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%.     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

A high-performance polycarbonate electrophoresis microchip with integrated three-electrode system for end-channel amperometric detection

A fully integrated polycarbonate (PC) microchip for CE with end-channel electrochemical detection operated in an amperometric mode (CE-ED) has been developed. The on-chip integrated three-electrode system consisted of a gold working electrode, an Ag/AgCl reference electrode and a platinum counter electrode, which was fabricated by photo-directed electroless plating combined with electroplating. The working electrode was positioned against the separation channel exit to reduce post-channel band broadening. The electrophoresis high-voltage (HV) interference with the amperometric detection was assessed with respect to detection noise and potential shifts at various working-to-reference electrode spacing. It was observed that the electrophoresis HV interference caused by positioning the working electrode against the channel exit could be diminished by using an on-chip integrated reference electrode that was positioned in close proximity (100 microm) to the working electrode. The CE-ED microchip was demonstrated for the separation of model analytes, including dopamine (DA) and catechol (CA). Detection limits of 132 and 164 nM were achieved for DA and CA, respectively, and a theoretical plate number of 2.5x10(4)/m was obtained for DA. Relative standard deviations in peak heights observed for five runs of a standard solution containing the two analytes (0.1 mM for each) were 1.2 and 3.1% for DA and CA, respectively. The chip could be continuously used for more than 8 h without significant deterioration in analytical performance.     

小型可连续进样微流控芯片分析仪的研制

报道了一种结构简单、可连续进样的小型微流控芯片分析仪的研制。顺序注射分析系统通过芯片上制作的接口将试样连续引入芯片 ,并采用自行设计的紧凑型光纤式激光诱导荧光检测器进行检测。该仪器用于芯片毛细管电泳分离实验室合成Cy5荧光染料 ,实现了连续进样和换样。峰高RSD为 1 .9% (n=1 1 ) ,试样通量 3 5 h ;相邻试样携出 <4%。     

Separation and quantification of lanthanides in synthetic standards by capillary electrophoresis: a new experimental evidence of the systematic "odd-even" pattern observed in sensitivities and detection limits

The systematic zigzag pattern of sensitivities and detection limits (LODs) of lanthanides, previously observed in mass spectrometric and chromatographic measurements, was once more investigated through the indirect photometric detection with capillary electrophoresis. Well-designed chemometric experiments were performed for the electrophoretic separation and detection of lanthanides using standards with similar concentrations. A fused silica capillary 355 mm x 75 microm was used. Complete separation for all 14 lanthanides was achieved in approximately 3 min at a capillary temperature of 15 degrees C. Indirect photometric detection at 214 nm using a voltage of +25 kV and a hydrostatic injection (100 mm for 20 s) were used. The background electrolyte used consisted of an optimum mixture of 0.004 M HIBA (as complexing agent) and 0.010 M UVCat-1 (as a UV-absorbing co-ion) with a pH 4.4. A good reproducibility in migration times (<2.7% RSD), peak areas (<3.8% RSD) and peak heights (2.7% RSD) were systematically obtained. Calibration curves based on both peak areas and peak heights (from seven replicates) were prepared using weighted least-squares regressions, which were employed for the correct estimation of individual sensitivities and LODs. For a better estimation of LODs, the lowest concentration standard was injected 30 times. A new experimental evidence of the systematic "odd-even" pattern was again observed in the lanthanide sensitivities (and therefore in LODs). The calculated sensitivities were greater for lanthanides with an odd-atomic number than for their corresponding neighboring element with an even-atomic number (i.e., (57)La-(58)Ce, (59)Pr-(60)Nd; (63)Eu-(64)Gd, etc.). Concerning the LODs, a systematic zigzag pattern was observed where the odd atomic number elements have lower LODs than the even atomic number neighbor elements (i.e., (57)La-(58)Ce; (59)Pr-(60)Nd; (63)Eu-(64)Gd, etc.). The possible origin of this "odd-even" effect is briefly discussed. Accuracy errors were less than 5% for lanthanide concentrations of three synthetic standard solutions, which were considered as "unknown" samples.     

C2-C8 hydrocarbon measurement and quality control procedures at the Global Atmosphere Watch Observatory Hohenpeissenberg

A new automated on-line GC-flame ionization detection system for long-term stationary measurements of atmospheric C2-C8 hydrocarbons in the lower ppt range is described. The system is operated at the Global Atmosphere Watch Observatory Hohenpeissenberg (47 degrees 48'N, 11 degrees 01'E) in rural south Germany. Atmospheric mixing ratios of more than 40 different hydrocarbons can be continuously measured in 80 min time intervals. Corresponding detection limits are below 3 ppt, except for propene, butenes and benzene (about 10 ppt). Detailed quality assurance and quality control protocols are described which are applied to routine operation and data analysis. The various error contributions, overall precision, and accuracy for all measured compounds are discussed in detail. Typical ambient air mixing ratios are in the range of a few ppt to a few ppb, and corresponding measurement accuracies are below 10% or 10 ppt. For less than 20% of the analyzed compounds measurement accuracies are worse, mainly because of insufficient peak separation, blank values or reduced reproducibilities. The present system was tested in international intercomparison experiments (NOMHICE, AMOHA). For most of the C2-C8 hydrocarbons analyzed, our results agreed better than +/- 10% (20% NOMHICE phase 5) or +/- 10 ppt with the corresponding reference values.     

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids

A microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related phenolic acids (including chlorogenic, gentisic, ferulic, and vanillic acids) is described. Relevant parameters of the chip separation and amperometric detection are examined and optimized. Under optimum conditions, the analytes could be separated and detected in a 15 mM borate buffer (pH 9.5, with 10% of methanol) within 300 s using a separation voltage of 2000 V and a detection voltage of +1.0 V. Linear calibration plots are observed for micromolar concentrations of the phenolic acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of phenolic compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption. An application on a commercial red wine was performed with minimal sample preparation and promising results.     

Influence of the peak height distribution on separation performances: Discrimination factor and effective peak capacity

Summary A new index of performance of the chromatographic separation between two adjacent peaks, the discrimination factor, d₀, is defined. It is normalized between 0 and 1 and is directly and easily determined from the chromatogram. It does not depend on any assumption regarding peak shape, except that the peak profiles of individual sample components have a single mode. Its value depends on the relative heights of the two peaks as well as on their separation. The separation power of a chromatographic system is classically measured by its peak capacity, defined on the basis of constant resolution between adjacent peaks. A previously developed statistical theory of the composition of mixtures makes it possible to extend the concept of peak capacity by taking into account the peak height distribution in typical average chromatograms. A new parameter, the effective peak capacity, is defined for this purpose on the basis of a constant discrimination factor between adjacent peaks. It allows to take into account the distribution of peak heights in statistical theories of the evaluation of complex chromatograms and in the measurement of the limit of determination in quantitative analysis. The characteristics of the two new parameters, the discrimination factor and effective peak capacity, are discussed and compared with those of their classical homologs, resolution and peak capacity, in the case of gaussian component peaks of equal widths.     

A chip-based immunoaffinity capillary electrophoresis assay for assessing hormones in human biological fluids

A chip-based capillary electrophoresis system has been designed for assessing the concentrations of four hormones in whole human blood, saliva, and urine. The desired analytes were isolated by immunoextraction using a panel of four analyte-specific antibodies immobilized onto a glass fiber insert within the injection port of the chip. Following extraction, the captured analytes were labeled prior to electro-elution into the chip separation channel, where they were resolved into four individual peaks in circa 2 min. Quantification of each peak was achieved by on-line LIF detection and integration of the area under each peak. Comparison to commercial high-sensitivity immunoassays demonstrated that the chip-based assay provided fast, accurate, and precise measurements for the analytes under investigation. As the availability of commercially available antibodies rapidly expands, the application of this system will greatly increase. Chip-based CE separations of multiple analytes from a single sample also provide a significant advantage in the analysis of small samples.     

A three-dimensionally adjustable amperometric detector for microchip electrophoretic measurement of nitroaromatic pollutants

A microchip capillary electrophoresis (CE)–amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an amperometric detection cell containing a one-dimensionally adjustable disc detection electrode in a Plexiglas holder. It facilitates the precise three-dimensional alignment between the channel outlet and the detection electrode without a complicated three-dimensional manipulator. The performance of this unique system was demonstrated by separating four nitroaromatic pollutants (nitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and p-nitrobenzene). Factors influencing their separation and detection processes were examined and optimised. The four analytes have been well-separated within 120 s in a 75 cm long separation channel at a separation voltage of +2000 V using an electrophoretic separation medium containing 15 mM borax and 15 mM sodium dodecyl sulfate (pH 9.2). Highly linear response is obtained for the four analytes over the range of 0–5 ppm with the detection limits ranging from 12 to 52 ppb. The present system demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n = 9). The new approach for the microchannel–electrode alignment should find a wide range of applications in other microfluidic analysis systems.     

Multisyringe flow injection analysis coupled to capillary electrophoresis (MSFIA-CE) as a novel analytical tool applied to the pre-concentration, separation and determination of nitrophenols

For the first time, a multisyringe flow injection analysis capillary electrophoresis system is described. The potential of the hyphenation for sample treatment including analyte pre-concentration is demonstrated by its successful application to the determination of mono-nitrophenols (NPs) in different water samples. The analytical system was used to automate in-line sample acidification, analyte pre-concentration, elution, hydrodynamic injection, electrophoretic separation, and detection as well as the maintenance and re-conditioning of the solid-phase extraction (SPE) column and the separation capillary. A pre-concentration factor of better than 115 and detection down to 0.11 micromol L(-1) were achieved. Detection was carried out at visible wavelength using a blue LED as a low baseline-noise light source. High repeatability was obtained each for migration times and for peak heights with relative standard deviations typically below 2.5 and 6% including the pre-concentration procedure, respectively. Three injections per hour were achieved by running in parallel the pre-concentrating and the electrophoretic separation procedures. Instrumental control and data registration and evaluation were carried out with the software package AutoAnalysis, allowing autonomous operation of the analytical system.     

Fabrication of a monolithic sampling probe system for automated and continuous sample introduction in microchip-based CE

A fabrication process for producing monolithic sampling probes on glass chips, with tip diameters of a few hundred micrometers was developed, using simple tools including a glass cutter and a bench drill. Microfluidic chips with probes fabricated by this approach were coupled to a linearly moving slotted-vial array sample presentation system for performing continuous sample introduction in the chip-based CE system. On-chip horizontal tubular reservoirs containing working electrolyte and waste were used to maintain a stable hydrostatic pressure in the chip channels during prolonged working periods. The performance of the system was demonstrated in the separation of FITC-labeled amino acids with LIF detection, by continuously introducing a train of different samples without interruption. Throughputs of 30-60/h were achieved with <1.0% carry-over and reproducibilities in peak height of 3.6, 3.3, and 3.5% RSD for arginine, FITC, and phenylalanine, respectively (n = 11). Continuous analysis of a mixture of FITC-labeled amino acids for 2 h, involving 60 analytical cycles, yielded an RSD of 7.5 and 6.8% for arginine and FITC (n = 60), respectively. An extremely low sample consumption of 30 nL for each analysis was obtained. Separation efficiencies in plate numbers were in the range of 0.8-2x10(5)/m. In addition to the application in sample introduction, the sample/reagent introduction system was also used to produce working electrolyte gradients during a CE separation to improve the separation efficiency. Comparing with isocratic electrophoresis separation, gradient CE demonstrated better separation efficiencies for a mixture of FITC-labeled amino acids.     

Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase,TniA

This work deals with zone electrophoresis (ZE) separations of proteins on a poly(methyl methacrylate) chip with integrated conductivity detection. Experiments were performed in the cationic mode of the separation (pH 2.9) with a hydrodynamically closed separation compartment and suppressed electroosmotic flow. The test proteins reached the detector in less than 10 min under these working conditions and their migration times characterized excellent repeatabilities (0.1–0.6% RSD values). The chip-to-chip agreements of the migration times, evaluated from the ZE runs performed on three chips, were within 1.5%. The conductivity detection provided for protein, loaded on the chip at 10–1000 μg/ml concentrations, detection responses were characterized by 1–5% RSD values of their peak areas. Such migration and detection performances made a frame for reproducible baseline separations of a five-constituent mixture (cytochrome c, avidin, conalbumin, human hemoglobin and trypsin inhibitor). On the other hand, a high sample injection channel/separation compartment volume ratio of the chip (500 nl/8500 nl) restricted the resolution of proteins of very close effective mobilities in spite of the fact that in the initial phase of the separation an electric field stacking was applied. A maximum macroconstituent/trace constituent ratio attainable for proteins on the chip was assessed for cytochrome c (quantifiable when its concentration in the loaded sample was 10 μg/ml) and apo-transferrin (containing a trace constituent migrating in the position of cytochrome c detectable when the load of apo-transferrin was 2000 μg/ml). This assessment indicated that a ratio of 1000:1 is attainable with the aid of conductivity detection on the present chip.     

Fully integrated PDMS/SU-8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole-channel imaging detection

A fully integrated polydimethylsiloxane (PDMS)/modified PDMS membrane/SU-8/quartz hybrid chip was developed for protein separation using isoelectric focusing (IEF) mechanism coupled with whole-channel imaging detection (WCID) method. This microfluidic chip integrates three components into one single chip: (i) modified PDMS membranes for separating electrolytes in the reservoirs from the sample in the microchannel and thus reducing pressure disturbance, (ii) SU-8 optical slit to block UV light (below 300?nm) outside the channel aiming to increase detection sensitivity, and (iii) injection and discharge capillaries for continuous operation. Integration of all these components on a single chip is challenging because it requires fabrication techniques for perfect bonding between different materials and is prone to leakage and blockage. This study has addressed all the challenges and presented a fully integrated chip, which is more robust with higher sensitivity than the previously developed IEF chips. This chip was tested by performing protein and pI marker separation. The separation results obtained in this chip were compared with that obtained in commercial cartridges. Side-by-side comparison validated the developed chip and fabrication techniques.     

Microfluidic chip capillary electrophoresis coupled with electrochemiluminescence for enantioseparation of racemic drugs using central composite design optimization

Optimization based on central composite design (CCD) for enantioseparation of anisodamine (AN), atenolol (AT), and metoprolol (ME) in human urine was developed using a microfluidic chip‐CE device. Coupling the flexible and wide working range of microfluidic chip‐CE device to CCD for chiral separation of AN, AT, and ME in human urine, a total of 15 experiments is needed for the optimization procedure as compared to 75 experiments using the normal one variable at a time optimization. The optimum conditions obtained are found to be more robust as shown by the curvature effects of the interaction factors. The developed microfluidic chip‐CE‐ECL system with adjustable dilution ratios has been validated by satisfactory recoveries (89.5–99% for six enanotiomers) in urine sample analysis. The working range (0.3–600 μM), repeatability (3.1–4.9% RSD for peak height and 4.0–5.2% RSD for peak area), and detection limit (0.3–0.6 μM) of the method developed are found to meet the requirements for bedside monitoring of AN, AT, and ME in patients under critical conditions. In summary, the hyphenation of CCD with the microfluidic chip‐CE device is shown to offer a rapid means for optimizing the working conditions on simultaneous separation of three racemic drugs using the microfluidic chip‐CE device developed.     

Automated high‐speed CE system for multiple samples

A high‐speed CE system for multiple samples was developed based on a short capillary and an automated sample introduction device consisting of a commercial multi‐well plate and an x‐y‐z translation stage. The spontaneous injection method was used to achieve picoliter‐scale sample injection from different sample wells. Under the optimized conditions, a 40 μm‐long sample plug (corresponding to 78‐pL plug volume) was obtained in a 50 μm id capillary, which ensured both the high separation speed and high separation efficiency. The performance of the system was demonstrated in the separation of FITC‐labeled amino acids with LIF detection. Five FITC‐labeled amino acids including arginine, phenylalanine, glycine, glutamic acid, and asparagine were separated within 15 s with an effective separation length of 1.5 cm. The separation efficiency ranged from 7.96 × 10⁵/m to 1.12 × 10⁶ /m (corresponding to 1.26–0.89 μm plate heights). The repeatability of the peak heights calibrated with an inner standard for different sample wells was 2.4 and 2.7% (n = 20) for arginine and phenylalanine, respectively. The present system was also applied in consecutive separations of 20 different samples of FITC‐labeled amino acids with a whole separation time of less than 6 min.     

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.     

Stacking and separation of coproporphyrin isomers by acetonitrile-salt mixtures in micellar electrokinetic chromatography

The effectiveness of the addition of salt and acetonitrile in the sample matrix to induce narrowing of the analyte zones is demonstrated for the first time in micellar electrokinetic chromatography (MEKC). Using coproporphyrin (CP) I and III isomers as test compounds, the use of sodium cholate (SC) as the micelle in the separation buffer and a high concentration of sodium chloride in the aqueous sample solution (without the presence of an organic solvent) were found to provide enhancement in peak heights for both CP I and III, but yielded very poor resolution of these two positional isomers at sample size of 10% capillary volume or larger. With the addition of acetonitrile as the organic solvent in the aqueous sample solution (acetonitrile-salt mixtures), baseline/partial resolution of CP I and III was obtained even at large injection volumes, along with significant increase in peak heights for both isomers. Possible mechanisms responsible for the narrowing of analyte zones are briefly discussed. The effects of experimental parameters, such as concentrations of salt and acetonitrile, on peak heights and resolution of the test compounds were studied. Importantly, the usefulness of the present method was demonstrated for the MEKC determination of endogenous CP I and III present in normal urine samples with good separation and detection performances.     

Three‐dimensional printed magnetophoretic system for the continuous flow separation of avian influenza H5N1 viruses

As a result of the low concentration of avian influenza viruses in samples for routine screening, the separation and concentration of these viruses are vital for their sensitive detection. We present a novel three‐dimensional printed magnetophoretic system for the continuous flow separation of the viruses using aptamer‐modified magnetic nanoparticles, a magnetophoretic chip, a magnetic field, and a fluidic controller. The magnetic field was designed based on finite element magnetic simulation and developed using neodymium magnets with a maximum intensity of 0.65 T and a gradient of 32 T/m for dragging the nanoparticle–virus complexes. The magnetophoretic chip was designed by SOLIDWORKS and fabricated by a three‐dimensional printer with a magnetophoretic channel for the continuous flow separation of the viruses using phosphate‐buffered saline as carrier flow. The fluidic controller was developed using a microcontroller and peristaltic pumps to inject the carrier flow and the viruses. The trajectory of the virus–nanoparticle complexes was simulated using COMSOL for optimization of the carrier flow and the magnetic field, respectively. The results showed that the H5N1 viruses could be captured, separated, and concentrated using the proposed magnetophoretic system with the separation efficiency up to 88% in a continuous flow separation time of 2 min for a sample volume of 200 μL.     

Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)-based microchips

MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.