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Ingeniería y Competitividad, Volumen 18, No. 1, p. 69 - 71 (2016)
INGENIERÍA DE MATERIALES
Synthesis and chemical and structural characterization of
hydroxyapatite obtained from eggshell and tricalcium phosphate
MATERIALS ENGINEERING
Síntesis y caracterización química y estructural de hidroxiapatita
sintetizada a partir de cáscara de huevo y fosfato tricálcico
Alejandro Arboleda*, Manuel Franco*, Julio Caicedo**,*, Liliana
Tirado***, Clara Goyes*§
*Universidad Autónoma de Occidente. Cali, Colombia.
**Facultad de Ingeniería Universidad del Valle, Cali-Colombia.
***Universidad del Quindío. Armenia, Colombia.
alejandro.arboleda@outlook.com, mfranco9009@hotmail.com, jcaicedoangulo1@gmail.com,
litirado@uniquindio.edu.co, §cegoyes@uao.edu.co
(Recibido: Abril 24 de 2015 – Aceptado: Agosto 20 de 2015)
Resumen
La cáscara de huevo es un residuo común que generalmente se desecha sin darle uso alguno. En este trabajo
se presenta una metodología experimental que usa la cáscara de huevo para obtener un biocerámico muy
conocido en el campo de la ingeniería de los materiales y la biomédica, llamado hidroxiapatita (HA). La
HA es un fosfato de calcio, su fórmula química es Ca10(PO4)6(OH)2 y tiene características físico-químicas
muy similares a la del hueso humano, lo cual la convierte en uno de los biomateriales más usados como
injertos o sustitutos para reparación ósea. Este material generalmente tiene un alto costo y se presenta en
forma micro y nanoestructurada, ta última, la opción en nanotecnología más promisoria para la ingeniería
de tejidos. La caracterización en este estudio incluye difracción de rayos X, en donde los difragtogramas
obtenidos permiten la identicación de hidroxiapatita, con fase cúbica fcc (111), (102), (211), y la fase
hexagonal hcp (h-211) y (h-322). Por otro lado se presentan resultados de espectroscopia infrarroja con
transformada de Fourier, en donde se determinaron los modos activos de vibración correspondientes a la
hidroxiapatita. Finalmente, mediante microscopia electrónica de barrido se observó la topografía del polvo
cerámico así como también la distribución morfológica obtenida.
Palabras Clave: Biocerámicas, cáscara de huevo, hidroxiapatita, regeneración ósea.
Abstract
The eggshell is a common residue that is usually discarded without giving any use to it. In this paper the
results obtained from a proposed procedure to get hydroxyapatite (HA) from eggshell are shown. The HA
is a calcium phosphate which has been widely used as implant material due to the close similarity of its
composition with the inorganic phase of natural bone. HA generally has a high cost and it is presented
as micro and nanostructured bioceramics; the last one is a promising option for tissue engineering
nanotechnology. In this study, results of X-ray diffraction (XRD) showed the hydroxyapatite production
exhibiting the characteristic peaks of this material for the cubic phase fcc (111), (102), (211), and for the
hexagonal phase hcp (h-211) and (h-322). From the results of Fourier transform infrared spectroscopy
(FTIR), it was possible to determine the active modes of vibration corresponding to hydroxyapatite (Ca10
(PO4)6(OH)2). From the results of scanning electron microscopy, it was determined the topography of the
ceramic powder as well as its morphological distribution.
Keywords: Bioceramics, bone regeneration, eggshell, hydroxyapatite.
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Ingeniería y Competitividad, Volumen 18, No. 1, p. 69 - 71 (2016)
1. Introduction
The eggshell is considered as residue and it is made
up of calcium carbonate (94%), calcium phosphate
(1%), magnesium carbonate (1%), and organic
matter (4%) (Stadelman, 2000). The eggshell is
basically unused after the production of the egg and
its derivates. In the literature, there are few reports
on the use of this residue and the companies that
generate it, considering it as an industrial residue
that might contribute to pollution, since it favors
the microbial proliferation in the environment
(Rivera et al., 1999). As an important source of
calcium carbonate, the eggshell has been used
as raw material in different elds of materials
engineering, particularly as biosensor and in
medicine (Balaz, 2014). In the biomedical eld,
there are some reports showing that the eggshell
has been used in the manufacture of bioceramics
to repair bone injuries (Rivera et al., 1999; Lee &
Oh, 2003). In Colombia, this residue is discarded
and it is not common to nd scientic alternatives
that promote its reuse; this is why this study is
aimed at exploring an alternative of use for the
manufacturing of a type of bioceramics, with the
purpose of contributing to the biomedical eld, in
case its production is optimized. In order to have
an idea of the yearly production of eggshell in
Colombia, it is estimated in 11’529.250 units in
2014, being Valle (Departments of Cauca, Nariño,
and Valle del Cauca) the second most productive
region at national level, with an estimate of
2’972’388 units in 2014 (FENAVI, 2014). At
national level, 691,756 tons were produced
(FENAVI, 2014).
The material that will be produced, known as
hydroxyapatite (HA), has a structure composed
of calcium, phosphorus, and hydroxyl ions
(Ca10(PO4)6(OH)2). Its biological importance
is due to its capacity of allowing a perfect
osseointegration, the absence of local and systemic
toxicity, and the null genotoxic activity (Ginebra
et al., 2006; Franco et al., 2014). HA associates
with the surrounding bone in a tight and stable
way, both chemically and physically, allowing the
repairing processes develop as if they were two
bone tissues in close contact (Ginebra et al., 2006).
Recent publications have demonstrated that HA
at nanostructured scale increases signicantly the
biocompatibility and bioactivity of the man-made
biodevices (Sadat-Shojai et al., 2013). Therefore,
this study includes the comparison of the product
obtained with commercial nano-hydroxyapatite,
with the purpose of nding similar characteristics
that lead to its optimization. One of the reported
sources to synthesize HA is the eggshell (Rivera
et al., 1999; Lee & Oh, 2003), which comprises
processes that take place in a high temperature
environment and using phosphate solutions
(Rivera et al., 1999; Balázsi et al., 2007; Lee &
Oh, 2003). In these reports, the obtaining of HA
has been done from precursors, either tricalcium
phosphate, Ca3 (PO4)2 (Rivera et al., 1999) or
phosphoric acid, H3PO4 (Balázsi et al., 2007; Lee
& Oh, 2003). In the case of tricalcium phosphate
(TCP), it has been reported for producing HA in
an already established relation of Ca/P (Rivera et
al., 1999) but it is recommended to optimize the
composition of the solutions used, the time, and
the temperature of annealing (Rivera et al., 1999).
This study reports an experimental methodology
for the synthesis of hydroxyapatite from eggshell
taking into consideration a variation in the content
of CaO and tricalcium phosphate Ca3(PO4)2, in
order to nd the parameters that generate a high
content of crystalline hydroxyapatite.
2. Methodology
The process for manufacturing hydroxyapatite
(HA) starts with an eggshell mechanic milling
stage to get a white powder. A rst stage of heating
eggshells at 450°C for two hours is carried out
in order for the organic residue to be destroyed
and CaCO3 to be obtained (Rivera et al., 1999).
In this study, the particle grain size was taken into
consideration; therefore, a sieving was done using
different lter sizes starting from an average grain
size of 54 µm up to 74 µm. Afterwards, a heat
treatment of calcium carbonate powder CaCO3
was done during two hours at 900°C, using a Type
30400 Thermolyne furnace in order to ensure its
transformation into CaO through the release of
carbon dioxide (CO2), as presented in Ec. 1.
CaCO3 → CO2 + CaO (1)
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Ingeniería y Competitividad, Volumen 18, No. 1, p. 69 - 71 (2016)
Then, HA is obtained using the precursors tricalcium
phosphate (Ca3(PO4)2, calcium oxide CaO and
water (H2O), as described in Ec. 2.
3Ca3(PO4)2+CaO+H2O→Ca10 (PO4)6(OH)2
(2)
In this work, an experimental study for
manufacturing HA was carried out; in it, particle
size, temperature ramp, and concentration of
precursors are varied, as shown in Table 1.
Figure 1 shows a ow chart that summarizes the
experimental methodology used in this study.
Sample No. Sieve
size
Ramp of
temperature
[CaO] [Ca3(PO4)2]
Final temperatures of
thermal treatment
Timing
M1 54 µm 10 °C/min 95 % 5 % 1060 °C 7 h
M2 54 µm 10 °C/min 85% 15 % 1060 °C 7 h
M3 54 µm 10 °C/min 75% 25% 1060 °C 7 h
M4 54 µm 10 °C/min 50% 50% 1060 °C 7 h
M5 74 µm 15 °C/min 95 % 5 % 1060 °C 7 h
M6 74 µm 15 °C/min 85% 15 % 1060 °C 7 h
M7 74 µm 15 °C/min 75% 25% 1060 °C 7 h
M8 74 µm 15 °C/min 50% 50% 1060 °C 7 h
M9 54 µm 10 °C/min 100% - 900 °C 5 h
M10 74 µm 15 °C/min 100% - 900 °C 5 h
M11 Commercial HA
In order to determine the crystalline structure
of the hydroxyapatite, a Bruker D8 Advance
diffractometer in the coupled mode θ-2θ was used.
The analysis of vibrational mode of the material
were determined through spectroscopy in the
infrared with Fourier transform (FTIR) through a
Shimadzu 8000 spectrometer (550 – 1000 cm-1) in
transmittance mode, which uses a ceramic source
Nerst type. The spectra of FTIR and diffractograms
of X-ray, for all the cases are compared with
commercial nano-hydroxyapatite samples,
known as FLUIDINOVA - nanoXIM-HAp202.
The morphology of the samples was observed
Figure 1. Summary of the process used
to obtain hydroxyapatite from eggshell.
Tabla 1. Experimental design for obtaining the samples.
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Ingeniería y Competitividad, Volumen 18, No. 1, p. 69 - 71 (2016)
through a Scanning Electron Microscope
(SEM) FEI QUANTA 200, equipped with an
optical light that has a magnifying range of
525-24,000 X and a high sensibility detector
(multi-mode) for dispersion electrons.
3. Results and discussion
3.1 Analysis of infrared spectroscopy
In the spectra obtained through FTIR, numerous
transmission bands in the region of near and
middle infrared can be observed; however, the
most relevant are associated with the vibrations
corresponding to P-O, Ca-O y O-H. Figure 2a
shows the FTIR spectra of the M1 and M4 samples,
corresponding to the hydroxyapatite obtained
with different percentages of CaO, with particle
size 54 µm, heating rate 10ºC/min, at a sintering
temperature 1060 ºC and 7 hours; for comparison
purposes, the spectrum of the commercial sample
is included. When we analyze Figure 2a, an active
vibration mode is found around 571 cm-1 and 601
cm-1 for bonds O–P–O of HA exion type (v4),
which are moderately visible for spectra of the
samples of HA obtained from eggshell. On the
other hand, it is visible when compared to the
commercial sample, a transmittance band around
1415 cm-1 regarding to C-O of CO32- stretching
type (Siriphannon et al., 2002). The spectrum
of the commercial sample presents a band in
3570 cm-1 corresponding to the groups O-H of
symmetric stretching according to the reports
by Siriphannon and Delgado (Siriphannon et al.,
2002; Delgado et al., 1996). This band is also
moderately evident for the samples manufactured
for this study. The vibration centered in the
band of 3570 cm-1 corresponds to O-H bonds of
stretching type (Pramanik et al., 2005). Figure 2b
shows the band of absorbance around 1080 cm-1
of the FTIR spectrum in the sample of commercial
nano-hydroxyapatite. These results allow
determining the contribution corresponding to the
symmetrical stretching type P-O bonds of HA (v3)
(Siriphannon et al., 2002) and to the secondary
phase vibrations such as O-Ca-O, Ca-O, P-O and
Ca-O-P, which characterize hydrated calcium
phosphates and synthesized hydroxyapatite with
the stoichiometric relation of Ca10 (PO4)6(OH)2.
Figure 2. Spectra of FTIR (the graphs have been displaced in the transmittance scale to improve their
comprehension): (a) Hydroxyapatite obtained with different percentages of CaO, with particle size
of 54 µm, heating ramp of 10°C/min, at a sintering temperature of 1060°C and 7 hours (M1-M4),
and commercial nano-hydroxyapatite. (b) Deconvolution of the absorbance spectrum FTIR with high
resolution in the region between 800 cm-1 and 1400 cm-1.
(a) (b)
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Ingeniería y Competitividad, Volumen 18, No. 1, p. 69 - 71 (2016)
Figure 3 shows the spectra of samples M5 to M8
corresponding to the hydroxyapatite obtained
with different percentages of CaO, with
particle size of 74 µm, heating ramp of 15°C/
min, under a sintering temperature of 1060°C
and 7 hours. Additionally, it is presented a
spectrum of commercial nano-hydroxyapatite
in which the band around 1080 cm-1 associated
to the transition corresponding to the O-P-O
bonds HA exion type (v4) (Siriphannon et al.,
2002) are mainly identied. Analyzing Figure
3, it is very clear the presence of an active
mode of vibration around 571 cm-1 and 601
cm-1 corresponding to the O-P-O bonds of HA
exion type (v4) (Siriphannon et al., 2002).
The vibration centered in the band of 3670 cm-1
corresponds to the OH- bonds of stretching type
(Pramanik et al., 2005).
Figure 3. Spectra of FTIR (the graphs have been
displaced in the transmittance scale to improve their
comprehension): Hydroxyapatite obtained with
different percentages of CaO, with particle size of
74 µm, heating ramp of 15°C/min, under a sintering
temperature of 1060°C and 7 hours (M5-M8),
and commercial nano-hydroxyapatite.
Figure 4 shows the spectra of samples M9
and M10 corresponding to the hydroxyapatite
obtained with different heating ramps of 10°C/
min and 15°C/min, a fixed percentage of CaO
100%, with particle size of 54 µm and 74 µm,
under a sintering temperature of 900°C and 5
hours; additionally, it is presented a spectrum
of commercial nano-hydroxyapatite in which
the band around 1080 cm-1 associated to the
transition corresponding to the O-P-O bonds HA
exion type (v3) already described (Siriphannon
et al., 2002) is presented. Figure 4 shows the
vibration centered in the band of 3670 cm-1
corresponding to the O-H bonds of stretching
type (Pramanik et al., 2005). A transition band
around 1415cm-1 which corresponds to the C-O
stretching type of CO32- (Pramanik et al., 2005)
present in the sample is shown as well.
Figure 4. FTIR spectra for the hydroxyapatite
obtained with different heating ramps of (10°C/min),
a xed percentage of CaO 100%, with particle size
of 54 µm and 74 µm, under a sintering temperature
of 900°C and 5 hours (M9, M10) (the graphs have
been displaced in the transmittance scale to improve
its understanding), and spectrum of the sample of
commercial nano-hydroxyapatite.
On the other hand, in Figure 4 it is evident a
signicant reduction in the signal associated
to the bands around 1080 cm-1 compared to the
previous spectra. The gure shows an active
mode of vibration around 571 cm-1 and 601 cm-1
for the O–P–O exion type bonds of HA (v4), as
well as a transition band around 1415 cm-1 related
to the symmetrical stretching type C-O of CO32-
(Siriphannon et al., 2002). The vibration centered
in the band of 3670 cm-1 corresponds to the bonds
O-H- of stretching type (Delgado et al., 1996).
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Ingeniería y Competitividad, Volumen 18, No. 1, p. 69 - 71 (2016)
3.2 X ray diffraction
A characterization through X ray diffraction
for the commercial HA (Ca10(PO4)6(OH)2) was
conducted to determine its structural nature. It is
compared to the X-ray diffractograms obtained
for the samples synthesized from solid residues
of eggshells. International patterns of indexation
taken from the JCPDF 01-074-0565 database
obtained from the “Inorganic crystal structure
database” (ICSD) were considered for the X-ray
diffraction analysis. Figure 5 shows the X-ray
diffractograms obtained for the samples that are
between M1 and M4 with different percentages
of CaO, with particle size of 54 µm, heating ramp
of 10°C/min, under a sintering temperature of
1060°C and 7 hours of treatment.
15 30 45 60 75 90
Si-331
420
422
511
HA-101
HA-110
HA-002
HA-211
HA-202
HA-220
HA-222
HA-321
HA-104
HA-210
HA-502
HA-520
HA-323
HA-006
HA-130
Intensity (u. a.)
2θ
ο
Commercial
CaO 95%
CaO 85%
CaO 75%
CaO 50%
54 µm
10 °C/min
2θ (o)
Figure 5. X-ray diffraction pattern for the hydroxyapatite
obtained with different percentages of CaO, with particle
size of 54 µm, heating ramp of 10°C/min, under a
sintering temperature of 1060°C and 7 hours (M1 –
M4). The diffraction pattern of the commercial nano-
hydroxyapatite sample is included.
Figure 6 shows the X-ray diffractograms obtained
for the samples that are between M5 and M8 with
different percentages of CaO, with particle size
of 74 µm, heating ramp of 15°C/min, under a
sintering temperature of 1060°C and 7 hours of
treatment. It can be analyzed from Figures 5 and 6
that the main peaks characteristic of the hexagonal
Hydroxyapatite with spatial group P63/m-176
appeared. The characteristic phases with the
high intensity crystallographic planes (110),
(210), (202), (220), (222), (321) y (104),
among others, are observed. It is also seen that
while the percentage of CaO decreases for both
sample groups, different crystallographic planes
appeared, which could imply defunctionalization
of the conjugated complexes conforming the
hydroxyapatite. For the particular case of CaO
50% the diffractograms of both samples (M4 and
M8) show variations in position and intensity
from the diffraction maximum values, suggesting
the emergence of secondary phases.
15 30 45 60 75 90
Si-331
420
422
511
HA-101
HA-110
HA-002
HA-211
HA-202
HA-220
HA-222
HA-321
HA-104
HA-210
HA-502
HA-520
HA-323
HA-006
HA-130
Intensity (u. a.)
2θ
ο
Commercial
CaO 95%
CaO 85%
CaO 75%
CaO 50%
74 µm
15 °C/min
2θ (o)
Figure 6. X-ray diffraction pattern for the Hydro-
xyapatite obtained with different percentages of CaO,
with particle size of 74 µm, heating ramp of 15°C/min,
under a sintering temperature of 1060°C and 7 hours
M5 – M8). The diffraction pattern of the commercial
nano-hydroxyapatite sample is included.
3.3 Scanning electron microscopy
Figure 7 shows two micrographs obtained by
scanning electron microscopy (SEM) for a sample
of hydroxyapatite (Ca10(PO4)6(OH)2) synthesized
from solid residues of eggshells. Sample M1
presents morphology of irregular grains forming
agglomerates. The images show that the particles
present an irregular shape, with size close to 10
μm, forming agglomerate until forming groups
of 50 μm. These agglomerates are due to the
porogenic agent in the manufacturing
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Ingeniería y Competitividad, Volumen 18, No. 1, p. 69 - 71 (2016)
process of the samples, which is strongly related
to the molecular formation registered in the
vibrational modes of the HA, analyzed in the
FTIR results (Figs. 2 – 4) and the corresponding
present phases observed by the results of the
X-ray diffraction (Figs. 5 and 6). Additionally,
the particle surface in the synthesized HA has
high roughness. This result is signicant since
the particle shape and its geometry can be
determinant in the nal response at the moment of
the biomaterial implanting.
Figure 7. SEM micrography for ceramic
powder of HA obtained from eggshell.
4. Conclusions
The results of the X-ray diffraction conrm the
synthesis of hydroxyapatite (HA) obtained from
eggshell, due to the presence of constituent
phases of the (HA). For the M4 and M8 samples a
percentage equal to 50% of the CaO and Ca3(PO4)2
components is taken into account. The results
presented show the diffractions corresponding
to the crystallographic planes of the cubic and
hexagonal structures, forming a mixture of phases
of the Ca, PO4 y (OH)2 components, with small
quantities of beta-tricalcium phosphate. Active
modes of vibration corresponding to the CO3
bonds and vibrational bands associated to PO4
were also determined.
5. Acknowledgements
This work has been developed thanks to the support
of the seedbed of Research in Advanced Materials
for Micro and Nanotechnology (IMAMNT) of
the Universidad Autonoma de Occidente, to the
technological support of the Interdisciplinary
Institute of Sciences (IIC) of the Universidad del
Quindio, and to the Center of Excellence in New
Materials (CENM) of the Universidad del Valle.
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