Development of an obidoxime chloride reference material: a metrological approach to the determination of chromatographic purity

A metrological approach to determination of the chromatographic purity of obidoxime chloride and the corresponding obidoxime chloride reference material (RM) with a certified chromatographic purity value have been developed. This value was defined as the ratio of the sum of peak areas of obidoxime chloride isomers to the total peak area of detected substances including impurities (%) under specified HPLC–UV conditions. The RM homogeneity and stability were studied using HPLC with UV detection and evaluated as satisfactory. The certified value calculated from the results of an interlaboratory trial was equal to 99.9% with the expanded uncertainty of 0.6% at the level of confidence 0.95 and the coverage factor 2. The RM certified value, like other results of chromatographic purity determination traceable to the reference measurement procedure, is not traceable directly to the SI mole. However, the results are comparable in metrologically traceable environments, i.e. when relevant measuring laboratory instruments are calibrated with traceability chains to the corresponding SI units. Therefore, the RM can be used as a measurement standard (calibrator) for analytical instruments and as a control sample for quality control of HPLC obidoxime chloride assay results.     

Determining the vibrational pattern via overtone cold spectra: C-H methyl stretches of propyne

Vibrationally mediated photodissociation and photoacoustic (PA) spectroscopy were employed for studying the intramolecular dynamics of propyne initially excited to the first through fourth overtone of methyl C-H stretching modes. Room-temperature PA and jet-cooled action spectra, monitoring the absorption of the parent and the yield of the ensuing H photofragments, respectively, were obtained. The PA spectra exhibit mainly broad features, while the action spectra, due to inhomogeneous structure reduction, expose multiple peaks of recognizable shapes in the differing overtone manifolds. Symmetric rotor simulations of the band contours of the action spectra allowed retrieving of band origins and linewidths. The linewidths of the bands in each manifold enabled estimates for energy redistribution times out of the corresponding states to the bath states, the times ranging from 18+/-6 ps for two quanta of C-H excitation to subpicosecond for five quanta. The data were also analyzed in terms of a normal-mode model and a joint local-/normal-mode model. These models enabled determination of harmonic frequencies, anharmonicities, and interaction parameters reproducing the observed data in all monitored regions and provided spectral assignments. The measured Doppler profiles were well fitted by Gaussians with widths suggesting low average translational energies for the released H photofragments. These low energies and their similarities to those for dissociation of propyne isotopomers preexcited to acetylenic C-H stretches were ascribed to an indirect dissociation process occurring after internal conversion to the ground electronic state and isomerization to allene.     

Persistence of embedded eigenvalues

We consider conditions under which an embedded eigenvalue of a self-adjoint operator remains embedded under small perturbations. In the case of a simple eigenvalue embedded in continuous spectrum of multiplicity m<∞ we show that in favorable situations, the set of small perturbations of a suitable Banach space which do not remove the eigenvalue form a smooth submanifold of codimension m. We also have results regarding the cases when the eigenvalue is degenerate or when the multiplicity of the continuous spectrum is infinite.     

Detection of tetracysteine-tagged proteins using a biarsenical fluorescein derivative through dry microplate array gel electrophoresis

The design of an extended-run 96-well sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and the development of protein detection technology based upon fluorescein derivatives that bind to peptide epitope tags, allows the creation of a truly high-throughput analysis of protein expression, where less than 20 min are needed to separate proteins and analyze results. We demonstrate the overall capabilities of such a method combination in a complex cell lysate background, while comparing the specific results obtained using a biarsenical fluorescein-derivative and tetracysteine epitope-tagged proteins with total protein staining using a fluorescent gel stain and with Western blotting where an anti-oligohistidine (His) tag antibody has been employed. When applied on purified target proteins without extraneous protein background, the demonstrated sensitivity of the assay on the extended-run 96-array precast SDS-PAGE system allows detection of quantities of tagged protein as low as 1 pmol per band.     

Characteristics of migration patterns of DNA oligomers in gels and the relationship to the question of intrinsic DNA bending

We have developed a methodology that is capable of quantitatively describing the electrophoretic mobility patterns of oligomeric B-DNA through polyacrylamide gels (PAG) in the presence of varying concentration of the organic solvent 2-methyl-2,4-pentanediol (MPD), used routinely to induce DNA crystallization. The model includes the ion atmosphere and its polarization, electrostatic excluded volume, hydrodynamic interactions, and fluctuation effects that characterize the overall size of the migrating polyion. Using this model, and by critically examining the mobility patterns of linear random-sequence B-DNA molecules in PAG as a function of MPD, we address the question of the discrepancy between current models used to explain the molecular origins of A-tract-induced DNA bending. Direct analysis of the mobility of B-DNA oligomers on PAG, and comparison to the mobility of A-tract-containing oligomers, shows a significant apparent effect of MPD on the mobility of generic B-DNA sequences, which is larger than the effect on A-tract-containing oligomers. The effect is chain-length dependent, especially at lower MPD concentration. Thus, the apparent reduction in gel mobility, as a function of MPD, is not unique to A-tract regions or A-tract-containing molecules. However, our analysis suggests that MPD molecules are probably excluded from the surface of both B-DNA and A-tract molecules. This is supported by circular dichroism studies on A-tract and B-DNA molecules in solutions containing various MPD concentrations.     

A case study of dynamic visualization and problem solving

This paper reports an example of a situation in which university students had to solve geometrical problems presented to them dynamically using the interactive computerized environment of the ‘MicroWorlds Project Builder’. In the process of the problem solving, the students used ten different solution strategies. The unsuccessful strategies were then classified into three main categories: distracting, reducing and confusing. One student group had to solve the same problem in its non-dynamic version. The results received from both groups were compared and analysed. Analysis of the solution strategies and the process of the categorization revealed that the percentage of success in both groups was similar and in the case of the given problem, the dynamic visual mode of the problem distracted the students’ attention away from proper handling of the solution of the problem.