Fep Frans Mikkers

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.

Isotachophoresis: The concepts of resolution, load capacity separation efficiency II. Experimental evaluation

Resolution, load capacity and separation efficiency in isotachaphoresis are experimentally evaluated and the results are compared with the theoretically expected values. The theoretical and experimental results show good agreement and confirm the reliability of the transient-state model. The importance of the dimensionless separation number and load capacity for the determination and standardization of experimental performance conforms with theory. It is shown that, in view of these two parameters, the pH of the leading electrolyte is the best rationale for optimization, whereas the pH of the sample has only restricted possibilities. Steady-state configurations in which constituents are not migrating in order of decreasing effective mobilities are shown and discussed.

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.

Determination of uric acid in serum using isotachophoresis

An operational system is described for the isotachophoretic determination of uric acid in serum, making use of column coupling. The method has been compared with a standard enzymatic procedure. With the present technique small amounts of serum (ca. 3 microliter) can be applied without any pretreatment. Urate recovery was 99.0-100.5%. Under the non-physiological measuring conditions used, 12-28% of control serum uric acid was bound to macromolecules of molecular weight exceeding 25,000. The day-to-day variations of the isotachophoretic procedure were smaller than those of the enzymatic method, whereas standard deviations were comparable. The isotachophoretic procedure is less influenced by certain metabolites.

Determination of theophylline binding to human serum proteins by isotachophoresis.

Free theophylline was isolated from human serum by ultrafiltration and analysed in a leading electrolyte of 7.5 mM morpholinoethanesulphonic acid with ammediol as a counter ion at pH 8.90 and alpha-alanine as a terminator. The UV (280 nm) absorbance of the theophylline spike between serine and bicine as spacers was integrated. Binding percentages to human pool serum, human albumin and alpha 1-acid glycoprotein (orosomucoid) were determined at physiological concentrations, and found to be 55, 44 and 12%, respectively. The calibration lines were straight from 0 to 30 mg/l, with a standard deviation of 0.2 mg/l. The detection limit was 1 mg/l. The time of analysis was 12 min at 40 microA in a 0.2 mm I.D. capillary.

Analytical isotachophoresis of uremic blood samples.

Uremic blood samples were analyzed for ionogenic substances using analytical isotachophoresis. Multicomponent separations proved that the uremic state shows significant differences from the normal state, especially with regard to anionic low-molecular-weight substances. As a quantitative parameter the ratio of anionic higher-molecular-weight substances to anionic low-molecular-weight substances is proposed: the HL ratio. Separation patterns and HL ratios were studied during nine weeks for one patient on chronic hemodialysis. The patient showed a low HL ratio due to excess of low-molecular-weight substances. Separation patterns before and after hemodialysis showed clear differences and the HL ratio increased. The method of analysis is neither time- nor sample-consuming and sample preparation is not needed. Experimental procedures are easily standardized and results are reliable.

Isotachophoresis of urinary purines and pyrimidines: the use of spacers and enzymes for identification.

An isotachophoretic system is described for the separation and identification of urinary purine and pyrimidine bases and nucleosides. For a better discrimination and interpretation of the UV profiles, well-defined non-UV-absorbing substances were introduced as spacers. Treatment of urine samples with purified enzymes before analysis resulted in specific shifts in the metabolite profiles, providing a sensitive and specific means of identifying a number of metabolites. With an injected volume of 3 microliters (untreated urine diluted 1:5) the present method allows reproducible separations within 20 min of at least twenty different nucleosides and bases.

Direct determination of valproate in serum by isotachophoresis. Comparison with a gas chromatographic method

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