Capillary electrophoresis separations on a planar chip with the column-coupling configuration of the separation channels

Some basic aspects of capillary electrophoresis (CE) separations on a poly(methyl methacrylate) chip provided with two separation channels in the column-coupling (CC) configuration and on-column conductivity detectors were studied. The CE methods employed in this study included isotachophoresis (ITP), capillary zone electrophoresis (CZE), and CZE with on-line ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed, and electrophoresis was a dominant transport process in the separations performed by these methods. Very reproducible migration velocities of the separated constituents were typical under such transport conditions, and consequently, test analytes could be quantified by various ITP techniques with 1-2% RSD. The CC configuration of the separation channels provides means for an effective combination of an enhanced load capacity of the separation system with high detection sensitivities for the analytes in concentration-cascade ITP separations. In this way, for example, succinate, acetate, and benzoate could be separated also in instances when they were present in the loaded sample (1.2 microL) at 1 mmol/L concentrations while their limits of detection ranged from 8 to 12 micromol/L concentrations. A well-defined ITP concentration of the analyte(s) combined with an in-column sample cleanup (via an electrophoretically driven removal of the matrix constituents from the separation compartment) can be integrated into the separations performed on the CC chip. These sample pretreatment capabilities were investigated in ITP-CZE separations of model samples in which nitrite, phosphate, and fluoride (each at a 10 micromol/L concentration) accompanied matrix constituents (sulfate and chloride) at considerably higher concentrations. Here, both the concentration of the analytes and cleanup of the sample were included in the ITP separation in the first separation channel while the second separation channel served for the CZE separation of the ITP pretreated sample and the detection of the analytes.

New leading electrolyte for the direct determination of chloride and other anions in analytical isotachophoresis.

In a number of experiments it was shown that the dithionate ion possesses a higher effective mobility than the chloride ion in aqueous solution, thus enabling the direct and simultaneous isotachophoretic determination of chloride and other anions. Using an acid-base titration, we found only one pKa value for dithionic acid, which is in contrast to the two pKa values stated in the literature. Based on this pKa value, theoretical calculations and the experimentally observed effective mobility of the dithionate ion indicate a higher effective mobility compared to the chloride ion from pH 3. Taking into account the physico-chemical properties of the dithionate, its unrestricted use as isotachophoretic leading ion was confirmed. Based on the dithionate ion, new electrolyte systems for the determination of chloride were used. One system was optimised for the determination of chloride and other low-molecular-mass anions and applied to the analysis of waste water and drinking water. The water samples were analysed in parallel by ion chromatography and compared with the isotachophoretic results.

Polyvinylpyrrolidone-coated silica packings for chromatography of proteins and peptides.

Polyvinylpyrrolidone (PVP)-coated silica sorbents were synthesized by interaction of a copolymer of vinyl-methyldiethoxysilane and vinylpyrrolidone with LiChrospher Si 300 and LiChrospher Si 500 silicas. The coating procedure retains the wide-pore structure of the starting silicas, as was shown by nitrogen adsorption, mercury porosimetry and inverse size-exclusion chromatographic measurements. Good selectivity and separation ability of the synthesized packings toward proteins and peptides were demonstrated in the hydrophobic interaction chromatographic mode of separation. Aromatic compounds undergo a specific interaction with bonded PVP chains which can be used for the preconcentration or selective recovery of polycyclic aromatic hydrocarbons.

Isotachophoretic determination of phosphate splitting from Amifostine and p-nitrophenyl phosphate in serum and neuroblastoma cells.

Amifostine [WR-2721; H2N-(CH2)3-NH-(CH2)2-S-PO3H2] is used as a protecting agent in the chemotherapy of neuroblastoma. It is supposed that Amifostine will be transformed into its active form, the free thiol (WR-1065), easier by normal cells than by tumour cells. Analytical capillary isotachophoresis was used to determine the dephosphorylation of Amifostine in serum and on neuroblastoma cells and peripheral blood cells. Furthermore, the biological effects of Amifostine and its free thiol, on cell proliferation of neuroblastoma cells were measured in combination with Carboplatin. It was found that neuroblastoma cells did not split phosphate less efficiently than normal peripheral blood cells. Furthermore, neither Amifostine (as expected) nor the free thiol (not expected according to the theory) were able to inhibit the effects of Carboplatin. Therefore, the current hypothesis concerning the mode of action of Amifostine must be questioned.

High-performance liquid chromatographic determination of hormonal peptides and their fluorenylmethoxycarbonyl derivatives.

Reference methods for the quantitation of peptide and protein hormones in blood are urgently needed and high-performance liquid chromatography (HPLC) is potentially applicable. Owing to the low concentrations of these substances in body fluids, very low detection limits have to be achieved. This can be done on the one hand by reducing the diameter of the separation column and increasing the number of theoretical plates, and on the other by derivatization. Angiotensin II was chosen as a model compound. Reduction of the inner diameter from 4 to 2 mm increased the peak height by a factor of 3.4 (theoretical value 4.0). The peptide was derivatized with 9-fluorenyl methylchloroformate in lithium carbonate-sodium hydrogencarbonate or sodium borate buffer at different pH values. The precision (coefficient of variation) was 10.4%, the linear range 1:40 (r = 0.998) and the detection limit 500 fmol of derivative on-column. The volume injected was 2 microliter. However, this is not sufficiently sensitive for the quantitation of most peptide hormones using an acceptable maximum specimen volume of 5 ml of serum or plasma.

Polymer support synthesis. XV. Behaviour of non-porous surface-coated silica gel microbeads in oligonucleotide synthesis.

Non-porous silica gel microbeads of diameter 1.5 microns have been investigated as supports for oligonucleotide synthesis. In the preparation of oligothymidylates of chain length up to 150 bases, with 5'-di-p-anisylphenylmethyl-3'-phosphoramidite as an intermediate, the average yields per chain elongation were up to 99%. Lower overall yields were observed in the case of a support which developed a strong tendency towards aggregation after the build up of an oligonucleotide coating.