Evaluation of polymeric methacrylate-based monoliths in capillary electrochromatography for their potential to separate pharmaceutical compounds

Polymeric methacrylate-based monoliths are evaluated in capillary electrochromatography (CEC) and pressurized capillary electrochromatography (p-CEC) for their potential in pharmaceutical analysis. Using a given polymerization mixture as a basis for the monolith synthesis, different mobile phase pH at constant organic modifier concentrations are tested in both CEC and p-CEC. The test set consists of basic, acidic, amphoteric, and neutral compounds, which are mainly pharmaceuticals. Because of the mainly hydrophobic character of the stationary phase, the interactions are largest when the compounds appear in an uncharged state, but some ion-exchange phenomena with negatively charged compounds can also be observed. In CEC, acidic substances are most retained at low pH. For amphoteric and neutral compounds, no preference regarding analyzing pH can be derived from these experiments. For basics, a high pH is chosen, but a reduced solvent strength is needed to enhance the retention of these compounds. The retention mechanism in p-CEC can also be assigned to both hydrophobic and ionic interactions. For acidic, amphoteric, and neutral compounds, acceptable retention is seen. For the basic compounds, the retention with a mobile phase containing 50% organic modifier is low, as in CEC. However, when the organic modifier content in the mobile phase is decreased, retention increases and the selectivity of the stationary phase is more pronounced. This mode of operation presents a possibility for separating some test mixtures, thus some potential for pharmaceutical analysis is seen. More efforts are needed to obtain higher efficiencies and better peak shapes, which might be solved by a further optimization of both the stationary phase synthesis and the mobile phase composition.     

Mössbauer spectroscopy and DFT calculations on all protonation states of the 2Fe-2S cluster of the Rieske protein

The Thermus thermophilus Rieske protein (TtRP) contains a 2Fe-2S cluster with one iron (Fe-Cys) coordinated by four sulfur atoms (2xS^2? and 2xCys) and one iron (Fe-His) by two sulfur and two nitrogen atoms (2xS^2?, His134 and His154). Here, the protein is investigated at three pH values (6.0, 8.5 and 10.5) in order to elucidate the protonation states of the His-ligands. Examination of the effect of protonation on the electronic structure of the cluster via Mössbauer spectroscopy gives a deeper understanding of the coupling of electron transfer to the protonation state of the His-ligands. Two components (1 referring to Fe-Cys and 2 to Fe-His) with parameters typical for a diamagnetic [2Fe-2S]²⁺ cluster are detected. The Mössbauer parameters and the protonation state clearly correlate: while δ remains almost pH-independent with δ ₁ (pH6.0) =?0.23 (±?0.01) mms^??1 and δ ₁ (pH10.5) =?0.24 (±?0.01) mms^??1 for Fe-Cys, it decreases for Fe-His from δ ₂ (pH6.0) =?0.34 (±?0.01) mms^??1 to δ ₂ (pH10.5) =?0.28 (±?0.01) mms^??1. ΔE _Q changes from ΔE _Q1 (pH6.0) =?0.57 (±?0.01) mms^??1 to ΔE _Q1 (pH10.5) =?0.45 (±?0.01) mms^??1 and from ΔE _Q2 (pH6.0) =?1.05 (±?0.01) mms^??1 to ΔE _Q2 (pH10.5) =?0.71 (±?0.01) mms^??1. Density functional theory (DFT)-calculations based on the crystal structure (pdb 1NYK) (Hunsicker-Wang et al. Biochemistry 42, 7303, 2003) have been performed for the Rieske-cluster with different His-ligand protonation states, reproducing the experimentally observed trend.     

High spatial resolution NMR imaging of polymer layers on metallic substrates

High spatial resolution NMR imaging techniques have been developed recently to measure the spatial inhomogeneity of a polymer coating film. However, the substrates of the polymer coatings for such experiments are generally required to be non-metallic, because metals can interact with static magnetic fields B(0) and RF fields B(1) giving rise to artifacts in NMR images. In this paper we present a systematic study on the effects of metallic substrates on 1D profiles obtained by high resolution NMR imaging. The off-resonance effect is discussed in detail in terms of the excitation profile of the RF pulses. We quantitatively show how the NMR signal intensities change with frequency offset at different RF pulse lengths. The complete NMR profiles were simulated using a Finite Element Analysis method by fully considering the inhomogeneities in both B(1) and B(0). The excellent agreement between the calculated and measured NMR profiles on both metallic and non-metallic substrates indicates that the experimental NMR profiles can be reproduced very well by numerical simulations. The metallic substrates can disturb the RF field of the coil by eddy current effect and therefore change the NMR profiles. To quantitatively interpret the NMR profile of a polymer layer on a metallic substrate, the profile has to be divided by the profile of a reference on the same metallic substrate located at the same distance from the coil.     

Magnetoresistance of Fe3O4/Au/Fe3O4 and Fe3O4/Au/Fe spin-valve structures

A detailed study of the in-plane magnetotransport properties of spin valves with one and two Fe₃O₄ electrodes is presented. Fe₃O₄/Au/Fe₃O₄ spin valves exhibit a clear anisotropic magnetoresistance in small magnetic fields but no giant magnetoresistance (GMR). The absence of GMR in these structures is due to simultaneous magnetization reversal in the two Fe₃O₄ layers. By contrast, a negative GMR effect is measured on Fe₃O₄/Au/Fe spin valves. The negative GMR is attributed to an electron spin scattering asymmetry at the Fe₃O₄/Au interface or an induced spin scattering asymmetry in the Au interfacial layers.     

Distortion product otoacoustic emissions provide clues hearing mechanisms in the frog ear

2f1-f2 and 2 f2-f1 distortion product otoacoustic emissions (DPOAEs) were recorded from both ears of male and female Rana pipiens pipiens and Rana catesbeiana. The input-output (I/O) curves obtained from the amphibian papilla (AP) of both frog species are analogous to I/O curves recorded from mammals suggesting that, similarly to the mammalian cochlea, there may be an amplification process present in the frog AP. DPOAE level dependence on L1-L2 is different from that in mammals and consistent with intermodulation distortion expectations. Therefore, if a mechanical structure in the frog inner ear is functioning analogously to the mammalian basilar membrane, it must be more broadly tuned. DPOAE audiograms were obtained for primary frequencies spanning the animals' hearing range and selected stimulus levels. The results confirm that DPOAEs are produced in both papillae, with R. catesbeiana producing stronger emissions than R. p. pipiens. Consistent with previously reported sexual dimorphism in the mammalian and anuran auditory systems, females of both species produce stronger emissions than males. Moreover, it appears that 2 f1-f2 in the frog is generated primarily at the DPOAE frequency place, while 2 f2-f1 is generated primarily at a frequency place around the primaries. Regardless of generation place, both emissions within the AP may be subject to the same filtering mechanism, possibly the tectorial membrane.     

Monolithic porous polymer stationary phases in polyimide chips for the fast high-performance liquid chromatography separation of proteins and peptides

Poly(lauryl methacrylate-co-ethylene dimethacrylate) and poly(styrene-co-divinylbenzene) stationary phases in monolithic format have been prepared by thermally initiated free radical polymerization within polyimide chips featuring channels having a cross-section of 200micromx200microm and a length of 6.8cm. These chips were then used for the separation of a mixture of proteins including ribonuclease A, myoglobin, cytochrome c, and ovalbumin, as well as peptides. The separations were monitored by UV adsorption. Both the monolithic phases based on methacrylate and on styrene chemistries enabled the rapid baseline separation of most of the test mixtures. Best performance was achieved with the styrenic monolith leading to fast baseline separation of all four proteins in less than 2.5min. The in situ monolith preparation process affords microfluidic devices exhibiting good batch-to-batch and injection-to-injection repeatability.