Vlasta Madajová

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.

Capillary zone electrophoresis of complex ionic mixtures with on-line isotachophoretic sample pretreatment

Separation modes provided by the column-coupling configuration of the separation unit in an on-line combination of capillary isotachophoresis (ITP) with capillary zone electrophoresis (CZE) were studied from the point of view of their potential in the (trace) determination of ions present in complex ionic matrices. Urine was arbitrarily chosen as such a matrix while sulphanilate and 3,5-dinitrosalicylate (currently not present in urine) served as model analytes. In one of these modes, ITP was employed to remove only the most abundant sample constituent (chloride) and concentrate the rest of those migrating between the leading and terminating zones for injection into the ZE stage. In the other mode, ITP was employed for maximum sample clean-up. Here, only the analyte(s) with a minimum of the matrix constituents was transferred for a final separation in the ZE stage. The fraction to be transferred was defined via a pair of discrete spacers added to the sample. Although a highly efficient sample clean-up was typical in this instance, the use of identical migration regimes in both stages (the separations according to ionic mobilities) did not prove the resolution of one of the analytes (sulphanilate) from the matrix constituent(s) in the ZE stage. A considerable improvement in this respect was achieved easily when the ITP clean-up was based on the separation according to pK values while the constituents present in the transferred fraction were finally separated via differences in their ionic mobilities. This two-dimensional approach provided a way to achieve a 150 ppb (10−6 mol 1−1) concentration detection limit for sulphanilate in a 1-μl volume of urine taken for the electrophoretic run.

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.

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.

Role of the charge number of the counter-ionic constituent in the separation of anions by isotachophoresis

Abstract The influence of the different charge numbers of the ionic forms of the buffering counter-ionic constituents on the effective mobilities of anions at pH 6.0 was investigated. It is shown that a proper choice of the charge number of the counter-ionic constituent can be used as an effective tool in optimization of the operating conditions in the separation of anions by isotachophoresis. The successful separation of a group of anions at pH 6.0 using 1,3-bis[tris(hydroxymethyl)methylamino]propane as the buffering counter constituent in the leading electrolyte (which could not be performed at this pH when other constituents were used for this purpose) illustrates the practical possibilities of this approach. Ca 2+ (a complex-forming cation) and doubly protonated diaminopropane (a non-complexing cation) were used as co-counterions in the leading electrolytes to show the different natures of their interactions with the same group of anions.

Determination of sorbic acid in food products by capillary zone electrophoresis in a hydrodynamically closed separation compartment

Abstract A simple, selective and rapid capillary zone electrophoresis method for the determination of sorbic acid in food products is described. The determination was carried out in a hydrodynamically closed separation compartment at pH 5.2 and for the samples taken into this study (soft drinks, wine, fruit and juice concentrates, margarine and marmalade) only simple sample preparation procedures were needed. A very good reproducibility in the migration time of the analyte (R.S.D. = 0.61%) was achieved and as could be expected no negative effect of matrix constituents on this performance parameter was detected. The recoveries of sorbic acid in the spiked samples ranged from 98-102% and 2-3% R.S.D.s in parallel determinations of the acid were typical.

Capillary zone electrophoresis in a hydrodynamically closed separation system with enhanced sample loadability

Some phenomena linked with capillary zone electrophoresis (CZE) performed in a hydrodynamically closed separation system with enhanced sample load capacity via the use of capillary tubes of larger I.D.s were studied. Calculations of the plate heights for varying amounts of the analytes loaded onto 50 and 300 μm I.D. columns under identical CZE separating conditions revealed that the analytical advantages of using columns of larger I.D. include significantly reduced contributions of electromigration dispersion. At higher concentrations of the carrier electrolytes this gain, however, can be partially lost due to increased thermal dispersive effects. Calculated resolutions for a varying ratio of a pair of the analytes loaded onto 50 and 300 μm I.D. columns favoured the use of the latter I.D. in situations when the ratio of the analytes was higher than ca. 102:1. CZE experiments were carried out in a 300 μm I.D. capillary tube made of fluorinated ethylene–propylene copolymer (FEP) with a porous cellophane membrane serving as a hydrodynamic barrier to prevent a flow of the solution in the separation compartment due to a pressure difference between the electrode vessels. Movement of the membrane was found to be a source of undesired flows in the separation compartment, which adversely affected both the reproducibilities of migration times of the analytes and their separation efficiencies. Mechanical support eliminated these problems. Dispersive phenomena associated with electroosmosis in the closed separation compartment were effectively suppressed by using high molecular weight derivatives of water soluble polymers (methylhydroxyethylcellulose and polyethyleneglycol) in the carrier electrolyte solutions. Examples from the separations of various groups of analytes (synthetic food colourants, some inorganic anions, alkali and alkaline earth metal cations and glycoforms of erythropoietin) are given to illustrate a practical utility of the studied CZE approach.

Analysis of asulam in soil by isotachophoresis and liquid chromatography

Abstract Capillary isotachophoresis, preparative capillary isotachophoresis and high-performance liquid chromatography were tested for the determination of the herbicide asulam in soil. Different configurations of analytical systems based on these techniques were evaluated with respect to detection limits, recovery and reliability of quantitative analysis. While capillary isotachophoresis used alone after a simple sample pre-treatment permitted reliable quantitation at concentrations down to 0.5 ppm, the use of preparative capillary isotachophoresis for the sample cleanup before the final isotachophoretic or chromatographic analyses gave, in general, much better results. A combination of preparative and analytical isotachophoresis permitted the reliable quantitation of asulam at fortification levels of 0.1–0.2 ppm with reliable detection at a concentration of 0.02 ppm. Similar results were achieved for the combination of preparative isotachophoresis and liquid chromatography. The recoveries of the complete analytical procedures were in the ranges 81–91% (0.2 ppm) and 95–120% (0.02 ppm).

Preparative capillary isotachophoresis as a sample pretreatment technique for complex ionic matrices in high-performance liquid chromatography

Abstract Preparative capillary isotachophoresis (ITP) was studied for sample pretreatment in the high-performance liquid chromatography (HPLC) of ionogenic analytes present in complex ionic matrices (urine and humic substances). Sulphanilate, methyl (4-aminobenzenesulphonyl)carbamate (asulam), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) served as model analytes. A high sample load of the ITP pretreatment was achieved by performing the preparative separations in tubes of 2.0 and 1.0 mm I.D. in the column-coupling configuration of the separation unit. The ITP separation according to ionic mobilities was combined with gradient elution HPLC in the ion-suppression mode to achieve highly dissimilar (orthogonal) separation systems in both techniques. The pretreatment provided the sample fraction for the HPLC analysis containing in addition to the analyte (sulphanilate) only ca. 2–3% of the urine matrix (spread along the complete elution profile) when a pair of discrete spacers defined the trapped constituents. Under these conditions the limit of detection for sulphanilate in urine could be reduced by more than two orders of magnitude. A high recovery of the pretreatment procedure [99 ± 1.5% for a 1.7 ppm (w/w) concentration of sulphanilate] was typical. For asulam, 2,4-D and MCPA present in a humic matrix it was shown that the ITP pretreatment may also be effective for multi-residue procedures while favourable analytical characteristics of the pretreatment such as recovery and efficient sample clean-up are maintained.

Photometric detection at 405 nm in trace analysis by capillary isotachophoresis

The possibilities of photometric detection at 405 nm were investigated for trace isotachophoretic analysis of various ionic constituents. Increased selectivity of the detection enabled, for example, the detection and determination of nitrophenols at 10(-8) M in a 25-microliter sample. 5-Nitrofurylacrylic acid, a wine stabilizer, could be detected with confidence at ca. 0.1 ppm in wine without any sample pretreatment. Approximately 250-fmol amounts of amino acids, labelled with 2,4-dinitrophenyl, were detectable with the technique employed. This is almost three decades lower in comparison to what is achievable with an high resolution universal detector. The adsorption of the analytes in the sample handling devices as well as in the separation compartment were problems in the analysis at this concentration level. The detection limit given above was achieved when these phenomena were suppressed by the addition of naphthalene-1,3,6-trisulphonate or pyrophosphate to the sample solution.