Adsorption of citrate ions on hydroxyapatite synthetized by various methods
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The specific adsorption of citric acid ions at hydroxyapatite interface was investigated by the means of radioisotope method (14C) as a function of citric acid ions concentration, NaCl concentration and pH. Application of the hydroxyapatite has become wide in the biomaterial field as the Ca10(OH)2(PO4)6 possess biocompatibility with human hard tissue. Hydroxyapatite was synthesized using three different methods. The physical properties of the resulting powder were characterized by DTA/TG, XRD, AFM and SEM microscopy. Physicochemical qualities characterizing the electrical double layer of the hydroxyapatite/NaCl solution interface were determined. The zeta potential and the adsorption of citric acid molecule were studied as a function of pH. The point of zero charge and the isoelectric point of samples were determined. Electrical double layer parameters of hydroxyapatite/NaCl interface are influenced by a synthesis method. The points pHpzc and pHIEP for sample 1 are pHpzc 7.5 and pHIEP 3; for sample 2 pHpzc 7.05 and pHIEP 3, for smaple 3 pHpzc 6.7 and pHIEP 3. Temperature has weak influence both on pure substance and with citric acid adsorbed, as derivatographic analysis has shown, and characterization of hydroxyapatite structure may be carried out by this thermal analysis. Two phenomena are responsible for citric acid adsorption: phosphate group’s replacement at hydroxyapatite surface by citric ions parallel to intraspherical complexes formation.
KeywordsHydroxyapatite Adsorption Citric acid Thermal analysis
Hydroxyapatite [Ca10(OH)2(PO4)6] has been studied for many years, due to its occurrence in natural environment and practical application. As a natural mineral it is present in igneous and metamorphic rocks. Moreover, it is a major inorganic component of bones and teeth. Calcium phosphate and calcium carbonate are human hard tissues constituents, with the same structure as the natural mineral. Thanks to its crystallographic similarity with natural bone minerals, biocompatibility with hard tissues and with skin or muscle tissues, bioactivity, no toxicity, and implants made of synthetic hydroxyapatite (bioceramic hydroxyapatite) are used as an artificial bone substitute in orthopedic, neurosurgery, dental surgery, plastic surgery and dental applications [1, 2, 3, 4].
The literature about sorption on hydroxyapatite using the isotope method includes the papers about adsorption of Ni, Cu, and Sr written by Rosskopfova et al. [11, 12, 13]. However of no less importance are problems related with anions, among others, citrate anions.
The adsorption process of citric acid on the surface of hydroxyapatite was described by Filgueiras, Mkhonto and de Leeuw using the computer modelling method. The structure and energy of the hydroxyapatite and citric acid complex was determined thanks to interatomic potentials. There was a series of computer simulations performed to investigate the adsorption of citric acid on different surfaces of hydroxyapatite. They showed that the intensity of the acid particle influence on the surface is conditioned by stability of the surface and possibility of repeated interaction of an acid molecule with the active adsorption places of hydroxyapatite, especially with two or more calcium atoms. Big adsorption energy causes the citric acid adsorption on the surface of hydroxyapatite to be exceptionally strong. Therefore it can be concluded that citric acid should serve as a good inhibitor for the hydroxyapatite increase if we look at it as to be used in the synthesis. Blocking the active places on the surface of hydroxyapatite inhibits the increase of its crystal .
While investigating the citric acid adsorption on hydroxyapatite, Vega, Narda and Ferreti  explained the process in a following way. The adsorption process occurred through the exchanges of phosphate groups with citrate anions. The adsorption isotherm equalled the Langmuir isotherm. The complex of citric acid and hydroxyapatite originated from combining one molecule of citric acid with two active places of hydroxyapatite. It was confirmed during research that the active places on the surface of hydroxyapatite are not equal and the surface itself is not uniform. The special structure of hydroxyapatite caused the fact that, the possibility of the ions adsorption depends on the occupied neighbouring spaces. In connection to that the adsorption of the first layer of citrate at the hydroxyapatite/nonelectrolyte solution interface can be treated as monolayer adsorption and the next layers become a part of the solution itself. As Lopez-Macipe, Gomez-Moralez and Rodriguez-Clemente  explain in their work, the adsorption of citric acid on hydroxyapatite depends to a great extent on pH value. However, the mechanism of interactions between citric acid and hydroxyapatite is not completely clear. There is a theory which states that citric acid ions influence hydroxyapatite through ionic exchange between phosphate groups and citrate ions. In reactions between citrate ions and hydroxyapatite, pH value has a crucial value because it determines the kinds of citric ions capable of adsorption on the hydroxyapatite surface. According to the authors’ assumptions citrate ions can have different forms e.g. Cit3−, HCit2− and H2Cit−. The dependence was determined on the basis of the analysis concerning the citrate ions on hydroxyapatite at of 25 and 37 °C. Amount of citric ions adsorbed was different in examined solutions because of different pH values. At pH value 6 the dominating form were H2Cit- ions whereas at pH value 8 there was the highest concentration of Cit3− ions. The adsorption process of citric acid on hydroxyapatite with low concentration of citric acid was described using the Langmuir adsorption model. This adsorption occurs here through replacement of the phosphate ions by citrate ions on the solid substance/solution interface. This is a result of higher relation of citrate ions to the hydroxyapatite surface. Presence of different forms of citrate ions caused different types of interactions. Cit3− ions react in a two-donor way (1 citrate ion occupies 2 sites of Ca) and reactions of HCit2− ions are stronger than the previous one (1 citrate ion replaces 1 Ca). Such dependence is possible owing to the resonance between the monodonor (using one group –COO−) and the surface of chelation (using two groups –COO−).To sum up, the adsorption decreases with the growth of pH value. Despite that the experiments showed that at pH 6 it is higher than in a solution at pH 8 . Chemical reactions between citric acid and hydroxyapatite in a human body have many consequences. The first one is partial dissolution of apatite, another one is precipitation of calcium citrate occurring when the concentration of the acid is over 10 mmol/dm3. According to Misra, interactions between citric acid and hydroxyapatite can be called chemical reactions. However, the exchange with phosphate ions is responsible for the adsorption of citrate ions. This is confirmed by short time of the balancing process during the reaction of citrate salts of alcaline metals with hydroxyapatite. The literature has largely demonstrated that the synthesis routes and related parameters could significantly affect not only hydroxyapatite particle size and morphology, but also phase composition, thermal stability as well as sintering behavior .
The information available in the literature concerning hydroxyapatite focuses mainly on two issues: thermal analysis of hydroxyapatite and electrochemical properties analysis. This work is combination of these two scientific currents using the example of hydroxyapatites obtained in three different syntheses, which makes a wider view possible on hydroxyapatite as biomaterial used by the human body.The goal of this paper is characterization of hydroxyapatite, determination of pHpzc and pHiep points, establishing zeta potential depending on pH. The specific adsorption of citric acid ions at the hydroxyapatite interface was investigated. The main part of the study was to investigate the thermal effects: caused by citric acid sorption on hydroxyapatite and methods of hydroxyapatite preparation.
Materials and method
In this paper hydroxyapatite was prepared using three methods:
Method 1 
Method 2 
In the reaction there were used the following reagents: calcium acetate (CH3COO)2Ca from Fluka and dipotassium hydrophosphate K2HPO4 from POCh, Gliwice. The solutions of concentrations 0.1 M K2HPO4 and 0.06 M –(CH3COO)2Ca were prepared. In the reaction there was taken 0.15 dm3 of each salt and both solutions were dropped into 0.2 dm3 of water placed in the reaction flask. The flask was immersed in a water bath heated up to 100 °C. The salt solutions were dropped in at the same time for 30 min and then the reaction mixture was boiled for 1 h. The mixture was stirred vigorously and the constant temperature was kept all the time. The obtained sediment was washed with redistilled water till the constant value of redistilled water conductivity was achieved.
Method 3 
Synthetic hydroxyapatite was prepared hydrothermally according to the reaction
In order to prepare hydroxyapatite there was applied the following procedure: 1.0324 g of CaHPO4 *2 H2O from Fluka and 0.2964 g of Ca(OH)2 from Aldrich were taken and mixed. 80 ml of double distilled water was poured in and the pH value was reduced to 9 using 80 % analytically pure acetic acid from POCh. The reaction mixture was put into a drier for 24 h at 120 °C. The obtained hydroxyapatite had to be purified from reagents so the sediment was washed many times with redistilled water and centrifuged.
Hydroxyapatite was investigated using X-ray diffraction (XRD), adsorption–desorption of nitrogen accelerated surface area and porosimetry (ASAP2405, Micromeritics Instruments, Co.), photon correlation spectroscopy (PCS), AFM and SEM microscopes. The classical thermogravimetric (aparatQ-1500D type made by MOM, Budapest, Hungary) TG and DTG curves of thermal analysis, which gave the dependence of the weight loss of a sample as a function of temperature or time, were measured over a temperature range from 20 to 1,000 °C with a furnace-heating rate of 10 °C/min. The TG and DTG curves were registered digitally under the control of the program Derivat running on PC.
The specific adsorption of citric acid ions at the hydroxyapatite interface was investigated by the means of the radioisotope method as a function of citric acid ions concentration, NaCl and pH. The initial concentration of citric acid ions ranged from 1 × 10−6 to 1 × 10−3 mol dm−3, pH was changed from 6 to 12. As a background electrolyte NaCl solution was used of the concentrations 0.001 mol dm−3. The adsorption measurements were complemented by the potentiometric titration of hydroxyapatite suspensions and electrophoresis measurements.
To remove ionic type contaminations, which might influence the ion adsorption measurements, the hydroxyapatite was washed with double distilled water until constant conductivity about 0.5 μS/cm was achieved. Adsorption and surface charge measurements were performed simultaneously in the suspension of the same solid content, to keep the identical conditions of the experiments in a thermostated Teflon vessel at 25 °C. To eliminate the influence of CO2 all potentiometric measurements were performed under nitrogen atmosphere. pH values were measured using a set of glass REF 451 and calomel pHG201-8 electrodes with the Radiometer assembly. Surface charge density was calculated from the difference of the amounts of added acid or base to obtain the same pH value of suspension as for the background electrolyte.
The zeta potential of hydroxyapatite dispersions was determined by electrophoresis with Zetasizer 3000 by Malvern. The measurements were performed at 100 ppm solid concentration ultrasonication of the suspension.
Results and discussion
Chosen structural parameters, sample 1, sample 2 and sample 3
BET surface area (m2/g)
Langmuir surface area (m2/g)
BJH cumulative adsorption surface area of pores between 1.7 and 300 nm diameter (cm3/g)
BJH cumulative desorption surface area of pores between 1.7 and 300 nm diameter (cm3/g)
Average pore diameter (4 V/A by BET) (nm)
BJH adsorption on average pore diameter (4 V/A) (nm)
BJH desorption on average pore diameter (4 V/A) (nm)
Investigations of the hydroxyapatite/electrolyte solution system are limited by dissolution of the minerals and pH range 7–11.
The synthesis method influences the parameters of electrical double layer at hydroxyapatite/NaCl.
The points pHpzc and pHIEP for sample 1 are pHpzc 7.5 and pHIEP 3; for sample 2 pHpzc 7.05 and pHIEP 3, for sample 3 pHpzc 6.7 and pHIEP 3.
Derivatographic analysis has shown small influence of temperature on pure substance as well as with citric acid adsorbed, but that the thermal analysis can be used for characterization of structural hydroxyapatite.
The adsorption of citric acid occurs as a result of the replacement of the phosphate groups on the surface of hydroxyapatite with the citric ones together with the formation of the intraspherical complexes.
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