Tpem Theo Verheggen

High-performance zone electrophoresis

An experimental approach to high-performance zone electrophoresis is given. It is shown that dispersion can be well controlled by the use of narrow-bore tubes made of chemically and electrically inert materials. The asymmetric concentration distributions that are frequently obtained in free zone electrophoresis are the result of migrational dispersion. This asymmetry only can be suppressed by the application of very small amounts of sample. High-performance separations with UV and conductimetric detection are shown. The time of analysis can be reduced to a few minutes by selecting the appropriate operational conditions. Plate heights smaller than 10 μm can easily be obtained.

Concentration distributions in free zone electrophoresis

Abstract The effect of electrophoretic migration on the concentration distributions in free zone electrophoresis is evaluated using a non-diffusional model. It is shown that sample constituents that have a mobility higher than that of the carrier constituent migrate with a concentration distribution that is diffuse at the front and sharp at the rear of the zone. The reverse holds for sample constituents that have a mobility lower than that or the carrier constituent. The conditions at which diffusional and migrational dispersion are of the same order of magnitude are discussed. It is shown that by a proper choice of operational conditions the adverse effect of a relatively large sample width can be reduced. Problems concerning retention behaviour and separability are discussed.

Determination of substances at low concentrations in complex mixtures by isotachophoresis with column coupling

Abstract A system is discussed that makes use of two PTFE narrow-bore tubes with different internal diameters. In the pre-separation narrow-bore tube, which has the larger internal diameter, a high preparation current is permitted. At a well defined distance from a conductivity detector (mounted in this pre-separation narrow-bore tube) the final separation narrow-bore tube, which has the smaller internal diameter, is coupled to the pre-separation narrow-bore tube via a T-piece. The smaller internal diameter of the final narrow-bore tube permits work at a higher current density during final separation and detection by means of conductivity and UV absorption detectors. The zones of interest can easily be separated from the sample zones, migrating isotachophoretically in the pre-separation narrow-bore tube via the detector. There are several advantages over conventional isotachophoretic equipment: (i) a high sample load is permitted; (ii) the analysis time is increased negligibly; (iii) high ratios of concentrations between the sample species we permitted; and (iv) different operational systems can be applied in separation and preparation compartment.

Effect of electroosmosis on detection in isotachophoresis

Abstract The effect of electroosmosis on the relative sharpness of the zone boundaries and hence the detection limit in isotachophoresis is discussed. A high concentration and a low pH of the leading electrolyte were found to be favourable. The magnitude and sign of the ζ-potential of the capillary wall was measured by streaming potential determinations as a function of concentration, pH and additives. Poly(vinyl alcohol), hydroxyethylcellulose and hydroxypropylmethylcellulose decrease the ζ-potential sufficiently, whereas cationic detergents reverse its sign and thus the direction of the electroosmotic flow. Both were found to be favourable in anionic separation. Fused-silica capillaries and the effect of methanol were also investigated.

Use of a double-detector system for the measurement of mobilities in zone electrophoresis

A double-detector system with which mobilities can be determined by capillary zone electrophoresis is described. Ionic species are detected by two detectors, mounted at a fixed distance from each other. In zone electrophoresis the velocity of an ionic species is proportional to its mobility. The time needed for an ionic species to pass both detectors is reversely proportional to that mobility, provided that the electric field strength is constant. From the ratio of the times required by a sample ionic species and a standard ionic species (with a known mobility) to pass from the first to the second detector, the mobility of the sample ionic species can be calculated. During the analyses parameters such as temperature, pH and ionic strength are constant, which simplifies the calculations substantially. From experiments with some standard ionic species, mobilities have been obtained comparable to those in the literature with a relative standard deviation of about 1%.

Feasibility of capillary zone electrophoresis with suppression of electroendosmotic flow in completely closed systems

Instrumental aspects of capillary zone electrophoresis in closed systems are reported. The combination of fixed-volume sample introduction, utilization of double-beam UV absorbance detection and the use of fixed membranes for the separation of the electrode reservoirs from the separation capillary proved to be a reliable instrumental configuration for capillary zone electrophoresis. Dispersion of eluted zones was studied as a function of capillary length. Repeatabilities of migration times and peak areas were determined. Several examples of separations of mixtures of organic acids and nucleotides are given. The separation of diluted and ultrafiltrated serum from patients with chronic renal failure proved to be promising.

Simple sampling device for capillary isotachophoresis and capillary zone electrophoresis

A simple sampling device for isotachophoresis and zone electrophoresis is described, whereby the sample solution is introduced directly into a broadened part of the capillary tube by two feeders, placed perpendicularly to the capillary tube. The advantage of this sampling device is that with the absence of moving parts, automation of the sample introduction can be carried out in an simple way, cleaning is simple and the device is inexpensive. Experiments showed that a reproducibility of less than 2% can be obtained, including the day to day variations using different sample solutions.

ISOTACHOPHORESIS IN NARROW-BORE TUBES INFLUENCE OF THE DIAMETER OF THE SEPARATION COMPARTMENT

The importance of the inside diameter of the separation compartment of electrophoretic equipment in which tubes are used in achieving optimal stabilization against conventive disturbances and for limiting the temperature increase is demonstrated. Both from a theoretical point of view and experimentally, smaller inside diameters than are commonly used are recommended. Results obtained with thermometric, conductivity and UV absorption detectors in narrow-bore tubes with inside diameters of ca. 0.2 mm are compared.

High resolution isotachophoresis by means of direct conductivity measurements with miniature sensing electrodes

Abstract A new detection system for analytical isotachophresis, the conductivity detector, is described. This detector excels in its high resolving power, by means of which the time required for analysis is reduced enormously and the detectable amount of sample is decrease by a factor of at least 10. A possible explanation is give for most artifacts that are connected with this method of detection.

DETERMINATION OF HEAVY METALS BY ISOTACHOPHORESIS

The suitability of isotachophoresis for the analysis of metals in, e.g., environmental samples was studied. In a cationic operational system the heavy metals Fe, Cu, Ni, Cd, Co, Zn, Pb and Mn were simultaneously determined. The separation was achieved through complex formation with one of the counter ions, hydroxyisobutyric acid. The other counter ion was acetic acid, the leading ion was 0.02 M potassium or sodium (pH 4.1) and the terminator was H+. The analysis time was 15 min at 60 microA in a 0.2 mm I.D. capillary. Aqueous samples containing ppm and ppb amounts were enriched on a cation exchanger with an extremely low affinity for sodium (Chelex 100). Good recovery, linearity, precision and accuracy were obtained even down to the ppb range. Although the sensitivity of the method is not greater than that of some of the more established methods for the individual metals, a great advantage of isotachophoresis is the simultaneous determination of the metals, with equal response factors. An example is given of the determination of metals, including aluminium, in serum.