effect of the sample preparation on the composition of...

Effect of the sample preparation on the composition of hydroxyapatite

derived from waste anchovy fish bone Hasan Daupor1*, Pateeroh Kuwae1, Anugrah Ricky Wijaya2, Isma-ae Chelong3,

Abd Naser Haji Samoh1 1Chemistry Major, Faculty of Science Technology and Agriculture, Yala Rajabhat University,

Yala 95000, Thailand 2Chemistry Department, Faculty of Mathematics and Natural Science, Universitas Negeri Malang,

State University of Malang, Jln. Semarang No. 5 Malang, Indonesia

3Biology Major, Faculty of Science Technology and Agriculture, Yala Rajabhat University,

Yala 95000, Thailand *E-mail: [email protected]

Abstract:

Calcium hydroxyapatite (HAp) powder was synthesized using waste anchovy fish

bone through maceration process and subsequently heat treatment. The percent yield of

obtaining product was 60%. On XRD analysis, the high purity of HAp phase was formed

after immersing of each HCl and NaOH for 12 h and followed by heat

treatmentat900°Cfor12h, resulting in thetriphasicmaterialofhydroxyapatite [Ca5(PO4)3OH],

magnesium phosphate [Mg2(P2O7)] and whitlockite [Ca5Mg2H2(PO4)14], which contained

weight percentage of 79:11:10 determined by the reference intensity ratio. The HAp powders

had the degree of crystallinity equal to 98% and crystal size equal to 56 mm. The lattice

parameter values of the HAp hexagonal crystal, a and c, were 9.4260 and 6.8845 Å,

respectively. Moreover, the results of the FTIR analysis showed vibrational modes of at

569, 962, 1036, and 1189 cm−1, and the weak peak located at 3571 and 633 cm−1 corresponds

to the vibration of ion in the HAp lattice. Meanwhile, there was also band at 371 cm-1

assigned to the bonding in calcium hydroxyl phosphates. This paper showed the great

potential for the conversion of this by-product into highly valuable compounds.

1. Introduction

Budu or fish sauce southern Thailand

style is a favorite side dish of the Southern

Border Provinces with various cooked forms

such as kneading rice, mixed with

vegetables, peper source and so on,

especially in Saiburi district, Pattani

Province Industrial production. The budu

bottle is sold in the form of One Tumbon

One Product (OTOP) there are two brands:

Budu Heng and Budu Yiseng. Budu is based

on digestion of enzymes and

microorganisms from naturally occurring

fish, which uses small fish such as

Stolephorus indicus, Clupeoides sp., Sardine

sp., Pinialo pingalo or Decapterus russelli.

These fishes are taken without removingit’s

tripe and by fermentation with sea salt in the

ratio of 3:1 for 8-12 months. Fish and bone

are fermentated and produced a liquid of

black color or rather blackcalled budu.

Liquid budu will leave the residue

containing fish bones, fins, and also

contained fish meat. Therefore, it is the

source of calcium, phosphorus, and other

proteins and nutrients which can be used to

prepare hydroxyapatite [Ca5(PO4)3(OH)-

Hap].1 The protein and other nutrients were

decomposed when calcined at high

temperature up to 900 °C resulting in HAp

growth.2

HAp has been synthesized from

various sources such as animal bone. Pulp

and paper tuna bones (Thunnus obesus) were

used to synthesis hydroxyapatite. It was

washed with hot tap water to remove the

remaining fish meat, followed with NaOH,

acetone to remove dirt, proteins, fat and

other organicsubstances. The collagen was

hydrolyzed with 2 M NaOH for 5 h at 250

© The 2018 Pure and Applied Chemistry International Conference (PACCON 2018) MN364

°C and finally burned at 900 °C for 5 h for

completely removing the organic matter

from the bone and furnish Hap.1 Calcium

phosphates are a class of compounds with

very high value due to their properties and

technological applications. Several calcium

phosphates are well known for their use as

biomaterials.3 Hydroxyapatite, the major

component of human bone, is probably the

most important one; it has very high

biocompatibility, and for this reason it is

widely used in many applications, e.g. fillers

for improving the properties of dental

adhesives, drug delivery agents, and

biosensor applications.4

Recently, HAp production from

different sources of bones such as Japanese

sea bream,5 Brazilian river fish6 and Atlantic

swordfish7using a subcritical water process

and alkaline hydrothermal hydrolysis was

reported.8 All possible sources were

successfully employed to obtain HAp using

thermal treatment methods. In this paper, we

report the use of anchovy fish bone collected

from the budu residue to convert into HAp,

an alkaline-hydrolysis process was

employed. The bones were also treated in

solution prior to calcination to change their

composition. This sample, never reported

before for this method of preparation, was

employed to achieve materials.

2. Materials and Methods

2.1 Materials

Anchovy fish bone as a waste was

obtained from the end of the budu

production process from Budu Yiseng

Factory, Amphor Saiburi, Pattani Province,

Thailand. Sodium hydroxide 1 M (BDH),

hydrochloric acid 1 M (BDH). All chemicals

were reagent grades and used without

further purification.

2.2 Synthesis of hydroxyapatite

The anchovy fish bone was collected

from budu Yiseng residue and washed with

water to remove the traces of meat and skin.

After thorough washing the bones were

dried at 60 ºC and ground in a mortar pestle.

To hydrolyze collagen and other organic

moieties, the alkaline hydrolysis method was

followed. Briefly, 20 g of grounded anchovy

bone was treated with 1 M HCl with

continuously stirring for 12 h (solid liquid

ratio 1:2). After that, the treated sample was

strirred in 1 M NaOH for 12 h. The mixture

was filtered in a suction pump with

continuous washing with water and dried in

an oven at 100 ºC. 1 g of the dried fish bone

was placed in a silica crucible and subjected

to a temperature of then calcined in an

electrical muffle furnace at 900 °C for 12 h.

The white powder of HAp was obtained.

2.3 Sample characterization

X-ray Diffraction (XRD) was

applied to monitor the phase composition

features of the samples after calcination at

900 ºC. The sample spectra were collected

using a Philips PW 3710 powder

diffractometer (PHILIPS X'Pert MPD, The

Netherlands), Cu Kα (Ni filtered) radiation

λ=1.5406 Å. Intensity data were collected by

the step counting method in the 2θ range of

= 10-90º. Fourier transform infrared

spectroscopy (FT-IR) was used to

investigate the chemical composition of the

control and treated samples. The FT-IR

spectra were performed using a Spectrum

JASCO 6800 spectrometer in the range of

400-4000 cm-1. The morphological and

microstructure analysis of all samples was

carried out using the instrument Quanta 400

scanning electron microscope (SEM, Quanta

400, FEI). The elemental composition was

analyzed by an energy dispersive

spectrometer (EDS) equipped with the SEM

microscope system.

3. Results and Discussion

3.1 XRD result

The relative content of HAp,

NaCaPO4 and whitlockite phases were the

ratio closest to the 79:11:10 ratios. Figure 1

showed powder XRD pattern of calcined

anchovy fish bones at 900 ºC. The

diffraction peaks can be assigned to the

hexagonal HAp (JCPDS No. 01-084-1998).


Figure 1. The XRD pattern of sample

Table 1. Calcination data and HAp crystal size D (nm) and lattice parameters calculated from

XRD using the Scherrer equation Initial

weigth

(g)

After

calcination

(g)

Yield

(%) a-exes (Å) c-exes (Å)

Average

Crystal size

(nm)

Unit cell

volume (Å3) Xc (%)

1.0 0.6 60 9.4260 6.8845 56 529.73 98

More specifically, the diffraction peaks at

= 25.8º, 31.8º, and 32.9º are consistent

with (002), (121), and (300) reflections of

hexagonal Hap9 respectively. In addition,

sodium calcium phosphate (NaCaPO4;

JCPDS No. 01-084-1998) and calcium

magnesium phosphate (Ca18Mg2H2(PO4)14;

JCPDS No. 01-084-1998), as well as HAp,

were detected.

The fraction of crystalline phase

(Xc%) in bioceramic powders can be

evaluated by the following equation:10

(

(1)

where Xc% is the crystallinity degree,

V112/300 is the intensity of the hollow between

(112) and (300) diffraction peaks, I300 is the

intensity of (300) diffraction peak.

The average crystallite size was

calculated from the broadening in the XRD

pattern according to the Scherrer’s

equation:11

(

(2)

where Dhkl is the average crystallite size, K

is the broadening constant, λ is the

wavelength of Cu Kα radiation (1.5406 Å),

β1/2 is the full-width at half-maximum of

(002) peak, and θ is the diffraction angle.

The lattice parameters (a and c) of

HAp were calculated by the method of least

squares using the following equation4:

(

(3)

where d is the spacing between the planes in

the atomic lattice. The volume (V) of the

hexagonal unit cell of each HAp formulation

was calculated using the below relation:9

(4)

The lattice parameters on a-axis

increased but on c-axis decreased upon

impurities addition, and also the volume of

the lattice undergoes a large contraction as

shown in Table 1. This behavior is due to

the many compositions contained in unit

cell, which is in agreement with the clearly

demonstrated NaCaPO4 and whitlockite


were structurally incorporated into HAp

crystals.

3.2 FTIR

In FTIR pattern (Figure 2c), the low

intensity peak around 3567-3574 cm-1 was

due to stretching vibration of O-H groups.

The bands corresponding to 1025 cm-1

signify the stretching vibration of P-O

bonds. The bands at 601 and 548 cm-1

representedthe bending mode of P-O groups.

The band of the carbonyl group appeared at

1744 cm-1. The band at 371 cm-1 was due to

the presence of Ca-O bonding. In the case of

uncalcined sample (Figure 2a), the peaks at

2926 and 2854 cm-1 were assigned to

vibrations of C-H groups, and the peaks at

1655, 1542, and 1165 cm-1 were assigned to

amide I and II bands that are not found in

the treated bone by the proposed methods.

These peaks were derived from the organic

compounds in bone.12

Figure 2. Infrared spectra of (a) raw bone,

(b) the reference spectrum of calcium

phospate from Sigma, (c) synthesized HAp

sample

3.3 SEM-EDX

Formation of HAp particles can be

observed in the SEM image presented in

Figure 3. It can be seen that HAp has formed

as much large irregular in the order of

microns. The average particle size of HAp

powders was determined from these

micrographs with approximately 0.5 µm. In

high magnification of Figure 3c, clearly,

some hexagonal morphologies of HAp were

obtained with a crystallite size of 0.5 µm

high, 1 µm wide corresponding to the lattice

paremeters of a-axes longer than c-axes.

(a)

(b)

(c)

(d)

Figure 3. (a)-(c) SEM images of sample

with different magnification, (d) EDX

spectrum of its sample


Figure 3d showed the typical EDX

spectrum of HAp powder, which confirmed

the impurity of the material by showing the

Na and Mg elements in the spectrum.

Moreover, the Ca/P atomic ratios in the

synthesized HAp powders determined

through EDS analysis closed to 1.67 of the

natural enamel.

4. Conclusion

This work showed the studied

anchovy fish bone from budu residue

samples calcined at 900 ºC exhibit a most

promising chemical composition and

structure that could be exploited to provide

good alternatives to synthetic

hydroxyapatite. The lattice parameter and

the volume began to increase, and this is

may be due to the crowdeness of the other

compound in the HAp crystal.

Acknowledgements

We acknowledge the Budu Yiseng

Factory, Amphor Saiburi, Pattani Province,

Thailand, for supporting the budu residue

samples. Our appreciation is also extended

to Mr. Tata Kwawi Mbinglo for assistance

with English corrections.

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