A ph study of calcium phosphate seeded precipitation

The formation of calcium phosphate phases over a range of pH has been investigated at 37°C using a constant composition method. At a controlled ionic strength of 0.10 mole/liter, and Ca/P = 1.333 the precipitation of octacalcium phosphate appears to be limited to a pH range of 6 to 7. At pH 5, dicalcium phosphate dihydrate is formed at all supersaturations whereas at pH 5.5, it occurs only at high supersaturation; octacalcium phosphate precipitating at low supersaturation. At higher pH (7.4 and 8.0), the results point to the formation of a hydroxyapatite-like material.     

Periodic Nucleation of Calcium Phosphate in a Stirred Biocatalytic Reaction

Highly ordered superstructures composed of inorganic nanoparticles appear in natural and synthetic systems, however the mechanisms of non‐equilibrium self‐organization that may be involved are still poorly understood. Herein, we performed a kinetic investigation of the precipitation of calcium phosphate using a process widely found in microorganisms: the hydrolysis of urea by enzyme urease. With high initial ratio of calcium ion to phosphate, periodic precipitation was obtained accompanied by pH oscillations in a well‐stirred, closed reactor. We propose that an internal pH‐regulated change in the concentration of phosphate ion is the driving force for periodicity. A simple model involving the biocatalytic reaction network coupled with burst nucleation of nanoparticles above a critical supersaturation reproduced key features of the experiments. These findings may provide insight to the self‐organization of nanoparticles in biomineralization and improve design strategies of biomaterials for medical applications.     

Nature identification and morphology characterization of anion-exchange membrane fouling during conventional electrodialysis

The aim of this work was to study the effect on the fouling of anion-exchange membranes (AEM) of (1) the pH value of the concentrate solution and (2) the composition in calcium, carbonate, and protein of the diluate solution to be treated by conventional electrodialysis. It appeared that after demineralization of solutions containing CaCl(2) using a concentrate solution maintained at a pH value of 7 or 12, mineral fouling appeared on the AEM surface in contact with the concentrate. The mineral deposits presented a cylindrical filament shape for conditions with a concentrate solution pH value of 7, while, for a pH value of 12, the mineral deposit had a crumbly and spongy texture formed by irregular aggregates. The nature of the fouling was identified as a calcium phosphate with or without calcium hydroxide. In addition, gel-like protein fouling was detected on the AEM surface in contact with the diluate after demineralization procedures using a concentrate pH value of 2 or 7, regardless of the mineral composition of the diluate.     

Formation of calcium phosphates in gelatin with a novel diffusion system

The present paper demonstrated a novel and simple diffusion system to precipitate calcium phosphates in gelatin gel. In this system, a gelatin cup was specially used as the membrane separating reservoirs of calcium and phosphate ions. Relative to the conventional diffusion system, the novel one in our experiment decreased the time required for the deposition from 5-7 days to 20 h and increased the amount of the precipitated mineral phases significantly. The influence of pH values and concentrations of calcium and phosphate solutions buffered with Tris-HCl and NaOH, respectively, was investigated. The results showed that precipitation of the mineral phase at low pH values (7 for calcium and 11 for phosphate) and concentrations (200 mM for calcium and 15 mM for phosphate) resulted in the formation of plate-like octacalcium phosphate (OCP) crystals. With increasing the pH values of calcium and phosphate solutions to 8 and 12, respectively, spherical amorphous calcium phosphate (ACP) particles were obtained uniquely. Furthermore, flower-like hydroxyapatite (HAP) aggregates composed of many nano-sized needles were formed from the solutions with high pH values (8 for calcium and 12 for phosphate) and concentrations (500 mM for calcium and 37.5 mM for phosphate). The novel diffusion system is proposed to play an important role in both studying the process of biological mineralization and synthesizing calcium phosphates in different forms.     

Calcium-phosphorus interactions at a nano-structured silicate surface

Nano-structured calcium silicate (NCS), a highly porous material synthesized by controlled precipitation from geothermal fluids or sodium silicate solution, was developed as filler for use in paper manufacture. NCS has been shown to chemisorb orthophosphate from an aqueous solution probably obeying a Freundlich isotherm with high selectivity compared to other common environmental anions. Microanalysis of the products of chemisorption indicated there was significant change from the porous and nano-structured morphology of pristine NCS to fibrous and crystalline morphologies and non-porous detritus. X-ray diffraction analysis of the crystalline products showed it to be brushite, CaHPO42H2O, while the largely X-ray amorphous component was a mixture of calcium phosphates. A two-step mechanism was proposed for the chemisorption of phosphate from an aqueous solution by NCS. The first step, which was highly dependent on pH, was thought to be desorption of hydroxide ions from the NCS surface. This was kinetically favoured at lower initial pH, where the predominant form of phosphate present was H2PO(-)4, and led to decreased phosphorus uptake with increasing pH. The second step was thought to be a continuing chemisorption process after stabilization of the pH-value. The formation of brushite as the primary chemisorption product was found to be consistent with the proposed mechanism.     

On the crucial importance of the pH for the formation and self-stabilization of protein microgels and strands

Stable suspensions of protein microgels are formed by heating salt-free β-lactoglobulin solutions at concentrations up to about C = 50 g·L(-1) if the pH is set within a narrow range between 5.75 and 6.1. The internal protein concentration of these spherical particles is about 150 g·L(-1) and the average hydrodynamic radius decreases with increasing pH from 200 to 75 nm. The formation of the microgels leads to an increase of the pH, which is a necessary condition to obtain stable suspensions. The spontaneous increase of the pH during microgel formation leads to an increase of their surface charge density and inhibits secondary aggregation. This self-stabilization mechanism is not sufficient if the initial pH is below 5.75 in which case secondary aggregation leads to precipitation. Microgels are no longer formed above a critical initial pH, but instead short, curved protein strands are obtained with a hydrodynamic radius of about 15-20 nm.     

Structural changes of casein micelles in a calcium gradient film

Calcium gradients are prepared by sequentially filling a micropipette with casein solutions of varying calcium concentration and spreading them on glass slides. The casein film is formed by a solution casting process, which results in a macroscopically rough surface. Microbeam grazing incidence small-angle X-ray scattering (microGISAXS) is used to investigate the lateral size distribution of three main components in casein films: casein micelles, casein mini-micelles, and micellar calcium phosphate. At length scales within the beam size the film surface is flat and detection of size distribution in a macroscopic casein gradient becomes accessible. The model used to analyze the data is based on a set of three log-normal distributed particle sizes. Increasing calcium concentration causes a decrease in casein micelle diameter while the size of casein mini-micelles increases and micellar calcium phosphate particles remain unchanged.     

Whisker nanohydroxyapatite synsthesis in casein medium and the effect of protein solution concentration on the morphology and structure of nanohydroxyapatite

The means of hydroxyapatite nanoparticles (n-HAP) preparation in biopolymer modifiers solutions under ultrasonic activation have been investigated. The use of casein as a modificator was suggested. It was shown that a protein solution with a concentration of 0.2% provides whisker n-HAP with a diameter of 2 nm and a length of 10 nm. This size of n-HAP is correlated to colloidal calcium phosphate in milk casein micelles. Synthesized nanoparticles can be used for the intensification of technologies processes in the dairy industry and for the fortification of food with calcium in the natural form.     

Formation and stability of poly-L-lysine/casein multilayers

The aim of our work was to investigate formation of multilayer films containing biocompatible polycation poly-L-lysine (PLL) and α- or β-casein. Since in the neutral pH casein is negatively charged, it has been used as a polyanionic layer for the film build-up. Casein containing films were formed at surface of Si/SiO₂ wafers and their thickness was measured by ellipsometry. The effect of ionic strength of PLL and casein solutions was investigated. After the multilayer films were formed, they were contacted with solutions having various pH and salts to determine film stability under these conditions. Additionally the response of the thickness of PLL/casein films to the temperature variation in the range of 5–45?°C was also analyzed. Formation and stability of casein containing films was also investigated on surfaces of titanium and stainless steel. We used fluorescently labeled protein to monitor the amount of casein in the film and its change after treatment with solutions containing calcium ions.     

Effects of the environmental factors on the casein micelle structure studied by cryo transmission electron microscopy and small-angle x-ray scattering/ultrasmall-angle x-ray scattering

Casein micelles are colloidal protein-calcium-transport complexes whose structure has not been unequivocally elucidated. This study used small-angle x-ray scattering (SAXS) and ultrasmall angle x-ray scattering (USAXS) as well as cryo transmission electron microscopy (cryo-TEM) to provide fine structural details on their structure. Cryo-TEM observations of native casein micelles fractionated by differential centrifugation showed that colloidal calcium phosphate appeared as nanoclusters with a diameter of about 2.5 nm. They were uniformly distributed in a homogeneous tangled web of caseins and were primarily responsible for the intensity distribution in the SAXS profiles at the highest q vectors corresponding to the internal structure of the casein micelles. A specific demineralization of casein micelles by decreasing the pH from 6.7 to 5.2 resulted in a reduced granular aspect of the micelles observed by cryo-TEM and the existence of a characteristic point of inflection in SAXS profiles. This supports the hypothesis that the smaller substructures detected by SAXS are colloidal calcium phosphate nanoclusters rather than putative submicelles.     

Monolayer studies of single-chain polyprenyl phosphates

The monolayer properties of some single-chain polyprenyl phosphates (phytanyl, phytyl, and geranylgeranyl phosphates), which we regard as hypothetical primitive membrane lipids, were investigated at the air-water interface by surface pressure-area (pi-A) isotherm measurements. The molecular area/ pressure at various pH conditions dependence revealed the acid dissociation constants (pKa values) of the phosphate. The pKa values thus obtained at the air-water interface (pKa1 = 7.1 and pKa2 = 9.4 for phytanyl phosphate) were significantly shifted to higher pH than those observed in the bilayer state in water (pKa1 = 2.9 and pKa2 = 7.8). The difference in pKa values leads to a stability of the phosphate as both monolayer and bilayer states in a pH range of 2-6. In addition, the presence of ions such as sodium, magnesium, calcium, and lanthanum in the subphase significantly altered the stability of the polyprenyl phosphate monolayers, as shown by the determination of monolayer collapse and compression/expansion hysteresis. Although sodium ions in the subphase showed only a weak effect on the stabilization of the monolayer, addition of magnesium ions or of a small amount of calcium ions significantly suppressed the dissolution of the monolayer into the subphase and increased its mechanical stability against collapse. In contrast, the presence of larger amounts of calcium or of lanthanum ions induced collapse of the monolayers. Based on these experimental facts, a plausible scenario for the formation of primitive cell membrane by transformation of a monolayer to vesicle structures is proposed.     

An unusual isotopic fractionation of boron in synthetic calcium carbonate precipitated from seawater and saline water

Inorganic calcium carbonate precipitation from natural seawater and saline water at various pH values was carried out experimentally. The results show the clear positive relationships between boron concentration and δ11B of inorganic calcium carbonate with the pH of natural seawater and saline water. However, the variations of boron isotopic fractionation between inorganic calcite and seawater/saline water with pH are inconsistent with the hypothesis that B(OH)4- is the dominant spe-cies incorporated into the biogenic calcite structure. The isotopic fractionation factors α between synthetic calcium carbonate precipitate and parent solutions increase systematically as pH increases, from 0.9884 at pH 7.60 to 1.0072 at pH 8.60 for seawater and from 0.9826 at pH 7.60 to 1.0178 at pH 8.75 for saline water. An unusual boron isotopic fractionation factor of larger than 1 in synthetic calcium carbonate precipitated from seawater/saline water at higher pH is observed, which implies that a substantial amount of the isotopically heavier B(OH)3 species must be incorporated preferentially into synthetic inorganic carbonate. The results propose that the incorporation of B(OH)3 is attributed to the formation of Mg(OH)2 at higher pH of calcifying microenvironment during the synthetic calcium carbonate precipitation. The preliminary experiment of Mg(OH)2 precipitated from artificial seawater shows that heavier 11B is enriched in Mg(OH)2 precipitation, which suggests that isotopically heavier B(OH)3 species incorporated preferentially into Mg(OH)2 precipitation. This result cannot be applied to explain the boron isotopic fractionation of marine bio-carbonate because of the possibility that the unusual environment in this study appears in formation of marine bio-carbonate is infinitesimal. We, however, must pay more attention to this phenomenon observed in this study, which accidentally appears in especially natural environment.     

Metastable Equilibrium Solubility Behavior of Carbonated Apatites in the Presence of Solution Fluoride

The aims of the present investigation were to assess the applicability of the metastable equilibrium solubility (MES) concept for the carbonated apatites (CAPs) over a range of pH and a wide range of solution fluoride concentrations and to examine the hypothesis that, in the presence of solution fluoride, a surface complex with the stoichiometry of fluorapatite (FAP) governs the MES behavior. Two CAP samples were prepared by precipitation from reaction media containing calcium nitrate (Ca(NO(3))(2).4H(2)O) and sodium phosphate (NaH(2)PO(4).H(2)O) at two different levels of sodium bicarbonate. The MES distributions of the two CAP preparations were determined by equilibrating approximately 10 mg of CAP powder in 2 L of 0.1 M acetate buffers (ionic strength=0.50 M) at pH 4.5 and 5.5 and at various levels of calcium, phosphate, and fluoride. The fluoride concentrations ranged from 0.03 to 12 ppm. From the compositions of the equilibrating buffer solutions, ion activity products based upon the stoichiometries of hydroxyapatite (HAP) and FAP were calculated in an attempt to determine the correct function governing the dissolution of the CAP preparations. The results of this study demonstrated that both CAP preparations exhibit the MES distribution phenomenon in solution media of varying pH and fluoride concentrations. Furthermore, the experimental MES data obtained with both CAP preparations at the lower pH (4.5) and at higher solution fluoride levels (>/=0.1 ppm) were essentially superimposable when plotted against the ion activity product based upon the stoichiometry of FAP, suggesting that in the presence of solution fluoride the MES governing surface complex may be an entity possessing a stoichiometry approximated by that of FAP. When the HAP stoichiometry was assumed to represent the surface complex, good superposition of the data was not possible. Copyright 2000 Academic Press.     

Comparison of stages in oil agglomeration process of quartz with sodium oleate in the presence of Ca(II) and Mg(II) ions

The oil agglomeration of quartz with sodium oleate in the presence of calcium and magnesium ions comprises three consecutive stages: adsorption of cations onto quartz surfaces, which leads to coagulation of the suspension, shear flocculation with sodium oleate and finally, agglomeration of flocs by kerosene. The effects of pH and cation concentration on these stages were investigated and the results were presented comparatively. It was found that all the stages of oil agglomeration of quartz exhibited sharp dependences on pH and cation concentration. That is, these stages generally took place in the pH and concentration ranges in which hydroxy complexes of the cations existed in the suspension. In the case of magnesium ion, the coagulation, shear flocculation and especially oil agglomeration of quartz improved after precipitation of hydroxide. These species of calcium and magnesium ions formed at high pH were adsorbed on the negatively charged surface of quartz, as a result of which the adsorption of sodium oleate became possible and thus the shear flocculation of the particles was achieved. Thereafter, the hydrophobic quartz flocs could be agglomerated by kerosene as bridging liquid. The increase in the shear flocculation efficiency depending on the increase of surface hydrophobicity enhanced the oil agglomeration of quartz with kerosene. The maximum recoveries for all the stages of the quartz were obtained in the presence of 10(-3) M magnesium and 5x10(-3) M calcium ions at pH 11. However, some differences in the behavior of shear flocculation and oil agglomeration of quartz suspension were observed above 10(-3) M concentration of magnesium ion.     

Interaction with Al and Zn induces structure formation and aggregation in natively unfolded caseins

Caseins are phosphoproteins that form the principal protein component of milk, their chief function being the transport of inorganic calcium and phosphate to the neonates. The four major members of the casein family are alpha(s1)-, alpha(s2)- (together referred to as alpha(s)-casein), beta- and kappa-casein, each having a characteristic high negative net charge as well as high hydrophobicity and preferring extended conformational states in solution. We have investigated the influence of the polyvalent metal cations Zn(II) and Al(III) on the structure of bovine caseins, using fluorescence and circular dichroic (CD) spectroscopy and light scattering. Changes in Trp and ANS fluorescence parameters (blue shifts of the emission maxima and enhancement of fluorescence intensity) and in the far-UV CD spectra of the caseins caused by the presence of both metals suggest that conformational changes are induced in them by low concentrations (20-40 microM) of the metal cations. These changes lead to formation of solvent-accessible hydrophobic clusters or cavities that, in turn, cause self-association and precipitation of caseins at higher concentration of the metals. These conclusions are supported by increased binding of ThT to the caseins, as well as enhancement of light scattering intensity, observed in presence of Al(III). The chaperonic property of alpha(s)-casein, which enables it to inhibit thermal aggregation of alcohol dehydrogenase, is shown to be partially destroyed by Zn(II)-induced structural alterations, due possibly to loss of flexibility of the natively unfolded casein chains.     

THE ROLE OF SURFACE FORCES IN FOULING OF STAINLESS STEEL IN THE DAIRY INDUSTRY

The major cause of fouling of processing equipment in the Dairy Industry used for the pasteurization of milk and whey resides in particle formation in the bulk of the dairy fluid. As a result of processing at elevated temperatures, the heat sensitive whey proteins aggregate and calcium phosphate becomes insoluble and precipitates. Hence, the process of fouling can be understood as a subsequent deposition of these particles onto the heating surface.

On the basis of model calculations it is demonstrated that at neutral pH both the calcium phosphate particles, and to a limited extent also the whey proteins, are attracted by stainless steel. This is the result of the monopolar character of steel, which leads in an aqueous environment to an attractive Lewis acid/base interaction term with both components. In combination with the Lifshitz-van der Waals contribution both contributions to the overall interaction are sufficiently large to overcome the electrostatic repulsion at the given pH.

This result points to the specific role of stainless steel in the process of fouling and has far reaching consequences for the way the process of fouling can be controlled as the behaviour of the contaminants, e.g. their heat sensitivity, can not be altered.     

Adsorption mechanism of polylysine on hydroxyapatite and its effect on dissolution properties of hydroxyapatite

Adsorbed amounts of poly-l-lysine (pLys) and bromide ion on hydroxyapatite (HAp) from aqueous solutions of poly-l-lysine hydrobromide, and amounts of calcium and phosphate ions liberated concurrently from HAp during the adsorption of pLys were determined at 25 degrees . The pLys was adsorbed on HAp by the mechanism of ion-exchange between its amino groups and calcium ions of HAp. The released amount of calcium ion increased, therefore, with the adsorbed amount of pLys. On the other hand, the released amount of phosphate ion first decreased and then increased after attaining a minimum with the equilibrium concentration of pLys. The analysis using an equilibrium dialysis method revealed that the released phosphate ions were mainly in the bound state to the amino groups of pLys remaining in the solution, and that the concentrations of calcium and phosphate ions free from both HAp and pLys were restricted by each other under the law of the solubility product of HAp. The first decrease in the released amount of phosphate ion was concluded to be attributed primarily to the increase in the released amount of calcium ion because pLys remaining in the solution was little in this region. When sodium hydroxide was added to the solution, the adsorbed amount of pLys increased and then slightly decreased with the equilibrium pH of the solution due to the increase or decrease of the electrostatic attractive force between the adsorbate and the adsorbent. However, conformational change in pLys around pH 10 seemed to have little effect on the adsorption.     

Calcium phosphate mineralization with linear poly(ethylene imine): a time-resolved study

We have earlier shown that linear poly(ethylene imine) (LPEI) is an efficient growth modifier for calcium phosphate mineralization from aqueous solution (Shkilnyy et al., Langmuir, 2008, 24 (5), 2102). The current study addresses the growth process and the reason why LPEI is such an effective additive. To that end, the solution pH and the calcium and phosphate concentrations were monitored vs. reaction time using potentiometric, complexometric, and photometric methods. The phase transformations in the precipitates and particle morphogenesis were analyzed by X-ray diffraction and transmission electron microscopy, respectively. All measurements reveal steep decreases of the pH, calcium, and phosphate concentrations along with a rapid precipitation of brushite nanoparticles early on in the reaction. Brushite transforms into hydroxyapatite (HAP) within the first 2 h, which is much faster than what is reported, for example, for calcium phosphate precipitated with poly(acrylic acid). We propose that poly(ethylene imine) acts as a proton acceptor (weak buffer), which accelerates the transformation from brushite to HAP by taking up the protons that are released from the calcium phosphate precipitate during the phase transformation.     

An understanding of renal stone development in a mixed oxalate-phosphate system

The in vivo formation of calcium oxalate concretions having calcium phosphate nidi is simulated in an in vitro (37 degrees C, pH 6.0) dual constant composition (DCC) system undersaturated (sigma DCPD = -0.330) with respect to brushite (DCPD, CaHPO 4 . 2H 2O) and slightly supersaturated (sigma COM = 0.328) with respect to calcium oxalate monohydrate (COM, CaC2O4 . H2O). The brushite dissolution provides calcium ions that raise the COM supersaturation, which is heterogeneously nucleated either on or near the surface of the dissolving calcium phosphate crystals. The COM crystallites may then aggregate, simulating kidney stone formation. Interestingly, two intermediate phases, anhydrous dicalcium phosphate (monetite, CaHPO4) and calcium oxalate trihydrate (COT), are also detected by X-ray diffraction during this brushite-COM transformation. In support of clinical observations, the results of these studies demonstrate the participation of calcium phosphate phases in COM crystallization providing a possible physical chemical mechanism for kidney stone formation.     

空心纳米磷酸钙载药体系的制备与表征

非离子表面活性剂Tween 80和PEG 6000在水溶液中以一定的比例混合可形成稳定的类磷脂囊泡结构,这些囊泡可以作为模板来合成磷酸钙纳米空球颗粒。所制备的磷酸钙材料的结构和形貌通过TEM,SEM,FTIR,XRD进行了表征,是尺寸为100～150 nm左右的无定形磷酸钙空心颗粒。磷酸钙具有良好的生物相容性,因此这些具有空心结构特征的磷酸钙可发展为理想的载药体系。我们以牛血清蛋白(BSA)为模型体系研究了材料的载药和释放性能,发现所获得的空心纳米磷酸钙不仅具有良好的蛋白质负载量而且还具有优异的可释放性,明显优于传统的羟基磷灰石体系。