Journal of Molecular Structure 1117 (2016) 283e286

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Journal of Molecular Structure

journal homepage: http://www.elsevier.com/locate/molstruc

Low temperature synthesis and characterization of carbonated

hydroxyapatite nanocrystals
Aneela Anwar a, *, Muhammad Nadeem Asghar b, Qudsia Kanwal a, Mohsin Kazmi c,
Ayesha Sadiqa a
a
Department of Basic Sciences and Humanities, University of Engineering and Technology, KSK Campus, GT Road, Lahore, Pakistan
b
Department of Chemistry, Forman Christian College (A Chartered University), Lahore, Pakistan
c
Department of Chemical Engineering, University of Engineering and Technology, KSK Campus, GT Road, Lahore, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Carbonate substituted hydroxyapatite (CHA) nanorods were synthesized via coprecipitation method
Received 30 January 2016 from aqueous solution of calcium nitrate tetrahydrate and diammonium hydrogen phosphate (with urea
Received in revised form as carbonate ion source) in the presence of ammonium hydroxide solution at 70
C at the conditions of
18 March 2016
pH 11. The obtained powders were physically characterized using transmission electron microscopy
Accepted 18 March 2016
Available online 21 March 2016
(TEM), X-ray powder diffraction analysis (XRD), and FTIR and Raman spectroscopy. The particle size was
evaluated by Dynamic light scattering (DLS). The chemical structural analysis of as prepared sample was
performed using X-ray photoelectron spectroscopy (XPS). After ageing for 12 h, and heat treatment at
Keywords:
Bioceramics
1000
C for 1 h, the product was obtained as highly crystalline nanorods of CHA.
Hydroxyapatite © 2016 Elsevier B.V. All rights reserved.
Nanoparticle
Carbonate
Bone regeneration
X-ray diffraction

1. Introduction
Hydroxyapatite (HA), [Ca10(PO4)6(OH)2] bioceramic has received
much attention as bone implants material because it is chemically
similar to the biological apatite. It is being extensively used in bone
regeneration [1] as it bonds physiochemically to bone and stimu-
lates bone formation that is essential for implant osteoconduction
and osseointegration. Bioactive and osteoconductive properties of
hydroxyapatite make it a good synthetic bioceramic [1,2].
The structure of regular bone has a varying carbonate content
from 4e8% besides trace elements (Naþ, Mg2, Kþ, F, Cl) [3e5].
Due to the presence of carbonate in natural bone, the need to
synthesize carbonated HA has acquired great importance among
researchers [6]. It is also reported that the solubility and resorption
of carbonated hydroxyapatite is greater than pure HA resulting in
an increased concentration of calcium and phosphate at the site of
bone formation [7]. The replacement of hydroxide and phosphate
ions by carbonate, forms A and B type carbonated hydroxyapatite
* Corresponding author.
E-mail address: a.anwar@uet.edu.pk (A. Anwar).
respectively [8]. The B-type carbonate reduces the crystallinity and
improves the solubility in the apatite lattice (both in vitro and
in vivo tests) [9].
A widespread research is being carried out to synthesize
nanocrystals of CHA due to their rapid solubility and stimulation of
bone growth rate [10]. In some studies carbonate substitution was
carried out in air and CO2 environment [11] while in some by
controlling temperature and HCO 3 concentration, two precipita-
tion variables acicular and spheroidal crystals were synthesized [8].
For dental and orthopedic-applications, nanosized substituted hy-
droxyapatite was prepared by using CO2 
3 and F by aqueous pre-
cipitation method [12].
In this paper, coprecipitation synthesis was used for the pro-
duction of carbonate substituted HA nanoparticles at low temper-
ature and atmospheric pressure. The aqueous solutions of calcium
nitrate tetrahydrate and diammonium hydrogen phosphate with
Urea ion were slowly mixed at 70
C in the pH range 11. It was
observed that nanoparticle properties are largely dependent on
reaction parameters such as particle size and shape, selection of
conditions such as the temperature, reaction pH, and variation in
Ca:P ratio.

http://dx.doi.org/10.1016/j.molstruc.2016.03.061
0022-2860/© 2016 Elsevier B.V. All rights reserved.
284 A. Anwar et al. / Journal of Molecular Structure 1117 (2016) 283e286
2. Materials and methods
Calcium nitrate tetrahydrate [Ca(NO3)2.4H2O, 99%] and dia-
mmonium hydrogen phosphate [(NH4)2HPO4, 98%] were purchased
by SigmaAldrich. Urea [(NH2)2CO, 99%] and ammonium hydroxide
solution (NH4OH, 28%) were purchased by VWR International.
Deionised water was used throughout all experiments.
2.1. Synthesis procedure
2.1.1. Carbonate(CO2
3 )-substituted hydroxyapatite
Carbonate substitution reactions were carried out by using
coprecipitation method in this study. In the first step, 0.25 M of
calcium nitrate were dissolved in 250 mL deionised water and the
pH of this solution was maintained to pH 11 by adding 15 mL neat
ammonium hydroxide solution. Urea was added to diammonium
hydrogen phosphate and this solution was then slowly added to
calcium nitrate solution at 70
C under constant stirring. A
Ca:[PO34þCO23 ] molar ratio of 1.67 was adjusted in all precursor
solutions. The aqueous suspension obtained by this process was
aged for 12 h. It was anticipated that carbonate substitutes phos-
phate ion in HA lattice leads to B-type carbonate substitution. The
wet residue obtained was washed, centrifuged and dried in oven at
90
C overnight.
2.2. Characterization
2.2.1. Chemical analysis
Chemical analysis of CHA samples was carried out using K-Alpha
X-ray photoelectron spectrometer (Thermo Scientific) with two
chamber vacuum. It uses a monochromated Al K- alpha source
(E ¼ 1486.6 eV) with maximum power of 72 W. The vacuum
chamber pressure was at a pressure of ~ 3  108 Torr. The spec-
trum involved an energy of 150 eV for survey scans. The detector is
a 128 channel sensitive detector. The XPS spectra were processed
using Casa ™ software.
2.2.2. Transmission electron microscopy
TEM images were collected using a JEOL JEM-1200EX II Electron
Microscope. Digital images were taken with a side mounted AMT
2K high sensitivity digital camera. A small amount of sample (less
than 10 mg) was dispersed in neat methanol and then ultra-
sonicated for 2 min to yield a very dilute suspension. A few drops of
the resulting suspension were then placed on a carbon-coated
copper grid (procured from Agar Scientific, UK), which was used
as the TEM specimen. The grid was dried prior to use in the double
tilt holder of the TEM. Image J software (version 5.0) was used for
assessing particle sizes.
2.2.3. Dynamic light scattering
DLS measurements were taken using a Malvern Instruments
Zetasizer operated in backscatter (173
) mode. The sample slurry
produced (solid content ~ 1% by volume) was diluted with meth-
anol then placed in an ultrasonic bath for 10 min to disperse the
sample. Square cuvettes with a path length of nominally 10 mm
were used for measurements.
2.2.4. Powder X-ray diffraction
Bruker AXS D4 Endeavour™ XRD diffractometer was used for
XRD collection of all samples. The data was collected in the 2q range
from 5 to 70
with a 0.05
scanning step and a count time of 2 s per
step using Cu-Ka radiation (l ¼ 1.5406 Å). DIFFRACplus Eva™
software was used for the phase analysis of the data by spectral
matching with standard patterns. The crystallite sizes were calcu-
lated by using the DebyeeScherrer equation [13].
2.2.5. FTIR spectroscopy
The functional groups present on the surface of HA were
examined using Fourier Transform Infrared spectroscopy (FTIR)
using a Nicolet 6700 FTIR (ThermoScientific, UK). The FTIR spectra
were collected in the range 400-4000 cm1 at resolution of 4 cm1
averaging 256 scans.
2.2.6. Raman spectroscopy
A Confocal Raman DXR Spectrometer (SP Thermo-Scientific)
was used for analysis. The powder Sample was deposited onto
316L stainless steel block with the help of a spatula. 316 L block was
wiped clean first with distilled water then with acetone prior to
sample analysis. The data was gathered using 780 nm laser, 10 X
Lens with the scan time of 90 s for each sample.
3. Results and discussion
A chemical analysis of as prepared CHA sample was done by
using XPS analysis as shown in Fig. 1. The peaks at 134 eV corre-
sponded to P 2p spectra of hydroxyapatite. While the binding en-
ergy values for O 1s and Ca 2p were measured as 532 and 347 eV,
respectively [14]. The peak at 285 eV corresponded to C 1s spectra
of carbonate substituted hydroxyapatite. The Ca/P ratio in the CHA
analysed sample (1.65) was determined using XPS analysis which is
found very closed to stoichiometric ratio of 1.67 in pure
hydroxyapatite.
Transmission electron microscopy was used to analyse the
particle size and morphology. The as prepared CHA sample syn-
thesized using coprecipitation process had a rod like morphology as
shown by TEM in Fig. 2(a) and average length along the longest axis
of each particle was ~110 ± 15 nm (200 particles sampled). TEM
image was also captured for heat-treated (1000
C, 1h) CHA sam-
ples as shown in Fig. 2 (b). It was observed that after heat treat-
ment, particles increased in size and became more agglomerated.
Particle size distribution was also conducted for CHA sample.
DLS measurements of CHA sample synthesized at 70
C reveals
average particle size of ca. 169 and PDI (polydispersity index) value
of 0.231. DLS measured diameter (100e300 nm) with PDI of 0.3 or
less shows good dispersion. It was also observed that DLS mea-
surement results are in good agreement with TEM determined
distributions (Fig. 3).
Powder X-ray diffraction data was obtained for the CHA sample
Fig. 1. XPS survey spectrum of CO2-
3 - substituted hydroxyapatite powders made using
coprecipitation method at 70
C.
 A. Anwar et al. / Journal of Molecular Structure 1117 (2016) 283e286 285

Fig. 2. Transmission electron microscope images of CO2-

3 - substituted hydroxyapatite nanorods of as prepared (a) and heat treated sample (1000 C, 1h) made using coprecipitation
method (bar size 100 nm with original magnification) at 70
C.

Fig. 3. DLS size distribution of as prepared CO2-

3 - substituted hydroxyapatite nanorods made using coprecipitation method at 70 C.

to investigate how carbonate substitution influenced phase

composition and phase purity. XRD patterns for CHA sample as
shown in Fig. 4 have a good match to JCPDS pattern 09-432, and
suggested no substantial effect of increasing carbonate content on
the phase-purity of the HA phase. But a variation in unit cell pa-
rameters of CHA powder has been observed compared to stoi-
chiometric hydroxyapatite powder. An increase in c parameter
from 6.8940 to 6.9126 and a decrease in a parameter from 9.4280 to
9.3880, exhibiting the formation of a B- type CHA as reported by
Landi et al. [15].
FTIR spectroscopy was accomplished in order to support the
observations made using XRD. The FTIR analysis (Fig. 5) revealed
strong absorption peaks at 872 cm1, 1430 cm1and 1450 cm1,
respectively for the presence of B-Type CHA. The characteristic
peaks at 880 cm1, 1450 cm1 and 1540 cm1 were not evident for
A-type CHA.
In FTIR analysis the stretching vibrations at 3510 cm1 revealed
the presence of stretching mode (ns) of the hydroxyl group in CHA
[16]. Peaks at 1430 cm1 and 1450 cm1 were assigned to the
stretching modes (n3) of vibrations for carbonate ions in CHA. Peaks
at 1100 cm1 and 1031 cm1 corresponded to the (PeO) asym-
metric stretching mode (n1) of phosphate, whilst the peaks at
602 cm1 and 564 cm1 revealed the presence of OePeO bending
modes (n4) as shown in Fig. 5.
Raman spectroscopy was carried out in order to supplement
XRD data and explore any structural changes with substitutions in
the apatite lattice. Fig. 6 demonstrates Raman spectra for all the
carbonate-substituted samples. The symmetric stretching mode
(n1) at 965 cm1 exhibited to the PeO bonds in the phosphate
groups [17]. The bending bands (n4) at 610 cm1 and 483 cm1
were observed for the OePeO linkage in the phosphate groups as
shown in Fig. 6. The asymmetric stretching modes (n3) of carbonate Fig. 4. Powder X-ray diffraction patterns of as prepared (a) and heat treated (b)
(1000
C, 1hr) CO2-
Appeared at 1070 cm1 in Raman spectrum and intensity of the 3 - substituted hydroxyapatite powders made using coprecipitation
method at 70
C.
286 A. Anwar et al. / Journal of Molecular Structure 1117 (2016) 283e286
Fig. 5. FTIR spectral data of CO2
3 substituted hydroxyapatite powders, presenting the
characteristic peaks of carbonate group (,) along with HA peaks.
Fig. 6. Raman data of CO2-
3 -substituted hydroxyapatite samples made using copreci-
pitation at 70
C.
peak was largely dependent on the amount of carbonate ions
present. Fig. 6(b) showed Raman spectra with increasing carbonate
concentration up to 2%. It was observed that intensity of carbonate
peak at 1070 cm1 increased significantly by increasing carbonate
contents as shown in Fig. 6(b).
In this study we synthesized very fine nanorods of carbonated
hydroxyapatite, which were confirmed by the use of different
characterization techniques. It was also observed that the phase
purity of the final product mainly dependent on reaction parame-
ters such as temperature, pH and Ca/P ratio. The reaction is carried
out at 70
C in order to enhance the precursor solubility by main-
taining the high concentration of calcium ions which ultimately
leads to lowering of reaction time up to 12 h. Because the tradi-
tional room temperature coprecipitation synthesis procedures are
very time consuming (have been taken 24 h or more). This research
presented the effective way to prepare very fine particles of ion
substituted bioactive bioceramics in relatively less time as
compared to literature methods. These nanobioceramics have a
great range of applications for use in replacement of living hard
tissues such as bone and teeth, as bone graft substitutes, as in-
jectables, coatings on metallic implants and as fillers for dental
composites.
4. Conclusions
Low temperature synthesis was used to successfully produce ion
substituted calcium hydroxyapatite from calcium nitrate and dia-
mmonium hydrogen phosphate (with Urea as carbonate ion
source) precursor solutions at 70
C, and atmospheric pressure at
relatively shorter time compared to traditional room temperature
coprecipitation method. Careful control of the quantities of re-
actants used, resulted in nano-sized carbonate substituted hy-
droxyapatite confirmed by TEM and DLS measurements. The
obtained product, with its unique features, is a promising material
and has potential to be used in bone regeneration applications
where fine particle size distribution control is beneficial.
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