of channels comprising a single separation channel with a 'double T' injection geometry. The device was produced in poly(methyl methacrylate) and incorporates an on-column conductivity detector. A new electrolyte system was developed to enable the rapid determination of chloride to be made. This electrolyte system uses a leading ion of 3.5 mM nitrate at pH 3.0 with 0.5 mM indium(III) added as a complexing agent. Use of this electrolyte system with the new separation device allowed chloride samples to be analysed in under 100 s, with a limit of detection (LOD) calculated to be 2.2 mg l(-1).     

Bidirectional isotachophoresis on a planar chip with integrated conductivity detection

The use of a miniaturised planar separation device with integrated conductivity detection for performing bidirectional isotachophoresis (ITP) is described. The chips were produced in poly(methyl methacrylate) (PMMA) using a milling procedure. To enable bidirectional ITP the devices were designed to inject samples into the centre of the section channel and incorporated two integrated on-column conductivity detectors, positioned at opposite ends of this channel. When used with a hydrodynamic sample transport system the devices were used for the analysis of a range of small ions: NH4+; Na+; Mg2+; Ca2+; Li+; NO3-; ClO4-; SO4(2-); F-. Results sucessfully achieved included the simultaneous separation of three anions and three cations.     

Miniaturised isotachophoretic analysis of inorganic arsenic speciation using a planar polymer chip with integrated conductivity detection

A new method allowing the analysis of inorganic arsenic species using isotachophoresis has been developed. This method has been shown to be suitable for use on both miniaturised planar polymer separation devices and capillary scale devices. A poly(methyl methacrylate) chip with integrated conductivity electrodes has been successfully used for the rapid analysis of inorganic arsenic species in under 600 s. Limits of detection of 1.8 mg l⁻¹ and 4.8 mg l⁻¹ for arsenic(V) and arsenic(III), respectively, have been achieved with the miniaturised device. The device has also been used to perform the simultaneous separation of arsenic(III), arsenic(V), antimony(III), molybdenum(VI) and tellurium(IV).     

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).     

Lab-on-a-Chip device with laser-patterned polymer electrodes for high voltage application and contactless conductivity detection

A laser-patterned microchip electrophoresis device with integrated polymer electrodes for DC high voltages and AC capacitively-coupled contactless conductivity detection was developed. Electrophoresis separations comparable to devices with metal electrodes were obtained, at approximately 20 times lower cost.     

Conductivity detection and quantitation of isotachophoretic analytes on a planar chip with on-line coupled separation channels

A poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors and intended, mainly, to isotachophoresis (ITP) and ITP-capillary zone electrophoresis (CZE) separations was developed recently. The present work was aimed at assessing its performance relevant to the detection and quantitation of the ITP analytes. Hydrodynamic (HDF) and electroosmotic (EOF) flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the ITP separations with model analytes carried out in this context. When the surfaces of the detection electrodes of the conductivity sensors on the chip were appropriately cleaned qualitative indices of the test analytes [relative step heights (RSHs)], provided by a particular detection sensor, agreed within 1% (expressed via RSDs of the RSH values). Their long-term reproducibilities for one sensor, as estimated from 70 ITP runs repeated in 5 days, were 2% or less. Sensor-to-sensor and chip-to-chip fluctuations of the RSH values for the test analytes were 2.5% or less. In addition, experimentally obtained RSH values agreed well with those predicted by the calculations based on the ITP steady-state model. Reproducibilities of the migration velocities attainable on the CC chips with suppressed EOF and HDF, assessed from the migration time measurements of the ITP boundary between well-defined positions on the separation channels of the chips (140 repeated runs on three chips), ranged from 1.4 to 3.3% for the migration times in the range of 100-200 s. Within-day repeatabilities of the time-based zone lengths for the test analytes characterized 2% RSDs, while their day-to-day repeatabilities were less than 5%. Chip-to-chip reproducibilities of the zone lengths, assessed from the data obtained on three chips for 100 ITP runs, were 5-8%.     

Comparison of different conductivity detector geometries on an isotachophoresis PMMA-microchip

Conductivity detection is one of the most often employed means of detection in isotachophoresis. In microanalytical devices, thin-film platinum electrodes can be used for conductivity detection and for other electrochemical methods of detection. The design and the performance of different electrode geometries for on-column contact conductivity detection with thin-film platinum electrodes integrated on an isotachophoresis PMMA-microchip is described. Three different electrode geometries for direct conductivity detection were used for the investigation of isotachophoretic separations. The influence of the width of the electrodes and their positioning relative to the separation channel was investigated. The performance of the different detectors is compared for the analysis of organic carboxylic acid anions.     

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.     

Use of miniaturised isotachophoresis on a planar polymer chip to analyse transition metal ions in solutions from metal processing plants

An electrolyte system, using malic acid as a complexing agent, has been developed to allow the determination of transition metal cations using miniaturised isotachophoresis. The method allowed the simultaneous determination of Mn2+, Cr3+, Fe2+, Co2+, Zn2+ and Ni2+ to be made without interference from other common ions. Limits of detection were calculated to be in the range 0.5-1.0mg l(-1) for Mn2+, Cr3+ Co2+ and Zn2+ and 2.0 mg l(-1) for Fe2+ and 4.7 mg l(-1) for Ni2+. The successful analysis of five industrial samples, containing a range of these metal ions, obtained from metal processing plants were achieved in under 13 min. The separations were performed on a poly(methyl methacrylate) chip with integrated platinum wire conductivity detection electrodes.     

Determination of bromate in drinking water by zone electrophoresis-isotachophoresis on a column-coupling chip with conductivity detection

The use of capillary zone electrophoresis (CZE) on-line coupled with isotachophoresis (ITP) sample pretreatment (ITP-CZE) on a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to the determination of bromate in drinking water was investigated. Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed and electrophoresis was a dominant transport process in the ITP-CZE separations. A high sample load capacity, linked with the use of ITP in this combination, made possible loading of the samples by a 9.2 microL sample injection channel of the chip. In addition, bromate was concentrated by a factor of 10(3) or more in the ITP stage of the separation and, therefore, its transfer to the CZE stage characterized negligible injection dispersion. This, along with a favorable electric conductivity of the carrier electrolyte solution, contributed to a 20 nmol/L (2.5 ppb) limit of detection for bromate in the CZE stage. Sample cleanup, integrated into the ITP stage, effectively complemented such a detection sensitivity and bromate could be quantified in drinking water matrices when its concentration was 80 nmol/L (10 ppb) or slightly less while the concentrations of anionic macroconstituent (chloride, sulfate, nitrate) in the loaded sample corresponding to a 2 mmol/L (70 ppm) concentration of chloride were still tolerable. The samples containing macroconstituents at higher concentrations required appropriate dilutions and, consequently, bromate in these samples could be directly determined only at proportionally higher concentrations.     

Analysis of chloride, bromide and iodide using miniaturised isotachophoresis on a planar polymer chip

A new method has been developed to allow the determination of the halide anions chloride, bromide and iodide using isotachophoresis. This method employs a new electrolyte system which incorporates the novel application of indium(III) as a complexing agent. This electrolyte system was devised based on the findings of an investigation into the potential for using indium(III) as a complexing counter ion to selectively manipulate the effective mobilities of halide ions. A leading electrolyte incorporating 3.5 mmol dm(-3) of indium(III) allowed the simultaneous determination of chloride, bromide and iodide to be successfully achieved. The new procedure allows such separations to be made without interference from common inorganic anions such as sulfate and nitrate. Separations were performed using a miniaturised planar poly(methyl methacrylate) chip with integrated platinum wire conductivity detection electrodes. Using this instrumentation the limits of detection were calculated to be 0.7 mg dm(-3), 1.7 mg dm(-3) and 2.2 mg dm(-3) for chloride, bromide and iodide respectively.     

Isotachophoresis and isotachophoresis--zone electrophoresis separations of inorganic anions present in water samples on a planar chip with column-coupling separation channels and conductivity detection

The use of a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to electrophoretic separations of a group of inorganic anions (chloride, nitrate, sulfate, nitrite, fluoride and phosphate) that need to be monitored in various environmental matrices was studied. The electrophoretic methods employed in this study included isotachophoresis (ITP) and capillary zone electrophoresis (CZE) with on-line coupled ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the separations performed by these methods. ITP separations on the chip provided rapid resolutions of sub-nmol amounts of the complete group of the studied anions and made possible rapid separations and reproducible quantitations of macroconstituents currently present in water samples (chloride, nitrate and sulfate). However, concentration limits of detection attainable under the employed ITP separating conditions (2-3 x 10(-5) mol/l) were not sufficient for the detection of typical anionic microconstituents in water samples (nitrite, fluoride and phosphate). On the other hand, these anions could be detected at 5-7 x 10(-7) mol/l concentrations by the conductivity detector in the CZE stage of the ITP-CZE combination on the CC chip. A sample clean-up performed in the ITP stage of the combination effectively complemented such a detection sensitivity and nitrite, fluoride and phosphate could be reproducibly quantified also in samples containing the macroconstituents at 10(4) higher concentrations. ITP-CZE analyses of tap, mineral and river water samples showed that the CC chip offers means for rapid and reproducible procedures to the determination of these anions in water (4-6 min analysis times under our working conditions). Here, the ITP sample pretreatment concentrated the analytes and removed nanomol amounts of the macroconstituents from the separation compartment of the chip within 3-4 min. Both the ITP and ITP-CZE procedures required no or only minimum manipulations with water samples before their analyses on the chip. For example, tap water samples were analyzed directly while a short degassing of mineral water (to prevent bubble formation during the separation) and filtration of river water samples (to remove particulates and colloids) were the only operations needed in this respect.     

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.     

Determination of the ascorbate content of photographic developer solutions using miniaturised isotachophoresis on a planar chip

The use of miniaturised isotachophoresis, performed on a planar poly(methyl methacrylate) device with integrated platinum conductivity electrodes, for the analysis of the ascorbate content of photographic developer solutions has been investigated. An electrolyte system has been developed which enabled the analysis to be made without interference from any of the other components in the developer solution, a number of which were present at significantly higher concentrations than that of the ascorbate ions. Using this system, the ascorbate content of the developer solutions could be analysed in under 6 min. The limit of detection for ascorbate using miniaturised isotachophoresis was calculated to be 0.011 mmol dm(-3).     

Enantiomeric resolution of amino acid derivatives on molecularly imprinted polymers as monitored by potentiometric measurements

Potentiometric measurements have been applied to the detection of enantiomeric separations on molecularly imprinted polymers. A flow-through column electrode, based on the use of polymers imprinted against L-phenylalanine anilide, is described. The electrode consisted of a glass column in which the polymer was packed and where the end frits constituted the electrodes. The flow stream potential across the column can be continuously recorded as solvent is pumped through the system. The column resolved the enantiomers of phenylalanine anilide as detected by both UV absorption and potentiometric measurements and the recorded signals could be correlated with the concentration of phenylalanine anilide. The calibration graphs obtained for the UV absorption of phenylalanine anilide were linear over the concentration range investigated, whereas the potentiometric signal was shown to be exponentially linear with concentration. The application of molecular imprints to the preparation of supports suitable for chromatographic separations of enantiomers and for the preparation of specific electrodes is discussed.     

Disposable Injection‐Moulded Cell‐on‐a‐Chip Microfluidic Devices with Integrated Conducting Polymer Electrodes for On‐Line Voltammetric and Electrochemiluminescence Detection

This work reports the construction and characterization of plastic electrochemical micro‐flow‐cells with integrated injection‐moulded polymer electrodes. The three electrodes (working, auxiliary, and reference) were fabricated by injection‐moulding from a conducting grade of polystyrene loaded with carbon fibers. On‐chip reference electrodes were prepared by coating one of the conducting polymer electrodes with a Ag/AgCl layer (implemented either by e‐beam evaporation of Ag followed by electrochemical formation of AgCl or by applying a Ag/AgCl paste). Working electrodes were either polymer electrodes coated with Au by e‐beam evaporation or bare conducting polymer electrodes. The electrodes were integrated into the micro‐flow‐cells by an over‐moulding process followed by ultrasonic welding. The devices were characterized by optical and electrochemical techniques. Studies by cyclic voltammetry (CV), anodic stripping voltammetry (ASV) and electrochemiluminescence (ECL) demonstrate ‘proof–of‐principle’ of the micro‐flow‐cells as electrochemical sensors.     

Separations of enantiomers by preparative capillary isotachophoresis.

The use of capillary isotachophoresis (ITP), operating in a discontinuous fractionation mode, for preparative separations of enantiomers of chiral compounds was studied. The ITP separations were carried out in the column-coupling configuration of the separation unit provided with the preseparation column of a 1.0 mm ID and the trapping column of a 0.8 mm ID. Such a configuration of the CE separation unit offers several working regimes suitable to preparative separations of enantiomers. 2,4-Dinitrophenyl-DL-norleucine (DNP-Norleu) was employed as a model analyte in our experiments with beta-cyclodextrin serving in the electrolyte solutions as a chiral selector. The preparative separations lasting about 20 min were evaluated by ITP and (more often) by capillary zone electrophoresis (CZE). It was found that one preparative run provided up to 14 microg of pure DNP-Norleu enantiomers. This corresponded to a 75 times higher production rate of ITP relative to a maximum value of this parameter as estimated for preparative CZE runs in cylindrical capillaries (0.5 pmol/s). About 75% of the DNP-Norleu enantiomers loaded into the preparative equipment could be recovered in pure enantiomer fractions. Contiguous natures of the zones in the ITP stack and adsorption losses of the enantiomers in the isolation step were found to set practical limits for a further enhancement of the recovery rates in the isolation of pure enantiomers.     

Design and optimization of a fused‐silica microfluidic device for separation of trivalent lanthanides by isotachophoresis

Elemental analysis of rare earth elements is essential in a variety of fields including environmental monitoring and nuclear safeguards; however, current techniques are often labor intensive, time consuming, and/or costly to perform. The difficulty arises in preparing samples, which requires separating the chemically and physically similar lanthanides. However, by transitioning these separations to the microscale, the speed, cost, and simplicity of sample preparation can be drastically improved. Here, all fourteen non‐radioactive lanthanides (lanthanum through lutetium minus promethium) are separated by ITP for the first time in a serpentine fused‐silica microchannel (70 µm wide × 70 µm tall × 33 cm long) in <10 min at voltages ≤8 kV with limits of detection on the order of picomoles. This time includes the 2 min electrokinetic injection time at 2 kV to load sample into the microchannel. The final leading electrolyte consisted of 10 mM ammonium acetate, 7 mM α‐hydroxyisobutyric acid, 1% polyvinylpyrrolidone, and the final terminating electrolyte consisted of 10 mM acetic acid, 7 mM α‐hydroxyisobutyric acid, and 1% polyvinylpyrrolidone. Electrophoretic electrodes are embedded in the microchip reservoirs so that voltages can be quickly applied and switched during operation. The limits of detection are quantified using a commercial capacitively coupled contactless conductivity detector (C⁴D) to calculate ITP zone lengths in combination with ITP theory. Optimization of experimental procedures and reproducibility based on statistical analysis of subsequent experimental results are addressed. Percent error values in band length and conductivity are ≤8.1 and 0.37%, respectively.     

Screen-printed poly(3,4-ethylenedioxythiophene) (PEDOT): A new electrochemical mediator for acetylcholinesterase-based biosensors

This work describes the use of a PEDOT:PSS-based conductive polymer for designing AChE-based biosensors. The transducers were obtained directly by screen-printing a PEDOT:PSS suspension on the surface of thick film carbon electrodes. The obtained working electrodes showed a high conductivity when compared with electrodes modified with conventional mediators like cobalt phthalocyanine or tetracyanoquinodimethane. The PEDOT:PSS polymer was shown to be suitable for thiocholine oxidation, allowing the measurement of AChE activity at 100 mV vs Ag/AgCl. The high conductivity of PEDOT:PSS allowed the accurate detection of the organophosphate insecticide chlorpyrifos-oxon at concentrations as low as 4 × 10⁻⁹ M, corresponding to an inhibition ratio of 5%.