Effect of chromatographic conditions and plasmid DNA size on the dynamic binding capacity of a monolithic support

DNA therapies are becoming recognized alternatives for the treatment and prevention of severe pathologies. Although most current trials have used plasmids <10 kbp, in the future larger plasmids would be required. The purpose of this work was to study the chromatographic behavior of nongrafted carbonyldiimidazole monolithic disks using plasmids with different sizes under hydrophobic conditions. Thereunto, the purification of several plasmids was performed. Higher size plasmids needed lower ammonium sulfate concentration, due to the greater number of interactions between the plasmids and monolith. The dynamic binding capacity experiments for the different plasmids revealed a lower capacity for bigger plasmids. It was also verified that the increase of salt concentration from 2.5 to 3 M of ammonium sulfate increased the capacity. At the highest salt concentration, a slight improvement in the capacity using lower flow rate was observed, possibly due to compaction of plasmid molecules and its better organization on the monolith channels. Finally, a low pH also had a positive effect on the capacity. So, this monolithic support proved to be appropriate to purify the supercoiled isoform of different plasmids with different sizes, providing a valuable instrument as a purification technique.     

Ethylene polymerization with imine and phosphine nickel complexes containing isothiocyanate

Three isothiocyanate complexes of nickel(II) containing diimine [ArN?C(Me)? C(Me)?NAr]Ni‐ (NCS)₂ (1), iminophosphine [Ph₂PC₆H₄CH?NAr]Ni(NCS)₂ (2), or diphosphine (dppe)Ni(NCS)₂ (3), [Ar = 2, 6‐ⁱPr‐C₆H₃; dppe = 1, 2‐bis(diphenylphosphine)ethane] were synthesized and examined for ethylene polymerization activated by methylaluminoxane (MAO). Their behavior was compared with those of the corresponding halide analogues [ArN?C(Me)? C(Me)?NAr]NiBr₂ (4), [Ph₂PC₆H₄CH?NAr]NiBr₂ (5), and (dppe)NiCl₂ (6). The diimines showed the highest polymerization activity. Replacement of the halide for the NCS pseudo halide affected the activity and decreased the molecular weight of the polymer formed. The highest molecular weights were obtained with the diimine complexes. Highly branched polyethylenes were obtained with the bulkier complexes 1 and 4. Replacement of the halide for NCS in the diimine complexes also caused an increase in the branching content, whereas the opposite occurs for the iminophosphine complexes. The different activities and behavior of the catalyst systems with halide versus NCS in the polymerization of ethylene and the characteristics of the final products suggest a modification in the active species caused by the non‐chelating ligand. Polymer molecular weight and branching content is dependent on the MAO/Ni molar ratio and on the working temperature. Copyright © 2004 John Wiley & Sons, Ltd.     

Atmospheric Corrosion Investigation in Industrial,Marine and Rural Environments in South-East Brazil

ASTM 283-C, AISI 304 and 316-L steel specimens (called coupons) were exposed in marine, industrial and rural area(s) for different periods ranging between 1–12 months, in four different season campaigns. The corrosion rate was determined by chemical loss measurements. Rust characterization was performed by XRD, SEM, optical, and Mössbauer spectroscopy (in transmission and backscattering geometry). Superparamagnetic maghemite and goethite were found as corrosion products. Magnetic goethite and feroxyhite decrease with time of exposure. Lepidochrosite is detected and its intensity increase with the atmospheric exposure time. The results obtained from XRD and Mössbauer are in good agreement.     

Trichloro-bridged diruthenium (III,III) complexes: X-ray isomorphous structures of [Ph3X=O···H···O=XPh3][Ru2Cl7(XPh3)2]·0.5(CH2Cl2)(H2O)(X = As or P)

The triply chloro-bridged binuclear complexes [Ph₃X=O···H···O=XPh₃][Ru₂Cl₇(XPh₃)₂]·0.5(CH₂Cl₂)(H₂O) (X = As or P) were obtained from [RuCl₃(XPh₃)₂DMA]·DMA (DMA = dimethylacetamide) CH₂Cl₂/Et₂O solution. The structures were characterized by X-ray diffraction studies. The complexes are formed from two Ru atoms bridged by three chloride anions. The two ruthenium atoms are also coordinated to two non-bridging Cl atoms and an AsPh₃ or PPh₃ ligand respectively. As an interesting feature, the cations of these complexes are protons, trapped in a very short hydrogen bond between two triphenylarsine or triphenylphosphine oxide molecules.     

Separation of different forms of proteose peptone 3 by hydrophobic interaction chromatography with a dual salt system

A panel of four hydrophobic adsorbents (butyl-, octyl-, phenyl- and epoxy-Sepharose) was used to examine the selectivity and fractionation of several proteose peptone 3 (PP3) forms from a freeze-dried extract of whey bovine milk. In particular, the effects of altering the ligand type and salt were investigated. The chromatographic studies suggest that PP3 strongly interacts among the three commercial hydrophobic resins leading to a drop off in selectivity, while a complete binding was achieved at low salt concentrations (below 0.5 m) and total elution only with phosphate buffer and/or water stepwise conditions. Only in epoxy-Sepharose was an appreciably selectivity of the several fractions of PP3 present in the initial feedstock attained. Despite the high salt concentration for a complete binding of PP3 (above 1.5 m ammonium sulfate) onto this support, the dual salt system (ammonium sulfate 1 m and sodium citrate 0.8 m) led to a high separation degree of high and low molecular weight forms of PP3.     

Electrochemical Determination of the Antioxidant Capacity: The Ceric Reducing/Antioxidant Capacity (CRAC) Assay

The CRAC assay is a direct electron transfer test of antioxidant capacity for several organic compounds. The ability of eight different compounds in reducing Ce⁴⁺ was studied by chronoamperometric measurements of the remaining Ce³⁺ species. The following antioxidant classification was observed: tannic acid quercetin>rutin>gallic acid≈catechin>ascorbic acid>BHA>Trolox. These results agree with others already published and a good correlation (R²=0.937) was found with the classical spectrophotometric FRAP assay. The CRAC assay is simple, fast, free from sample pretreatment and applicable to nontransparent samples.     

Amphimedonoic acid and psammaplysene E,novel brominated alkaloids from Amphimedon sp.

Examination of the CH₂Cl₂-MeOH (1:1) extract from the Madagascan sponge Amphimedon sp. highlighted two new brominated alkaloids, amphimedonoic acid (1) and psammaplysene E (2), along with the known 3,5-dibromo-4-methoxybenzoic acid (3). Their structures were elucidated by 1D and 2D NMR spectroscopy and HRESIMS data.     

Purification of Membrane-Bound Catechol-O-Methyltransferase by Arginine-Affinity Chromatography

Affinity chromatography strategies using amino acids as immobilize d ligands have been successfully applied for the purification of different biomolecules from complex mixtures. Therefore, in this work, several supports with immobilized amino acids were applied for the purification of membrane-bound catechol-O-methyltransferase (MBCOMT) from Pichia pastoris lysates and it was verified that l-arginine provided the required selectivity for MBCOMT isolation. The optimization of the binding and elution buffers composition allowed the recovery of purified MBCOMT in a biological and immunologically active state from the arginine support. Additional optimization experiments varying the mobile phase pH, temperature and the concentration of the injected sample were carried out and an improvement of MBCOMT adsorption and purity was observed. Indeed, the optimized conditions for MBCOMT isolation and purification consisted in: loading of 4 mg of total protein onto the column previously equilibrated at 20 °C where the target enzyme was recovered in a purified fraction using 500 mM NaCl, 10 mM DTT and 0.5 % (v/v) Triton X-100 in 10 mM Tris buffer (pH 7) with a total bioactivity recovery of 24 ± 2.2 % and a purification fold of 4.95 ± 0.23, a value that is consistent with the best values ever reported for MBCOMT. Moreover, the l-arginine support demonstrated the ability to bind the target protein in a wide range of pH values (above and below the pI of the target protein) and the MBCOMT elution occurs in a single peak pattern. Finally, the strategy here reported can aid in the implementation of crystallization studies with MBCOMT in complex with clinically relevant inhibitors since it is obtained in a purified form with biological activity. In conclusion, a novel affinity chromatography strategy was developed and implemented for recombinant MBCOMT purification in a highly immunological and biologically active state.

     

Bioanalytical procedures and recent developments in the determination of opiates/opioids in human biological samples

The use and abuse of illegal drugs affects all modern societies, and therefore the assessment of drug exposure is an important task that needs to be accomplished. For this reason, the reliable determination of these drugs and their metabolites in biological specimens is an issue of utmost relevance for both clinical and forensic toxicology laboratories in their fields of expertise, including in utero drug exposure, driving under the influence of drugs and drug use in workplace scenarios. Most of the confirmatory analyses for abused drugs in biological samples are performed by gas chromatographic–mass spectrometric methods, but use of the more recent and sensitive liquid chromatography–(tandem) mass spectrometry technology is increasing dramatically. This article reviews recently published articles that describe procedures for the detection of opiates in the most commonly used human biological matrices, blood and urine, and also in unconventional ones, e.g. oral fluid, hair, and meconium. Special attention will be paid to sample preparation and chromatographic analysis.