W.Th. Kok

Capillary electrochromatography with 1.5 μm ODS-modified non-porous silica spheres

SummaryThe particle realization of electrochromatography on capillaries packed with 1.5 μm ODS-modified non-porous silica spheres is demonstrated. In order to realize stable separation conditions it is crucial to add sodium dodecylsulphate (SDS) to the mobile phase and to pressurize both buffer vials at 10 bar. The presence of SDS stabilizes the current and makes the electro-osmotic flow in the packing more uniform so that no air bubbles are generated at high field strengths. The capillary columns are extremely efficient and on a 24 cm long column about 120,000 plates can be generated (a reduced plate height of about 1.3). The columns are very stable and no loss in efficiency was found after using a column continuously for two months.

Determination of aflatoxins in cattle feed by liquid chromatography and post-column derivatization with electrochemically generated bromine

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Amperometric detection of amino acids in high-performance liquid chromatography with a copper electrode

A copper electrode has been used as an amperometric detector for amino acids in high-performance liquid chromatography. The amino acids are separated in a reversed-phase system, using silica-based and polymer-type column materials. Neutral or alkaline buffer solutions of phosphate and carbonate can be used as mobile phases. Borate buffers are less suitable. The detection method is characterized by a high linear dynamic range, good reproducibility, the absence of electrode poisoning and a sensitivity comparable to that of UV absorption methods after derivatization of the amino acids. Detection limits with conventional-scale columns are in the range 10-100 pmoles. A reduction in the flow-rate in the flow-through cell improves the sensitivity for amino acids that give relatively low signals, such as proline. Therefore, the use of microbore columns is especially advantageous for these compounds. The absolute detection limits decrease by about one order of magnitude on changing to a miniaturized system.

Determination of thiols by capillary electrophoresis with electrochemical detection using a palladium field-decoupler and chemically modified electrodes

Abstract Off-column electrochemical detection has been used for the determination of cysteine and related compounds after separation by capillary electrophoresis. For the decoupling of the high-voltage field, an improved palladium decoupler was developed with a dead volume of less than 10 nl. Micro-electrodes, bulk-modified with cobalt phthalocyanine, were used in a nanoliter-volume cell for detection of the thiol compounds. With a detection potential of +0.6 V vs. Ag/AgCl a high selectivity and sensitivity was obtained. Baseline separation of cysteine, homocysteine and glutathione could be realized in 25 min, using a formate buffer with pH 2.7 as background electrolyte. Detection limits of 0.5 μmol l −1 for homocysteine, 1 μ mol l −1 for cysteine and 5 μ mol l −1 for glutathione were obtained. The concentrations of free cysteine and total cysteine (after treatment with dithiothreitol) in urine samples could be measured.

Use of conventinal instrumentation with microbore column in high-performance liquid chromatography

A method to determine extra-column band broadening in microbore high-performance liquid chromatography (HPL) (column diameter, ca. 1 mm) is developed. The extra-column band broadening of several commercially available UV detectors is compared to that of a home-made electrochemical detector. A trace-enrichment method for microbore HPLC is presented and applied to the determination of a series of aromatic nitro compounds. The effect of miniaturization of HPLC on detection limits is discussed.

Amperometric detection of amino acids with a passivated copper electrode

Abstract The amperometric behaviour of a copper electrode towards amino acids is studied by means of a rotating disc electrode. A theoretical model describing the anodic background current in a buffer solution and the increase of the current caused by amino acids is checked experimentally. The influences of the amino acid concentration, the rotation speed of the electrode and the composition of the buffer solution are studied. It is proved that chemical dissolution of a passivating film on the electrode surface, which is enhanced by the complexation reaction between the amino acid and copper(II) ions, is the principle of the phenomena observed. The applicability to flow systems is demonstrated.

Determination of aromatic amines in water samples by capillary electrophoresis with electrochemical and fluorescence detection

Two capillary electrophoresis methods have been compared for the determination of aniline derivatives in environmental water samples. With the first method the anilines were separated as cations by free zone electrophoresis at low pH, and detected by amperometry. For this, the separation capillary was connected through a palladium field decoupler to an electrochemical detection cell which had been modified to match the volume scale of the separation. Most anilines tested, except chlorinated compounds, could be detected with full sensitivity at a detection potential of +0.7 V. Detection limits with this detection scheme were on a low microg/l level. The alternative method involved the derivatization of the anilines with fluorescamine, the separation of the derivatives formed by micellar electrokinetic chromatography, and fluorescence detection. For detection a lamp-based, fibre optics instrument was used. Detection limits with fluorimetry were comparable with those obtained with amperometric detection (in the order of 1 microg/l). Still, this method was preferred since it gave a higher separation efficiency and shorter analysis times (approximately 4 min). The most important argument, however, was its higher reliability and ease-of-handling. Preliminary experiments with water samples collected in areas where pollution with anilines may be expected showed that the method is highly specific, with few interferences showing up in the electropherograms.

Retention behaviour of amylopectins in asymmetrical flow field-flow fractionation studied by multi-angle light scattering detection

Asymmetrical flow field-flow fractionation (FFF) with multi-angle light scattering (MALS) detection was applied for the fractionation of amylopectins from four different sources. Samples originated from genetically modified potatoes and waxy maize. Amylopectins were dissolved in a 1 mol l(-1) sodium hydroxide solution or water. With an injected mass of 0.2 microg, well below overloading conditions, a decrease of the apparent hydrodynamic radius with increasing inlet flow-rate was observed. Moreover, a decrease of the radius of gyration with increasing elution volume was recorded by the MALS detector. Steric/hyperlayer effects are a feasible explanation for this behaviour. The observed radius of gyration at the steric inversion point was in the order of 0.3 microm, which is smaller than the theoretically calculated inversion point. Apparently, the amylopectin behave as macromolecules with a larger hydrodynamic radius than expected on basis of their radius of gyration and are subjected to significant lift forces. The results were confirmed by four fractionations with varying flow-rates but constant ratio of cross to outlet-flow. In contrast to the normal mode operation, the retention of the amylopectins depended strongly on the applied flow-rates and was close to that of a much smaller 10 kDa dextran. Apparent molar masses in the order of between 10(7) and 10(9) g mol(-1) were obtained. The results are contrasted with enzymatically degraded and oxidised starch samples that were fractionated in the normal mode.

Capillary electrochromatography with 1.8-μm ODS-modified porous silica particles

The feasibility of capillary electrochromatography with 1.8-μm ODS-modified porous Zorbax particles was studied. It appeared not possible to prepare stable frits from this material by in situ sintering; therefore, frits were made from another packing material. Addition of sodium dodecyl sulfate to the mobile phase was not necessary for operation of the columns without bubble formation. The columns could be operated with field strengths up to 125 kV m−1. The effect of the mobile phase composition on electroosmotic mobility and plate heights was investigated. The electroosmotic mobility was almost constant in the pH range from 6 to 9, but declined steeply to half the value in the pH range 3–5. The ionic strength of the mobile phase had only a small effect on the plate heights and electroosmotic flow. Efficiencies up to 310 000 plates m−1 (h=1.7) were obtained with 80% (v/v) acetonitrile in the mobile phase. Compared to previous experiments with 1.5-μm non-porous ODS modified particles, capacity factors were 12–15-times higher, while the observed loading capacity of the particles was approximately 50-times larger.

Change of pH in electrophoretic zones as a cause of peak deformation

Electromigration dispersion (EMD) was studied theoretically with comparison of the results to experimental findings. The EMD behavior of a sample constituent in a given background electrolyte (BGE) could be described by an EMD constant, which determines uniquely the direction and the degree of a peak deformation into a triangular shape in a strong or a weak ion BGE system in capillary zone electrophoresis (CZE). The EMD constant was found to be proportional to the linear sum of the relative change of the electric field strength (conductivity effect) and the relative change of the effective mobility of the sample constituent (pH effect) across the steep boundary between sample and BGE zones. Based on the moving boundary model or equations, the two effects, as well as the EMD constants for different BGE types, can be calculated separately. Analytical solutions for those effects were also obtained for some simple cases. Computational results have shown that the conductivity effect and the pH effect for an analyte can be quite different with different BGE types. In some cases, the effects of conductivity and pH on EMD act in the same direction, and reinforce the peak broadening. In other cases they act in opposite directions, and therefore counteract each other partially or completely, leading to a relatively symmetric and narrower peak. To compare the contribution of each dispersion source, a variance of EMD, which is proportional to the EMD constant and time, was defined. It was found that the total variance of a peak can be approximated as the sum of the variance due to EMD and the variances due to other dispersion sources.