Marián Masár

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.

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.

Isotachophoresis and isotachophoresis‐zone electrophoresis of food additives on a chip with column‐coupling separation channels

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 isotachophoresis (ITP) and ITP-ZE separation and determination of food additives was studied. A group of preservatives and taste intensifying components examined in this study included benzoate, sorbate, p-hydroxybenzoic acid esters (parabens), and glutamate, while various food products and cosmetics represented different matrices (proteins, fat, organic acids, carbohydrates, salts). ITP on the CC chip was found suitable for the determination of glutamate in the food products with only a minimum sample preparation (dilution, filtration). It also provided a rapid and simple procedure for the determination of parabens in cosmetics. On the other hand, ITP experiments with benzoate and sorbate revealed that sample preparations providing high analyte/matrix concentration ratios are essential when these food preservatives are to be determined by ITP on the chip. ITP-ZE combination on the same chip provided a solution to this problem by integrating an efficient ITP sample preparation (concentration of the preservatives and removal of the main part of the matrix), capable of processing μL sample volumes, with a final ZE separation and sensitive detection (low μmol/L limits of detection) of the preservatives. In both ITP and ITP-ZE separations on the CC chip no interference from food matrices was found.

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%.

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 νL sample injection channel of the chip. In addition, bromate was concentrated by a factor of 103 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.

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.

Separation of synthetic food colourants by capillary zone electrophoresis in a hydrodynamically closed separation compartment

Abstract Separation conditions enabling the complete resolution of eleven permitted synthetic food colourants and some of their subspecies by capillary zone electrophoresis (CZE) were determined. Those conditions involve combining a suitable pH of the carrier electrolyte (pH 6.8) with host-guest complexation effects of β-cyclodextrin. A 300 μm I.D. capillary tube made of fluorinated ethylene-propylene copolymer in a hydrodynamically closed separation compartment was used for the CZE separations. The capillary could accommodate 90-nl sample injection volumes, thus providing limits of detection for the dyes of 11–300 ppb using a photometric detector operating at a wavelength of 254 nm. R.S.D.s of 0.4–3.0% were typical for the determinations of the dyes present in samples at 16 ppm concentrations. Erythrosine, exhibiting residual adsorption, gave more-scattered results under identical working conditions (R.S.D. of ca. 9.0%). The utility of this rapid CZE procedure (migration times of the dyes were 2.5–10.5 min) is illustrated for several practical samples, including soft drink concentrate and liqueur and monitoring of the stability of aqueous solutions of indigo carmine.

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).

Separations of inorganic anions based on their complexations with α-cyclodextrin by capillary zone electrophoresis with contactless conductivity detection

Abstract Capillary zone electrophoresis (CZE) separations of inorganic anions based on their host–guest complex equilibria with α-cyclodextrin (α-CD) were investigated. α-CD employed as a host was found to influence selectively the effective mobilities most of the studied anions (chloride, bromide, iodide, sulfate, nitrite, nitrate, fluoride, phosphate). Its complexing ability combined with a low pH of the carrier electrolyte solution provided working conditions suitable for rapid CZE separations of the anions. For example, iodide and chloride present in the injected sample in a concentration ratio of ca. 1:2·103 could be separated in less than 100 s and seven of the above anions (α-CD failed to resolve chloride and bromide) were baseline-resolved in less than 120 s. A high overall selectivity of the carrier electrolytes combining the host–guest equilibria with a low pH in the analysis of highly complex samples is illustrated by CZE separations of inorganic anions present in milk. Here, no interferences to the separations of inorganic anions due to co-migrations of organic acids present in milk were observed also when a 200 nl volume of a diluted (1:10) milk sample was loaded onto the column. A contactless conductivity detector used for the detection of anions in this work proved very reliable. Under our separating conditions it provided for the studied anions concentration limits of detection in the range of 0.5–1.4 μmol/l (a 200-nl sample injection volume) when the separations were carried out in a 300 μm I.D. capillary tube made of polytetrafluoroethylene.

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%.