Isotachophoresis separations of enantiomers on a planar chip with coupled separation channels.

The use of a poly(methylmethacrylate) chip, provided with a pair of on-line coupled separation channels and on-column conductivity detectors, to isotachophoresis (ITP) separations of optical isomers was investigated. Single-column ITP, ITP in the tandem-coupled columns, and concentration-cascade ITP in the tandem-coupled columns were employed in this investigation using tryptophan enantiomers as model analytes. Although providing a high production rate (about 2 pmol of a pure tryptophan enantiomer separated per second), single-column ITP was found suitable only to the analysis of samples containing the enantiomers at close concentrations. A 94-mm separation path in ITP with the tandem-coupled separation channels made possible a complete resolution of a 1.5 nmol amount of the racemic mixture of the enantiomers. However, this led only to a moderate extension of the concentration range within which the enantiomers could be simultaneously quantified. The best results in this respect were achieved by using a concentration-cascade of the leading anions in the tandem-coupled separation channels. Here, a high production rate, favored in the first separation channel, was followed by the ITP migration of the enantiomers in the second channel under the electrolyte conditions enhancing their detectabilities. In dependence on the migration configuration of the enantiomers, this technique made possible their simultaneous determinations when their ratios in the loaded sample were 35:1 or less (D-tryptophan a major constituent) and 70:1 or less (L-tryptophan a major constituent).

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%.

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 organic acids and inorganic anions in wine by isotachophoresis on a planar chip.

Isotachophoretic (ITP) separation and determination of a group of 13 organic and inorganic acids, currently present in wines, on a poly(methyl methacrylate) chip provided with on-column conductivity detection was a subject of a detailed study performed in this work. Experiments with the ITP electrolyte systems proposed to the separation of anionic constituents present in wine revealed that their separation at a low pH (2.9) provides the best results in terms of the resolution. Using a 94 mm long separation channel of the chip, the acids could be resolved within 10-15 min also in instances when their concentrations corresponded to those at which they typically occur in wines. A procedure suitable to the ITP determination of organic acids responsible for some important organoleptic characteristics of wines (tartaric, lactic, malic and citric acids) was developed. Concentrations of 2-10 mg/l of these acids represented their limits of quantitation for a 0.9 microl volume sample loop on the chip. A maximum sample load on the chip, under the preferred separating conditions, was set by the resolution of malate and citrate. A complete resolution of these constituents in wine samples was reached when their molar concentration ratio was 20:1 or less. ITP analyses of a large series of model and wine samples on the chip showed that qualitative indices [RSH (relative step height) values] of the acids, based on the response of the conductivity detector, reproduced with RSD better than 2% while reproducibilities of the determination of the acids of our interest characterized RSD values better than 3.5%.

Conductivity detection cell for capillary zone electrophoresis with a solution mediated contact of the separated constituents with the detection electrodes.

A contact conductivity detection cell for capillary zone electrophoresis (CZE) with an electrolyte solution mediated contact of the separated constituents with the detection electrodes (ESMC cell) was developed in this work. This new approach to the conductivity sensing in CZE is intended to eliminate detection disturbances due to electrode reactions and adsorption of the separated constituents when these are coming into direct contact with the detection electrodes. An optimum detection performance of the cell was achieved when the carrier electrolyte solution mediated the electric contact of the detection electrodes with the separated constituents. Different compositions of the mediator and carrier electrolyte solutions led to large drifts of the detection signals. Isotachophoresis experiments performed in this context with the ESMC cell revealed that origins of these drifts are in transport processes (diffusion and electromigration) between the detection compartment and the detection electrodes in the cell. These processes affected, to some extent, other analytically relevant performance parameters of the ESMC cell of the present construction as well [e.g., concentration limits of detection (LODs), a contribution of the cell to the band broadening]. For example, the ESMC cell gave, under optimum operating conditions, 3-4 times higher concentration LODs for the test analytes than a current on-column conductivity cell employed under identical working conditions. On the other hand, these LOD values (25-150 nmol/l) were still 20-25 times lower than those estimated from reference experiments for a contactless conductivity detector. CZE experiments with iodide, carried out under working conditions leading to electrochemical reactions of this anion on the detection electrodes of current conductivity cells, did not occur in the ESMC cell. In addition, this cell, contrary to a reference contact conductivity cell, required no special care (e.g., cleaning of the surfaces of the detection electrodes by chemical or electrochemical means) to maintain its reliable long-term performance. Anionic CZE analyses of tap and mineral water samples monitored by the conductivity detector provided with the ESMC cell demonstrated a practical applicability and certain limitations of this detection approach in the analysis of ionic constituents present in high ionic strength sample matrices.

Capillary electrophoresis separations on a planar chip with the column-coupling configuration of the separation channels

Some basic aspects of capillary electrophoresis (CE) separations on a poly(methyl methacrylate) chip provided with two separation channels in the column-coupling (CC) configuration and on-column conductivity detectors were studied. The CE methods employed in this study included isotachophoresis (ITP), capillary zone electrophoresis (CZE), and CZE with on-line ITP sample pretreatment (ITP-CZE). 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 separations performed by these methods. Very reproducible migration velocities of the separated constituents were typical under such transport conditions, and consequently, test analytes could be quantified by various ITP techniques with 1-2% RSD. The CC configuration of the separation channels provides means for an effective combination of an enhanced load capacity of the separation system with high detection sensitivities for the analytes in concentration-cascade ITP separations. In this way, for example, succinate, acetate, and benzoate could be separated also in instances when they were present in the loaded sample (1.2 microL) at 1 mmol/L concentrations while their limits of detection ranged from 8 to 12 micromol/L concentrations. A well-defined ITP concentration of the analyte(s) combined with an in-column sample cleanup (via an electrophoretically driven removal of the matrix constituents from the separation compartment) can be integrated into the separations performed on the CC chip. These sample pretreatment capabilities were investigated in ITP-CZE separations of model samples in which nitrite, phosphate, and fluoride (each at a 10 micromol/L concentration) accompanied matrix constituents (sulfate and chloride) at considerably higher concentrations. Here, both the concentration of the analytes and cleanup of the sample were included in the ITP separation in the first separation channel while the second separation channel served for the CZE separation of the ITP pretreated sample and the detection of the analytes.

Capillary electrophoresis of inorganic anions.

This review deals with the separation mechanisms applied to the separation of inorganic anions by capillary electrophoresis (CE) techniques. It covers various CE techniques that are suitable for the separation and/or determination of inorganic anions in various matrices, including capillary zone electrophoresis, micellar electrokinetic chromatography, electrochromatography and capillary isotachophoresis. Detection and sample preparation techniques used in CE separations are also reviewed. An extensive part of this review deals with applications of CE techniques in various fields (environmental, food and plant materials, biological and biomedical, technical materials and industrial processes). Attention is paid to speciations of anions of arsenic, selenium, chromium, phosphorus, sulfur and halogen elements by CE.

Capillary zone electrophoresis of nitrophenols with off-line isotachophoretic sample pretreatment.

Preparative capillary isotachophoresis (ITP) operating in a discontinuous fractionation mode was studied as a sample pretreatment technique for capillary zone electrophoresis (CZE) trace analysis of a group of ten nitrophenols. Different separation mechanisms employed by these capillary electrophoresis techniques made possible a group isolation of the studied analytes by ITP while their CZE resolutions based on differences in the interactions with polyvinylpyrrolidone (PVP) provided suitable conditions for a final analytical evaluation. Experiments with model and practical water samples revealed high and reproducible recovery rates of the ITP pretreatment of nitrophenols when sample loadability limits of the preparative ITP equipment were met. A main disadvantage of the studied ITP-CZE combination was an inefficient use of the pretreated sample as only 0.5% of the fraction containing nitrophenols was used in the final CZE step. Despite these limitations associated with the CZE sample injection, the concentration limit of detection (LOD) for nitrophenols in practical water samples could be reduced to 2-8 ppb concentrations (photometric absorbance detector operating at a 254 nm wavelength) when 200 microL sample volumes were taken for the ITP pretreatment. This was accompanied by an efficient sample clean-up as no other trace ionic constituents originating from the samples were detected on CZE.

Electroosmotic flow suppressing additives for capillary zone electrophoresis in a hydrodynamically closed separation system.

Electroosmotic flow in a hydrodynamically closed capillary zone electrophoresis (CZE) separation compartment must be minimized to achieve high efficiency CZE separations. A group of eight potential electroosmotic flow suppressors was investigated in this context for the separations in fluorinated ethylene-propylene capillary tubes. The suppressors included water soluble methylhydroxyethyl derivatives of cellulose, polyvinylalcohol, polyvinylpyrrolidones and polyethyleneglycols of different molecular masses and Triton X-100. Methylhydroxyethylcellulose derivatives and polyvinylalcohol were found to provide the highest separation efficiencies for a group of model anions when the electroosmotic flow suppressors were used as the carrier electrolyte additives. Using a methylhydroxyethylcellulose coated separation compartment very significant improvements in the separation efficiencies were achieved for polyvinylpyrrolidones and polyethyleneglycols applied in the carrier electrolyte solutions. For example, polyvinylpyrrolidone K 90 applied in this way gave for some of the model analytes the plate height values approaching those estimated in the calculations as theoretical limits for our experimental conditions (H approximately 3.5 microns). CZE experiments with albumin and gamma-globulin showed that the use of methylhydroxyethylcellulose derivative in the carrier electrolyte solution at pH = 9.2 was effective in eliminating potential disturbances in the separation efficiencies of the analytes due to adsorption of the proteins.

Determination of synthetic dyes in food products by capillary zone electrophoresis in a hydrodynamically closed separation compartment.

The application of capillary zone electrophoresis (CZE) in a hydrodynamically closed separation system to determine synthetic food colorants added to food products was investigated. The CZE separations were carried out in a 300-micron-i.d. capillary tube made of fluorinated ethylene-propylene copolymer. The inner diameter of the capillary tube made it possible to enhance sample loads (100-nL injection volumes) so that 10-300 ppb limits of detection (LOD) values could be achieved for the studied dyes by a photometric absorbance detector operating at a 254-nm detection wave-length. With the exception of erythrosine (which exhibited a residual adsorption), very good reproducibilities of the determination were typical for 4- and 32-ppm concentrations of the dyes. This rapid CZE procedure (migration times of the resolved analytes were between 2.5 and 10.5 min) provided good selectivities in the determination of the dyes in various food matrices (soft drink concentrates, liqueurs, and chewing gums). Simple sample preparation steps were effective for the sample matrices used in the investigation.