Synthesis and characterization of

eggshell-derived hydroxyapatite via

mechanochemical method: A comparative
study
Cite as: AIP Conference Proceedings 1835, 020045 (2017); https://doi.org/10.1063/1.4981867
Published Online: 26 April 2017

A. A. Hamidi, M. N. Salimi and A. H. M. Yusoff

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AIP Conference Proceedings 1835, 020045 (2017); https://doi.org/10.1063/1.4981867 1835, 020045

© 2017 Author(s).
 Synthesis and Characterization of Eggshell-Derived
Hydroxyapatite via Mechanochemical Method: A
Comparative Study
AA Hamidi1,a), MN Salimi1,b) and AHM Yusoff1,c)
1
School of Bioprocess Engineering, Universiti Malaysia perlis (UniMAP), 02600 Jejawi, Perlis, Malaysia.
a)
Corresponding author: asadulhaq.ali@gmail.com
b)
nabil@unimap@unimap.edu.my
c)
huzaifah.usm@gmail.com

Abstract. The focus of bone graft properties has developed through generations, from the ability to withstand
mechanical stress to the ability to integrate with the biological structure.In recent years, the use of
hydroxyapatite (HA) as bone graft material in orthopedic and dental applications has been increasing. HA is
a natural occuring mineral with excellent bioactivity but relatively poor mechanical properties. It constitutes
96% portion of enamel in teeth and 67% portion of bone. HA can be extracted from animal bones or
fabricated from synthetic or biologic sources. In this study, eggshells were used as raw material to synthesize
eggshell-derived HA (EHA) via mechanochemical method. The synthesis of EHA involved CaO, which was
obtained from the calcination of eggshells, and reaction with dicalcium hydrogen phosphate dihydrous
(DCPD) or phosphoric acid (H3PO4). The effects of rotational speed and heat treatment temperature on
EHA’s characteristics were investigated. The characterization studies were carried out by using the Fourier
Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD) analysis and Scanning Electron
Microscopy (SEM). HA powder was successfully synthesized with crystallite and particle sizes in the range
of 8–47 nm and 250–550 nm respectively. It was observed from this study that the increase of milling
rotational speed had increased the phase purity of EHA samples. Furthermore, the higher heating temperature
of HA samples resulted in higher degree of crystallinity of HA and the appearance of β-tricalcium phosphate
(β-TCP) as secondary phase.

INTRODUCTION
In the past few decades, researches on engineered implants in biomedical applications have shown promising
development. Implant is defined as material inserted (grafted) into an organ or structure of the body [1]. The implant
material is also often referred as biomaterial. Generally, biomaterials have a wide range of applications, and one of
the most important applications of biomaterials in medical field is to repair bone defects. In the USA alone, it is
estimated that 600,000 bone grafts are performed annually, making it the second most implanted material in the
human body after blood transfusion [2].

Bone grafts can be classified in different manner. According to the implant source, they are divided into (i)
autograft: bone graft harvested from different site within the same individual; (ii) allograft: bone tissue harvested
from different individuals of the same species; and (iii) various biomaterial substitutes [3,4].Furthermore,
biomaterial substitutes for bone replacement have four major classes: (i) metal (titanium, magnesium alloy, stainless
steels); (ii) ceramic and glass (alumina, calcium phosphates, bioactive glass); (iii) polymer (polylactides, collagen,
chitosan); and (iv) the combination of the aforementioned classes called composite (carbon-reinforced
hydroxyapatite (HA), HA-coated metals) [5]. Among them, HA, a calcium phosphate mineral, is the most prominent
bioceramic used in recent years due to its ability to bond strongly to bond tissue at their surface [6,7].

Advanced Materials Engineering and Technology V

AIP Conf. Proc. 1835, 020045-1–020045-12; doi: 10.1063/1.4981867
Published by AIP Publishing. 978-0-7354-1505-8/$30.00

020045-1
 Not only as bone graft substitute, HA has been used in a variety of practices such as metal coating, drug delivery
system and protein purification. Various methods and routes to synthesize HA are developed depending on the
purpose of the application. For bone graft application, the most frequently cited methods in the literature are wet
precipitation, sol-gel, hydrothermal and mechanochemical methods [8]. Each method has its characteristic
parameters that can control the physical structure and properties of the yield, such as the shape and crystallinity.

With the emerging of biorenewable resources usage as raw materials, HA synthesized from natural-based
materials has also been studied. HA precursors can be extracted from abundantly available waste of biological
reservoirs such as mammalian and fish bones, eggshells, sea shells and even from corals as well as plants [9].
Eggshell-derived HA (EHA) has been successfully fabricated to form nanocrystalline HA powder which is highly
biocompatible to bone. The bone-like properties of EHA is generally due to eggshell’s composition which mainly
consists of calcium carbonate (94%), as well as calcium phosphate (1%), magnesium carbonate (1%), organic
substances (4%) and trace ions such as Na+, Mg2+ and Sr2+ [10,11].

In 2013, roughly 10,441 tonnes of eggs were produced daily in Southeast Asia only[12]. Eggshells, which make
up 11% weight of eggs [11], are basically useless after the use of eggs and are usually discarded. With 1,148.51
tonnes of eggshells thrown away daily from various industries, eggshells become cheap, promising precursors for
the production of HA as bioceramic bone replacement.

METHODOLOGY
Firstly, hen eggshells collected from cookery waste were rinsed with water and dried in an oven at 60 °C for 3
hours. Afterwards, the dried eggshells were crushed into powder using a grinder. The ground eggshell were sieved
with a 325-mesh sieve to make uniform powder. The powder was calcined at 900 °C for 3 hours at a heating rate of
5 °C/min to decompose CaCO3 into CaO as well as the organic matters contained in the eggshell powder.

Afterwards, the calcined eggshell was milled using a planetary ball mill (Fritsch, Germany) with dicalcium
phosphate dihydrous (CaHPO4.2H2O, DCPD, R&M Chemicals, Malaysia) or phosphoric acid (H 3PO4, Bendosen,
Malaysia) to undergo mechanochemical activation as carried out by Adzila et al [13].

4 CaO + 6 CaHPO4.2H2O Ca10(PO4)6(OH)2 + 14 H2O (1)

10 CaO + 6 H3PO4 Ca10(PO4)6(OH)2 + 8 H2O (2)

The mechanochemical activation process was performed in a quartz-medium jar using alumina balls (20 mm) for
15 hours, with powder-to-ball mass ratio of 1:5. To begin with, the two reactions were carried out at 200 rpm. The
HA samples produced were analyzed by XRD to observe which reaction yields more amount of HA. Afterwards, the
preferred reaction was carried out in higher rotational speed (400 and 600 rpm) to observe its effects on the
characteristics of HA powder.

The milled powders were heat treated in different temperatures at 800 °C and 1100 °C [14] to observe the effects
of heating temperature. The heating processes were done in box furnace (Carbolite, UK) with maintained heating
and cooling rates of 5 °C/min.

In order to distinguish the unique characteristics of the synthesized EHA samples, a commercial HA (CHA,
Sigma-Aldrich, Malaysia) sample was also analyzed as a comparison and standard reference. The details of
processing parameters of the samples are shown in Table 2.1.

The FTIR (PerkinElmer, USA) was used to identify the functional groups of the HA samples. The FTIR spectra
were observed in the range of 4000-400 cm-1 region with 4 cm-1 spectral resolution using KBr pellets. The phase
composition and crystallite size of the synthesized EHA powders were analyzed by X-ray powder diffraction (XRD;
LabX XRD-6000, Shimadzu, Japan) with monochromatic Cu-Kα radiation (λ = 1.5406 Å). The XRD patterns were
analyzed by “PANalytical X’Pert Highscore” software and compared to the standard references compiled by the
Joint Committee on Powder Diffraction Standards (JCPDS). The reference cards used are PDF #24-0033 (HA),

020045-2
#002-0789 (β-TCP), #70-2065 (α-TCP), #09-0080 (CaHPO4), #01-0837 (CaCO3) and #04-0733 (Ca(OH)2). The
crystallite size was estimated by using Scherrer equation:

‫ߣܭ =ݐ‬/‫ܤ‬. ܿ‫ߠݏ݋‬ (3)

where ‫ ݐ‬is the average crystallite size (nm); ‫ ܭ‬is the shape factor (‫ = ܭ‬0.9); ߣ is the wavelength of the X-rays (ߣ =
1.5406 Å for Cu Kα radiation); ‫ ܤ‬is the full width at half maximum (radian); and ߠ is the Bragg’s diffraction angle
(°) [15].The particle size and morphology of the EHA particles was observed using scanning electron microscopy
(SEM; JSM 6460LA, JEOL, Japan) operating with accelerating voltage of 15 kV. The powder samples were coated
(Fine Coater 1600, JEOL, Japan) prior to SEM analysis. The size of particles observed in the SEM micrograph was
measured by using “ImageJ” software.

TABLE 1. Details of processing parameters of each sample.

Rotational speed Heat
Samples Reaction
(rpm) treatment (°C)
EHA1 (1) 200 -
EHA1_800 (1) 200 800
EHA1_1100 (1) 200 1100
EHA2 (1) 400 -
EHA2_800 (1) 400 800
EHA2_1100 (1) 400 1100
EHA3 (1) 600 -
EHA3_800 (1) 600 800
EHA3_1100 (1) 600 1100
EHA4 (2) 200 -
EHA4_800 (2) 200 800
EHA4_1100 (2) 200 1100
CHA - - -

RESULTS AND DISCUSSION

Fourier Transform Infrared Analysis
The FTIR absorption spectra of the synthesized HA powders before and after heat treatment were obtained.
Figure 3.1 shows the FTIR spectra of the four samples after undergoing mechanochemical activation in a planetary
ball mill with different rotational speed. A wide absorption band corresponding to the IR adsorption of H 2O [16,17]
was observed between 3600 and 2600 cm-1 in sample EHA1, with an explicit peak at 3435 cm-1. Similar water
adsorptions were also observed in samples EHA2 and EHA3 at 3436 and 3435 cm-1 respectively. In contrast, sample
EHA4 showed lower water adsorption intensity at 3448 cm -1 which indicates that the sample contained less water
molecule. Furthermore, a much higher intensity peak at 3642 cm-1 was observed in sample EHA4, which
corresponds to the stretching of OH- group. Peak of OH- group of different mode showed up in EHA1, EHA2 and
EHA3 but and in very weak intensity at 3566, 3567 and 3569 cm-1 respectively. These absorption bands, which is
also known as OH- characteristic peak of HA [16], appeared as shoulders as they overlaped with water adsorption
bands. However, over the three samples, EHA3 showed the most significant stretching peak at 3569 cm -1, followed
by samples EHA2 and EHA1.Although there are many characteristic peaks of HA, the presence of OH- stretching
band in the samples strongly indicates that HA is present in the sample. The presence of significantly broad water
adsorption band in EHA1, EHA2 and EHA3 also proves that reaction (1) produces more water molecules compared
to reaction (2).

Other characteristic peaks of HA were also observed in all the four samples. In HA, phosphate groups exist in
asymmetrical stretching vibration modes, namely v1, v2, v3 and v4[18]. In sample EHA1, vibrating peaks of v3–PO43-,
v4–PO43- and v2–PO43- were significantly observed at 1040, 602 and 566 and 471 cm-1respectively. While v1

020045-3
vibration band was observed in a weak stretching peak at 963 cm-1. The same peaks with higher intensity can also be
observed in samples EHA2 and EHA3. The higher intensity of HA characteristic peaks in samples EHA2 and EHA3
proves that the higher rotational speed forms chemically more complete HA groups.

FIGURE 1. FTIR spectra of samples (a) EHA1, (b) EHA2, (c) EHA3 and (d) EHA4.

The EHA samples showed to contain substitutional group of CO32- between 1460-1420 cm-1 which could be due
to either the trace presence of CaCO3 from eggshell powder or the presence of CO2 as a result of calcination. CO32-
substitutional group of HA has been classified into type A and type B apatites, depending on the functional group
being substituted: CO32- for OH- (type A); and CO32- for PO43- (type B). It has been reported that the type B apatite
has better bioactivity and is better for bone replacement due to its similarity to biological apatites in human bone
[19]. All the four samples exhibited the presence of CO32- groups in the region of which the type B apatite is
characterized. Therefore, it can be inferred that all the synthesized EHA samples before heat treatment were non-
stoichiometric HA and more similar to the structure of biological apatites.

After heating at 1100 °C in a furnace, all the four HA samples were again characterized by FTIR. The
corresponding FTIR absorption spectra of heat treated samples are shown in Figure 3.2. Compared to the absorption
spectra of the samples before heat treatment, spectra in Figure 3.2 were rather well defined since several absorption
peaks had disappeared. The most significant absorption bands to disappear were water adsorption bands which were
previously most distinguished between 3600 to 2600 cm-1 and 1700 to 1600 cm-1 in Figure 3.1. The heat treatment
had evidently removed the water molecules in previous samples. As a result, OH- libration peaks around 3570 cm-1
became more explicit.

Generally, heat treated EHA samples made by reaction (1) had chemically purer HA phase as can be seen in
Figure 3.2(a), 3.2(b) and 3.2(c). All characteristic functional groups of HA, namely OH- and PO43- groups, were

020045-4
observed explicitly. In addition, substitutional groups such as CO 32- had disappeared due to heat treatment. This
highly indicates that the heat treatment of powders made by reaction (1) up to 1100 °C results in stoichiometric HA.

FIGURE 2. FTIR spectra of samples (a) EHA1_1100, (b) EHA2_1100, (c) EHA3_1100 and (d) EHA4_1100.

Phase Determination
XRD analysis was necessary as a complementary of the findings from FTIR analysis. It is used to determine
whether the product is single-phase or multi-phase. The phase composition of sample EHA1 before and after heat
treatment was illustrated in Figure 3.3. It can be seen from Figure 3.3(a) that the milling of reaction (1) at 200 rpm
produced HA along with CaHPO4 and Ca(OH)2 phases. The presence of CaHPO4 could be due to uncomplete
reaction of the initial powder, which is also supported by the presence of Ca(OH) 2 as intermediate phase. This
suggests that the HA produced was calcium defficient (Ca/P ratio < 1.67) [18].

After heat treatment at 800 °C, the crystallinity of sample EHA1 increased significantly, which is indicated by
the sharpening of the HA characteristic peaks in sample EHA1_800. CaHPO 4 phase was still observed with lesser
peaks, while Ca(OH)2 completely disappeared. As a consequence, peaks corresponding to β-TCP and α-TCP phases
started to appear. Further heating until 1100 °C resulted in higher intensity of both HA and β-TCP peaks as can be
seen in Figure 3.3(c). This indicates that heating as high as 1100 °C effectively produced highly crystalline HA
powder with β-TCP as secondary phase. Out of the three samples above, EHA1_1100 would be the most fitted HA
product for the use of bone replacement, since it would provide osteoconductivity as well as bioresorbability due to
the presence of HA and β-TCP respectively.

In contrast to samples EHA1, milling of reaction (1) at 600 rpm resulted in a single-phase HA product with
relatively higher degree of crystallinity as can be seen in Figure 3.4(a). This could be due to the heat generated
during the high speed milling that heated up the HA phase formed to some extent. In other words, partial heat
treatment also occurred during the milling process, and the higher rotational speed the higher heat would be

020045-5
generated. Accordingly, heat treatment to 800 °C and 1100 °C also yielded better crystallized products. However, in
Figure 3.4(c) there was a peak at 20.44° that was suggested to belong to CaHPO4 which could be due to the remain
of initial powder.

FIGURE 3. XRD profile of samples (a) EHA1, (b) EHA1_800 and (c) EHA1_1100.

FIGURE 4. XRD profile of samples (a) EHA3, (b) EHA3_800 and (c) EHA3_1100.

020045-6
 Figure 3.5 shows the XRD profile of samples after milling of reaction (2) at 200 rpm. As opposed to the previous
XRD profiles, HA phase was not observed after milling as can be seen in Figure 3.5(a). Many peaks were assigned
to CaHPO4 instead, which is the main product of reaction (2). Peaks in accordance to Ca(OH) 2 and CaCO3 patterns
were also observed, which could be the products of other reactions that occur. The reactions that took place during
the milling process of reaction (2) could be suggested as written below.
CaO + H3PO4 CaHPO4 + H2O (4)

2 CaO + H3PO4 CaHPO4 + Ca(OH)2 (5)

CaO + CO2 CaCO3 (6)

HA phase was only observed after the powder was heated. β-TCP phase also appeared along with CaHPO4 in
trace amount. The presence of CaHPO4 might be due to the excess amount of CaHPO4 that did not get to react. This
agrees to the suggested reactions that might have occurred as written below.
6 CaHPO4 + 4 Ca(OH)2 Ca10(PO4)6(OH)2 + 6 H2O (7)

2 CaHPO4 + Ca(OH)2 Ca3(PO4)2 + 2 H2O (8)

As a comparison, the XRD profile of commercial HA product was shown in Figure 3.6. The figure shows a clear
single-phase HA pattern. This pattern was almost the same as the pattern of sample EHA3 (Figure 3.4(a)), only that
sample CHA had slightly lower intensity.

FIGURE 5. XRD profile of samples (a) EHA4, (b) EHA4_800 and (c) EHA4_1100.

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 FIGURE 6. XRD profile of sample CHA.

Crystallite Size
The crystallinity of the powder samples was analyzed by using Scherrer equation and X’Pert Highscore Plus
software. The average crystallite size of the HA phase was calculated and summarized in Figure 3.7. It is necessary
to note that the HA crystallite size in EHA4 was set to 0 nm because there was no sign of HA phase found based on
the phase determination of the XRD profile.

According to Aoki [20] and Seckler et al. [21], HA with high degree of crystallinity or high crystallite size
showed lower or no activity towards bone resorption and is insoluble in physiological environment. For the
application of bone replacement, therefore, synthesized HA with low degree of crystallinity has been sought by
researchers. Based on Figure 3.7, the lowest degree of HA crystallinity was found to be in sample EHA1 with
crystallite size of 8.14 nm. However, according to the phase determination by XRD profile, sample EHA1 only had
few amounts of HA with many impurities of CaHPO4 and Ca(OH)2, which would not be suitable for the
physiological environment. Even after heat treatment at 800 °C, sample EHA1 showed low crystallite size of 19.00
nm but still contained trace amounts of CaHPO4 and α-TCP. After being heated at 1100 °C, BPC was formed with
relatively high HA crystallite size of 34.89 nm. Even though the crystallite size significantly increased, the presence
of β-TCP has been known acknowledged to support the bioresorption ability, thus can be good for bone replacement
application as well.

FIGURE 7. The effect of heat treatment and milling rotational speed on the crystallite size of HA.

020045-8
 Morphology Study
Figures 3.8(a) and 3.8(b) show the SEM micrographs of sample EHA1’s morphology. It can be observed from
the figure that the as synthesized powder was in irregular shape. Furthermore, the particles tended to form
aggregates which leads to non-uniform size of particles. This indicates that the ball milling process did not succeed
in crushing the powder uniformly. It could be due to the reaction that occurred during the milling process that
prevented the complete size reduction process. When the balls collided, the particles of the initial powders formed a
new molecule that could be bigger than the previous ones. The particle size difference could also be due to the phase
difference, since CaHPO4 and Ca(OH)2 phases were observed in the XRD profile.

Heating of sample EHA1 resulted in more well shaped particles as shown by Figures 3.8(c) and 3.8(d).
Significant morphological difference can be observed compared to Figure 3.8(a). The HA particles formed spherical
morphology with irregular form. It also consisted of small and large particles that form clusters. The mean particle
size was calculated to be 263 nm.Further heating to 1100 °C resulted in significantly bigger size of HA particles.
Particles of 513 nm mean particle size were observed in Figure 3.8(d). The particle size of sample EHA1_1100 was
almost two times of sample EHA1_800. This agrees to other works that reported heating of HA powder promoted
the particles to agglomerate and form bigger particles due to the increase of degree of crystallinity [22,23].

FIGURE 8. SEM micrographs of samples (a) EHA1 in 5000x magnification, (b) EHA1 in 10,000x magnification, (c) EHA1_800
and (d) EHA1_1100.

As synthesized sample EHA2 was found in agglomerated condition on the wall of milling jar with little amount
of powder at the bottom of the jar and on the surface of the milling balls. The agglomerated HA was crushed again
by using mortar and pestle to obtain smaller grain size. As a result, non-uniform EHA2 powder was obtained as
shown in Figure 3.9(a). Large agglomerates were the majority particles with fine particles spreading on their surface.
The particles were measured by excluding significantly large agglomerates. It was calculated that the mean particles
were in the size of 257 nm, bigger than sample EHA1. As opposed to the report by Zahrani & Fathi [24] and Adzila
et al. [13], increasing the rotational speed turned out to result in bigger particle size.

The agglomerated HA product on the jar wall was suggested to be caused by the reaction that occurred between
the initial powders. It was evident that reaction (1) produced by-product of water in relatively high amount. When
water was produced, the product became slurry and spread all over the jar wall. Afterwards, the heat generated
during the milling process dried up the water and the slurry was hardened. In order to overcome this, the milling
parameters should be evaluated. The choice of milling balls and jar media; increasing ball to powder ratio; or
extending the milling time so that the water molecule could be completely dried up by the heat, are possible

020045-9
alternatives. However, the milling pause interval should also be considered to avoid excessive heat due to the long
milling process [18]. Otherwise, secondary milling after drying the milled powder to some extent could be
employed.

FIGURE 9. SEM micrographs of samples (a) EHA2, (b) EHA2_800 and (c) EHA2_1100.

As a comparison, CHA powder was physically rather moist and sticky compared to the synthesized powders. It
tended to stick to other particle’s surface as can be seen in Figure 3.10, which shows highly agglomerated powder of
sample CHA. Even so, fine grains in irregular shape could also be observed. The measurement of particle size
included only the agglomerates as it was not possible to measure the fine grain without higher magnification. The
calculated mean particle size was 534 nm.

FIGURE 10. SEM micrograph of sample CHA.

CONCLUSION

HA samples based on eggshell precursor were successfully synthesized. Furthermore, the effect of milling
rotational speed and heating temperature on the mechanochemical synthesis of EHA was investigated, involving two
reactions of HA synthesis as a comparison. Based on the results, it was evidenced that reaction (1) yielded more
amount of HA compared to reaction (2), hence reaction (1) is preferred. It was also indicated that increasing the
milling rotational speed resulted in more stable, purer and higher degree of crystallinity of the HA phase. However,
as the rotational speed increased, HA tended to agglomerate as a result of heat generated in the milling process.
Consequently, HA particles formed after heat treatment at 800 °C were irregular in shape and stacked in submicron-
sized cluster of particles. After further heating at 1100 °C, the agglomerate’s surface became smoother and the
stand-alone particles were observed to be in spherical shape. Measurement of mean particle size for reaction (1)

020045-10
showed that the synthesized EHA samples were in the range of 250–550 nm. Compared to CHA sample which
owned 534 nm of mean particle size, EHA samples were considered to be in acceptable size for bone replacement
application.XRD analysis showed that HA phase was formed in all samples except sample EHA4. Furthermore,
heating EHA samples produced secondary phase of β-TCP. In terms of crystallite size, EHA samples were found in
relatively low size (8–47 nm). As has been reported, heating HA powder resulted in significantly higher degree of
crystallinity. Likewise, increasing the rotational speed also increased the degree of crystallinity but with relatively
lower increment.In addition, the FTIR study indicated that EHA synthesized by reaction (1) was non-stoichiometric
HA with carbonate substitute of type B. However, after heating at 1100 °C the carbonate group disappeared, with
peaks of HA characteristics only observed.

RECOMMENDATION

In order to synthesize smaller size of HA particles, the milling parameters should be evaluated. The choice of
milling media such as type of balls and media of milling jar should be considered to avoid the formation of
agglomerates during the reaction. Ball to powder mass ratio was also reported to have an effect on mechanochemical
synthesis of HA, hence should also be considered. Furthermore, morphology study of the EHA samples can be
improved by using FE-SEM or TEM to increase the magnification and observe the particle structure more
comprehensively. In vitro and in vivo studies should also be done to investigate the performance of EHA as bone
replacement.

ACKNOWLEDGEMENT

The authors would like to thank Universiti Malaysia Perlis (UniMAP) and Ministry of Higher Education
Malaysia (KPT) for financial support.

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