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Received: 9 May 2022 Revised: 24 July 2022 Accepted: 20 August 2022 Micro & Nano Letters
DOI: 10.1049/mna2.12143
LETTER
Microstructural analysis of biowaste-derived
hydroxyapatite-chitosan nanocomposites
Sambridhi Shah1Rajendra Joshi1Nelson Rai2Rameshwar Adhikari2,3
Rajesh Pandit1
1Department of Chemistry, Tri-Chandra Multiple
Campus, Tribhuvan University, Ghantaghar,
Kathmandu, Nepal
2Central Department of Chemistry, Tribhuvan
University, Kirtipur, Kathmandu, Nepal
3Research Centre for Applied Science and
Technology (RECAST), Tribhuvan University,
Kirtipur, Kathmandu, Nepal
Correspondence
Rajesh Pandit, Department of Chemistry,
Tri-Chandra Multiple Campus, Tribhuvan University,
Ghantaghar, Kathmandu 44600, Nepal.
Email: panditrajesh02@gmail.com
Abstract
In recent years, considerable attention is given in various nanomaterials development
using biodegradable wastes for establishing more eco-friendly technologies. Due to the
biocompatible, biodegradable, and non-toxic nature hydroxyapatite (HAp) and chitosan
(CS) are highly studied in recent times. In this research work, HAp and CS were syn-
thesized in a nanometric range using the bio-wastes obtained from chicken bones and
pila shells, respectively. In addition, HAp/CS nanocomposites were also prepared through
the co-precipitation method in various weight ratios. The synthesized nanomaterials and
nanocomposites were characterized by X-ray diffraction (XRD) and Fourier Transform
Infrared (FTIR) spectroscopy. The XRD pattern analysis revealed the hexagonal crystalline
structure of HAp and orthorhombic crystallite structure of CS with crystallite sizes of 40.52
and 39.51 nm, respectively. The physical parameters such as d-spacing, dislocation density,
stacking fault probability, and lattice strain of HAp and CS were also analyzed. The FTIR
spectra analysis confirmed the formation of HAp and CS. Likewise, the broadening and
weakening of the chemical bond between two phases of HAp and CS indicated the bond
formation and compatibility between HAp and CS.
1 INTRODUCTION
Nanomaterials have been employed in various fields such
as medicine, agriculture, cosmetics, wastewater management
etc., as a prominent solution to many long-existing prob-
lems due to their tailored properties, such as large surface
area-to-volume ratio and high surface area, low density [1].
Due to its versatile features, hydroxyapatite (HAp), an inor-
ganic bioceramic also known as calcium phosphate ceramic
[Ca10(PO4)6(OH)2], is gaining increasing attention. Due to its
biocompatibility, biodegradability, and osteoconductive charac-
teristics, HAp is commonly used in biomedical applications
[2, 3]. HAp has also been used in wastewater treatment,
chromatography, and electrodeposition activities [3]. Likewise,
chitosan (CS), a deacetylated product of chitin, is widely used
in wastewater treatment, membrane separation, food packaging,
biotechnology, and medicine, including as a potential medicine
against hypertension [4].
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is
properly cited.
© 2022 The Authors. Micro & Nano Letters published by John Wiley & Sons Ltd on behalf of The Institution of Engineering and Technology.
To enhance the mechanical properties (e.g. strength, elas-
ticity, and toughness) of HAp, recent studies have attempted
to form its nanocomposite with polymers [3, 5, 6]. Among
the various biopolymers (e.g. dextran, CS, alginate, gelatin, car-
boxymethyl cellulose) [5], CS is known to form an excellent
composite with HAp due to their reconcilable properties such as
biocompatibility, biodegradability [5], chelation, and adsorption
properties [6]. Several methods for synthesizing HAp and CS
such as the green method [7–10] and chemical method [11–14]
have been reported in the literature. The majority of these
studies are focused on the influence of synthesis routes and
precursors on the biological and physicochemical properties of
HAp and CS, such as thermal stability [15, 16], and solubility
[17, 18], crystallinity [19, 20], and hardness [21, 22]. However,
microstructural parameters that provide a better understanding
of the observed difference in biological and physicochemi-
cal properties of HAp and CS are poorly understood. Thus,
this research focuses on synthesizing HAp, CS, and HAp/CS
Micro Nano Lett. 2022;1–8. wileyonlinelibrary.com/iet-mnl 1
2SHAH ET AL.
nanocomposites and studying their microstructural parameters
such as lattice strain, dislocation density, and stacking fault
probability.
2MATERIALS AND METHODS
2.1 Materials
The chicken bones and pila shells were collected from the
local markets in Kathmandu, Nepal. Chemicals such as Sodium
hydroxide (97%), Sulfuric acid (98%), Hydrochloric acid (36%),
and bleaching powder (24%) were purchased from local sup-
pliers of HiMedia Laboratories Pvt Ltd (Mumbai, India) in
Kathmandu and were used without further modification.
2.2 Methods
2.2.1 Synthesis of hydroxyapatite (HAp) from
chicken bones
Muscles and unwanted substances such as tissues and bone
marrow on the bones were cleaned using tap water and boiled
for 1.5 h for defatting and deproteinizing [23, 24]. The bones
were then dried, ground using a rotary mill, and sieved using a
450-μm sieve. Finally, the obtained powder was calcined at two
different temperatures, first at 600◦C for 3 h and then at 900◦C
for 3 h to ensure removal of the organic constituents and the
carbonate apatite hence obtain a white powder of HAp [25, 26].
2.2.2 Extraction of chitosan (CS) from pila
shells
The pila shells were washed using distilled water, dried, ground
using a rotary mill, and sieved at 200 μm. Then, chitin was
isolated from shell powder by wet chemical processes via dem-
ineralization (1 M HCl), deproteinization (2.5 M NaOH), and
decolourization (2% bleaching powder). Finally, CS powder was
isolated from the chitin by deacetylation (12.5 M NaOH) [27].
2.2.3 Preparation of HAp/CS nanocomposites
The HAp/CS nanocomposites of various weight ratios, that
is, 20/80, 40/60, and 60/40 were synthesized by the co-
precipitation method. For the synthesis of 20/80 HAp/CS
composite, 8% CS was dispersed in a 1% aqueous solution of
acetic acid with continuous stirring until fully dissolved. Then,
2% HAp was added to the mixture and stirred vigorously with
a magnetic stirrer. The HAp and CS precipitated out gradu-
ally at the maintained temperature of 40◦C. After the mixing
process was completed, 1 M NaOH was added to the mixture,
drop wisely, to increase its pH to 12, for complete precipitation.
After 24 h, the composite was washed with distilled water, fil-
tered off, and dried in an air oven at 150◦C. A similar procedure
was followed for the preparation of two other nanocomposites
containing 40/60 and 60/40 of HAp/CS [28]. The chemical
reaction between HAp and CS is shown in Figure 1.
It shows that the ─OH group of HAp binds with the ─OH
groups of CS to form an H─bond in the formation of HAp/CS
nanocomposites.
2.3 Characterization techniques
2.3.1 X-ray diffraction analysis
The crystalline size and the structure of the samples were deter-
mined by an X-ray diffractometer (Bruker D2Phaser, USA) with
a monochromatic CuKαradiation source (λ=0.15418 nm) at
angle 2θranging from 10◦to 80◦with the accelerating voltage
of 30 kV and emission current of 10 mA.
Crystallite size
From the X-ray diffractometer (XRD) patterns, the crystalline
size of HAp, and CS were calculated using the Debye-Scherrer
formula [29]
D=k𝜆
𝛽Cos𝜃(1)
where K=shape factor (K=0.94 for most of the spherical
crystals), λ=wavelength of the monochromatic X-ray beam
(λ=1.54060◦A for CuKαradiation), β=Full Width of Half
Maximum (FWHM) of the peak at the maximum intensity (in
radians), θ=peak diffraction angles that satisfy Bragg’s law for
the (hkl) plane and Dis the crystalline size.
d-spacing
The d-spacing is the distance between planes of atoms that gives
diffraction peaks, where each peak in a diffractogram results
from a corresponding d-spacing. The planes of atoms can be
referred to as a 3D coordinate system with a plane direction hkl
and so can be described as per the direction within the crystal
structure. The d-spacing is usually denoted in Ångstroms. The
d-spacing is calculated by using Bragg’s law [30]
2dSin𝜃=n𝜆(2)
where
d=n𝜆
2Sin𝜃(3)
Wavelength (λ)=1.5418 Å for CuKα
Dislocation density (𝛿)
A dislocation is a crystallographic defect or irregularity inside
the crystal construction that can have a powerful effect and can
alter many properties of materials. The dislocation density is the
number of dislocations in a unit volume of a crystalline material
and is measured by the length of the dislocation lines per unit
SHAH ET AL.3
FIGURE 1 Schematic representation of
hydrogen bonding involved in the reaction between
HAp and chitosan compounds [28]
volume of the crystal. The values for 𝛿HAp and CS were calcu-
lated by using the following relationship with the crystallite size
[31]:
Dislocation density (𝛿)=1
D2(4)
where D=crystallite size of HAp and CS
Stacking fault probability (α)
In crystallography, the stacking fault probability (α) is a type
of planar defect which defines the disorder of crystallographic
planes, and one fault is expected to be found in 1/αlayers. Peak
positions of different reflections can alter from the ideal posi-
tion due to the presence of stacking faults. The stacking fault
probability of HAp and CS was calculated by using the following
relation [32]:
𝛼=
2𝜋2
453Δ(2𝜃)
Ta n 𝜃(5)
where Δ(2θ)=Calculated two theta – Reference two theta
Estimation of lattice strain (𝜀) and crystalline size (D) using
Williamson–Hall Method
The internal stress can be calculated by using the Uni-
form Deformation Model (UDM) as suggested by using
the Williamson–Hall equation. The lattice strain induced in
nanomaterials due to crystal imperfection and distortion was
calculated by the W–H equation [33]
𝜀= 𝛽hkl
4Ta n 𝜃(6)
Equations (1)and(6) show the dependence of strain and size
on θvalues. Now the total dependence can be expressed in the
form of Williamson–Hall equation as shown below.
𝛽hkl =k𝜆
DCos 𝜃+4𝜀Ta n 𝜃
On rearranging we get
𝛽hkl Cos𝜃=
k𝜆
D+4𝜀sin𝜃(7)
2.3.2 Fourier transform infrared (FTIR)
spectroscopy
FTIR spectroscopy analysis was carried out to identify func-
tional groups and vibration modes associated with each peak
by using the IR Prestige-21 FTIR Spectrometer (SHIMADZU,
Japan) where spectra were collected in the spectral range of 4000
to 400 cm−1using the KBr pellet method.
3RESULTS AND DISCUSSION
3.1 Structural analysis using X-ray
diffractometer (XRD)
3.1.1 Peak indexing of HAp, CS, and their
nanocomposites
The peak indexing is the most important step in diffraction pat-
tern analysis where the peaks of samples were assigned to the
diffraction angle 2θranging from 20◦to 80◦corresponding to
their diffraction planes. Figure 2a shows the diffraction pattern
of HAp, CS, and their nanocomposites.
The peaks for HAp at 2θvalues of 26.1◦, 28.2◦, 32.0◦,
33.2◦, 40.1◦, 46.9◦, and 48.5◦were indexed with correspond-
ing diffraction plane (002), (012), (211), (300), (310), (222),
and (320), respectively, having hexagonal space group found in
4SHAH ET AL.
FIGURE 2 (a) XRD pattern of synthesized HAp, CS, and HAp/CS nanocomposites of various HAp/CS weight ratios as indicated (20/80, 40/60, and 60/40).
(b) Schematic hexagonal molecular geometry of HAp where blue ball represents Ca and purple ball represent P. (c) Schematic diagram showing the orthorhombic
molecular geometry of the CS where the red, pink, purple, and brown ball represents O, H, C, and N, respectively. (d) Plot of 4sinθv/s βcosθofHAp.(e)Plotof
4sinθversus βcosθof CS. CS, chitosan; XRD, X-ray diffraction.
standard reference data (JCPDS 96-901-3628). Further, the unit
cell values for HAp were calculated to be a=b=9.77 A◦and
c=6.22 A◦. Using the values of these unit cells, the crystal struc-
ture of HAp can be drawn using Visualization for Electronic
and Structural Analysis (VESTA) 3.5.3 version [34], as shown in
Figure 2b.
Similarly, peaks of CS at 2θvalues of 26.69◦, 27.68◦,
29.87◦, 31.59◦, 33.6◦, 36.58◦, and 38.36◦were assigned with
corresponding diffraction planes (013), (202), (123), (241),
(152), (104), and (124), respectively, found in standard reference
(JCPDS 00-039-1894). The XRD analysis indicates the synthe-
sized CS shows an orthorhombic crystal structure and the unit
shell values were calculated as a=8.06 A◦,b=17.09 A◦,and
c=10.01 A◦. Using the values of these unit cells, the crystal
structure of CS can be drawn using VESTA 3.5.3 version [34],
as shown in Figure 2c.
SHAH ET AL.5
TAB L E 1 Calculations of crystalline size, d-spacing, dislocation density, and stacking fault probabilityfor hydroxyapatite (HAp) using X-ray diffractograms
Hkl 2θFWHM
Crystalline
size (nm)
d-spacing
(nm)
Dislocation
density (10−3
line/nm2)
Stacking fault
probability (α)
(002) 26.1 0.21 40.36 0.017 0.61 0.24
(012) 28.2 0.29 29.51 0.019 10.08 0.074
(211) 32.0 0.22 40.10 0.021 10.20 0.234
(300) 33.2 0.19 44.71 0.022 9.88 0.219
(310) 40.1 0.17 51.60 0.026 10.07 0.117
(222) 46.9 0.16 55.12 0.030 9.70 0.098
(320) 48.5 0.41 22.25 0.032 9.64 0.071
TAB L E 2 Calculation of crystalline size, d-spacing, dislocation density, and stacking fault probability for chitosan (CS) using X-ray diffractograms
Hkl 2θFWHM
Crystalline
size (nm)
d-spacing
(nm)
Dislocation
density (10−3
line/nm2)
Stacking fault
probability (α)
(013) 26.7 0.2 42.6 0.33 0.55 0.53
(202) 27.7 0.25 34.2 0.32 0.85 0.27
(123) 29.9 0.3 28.6 0.29 1.22 0.09
(241) 31.6 0.14 61.6 0.28 0.26 0.31
(152) 33.6 0.22 39.4 0.27 0.64 0.28
(104) 36.6 0.26 33.6 0.25 0.88 0.18
(124) 38.4 0.24 36.6 0.23 0.75 0.31
In Figure 2a, the XRD pattern of HAp and CS shows a peak
at 26.1◦and 26.62◦, respectively, and however, in the XRD pat-
tern of HAp/CS, it can be seen that these peaks shifted to 26.4◦
which indicates the composite formation of HAp and CS. The
broadening and weakening of characteristic peaks of HAp and
CS after composite formation indicate a bond between them.
The peak of CS was dominant over the HAp in nanocomposite
(20/80) due to the high amount of CS powder [26]. How-
ever, as the HAp content increases in the nanocomposites, the
crystallinity increases in the nanocomposites, and peak inten-
sity shifts towards HAp as shown in the XRD diffractogram of
nanocomposites (40/60 and 60/40) [28, 29, 35].
3.1.2 Crystalline size
By using Equation (1), the average crystalline size of HAp, and
CS were found to be 40.52 and 39.51 nm, respectively. The data
obtained from the calculation of crystallite sizes of HAp and CS
is shown in Tables 1and 2, respectively.
3.1.3 d-spacing
The averaged spacing calculated for HAp and CS was found
to be 2.6 Å and 2.8 Å, respectively by using Equation (3). The
data obtained from the calculation of d-spacing are shown in
Tables 1and 2, respectively.
3.1.4 Dislocation density
The average dislocation density for HAp and CS was calculated
to be 8 ×10−4nm−2and 7 ×10−4nm−2respectively using
Equation (4). The values of dislocation density for both HAp
and CS are attributed to the temperature. The data obtained
from the calculation of dislocation density of HAp and CS are
shown in Tables 1and 2, respectively.
3.1.5 Stacking fault probability (α)
By using Equation (5), the average stacking fault probability
of HAp and CS was calculated to be 0.15 and 0.28, respec-
tively. The data obtained from the calculation of stacking fault
probability of HAp and CS are shown in Tables 1and 2,
respectively.
3.1.6 Estimation of lattice strain (”) and
crystalline size (D) using Williamson–Hall method
A linear plot of HAp and CS is drawn by taking 4sinθalong the
x-axis and βcosθalong the y-axis using Equation (6)asshown
6SHAH ET AL.
FIGURE 3 FTIR spectra of HAp, CS, and their
nanocomposite of various HAp/CS weight ratios as indicated
(20/80, 40/60, and 60/40)
in Figures 2d and 2e, respectively. In the W–H model, the lattice
strain values, and the crystallite size values of HAp and CS are
respectively extracted from the slopes and the intercepts of the
respective linear fits made through experimental data. The esti-
mated values of the lattice strain for HAp and CS were found
to be 0.0008 and 0.0003, respectively. Similarly, the size of HAp
and CS was found to be 49.95 and 41.39 nm, respectively, by this
method. There is a difference in crystallite size calculated from
the Scherrer equation and the Williamson–Hall UDM model.
This difference is because the Williamson-Hall method takes
into account some important factors affecting the peak broad-
ening such as inhomogeneous strain and instrumental effects
[35].
It is acknowledged that the complementary and generally
direct information on the crystallites size and shapes could be
correlated to the materials properties can be obtained by elec-
tron microscopy. This aspect of the work will be followed in the
future research.
3.2 Fourier transform infrared (FTIR)
spectroscopy
The chemical modification was analyzed by using FTIR spec-
troscopy. The FTIR spectra of synthesized HAp, CS, and their
nanocomposite are shown in Figure 3.
The FTIR spectrum of synthesized HAp is shown in Figure 3
where peaks centred at 555.50, 594.08, and 1018.14 cm−1were
recorded. The strong and sharp peak at 1018.41 cm−1is due to
the asymmetrical stretching of the phosphate group, while two
relatively sharp peaks at 555.50 and 594.08 cm−1are due to
the symmetrical stretching of the phosphate group which gives
authenticated information about the vibrational origin of the
phosphate to indicate the production of HAp [25, 36].
The spectrum of synthesized CS assigned the peaks around
1023.13, 1149.57, and 1056.99 cm−1corresponding to the
C─O─C stretching, and strong C─O bond and the peak at
1381.03 cm−1represents the alkane bending vibration of the
C─H group. Similarly, the peak at 1566.19 cm−1is assigned
to the ─NH2group and the peak at 2870.08 cm−1shows
C─H stretching with strong intensity, whereas the peak at
3255.84 cm−1indicates N─H stretching and at 3356.16 cm−1
shows symmetric stretching of vibration of O─H[27, 36–39].
On comparing the FTIR spectra, the peak related to
the ─CH backbone vibrations of CS decreases in inten-
sity as HAp content increases. The peak at 763.81, 856.39,
and 925.83 cm−1indicates the strong deformation of the β-
1,4-glycosidic linkage in the composite which confirms the
hydrogen interactions between HAp and CS and the deforma-
tion of the ether bond in the pyranose ring at 1149.57 and
1365.60 cm−1serves evidence for the chemical interconnection
of the two phases. Similarly, the peaks that referred to stretching
vibrations of hydroxyl groups show a slight shift towards lower
wavenumbers [28, 40].
4 CONCLUSIONS
HAp and CS were isolated successfully from bio-waste, that
is, chicken bones and pila shells, respectively. Furthermore,
the synthesized nanomaterials were mixed to form HAp/CS
nanocomposites via the co-precipitation method. The XRD
spectra show the presence of HAp and CS in a nanometric
range with a hexagonal and orthorhombic crystalline struc-
ture. The broadening and weakening of the XRD spectra of
HAp and CS confirmed the formation of their nanocompos-
ite. The d-spacing, dislocation density, stacking fault probability,
micro-strain, and energy of HAp and CS were calculated from
diffraction patterns. The FTIR spectra of synthesized HAp, CS,
and HAp/CS nanocomposite confirmed their formation.
ACKNOWLEDGEMENTS
The authors are thankful to the Nepal Academy of Science
and Technology (Khumaltar, Lalitpur, Nepal), for XRD analysis
SHAH ET AL.7
and the Department of Plant Resources (Thapathali, Kath-
mandu, Nepal) for FTIR spectroscopic analysis. RA and RP
further thank the University Grants Commission (UGC), Nepal
for supporting the collaborative research project ‘From waste
to biomaterials: preparing hydroxyapatite nanomaterial from
ostrich bone and biomedical application’ (CRG73/74-ST-02).
We are also thankful to Dr. Santosh Thapa (Baylor College of
Medicine, Houston, Texas, USA) for his support in editing the
manuscript.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
FUNDING INFORMATION
Rameshwar Adhikari and Rajesh Pandit thank the University
Grants Commission (UGC) for supporting the collabora-
tive research project ‘From waste to biomaterials: preparing
hydroxyapatite nanomaterial from ostrich bone and biomedical
application’ (CRG73/74-ST-02).
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
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How to cite this article: Shah, S., Joshi, R., Rai, N.,
Adhikari, R., Pandit, R.: Microstructural analysis of
biowaste-derived hydroxyapatite-chitosan
nanocomposites. Micro Nano Lett. 1–8 (2022).
https://doi.org/10.1049/mna2.12143
