A fluorescent derivatization method of proteins for the detection of low‐level impurities by microchip capillary gel electrophoresis

A novel pre‐chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water‐soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on‐chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver‐stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre‐column derivatization include a shorter analysis time and an increased robustness and ease of use.     

Composition and structure of iron oxidation surface layers produced in weak acidic solutions

Although oxidation/passivation of iron in basic solution has been extensively investigated, there is very little information on iron corrosion in weak acidic solutions. In this work, iron surface composition and structure, produced in aerobic aqueous solutions ranging from pH 2 to 5, were determined in detail by the use of infrared external reflection spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. The most striking observation is that at pH 2 and 3 almost all oxidized iron is dissolved in solution, whereas at pH 4 and 5 the product of iron oxidation is deposited on the iron surface in the form of lepidocrocite, gamma-FeOOH. Detailed iron surface and solution analyses allow the proposition of the following overall oxidation reactions: [EQUATION: SEE TEXT]. At pH 2 and 3, only a very thin surface layer consisting of FeO and Fe(OH)2 with polymeric structure is observed on the iron surface. The amounts of these surface species remain almost constant (2-5 nm) from the first minutes to a few hours of reaction, if pH is kept constant. Nevertheless, with time the akaganeite-like, beta-FeOOH structure is also detected. At pH 4 and 5, the amount of lepidocrocite deposited on the iron surface increases with reaction time. Detailed quantitative evaluation of the lepidocrocite produced at pH 5 and its surface distribution on iron was performed based on the comparison of infrared spectroscopic data with spectral simulation results of assumed surface structures. At pH 4 and 5 and a temperature of 40-50 degrees C, in addition to a very large amount of lepidocrocite other oxy-hydroxide surface species such as goethite (alpha-FeOOH) and feroxyhite (delta-FeOOH), were identified. Addition of Cl- ions to solution at 10(-3) M concentration at pH 5 increases the oxidation rate of iron by about 50%, and lepidocrocite remains the only oxidation product. Similarly, an addition of Fe2+ ions to solution at pH 5 very strongly enhances lepidocrocite formation as well as its conductivity. The latter finding is important for the possible application of metallic iron as a catalyst in redox reactions, for example, for decomposition of difficult-to-biodegrade water pollutants.     

Sodium ibuprofen dihydrate and anhydrous

(R,S)-(±)-ibuprofen sodium salt (racemate) dihydrate (SID) was dehydrated and the physicochemical properties of SID and the anhydrous forms (SIA) were compared by different analytical techniques (scanning electron microscopy, helium pychnometry, differential scanning calorimetry, X-ray powder diffractometry). The dehydration of SID, followed by thermogravimetry in isothermal conditions, allowed to calculate the activation energy of the dehydration process and to predict the mechanism of dehydration. Dehydration occurred in one step and the activation energy was rather low, indicating the ease of water removal from the crystal. The mechanism of dehydration followed a three dimensional diffusion (Jander equation). Similarly to the dehydration, the hydration process was followed under isothermal conditions by exposing the anhydrous powder at 64% RH at different temperatures. The mechanism of hydration was governed by a two dimensional diffusion and the energy associated to the process was very low, indicating the ease of crystal hydration. The driving force for the hydration is higher than that for the dehydration. From a thermodynamic point of view this fact may explain why the hydrated form is more stable than the anhydrous one. Solubilities, determined at different temperatures in water and in phosphate buffer (pH 6.8), showed that SID is more soluble in water than SIA for temperatures higher than approximately 283 K. On the contrary, in phosphate buffer SIA is always more soluble than SID in the temperature range considered for the experiments. Drug release reflects the solubility in water and phosphate buffer previously reported.     

Physicochemical characterization of nicergoline and cabergoline in its amorphous state

Formulation of poorly water-soluble crystalline drugs into their more soluble amorphous form is a common approach for improving their bioavailability. In this study, the amorphous forms of nicergoline (NIC) and cabergoline (CAB) were obtained by different methods (melting and precipitation under solvent evaporation). The physicochemical characteristics of the samples were determined by HPLC, differential scanning calorimetry (DSC), thermogravimetry, and X-ray powder diffractometry. The physical stability of the amorphous forms was investigated by DSC by considering how the onset temperature and the enthalpy content at the glass transition vary with aging time and temperature. Using the Kohlrausch–Williams–Watts equation on the data obtained from the experiments, the “mean molecular relaxation time constant” (τ) was estimated. This parameter was used to understand the stability of NIC and CAB in their glassy state at different temperatures, and results showed that their stability is adequate to enable the formulation of these drugs into solid dosage forms.     

Sucrose cryo-protection facilitates imaging of whole eye sections by MALDI mass spectrometry

Sucrose is used as a cryo-preservation agent on large mammalian eyes post formalin fixation and is shown to reduce freezing artefacts allowing the collection of 12-μm thick sections from these large aqueous samples. The suitability of this technique for use in MALDI imaging experiments is demonstrated by the acquisition of the first images of lipid distributions within whole sagittal porcine eye sections.     

Synthesis, testing, and characterization of a novel Nafion membrane with superior performance in photoassisted immobilized Fenton catalysis

A new type of Nafion/Fe structured membrane ensuring faster kinetics, higher efficiency, and mechanical properties has been prepared and will be compared in its performance with the Fe-exchanged commercial Dupont 117 Nafion/Fe membrane during the abatement of model organic compounds. During the casting of the laboratory Nafion sample, the iron ions were introduced directly into the Nafion oligomer solution. This novel laboratory Nafion/Fe was tested as an immobilized catalyst in the degradation of several toxic pollutants showing a faster photoassisted degradation kinetics and a wider effective photocatalytic pH range compared to the Fe-exchanged commercial Dupont 117 Nafion/Fe membrane. When carrying out Ar ion sputtering of the 50 topmost catalyst layers, evidence is presented by X-ray photoelectron spectroscopy that Fe ions are found in the inner Nafion layers and seem to be responsible for the immobilized photoassisted Fenton processes leading to the degradation of 4-chorophenol (4-CP) taken as a model organic pollutant for the degradation process reported in this study. In the laboratory sample, the iron oxy/hydroxy Nafion moiety undergoes a transition to a more stable Nafion/Fe species during 4-CP degradation as determined by X-ray diffraction. This more stable form shows a higher iron dispersion and crystallinity compared to the fresh sample and is stabilized by the Nafion matrix avoiding the formation of separate iron phases. By infrared absorption (Fourier transform infrared), evidence is presented for the band of akaganeite-like species at 870 cm(-1) on the laboratory Nafion/Fe sample. This band disappears after 4-CP degradation because of the formation of the more highly dispersed iron species. Sputtering experiments show a decrease of F-containing groups in the laboratory Nafion/Fe samples closer to the catalyst upper layer while the amounts of Fe, C, and in particular O species increase in the topmost layer(s). In particular, the oxygenated species develop in the Nafion/Fe up to approximately 50 A below the catalyst surface. These species remain stable during the long-term Nafion/Fe degradation of 4-CP. Dynamo-mechanical analysis performed on laboratory Nafion/ Fe membrane samples revealed that these membranes possessed a greater mechanical modulus and resistance than the commercial Dupont 117 Nafion membrane.     

Efficient diverse approach for quinoxaline‐derived glycosylated and morphinylated analogs

Sulfanyl‐glycosides have been synthesized by reaction of 2,3‐dimercaptoquinoxaline ( 1 ) with acetohalo sugars in presence of base to give the thioglycosides‐derived quinoxalines 5 , 6 , 7 and 9 . Similarly, the acyclic analogs 23 , 24 , 25 , 26 were prepared by coupling of 1 with different acyclo‐alkylating agents. The preparation of 3‐morpholinyl‐quinoxalines 10 and 11 allowed the synthesis of 3‐glycosylsulfanyl‐2‐morpholinyl‐quinoxalines 12 , 13 , 14 and 17 as well as the acyclic analogs 27 , 28 , 29 . Microwave irradiation of the reactants turned out to be preferred over the conventional method for achieving the synthetic goals. This study made an available venue to the synthesis of diverse quinoxaline derivatives. J. Heterocyclic Chem., 2011.     

Real time evaluation of composition and structure of concanavalin A adsorbed on a polystyrene surface

In situ qualitative and quantitative evaluations of adsorbed submonolayers and multilayers of the protein Concanavalin A (ConA) on a polystyrene surface were carried out by attenuated total reflection infrared spectroscopy. The influence of pH and adsorption time on the composition and structure of the adsorbed protein layers was investigated by comparison of the experimental spectra with simulated spectra of hypothetical multilayer systems with the assumed composition, thickness, and structure. This methodology allows the differentiation of observed spectral changes that result from pure optical effects, associated with the passing of an incident beam through the multilayer system, from the chemical and structural changes caused by physicochemical interactions of proteins with polymer surfaces. This represents significant progress since small variations in the band positions or intensities of amide I and amide II infrared absorbance bands have an important interpretation consequence. The applied methodology significantly reduces the misinterpretation of recorded spectra of protein layers and is rewarded by a deep insight of the structure and composition of the samples. The composition, structure, and kinetics of the adsorption of ConA and hydration level of the adsorbed layers were evaluated in detail. Competitive adsorption of bovine serum albumin on pre-adsorbed ConA layers also was investigated to characterize the ConA surface distribution. Parallel studies using X-ray photoelectron spectroscopy support the conclusions drawn from infrared spectroscopic investigations on ConA molecular distributions at the polymer surface. Two-step models that describe ConA submonolayer formation at pH 4.8 and multilayer formation at pH 7.8 are proposed.     

Electrical properties and thermal conductivity of AgTlTe2 in the solid and liquid phases

Measurements of the electrical conductivity, thermelectric power and thermal conductivity of an AgTlTe₂ semiconductor in the solid and liquid states were carried out in a wide range of temperatures. In the liquid state the data analyzed in terms of a model developed for the density of states and electrical transport in solid amorphous semiconductors. Positive thermoelectric power suggests a large predominance of holes in electrical transport.