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Investigation of sintering temperature and concentration effects on Zn substituted HA.


The flexible structure of Hydroxyapatite (HA) welcomes a variety of cationic and anionic substituents in its structure [1-3]. Such substitution greatly enhanced the physical, chemical, mechanical and physiological properties of this bio-ceramic apatite [4, 5]. Moreover, natural bone is non-stoichiomertic with variety of substituents such as [Na.sup.+], [Mg.sup.2+], [Zn.sup.2+], [Mn.sup.2+] etc. [6-8]. Thus interest is now turning towards producing doped apatite mimics mineral component of natural bone apatite. These substituted biomaterials are now being extensively used for medical purposes, such as in diagnostic and therapeutic purposes [4]. For instance, Fe doped apatite shows promising criterion in Hyperthermia treatment causes delay of tumor growth or completely damage the tumor [9, 10]. Incorporation of doping elements also plays a significant role in improving the suitability of HA for restoration of hard tissue such as bone and teeth [4]. Among different cationic substituents Zn shows the greater potentiality for various reasons.

Zn is the most abundant trace element found in all biological hard tissue [11]. It exhibits better response in terms of biological function such as nucleic acid metabolism, DNA replication, gene expression, in maintenance of membrane structure and function [12,13]. Zn acts as a co-factor for more than 300 enzymes implied in enzyme activity, carbohydrate and protein synthesis [6, 13]. Incorporation of Zn in HA lattice promotes bone formation whereas inhibits osteoclastic bone resorption [14]. Zn stimulates the activity of vitamin D3-dependent promoters in osteoblasts [7]. Bone acts as Zn reservoir and mediates its release in body fluid. With these points as background, synthesis of Zn-releasing HA has now received significant attention of researchers throughout the world. Hardly some experimental studies have dealt with the effect of sintering temperatures as well as concentration on Zn-doped HA. Thus our aim was to address these effects on phase properties of Zn-doped apatites derived from waste egg shell material.

Materials and Methods

Analar grade chemicals ZnC[O.sub.3], N[H.sub.4]OH, HN[O.sub.3], [(N[H.sub.4]).sub.2]HP[O.sub.4] (99.99% pure) were used in this method, obtained either from E. Merck. All the solutions were prepared using double distilled water.

Raw egg shells, collected from a local restaurant were first thoroughly washed, calcined, then powdered and finally characterized as mentioned earlier [15]. The synthesis procedure (precipitation method) of different mol% Zn substituted HA (Zn4HA containing 4 mol% Zn and Zn8HA containing 8 mol% Zn) was the same as described earlier [15]. The synthesized sample (pure and Zn-doped apatite) was then crushed to fine powder and subjected to calcination at four different sintering temperatures for observing the temperature effects. Molar percent concentrations of all precursors are summarized in Table 1. Pure HA was also prepared and calcined at 900[degrees]C for comparison.

Synthesized apatites were characterized by different analytical procedure. Atomic absorption spectroscopy (AAS) and ultra violet spectrophotometric methods (UV) were used to analyze the presence of Ca and P respectively. Presence of Zn was confirmed through EDS and XRF analysis. The functional groups were determined by Fourier transform infrared spectroscopy (FT-IR, Model no. FTIR -8900, SHIMADZU). Experimental spectra were obtained by using KBr disks with a 1:100 "samples-to-KBr" ratio and the samples were scanned in the wave number range of 4000 [cm.sup.-1] - 400 [cm.sup.-1] with an average of 30 scans. The resolution of the spectrometer was 4 [cm.sup.-1].

X-ray diffraction analyses of both pure and doped apaties (calcined at various temperatures) were performed to observe the sintering effects. Phase properties of the prepared samples were investigated by using PANalytical (X'Pert PRO XRD PW 3040). The intensity data were collected in 0.02[degrees] steps following the scanning range of 2e = 20[degrees]-80 using CuKa (e = 1.54178A) radiation. The observed phases were compared and confirmed using standard JCPDS files [15].

The crystallite sizes from Scherrer's relationship and crystallinity of apatites were calculated using the following equation.

D = 79.5/[DELTA] cos[theta]

Where, D = crystal size ([degrees]A), [DELTA] = FWHM in degree.

The Bragg reflection at (211), (112) and (300) planes of this sample were considered to calculate the crystallite size [16].

Crystallinity, [X.sub.c] = ([k.sub.[alpha]]/[DELTA])3 [11]. Where, D= FWHM of the (002) reflection, ka=0.24

Calcination up to 900[degrees]C of all apatites causes appearance of new phase e.g.; [??]-TCP ([Ca.sub.3] [(P[O.sub.4]).sub.2]).Thus volume fraction([X.sub.[beta]]) of [??]-TCP was calculated according to following equation.

[W.sub.[beta]] = [I.sub.[beta](0210)]/[I.sub.[beta](0210)] + [I.sub.H(211)],

[X.sub.[beta]] = [PW.sub.[beta]/1 + (P - 1)[W.sub.[beta]]

Here, [I.sub.[beta](0210)] and [I.sub.H(211)] are XRD integrated intensity of [beta]=TCP at planes(0210) and HA at planes (211).The coefficient P was the intensity integrated ratio of planes (211) to planes (0210) [3].

Results and Discussion

FT-IR analysis

The FT-IR spectra for both pure and Zn doped HA (4mol% and 8 mol% ) calcined at three different temperatures 110[degrees]C, 300[degrees]C, 600[degrees]C representing the characteristic peaks for the phosphate (P[O.sub.4.sup.3-]) and O[H.sup.-] groups. The bands appeared (calcined at 110[degrees]C-600[degrees]C) in broad fashion supported the formation of apatite but indicates its poor crystalline nature (Fig. 1). The intensities of all peaks increased with increasing calcination temperature and this observation was confirmed from the spectra of the apatite sintered at 900[degrees]C (Fig. 2). Characteristic band position at 3570[cm.sup.-1] is due to stretching mode of O[H.sup.-] group. Upon calcination this mode disappeared for doped apatite whereas for pure HA intensity of this peak is quite high (Fig. 2 and 3) [2, 3]. The band for adsorbed water at 3431.36 [cm.sup.-1] was appeared for all apatites (Zn-HA and pure HA) sintered at even 900[degrees]C but in broad fashion. Particularly, the increased gap between the band positions of P[O.sub.4.sup.3-] group at 563.1[cm.sup.-1] and 630.7 [cm.sup.-1] for Zn doped apatite with respect to HA (where, P[O.sub.4.sup.3-] group at 563.1 [cm.sup.-], 1,601.79 [cm.sup.-1] and 630.7 [cm.sup.-1]) suggested the formation of less crystalline apatitic phase, which also supports the XRD observation. Vibrational modes of C[O.sub.4.sup.3-] group appears at 1423 [cm.sup.-1] for doped apatite even upon calcination at 900[degrees]C could be assigned to the presence of Zn, because FT-IR spectra of pure HA shows complete absence of this group (Fig. 3). Disappearance of C[O.sub.4.sup.3-] group for Zn doped HA due to volatilization may be expected on calcination above 1000[degrees]C. Furthermore, presence of some extra broad peaks at 962[cm.sup.-1] and 1018-1091 [cm.sup.-1] for all calcined apatites supported the notion of [??]-TCP formation at 900[degrees]C [3]. The FT-IR band positions and their corresponding assignments for Zn4HA and Zn8HA at various temperatures were tabulated in Table 2 and Table 3.




XRD analysis

The XRD analysis provides crystallographic information and phase composition of all apatites. In this paper we had studied the structural changes of doped and un-doped apatites in terms of both concentration variation and different sintering temperatures (Fig. 4a, 4b, 4c, 4d) has represented the XRD spectra for Zn8HA. Here spectra (Fig. 4a) for oven dried (at 110[degrees]C) doped apatite appeared with broad peaks resembles poor crystalline nature. This observation was similar for apatite calcined at 300[degrees]C and 600[degrees]C (Fig. 4b and 4c) where most of the peaks are of lower intensities supporting the observed FT-IR data. This behavior can be attributed to the temperature effect. It is now well established that the increase in sintering temperature increases the crystallinity degree resulting several distinct and sharp peaks. This low crystalline and amorphous nature has been dramatically changed to well-defined crystalline phase due to the thermal treatment at 900[degrees]C (Fig. 4d). This observation was further satisfied by plotting the peak height (at 2 positions ~ 31.8228) as a function of the calcination temperatures. The graph (Fig. 5) clearly showed that the peak height significantly increased at 900[degrees]C ultimately supported the well crystalline behavior of the doped sample at this sintering temperature. However, the observed intensity and d-spacing values were in excellent agreement with the JCPDS standard data (ref. code: 09-0432) for HA. Crystal size and crystallinity changes with sintering temperature effects were presented in Table 4 also strengthen this statement.



Lattice parameter changes

Lattice parameter of pure HA is slightly higher than doped apatites (Zn4HA and Zn8HA).This suggest that Zn incorporation lowers cell parameter a up to 8 mol% for calcined apatite (Fig. 6) which was consistent with the findings of Fumiaki Miyaji et al. This behavior can also be explained as ionic radius of [Zn.sup.2+] (0.74%A) is lower than that of Ca (0.99%A) [1, 11]. On the other hand, lattice parameter c shows a consistent value, not greatly affected by Zn content but slightly increase with temperature effects (Table 5). Cell volume calculated from these parameters is highest for HA while gradual decrease is observed with increased Zn content (Fig. 7).










Effects of Zn content on crystallographic properties

As we previously studied lattice parameter changes with Zn content reveal that crystallographic properties would also alter. But such alteration was not linear with cell parameters. Increased 8mol% of Zn did not shrink the crystal size rather this value is quite higher than that of 4mol% Zn. Although Crystal size and crystallinity for pure HA were high and Zn incorporation lowered this value but this result is contradictory to the findings of Fuzeng Ren et al. This may due to different preparation procedure and for heat treatment (Fig. 8). Crystallinity, on the other hand was measured only for well crystal sized apatite and as mentioned earlier substitution also lower this value.

Phase determination with calcination

It is well established that HA prepared by means of precipitation route decompose on calcination near 800[degrees]C to 900[degrees]C [1, 3]. Appearance of [??]-TCP phase in addition to the HA in XRD spectra of both calcined pure and substituted apatites supports this notion. Moreover parascholzite (Ca[Zn.sub.2] [(P[O.sub.4]).sub.2].2[H.sub.2]O) also appeared for Zn doped calcined apatite (at different [2% Theta] values, e.g.; 26.938, 31.481, 33.928 etc.).

The Diffraction angles and d-spacing values of (0210) plane for [??]-TCP phase were presented in Table 6 and compared with the JCPDS value of [??]-TCP (JCPDS#090169). Furthermore, crystal size and volume fraction of [??]-TCP were also tabulated in Table 7. Thus it can be suggested that crystal size of this 2nd phase for pure HA is smaller than that of doped HA. Moreover, ci-spacing value for un-doped HA is somewhat closer to the reported JCPDS value than those of Zn substituted apatites. This observation support stabilized [??]-TCP phase for Zn substitution.

SEM analysis

The crystallinity of apatite strictly depends on the sintering temperature. It is obvious that a well defined crystalline structure of Zn doped apatites appeared only after sintering at 900[degrees]C, so the morphology and micro structural features of different mol% Zn -HA sintered at this temperature were further examined by capturing their SEM micrographs. The recorded SEM pictures (Fig. 9a, 9b, 9c) for all apatites appeared with a combination of different regular but agglomerated shapes, such as hexagonal, spherical, etc. However, no significant morphological changes observed between 4 and 8 mol% Zn from SEM images (Fig. 9b and 9c), but micrograph for 0 mol% Zn (Fig. 9a) content is evident of comparably more sharp hexagonal phases. Such morphological analyses strengthen our previous report on the basis of FT-IR and XRD analysis.


Our effort was to characterize the prepared Zn doped apatite (4 and 8 mol% Zn-HA) from waste egg shell (as Ca precursor) with respect to un-doped HA. From overall observation it can be concluded that Zn was successfully substituted in the HA lattice. Cell parameter, cell volume, crystal size and crystallinity changes with Zn content and confirmation of the presence of Zn from XRF prove this determination. Furthermore, all these values were inferior than those of pure HA, means addition of Zn deteriorate the crystalline nature of the mentioned apatite. Since mechanical strength of un-doped apatite increases with doping and thus increases its suitability for load bearing application and the investigation is going on in this direction.


The authors gratefully acknowledge the financial support from IGCRT, BCSIR. The assistance from Mr. F. U. Farhad (SO), IPD, BCSIR and Dr. M. M. Rahman, Associate Professor, ACCE, DU for SEM and FTIR are gratefully acknowledged. Thank is also due to the Ministry of Science and Information & Communication Technology, Government of Bangladesh for granting NSICT fellowship to S. Kabir.


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[15.] S. F. Kabir, S. Ahmed, M. Ahsan and A. I. Mostafa, Zn substituted hydroxyapatite from waste Ca source, Dhaka Univ. J. App. Sci. and Engg., 1(2), 81-86 (2011)

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S.F. Kabir (a), S. Ahmed * (b), M. Ahsanb and A.I. Mustafa * (a)

(a) Department of Applied Chemistry and Chemical Engineering, University of Dhaka, Dhaka 1000, Bangladesh,

(b) Institute of Glass and Ceramic Research and Testing (IGCRT), Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka 1205, Bangladesh

Received 19 April 2011; Accepted 8 September 2011; Available online 8 September 2011
Table 1: Molar percentage concentration of precursors in HA

Sample                                         Zn/(Zn+Ca)
apatites     Ca (mol%)   P (mol%)   Zn(mol%)     (mol%)     Ca/P

HA             62.43      37.56        0           0        1.66
Zn8HA          62.43      37.56       5.23        8.13      1.66
Zn4HA          62.43      37.56       2.68        4.2       1.66

Table 2: FT-IR band positions and their corresponding assignments of

          Observed band positions ([cm.sup.-1]) of Zn8HA

110[degrees]C    300[degrees]C   600[degrees]C   900[degrees]C

   560.2             561.2           562.9           565.1
   601.3             603.3           602.7           603.7
   626.72            628.72          629.3           629.5
   962.3             960.3           963.4           962.4
  1418.3            1419.3          1421.4          1423.47
  3230.5            3233.5          3432.3          3431.5
  3569.24           3567.24         3568.24           --

              Corresponding assignments

        P[O.sub.4.sup.3-] bending ([v.sub.4])
        P[O.sub.4.sup.3-] bending ([v.sub.4])
 P[O.sub.4.sup.3-] asymmetric stretching ([v.sub.1])
 P[O.sub.4.sup.3-] symmetric stretching (([v.sub.3])
         C[O.sub.3.sup.2-] group ([v.sub.3])
                Structural O[H.sup.-]
           [H.sub.2]O adsorbed ([v.sub.2])

Table 3: FT-IR band positions and their corresponding
assignments of Zn4HA

         Observed band positions ([cm.sup.-1) of Zn4HA

110[degrees]C    300[degrees]C    600[degrees]C    900[degrees]C

    561.2            561.2            562.9            561.2
    601.3            602.3            603.7            602.3
    626.5            626.4            627.8            629.9
    960.3            962.3            963.4            961.3
    1417.3           1421.3           1418.4           1419.3
    3231.5           3232.5           3433.3           3231.5
    3569.2           3567.4           3569.2           3569.8

             Corresponding assignments

       P[O.sub.4.sup.3-] bending ([v.sub.4])
       P[O.sub.4.sup.3-] bending ([v.sub.4])
 P[O.sub.4.sup.3-] symmetric stretching ([v.sub.1])
P[O.sub.4.sup.3-] asymmetric stretching ([v.sub.3])
        C[O.sub.3.sup.2-] group ([v.sub.3])
               Structural O[H.sup.-]
          [H.sub.2]O adsorbed ([v.sup.2])

Table 4: Crystallographic information of apatite

                              Crystal        Crystallinity
   Sample apatites         size (A[??])

Pure HA 900[degrees]C         690.90             5.03
Zn8HA 110[degrees]C           344.72              --
Zn8HA 300[degrees]C           516.65              --
Zn8HA 600[degrees]C           517.10             0.42
Zn8HA 900[degrees]C           679.65             3.37
Zn4HA 110[degrees]C           206.59              --
Zn4HA 300[degrees]C           413.63              --
Zn4HA 600[degrees]C           413.61             0.63
Zn4HA 900[degrees]C           459.22             2.37

Table 5: Cell parameters as of all apatites

   Sample apatites         Lattice parameter         Cell
                         a(A[??])     c(A[??])     (A[??])

Pure HA 900[degrees]C      9.42         6.88       1580.60
Zn8HA 110[degrees]C        9.33         6.86       1546.03
Zn8HA 300[degrees]C        9.39         6.86       1569.65
Zn8HA 600[degrees]C        9.40         6.87       1570.60
Zn8HA 900[degrees]C        9.40         6.87       1573.89
Zn4HA 110[degrees]C        9.33         6.86       1548.03
Zn4HA 300[degrees]C        9.40         6.86       1570.89
Zn4HA 600[degrees]C        9.40         6.87       1571.60
Zn4HA 900[degrees]C        9.41         6.87       1574.95

Table 6: Diffraction angles and d-spacing values for 2nd
phase ([??]-TCP) appeared on calcined at 900[degrees]C

  Sample apatites                      (0210 plane)

                             d-spacing(A[??])   2([??]Theta)

Pure HA                           2.8594          31.25540
Zn8HA HA                          2.8532          31.3525
Zn4HA HA                          2.8557          31.3240
JCPDS#09-0169([beta]-TCP)         2.8800          31.0260

Table 7: Crystal size and volume fraction of 2nd phase
([??]-TCP) at 900[degrees]C.

Sample apatites    Crystal size   Volume fraction
                       (nm)            (%v)

Pure HA                43.5            28.1
Zn8HA                  59.9            28.2
Zn4HA                  59.9            27.1
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Title Annotation:Original Article
Author:Kabir, S.F.; Ahmed, S.; Ahsanb, M.; Mustafa, A.I.
Publication:Trends in Biomaterials and Artificial Organs
Article Type:Report
Geographic Code:9BANG
Date:Oct 1, 2011
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