Complex Formation of Phytic Acid With Selected Monovalent and Divalent Metals.

The formation of metal complexes with phytic acid is a complex process that depends strongly on the metal-to-ligand molar ratio, pH value and consequent protonation level of the phytate ligand as well as accompanying side reactions, in particular metal hydrolysis and precipitation of the formed coordination compounds. In the present work, the potentiometric titration technique was used in combination with a detailed analysis of the equivalent point dependencies for selected biologically relevant monovalent and divalent cations from the groups of alkaline earths and transition metals, namely: Mg(II), Zn(II), Fe(II), Cu(I), and Cu(II) ions. The investigation of complex formation mechanism, the evaluation of the species formed, and the identification of other side reactions was based on the examination of three distinct equivalent points, which were detectable by alkalimetric titrations of phytic acid in the presence of selected metal ions. It has been demonstrated that alkaline earth metals interact with different binding site(s) than the transition metals, and experiments with both oxidation states of copper revealed similar complexing characteristics, which depend mainly on the ionic radius (and not on the ionic charge as initially expected). Quantitative data on phytate complexation, hydroxide formation and complex precipitation are presented herein for all metals studied, including Cu(I), which was investigated for the first time by means of alkalimetric titration.

Optimization of High Performance Liquid Chromatography Method for Simultaneous Determination of Some Purine and Pyrimidine Bases.

Nine purine and pyrimidine bases were separated and determined simultaneously using reversed phase (RP) high performance liquid chromatography (HPLC) in some food samples and biological fluids. Chromatographic behavior of these ionizable compounds highly depends on the interactions with the solvent as confirmed experimentally and by calculation of distribution of this species as a function of pH. Chromatograms show the optimal separation of five purine (uric acid, hypoxanthine, xanthine, adenine, and guanosine), and four pyrimidine (cytosine, uracil, cytidine and tymine) bases at pH around four. Accordingly, acetate buffer was selected due to high buffer capacity in this region. By variation of pH, concentration of buffer and volume ratio between buffer and methanol, we found that a mixture of 50 mM acetate buffer of pH 4.0 ± 0.1 with 3 % of methanol ensures reproducibility, complete separation in less than 15 minutes and compatibility with MS detection. Developed method was validated and applied for the analysis of complex clinical and beverage samples.

Potentiometric Determination of Phytic Acid and Investigations of Phytate Interactions with Some Metal Ions.

Determination of correct amount (concentration) of phytic acid is of vital importance when dealing with protonation and/or metal complexation equilibria. A novel approach for precise and reliable assay of phytic acid, based on the difference between end points by potentiometric titration, has been presented. Twelve phytic acid protons are classified into three groups of acidity, which enables detection of 2 to 3 distinct equivalent points (EPs) depending on experimental conditions, e.g. counter-ion concentration. Using the differences between individual EPs enables correct phytate determination as well as identification of potential contamination and/or determination of initial protonation degree. Impact of uncertainty of phytate amount on the calculation of protonation constants has been evaluated using computer simulation program (Hyperquad2013). With the analysis of titration curves different binding sites on phytate ligand have been proposed for complexation of Ca2+ and Fe3+ ions.

Protection of copper surface with phytic acid against corrosion in chloride solution.

Phytic acid (inositol hexaphosphate) was tested as a corrosion inhibitor for copper in 3% sodium chloride. Phytic acid is a natural compound derived from plants, it is not toxic and can be considered as a green inhibitor. Electrochemical methods of linear polarization and potentiodynamic polarization were used to study the electrochemical behaviour and evaluate the inhibition effectiveness. To obtain the optimal corrosion protection the following experimental conditions were investigated: effect of surface pre-treatment (abrasion and three procedures of surface roughening), pre-formation of the layer of phytic acid, time of immersion and concentration of phytic acid. To evaluate the surface pre-treatment procedures the surface roughness and contact angle were measured. Optimal conditions for formation of phytic layer were selected resulting in the inhibition effectiveness of nearly 80%. Morphology and composition of the layer were further studied by scanning electron microscopy, energy dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy. The layer of phytic acid with thickness in the nanometer range homogeneously covers the copper surface. The obtained results show that this natural compound can be used as a mildly effective corrosion inhibitor for copper in chloride solution.

The Effect of pH Value of a Simulated Physiological Solution on the Corrosion Resistance of Orthopaedic Alloys.

Metals and alloys used in orthopaedics and dentistry are exposed in vivo to various agents and environmental conditions. One of the important factors that determine the corrosion behaviour of metallic biomaterials is the pH of the environment. The corrosion resistance of stainless steel 316L (Fe/Cr18/Ni10/Mo3), titanium and titanium alloy Ti-6Al-4V (Ti90/Al6/V4) was studied in terms of their electrochemical properties and biodegradation in simulated physiological solutions of different pH values (4.5, 6.5, 7.5 and 8). The electrochemical characteristics of individual metal components were also investigated using cyclic voltammetry, linear polarization and potentiodynamic polarization methods. The concentration of dissolved metal ions released during 32 days immersion under simulated physiological conditions was determined by inductively coupled plasma mass spectroscopy. The corrosion behaviour of stainless steel 316L is strongly affected by the pH of the physiological solution in the range from 4.5 to 8.0. The corrosion resistance was enhanced at higher pH and the concentrations of released metal ions lower. The behaviour of titanium and its alloy however is almost independent of the pH.

Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns.

Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples.

Potentiometric and ³¹P NMR studies on inositol phosphates and their interaction with iron(III) ions.

Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP(6) ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P-O-Fe-O-P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)-InsP(6) aggregates. (31)P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T(1) relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP(6) molecule results in an unstable heterogeneous system.

Reversed phase monolithic analytical columns for the determination of HA1 subunit of influenza virus haemagglutinin.

Monoliths are chromatographic stationary phases, which were specially designed for efficient purification of large biomolecules, like proteins, viruses and DNA. In this work, the small scale monolithic butyl (C4) and styrene-divinyl benzene (SDVB) columns were applied for reversed phase analyses of various degraded influenza viruses. The binding of the HA1 subunit of haemagglutinin to the monolithic columns was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot. The working linear range was determined as 1.60×10(10) viral particles/mL to at least 1.64×10(11) viral particles/mL, the limit of detection was found to be 2.56×10(9) virus particles/mL and the limit of quantification was 5.12×10(9) virus particles/mL. The analytical HPLC method developed with the H1N1 virus was also applicable for the analytics of the HA1 subunit of H3N2 influenza virus and the influenza B virus.

Hard modeling of ion chromatography separations on hydroxide-selective stationary phase.

In the present paper the development and testing of the computer software for the simulation of the on-column ion chromatography separation processes are presented. The computer algorithm based exclusively on the selectivity coefficients of the tested analytes has been upgraded to cope with the 2:1 and 1:1 ratios of the analyte-to-eluent ion charge. The analytical solution of the cubic equation, which is needed for calculation of chromatograms for doubly charged analytes in the presence of singly charged eluent, is presented. The developed modeling approach was tested on the data sets produced by the system composed of hydroxide-selective stationary phase in combination with on-line electrolytically generated OH(-)-based eluents. Retention behavior of the selected anions on the AS15 (DIONEX, USA) stationary phase was investigated. The study of the dependence of the peak widths on the number of theoretical column segments considered in the calculated chromatograms enabled us to choose the optimal number of column segments. The average error in the retention time of the calculated chromatograms for the data set used in the study, i.e. seven different ions at eight different eluent concentrations was found to be 1.4%. A good match with the experimental chromatograms allows us to use the information of the intermediate states of calculations to get a detailed insight into the time-dependent on-column analyte distribution.

Nondestructive analysis and dating of historical paper based on IR spectroscopy and chemometric data evaluation.

Sampling restrictions in analysis of cultural heritage materials narrow the choice of appropriate analytical methods considerably. In this work, near- and mid-FT-IR reflectance data were related to paper properties determined with classical analytical methods using partial least-squares. Nondestructive determination of properties, which are of importance for evaluation of the long-term stability of historical paper, i.e., ash content, lignin content, degree of polymerization of cellulose, pH, and aluminum content, is possible. With the use of a considerable sample set, satisfactory reliability was achieved for all properties but aluminum content. Considering that with age, chemical properties of paper change, dating of historical documents was attempted for the first time, also with success.

Non-Destructive Evaluation of Historical Paper Based on pH Estimation from VOC Emissions.

Volatile organic compounds (VOCs) emitted from materials during degradationcan be a valuable source of information. In this work, the emissions of furfural and aceticacid from cellulose were studied using solid-phase micro-extraction (SPME) incombination with gas chromatography-mass spectrometry. Two sampling techniques wereemployed: static headspace sampling using SPME for 1 h at 40 oC after 18-h samplepreparation at 80 oC in a closed glass vial, and contact SPME in a stack of paper (or abook). While a number of VOCs are emitted from paper under conditions of natural oraccelerated degradation, two compounds were confirmed to be of particular diagnosticvalue: acetic acid and furfural. The emissions of furfural are shown to correlate with pH ofthe cellulosic environment. Since pH is one of the most important parameters regardingdurability of this material, the developed method could be used for non-destructiveevaluation of historical paper.

Mixed-valence Cu(II)/Cu(I) complex of quinolone ciprofloxacin isolated by a hydrothermal reaction in the presence of L-histidine: comparison of biological activities of various copper-ciprofloxacin compounds.

A new quinolone-metal complex was prepared by a hydrothermal reaction in the presence of L-histidine that served as a reducing agent for a metal. The title compound [Cu(II)(cfH)(2)(Cu(I)Cl(2))(2)] (1) is a mixed-valence Cu(II)-Cu(I) complex, which contains two ciprofloxacin (cfH) molecules bonded to the central copper(II) atom and two almost planar [Cu(I)Cl(2)](-) moieties. Both metal centers are connected through two bridging atoms (chloride and quinolone oxygen). The electrochemical methods (differential-pulse polarography and cyclovoltammetric measurements) confirmed the presence of various copper-ciprofloxacin complex species in aqueous solution at low concentrations used in biological activity tests and also indicated that the equilibria in this system are very complex. The biological properties of the title compound and some previously isolated copper-ciprofloxacin complexes ([Cu(cfH)(2)Cl(2)].6H(2)O (2) and [CuCl(cfH)(phen)]Cl.2H(2)O (3)) (phen=1, 10-phenantroline) were determined and compared. The DNA gyrase inhibition tests and antibacterial activity tests have shown that the effect of copper complexes is comparable to that of free quinolone. Additionally, an interesting DNA cleavage activity of the title compound was also discovered.

Cobalt-based alloys for orthopaedic applications studied by electrochemical and XPS analysis.

The composition of the passive layers formed by electrochemical oxidation at different passivation potentials on Co-Cr-Mo and Co-Ni-Cr-Mo alloys in simulated physiological solution (SPS), with and without the complexing agent EDTA, was studied by X-ray photoelectron spectroscopy. Composition as a function of depth, cationic fraction and thickness of the passive film was determined. Chromium oxide is shown to be the major constituent of the passive layer on both Co-Cr-Mo and Co-Ni-Cr-Mo alloys. The minor constituents of the passive layers, Co- and Mo-oxide in the case of Co-Cr-Mo alloy and Ni-, Co- and Mo-oxides in the case of Co-Ni-Cr-Mo alloy, are also located in the outer part of the layer. EDTA affects the formation of the passive layer on each alloy. The content of Co-, Ni- and Mo-oxide in the passive layer is lower in the presence of EDTA, thus indicating increased solubility associated with higher stability constants for complexes of metal cations with EDTA.

Determination of 210Pb and 210Po in sediment and soil leachates and in biological materials using a Sr-resin column and evaluation of column reuse.

In this work we present a suitable way to release lead and polonium from sediments and other samples. Results obtained by hot-acid dissolution and cold leaching showed that while good recoveries of 210Pb were obtained in both the cases, 210Po recovery in hot dissolution was poor due to volatility of polonium, and might lead to erroneous results due to non-equilibration of added 208Po tracer and 210Po in the matrix. A Sr-resin column was used for separation of Pb and Po. We also studied the possibility of reusing columns used earlier. The most significant problem in reuse is the reduction in column capacity, resulting in a reduction to half of its initial capacity. Other natural radionuclides do not interfere in the determination of 210Pb and 210Po and we found no measurable contamination of the column on repeated usage.

Interactions of oxovanadium(IV) and the quinolone family member--ciprofloxacin.

The interactions of quinolone ciprofloxacin (cfH) and oxovanadium(IV) were studied by various methods. Green crystals of a complex [V(IV)O(cf)(2)(H(2)O)] were isolated and the molecular connectivities established, although the crystal structure was not perfectly refined due to the instability of the crystals. Based on a plausible interpretation of the data sets, two cf anions bidentately coordinate to a vanadyl cation through carboxylate and carbonyl oxygen atoms; in addition, there is a water molecule in the coordination sphere. Solution techniques (cyclic voltammetry, electronic and electron paramagnetic resonance spectroscopy, potentiometric measurements) confirmed the presence of various species in the solution, the composition of which strongly depends on the conditions in the system. The antibacterial activity of the complex against various microorganisms was tested and it was established that its activity is similar to that of free ciprofloxacin.

Transformations of mercury in the terrestrial isopod Porcellio scaber (Crustacea).

The biological cycle of mercury in the terrestrial isopod Porcellio scaber was investigated. Testing the possibility of in vivo Hg(2+) methylation was divided into two methodologically different parts. Firstly, concentrations of total mercury and MeHg in isopods P. scaber and their environment from a Hg-unpolluted area were measured by the use of validated methods (CV AAS, CV AFS). The data obtained show that the percentage of MeHg in leaves, soil and faeces was less than 1%. In contrast, the percentage of MeHg in gut and hepatopancreas was increased to 14 and 77%, respectively, indicating methylation of Hg(2+) in the gut and its further accumulation in glands. To confirm this assumption, the second methodology was applied-a radiotracer technique with 203Hg(2+) of high specific activity. There are few radiotracer techniques for Hg-methylation assays; for our work we chose the method of Czuba et al. which includes alkaline leaching of Hg species, their extraction into dithizone-toluene, followed by specific separation of Hg dithizonates by thin-layer chromatography and gamma counting. All steps of the analytical protocol were checked and optimised by the use of aqueous solutions of 203Hg(2+) and Me(203)Hg(+). The most important finding was that cleaning-up the extract through a florisil column is not appropriate, because the column retains different percentages of Hg(2+) and MeHg(+) and consequently affects the accuracy of the final result. This optimised protocol was then applied to Hg transformation studies in the terrestrial isopod P. scaber. Leaching Hg species from P. scaber fed with 203Hg(2+) or Me(203)Hg(+) dosed food was completely efficient only at elevated temperatures. Preliminary results of methylation/demethlytion studies are rather variable but they show that both processes (Hg(2+)<-->MeHg(+)) take place in the isopod P. scaber. Additionally, an assessment of the mass balance of Hg in isopods P. scaber exposed to 203Hg(2+) indicates that volatile Hg species are also formed.

The influence of complexing agent and proteins on the corrosion of stainless steels and their metal components.

The present work is devoted to the problem of biodegradation of orthopaedic implants manufactured from stainless steel. In vitro simulations of the biocompatibility of two types of stainless steel, AISI 304 and AISI 316L, and their individual metal components, i.e. iron, chromium, nickel and molybdenum, were carried out in simulated physiological solution (Hank's) containing complexing agents. Knowledge of the effects of the chemical and biological complexing agents, EDTA and proteins, respectively, on the corrosion resistance of a metal should provide a better understanding of the processes occurring in vivo on its surface. The behavior of stainless steels and metal components was studied under open circuit and under potentiostatic conditions. The concentration of dissolved corrosion products in the form of released ions was determined by differential pulse polarography (DPP) and atomic emission spectrometry using inductively coupled plasma (ICP-AES). The composition of solid corrosion products formed on the surface was analyzed by energy dispersive X-ray spectroscopy (EDS) and their morphology was viewed by scanning electron microscopy (SEM). The addition of EDTA and proteins to physiological solution increased the dissolution of pure metals and stainless steels. The effect of particular protein differs on different metals and alloys.

Determination of trace cobalt concentrations in human serum by adsorptive stripping voltammetry.

The goal of our study was to develop an accurate and reliable method for determining trace cobalt concentrations in human serum. The method was used to determine cobalt in the sera of healthy persons and patients with orthopaedic implants containing cobalt - a possible source of systemic release of cobalt into the human body. This goal is of vital interest since cobalt and its compounds are classified by IARC as potentially carcinogenic to humans. We used an electrochemical method, adsorptive stripping voltammetry (AdSV), which made possible the low detection limit and high sensitivity needed for measurements in human serum. The serum was acid digested by a combination of H2SO4, HNO3 and H2O2 in a 10 mL Kjeldhal flask. The digested sample was then dissolved in 0.1 mol/L ammonia buffer, pH 9.0 +/- 0.2. The determination is based on the adsorptive collection of the complex of cobalt (II) with dimethylglyoxime on a hanging mercury drop electrode (HMDE). The optimum values of adsorption potential and time were determined to be -0.8 V and 60 s. The optimisation of the sample digestion protocol and measurement procedures ensured the reliable assessment of low cobalt concentrations, down to 0.03 microg/L. The mean concentration of serum cobalt in four healthy persons was 0.11 +/- 0.06 microg/L, and in four patients with total hip replacements 0.34 +/- 0.07 microg/L. This method will be used routinely for measuring serum cobalt levels in patients with total hip replacements.

Optimisation of a dialytic set-up for liquid chromatography: automated separation and preconcentration of ciprofloxacin.

Continuous-flow and static dialysis coupled on-line to liquid chromatography was evaluated and an automated method for determination of ciprofloxacin in biological samples developed. A trace enrichment column packed with C18 material and coupled with a continuous dialysis and reversed-phase HPLC system with fluorescence detection enabled determination of ciprofloxacin in human blood serum at the 0.1-nmol/l level. The amount of analyte preconcentrated and loaded on the HPLC system was linearly proportional to the concentration in the dialysed sample over more than 4 orders of magnitude (up to 1 x 10(-6) M). Data for linearity, repeatability and detectability for each particular set-up are given. The trace enrichment step eliminates band broadening caused by solvents different from those of the eluent and affecting retention of ciprofloxacin on the analytical column (increase in k') due to the on-column change of eluent composition. In analysis of human serum samples phthalates leached from plastic materials may interfere due to coelution with the analyte.

Simultaneous determination of inorganic mercury and methylmercury compounds in natural waters.

The purpose of the present work was to develop a simple, rapid, sensitive and accurate method for the simultaneous determination of inorganic mercury (Hg(2+)) and monomethylmercury compounds (MeHg) in natural water samples at the pg L(-1) level. The method is based on the simultaneous extraction of MeHg and Hg(2+)dithizonates into an organic solvent (toluene) after acidification of about 300 mL of a water sample, followed by back extraction into an aqueous solution of Na(2)S, removal of H(2)S by purging with N(2), subsequent ethylation with sodium tetraethylborate, room temperature precollection on Tenax, isothermal gas chromatographic separation (GC), pyrolysis and cold vapour atomic fluorescence spectrometric detection (CV AFS) of mercury. The limit of detection calculated on the basis of three times the standard deviation of the blank was about 0.006 ng L(-1) for MeHg and 0.06 ng L(-1) for Hg(2+)when 300 mL of water was analysed. The repeatability of the results was about 5% for MeHg and 10% for Hg(2+). Recoveries were 90-110% for both species.