Synthesis and optimization of the production of millimeter-size

hydroxyapatite single crystals by Cl--OH- ion exchange

Esther García-Tuñon1,3, Jaime Franco2, Salvador Eslava3, Vineet Bhakhri3, Eduardo

Saiz3, Finn Giuliani3, Francisco Guitián1

1Instituto de Cerámica de Galicia, Universidad Santiago de Compostela, Santiago de

Compostela, Spain2Keramat S. L., Spain

3Centre for Advanced Structural Ceramics, Materials Department, Imperial College London,

London, United Kingdom

Corresponding Author: *Esther García-Tuñón

Instituto de Cerámica de Galicia, Universidad de Santiago de Compostela

Avda Mestre Mateo S/N, 15706 Santiago de Compostela (Spain)

*Current address: Imperial College London, CASC, London, United Kingdom,

+44(0)2075895111, esther.garcia-tunon@imperial.ac.uk

RECEIVED DATE

ABSTRACT

Millimeter-size hydroxyapatite single crystals were synthesized from chlorapatite

crystals via the ionic exchange of Cl- for OH- at high temperature. X-ray diffraction,

Fourier-transform infrared spectroscopy, and chloride content measurements were used

to follow the progress of this conversion, and to assess the effect of the experimental

conditions (temperature, time and atmosphere). Cl-OH- exchange took place

homogeneously and was enhanced by firing in wet air. After firing at 1425ºC for 2

hours 92% of the Cl- ions were exchanged by OH- while maintaining crystal integrity.

Temperatures above 1450ºC damaged the surface of the crystals, destroying the

hexagonal habit at 1500ºC. The composition of these apatite crystals was close to bone

mineral content. Their hardness (8.71.0GPa) and elastic modulus (12010GPa) were

similar to those of the starting chlorapatite (6.61.5GPa, and 11015GPa respectively).

However, their average flexural strength was 25% lower due to the formation of

defects during the thermal treatments.

1. Introduction.

Calcium orthophosphates are chemical compounds of wide interest in different fields of

research such as geology, chemistry, biology and medicine. Calcium apatites are the

main ore of inorganic phosphorous in nature. Hydroxyapatite (HA) is a calcium

orthophosphate with a chemical composition similar to the inorganic component of

bone and teeth. Bone serves as a structural support for the body and is the major

reservoir of calcium and phosphate ions necessary for several metabolic functions [1].

Bone is a composite material with a complex and hierarchical structure composed by

inorganic apatite mineral, type I collagen fibrils (organic part) and cells (osteoblasts,

osteoclasts and osteocytes) [2]. Synthetic calcium phosphate ceramics have been

successfully employed as bone replacement materials for more than 30 years [3, 4].

These materials have good biocompatibility and bioactivity and are being used in dental

and medical applications. It is also well known that calcium phosphate ceramics exhibit

relatively weak mechanical properties. They show low reliability and brittle behavior,

i.e. low Weibull modulus [5, 6], especially in wet conditions [6, 7]. The fracture

toughness of sintered hydroxyapatite and tricalcium phosphate materials are of the order

of 1 MPa·m1/2, while, for example, reported values for bone range between 2 to 12

MPa·m1/2 [8, 9]. Due to their poor mechanical performance, their application is limited

to non load-bearing applications. One possibility to overcome this could be the use of

high aspect ratio single crystal fibers as biocompatible reinforcement to obtain bio-

composites [10, 9].

Also, large apatite crystals, unlike polycrystalline bulk materials, can be used to

determine intrinsic properties and analyze basic surface chemical and biological

processes. Millimeter-size crystals with a similar composition to the mineral component

of bone are the best substrate to perform basic dissolution studies [11-14], and to study

how extracellular matrix proteins adsorb and interact on their surfaces [15, 16]. They

can also be used to study the dependence of the mechanical properties depending with

crystal orientation [17].

To the best of our knowledge, large amounts of millimeter-size HA single crystals have

not yet successfully been obtained by using either the molten salts method or

hydrothermal synthesis. Following the molten salt approach several authors have

reported the synthesis of micro-sized whiskers of HA doped with K+ [18, 19] and

millimeter-size carbonate apatite (with formula Ca9.8[(PO4)5.6(CO3)0.4](CO3)) using

CaCO3 as flux [20], where the carbonates are allocated in both anionic channel and

phosphate positions [21]. Using the hydrothermal route several authors have obtained

micrometer-sized HA single crystals with needle, platelet, flake or whisker

morphologies [22-25]. Some hydrothermal and combined procedures to obtain

millimeter-sized apatite single crystals have been reported. Ito et al. obtained carbonate-

containing hydroxyapatite single-crystals with 4mm in length by CaHPO4 hydrolysis

[26]. The disadvantages of this approach are that only 4-8 large apatite single crystals

per experiment are obtained, the set-up is complicated and long times are needed (it

requires heating rates of 0.005ºC·min-1) [26]. Following a similar approach under

natural convection Teraoka et al. obtained Ca-deficient hydroxyapatite whiskers with

length up to 8.3mm [27]. Again, the main drawback is the low number of crystals

obtained per experiment and the time needed [27]. Given the difficulties in synthesizing

large HA single crystals, other authors have investigated the conversion of other

apatites, such as chlorapatite or fluorapatite into HA. Elliot and Young provided the first

evidence of the solid-state ion exchange in the apatite structure [28]. An alternative

procedure was proposed by Yanagisawa et al. and Rendón-Ángeles et al., who

investigated the ion exchange of Cl- and OH- with F- in synthetic calcium ClAp and

calcium hydroxyapatite single crystals under hydrothermal conditions [29, 30].

In this work we aim to synthesize and optimize the production of large HA single

crystals by delving into the time and temperature dependent evolution of the ionic-

exchange process (between Cl- and OH- species along the whole structured apatite

system) during the conversion of chlorapatite (ClAp) into HA following a similar

approach to that proposed by Elliot and Young [28, 31]. Starting from millimetre-size

ClAp single crystals obtained by the molten salt synthesis method [32] we follow the

conversion as a function of time, temperature and atmosphere and its effects on the

mechanical properties of the material. The analysis allows us to identify optimum

processing conditions to maximize the exchange and synthesize large HA crystals.

2. Experimental Procedure.

2.1. ClAp Growth.

Calcium chloride (Merck, PA, CaCl2·2H2O CAS 10043-52-4) and β-tricalcium

phosphate, (β-Ca3(PO4)2, β-TCP, D50=0.5m, >99wt%, Keramat®, Spain) powders were

mechanically blended in different proportions in an agate mortar (Restch, RM-100,

Germany) for 12 min. Prior to mixing the CaCl2 was dehydrated at 110oC for 2 weeks.

Thermo-gravimetric analysis (PL-STA, Polymer Laboratories) of the dehydrated flux

showed that the remaining H2O percentage was 8wt%. Water was fully eliminated at

300oC. After milling, the mixtures were uniaxially pressed at 60 MPa into cylindrical

pellets (2.54cm diameter) using a stainless steel dye. For the growth process, pellets

(12-15g per batch) were placed inside a platinum crucible and heated up to 1100oC for

30 min in a rapid melting furnace. The heating rate was 10oC·min-1 and the quenching

was done in air. After cooling, the sample was rinsed with deionized water to dissolve

the unreacted calcium chloride. The crystals were vacuum-filtered and washing was

repeated three times. Subsequently they were sieved trough a 63 µm mesh, and dried at

110ºC for 24 hours. The final samples consist mostly of millimeter size ClAp single

crystals with needle-shape and characteristic microscopic roughness [32].

2.2. Chlorapatite conversion to hydroxyapatite.

ClAp millimetre-size crystals were converted into HA by thermal annealing under

different atmospheres. For the first set of experiments, we used a lift furnace to anneal

the samples in air for 2 hours at temperatures ranging from 1300ºC to 1500ºC. For the

second set, an alumina tube furnace with moisture atmosphere control was used. A flow

of wet air was fed into the furnace during the annealing process to increase the humidity

and enhance the Cl- OH- exchange. Synthetic air was pumped through a water

bubbler into the furnace (gas pressure 1.5MPa) and a second bubbler was placed in the

exit to monitor the flow during the thermal treatment. To study the effect of

temperature, the samples were fired for two hours at temperatures ranging from 1375ºC

to 1490ºC. A set of samples was also fired at 1425ºC for times ranging from 2 to 8

hours. All the samples were placed on alumina crucibles and heating and cooling rates

were set at 10ºC·min-1 for all the experiments.

2.3. Characterization.

The samples were analyzed by powder X-ray diffraction (XRD). In order to get the best

peak profile and minimize the preferred orientation effect, the samples were milled in a

zirconia ball mill and the powder was sieved through a 63μm mesh. The XRD analysis

(D5005, Siemens, Germany) was performed using Cu(Kα1) radiation (λ=1.5406Å). The

powder X-ray diffraction pattern was collected using a step size of 0.02° and counting

time of 4s per step. Single-crystal XRD measurements were also carried out as

described in Ref. [33].

The crystal morphology and chemical composition were analyzed by scanning electron

microscopy (SEM) on a JEOL 1640 equipped with an energy dispersive spectroscopy

(EDS) microprobe (INCA Sight Oxford-instruments, UK). The samples were also

characterized by Fourier transform infrared spectroscopy (FTIR Bomem, MB-100

series, USA), in transmission mode, in the range of 400-4000cm -1, and with a resolution

of 2cm-1. For this characterization, pellets were prepared by vacuum dye pressing

mixtures of 1mg of each sample with 100mg of dry KBr.

Chloride contents were measured by using an ion selective electrode (9617BNWP)

attached to Thermo Benchtop pH/ISE Model 720A. Standard solutions (0.1M NaCl,

100ppm, and 1000ppm Thermo) and an ionic strength adjustor solution (Thermo ISA)

were employed during the analysis. Measurements were performed on 0.5mg of each

sample dissolved in nitric acid solution (5wt%).

Room temperature (20 °C) nanoindentation tests were performed by using a Nano

Test instrument (Micro Materials Ltd. Wrexham, UK) on the millimeter-sized crystals

embedded in Bakelite resin. Samples were polished to 6 µm finish with SiC paper, to

obtain flat surfaces and avoid the effect of angled indentation [34]. During each

experiment, load was increased at a fixed rate of 1.25mN.s-1 to a predetermined

maximum value of 50 mN. The maximum load was then maintained for 60 s before

unloading at the original loading rate. Thermal drift contribution to the depth signal was

estimated by monitoring the depth signal over 30 seconds at 10% of the maximum load

during unloading and final indentation data was corrected for this estimated artefact. An

average of 80 indentations placed on 30 crystals per sample, were used to obtain the

elastic modulus and hardness. The inter-indent distance was chosen as ten times the size

of the residual impression (Berkovich shape). The hardness (H) and the reduced

modulus (Er) were calculated by using the procedure outlined by Oliver and Pharr

method [35, 36]. The values of elastic modulus (Es) were calculated using Er and

Poisson’s ratios of diamond (Ei=1141GPa, νi=0.07), chlorapatite and hydroxyapatite

(νi=0.27) [37-39]. All the load-displacement curves were checked and also the

morphology of the indents was observed by SEM, in order to identify possible sources

of error such us pile up or pop in among others.

Flexural strength of 20 single-crystals of selected samples was measured by three-point

bend test in a horizontal nanoindenter (Micro Materials Ltd. Wrexham, UK). The

single-crystals were mounted on the top of a custom-built aluminium holder with

0.5mm wide deep trenches. The crystals were subjected to bending with a constant load

of 200mN·s-1, using a diamond cone-spherical probe with a 25m tip radius. The

average diameter of the crystals analyzed was 50m, being the ratio of trench width to

crystal diameter 10.

Results and Discussion

Searching for an optimum environment to facilitate the anionic exchange in the ClAp

conversion to HA, we assessed the annealing under a wet atmosphere and compared it

to the conversion in air. Figures 1 and 2 show the evolution of the powder XRD patterns

and FTIR spectra of the single crystals under these two conditions for temperatures

between 1300 and 1500 ºC. Figure 1 includes the plots of the Le Bail refinements that

we perform in the samples annealed in air to determine how the unit cell parameters (a,

c and therefore dhkl) evolve with temperature. We highlight in grey the 2θ range (29º-

36º) that we use to follow the conversion by powder XRD in Figures 2a and 3a. In this

2θ interval, we can identify the main diffractions of the phases involved in the process

and therefore monitor the conversion. In both cases, in air and wet atmosphere (Figure

1a and 2a), powder XRD shows systematic peak displacements with temperature

towards the HA structure above 1450C [40]. There is also a secondary transformation

to a monoclinic calcium phosphate without Cl- or OH- in the structure, identified by

diffractions at 2θ=30.7 and 34.5º [33]. Comparison of the XRD pattern evolution in the

two different conditions (room and wet air) shows that the HA structure is achieved at

lower temperatures in wet air (Figures 1a and 2a). Thus, vapor accelerates the Cl-

OH- exchange. FTIR spectroscopy confirms it, since OH- vibration bands at 630, 3570

and 3640cm-1 appear at lower temperature and with higher intensity in samples prepared

in wet air conditions (Figures 1B and 2B). A temperature of 1400ºC in wet air is enough

for a clear OH- incorporation in the apatite structure (Figure 2B). In view of these

results, firing at 1425 ºC in wet air was selected as the optimum condition to follow the

degree of conversion with time (Figure 3). FTIR spectra also show a small absorption

band in the vicinity of 1600cm-1, which may indicate the presence of CO32- in the

samples [41, 42]. Carbonate incorporations in the structure either in the anionic channel

or PO43- substitutions were ruled out by SXRD. This band may be justified by the

carbonation of spodiosite remaining on the surfaces of the ClAp from the fabrication

process [33, 40].

Chloride content measurements allowed us to quantify the conversion to HA. From this

measurements and considering the general formula Ca5(PO4)3Cl1-x(OH)x, we have

calculated the average hydroxyl incorporation in the structure, x. The analysis shows

that the Cl- content is close to saturation after firing for 2 hours (Fig 4A). The overall

reaction could be written:

Ca5(PO4)3Cl + xH2O(g) Ca5(PO4)3Cl1-x(OH) x + xClH(g) (1)

The saturation values of x increase with temperature and are higher for wet atmospheres

(Fig. 4B). When the experiments are carried out in room air, a limit on the conversion is

reached at 1450ºC, above which the chloride content of the samples remains unaltered

(Figure 4B). In wet air, however, the hydroxyl incorporation continues increasing above

1450ºC, reaching values close to the OH- content in HA above 1475 ºC (x=1, dashed

line in Figure 4B), which indicate a high conversion. In wet air values of x>0.9 are

reached after 2 hours at 1425ºC (Figure 4A). Therefore, a high conversion can be

achieved in wet air by different paths, either increasing the temperature or the time.

In addition to high conversion, we also aimed to obtain best mechanical properties,

while maintaining the quality of the crystals (in terms of quality of the data during

SXRD measurements), their apatite structure and avoiding damage to the surfaces.

Starting ClAp single crystals have a clear hexagonal habit and a characteristic roughness

on the surface (Fig 5). This roughness is formed during fabrication due to a peritectic

reaction between ClAp and the flux [32].

Thermal treatments have an effect not only on the ionic exchange but also on the

morphology of the crystals. SEM was used to inspect the surfaces and morphology of

transformed single crystals (Figure 6 and 7). Above 1050ºC the incongruent melting of

small amounts of spodiosite remaining on the crystal surfaces takes place according to:

Ca2(PO4) Cl Ca5(PO4)3Cl + CaCl2 (2)

This leads to the formation of small CaCl2 deposits on the surfaces (Figure 6A). Thus

the decomposition of small amounts of spodiosite on the surfaces (equation 2), the re-

crystallization of chlorapatite (equation 2), and also the formation of small amounts of

HCl(g) in the furnace atmosphere (equation 1) promote changes in the morphological

features. The surface of the crystals presents more defects after the heat treatments

(Figure 6A-B) and the hexagonal habit is damaged at 1480ºC and above (Figure 6C).

The presence of defects increases with temperature and time in any conditions.

Hexagonal defects were spotted on the prism surfaces after treatment in room air at

1400ºC (Fig. 6B), while the hexagonal habit and surface of the crystals were damaged at

higher temperatures (1480ºC, Figure 6B, D). At even higher temperature, 1500ºC,

displacements and bending of the crystals along the hexagonal axis were observed

(Figure 6E), the crystals are also considerably rounded due to the closeness to the

melting point of chlorapatite (Tf = 1530ºC). After firing in wet air the crystals preserved

their hexagonal habit, the formation of surface defects was minimized and their

roughness was softened (Figures 7A-7B). Although, firing at 1475ºC and above in wet

air also led to a certain degree of mass transport and chlorapatite recrystallization on the

surfaces due to the decomposition of spodiosite. Thus, control of the furnace

atmosphere allows not only a higher percentage of conversion but also minimization of

morphological changes.

EDX analyses also confirmed the chloride loss in the surface of different single crystals

(Figures 6F and 7C-D). To assess if the loss of chloride content was homogeneous from

the surface to the inner core, a single crystal prepared in wet air for 4 hours at 1425 ºC

was embedded into resin and polished perpendicularly (Figure 7B). EDX analysis on

the transversal section showed that the ion exchange is homogeneous across the

structure and the chloride content is below the detection limit (Figure 7B). EDX also

served to assess the composition homogeneity among different single crystals for each

treatment. The homogeneity was confirmed in all the samples treated in room air.

However, for wet air treatment, the concentration is slightly different from one crystal

to another. This may be explained by the lack of laminar flow in the furnace fluxed with

wet air, resulting in positions with slightly different humidity.

SXRD analyses indicated an increase of the mosaicity (higher tension in the network of

unit cells due to the enhanced chemical changes in the structure) of the single crystals

when using times ≥4 hours at 1425ºC (Figure 7D), being the SXRD data not as good as

for any sample annealed for 2 hours (Figure 7C). Therefore, it is necessary to find a

compromise between the percentage of conversion and the quality of the crystals.

Considering SXRD, XRD, FTIR analyses and chloride content measurements, the best

samples (those that showed a degree of conversion above 85% and up to 96%, while

maintaining the surface roughness and the single crystal structure) were selected for

mechanical characterization (Table 1). Table 1 also shows the chloride content of the

selected samples relative to the measured Cl- content of stoichiometric ClAp [43].

The results of the mechanical characterization are summarized in Table 1, including the

values of hardness (H) and elastic modulus (Es). Figure 8A shows typical load-

displacement curves obtained in the nanoindentation tests to measure the elastic

modulus and hardness. It must be pointed out that hollow core defects and the formation

of micro-tubes is a common phenomenon that takes place during the synthesis of these

crystals [25]. The presence of these defects affects the mechanical behavior. Most of the

curves are continuous, i.e. without any visible pop in or pop out, both in loading and

unloading. Nevertheless, some of them present discontinuities likely due to the presence

of defects under the site of the indentation. Data obtained from these curves were

rejected. The morphology of the indents was analyzed by SEM in order to identify

sources of error (Figure 8B). No signs of pile-up, sink in or cracking were detected.

Table 1 shows the values of H and Es obtained for the selected samples ordered with

decreasing chloride content. All the samples have higher H after the conversion

treatment compared to starting ClAp crystals however there is not a clear trend with

remaining Cl content, while the elastic modulus appears to remain constant. These

results suggest no significant differences associated with the ionic exchange. All these

values are of the order of those typically reported for hydroxyapatite single crystals and

thin films as well as those calculated using ‘ab-initio’ models [17, 44-47]. Viswanath et

al. [47] and Saber-Samandari & Gross [17] reported certain degree of anisotropy

(smaller than 10%) from measurements on synthetic and natural single HA crystals.

However, it must be pointed out that due to the complex multi-axial stress field

generated during nanoindentation the measured values are weighted averages over

several directions. In addition the surface roughness and the introduction of defects

during the exchange process can add to the variability of the results.

We also carried out three point bending test on ClAp single crystals and on the

converted samples with highest H and Es (those fired in wet air at 1425ºC for 2 and 4

hours, Figure 9A). The addition of defects during the thermal treatments decreases the

average flexural strength (D50, Figure 9A). As it could be expected, the measured

flexural strength (f) also decreases in crystals with larger diameter (Figure 9B). In any

case, the average values are in the same order of magnitude reported in literature for

hydroxyl apatite and carbonate-hydroxyapatite synthesized by hydrothermal methods

and natural convection (300-500MPa) [36, 27]. However, our results indicate that the

smaller crystals can exhibit high strengths, reaching values up to 2GPa. These values

are comparable to the tensile strengths reported for single crystalline fibers of technical

ceramics such as Al2O3 or SiC (2-6 GPa) [48, 49] and will likely correspond to crystals

with very low defect concentrations. The results illustrate how one of the main

limitations in the synthesis of apatite fibers and crystals is the need to develop processes

for the reliable fabrication of “pristine” materials.

Conclusions

We have successfully obtained apatite single crystals with workable sizes through the

thermal treatment of Ca5(PO4)3Cl to promote Cl-OH- exchange. The crystals have the

general formula Ca5(PO4)3(OH)xCl1-x and hydroxyapatite structure. There is a range of

firing conditions that results in samples with low chloride content, close to the one of

biological apatites (~0.13wt%) [2]. By adjusting the heat treatment it is possible to

reach values of 1-x as low as 0.04. However, the process can introduce defects that are

detrimental to the mechanical response. In general “softer” firing conditions (lower

temperature under a flow of wet air) preserve the crystal structure and morphological

features while reaching higher exchange. The best combination of larger OH - content

and higher mechanical properties was achieved after firing the chlorapatite crystals for 2

hours at 1425 ºC in wet air. In this way it is possible to get crystals with average

formula Ca5(PO4)3(OH)0.920.03Cl0.080.03, H=8.71.0, Es=12010 GPa, and f=644±389.

Acknowledgements

This work has been partially supported by the Galician Government project

number PGIDT09TMT003239PR. ES acknowledges support from the National

Institutes of Health/National Institute of Dental and Craniofacial Research

(NIH/NIDCR) Grant No. 1 R01 DE015633 and 2 R01 DE015821.

TABLES

Table 1. Mechanical properties on ClAp and HA single crystals. Large standard

deviations are expected due to the different size and distribution of defects in ClAp

crystals.

Firing

conditions

Average Formula

Cl- content

(Relative %

to ClAp)

H

(GPa)

Es

(GPa)

Chlorapatite Ca5(PO4)3Cl 100 6.61.5 11015

145ºC 2h

(room air)

Ca5(PO4)3Cl0.14±0.02(OH)0.86±0.02

142 7.42 11530

1425ºC 2h

(wet air)

Ca5(PO4)3Cl0.08±0.03(OH)0.92±0.03 83 8.71 12010

1450ºC 2h

(wet air)

Ca5(PO4)3Cl0.07±0.03(OH)0.93±0.03

73 7.01.5 10525

1425ºC 4h

(wet air)

Ca5(PO4)3Cl0.04±0.03(OH)0.96±0.03 43 8.21.6 10615

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FIGURE CAPTIONS

Fig. 1. Effect of the temperature on the structure of the crystals after firing in room air:

Le Bail refinements of powder XRD (A) and FTIR (B). A) The interval highlighted in

grey shows the range that we use to study the conversion in wet atmosphere. There is a

good agreement between the experimental intensities (black dots) and the calculated

after refinement (red line). The position of the peaks shifts to hydroxyapatite (HA) dhkl

values as temperature increases, at temperatures ≥1450C a secondary monoclinic phase

is also identified and highlighted with filled squares (2=30.7 and 34.5). Filled and

open circles show the main diffractions of HA and ClAp patterns respectively. B) FTIR

scans showing weak vibration bands at 3570 and 630cm-1 with increasing intensity

depending on the annealing temperature. Bands due to carbon dioxide, vibrational

overtones and carbonates (CO32-) are highlighted in the graph.

Fig. 2. Effect of the temperature on the structure of the crystals after firing in wet air:

powder XRD (A) and FTIR (B) of ground and sieved samples. The position of the

peaks shifts from chlorapatite (ClAp) to hydroxyapatite (HA) dhkl values as temperature

increases, at temperatures ≥1450C a secondary phase is also identified (2=30.7 and

34.5). Vertical lines show the HA pattern (ICDD# 09-432). B) FTIR scans showing

clear vibration bands at 3570 and 630cm-1 at 1425C, with increasing intensity

depending on the annealing temperature, thus confirming a higher OH- incorporation in

wet air. Bands due to carbon dioxide, vibrational overtones and carbonates (CO32-) are

highlighted in the graph.

Fig. 3. Effect of firing time on the structure of the crystals annealed in wet air at

1425ºC: powder XRD (A) and FTIR (B) of ground and sieved samples. A) Firing times

≥2 hours lead to the formation of the secondary monoclinic phase (2=30.7 and 34.5).

B) FTIR analyses confirm the OH- incorporation in the structure (vibration bands at

3570cm-1 and 630cm-1); bands due to carbon dioxide, vibrational overtones and

carbonates (CO32-) are highlighted in the graph.

Fig. 4. Hydroxyl content in the crystals, x in Ca5(PO4)3Cl1-x(OH)x, as a function of

annealing temperature and time. The dashed line corresponds to HA, x=1, in both

graphs. The shaded region highlights the conversion to hydroxyapatite. A) Hydroxyl

incorporation with time at 1425C in wet air. This incorporation reaches values close to

saturation after 2 hours, reaching x ≥0.9. B) Hydroxyl content as a function of

temperature. Higher x values are reached with increasing temperatures and in wet

atmosphere. This ionic exchange reaches a plateau in air at 1450C while it keeps

progressing to a maximum in wet air. It must be pointed out that the chlorine

measurements are very close to the detection limit of the electrode, being difficult to

quantify minute chloride concentrations.

Figure 5. SEM images showing features of the starting ClAp single crystals before

annealing (A, B). Image in A shows a crystal with a clear hexagonal habit and no

defects on the basal plane; the characteristic microscopic roughness is also appreciated

on prism planes. Image in B shows a ClAp twin, where the hexagonal habit is also well

defined, and prism surfaces exhibit microscopic roughness.

Fig. 6. SEM images (A-E) showing the effect of temperature on the morphology of the

crystals after heating in room air. (F) EDX analysis on the crystal in (D) showing low

chloride content. Image in A shows how the basal surface of the crystal presents defects

and small depositions that may be associated with CaCl2 formation due to the

decomposition of the spodiosite remaining on the surface. Image in B shows some of

the hexagonal defects spotted on prism planes after annealing at 1400C. Higher

annealing temperatures damage the surfaces as images in C and D show, at 1480C

crystals lose the hexagonal habit and the surfaces are significantly damaged. At 1500C

the crystals are distorted along the hexagonal axis; surfaces show signs of mass

transport due to the proximity to the melting point of chlorapatite, 1530ºC. Graph in F

shows a representative EDX analysis performed on the crystal in D, where the chloride

content is very low.

Fig. 7. (A, B) SEM images and EDX analysis for two single crystals transformed into

low chloride content HA after firing in wet air. Image in A shows how after 2 hours at

1450C the hexagonal habit is still appreciated and the surfaces are not as damaged as

after firing at similar temperatures in air. The EDX analysis for this crystal shows how a

residual chlorine peak is still visible. Some of the crystals were embedded in resin and

polished to carry out EDX in the transverse section. Image in B shows one of them

obtained at 1425C for 4 hours. Several EDS-analyses on the transversal surfaces of

fractured and unpolished crystals were carried out to confirm these results. The

composition measured by EDX is homogenous across the section, where the chloride

value is below the detection limit of the equipment. Synthetic precession images in C

and D were generated from SXRD data collection. After 2 hours at 1475ºC in air (C),

the image shows well-defined spots, while the same image for crystals obtained after 8h

in wet air at 1425ºC (D) exhibit splitting and lack of definition due to tension between

unit cells (mosaicity).

Fig. 8. A) Representative load-displacement curves. B) SEM image of one indent on a

single-crystal. Sources of error, such as pile up, sink in, or crack in the edges of the

indent, were not detected.

Fig. 9. A) Cumulative frequency distribution and D50 values of the flexural strength for

the samples tested by three point bending. Where x represents the Hydroxyl content in

the crystals, x in Ca5(PO4)3Cl1-x(OH)x. B) Representative flexural strength of single-

crystals vs diameter for single crystals prepared at 1425ºC for two hours in wet air. The

flexural strength decreases with increasing diameter as expected.