ARTICLE IN PRESS

Journal of Crystal Growth 263 (2004) 517–523

Synthesis of stoichiometric nano crystalline hydroxyapatite by

ethanol-based sol–gel technique at low temperature
T. Anee Kuriakosea, S. Narayana Kalkuraa,b,*, M. Palanichamyc, D. Arivuolid,
Karsten Dierkse, G. Bocellif, C. Betzelb
a
Crystal Growth Center, Anna University, Chennai 600025, India
b
. Medizinische Biochemie und Molekularbiologie, Universitatsklinikum
Institut fur . Hamburg-Eppendorf,
Arbeitsgruppe fur. Makromolekulare Strukturanalyse, c/o DESY, Geb. 22a NotkestraX e 85, 22603 Hamburg, Germany
c
Department of Chemistry, Anna University, Chennai 600 025, India
d
Department of Physics, Anna University, Chennai 600025, India
e
Dierks & Partner, Systemtechnik, Pinneberger Weg 22, 20257 Hamburg, Germany
f
C.S. Strutturistica Diffrattometrica CNR, CSSD-Palazzo Chimico, Parco Area delle Scienze 17/a, I-43100 Parma, Italy

Received 5 October 2003; accepted 11 November 2003

Communicated by M. Schieber

Abstract

A relatively easy method to synthesize pure, stable, stoichiometric nano crystalline hydroxyapatite (Ca10
(PO4)6(OH)2, HAP) at low temperature by sol–gel route is presented. Equimolar solutions of Ca(NO3)24H2O and
(NH4)2HPO4 dissolved in ethanol were used in the synthesis at 85
C. The product was sintered at 400
C, 750
C and
1200
C. They were characterized by chemical analysis, XRD, FT-IR, SEM and TGA/DTG. The synthesized HAP was
found to be stable upto 1200
C without any by-products. The SEM analysis indicated presence of pores in the crystal
planes. These pores might be important for biomedical applications as they could enhance the adhesion between the
natural and synthesized bone apatite. Dynamic Light Scattering analysis revealed the presence of nanocrystalline HAP
of size 1.3 nanometer radius in the samples synthesized using this technique.
r 2003 Elsevier B.V. All rights reserved.

Keywords: A1. Biomaterials; A1. Nanocrystalline; A2. Sol–gel; B1. Stoichiometric hydroxyapatite

1. Introduction sintered HAP has many bone replacement appli-

Hydroxyapatite (Ca10(PO4)6(OH)2, HAP) is an
important biomaterial used in hard tissue surgery
[1,2] due to its bioactive nature [3,4]. Dense,
*Corresponding author. Tel.: +49-40 8998 4749/4745; fax:
+49-40 8998 4747.
E-mail address: kalkura@yahoo.com (S.N. Kalkura).
0022-0248/$ - see front matter r 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcrysgro.2003.11.057
cations and is used for repairing bone defects in
dental and orthopaedic sites, immediate tooth
replacement, augmentation of alveolar ridges, pulp
capping material and maxillo facial reconstruc-
tion, etc [5]. Various techniques have been
developed for the synthesis of HAP due to its
growing importance and applications [6,7]. Main
techniques being used are solid-state reaction [8],
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518 T.A. Kuriakose et al. / Journal of Crystal Growth 263 (2004) 517–523
co-precipitation [9,10], and hydrothermal method
[11] and sol–gel route [12–16]. Sol–gel route has
more advantages than the others such as high
product purity, homogeneous composition and
comparatively low synthesis temperature. Reports
till date on the sol–gel derived HAP indicate the
synthesis of HAP is always accompanied by
secondary phase of calcium oxide [13–16]. In
certain literatures on synthesis of HAP by sol–gel
process, phosphorous alkoxide was used as a
precursor for phosphorous [17–27]. Liu et al. [28]
used tri ethyl phosphate and calcium nitrate as the
precursors respectively for P and Ca for HAP
synthesis. In our earlier reports gels were used to
synthesize HAP at low temperature [16,29]. It is
reported that HAP transforms into other phases
during heating up to 1200
C [30,31]. We could
also synthesize pure HAP crystals by a novel sol–
gel technique using agars and these were comple-
tely transformed into a-calcium pyrophosphate at
1200
C [16]. Report is available on the prepara-
tion of carbonated HAP of 8–10 nm sizes, forming
into aggregates [32]. Sarig and Kahana highlighted
the importance and advantages of nano crystalline
HAP and they could synthesize HAP with 300 nm
edges, which were loosely aggregated into spher-
ulites of 2–4 mm dimensions [33]. However, often
these sintering techniques produce calcium
phosphates that are weak and composed of
many other phases and further they have the
disadvantage of requiring high temperatures to
synthesize pure HAP. In the present investigation,
instead of phosphorous alkoxides and gels,
ethanol was used as a solvent under alkaline
conditions to obtain stoichiometric, nanocrystal-
line HAP. Here we report the synthesis of
pure, stoichiometric, nano crystalline HAP with-
out any other additional phase, which is stable
even at 1200
C.
2. Materials and methods
0.5 M Ca(NO3)24H2O (Merck) in ethanol with a
pH 10.5 was added to 0.5 M (NH4)2PO4 (Merck)
slowly at a rate of 5 ml/min. and constant
temperature (85
C) with vigorous stirring. The
experimental procedure was as shown in the flow
diagram given below.
Flow sheet
Ca(NO3)2.4H2O
+Ethanol (pH =10.5)
(NH4)2HPO4
Vigorous stirring and heating
+Ethanol.(pH=10.5)
Sol-Gel( pH = 10)
Heating at 85°C
Sintering
Powder at 400°C
(2 Hrs)
Sintering
Powder at 750°C
(2 Hrs)
Sintering
Powder at 1200°C
(2 Hrs)
The resultant sol–gel was continuously stirred at a
constant pH of 10 (pH was kept constant by
adding Ca(OH)2 solution) and at a constant
temperature of 85
C for 4 h. After allowing the
product to cool, it was kept inside the oven at
40
C overnight. The product was sintered for 2 h
at 400
C, again at 750
C and 1200
C as shown in
the flow sheet.
The stoichiometric ratio of the samples was
determined by chemical analysis. The samples were
further characterized by FT-IR spectroscopy in the
range of 400–4000 cm1 using a Nicolet, avatar
360 (ESP) FTIR Spectrophotometer using KBr
pellet technique. The XRD patterns were recorded
using Siemens D5005 Diffractometer (Siemens,
Germany) with CuKa radiation (l ¼ 0:154 nm).
Scanning electron micrographs were obtained with
Scanning Electron Microscope LEO STEREO-
SCAN 440. Thermo gravimetric analysis (TGA)
coupled with differential thermal analysis (DTA)
of the material was performed between 35
C and
750
C in a METTLER TA 4000 System in air at a
heating rate of 20
C per minute.
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T.A. Kuriakose et al. / Journal of Crystal Growth 263 (2004) 517–523
The Dynamic Light Scattering analysis (DLS)
was performed using a Spectroscatter 201 (RiNA
GmbH, Berlin). A diode laser of wavelength
680 nm was used as the source. The scattered light
was collected at a fixed angle of 90
by a lens
system and directed through a glass fibre to a
photo-multiplier. It’s output pulses are processed
by the auto-correlator. The resulting auto correla-
tion functions were analysed with the program
CONTIN to obtain radius distributions. For the
DLS analysis, the HAP was prepared by disper-
sing small amounts of the synthesized powder in
ethanol followed by immersion in an ultrasonic
bath for 10 min [34]. Subsequently, these samples
were diluted again using ethanol and further
sonicated for 3 min. A series of measurements
with sampling time 30 s and wait time 1 s was
carried out.
3. Results and discussion
3.1. Determination of Ca/P ratio
The stoichiometric (Ca/P) ratios of the as-
synthesised sample and the samples sintered at
different temperatures were determined. The
amount of Ca was estimated using EDTA titration
method and P by UV-Spectrophotometer (UV-
1601, Shimadzu) using ascorbic acid [35]. All these
Ca/P ratios coincide with the theoretical value
(1.67) of HAP.
3.2. X-ray diffraction patterns (XRD)
The XRD patterns of the as-prepared material
at 40
C and its sintered products at 400
C, 750
C
and 1200
C are shown in Figs. 1a–d, respectively.
The patterns due to the as-prepared material bears
with it the characteristic patterns of HAP but not
with much resolution and intensity. It contains no
other crystalline phase other than HAP. The broad
patterns around at (0 0 2) and (2 1 1) indicate that
the crystallites are very tiny in nature with much
atomic oscillations [36]. Existence of such crystal-
lites was also noticed by van der Houwen et al. [37]
during the organic ligands-assisted growth of
calcium phosphate. Here the mother liquor is
519
Fig. 1. XRD patterns of the material (a) as-synthesized at
40
C, (b) sintered at 400
C, (c) sintered at 750
C, and (d)
sintered at 1200
C.
expected to permit selective growth of multitudes
of HAP crystallites with high rate thus avoiding
orderly growth of large crystals.
The XRD patterns of the sintered material at
400
C (Fig. 1b) show increase in intensity due to
planes around (0 0 2), (2 1 0), (2 1 1), (3 1 0), (2 2 2),
(2 1 3) and (4 1 1). Again it rules out the formation
of any new crystalline phase other than HAP. As
the patterns appear still broad, the crystals are
expected to be still tiny, but bigger than the parent
crystallites. Here also there must be significant
atomic oscillations. As the growth of many planes
is evident, there must be orderly transport of mass
to the central growing crystal from the neighbour-
ing ones. In order to verify this, heat-assisted mass
transport sintering was also done at 750
C and
1200
C. In correspondence with the view, there is
gradual increase in the intensity with enhanced
resolution was observed for all the diffraction
patterns obtained.
Again at the sintering temperature of 1200
C,
new crystalline phases are not seen. This precludes
even amorphous phases, as the patterns appear
very well resolved. As the increase in intensity is
noticed for every pattern with increase in sintering
temperature, the growth of crystal need not be due
to particle coalescence at the planes of maximum
density. In our previous studies we reported
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520 T.A. Kuriakose et al. / Journal of Crystal Growth 263 (2004) 517–523
transformation of HAP into calcium pyropho-
sphate at 1200
C [16]. It was suggested to be due
to CaO. The decomposition of HAP partly into tri
calcium phosphate was also reported by Raynaud
et al. [30]. This decomposition into tri calcium
phosphate was also not observed in the present
study when the material was sintered at 750
C.
Zhou et al. reported transformation of HAP into
oxyapatite between 1200
C and 1400
C [31], but
at 1200
C no such transformation is observed in
the present study.
3.3. Infrared spectroscopy
The FTIR spectrum of the material prepared at
85
C is shown in Fig. 2a. There is a broad
envelope between 3800 and 2600 cm1 due to the
O–H stretch of water and HAP. The O–H groups
are hydrogen bonded. The sharp peak at
Fig. 2. FTIR Spectra of the material at (a) as-synthesized at
40
C, (b) sintered at 400
C, (c) sintered at 750
C, and (d)
sintered at 1200
C.
3566 cm1 is assigned to unhydrogen bonded free
O–H stretch which may be present at the surface of
the crystallites. The peak at 1636 cm1 is assigned
to bending mode of water. The absorption band at
1382 cm1 is attributed to the presence of carbo-
nate and/or hydrogen phosphate in the sample
[37]. The intense broad peak between 900 and
1100 cm1 is assigned to PO3 4 . The stretching and
the bending modes of PO3 4 appeared at 602 and
562 cm1 as intense sharp peaks. van der Houwen
et al. reported identification of HPO2 4 based on its
water absorption bands at 1210 cm1 together
with one at 1130 cm1 (shoulder to the absorption
band at 1099 cm1) and a clear absorption band at
879 cm1. But these features are not clearly evident
in the spectrum, as the absorption band due to
PO34 of HAP is intense and broad. But the
distinct shoulder at 868 cm1 indicates the pre-
sence of HPO2 4 in the structures. van der Houwen
et al. reported dissolving of atmospheric CO2
yielding CO2 3 based on the peaks at 1456 and
873 cm1 as the crystallization was carried out in
alkaline range (pH=10.5) [37]. Since the present
experiment was also done in the alkaline range
similar process should also occur. But in the IR
spectrum, there is no sharp peak at 1456 and
873 cm1. But a close observation of the spectrum
shows a shoulder at about 1456 cm1 in the
spectrum of the as-prepared material. But this
peak is somewhat more evident in the spectra of
sintered materials. Since the decomposition of
CaCO3 is reported to occur at 580
C [32],
carbonate stretching should be absent in the
spectra of the materials taken at 750 and
1200
C. But even in these spectra the peak is
evident. It might be due to chemisorption of
atmospheric CO2 on the surface of HAP. But there
are no separate patterns for CaCO3 in XRD
spectra of the sintered materials at 750 and
1200
C. Hence it could be expected to be present
in traces in the sample. Elliot et al. reported
absorption of non-structural carbonate at the
surface of HAP at 1465, 1412 and 873 cm1 [38].
In the IR spectra of the materials sintered at 400
and 750
C (Figs. 2b and c), the first two peaks are
partly evident although the last is common HPO2 4
vibration. The peaks at 624 and 3566 cm1 are
assigned to OH group of HAP. For the sintered
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sample at 1200
C (Fig. 2d), the peaks due to CO23
(892 and 1406 cm1) are very evident and it might
be due to the absorption of atmospheric CO2 on
the surface of HAP. The peaks at 630 and
3570 cm1 are the characteristic peaks for stoichio-
metric HAP [34]. These are present in the samples
sintered at all the temperatures, confirming the
XRD results for pure stoichiometric HAP.
3.4. Thermogravimetric analysis
The thermogravimetric analysis of the as-pre-
pared material was carried out between 35
C and
700
C in air at a heating rate of 20
C/min. The
thermogram and its differential thermogravimetric
trace are shown in Fig. 3. There is a minute weight
Fig. 3. TGA/DTG of the as-synthesized sample.
521
loss (7%) around 130
C. It is assigned to weakly
entrapped water in the material. It is followed by
major weight loss (50%) between 200
C and
350
C. It is assigned to the dehydration of calcium
hydroxide [16]. The decomposition trace does not
bear any inflection, and this process is also not
accompanied by any other additional decomposi-
tion. Hence there might be only one phase
involved in the decomposition. There is a minute
weight loss between 450 and 550
C. It is assigned
to decomposition of calcium hydroxide and
carbonate to water and carbon dioxide [32].
3.5. Scanning electron microscopy (SEM)
The SEM picture of the sintered material at
750
C is shown in Fig. 4. There are many spherical
agglomerations and crystallites of submicrometric
(0.9 mm) in size with a tendency to agglomerate
leaving submicrometric pores in between. To test
this the material was sintered at 1200
C and
subjected to SEM analysis. The SEM picture is
shown in Fig. 5. There are dense smooth plates
with distributed pores in them. The formations of
pores are beneficial, as they would permit the
circulation of the physiological fluid throughout
the coatings when it is used as biomaterial.
Fig. 4. Scanning electron micrograph of the sample at 750
C.
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522 T.A. Kuriakose et al. / Journal of Crystal Growth 263 (2004) 517–523
Fig. 5. Scanning electron micrograph of the sample at 1200
C.
3.6. Dynamic light scattering analysis
The investigation of particle size distribution
of the as-synthesized HAP by DLS technique
revealed a non-uniform distribution of grains
(Table 1). There are grains of 183, 213, 512, 797,
936, 1822 and 2246 nm radius together with grains
of nanosize 1.3 nm. The formation and retention
of nanoparticles together with large grains are
interesting observations made in this study; as
such observation has not been reported in the
literature to the best of our knowledge. Their
importance lies in the fact that the nano particles
improve the contact reaction and the stability at
the artificial natural bone interfaces. Enhanced
osteoclast like cell functions on nanophase cera-
mics was also reported by Webster et al. [39].
At 750
C temperature, the nanophase of as-
synthesized HAP remained deagglomerated, while
the particles of 183, 213 and 512 nm agglomerated.
Particles of 1822 nm were desized to 1077 nm,
where as the size of 2246 nm was presumed to be
increased to 2987 nm.
A very narrow distribution of grain size is
evident due to agglomeration of nano phase and
deagglomeration of bulk phase obtained by
sintering of the previous materials (sintered at
750
C) at 1200
C. Increase in the local tempera-
Table 1
DLS analysis of HAP
Sample Temperature Radius (nm)
(
C)
85 1.3, 183, 213, 512, 797, 936,
1822, 2246
HAP prepared 750 1.3, 467, 552, 741, 917, 1077,
with ethanol 2987
1200 50 (predominant), 127,175
ture at the collision points are the likely key
contributing factors in triggering the solid state
reactions in a nano meter scale. Similarly increase
in the thermal energy might be the driving force
for deagglomeration of bulk phase E468 nm
(obtained at 750
C) to 50–175 nm ranges with
predominant 50 nm-sized particles. Transmission
electron microscopic (TEM) studies [40,41] prove
that bone HAP crystals are having a length of
around 50 nm. Presence of large amount of 50 nm
size particles at this temperature is an important
observation in this study.
4. Conclusions
This study presents an alternative method to
form pure, stoichiometric, stable, crystalline HAP
phase at lower temperature than the other existing
methods in literature. This method provides the
synthesis of pure, porous stoichiometric HAP at
85
C at alkaline pH via an alcohol based sol–gel
process. The presence of alcohol seems to provide
a thermally stable HAP. The pores in the crystal
planes itself will help the material to attain more
biocompatibility and enables the circulation of
physiological fluids. This method of synthesis also
produced nanocrystalline HAP in the range of
1.3 nm in radius, which would make them ideal
bone replacement material. By this process, we can
prepare pure, stoichiometric, nanocrystalline
HAP, needed for various biomaterial applications.
Acknowledgements
One of the authors, T.K. Anee gratefully
acknowledges University Grants Commission,
 ARTICLE IN PRESS
T.A. Kuriakose et al. / Journal of Crystal Growth 263 (2004) 517–523
India, for the award of Teacher Fellowship under
FIP. This study was also supported by a research
grant from the University Grants Commission,
India and by a grant from the RiNA GmbH,
Berlin and the DeutscheForschungsgemeinschaft
(DFG) Germany, under contract BE1443/9-1.
The authors wish to thank Mr. A. Vinu,
Department of Chemistry, Anna University, India
for his help in the analysis.
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