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[en] Complete text of publication follows. The use of Laser Ablation (LA) Inductively Coupled Plasma (ICP) Mass Spectrometry (MS) has undergone a considerable increase as a detection system of metals in proteins after separation by both one dimensional (1D) and bidimensional (2D) Polyacrylamide Gel Electrophoresis (PAGE). In this regard, it is very important to maintain the integrity of metal-protein binding during all the process. When the object of the study is a heteroatom-containing protein, such as P and Se, the element is strongly bound to the protein and the binding is not broken during the electrophoretic procedure. However, metal-binding proteins in which metal-protein interactions are weaker can loss the metal during electrophoretic separation, especially when using a denaturing one. Because of this, great efforts must be made to develop new strategies to separate proteins without breaking the metal-protein binding. We have developed some experiments to investigate the influence of electrophoretic conditions on the subsequent detection of metals by LA-ICP-MS of two metal-binding proteins , superoxide dismutase (SOD), containing Zn and Cu, and alcohol dehydrogenase (ADH), containing Zn. We have studied not only the nature of the electrophoretic method, but also the influence of the intensity applied, the trailing ion and the post-separation gel treatment. We recommended the use of tricine as trailing ion for SOD in SDS-PAGE. Non denaturing PAGE based in tricine is preferred to preserved metals-protein binding for ADH. With respect to the intensity applied, it has been observed that as higher intensity is applied the possibility of metal-protein binding losses is higher. The results showed that it is better to avoid staining procedures because they can alter the stability of the metal-protein complexes and prevent detecting metals bound to proteins.
[en] Complete text of publication follows. Low-molecular-mass (LMM) thiols, such as cysteine or glutathione, and thiolic proteins are important in cellular antioxidant defences, redox signalling, enzymatic activity and catalysis. From the analytical point of view the specific reactivity of -SH groups toward a large number of chemical probes represents a useful property allowing a wide choice of detectors for their determination. Mercurial probes (organic RHg+ and inorganic mercury Hg2+) interact with -SH groups with high affinity and specificity, and can be revealed by sensitive instrumental techniques. Cold vapour generation (CVG) coupled with atomic spectrometric (AS) techniques represents the most popular method for ultra-trace determination of mercury. In the past ten years we applied CVGAS, in particular atomic fluorescence spectrometry (AFS), for characterization of thiols and thiolic proteins following their derivatization with inorganic or organic mercury. While Hg(II) is suitable for flow injection-AS hyphenated systems, p-hydroxymercurybenzoate (PHMB) is a suitable probe for liquid chromatography experiments (LC-CVGAFS) (E. Bramanti et al., J. Anal. Atom. Spectrom., 16 (2001) 166-171). PHMB is a mono-functional organic mercurial probe that interacts at pH 7.4 with high affinity and specificity at room temperature in a time <90 s with LMMthiols and reactive -SH groups of proteins, giving stable, soluble complexes. Unlike Hg(II), free or S-bound PHMB does not interact with LC stationary phases. Pre-column derivatization of -SH groups with PHMB coupled with LC-CVGAFS system allowed us -SH characterization studies in native, denatured, denatured and reduced proteins as well as speciation and determination of thiolic compounds of clinical and environmental interest: LMMthiols in human blood and wines, metallothioneins, phytochelatins. These studies represent a fundamental work for future applications in the field of peptide/protein labelling. Most recently, LC-CVGAFS has been applied to a challenging topic, such as the speciation and determination in human plasma of LMM S-nitrosothiols, which are molecules involved in the transport and metabolism of NO radical (E. Bramanti et al., Talanta, 77 (2008) 684-694).
[en] Complete text of publication follows. Since 1988, when LECO took over Spectruma, advantages were made in optical-, source- and software-design. QDP quickly identifies: Contamination and cleanliness at the surface and interfaces; Migration and diffusion at interfaces; Heterogeneity of coating/substrate; Adherence issues; Oxidation/corrosion; Inclusion/blister; Chemical composition; Layer thickness/coating weight accurate in an area of 1nm to app. 150μm. Although many people think of GD-AES only as a tool for depth-profile, 65% of our European installations are just only for bulk analysis in foundries and quality labs. 10 advantages GDS has over other analytical techniques: Separation of sputtering and excitation; Linear calibration curves with wide dynamic range; Lowered self absorption and material re-deposition; Excitation of almost exclusively atom lines; Fewer and narrower emission lines reduce interferences; Freedom from metallurgical history; Fewer standards required for calibration; Minimal memory effects from a quick matrix change; Low Ar gas consumption (less than 1 litre per analysis); Automatic cleaning between samples. Recent applications will be discussed during the presentation.
[en] Complete text of publication follows. Independent, but small research projects at the arts and science interface, that focus on colourant determination, gain success by simple, yet powerful methods whose use supports the analytical approach. The presentation discusses the recent application of such a method: iterative target testing (ITT). ITT has been successfully adopted for pre-evaluation of multi-component FT-Raman spectra of paint formulations used in modern art. In particular, if just insufficient information about a paint sample under investigation are available, the group of necessary references can be marked off by iterative target testing. It has been shown previously that multi-component Raman spectra can be interpreted by the means of target transformation factor analysis (TTFA) (K. Petzold, GeoRaman '08, (2008) 41). Both TTFA and ITT require reference spectra, i.e. targets, to run the routine. However, ITT benefits from published spectra and, especially, the tabulated characteristic peak assignments. Since given descriptive intensities (weak, middle etc.) can be replaced by fixed numerical values on the one hand and constants as zero can be substituted for missing data points on the other hand, the incompleteness of reference vectors does not hamper the routine. If a test vector is a factor the vacant data points are correctly calculated automatically. The assessment of likeliness between reconstructed and published spectrum completes the iterative target testing procedure: A close spectral shape points to the presence of a particular colourant. Although the comparison of spectra forms one of the most time consuming steps, ITT presents a quick and reliable pre-evaluation tool for paint analysis. The author kindly acknowledge the grant from the FAZIT Stiftung, Frankfurt/M. Additionally, the author is indebted to the Wissenschaftliche Gesellschaft Freiburg for their financial support as well as to Mrs Anita Becherer for recording the Raman spectra.
[en] Complete text of publication follows. Trace amount of lead at ng/mL level was preconcentrated by using homogeneous liquid-liquid extraction method of lead diethyldithiocarbamate (Pb-DDTC) complex with perfluorooctanoate ion (PFOA-) dissolved in lithium hydroxide under acidic conditions. The effects of type and concentration of acid solution, amount of DDTC, pH of the sample solution, type and volume of organic solvent, and concentration of PFOA- on the extraction method of Pb(II) were investigated in detail. Under the optimized conditions (0.01M DDTC, 2.5 x 10-2M PFOA-, 8.0M HCl), 5 μg of Pb(II) in 60 mL aqueous phase could be extracted quantitatively into 100 μL sedimented phase. The sedimented phase dissolved in 3 mL of 1%(v/v) HNO3 was then subjected into a conventional measurement of flame atomic absorption spectrometry (FAAS). The enrichment factor of 20-fold for this method was obtained. The proposed method was applied to the extraction and determination of Pb(II) in drinking water, distilled spirit and wine samples.
[en] Complete text of publication follows. We have found that several species of probiotic bacteria also used in food industry for making yogurts is capable of producing spherical elemental selenium nanospheres having an average diameter in the range of 50-500 nm when 1-1000 mg/L selenium was added to the medium in the form of selenite ions. Elemental selenium produced thereby has a high degree of purity, is spherical, and its size and crystalline form depends on the bacterium species applied. We have found that some species of the probiotic yogurt bacteria (e.g. Bifidobacterium bifidum or Bifidobacterium longum) are capable of producing the grey crystalline form of elemental selenium which is so far unprecedented in the art for any bacteria capable of producing elemental selenium nanoparticles. Our finding, therefore, enables the first time the development of economical industrial bacterial fermentation based processes for the production of a high quality elemental selenium material comprising uniformly sized grey or red nanospheres having an average diameter in the range of 50-500 nm. The size and the crystalline form of the produced nanosized (50-500 nm) spherical particles is defined by the selected microorganism. This genetic pre-determination result in reproducible production of a material having individual characteristics which can be used in numerous fields of industry and research. The produced nano selenium could be a good raw material for a production of certified reference materials. The measurement of the produced elemental nanoselenium is a new challenge for the speciation analysis. A sample preparation and measurement method was developed and investigated for the analysis of different selenium forms by HPLC-AFS system. The atomic fluorescence (AFS) was a reliable and simple detection method for the elemental nano selenium. The elemental selenium can produce hydride in the system or can be converted to selenite with HCl/H2O2 digestion.
[en] Complete text of publication follows. It is a general aim of modern catalysis to develop substances that are able to act as highly active and, more importantly, highly selective catalysts. In order to achieve this goal one of the most promising ways is attempting to mimic Nature's most efficient catalysts, the enzymes. A method of doing it is the immobilisation of the active site over various supports. In our laboratory we have chosen mimicking electron-transfer enzymes, mainly superoxide dismutase (SOD). In this contribution an account is given on the results of the work, when copper complexes were covalently attached to surface-modified silica gel, and the ligands were mixtures of N- or C-protected histidine and tyrosine. The central ion for the complexes was Cu2+, the ligands were protected amino acids as tert-butoxycarbonyl-L-tyrosine, L-tyrosine methyl ester, tert-butoxycarbonyl-L-histidine, L-histidine methyl ester. The host material was chloropropylated silica gel. The first step of immobilisation was the reaction of the appropriately protected amino acid mixture (1:1 molar ratio) with the chloropropylated silica gel (24 hs reflux in isopropanol). The resulting material was soaked in Cu(NO3)2 solution under stirring for another day at room temperature (covalent grafting under ligand-poor conditions complexes present only with surface-grafted amino acid ligands). To a portion of this complex isopropanolic solution of the appropriate amino acid mixture (1:1 molar ratio) was added in excess and the suspension was stirred at room temperature for 24 hs (covalent grafting under ligand-excess conditions). Structural information on each step of the synthesis procedure was obtained by mid-range infrared spectroscopy, measuring diffuse reflectance and the SOD activities of the materials were also measured. The IR spectra revealed that anchoring of both amino acids was successful. The coordination sites were also identified through studying the spectra and using chemical reasoning. All four anchored complexes performed well in the SOD activity test, however, complexes prepared under ligand-poor conditions were more active than those made in the presence of ligand excess. Acknowledgement This work was supported by the National Science Fund of Hungary through grant K62288. The financial help is highly appreciated.
[en] Complete text of publication follows. Glow discharge (GD) atomic spectrometric techniques, based on mass spectrometry (MS) or optical emission spectroscopy (OES), have been extensively developed during the last years because of their important applications to direct solid analysis of bulk and coated samples. In this work a new experimental set-up has been designed to allow spatial resolved measurements of the optical emission in the radiofrequency GD plasma. The discharge source set-up mimics a typical GD-MS configuration. In contrast to usual GD-OES analysis in which the average emitted light is frontally detected, in this case the measurements are also done along the plasma plume. The light is observed through a quartz cylinder that is placed next to the GD source, and detected with an ICCD (intensifier-charge coupled device) coupled to a spectrometer. Two plano-convex lenses are used to focus this light from the plasma into an optical fiber, which provides the experimental system with a spatial resolution of 1 mm. Two positioners with micrometric precision translate the fiber and the lenses along the plume. The investigation of the spatial distribution of the species in the rf-GD, has been carried out to characterize the ionization and excitation processes within the plasma, as a function of different parameters such as the argon flow rate, the pressure or the applied power. Atomic and ionic emission lines (emitted by the analyte and by the discharge gas, argon) have been measured at different operating conditions and at different distances from the cathode. Depending on the involved levels in the transition, the emission intensity provides information on the population of ionic, atomic or metastable states in different regions of the plasma (C.L. Lewis et al., Spectrochimica Acta Part B, 56 (2001) 487-501). Therefore, these results will be used to optimize the ion extraction in GD-mass spectrometry. This work was supported by the Ministry of Science and Innovation (Spain) within the framework of the project no. MAT 2007-65097-C02. R. Valledor acknowledges 'FPU' Ph. D. Grant (Ministry of Science and Innovation, Spain). J. Pisonero acknowledges 'Ramon y Cajal' Research Contract (Ministry of Science and Innovation, Spain).
[en] Complete text of publication follows. Applied as ionization and excitation source, glow discharge (GD) is very often used in analytical spectrometry because of its stability and homogeneous sputtering. Pulsed power supply of the GD has a number of advantages in comparison with the continuous one and may broaden the field of GD spectrometry application. In spite of the numerous advantages of pulsed GD (PGD), it is not yet used in routine analysis. This is because of the large number of primary regulating parameters: voltage, current, pressure, frequency and pulse length. Secondary parameters as duty cycle, instantaneous and average power are also essential to consider and optimize. Their influence on the sputtering rate, emission yield and crater shape is not fully understood. Therefore, it is very important to investigate the GD processes in dependence on the pulse parameters. The dependence of the current on the voltage was investigated, and it was concluded that the impedance is decreasing with increasing power (V. Hoffmann et al., Journal of Physics: Conference Series 133(2008), 12017/1-12). This can be explained by a heating of the discharge gas. Crater shapes and sputtering rates were studied for various pulse parameters. A significant influence of the pulse duration on the crater shapes was found for both, pulsed radio frequency and pulsed direct current discharges. By use of radio frequency GD, non-conductive samples can be analysed. The results of fundamental and experimental investigations were applied to improve the GD optical emission spectrometry (GDOES) analysis of thin-film solar cells based on Cu(In,Ga)Se2 and Cu(In,Ga)S2 absorber layers. The elemental distributions in these layers affect considerably the solar-cell performance. GDOES was found to be the fastest method to study the elemental distribution in Cu(In,Ga)Se2 (D. Abou-Ras et al., Proc.EMC 2008). The difficulty in analysis of these solar cells is that in most cases, glass is used as substrate, which is very heat sensitive and can be broken during the analysis owing to thermal stress. The reduction of sample heating may be achieved by the application of PGD. From the GDOES results obtained on ZnS/CuInS2/Mo/glass stacks, the temperature dependence of the diffusion coefficient of Zn into CuInS2 was studied. The authors kindly acknowledge the financial support from EC funded Research Training Network GLADNET (No. MRTN-CT-2006-035459).
[en] Complete text of publication follows. An experimental algorithm comprising FTIR spectroscopic measurements of the hydration of oriented films (W. Pohle et al., Biospectroscopy, 4 (1998) 267-280.) was used to explore structural and phase properties of a series of amphiphiles which are inter-related by having the same chain length of 18 carbon atoms. The set of compounds involved the surfactants stearylalcohol, stearylamine and stearyltrimethylammonium chloride (STMAC) and the biologically more relevant dioleoylglycerol (DOG). These compounds can be regarded as reasonable phospho- and glycolipid models, respectively. The spectroscopic data revealed considerable differences in the chain fluidity as well as, rather unexpectedly, a qualitatively distinct water-uptake capacity estimated at 98% relative humidity. While STMAC imbibes some water, stearylalcohol, stearylamine and DOG do not so. This different behavior is primarily referred to the way the monomeric amphiphiles assemble at the supramolecular level under the ambient conditions. Peculiar behavior was found for STMAC which undergoes a novel lyotropic transition between an interdigitated crystalline and a strongly tilted gel phase. This transition is accompanied by a number of spectral features not observed so far. The interpretations are supported by the results of complementary synchrotron X-ray diffraction measurements performed according to our previously reported protocol (W. Pohle, et al. J. Biomol. Struct. and Dynam., 19 (2001) 351-364.) as well as those of quantum-chemical calculations of related model compounds. The IR spectra of stearylamine demonstrate the presence of NH and NH3+ groups instead of the NH2 groups normally expected for a primary amine and, thus, appear to indicate a proton transfer occurring under the applied experimental conditions and resulting in an ammonium amide (W. Pohle, D.R. Gauger, J. Mol. Struct. 924-926 (2009) 144-147.). Stearylalcohol and DOG together cover a wide range of chain fluidity and O-HO hydrogen bonds. Experimental data as well as molecular- dynamics and quantum-chemical calculations suggest that their hydroxyl groups are locked inside the formed supramolecular aggregates and become therefore excluded from binding outside water.