Rapid wet chemical synthesis for 33P-labelled hydroxyapatite – An approach for environmental research

Accepted Manuscript Rapid wet chemical synthesis for environmental research

33 P-labelled hydroxyapatite – An approach for

J. Wolff, D. Hofmann, W. Amelung, H. Lewandowski, K. Kaiser, R. Bol PII:

S0883-2927(18)30227-0

DOI:

10.1016/j.apgeochem.2018.08.010

Reference:

AG 4147

To appear in:

Applied Geochemistry

Received Date: 22 May 2018 Revised Date:

13 August 2018

Accepted Date: 20 August 2018

Please cite this article as: Wolff, J., Hofmann, D., Amelung, W., Lewandowski, H., Kaiser, K., Bol, 33 R., Rapid wet chemical synthesis for P-labelled hydroxyapatite – An approach for environmental research, Applied Geochemistry (2018), doi: 10.1016/j.apgeochem.2018.08.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Rapid wet chemical synthesis for 33P-labelled hydroxyapatite

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– an approach for environmental research J. Wolff1,2,*, D. Hofmann2, W. Amelung1,2, H. Lewandowski2, K. Kaiser3, R. Bol2

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Ecology, University of Bonn, Nussallee 13, 53115 Bonn, Germany

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Institute for Crop Science and Resource Conservation (INRES) – Soil Science and Soil

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GmbH, 52425 Jülich, Germany

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Institute for Bio- and Geosciences – IBG-3: Agrosphere, Forschungszentrum Jülich

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Seckendorff-Platz 3, 06120 Halle (Saale), Germany

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Soil Science and Soil Protection, Martin Luther University Halle-Wittenberg, von

*Corresponding author:

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Jan Wolff

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Institute for Crop Science and Resource Conservation (INRES) – Soil Science and Soil

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Ecology, University of Bonn

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Nussallee 13

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53115 Bonn, Germany

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Tel.: +49 228 73 2981

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Fax: +49 228 73 2782

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[email protected]

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Abstract Apatite is the principal primary phosphorus (P) source in the environment; yet there

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is no consensus on how it can be synthesized for controlled microcosm studies,

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particularly not in labelled form. Here, we present a methodology that allows for the

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production of stoichiometric

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produced by a simple and fast wet chemical procedure, with different precursor

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compounds and at different reaction (25, 40, 60 and 80°C) and calcination (100 and

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200°C) temperatures. The resulting morphological structures were analysed by Raman

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spectroscopy, X-ray diffraction (XRD), and scanning electron microscopy (SEM). The

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results showed that rapid synthesis of hydroxyapatite is successful using 33P-labelled di-

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ammonium hydrogen phosphate and calcium nitrate with a Ca/P ratio of 1.67 in less

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than 30 h. Crystallinity increased with increasing reaction temperatures. Solubility tests

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confirmed a strong pH dependency for all hydroxyapatites at pH values <3.7. To our

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knowledge this is the first procedure that can rapidly synthesize radioactive labelled and

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chemically pure hydroxyapatite of different crystallinities: It can be easily modified to

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allow for labelling with other isotopes, such as

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hydroxyapatite in reproducible manner for investigating the availability and uptake of P

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from apatite in future soil and environmental studies and beyond.

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Phosphorus (33P)-labelled hydroxyapatite powders

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Ca or

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O, in order to provide

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Keyword:

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Hydroxyapatite; 33P labelling; mineral solubility; crystallinity variations; mineral P tracer

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1. Introduction Apatites are known to be the primary mineral source of phosphorus (P) in soils and

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sediments (Chen et al., 1997). Fluor-, chlor- and hydroxyapatites may rarely occur in

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form of individual grains but mostly as accessory phases which are omnipresent in

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metamorphic and igneous rocks (McClellan and Van Kauwenbergh, 1990; Nezat et al.,

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2007; Syers et al., 1967). In addition to mineral apatites, biological apatites are present

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in the environment, e.g., in mammal residues of bones and other hard tissues,

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consisting of about 65 wt% apatite (Wang et al. 2006). Even though both forms are

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members of the ‘apatite’ family, mineral and biological forms differ in certain

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characteristics (Tas, 2000a; 2000b). Biological apatite is formed by biomineralization,

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which, in contrast to mineral apatite, leads to very small crystals (250 Å × 30 Å) of low

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crystallinity and non-stoichiometric Ca/P ratios (Kalita and Verma, 2010; Villacampa et

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al., 2000).

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Hydroxyapatite (Ca10(PO4)6(OH)2), an isomorph of

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the naturally abundant

fluorapatite, is widely used as a biomaterial because of its bioactive, biodegradable, and

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osteoconductive properties (Chen et al., 2002; Narasaraju and Phebe, 1996). In the

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past, researchers were targeting the preparation of hydroxyapatite for biomedical

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implant applications and bone regeneration purposes (Chen et al., 2002; Hench, 1991;

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Mengeot et al., 1973). To synthesize the different forms of hydroxyapatites, numerous

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methods have been developed over the last decades, including the solid-state (Sonoda

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et

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(Zhang and Zhang, 2011). None of these techniques were optimised for the specific

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requirements as needed for production of radioactively labelled apatite forms that mirror

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natural (Ca/P ratio of 1.67) hydroxyapatites. While synthesis of hydroxyapatites has

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al.,

2002),

sol-gel

(Fathi and Hanifi, 2007)

and

precipitation

techniques

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been achieved for medical purposes, their application and quantification in soils and

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other environments is still challenging. Methods for mineralogical analysis in soils are, e.g., X-ray diffractions (XRD) and

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near-edge synchrotron-based X-ray spectroscopy. XRD can be used to identify apatites

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in petrographic thin sections of rocks (Nezat et al., 2007). This method is however

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insufficient for apatite quantification in soils because they only occurred in trace

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amounts, i.e., this technique can hardly provide information on soil apatites and thus be

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utilized as method to derive P dynamics in soils. Further, quantification limitations also

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apply to bulk soil digestions due to apatites being included in weathering-resistant

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minerals such as silica (Nezat et al., 2007). Near-edge synchrotron-based X-ray

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spectroscopy may identify apatite hotspots in soil (Kruse et al., 2015); yet, this

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methodology is laborious and far from being routine and thus not widely applicable.

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Werner et al. (2017) recently highlighted that there is still only little scientific

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information about the spatial micro-distribution patterns of soil P minerals due to the lack

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of suitable analytical methods. In this context, labelling with the two distinct radioactive

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isotopes of P; i.e.

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phosphate ions from soil into soil solution and further trace the uptake by plants

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(Frossard et al., 2011). Therefore, labelling apatite minerals with radioactive P isotopes

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as a tracer for plant P acquisition, especially in combination with novel methods such as

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radioactive imaging (Bauke et al., 2017), may greatly enhance our knowledge on P

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cycling in soils and its uptake into biota.

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P, has proven to be a useful tool to trace the release of

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The principal goal of this research was to synthesize

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P-labelled hydroxyapatite of

different degrees of crystallinity by wet chemical precipitation for environmental 4

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research. Relevant literature (> 80 research articles) was assessed according to

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important criteria for a simple, safe and fast production of radioactive labelled

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hydroxyapatite with a Ca/P ratio of 1.67 [cf. Table S2; Supplementary material]. Based

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on this selection, a modified method of Wang et al. (2010) seemed most suitable for our

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purposes. Using this modified method, we were able to rapidly synthesize

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hydroxyapatite powders of different degrees of crystallinity at various temperatures (25–

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80 °C). The effects of controlling factors during the synthesis were recorded and the

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produced powders were subsequently analysed using RAMAN spectroscopy, X-ray

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diffraction and scanning electron microscopy (SEM). The different hydroxyapatite

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powders were tested for their solubility kinetics at different pH values by photometric

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determination of the amount of P released into solution. To the authors’ knowledge, this

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is the first attempt to synthesize

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tracer for environmental P cycling.

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2.1

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P-labelled

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P-labelled hydroxyapatite of different crystallinity as

Experimental section Materials CaO

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(Sigma

Aldrich,

Germany),

H3PO4

(Sigma

Aldrich,

Germany),

HNO3 (Sigma Aldrich, Germany), NH3 · H2O (Merck, Germany), NH2CH2CH2OH (Sigma

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Aldrich, Germany), C2H5OH (Merck, Germany), HOC(COOH)(CH2COOH)2 (Sigma

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Aldrich, Germany) and HOC(COONa)(CH2COOH)2 (Sigma Aldrich, Germany) were used

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without any further purification. 700 µl of H333PO4, equal to an activity of 295 MBq with

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420 kBq/µl, were purchased from Hartmann Analytics, Germany.

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2.2

Methods

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2.2.1 Method selection In order to find the most suitable method for our purposes, 87 papers looking at

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different synthesis methods of hydroxyapatite were reviewed [cf. Table S2;

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Supplementary material]. Most frequently, precipitation methods (27% of papers),

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followed by hydrothermal methods (11%) and sol–gel methods (9%) were used in the

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past. The most common precipitation methods, also known as chemical precipitation,

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aqueous precipitation or wet chemical precipitation, were based on a chemical reaction

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of aqueous calcium solution with aqueous orthophosphoric acid solution. These

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reactions could be performed at different conditions (e.g., pH, temperature, etc.) in order

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to produce large quantities of hydroxyapatite at reasonable costs (e.g. Nayak 2010).

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Due to the fact that we aimed at producing

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methods with short production periods and particularly on the absence of methodical

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steps that preclude work with radioactive substances for safety reasons, such as milling

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the samples into fine powders (risk of inhalation) or heating of liquids (risk of radioactive

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steam formation). We also excluded methods that did not clearly result in products

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stoichiometrically similar to the required Ca/P ratio of 1.67. We preferred methods

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synthesizing powders to those synthesizing gels to facilitate a uniform distribution of the

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apatites in soil matrices.

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P-labelled hydroxyapatite, we focused on

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All these preconditions led to the wet chemical method described by Wang et al.

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(2010). They described a preparation of hydroxyapatite powders with “various

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morphologies” under controlled synthesis conditions, relying on different “drying

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methods, solvent and dispersant species, initial pH, and reaction temperatures”. They

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reported that at an initial pH of 10, hydroxyapatite powders of different degrees of 6

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crystallinity can be obtained. We modified the described synthesis pathways to comply

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with radioactive work requirements by excluding elevated pressures, and, above all, by

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changing the preparation of solutions involved, the calcination temperatures, and the

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duration of different synthesis steps.

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2.2.2 Synthesis of hydroxyapatite

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As calcium and phosphorus sources for apatite synthesis, calcium nitrate tetra-

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hydrate ((Ca(NO3)2·4H2O) and diammonium hydrogen phosphate ((NH4)2HPO4)

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solutions were prepared. For a 1 M calcium nitrate tetra-hydrate solution, 100 mL of 2 M

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HNO3 were slowly added to 100 mL 1 M CaO suspension under continuous stirring. For

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the preparation of a 1 M diammonium hydrogen phosphate solution, 50 mL 1 M H3PO4

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were slowly added into 50 ml 2 M NH3 and stirred for 1 h. To label the phosphate with

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P, a defined amount of H3PO4 was replaced by H333PO4 with an activity of 295 MBq. For the synthesis, 100 ml of the Ca(NO3)2 solution was added into a 500 ml

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double-walled three-necked synthesis reactor placed on a magnetic stirrer. A water heat

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pump, a thermometer, and a pH meter were connected to the reactor in order to control

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pH and reaction temperature. To prevent aggregation, 3 wt.% of ethanolamine were

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added as a dispersant to the Ca(NO3)2 solution. Additional aqueous ammonia (NH3 ·

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H2O) was used to adjust the pH to 10 if necessary. Before further proceeding with the

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synthesis, the Ca(NO3)2 mixture was heated to the intended temperature (cf. Fig. 1)

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under continuous stirring at 300 rpm. A dropping funnel was filled with 100 ml of the 33P-

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labelled diammonium hydrogen phosphate solution. As soon as a constant temperature

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of the Ca(NO3)2 mixture was achieved, the

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a rate of 2 mL/min. During the synthesis process, additional aqueous ammonia was

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P-labelled solution was added dropwise at

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added to maintain a pH ≥ 9. The resulting suspension was stirred for 1.5 h without

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additional heating and then aged for another 12 h at room temperature. Thereafter, the

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suspension was filtered and washed twice with 30 mL deionized H2O to remove any

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residues and impurities. To remove the remaining water, the filtrate was washed with

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15 mL ethanol before drying for 5 h at 60 °C. The final calcination step was performed at

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100 and 200 °C for 1 h in a muffle furnace.

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Raman measurements were performed using a Bruker RFS 100/S FT-Raman

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spectrometer and the OPUS software. The excitation source was a Nd:YAG laser

100 mL, 1M Ca(NO3)2

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3%-w. C2H7NO (add NH3 (32%) for adjustment of pH ≥ 10)

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Stirring, reaction temp.: 25, 40, 60, 80°C (synthesis reactor)

Formation of HA-Suspension

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(with 30 mL diH2O)

(dropping funnel)

Readjustment of pH (with NH3 (32%); pH ≥ 9)

(alter for 2h)

Filtration & 2x Washing

100 mL, 1M (NH4)2HPO4

Drying for 5h 60 °C

HA powder

Filtration & 1x Washing

Calcination

Analytics

(with 15 mL C2H6O)

(1h at 100 or 200 °C)

(e.g. Raman, XRD)

Figure 1: Schematic flow chart showing the wet chemical procedure for 33 synthesizing P labelled hydroxyapatite powders.

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(1064 nm, 500 mW). All spectra were recorded at a resolution of 2° cm-1. The nature of

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the precipitates was analysed via X-ray diffraction (X’Pert Pro, PANalytical B.V., Almelo,

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The Netherlands) with Cu Kα-radiation (λ = 1.541 nm) in step scan mode with a step

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size of 0.02° 2θ and fixed slits, using random powder mounts. Identification of mineral

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phases was carried out with the HighScore 4.2 software (PANalytical). Images were

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taken with a scanning electron microscope (Merlin, Carl Zeiss Microscopy GmbH,

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Germany) with acceleration voltages between 2 kV and 5 kV.

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2.2.3

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Solubility tests

Solubility tests were performed using deionized water (pH > 5), acetate/lactate

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buffer (pH 4.3) and citric acid/citrate buffers, ranging from pH 2.6-4.1 (intermediate steps

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of 0.3) representing components of root exudates (Jones 1998). Tests were performed

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with one measurement per minute, using the spectrophotometer Spectramax Plus 384

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microplate reader (Molecular Devices, California) with an integrated shaker (shake

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duration 50 seconds before each analysis). The synthesized hydroxyapatite powders

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were evenly distributed over a 96-well plate, with a spatula of hydroxyapatite in each

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well. 250 µL of the prepared solutions were given into the previously assigned wells. The

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data acquisition was carried out with the Softmax Pro v4.8 software (Molecular Devices,

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California).

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Results and discussion

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Previous studies applying Raman spectroscopy to ‘pure’ apatites revealed scattering

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signals of different vibrational modes in the spectral range of 0–1200 cm-1 (Li et al. 2004;

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O´Shea et al. 1974). Carbonate, phosphate, and hydroxyl ions within hydroxyapatite

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appear at ranges >~400 cm-1 (Klinkaewnarong et al. 2010). The Raman spectra confirm 9

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major characteristics of the four different hydroxyapatite powders synthesised via our

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wet chemical method (cf. Fig. 2).

)

4(PO4) 2(PO4)

d c

b a

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3(PO

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Figure 2: Raman analysis of hydroxyapatite, synthesized at different temperatures: a= 25°C; b = 40°C; cc = 60°C; d = 80°C. Highlighted are the 1, 2, 3, and 4 phosphate mode regions of the hydroxyapatite compounds.

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The bands at vibration mode ~150 cm-1 can be referred to the lattice mode region of

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hydroxyapatites, where three typical lines, appearing at ~68 cm-1, ~144 cm-1 and

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~160 m-1, often overlap. The bands appearing at ~430 cm-1 are associated to the ( v2)

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O-P-O stretching whereas the bands at ~580 cm- 1 and ~590 cm-1 are due to the

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symmetric stretching of the ( v4) O-P-O. The most characteristic and most intense band

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of hydroxyapatite at ~963 cm-1 is due to the total stretching of ( v1) P-O-P along with free

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ions of tetrahedral phosphate. The bands at vibration mode at ~1046 cm-1 can be

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referred to the ( v3) P-O-P stretching symmetry (Klinkaewnarong et al. 2010; Li et al.,

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2004; O´Shea et al. 1974). The increasing band at mode ~770 cm-1 could be due to

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necessary additions of liquid ammonia with increasing synthesis temperatures.

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Figure 3: X-ray diffraction patterns of hydroxyapatites synthesized at different reaction temperatures (25, 40, 60, 80°C). Calcination temperature: (A) 100 °C and (B) 200°C.

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The X-ray diffraction (XRD) patterns of the different hydroxyapatite powders are

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shown in Fig. 3. The diffractograms of all synthesized powders produced at 25, 40, 60,

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and 80 °C showed no other reflexes than those of hydroxyapatite.

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Figure 4: Scanning electron microscopy (LSM) images of synthesized apatite powders. Reaction temperatures: (a) = 25°C; (b) = 40°C; (c) = 60°C; (d) = 80°C. Calcination temperatures: (a1) - (d1) = 100°C; (a2) - (d2) = 200°C. Images shown here were taken with acceleration voltages between 2 kV and 3 kV, respectively. The scales shown for images b2 and d1 is 2 µm, for the others 1 µm, respectively.

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The samples thus represent rather pure hydroxyapatite phases. The resolution of

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the XRD diffractograms increased with increasing with increasing crystallinity of the

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produced hydroxyapatite phases and increasing reaction temperature of the synthesis,

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due to more well-defined reflexes of decreasing half-height width and an overall increase

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in signal-to-noise ratios. The increasing crystallinity of the produced hydroxyapatite

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phases was best reflected by the increasing resolution of reflexes at the positions

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between 31 and 34 °2Θ.

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The scanning electron microscopy (SEM) images of non-ground hydroxyapatite

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powders, produced at 25, 40, 60, and 80 °C and calcinated at 100 and 200 °C,

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respectively are shown in Fig. 4. In comparison to the hydroxyapatite powders described

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by Wang et al. (2010), we did not produce nano-sized powders but rather aggregate-

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type clusters with sizes of several micrometers. The different morphology might be due

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to a previous grinding of the samples by Wang et al. (2010), though this is not

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mentioned in their publication. SEM revealed that the hydroxyapatite powders

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synthesized at low temperature (cf. Fig. 4a1 and 4a2) comprised of smaller aggregates

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(1–6 µm) with a porous surface structure and SEM analyses also revealed that all

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aggregates within a given fraction were of similar shape. At high reaction temperatures

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larger aggregates form and more needle-type crystals can be observed at the surfaces

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as opposed to at lower temperatures. As to be seen in Fig. 4b1 to 4d2, more distinct

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needles develop with increasing reaction temperatures, which matches the increasing

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crystallinity of these samples. Aggregates synthesized at 60 and 80 °C (cf. e.g. Fig. 4c1

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and 4d1) also show plane, smooth surfaces, which likely reflect higher crystallinity at

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higher reaction temperatures. The temperature of calcination caused only minor

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morphological differences between hydroxyapatites produced at the same temperature

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but did not result

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in major visible changes. Fig. 5 displays the dissolution kinetics of the hydroxyapatite powders in different

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citric acid/citrate buffer solutions within the pH range 2.9 to 3.8. In contrast there was no

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dissolution of hydroxyapatite in water and lactate-acetate buffer [cf. Figure S1;

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Supplementary material].

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Average solubility of apatite (25°C) in citric acid/ citrate buffer pH 3,5

pH 3,8

pH 2,9

ABSORBANCE

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

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pH 2,9

Average solubility of apatite (40°C) in citric acid/ citrate buffer

Average solubility of apatite (80°C) in citric acid/ citrate buffer

TIME [HH:MM]

pH 2,9

pH 3,8

ABSORBANCE

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ABSORBANCE

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

pH 3,5

pH 3,8

TIME [HH:MM]

Average solubility of apatite (60°C) in citric acid/ citrate buffer pH 3,2

pH 3,5

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

TIME [HH:MM]

pH 2,9

pH 3,2

pH 3,2

pH 3,5

pH 3,8

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

TIME [HH:MM]

Figure 5: Solubility tests of different synthesized hydroxyapatite powders in 0.1 M citric acid/citrate buffers of pH 2.9 to 3.8. No deviating changes have been detected at lower (pH 2.6) and higher pH values (4.1), respectively (data not shown).

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All hydroxyapatite powders are stable at pH values >3.8, which is in agreement

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with previous findings (Koutsopoulos, 2002). When hydroxyapatites are exposed to pH 14

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values < 3.7, the minerals dissolve within the first hour except for hydroxyapatites

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synthesized at 80 °C. Moreover, Figure 5 shows that less crystalline hydroxyapatites

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dissolve faster than hydroxyapatites of higher crystallinity. These results support the

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assumption that poorly crystalline hydroxyapatites might be easier available to plants

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than more crystalline forms in soil, due to a more rapid dissolution.

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Conclusion

We successfully synthesized

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P-labelled hydroxyapatite powders using a

modified approach of an established wet-chemical method (Wang et al. 2010). The new

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method allows a rapid synthesis of hydroxyapatites of varying crystallinity and thus is

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suitable to produce radioactively labelled apatite specimen for environmental research.

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Raman spectroscopy, XRD, and SEM confirmed that increasing reaction temperatures

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resulted in increasing crystallinity of the produced hydroxyapatites while calcination

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temperature hardly affected the surface features of the hydroxyapatites. Solubility tests

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confirmed that the synthesized hydroxyapatite powders dissolve at pH values <3.7 and

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that dissolution rates decrease with increasing crystallinity. The (i) short preparation

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time, (ii) resulting close-to-natural pure hydroxyapatite phases of different crystallinity,

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and (iii) the absence of special equipment make this modified method suitable for

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producing radio-labelled hydroxyapatites of different reactivity to test biological P uptake

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processes under near-natural conditions. Due to the simple protocol of the method, any

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kind of additional labelling such as with 44Ca or 18O is possible.

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Acknowledgements

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This work has been supported by Forschungszentrum Jülich GmbH (FZ Jülich) and

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the PAK 888 project. The authors gratefully acknowledge the help of Ursula Pfaffen (IBG 15

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3, FZ Jülich) for the Raman measurements and laboratory support, Martina Krause

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(IBG-3, FZ Jülich) for laboratory support in the radioactive laboratory area, and Dr.

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Wolfgang Tappe (IBG 3, FZ Jülich) and Sirgit Kummer (IBG 3, FZ Jülich) for constructive

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comments and their help with the solubility measurements. X-ray diffraction

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measurements were carried out by Dr. Stefan Stöber (Mineralogie and Geochemistry,

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Martin Luther University Halle-Wittenberg). We thank Prof. Dr. L. Singheiser (IEK 2, FZ

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Jülich) and Dr. E. Wessel (IEK 2, FZ Jülich) for conducting the SEM images of the

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hydroxyapatite powders.

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Highlights: First chemical synthesis to produce 33P-labelled hydroxyapatites

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Synthesizing powders with various degrees of crystallinity in less than 30 h

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Hydroxyapatites of varying crystallinity, and thus, solubility were produced

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Near-natural hydroxyapatites suitable for tracer-based environmental research

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