pH-stat Apparatus Research Articles

Small-volume in vitro lipid digestion measurements for assessing drug dissolution in lipid-based formulations using SAXS

Lipid-based formulations improve the absorption capacity of poorly-water-soluble drugs and digestion of the formulation is a critical step in that absorption process. A recent approach to understanding the propensity for drug to dissolve in digesting lipid-based formulations couples an in vitro pH-stat lipolysis model to small-angle X-ray scattering (SAXS) by means of a flow-through capillary. However, the conventional pH-stat apparatus used to measure the extent of lipid digestion during such experiments requires digest volumes of 15–30 mL and drug doses of 50–200 mg, which is problematic for scarce compounds and can require excessive amounts of formulation reagents. This manuscript describes an approach to reduce the amount of material required for in vitro lipolysis experiments coupled to SAXS, for use in instances where the amount of drug or formulation medium is limited. Importantly, this was achieved while maintaining the pH stat conditions, which is critical for maintaining biorelevance and driving digestion to completion. The digestibility of infant formula with the poorly-water-soluble drugs halofantrine and clofazimine dispersed into it was measured as an exemplar paediatric-friendly lipid formulation. Halofantrine was incorporated in its powdered free base form and clofazimine was incorporated both as unformulated drug powder and as drug in nanoparticulate form prepared using Flash NanoPrecipitation. The fraction of triglyceride digested was found to be independent of vessel size and the incorporation of drug. The dissolution of the two forms of clofazimine during the digestion of infant formula were then measured using synchrotron SAXS, which revealed complete and partial solubilisation over 30 min of digestion for the powdered drug and nanoparticle formulations, respectively. The main challenge in reducing the volume of the measurements was in ensuring that thorough mixing was occurring in the smaller digestion vessel to provide uniform sampling of the dispersion medium.

Evaluation of the digestibility of solid lipid nanoparticles of glyceryl dibehenate produced by two techniques: Ultrasonication and spray-flash evaporation

ObjectiveTo evaluate the digestibility of Solid Lipid Nanoparticles (SLN) of glyceryl dibehenate prepared either with surfactants by ultrasonication or without surfactant by spray-flash evaporation. MethodsSLN of glyceryl dibehenate (Compritol® 888 ATO) were produced by two processes: (i) high-shear homogenization with a solution of water-soluble surfactants followed by ultrasonication (ii) and Spray-Flash Evaporation (SFE) of the pure lipid. The digestibility of these nanoparticles was then tested by in vitro lipolysis using a pH-stat apparatus and the assay of glycerides by gel phase chromatography. ResultsSLN of glyceryl dibehenate prepared by ultrasonication exhibited a mean particle size of 180nm and showed a limited digestion of the lipid excipient. SLN comprising only glyceryl dibehenate produced by SFE have a mean particle size between 235 and 411nm depending on process parameters. These nanoparticles were not digested by lipases. The presence of surfactant at the lipid/water interface of the SLN seems to be mandatory to allow the adsorption of the lipase and degradation of glyceryl behenate. ConclusionsGlyceryl dibehenate as a solid particle – even as a SLN – is not digested by pancreatin during in vitro lipolysis test.

Effect of Fly Ash as an Additive on the Limestone Dissolution Rate Constant

Limestone dissolution is a very important factor in flue gas desulfurization systems because it determines its reactivity toward SO2. Fly ash, a siliceous material, has been reported to improve sorbent reactivity. This study investigates the effect of adding fly ash to limestone on its dissolution rate constant. The experiments were carried out using a pH stat apparatus where the effects of the reaction variables, fly ash/limestone ratio, slurry pH, reaction temperature, and concentration of acid, used were investigated. The central composite design (CCD) of the experiment was used to develop a model that correlates the dissolution rate constant and the reaction variables. It was found that fly ash had a positive effect on the dissolution rate constant of limestone, with the pH having the most significant effect. The dissolution rate constant was found to increase with an increase in the temperature and acid concentration. X-ray diffraction (XRD) analysis showed products of hydration formed, which are mainly calcium silicate hydrates, on the samples. This led to an increase in the specific surface area, as observed in the Brunauer–Emmett–Teller (BET) analysis.

In vitro lipolysis tests on lipid nanoparticles: comparison between lipase/co-lipase and pancreatic extract

Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLC) are lipid nanocarriers aimed to the delivery of drugs characterized by a low bioavailability, such as poorly water-soluble drugs and peptides or proteins. The oral administration of these lipid nanocarriers implies the study of their lipolysis in presence of enzymes that are commonly involved in dietary lipid digestion in the gastrointestinal tract. In this study, a comparison between two methods was performed: on one hand, the lipase/co-lipase assay, commonly described in the literature to study the digestion of lipid nanocarriers, and on the other hand, the lipolysis test using porcine pancreatic extract and the pH-stat apparatus. This pancreatic extract contains both the pancreatic lipase and carboxyl ester hydrolase (CEH) that permit to mimic in a biorelevant manner the duodenal digestive lipolysis. The test was performed by means of a pH-stat apparatus to work at constant pH, 5.5 or 6.25, representing respectively the fasted or fed state pH conditions. The evolution of all acylglycerol entities was monitored during the digestion by sampling the reaction vessel at different time points, until 60 min, and the lipid composition of the digest was analyzed by gas chromatography. SLN and NLC systems obtained with long-chain saturated acylglycerols were rapidly and completely digested by pancreatic enzymes. The pH-stat titration method appears to be a powerful technique to follow the digestibility of these solid lipid-based nanoparticles.

Dissolution kinetics of magnesium hydroxide for CO2 separation from coal-fired power plants

The dissolution of magnesium hydroxide in water for the release of magnesium and hydroxyl ions into the solution to maintain suitable alkalinity is a crucial step in the Mg(OH)2-based CO2 absorption process. In this study, the rate of dissolution of Mg(OH)2 was investigated under different operating conditions using a pH stat apparatus. The dissolution process was modeled using a shrinking core model and the overall Mg(OH)2 dissolution process was found to be controlled by the surface chemical reaction of Mg(OH)2 with H+ ions. Under the chemical reaction control regime, the dissolution of Mg(OH)2 in alkaline conditions was found not to follow a first-order reaction, and the fractional order of reaction was estimated to lie between 0.20 and 0.31. This suggests that the dissolution reaction is a non-elementary reaction, consisting of a sequence of elementary reactions, via most likely forming a surface magnesium complex. The true activation energy value of 76±11kJ/gmol was found to be almost twice as much as the observed activation energy value of 42±6kJ/gmol determined at pH 8.6, and was comparable with the previously reported values. The particle sizes predicted from the intrinsic kinetics determined from the model were in good agreement with the experimentally measured particle sizes during the dissolution process.

Improving Lime’s Reactivity Towards Flue Gas Desulfurization by using Fly Ash, Bottom Ash and Waste Activated Sludge

Lime reactivity was improved by blending it with either fly ash, bottom ash or waste activated sludge (W.A.S). The reactivity was tested using a pH-stat apparatus which simulates wet flue gas desulfurization (FGD). Temperature, solid to liquid ratio and stirring speed were varied in addition to lime to additive ratio in optimization tests. Design expert’s central cubic design was used in the design of experiments to aid with regression analysis. Temperature had the highest effect whereas W.A.S was the best additive then fly ash and finally bottom ash. The results were confirmed by the use of fixed bed apparatus where the best sorbents from each additive was tested in dry FGD. In a bid to explain the results, BET surface area analysis was used where the sorbent from the blend of lime and W.A.S had the highest improvement in surface area.

Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations, Part 1: Method Parameterization and Comparison of In Vitro Digestion Profiles Across a Range of Representative Formulations

The Lipid Formulation Classification System Consortium is an industry-academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation.

Blending lime and iron waste to improve sorbents reactivity towards desulfurization

Lime and iron waste were blended in various ratios and the resulting sorbents tested in two equipments namely; pH-stat apparatus simulating wet flue gas desulfurization (FGD) and fixed bed reactor for dry FGD, to investigate the effect of the blends in the FGD process. The best blend was found at a ratio of 1:2 (lime to iron waste) by mass. Brunauer, Emmett and Teller (BET) surface area analysis and Scanning Electron Microscopy (SEM) were used to assist in characterizing the resulting sorbents. Temperature, solid to liquid ratio and stirring speed were varied in addition to lime to iron waste ratio in optimization tests that followed. Design expert’s central cubic design was used in the design of experiments to aid with regression analysis. Temperature had the highest effect, followed by lime to iron waste ratio. Solid to liquid ratio had the least effect on the overall reactivity of the sorbents. The effects were more pronounced at higher levels of these variables.

Use of fly ash, bottom ash and zeolite as additives for enhancing lime reactivity towards flue gas desulfurization

Lime reactivity toward desulfurization was examined by blending lime with pozzolanic additives namely fly ash, bottom ash, and zeolite. The reactivity was tested in a pH-stat apparatus. Apart from lime to additive ratio, other important operating conditions, namely, temperature, solid to liquid ratio, and stirring speed, were varied accordingly using the design expert's design of experiments. Central cubic design was used to investigate linear, quadratic, cubic, and cross-product effect of these operatives. Sorbents made from lime-zeolite blends were found to have the highest reactivity followed by fly ash then bottom ash. These results were confirmed through sorption capacities of the best sorbents using a fixed bed reactor. BET surface area analysis of the best sorbents indicated that surface area improvement is the root cause of improvements in reactivity and sorption capacities. Temperature had the largest effect on reactivity. The other operative's effect varied from one additive to the other. © 2011 American Institute of Chemical Engineers Environ Prog, 32: 75–83, 2013.

Enhancement of lime reactivity by addition of diatomite

Diatomite was used as a Pozzolan to augment lime reactivity. This was confirmed through laboratory wet and dry flue gas desulfurization (FGD) experiments using pH-stat apparatus (representing wet FGD) and fixed bed reactors (representing dry FGD). BET surface area analysis and SEM imaging were used in characterization and to aid in understanding the behavior of the sorbent. After affirming that diatomite improves lime's reactivity, optimization tests were conducted to maximize the reactivity output. The effect of various variables namely; temperature, lime to diatomite ratio, solid to liquid ratio and stirring speed, were maximized linearly, quadratically and interactively. The design of optimization experiments was done through design expert software. Central cubic design was used for regression analysis. As expected, temperature had the highest effect towards reactivity. Some effect showed a mini-max behavior while others had a sinusoidal trend.

Investigating Effects of Zeolites As an Agent to Improve Limestone Reactivity toward Flue Gas Desulfurization

Natural zeolite was utilized as an agent to increase the reactivity of lime for desulfurization. Due to the presence of hydrous silica−aluminates in zeolite, a pozzolanic reaction ensued which ended up with an increase in the surface area of lime thus making it more reactive. Tests were done in a pH-stat apparatus and a fixed bed reactor, representing wet and dry flue gas desulfurization (FGD), respectively, to check if lime reactivity was augmented by the addition of zeolite. It was found that zeolite amplifies reactivity of lime with the highest reactivity being achieved with a lime to zeolite ratio of 1:1 (by mass). This was confirmed by Brunauer−Emmett−Teller (BET) surface area analysis (surface area increment with the reactivity) and scanning electron microscopy (SEM) imaging (rough morphology being formed). Further optimization tests were conducted in the pH-stat apparatus with the aid of design expert software to quantify linear, quadratic, and interactive effects of four variables (temperature, lime to zeolite ratio, liquid to solid ratio, and stirring speed). Temperature as a variable had the highest effect, followed by lime to zeolite ratio. Stirring speed had the least effect on the reactivity.

Biochemical Characterization of SHV-55, an Extended-Spectrum Class A β-Lactamase from Klebsiella pneumoniae

We biochemically characterized the Klebsiella pneumoniae extended-spectrum SHV-55 enzyme carrying the amino acid substitutions Tyr7→Phe (as in SHV-28) and Gly238→Ser and Glu240→Lys (both found in SHV-5) identified in a previous study (5). The SHV-55 extended-spectrum β-lactamase differed from SHV-5 only in the signal peptide region (1). The blaSHV-55 gene was obtained as described by Mendonca et al. (6), and transformants were selected on Luria broth agar supplemented with 30 μg of kanamycin/ml and 16 μg of amoxicillin/ml. SHV-55 was extracted and purified according to the previously described protocol (6). The Michaelis constant (Km) and catalytic activity (kcat) of purified extracts of SHV-55 were obtained by using a computerized microacidimetric method and a 702 SM Titrino pH-stat apparatus (Metrohm, Herisau, Switzerland) (3). The complete hydrolysis time courses were analyzed, and the kinetic progress curves were fitted by nonlinear least-squares regression. These kinetic parameters were determined and compared to those of the SHV-1 enzyme for 10 β-lactams (Table ​(Table11). TABLE 1. Kinetic constants of SHV-55 and SHV-1 β-lactamasesa SHV-55 has a high affinity (Km, 5 to 10 μM) for penicillins, similar to that of SHV-5 (1) and higher than that of SHV-1 (Km, 11 to 31 μM). SHV-55 presented higher affinity values (Km, 9 to 58 μM) than SHV-1 (Km, 40 to 257 μM) for narrow-, extended-, and broad-spectrum cephalosporins and monobactams. This finding may be a consequence of the Gly238→Ser substitution present in the active sites of both SHV-55 and SHV-5, which pushes the β-strand out and away from the reactive Ser70 (2). This effect results in a slightly expanded active site that may improve binding and accommodate cephalosporins with bulky side chains (4). SHV-55 presented a higher affinity for cefotaxime than for ceftazidime (Kms, 21 and 58 μM, respectively), as did SHV-5 (1, 7). This finding is surprising because both enzymes possess the Glu240→Lys substitution, which increases hydrolytic activity against ceftazidime (8) due to the change in the electrostatic charge of the exposed group at position 240 (2). The enzymatic activities (kcats) of SHV-55 for penicillin G and amoxicillin were 84- and 45-fold lower, respectively, than those of SHV-1, and the catalytic efficiency (kcat/Km ratio) against penicillins was more than 10-fold higher for SHV-1 (kcat/Km ratio, 20 to 84 μM−1·s−1) than for SHV-55 (kcat/Km ratio, 2 to 5 μM−1·s−1). However, the enzyme activity and catalytic efficiency against extended- and broad-spectrum cephalosporins were higher for SHV-55 (kcat, 7 to 24 s−1, and kcat/Km ratio, 0.2 to 1 μM−1·s−1) than for SHV-1 (note, however, that the values for monobactam were undeterminable), although the catalytic efficiencies of the two enzymes against cephalothin were similar (kcat/Km ratios, 3.2 and 4.4 μM−1·s−1). This result may be due to the amino acid substitutions in SHV-55 causing conformational modifications in the active site. Fifty percent inhibitory concentrations (IC50s) indicated that SHV-55 was ninefold more susceptible to the inhibitor activity of clavulanate than SHV-1 (IC50s of clavulanate, 0.02 versus 0.17 μM). In conclusion, these results confirmed the extended-spectrum activity of the SHV-55 enzyme, which is important due to the magnitude of extended- and broad-spectrum SHV β-lactamases described to date and not biochemically characterized, in spite of the ease of sequencing genes (http://www.lahey.org/studies).

Production of medium-chain-length hydroxyalkanoic acids from Pseudomonas putida in pH stat

Pseudomonas putida GP01 cells that had accumulated medium-chain-length polyhydroxyalkanoates (PHA(MCL)) secreted 3-hydroxyoctanoate and 3-hydroxyhexanoate when incubated in alkaline buffers. The release of acids strongly decreased the pH resulting in less efficient secretion of 3HA(MCL) at neutral pH. To increase the yield of secreted MCL-hydroxyalkanoates, experiments at constant pH in a pH stat apparatus were performed. High acid releasing rates were recorded for the wild type GP01 at pH 9.2 (0.60 mmol acid h(-1) g(-1) cellular dry weight [cdw]). At more alkaline constant pH values (pH 9.3-11), the initial acid secretion rates were even higher but rapidly decreased by time. When acid secretion of PHA depolymerase mutant GPo500 was tested (pH 9.2), considerably lower rates compared to wild type were recorded (0.18 mmol acid h(-1) g(-1) cdw). Determination of dissolved oxygen during acid release indicated different respiratoric activity in wild type (low) and mutant (high). Acid release of mutant, but not of the wild type, could be enhanced by aeration. Determination of PHA content of cells after alkaline incubation showed that the wild type had lost most of its accumulated PHA, whereas the PHA content of the depolymerase mutant was not significantly reduced. Considerable amounts of 3HA(MCL) were secreted by the wild type, but only little 3HA(MCL) were found for the depolymerase mutant. In summary, 3HA(MCL) can be more efficiently produced at constant high pH than by incubation without pH control. High PHA depolymerase activity enabled the wild type to compensate for the high external pH by secretion of PHA hydrolysis products, whereas production of protons at aerobic conditions presumably was responsible for the major portion of the observed acid releasing rates in the depolymerase mutant.

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.

Growth kinetics of CaF 2 in a pH-stat fluidized-bed crystallizer

In this study, experiments were first conducted in a stirred vessel to establish the metastable region of calcium fluoride, using a pH-stat apparatus for controlling the pH value of solution. The effects of pH value on the solubility and metastable region were observed. Then, operating variables, including supersaturation, superficial velocity, pH value, seed size, and types of seed and solution, that influenced the crystal growth of the same system were investigated in a fluidized-bed crystallizer. The crystal growth rates were determined from the consumption rates of fluoride ions. The growth kinetics was explored by using the two-step growth model. The effectiveness factor was used to judge whether a controlling step was existing. The kinetics of crystal growth in a lean bed and a dense bed was also discussed.

Oxidation of Good’s buffers by hydrogen peroxide

Good’s zwitterionic buffers are widely used in biological and biochemical research in which hydrogen peroxide is a solution component. This study was undertaken to determine whether Good’s buffers exhibit reactivity toward H 2O 2. It is found that H 2O 2 oxidizes both morpholine ring-containing buffers (e.g., Mops, Mes) and piperazine ring-containing zwitterionic buffers (e.g., Pipes, Hepes, and Epps) to produce their corresponding N-oxide forms. The percentage of oxidized buffer increases as the concentration of H 2O 2 increases. However, the rate of oxidation is relatively slow. For example, no oxidized Mops was detected 2 h after adding 0.1 M H 2O 2 to 0.1 M Mops (pH 7.0), and only 5.7% was oxidized after 24 h exposure to H 2O 2. Thus, although all of these buffers can be oxidized by H 2O 2, their slow reaction does not significantly perturb levels of H 2O 2 in the time frame and at the concentrations of most biochemical studies. Therefore, the previously reported rapid loss of H 2O 2 produced from the ferroxidase reaction of ferritin is unlikely due to reaction of H 2O 2 with buffer, a conclusion supported by the fact that H 2O 2 is also lost rapidly when the solution pH of the ferroxidase reaction is controlled by a pH stat apparatus in the absence of buffer.

Development of myofibrillar ATPase assay system on pH stat

The ATPase assay system on pH stat apparatus was developed. For the ATPase activity measurement, hydrogen ion (H+) concentration delivered from inorganic phosphate (Pi) as a hydrolysis production of ATP was estimated by titrating with 20 mM NaOH solution instead of colorimetric measurement of Pi. Inclusion of 0.5 M KCl in the ATP stock solution and 2 mM Tris-maleate (pH 7.0) buffer in the reaction medium reduced the extent of pH change upon addition of ATP to initiate the ATPase reaction. The amount of H+ liberated from Pi was strongly affected by the set pH for the ATPase assay with a promotion at alkaline pH. Thus, it was required to estimate the coefficient to convert H+ to a Pi concentration at a specific pH. The specified coefficient at pH 7.0 was 1.248. ATPase assay on pH stat allowed us to follow the ATP hydrolysis by myofibrils continuously showing a curvature profile at low salt medium (≤0.2 M KCl) or a linear profile at high salt (≥0.3 M KCl).

Mineralogical and isotopic properties of inorganic nanocrystalline magnetites

Inorganic magnetite nanocrystals were synthesized in an aqueous medium at 25°C, atmospheric pressure, ionic strength of 0.1 M, oxygen fugacity close to 0, and under controlled chemical affinity, which was maintained constant during an experiment and varied between different experiments. The total concentration of iron in the initial solutions, with Fe(III)/Fe(II) ratios of 2, was varied in order to measure the role of this parameter on the reaction rate, particle morphology, and oxygen isotopic composition. The reaction rates were followed by a pHstat apparatus. The nature and morphology of particles were studied by transmission electron microscopy and electron energy loss spectroscopy. Fractionation factors of oxygen isotopes were determined by mass spectrometry after oxygen extraction from the solid on BrF 5 lines. At low total iron concentrations, goethite and poorly crystalline iron oxides were observed coexisting with magnetite. At higher concentrations, euhedral single crystals of pure magnetite with an average characteristic size of 10 nm were formed, based on a first-order rate law with respect to total iron concentration. These results confirm that, under high supersaturation conditions, low-temperature inorganic processes can lead to the formation of well-crystallized nanometric magnetite crystals with narrow size distribution. The observed oxygen isotope fractionation factor between magnetite crystals and water was of 0–1‰, similar to the fractionation factor associated with bacterially produced magnetite. We suggest that the solution chemistry used in this study for inorganic precipitation is relevant to better understanding of magnetite precipitation in bacterial magnetosomes, which might thus be characterized by high saturation states and pH.

Effects of dissolved carbonate on arsenate adsorption and surface speciation at the hematite--water interface.

Effects of dissolved carbonate on arsenate [As(V)] reactivity and surface speciation at the hematite-water interface were studied as a function of pH and two different partial pressures of carbon dioxide gas [P(CO2) = 10(-3.5) atm and approximately 0; CO2-free argon (Ar)] using adsorption kinetics, pseudo-equilibrium adsorption/titration experiments, extended X-ray absorption fine structure spectroscopic (EXAFS) analyses, and surface complexation modeling. Different adsorbed carbonate concentrations, due to the two different atmospheric systems, resulted in an enhanced and/or suppressed extent of As(V) adsorption. As(V) adsorption kinetics [4 g L(-1), [As(V)]0 = 1.5 mM and I = 0.01 M NaCl] showed carbonate-enhanced As(V) uptake in the air-equilibrated systems at pH 4 and 6 and at pH 8 after 3 h of reaction. Suppressed As(V) adsorption was observed in the air-equilibrated system in the early stages of the reaction at pH 8. In the pseudo-equilibrium adsorption experiments [1 g L(-1), [As(V)]0 = 0.5 mM and I = 0.01 M NaCI], in which each pH value was held constant by a pH-stat apparatus, effects of dissolved carbonate on As(V) uptake were almost negligible at equilibrium, but titrant (0.1 M HCl) consumption was greater in the air-equilibrated systems (P(CO2) = 10(-3.5) atm) than in the CO2-free argon system at pH 4-7.75. The EXAFS analyses indicated that As(V) tetrahedral molecules were coordinated on iron octahedral via bidentate mononuclear ( 2.8 A) and bidentate binuclear (approximately equal to 3.3 A) bonding at pH 4.5-8 and loading levels of 0.46-3.10 microM m(-2). Using the results of the pseudo-equilibrium adsorption data and the XAS analyses, the pH-dependent As(V) adsorption under the P(CO2) = 10(-3.5) atm and the CO2-free argon system was modeled using surface complexation modeling, and the results are consistent with the formation of nonprotonated bidentate surface species at the hematite surfaces. The results also suggest that the acid titrant consumption was strongly affected by changes to electrical double-layer potentials caused by the adsorption of carbonate in the air-equilibrated system. Overall results suggest that the effects of dissolved carbonate on As(V) adsorption were influenced by the reaction conditions [e.g., available surface sites, initial As(V) concentrations, and reaction times]. Quantifying the effects of adsorbed carbonate may be important in predicting As(V) transport processes in groundwater, where iron oxide-coated aquifer materials are exposed to seasonally fluctuating partial pressures of CO2(g).

Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidation

It is widely accepted that iron deposition in the iron storage protein ferritin in vitro involves Fe(II) oxidation, and that ferritin facilitates this oxidation at a ferroxidase site on the protein. However, these views have recently been questioned, with the protein ferroxidase activity instead being attributed to autoxidation from the buffer alone. Ligand exchange between another protein with ferroxidase activity and ferritin has been proposed as an alternative mechanism for iron incorporation into ferritin. In the present work, a pH stat apparatus is used to eliminate the influence of buffers on iron(II) oxidation. Here we show that the recent experiments questioning the ferroxidase activity of ferritin were flawed by inadequate pH control, that buffers actually retard rather than facilitate iron(II) oxidation, and that horse spleen ferritin has ferroxidase activity when measured under proper experimental conditions. Furthermore, high pH (7.0), a high Fe(II) concentration and the presence of Fe(III) all favour Fe(II) autoxidation in the presence or absence of ferritin.