Gerard J. M. Bruin

Integrated Capillary Electrophoresis on Flexible Silicone Microdevices : Analysis of DNA Restriction Fragments and Detection of Single DNA Molecules on Microchips

Microchips for integrated capillary electrophoresis systems were produced by molding a poly(dimethylsiloxane) (PDMS) silicone elastomer against a microfabricated master. The good adhesion of the PDMS devices on clean planar surfaces allows for a simple and inexpensive generation of networks of sealed microchannels, thus removing the constraints of elaborate bonding procedures. The performance of the devices is demonstrated with both fast separations of φX-174/HaeIII DNA restriction fragments labeled with the intercalating dye YOYO-1 and fluorescently labeled peptides. Detection limits in the order of a few zeptomoles (10(-)(21) mol) have been achieved for each injected DNA fragment, corresponding to a mass detection limit of ∼2 fg for the 603 base pair fragment. Single λ-DNA molecules intercalated with YOYO-1 at a base pair-to-dye ratio of 10:1 could be detected with an uncomplicated laser-induced fluorescence detection setup. High single-molecule detection efficiency (>50%) was achieved under electrophoretically controlled mass transport conditions in PDMS microchannels.

Capillary zone electrophoretic separations of proteins in polyethylene glycol-modified capillaries

Abstract Fused-silica capillaries were wall modified with γ-glycidoxypropyltrimethoxysilane and polyethylene glycol 600 in order to decrease the influence of wall adsorption in capillary zone electrophoretic separations of proteins. It is shown that a significant decrease in adsorption is obtained and electro-osmotic flow is also diminished. For the proteins studied, symmetrical peaks were obtained in the pH range 3–5. However, some adsorption still occurs as the plate numbers are below theoretical expectations. At higher pH values appreciable peak deformations and drastic decreases in resolving power are observed. The procedure allows the rapid and efficient separation of protein mixtures suitable for separation in the indicated pH range, and the coating shows a good stability.

Performance of carbohydrate-modified fused-silica capillaries for the separation of proteins by zone electrophoresis

The walls of fused-silica capillaries were chemically modified with small carbohydrate moieties in order to diminish the wall adsorption of proteins in capillary zone electrophoresis. A diol-type coating, prepared by bonding of gamma-glycidoxy propyltrimethoxysilane to the wall followed by acidic hydrolysis, shows for proteins a similar electrophoretic behaviour as various pH values to a polyethylene glycol (PEG) coating tested previously. Although good peak shapes were obtained for proteins in the pH range 3-5, the efficiency on the diol coating is worse than that on the PEG coating. At higher pH values the peaks are deformed and the efficiency is lost. A maltose coating appears to shield the silica surface well for proteins up to pH 7. The peak shapes of proteins are acceptable, but the efficiency of the maltose coating is smaller than that on the diol coating. The diol coating is stable in the indicated pH range. However, with the maltose coating good stability is obtained only on adding an antimicrobial agent to the buffers.

Electrically driven open-tubular liquid chromatography

Abstract Electrically driven (ED), as opposed to pressure-driven (PD), open-tubular liquid chromatography (OTLC) was evaluated for different types of open-tubular columns, with inner diameters in the range 5–25 μm. The efficiency of ED-OTLC was found to be better than that of PD-OTLC by a factor of ca. 2, in agreement with theory. Injection of the sample in ED-OTLC by electromigration appears to be much simpler than that with the split-injection procedures in PD-OTLC. Electroosmotic mobility was found to depend little on the application of an ODS coating.With the mobile phase used at 60 000 V/m, the maximum linear velocity was 1.4 mm/s. The potential of ED-OTLC in 10–25 μm I.D. capillaries is demonstrated.

Theoretical and experimental aspects of indirect detection in capillary electrophoresis

Abstract Theoretical and experimental aspects of indirect UV detection are considered. Based on a mathematical treatment of the transport of ions through the capillary, resulting in an eigenvector-eigenvalue problem, some guidelines are formulated about how to increase efficiency and sensitivity in an indirect (UV) detection system. Also, the existence of system peaks can be explained properly. An experimental system consisting of seven amino acids as sample ions and salicylate at pH 11.0 as the UV-absorbing ion was chosen in order to compare theoretical and experimental results. Constructed electropherograms, produced with a computer program based on the above-mentioned mathematical treatment, are also presented and compared with experimental electropherograms.

Optimization and evaluation of the performance of arrangements for UV detection in high-resolution separations using fused-silica capillaries

Abstract The evaluation of arrangements for UV measurements in fused-silica capillaries as applied in, e.g., high-performance capillary electrophoresis, capillary liquid chromatography and hydrodynamic chromatography is described. The various designs are characterized with respect to sensitivity, noise, linearity, contribution to the peak width and sensitivity to refractive index effects. Some guidelines are formulated, based on theoretical and experimental observations, in order to maximize the performance. A cell with a focusing lens in front of the capillary resulted in a higher sensitivity and linear range than a cell with an adjustable aperture width. With the U-cell, having a design which is comparable to Z-shaped cells and having a longer longitudinal light path, a substantial increase in signal-to-noise ratio was achieved. Some applications of the UV cells are shown.

Pharmacokinetics, Distribution, Metabolism, and Excretion of Deferasirox and Its Iron Complex in Rats

Deferasirox (Exjade, ICL670, CGP72670) is an iron-chelating drug for p.o. treatment of transfusional iron overload in patients with β-thalassemia or sickle cell disease. The pharmacokinetics and disposition of deferasirox were investigated in rats. The animals received single intravenous (10 mg/kg) or p.o. (10 or 100 mg/kg) doses of 14C-radiolabeled deferasirox. Biological samples were analyzed for radioactivity (liquid scintillation counting, quantitative whole-body autoradioluminography), for deferasirox and its iron complex [high-performance liquid chromatography (HPLC)/UV], and for metabolites (HPLC with radiodetection, liquid chromatography/mass spectrometry, 1H and 13C NMR, and two-dimensional NMR techniques). At least 75% of p.o.-dosed deferasirox was absorbed. The p.o. bioavailability was 26% at the 10 mg/kg dose and showed an overproportional increase at the 100 mg/kg dose, probably because of saturation of elimination processes. Deferasirox-related radioactivity was distributed mainly to blood, excretory organs, and gastrointestinal tract. Enterohepatic recirculation of deferasirox was observed. No retention occurred in any tissue. The placental barrier was passed to a low extent. Approximately 3% of the dose was transferred into the breast milk. Excretion of deferasirox and metabolites was rapid and complete within 7 days. Key clearance processes were hepatic metabolism and biliary elimination via multidrug resistance protein 2. Deferasirox, iron complex, and metabolites were excreted largely via bile and feces (total ≥90%). Metabolism included glucuronidation at the carboxylate group (acyl glucuronide M3) and at phenolic hydroxy groups, as well as, to a lower degree, cytochrome P450-catalyzed hydroxylations. Two hydroxylated metabolites (M1 and M2) were administered to rats and were shown not to contribute substantially to iron elimination in vivo.

Use of a microplate scintillation counter as a radioactivity detector for miniaturized separation techniques in drug metabolism

In miniaturized separation techniques, such as capillary electrophoresis (CE) or capillary liquid chromatography (LC), conventional on-line radioactivity detection of labeled compounds is restricted, because of insufficient sensitivity. It will be shown that a microplate scintillation counter for 96-well plates (TopCount) can be used as a sensitive and easy-to-handle radioactivity detector for capillary LC and CE. The attractive combination of capillary LC, eluent fractionation, and subsequent off-line counting is described. The new method is applied for rapid and sensitive separation and detection of 3H-labeled parent drug and its metabolites at levels between 25 and 700 cpm in rat urine. The advantages of capillary LC coupled to the TopCount, and combined with LC-MS data, can be of benefit in many analytical areas, including the characterization of metabolites at low concentration within complex biological fluids. With the same setup, the fractionation with subsequent off-line counting is equally applicable to CE. This is demonstrated with electrophoretically separated 14C-labeled impurities, nicely resolved from a negatively charged main compound, at low levels.

Post-column reaction detection for open-tubular liquidchromatography using laser-induced fluorescence

Abstract The applicability of post-column reaction detection to open-tubular liquid chromatography is investigated, using the o -phthalaldehyde—amino acid reaction and laser-induced fluorescence detection. Two mixing devices were designed and evaluated in terms of external peak broadening on the nanolitre scale. It was found that mixing devices having a volume of about 10 nl resulted in an acceptable loss in plate numbers for unretained peaks using 25 μm I.D. fused-silica columns. A method was developed for determining the flow-rates and residence times in the various parts of the system, based on the correlation of observed total residence times and the pressures on the column and the reagent delivery system.

Analysis of Oligonucleotides and Antisense DNA Analogs

Single stranded oligonucleotides comprise short strands of both natural and chemically modified nucleic acids. Although there is no fixed terminology, it is generally understood that the length of synthetic oligonucleotides is in the range of 5 to 150 bases. DNA sequencing reaction mixtures contain single stranded oligonucleotides, most often 300 up to more than 1000 bases long. In terms of the separation problem, DNA sequencing mixtures are not at all that different to synthetic oligonucleotides and the same or similar electrophoretic conditions can be used for both types of compounds. However, the strategies for large volume sequencing require a unified approach which includes instrumental parameters such as detection and multiplexing and separation parameters such as speed and resolution. DNA sequencing with CE will be discussed in Chapter 10.