Preparation and comprehensive characterization of a calcium hydroxyapatite reference material

Journal of Research of the National Institute of Standards and Technology, Nov-Dec, 2004 by Milenko Markovic, Bruce O. Fowler, Ming S. Tung

Numerous biological and chemical studies involve the use of calcium hydroxyapatite (HA), [Ca.sub.10](P[O.sub.4])[.sub.6](OH)[.sub.2]. In this study detailed physicochemical characterization of HA, prepared from an aqueous solution, was carried out employing different methods and techniques: chemical and thermal analyses, x-ray diffraction, infrared and Raman spectroscopies, scanning and transmission microscopies, and Brunauer, Emmett, and Teller (BET) surface-area method. The contents of calcium ([Ca.sup.2+]), phosphate (P[O.sub.4.sup.3-]), hydroxide (O[H.sup.-]), hydrogenphosphate (HP[O.sub.4.sup.2-]), water ([H.sub.2]O), carbonate (C[O.sub.3.sup.2-]), and trace constituents, the Ca/P molar ratio, crystal size and morphology, surface area, unit-cell parameters, crystallinity, and solubility of this HA were determined. This highly pure, homogeneous, and highly crystalline HA is certified as a National Institute of Standards and Technology (NIST) standard reference material, SRM 2910.

Key words: chemical analysis; crystal size; crystallinity; hydroxyapatite; infrared; morphology; preparation; Raman; solubility; surface area; thermal analysis; unit-cell parameters; x-ray diffraction.

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1. Introduction

Calcium hydroxyapatite (HA), [Ca.sub.10](P[O.sub.4])[.sub.6](OH)[.sub.2], is an important inorganic material in biology and chemistry [1-3]. Biological apatites, which are the inorganic constituents of bone, tooth enamel and dentin, are typically very variable in their composition and morphology, and contain different impurities ([Mg.sup.2+], [K.sup.+], [Na.sup.+], C[O.sub.3.sup.2-], HP[O.sub.4.sup.2-], [Cl.sup.-], [F.sup.-], etc.) [1]. In general, these impure biological apatites are designated as calcium deficient or non-stoichiometric apatites.

Synthetic HAs are frequently used as reference materials in biomineralization and biomaterial studies. The composition, physicochemical properties, crystal size and morphology of synthetic apatites are extremely sensitive to preparative conditions. Common impurity phases in synthetic apatites prepared by precipitation from supersaturated aqueous solutions are calcium phosphate compounds such as amorphous calcium phosphates (ACP) with variable compositions of [Ca.sub.3](P[O.sub.4])[.sub.2-2x](HP[O.sub.4])[.sub.3x] * n[H.sub.2]O, octacalcium phosphate (OCP), [Ca.sub.8](HP[O.sub.4])[.sub.2](P[O.sub.4])[.sub.4] * 5[H.sub.2]O, and calcium hydrogenphosphate dihydrate (DCPD), CaHP[O.sub.4] * 2[H.sub.2]O. In addition, the incorporation of various ions as trace impurities (hydrogenphosphate, carbonate, silicate ions, etc.) is very difficult to prevent in any preparative procedure of HA [3].

For control and reference purposes, it is important to have available pure and stoichiometric HA, or nearly stoichiometric HA, characterized in detail with respect to its chemical composition and numerous other important properties. To meet this need, a large amount of highly pure, homogeneous and highly crystalline HA was synthesized by precipitation from aqueous solution of calcium hydroxide and phosphoric acid and then rigorously characterized by chemical and thermal analyses, infrared (IR) and Raman spectroscopies, powder x-ray diffraction (XRD), scanning and transmission microscopies, and surface area and solubility product [4] measurements. The chemical composition and other analyzed properties of this HA qualify it as a standard reference material (NIST, SRM 2910) [5] and it is here-after denoted as HA-SRM.

Synthetic HA occurs in two structural forms, hexagonal and monoclinic, which have minor structural differences [2]. The hexagonal HA form is usually formed by precipitation from supersaturated solutions at 25 [degrees]C to 100 [degrees]C and the monoclinic form of HA is primarily formed by heating the hexagonal form at 850 [degrees]C in air and then cooling to room temperature [6]. The overall XRD patterns of hexagonal and monoclinic HA are almost identical; however the pattern of monoclinic HA has additional weak lines whose intensities are less than 1% of the strongest hexagonal HA line [7]. The HA-SRM analyzed here is composed of the hexagonal form (mass fraction of about 75%) and of the monoclinic form (mass fraction of about 25%) as determined by normalized additional XRD measurements of the weak line of monoclinic HA at 2[theta] = 36.28[degrees] [6-8]. Only the hexagonal form, the major component in HA-SRM, is discussed in this paper. Preparation and characterization of the monoclinic form of HA and differences between the hexagonal and monoclinic HA will be discussed in a separate paper [8].

2. Experimental Section

2.1 Preparation

Calcium hydroxyapatite-standard reference material (HA-SRM) was synthesized by solution reaction of calcium hydroxide and phosphoric acid in accordance with the preparation of McDowell et al. [9]. In brief, about 5 L of distilled water was boiled for 60 min in a 7.5 L Teflon-coated pot equipped with an electric stirring paddle, a reflux condenser with a C[O.sub.2]-absorbing NaOH trap to protect from atmospheric C[O.sub.2], and ports for introducing titrant and nitrogen gas. Calcium oxide (prepared from calcium carbonate heated for 3 h at 1100 [degrees]C) was added to the water. Phosphoric acid (concentration 2 mol/L) was added to the calcium oxide/calcium hydroxide slurry at a rate of 0.3 mL/min to 0.6 mL/min and to a final Ca/P molar ratio of 1.67. The reacting mixture was boiled for 2 d. The precipitated solid phase was allowed to settle, the supernatant decanted, and an equal volume of boiled distilled water was added. This suspension was boiled for another 2 d. These washing and boiling procedures were repeated four times until the pH of the supernatant was [approximately equal to]6; at pH 6, any possible traces of anhydrous dicalcium hydrogenphosphate (DCPA) are converted into HA. The precipitate, collected by filtration, was thoroughly washed with acetone, and then dried at 105 [degrees]C for 1 d. The yield was about 1 kg.