Chapter III Characterization of Hydroxyapatite Nanoparticlesshodhganga.inflibnet.ac.in/bitstream/10603/34676/8/08... · 2018-07-02 · Department of Materials Science Page 71 Chapter

Department of Materials Science Page 71

Chapter III

Characterization of Hydroxyapatite Nanoparticles

III.1 Introduction

The hydroxyapatite is known to be an excellent adsorption material into the

human body. In addition to high adsorption capacity for heavy metals, it has low water

solubility, high stability under reducing and oxidizing conditions. It can be processed

with ease and method is cost effective. Therefore, in the present work hydroxyapatite was

prepared and efforts were made to optimize the processing parameter of hydroxyapatite

having high surface area and mesoporosity have been optimised.

III.2 Characterization of HA Nanoparticles

The preparation conditions used to prepare HA nanoparticles for which the

various analysis’s and tests mentioned in chapter two have been carried out are given in

Table III.1.

Table III.1 Experimental parameters used to prepare HA nanoparticles via chemical precipitation method

III.2.1 Physical properties

III.2.1.a Particle size and shape

TEM micrographs of the prepared samples after calcination are shown in Figure

Sample pH of CN pH of AHP

Reaction Temperature

A 9.5 9.5 Ambient

B 10.5 10.5 Ambient

C 12 12 Ambient

Chapter III


III.1. The particles are mainly composed of aggregates of nanoparticles with different

Ca/P ratio.

TEM micrographs of prepared samples after calcination are shown in Figure

III.1. These micrographs indicate nanometer scale size of precipitated HA particles

prepared by chemical precipitation method. The comparison of the relative micrographs

of figure reveals some significant changes between morphology and size of the particles.

The particles of all the three samples show two different shapes (i) nanorods and

(ii) nearly spherical nanoparticles. Irrespective of the shape of particles, the particle size

varies from 20-50 nm.

Sample A is composed mainly of from nanoparticles having low aspect ratio

represents the HA nanomaterials. In Figure III.1.a, HA particles hold spherical crystals

and congregation also can be observed. HA particles synthesized at pH 9.5 were spherical

or close to spherical in shape with different size. The results indicate that the pH has an

obvious influence on the morphology and size of HA particles. The morphology of HA

shown in Figure III.1.b presents needle-like crystals with an average size of

approximately 20-50 nm in width.

As it is seen in Figure III.1.c, nanoparticles indicated by arrow-head, is

containing some fine holes in their infrastructure. These holes are fundamentally some

structural defects that can be related to the kind of synthesis of them. Since, synthesis of

HA was carried out in alkaline environment, so, there are some high corrosive hydroxyl

groups in the synthesis reactor which they because the defects in the structure. It can be

concluded that the low pH may prevent the growth of HA particles and provide a more

uniform size distribution.

Chapter III


Figure III.1.a TEM micrographs of sample A (length of scale bar are 200 and 100nm).

Chapter III


Figure III.1.b TEM micrographs of sample B (length of scale bar are 50 and100nm).

Chapter III


Figure III.1.c TEM micrographs of sample C (length of scale bar are 100nm).

Chapter III


III.2.2 Chemical composition

III.2.2.a Fourier transform infrared spectroscopy

The FT-IR spectra of samples A, B and C before and after calcination are shown

in Figure III.2.a & b respectively. In both spectra, the absorption due to the vibration

modes from phosphates and hydroxyl groups, which represent the HA structure in IR

spectra are present.

Figure III.2.a FT-IR spectra for samples A, B and C before calcination

Figure III.2.b FT-IR spectra for samples A, B and C after calcination.

Chapter III


The FT-IR spectra of samples A, B and C before calcination and after calcination

are shown in Figure III.2.a and Figure III.2.b respectively.

In both spectra the absorption due to the vibration modes from phosphates and

hydroxyl groups, which represent the hydroxyapatite structure in IR spectra are seen. The

bands at 3570 cm-1and 634 cm-1 arise from stretching and librational modes of OH– ions

respectively. The bands at 1095 cm-1 and 1035 cm-1 arise from ν3 PO4, the 962 cm-1 band

arise from ν1 PO4, the 601 cm-1 and 565 cm-1 band arise from ν4 PO4 1-2. The group of

weak intensity bands in the 2200 cm-1 to 1950 cm-1 region derives from overtones and

combinations of the ν3 and ν1 PO4 modes.

The bands at 1407 cm-1 and 1458 cm-1 are attributed to components of the ν3 mode

of a trace amount of CO32–, the band at 872 cm-1 is attributed to components of the ν2

mode of CO32-, and bands at 1540 cm-1 derived from CO3

2– that replace OH– ions in the

hydroxyapatite lattice, were detected suggesting the substitution of PO4 group in the

structure of hydroxyapatite by CO32– 3-4. The broad peaks at 1650 cm-1 and 3200-3350

cm-1 show the presence of water5-6. Result shows that after calcination, the peaks become

sharper and more distinguishable because of the reduction of the number of phases.

Presence of carbonated group in spectra confirms formation of carbonated

hydroxyapatite.

III.2.2.b Inductively coupled plasma method

The results of the ICP analysis of the prepared samples are given in Table III.2.

The aim of this analysis was to determine the Ca/P molar ratio to be used to find

stoichiometric status of the prepared HA. It has been observed that all prepared HA is

Chapter III


Carbonated HA, i.e., having Ca percent greater than the ideal status, as can also be

observed in FT-IR spectra.

Comparing the Ca/P ratio of samples A, B and C having different pH shows that

with increasing pH, the Ca/P ratio increases up and is highest in case of sample C. This

can be explained by the structural tendency of HA to have the lowest level of stability at

pH 12, which is represented by the stoichiometric status.

Table III.2 Molar ratio obtained from ICP Analysis

III.2.2.c X-Ray Diffraction Analysis

the XRD patterns of the samples A, B and C after calcinations are shown in

Figure III.3.a, b and c. The observed positions of the diffraction lines (2θ and

corresponding d2θ) for the patterns of all samples are found to be in full agreement with

the corresponding values reported for hexagonal hydroxyapatite. The major expected

phase is hydroxyapatite, which is confirmed by comparing data obtained with the JCPDS,

Card No. 9-432.

The formation of hydroxyapatite was indicated by the appearance of

characteristics peak at 2θ =31.8˚ which is seen in all HA samples. The narrow peaks in

XRD spectra indicate high degree of crystallinity in the hydroxyapatite particles 7.

The occurrence of the two peaks at 2θ =32˚ in the diffraction spectrum and the

diffraction peaks at 2θ= 33.5˚, 34.6˚ and 49.9˚, respectively, became narrower and

No Sample ID Molar Ratio

1 Sample A 1.6787

2 Sample B 1.6815

3 Sample C 1.7001

Chapter III


sharper, and the diffraction peaks at 2θ =26˚, 28.8˚ and 48.1˚ appear with increasing pH.

These suggested that higher pH HA with larger proportion of crystalline phase. The XRD

pattern of samples show that secondary phase present is calcium oxide (CaO), identified

at 53.67 (2θ)8.

These result shows formation of hydroxyapatite. The occurrence of sharp wide

and high peaks reveals very small size and excellent crystal quality of HA. In all the

cases intense peaks were observed, showing formation of nano-HA powders with

superior quality by chemical precipitation method 9. These results are found to be in

agreement with the results reported in literature which suggested that the initial

precipitate was an amorphous calcium phosphate (ACP), which underwent stages of

aging to form a poorly crystalline apatite phase, and then a crystallized HA phase10-11 on

sintering at 550˚C.

Figure III.3.a XRD patterns of prepared HA sample A

Chapter III


Figure III.3.b XRD patterns of prepared HA sample B

Figure III.3.c XRD patterns of prepared HA sample C

Department of Materials Science

III.2.3 Surface characteristics

III.2.3.a Nitrogen adsorption stud

The adsorption and desorption of nitrogen isotherms of the calcined samples are

shown in Fig. III.4. The shape of these plots match with ‘type IV’ of the six principal

classes of isotherm shapes.

Fig III.4 Adsorption and desorption isotherms of

Department of Materials Science

II.2.3 Surface characteristics

II.2.3.a Nitrogen adsorption study


The shape of these plots match with ‘type IV’ of the six principal

classes of isotherm shapes.

Adsorption and desorption isotherms of the calcined HA samples.

Chapter III

Page 81


The shape of these plots match with ‘type IV’ of the six principal

HA samples.

Chapter III


Type IV isotherms possess a hysteresis loop, the shape of which varies from one

adsorption system to another. Hysteresis loops are associated with mesoporous solids,

where capillary condensation occurs. The initial part of the Type IV isotherm is attributed

to monolayer-multilayer adsorption since it follows the same path as the corresponding

part of a Type II isotherm obtained with the given adsorptive on the same surface area of

the adsorbent. Thus the samples have mixed porosities 12.

The surface characteristics of three samples are tabulated in Table III.3. These

results show that changes in reaction pH results in the decrease in surface area but at

higher pH the average pore diameter suddenly increase may be due to the agglomeration

of particles at higher pH. Samples prepared at pH 12, 10.5 and 9.5 gives 10.74%, 8.21%

& 0.486% micro porosity and surface area as 31.35 m2/g, 52.68 m2/g & 61.85 m2/g

respectively.

Table III.3 Surface characteristics of hydroxyapatite samples.

No Sample ID Surface Area (m2/g)

BJH Adsorption Average Pore Diameter (nm)

1 A 61.85 34.21

2 B 52.68 37.22

3 C 31.35 42.30

Samples B and C have the lowest surface area among the three samples because

they have larger particle size as evidenced by TEM observations. Sample A have the

highest surface area because of high percentage of small particle size.

Chapter III


III. 2.4 Determination point of zero charge (PZC) of HA

The point of zero charge (PZC) was determined in order to find pH at which the

solution had been prepared to remove heavy metal ions. The pH of PZC of the HA is

shown in Fig III.5. The PZC of HA was found to be 6.86. It was observed that at pH <

6.86, the surface of the HA is predominated by positive charges while at pH > 6.86, the

surface is predominated by negative charges.

Table III.4 Point zero charge for HA at different pH

No Initial pH (pH0) Final pH (pHf) Difference (pH)

1 8 9.25 1.25

2 7 7.20 0.20

3 6 5.60 -0.40

4 5 3.75 -1.25

5 4 1.75 -2.25

Fig III.5 Point zero charge for HA

y = 0.843x - 5.542R² = 0.988

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

0 2 4 6 8 10

(p

H)

pH0

Chapter III


III.3 Characterization of HA Pellets

III.3.1 Particle size and shape

TEM micrographs of the HA powders are shown in Fig III.6. As seen from the

morphologies of particles, there is a distribution of small particles and large agglomerates

with their size in the range 20 to 50 nm. These agglomerates consist of fine particles that

are cold welded together. The powders are composed of aggregates of nanosized HA

particles smaller than 50 nm There are two types of nanoparticles seen in the

micrographs: (i) nanorods and (ii) nearly spherical nanoparticles.

Fig III.6 TEM micrograph of the HA nanoparticles.

III.3.2 Chemical composition

III.3.2.a Fourier transform infrared spectroscopy

The FT-IR spectra of dense HA are shown in Figure III.7. The bands at 3568 cm-1,

and 635cm-1 arise from stretching, and librational modes of OH– ions, respectively. The

peaks at 567 cm-1 and 1039 cm-1 show the presence of PO4 group, the peaks at 982 cm-1

Chapter III


and 1091 cm-1 show the presence of HPO4 group. The sharpness of bands, especially after

calcination, indicates the well crystallinity of the prepared nanoparticles. Bands at

1455cm-1 and 1540cm-1 were detected suggesting the substitution of PO4 group in the

structure of HA by CO3. This type of substitution is commonly occurred during the

preparation of HA.

Figure III.7 FT-IR spectra for dense HA samples

The same observation recorded for sample A and has been observed for the

sintered samples. However, the peak attributed to HPO4 group has disappeared because

of their conversion to PO4. Moreover, the intensity of the peaks owing to CO3 groups has

been reduced. As expected, this indicates that the amount of the carbonate groups was

reduced.

Chapter III


III.3.2.b Inductively coupled plasma method

The Ca/P molar ratio was determined as 1.678. The measured Ca/P ratio for this

produced powder was higher than stoichiometric ratio (1.667) expected for a pure HA

phase that can arise from two matters: (a) local presence of carbonate apatite in which the

Ca/P ratio can be as high as 3.33 or (b) presence of impurities such as CaO. According to

the XRD patterns & FT-IR spectra that showed existence of carbonate apatite, the first

matter is much more reasonable.

III.3.2.c X-Ray Diffraction Analysis

The XRD patterns for the dense HA samples are shown in Figure III.8. It has

been observed that obtained peaks match perfectly with the corresponding values

reported for hexagonal hydroxyapatite (JCPDS, Card No. 9-432). The result shows a

crystalline nature of typical apatite crystal structure with broad diffracted peaks and does

not show any extraneous phases, which suggests that the chemical precipitation reaction

has produced phase pure or homogeneous HA.

Figure III.8 The XRD patterns for the Dense HA.

Chapter III


III.3.3 Surface characteristics

III.3.3.a Nitrogen & Methylene blue adsorption studies

The surface characteristics determined by nitrogen adsorption on HA pellets

samples are given in Fig III.9.

Fig III.9 Adsorption isotherms of the prepared HA Pellets.

0.0 0.2 0.4 0.6 0.8 1.0

0

1

2

3

4

5

6

7

Vo

lum

e A

dso

rbe

d (

cm3/g

ST

P)

Relative Pressure (P/Po)

Compaction Pressure 100 kg/cm2

0.0 0.2 0.4 0.6 0.8 1.0

0

2

4

6

8

10

Volu

me A

dso

rbed

(cm

3/ g S

TP

)


Compaction Pressure 75 kg/cm2

0.0 0.2 0.4 0.6 0.8 1.0

0

1

2

3

4

5

6

7

Volu

me A

dso

rbed

(cm

3/ g S

TP

)


Compaction Pressure 125 Kg/cm2

0.0 0.2 0.4 0.6 0.8 1.0

0

2

4

6

8

10

12

Vo

lum

e A

dso

rbe

d (

cm

3/g

ST

P)

Relative Pressure (P/P0)

Compaction Pressure 50 Kg/cm2

Chapter III


These isotherms show mesoporous nature showing multilayer adsorption process

where complete filling of the smallest capillaries has occurred corresponding to type IV

adsorption isotherms which are characteristics of mesoporous materials. Type IV

isotherms describe a multilayer adsorption process where complete filling of the smallest

capillaries has occurred.

The Surface characteristic properties of the dense HA prepared in different

packing condition are given in Table III.5. As compaction pressure increases the pores

got closed resulting in enlarging the pore diameter. BET surface area also decreased from

10.94 to 5.98 m2/g as compaction pressure increases from 50 to 125 Kg/cm2. These

results clearly show that compaction pressure is important parameter for formation of

porosity. Surface area measured using methylene blue method also show a similar kind of

nature. It decreases from 12.42 to 7.83 m2/g as compaction pressure increases from 50

to 125 Kg/cm2.

Table: III.5 Surface characteristic properties of HA pellets.

Sample No.

Compaction pressure (Kg/cm2)

Surface Area BET Method

(m2/gm)

Surface Area Methylene Blue

Method (m2/gm)

Average Pore Diameter

(nm)

1 50 10.94 12.42 5.90

2 75 9.32 11.05 5.80

3 100 6.51 8.68 5.78

4 125 5.98 7.83 2.39

Chapter III


III.3.4 Physical properties

The physical properties of the HA pellets prepared at different conditions are

given in Table III.6. The kerosene porosity and hence water absorption, decreased by

using higher applied pressure. In contrast, the density found to be increased through

increasing the packing pressure. Comparing the moisture content values of the prepared

samples shows that it decreases with increasing the packing pressure.

Table III.6 The physical properties of dense HA packed at different conditions.

Compaction pressure (Kg/cm2)

50 75 100 125

Kerosene Porosity (%) 53.73 49.06 44.80

42.64

Water Absorption (%) 51.77 46.39 41.99

39.02

Bulk Density (g/cc) 1.2690 1.3747 1.5315

1.6240

Moisture content (%) 0.9393 0.7830 0.6945

0.6442

III.3.5 Mechanical Testing

III.3.5.a Compressive strength

The results of compressive strength of HA samples are given in Table III.7. It has

can be seen that by increasing the compaction pressure improves the compressive

strength of HA. The result suggests that compaction pressure has an important role in

controlling the mechanical properties of HA. With the applied pressure porosity of

samples gets reduced, enhancing compressive strength of HA pellets.

Chapter III


Table III.7 The Mechanical properties of dense HA packed at different conditions.

No Pressure (Kg/cm2 )

Compressive Strength (MPa)

Compressive Modulas (GPa)

1 50 27.92 0.64

2 75 48.09 0.70

3 100 87.92 0.89

4 125 128.96 1.02

III.4 Conclusions

It has been found that carful adjustment of the processing parameters during the

preparation of HA nanoparticles is very necessary to control the microstructural

characteristics of the HA which is a complex ceramic system. This can successfully solve

many processing difficulties associated with the use of the HA and allowing

nanocrystalline particles of perfect phase purity to be achieved. The chemical

precipitation method with high pH precursors yield crystalline HA nanoparticles which

compose mainly from agglomerated nanorods having thermal stability. It has been

observed that the chemical and mineral composition as well as the morphology of the HA

nanoparticles prepared by chemical precipitation method are highly affected by the pH of

the starting solutions. The pH has been found to have significant role in the shape and

crystallinity of the nanoparticles. Equiaxed nanoparticles, nanowhisker, and nanorods can

be prepared from the same starting solutions by controlling the pH.

Chapter III


III.5 References

1) G. Gergely, F. Wéber, I. Lukács, L. Illés, A. L. Tóth, Z. E. Horváth, J. Mihály, C. Balázsi, Central European Journal of Chemistry, Vol. 8,375–381,(2010) 2) S. K. Ghosh, A. Prakash, S. Datta, S. K. Roy and D. Basu, Bulletin of Materials Science,Vol. 33, 7–16, (2010) 3) M. Shahmohammadi , R . Jahandideh , A. Behnamghader, M. Rangie, Int.J.Nano.Dim, Vol.1,41-45, (2010) 4) T.V. Thamaraiselvi, K. Prabakaran and S. Rajeswari, Trends Biomater. Artif. Organs, Vol. 19, 81-83,( 2006) 5) E. Bouyer, F. Gitzhofer, M. I. Boulos, Journal of Materials Science: Materials in Medicine, Vol.11, 523 – 531, (2000) 6) K.C. Blakeslee and R. A. Condrate, Journal of the American Ceramic Society, Vol. 54, 559- 564, (1971) 7) K. Kieswetter, TW. Bauer, SA. Brown, F. Van Lette, K Merrit, Biomaterials,15,183-188, (1994) 8) Recent Researches in Communications, Automation, Signal Processing, Nanotechnology, Astronomy and Nuclear Physics. ISBN: 978-960-474-276-9 9) Smičiklas, I. Onjia, A. and Raičevič, S., Sep.Purif. Technol. 44 : 97-102, (2005) 10) E. D. Eanes, I. H. Gillessen, and A. S. Posner, Nature, 208: pp365, (1965) 11) A. L. Boskey and A. S. Posner, J. Phys. Chem., 77: pp 2313 (1973) 12) S. J. Gregg and K. S. W. Sing, Adsorption, Surface Area and Porosity, London:

Academic Press (1982)

Chapter III Characterization of Hydroxyapatite Nanoparticlesshodhganga.inflibnet.ac.in/bitstream/10603/34676/8/08... · 2018-07-02 · Department of Materials Science Page 71 Chapter

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