Numerical Study of a Free-Burning Argon Arc with Copper Contamination from the Anode

The present modeling of a free-burning argon arc accounts for copper vapor contamination from the anode. Simulations are made for an atmospheric arc that has a length of 10 mm and an electric current of 200 amps. Predicted results for two different anode evaporation rates are compared to those from a pure argon arc with no copper vapor contamination. Copper vapor concentration, temperature, electric potential, and current density profiles are presented. Included in this analysis are radiation losses from both the argon and copper by using recently calculated net emission coefficients. It was found that evaporation of copper from the anode results in a cooling of the arc in a region close to the anode, but has an insignificant influence on the arc close to the cathode. Due to the arc flow characteristics most of the copper vapor tends to be confined to the anode region.     

Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha.

In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor alpha (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-alpha molecule. Using low cultivation temperature and appropriate protease deficient E. coli strains, it was possible to obtain intact, full-length proteins with NHis2Xa and HisArg tags, which could be purified to over 95% purity in a single step. However, in comparison to model proteins bearing a surface histidine cluster, accumulation of the histidine-tagged proteins in E. coli was significantly reduced, even in protease deficient strains. In addition, the histidine tagged TNF-alpha proteins never displayed good chromatographic behavior, which was otherwise easily achieved with model proteins. Although the most popular hexa-histidine tag is generally recognized as very convenient for single step isolation of monomeric proteins, our results with trimeric TNF-alpha indicate that oligomeric proteins may require further optimization of the tag, with respect to its length, composition, and location. Histidines, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags with the same total number of histidines.     

Chemometric approach in quantification of structural identity/similarity of proteins in biopharmaceuticals

We present a chemometrics study in which we show the identity or degree of similarity of 3D protein structures of various G-CSF (Granulocyte Colony-Stimulating Factor) isolates. The G-CSF isolates share the same amino acid sequence, but the preparation was carried out by somehow diverse technologies. The comparison of 3D structures was made on the basis of 2D NMR NOESY (Nuclear Overhauser Enhancement Spectroscopy) spectra of proteins. In searching for the most appropriate criteria to determine the identity or degree of similarity of selected spectral regions of different isolates, two methods for quantitative evaluation of identity/similarity were used. The first method compares all peaks in the two investigated protein spectral regions; the extent of peaks that overlap is determined. The second method includes spectral invariants originating from graph theory. The criteria of identity/similarity were calculated from graphs, derived from a collection of up to 200 peaks of investigated 2D NMR spectral region. The peaks were linked into a graph according to the sequential nearest neighborhoods. According to the first method all peaks were relevant, considering that spectral noise was previously removed; the largest similarity was found between the protein of a commercially available G-CSF drug and one of the three new isolates produced in the laboratory. The second method indicated that the pairwise similarity of the three new isolates is larger than the similarity of any of the new isolates with the commercially available drug. This is an expected result taking into account that the new isolates are produced by the same technology, while the commercial product has additives for long-term storage that could not be completely compensated. The proposed measure of similarity may help the developers of biosimilar products to optimize the controllable parameters of the production technology and eventually to argue the identity of the new isolate in comparison with the originator commercial product.     

Classification of iron‐based inks by means of micro‐Raman spectroscopy and multivariate data analysis

In this work, multivariate data analysis methods were applied to the analysis and interpretation of micro‐Raman spectra, collected from a broad set of historical iron‐based ink samples, previously characterised for the content of organic acids (gallic acid, ellagic acid and protocatechuic acid). The proposed method relies on principal component analysis of the noisy spectra typically obtained on original, degraded, organic samples, where fluorescence could affect the Raman signal. The signal components could be distinguished from the noise components and then used to build a linear discriminant analysis (LDA) model, achieving separation of the spectra into three classes. Selection of pure signal factors also improved effectiveness and performances of partial least square regression (PLS) algorithms, allowing quantification of condensed tannic acid residuals. Application of multivariate methods to discriminate signal from noise removes the need for spectral data manipulation (filtering, smoothing and differentiating). The obtained classification method for discrimination of historic inks and the regression method for determination of condensed tannic acid residuals supports the use of Raman analysis of fluorescing organic materials, and may provide information to scholars on ink composition and potentially on its provenance. Copyright © 2013 John Wiley & Sons, Ltd.     

Theoretical radiative emission results for argon/copper thermal plasmas

Theoretically determined radiative properties for argon and argon/copper plasmas at one atmosphere, in the temperature range from 3000 to 25000 K, are presented. The spectrum from 0.03 to 25 μm is covered. The spectral emission coefficient, integrated emission coefficient, and net emission coefficient are the properties considered. To determine the limits of the optically thin regime, the intensity for a line-of-sight in an isothermal, homogeneous plasma versus pathlength is compared to the intensity calculated with the optically thin assumption. To obtain information on the dominating emission mechanisms, the fractional contributions of the bound bound (line), free-bound, and free-free emission are shown. To obtain information on the dominating species, plots of the fractional contributions of each of the atom and ion types is presented. Because it is of interest to know how much of the total radiation is in the vacuum ultraviolet wavelength region, fractional plots of this are presented too.     

Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.     

Emission of reactive oxygen species during degradation of iron gall ink

Iron gall inks are characterised by high contents of acids and transition metals, promoting degradation of cellulose due to hydrolysis and oxidation, respectively. Their chemical interaction with the environment is not well understood, especially in view of emissions of degradation products which could lead to spread of degradation processes.In order to study the emissions, we employed gas chromatography/mass spectrometry following headspace micro-extraction, and liquid chromatography following hydroxyl radical scavenging with appropriate probes. We also studied chemiluminescence of cellulose affected by ink degradation.We show that while the emissions of organic volatile degradation compounds by inks are less intense than those of surrounding paper, ink does promote the degradation of cellulose across big distances (from object to object). We were able to link this to emission of reactive oxygen species, probably hydrogen peroxide. Its emission from ink is considerably more intensive than from paper.     

Characterisation of metal-chelate methacrylate monoliths

Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.     

Effects of NO2 and acetic acid on the stability of historic paper

This research investigates degradation of historic paper in polluted environments during long-term dark storage. In an innovative experiment, degradation rates at realistic pollution levels are compared with degradation rates in the absence of pollution, using a set of real historic papers. The most abundant pollutants in repositories in post-industrial environments are taken into account: acetic acid and nitrogen dioxide. Their action was assessed in terms of reduction of ‘handling’ (as defined by decrease in degree of polymerisation) and ‘display’ (as defined by discolouration) lifetimes. Extrapolations to room conditions enabled lifetime predictions in conditions that are comparable to a real archival or library repository environments while prediction uncertainties were analytically evaluated to assess the significance of conclusions. While 10 ppb of NO₂ does reduce the handling lifetime of almost all types of paper, their predicted lifetimes were still assessed to be several millennia, with the exception of acidic paper. Acetic acid at concentrations that are typical for archival and library repositories (<100 ppb) has significantly less effect than NO₂ while it does not affect display lifetimes. From a conservation management perspective, it needs to be addressed whether the predicted reductions in otherwise significant handling lifetimes are of real concern and whether air filtration in archival and library repositories is justified.     

Three-dimensional boundary layer and vortex wake over a cone at high angle of attack: study of asymmetries

An experimental investigation of the flow over a one at a large angle of attack is reported. First, the study was focused on the wall shear stress measurement, including the localization of the separation. Secondly, the mean flow field in the whole wake of the cone was measured, as well as the velocity fluctuations. Results indicate that the separation and the fluctuations are asymmetrical in a certain way, whereas the mean flow field is approximately symmetrical. Finally, the different parts of the flow can be easily determined using vorticity calculations.List of symbols C vortex core - D diffusion coefficient for the polarographic solution - D cone diameter for the rotation plane of the electrochemical probes - D separation point - F function F (sin ) = (K ₁-K ₂)/(K ₁+K ₂) - G function G(sin ) = (K ₁+K ₂)/(K ₁+K ₂)( = 90dg) - g bidimensional gain of the electrochemical probe (constant for each probe) - K ₁, K ₂ mass transfer coefficients for differential probes - Re _x Reynolds number based on the X length, and relative to the forward upstream velocity - wall velocity gradient vector - S wall velocity gradient modulus - S enclosing saddle point - S _x azimuthal component of the wall velocity gradient (perpendicular to a generator) - S _z longitudinal component of the wall velocity gradient (along a generator) - U mean value of the forward upstream velocity - U _i component number i of the velocity vector in the (X, Y, Z) coordinates - X, Y, Z cone cartesian coordinates - non-dimensional cone cartesian coordinates (relative to D) Greek symbols incidence (part 1) angle between the wall velocity gradient and the neutral axis of the electrochemical probe (except part 1) - _r relative incidence /0 _c - velocity circulation - wavelength of the laser beam - kinematic viscosity - azimuthal angle - _c cone semi-apex angle