Fm Frans Everaerts

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 absolute mobilities, pK values and separation numbers by capillary zone electrophoresis : effective mobility as a parameter for screening

Migration times or apparent mobilities can never be used for the identification of ionic species in capillary zone electrophoresis if an electroosmotic flow (EOF) is present, because the velocity of this flow varies considerably with the “state” of the capillary. From the migration times of the EOF and the ionic species, the effective mobilities can be calculated. These effective mobilities are nearly independent of the concentrations of the sample ionic species. Although a large excess of one of the sample components can cause different values of the calculated effective mobility, they are reproducible if the matrix has a constant composition and in this way effective mobilities can be used for screening purposes. In the determination of effective mobilities the use of a “true” EOF marker is extremely important. If effective mobilites are measured in two different electroyte systems at different pH values, at which the degrees of dissociation differ sufficiently, the absolute ionic mobilities and pK values of ionic species can be calculated. Values obtained in this way, for mobility and pK were compared with data obtained isotachophoretically, showing good agreement. Theoretically, the separation number in zone electrophoresis, defined as the number of components that can be separated within a unit of mobility, varies widely with the mobilities of the ionic species and the EOF. Experimentally obtained values of the separation number are significantly lower than the calculated values owing to the method of injection, temperature effects during analysis and amount of sample. For low-molecular-weight ionic species separations are possible if the effective mobilities differ by about one unit for cations and 0.2–0.3 for anions. A negative wall charge (at higher pHs) diminishes the separation number of cations considerably, especially on applying small diameter capillaries, owing to attractive forces between the wall and analytes.

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.

Quantitative analysis in capillary zone electrophoresis with conductivity and indirect UV detection

An interesting point in quantitative capillary zone electrophoresis, when applying conductivity detection or indirect UV detection with non-UV absorbing components, is the existence of a relationship between effective mobilities and peak area, independent of the kind of ionic species. This relationship is theoretically considered for fully ionized monovalent ions resulting in a linear relationship, passing through the origin, between temporal peak area and the product of a correction factor (dependent only on the effective mobilities of the ionic species) and migration time for an equimolar sample composition. A good correlation between theory and practice could be established by applying experimental measured data.

Diffusion coefficients of oxygen, hydrogen peroxide and glucose in a hydrogel

For the design of a new glucose sensor, a knowledge of the diffusion of all participating compounds is needed. A rotating disc electrode covered with hydrogel layer was used to determine the effective diffusion coefficients (Deff) of oxygen, hydrogen peroxide and hydroquinone in a hydrogel, which is used in the sensor. Measurements were carried out under steady-state conditions. The three compounds appeared to be slowed by the gel to the same extent. A comparison was made between the Deff values of glucose and hydroquinone by simultaneous diffusion through a hydrogel membrane. In this case glucose diffusion was slowed to a larger extent than hydroquinone diffusion. The effect, however, was independent of the degree of cross-linking of the hydrogel.

Determination of aminoglycoside antibiotics in pharmaceuticals by capillary zone electrophoresis with indirect UV detection coupled with micellar electrokinetic capillary chromatography

Aminoglycoside antibiotics can be determined by capillary zone electrophoresis (CZE) with indirect UV detection in the anionic mode with a reversed electroosmotic flow (EOF) by addition of FC 135 to the background electrolyte. The effective mobilities of thirteen aminoglycoside antibiotics were determined as a function of pH. Applying CZE with indirect UV detection in the anionic mode and reversed EOF coupled with micellar electrokinetic capillary chromatography with the cationic surfactant cetyltrimethylammonium bromide, both neutral and charged antibiotics can be determined in combined pharmaceuticals. As an example, neomycin and hydrocortisone were determined in Otosporin eardrops.

Detection of point mutations in DNA using capillary electrophoresis in a polymer network

The use of capillary electrophoresis (CE) in a polymer network for single-strand conformation polymorphism (SSCP) is investigated. SSCP is a method to detect DNA point mutations, essential in the diagnosis of several diseases. The PCR (polymerase chain reaction) amplified p53 gene, a tumour suppressor gene known to be frequently mutated in malignant cells, was subjected to CE analysis. Two single-strand DNA fragments of 372 bp in length differing in only one nucleotide could be separated. We conclude that SSCP using CE in a polymer network is a powerful method for the detection of point mutations in DNA sequences.

Isotachophoresis : The qualitative separation of cation mixtures

Abstract The formulae needed for computations on buffered systems are given. Using these formulae, a computer program was evaluated and calculations were correlated with the results of some experiments. The possibility of the simultaneous separation of cations depends on the differences between the effective mobilities, which can be affected by the choice of pH, counter-ion and solvent. The qualitative simultaneous separation of cations was studied in eight electrolyte systems using methanol and water as solvents. In each system, simultaneous separations of 7–10 cations were possible, which could be increased by combining electrolyte systems.