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Effects of organic modifiers and temperature on the synthesis of biomimetic carbonated hydroxyapatite
Journal Pre-proof Effects of organic modifiers and temperature on the synthesis of biomimetic carbonated hydroxyapatite Taufiq Hasan Aneem, Sushanto Kumar Saha, Rumana A. Jahan, Siew Yee Wong, Xu Li, M. Tarik Arafat PII:
S0272-8842(19)32408-3
DOI:
https://doi.org/10.1016/j.ceramint.2019.08.211
Reference:
CERI 22684
To appear in:
Ceramics International
Received Date: 27 May 2019 Revised Date:
10 August 2019
Accepted Date: 22 August 2019
Please cite this article as: T.H. Aneem, S.K. Saha, R.A. Jahan, S.Y. Wong, X. Li, M.T. Arafat, Effects of organic modifiers and temperature on the synthesis of biomimetic carbonated hydroxyapatite, Ceramics International (2019), doi: https://doi.org/10.1016/j.ceramint.2019.08.211. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Effects of organic modifiers and temperature on the synthesis of biomimetic carbonated hydroxyapatite Taufiq Hasan Aneema, Sushanto Kumar Sahaa, Rumana A Jahanb, Siew Yee Wongc, Xu Lic, M Tarik Arafata,* a
Department of Biomedical Engineering, Bangladesh University of Engineering and
Technology (BUET), Dhaka 1205, Bangladesh b
Center for Advanced Research in Science (CARS), University of Dhaka, Dhaka 1000,
Bangladesh c
Institute of Materials Research and Engineering (IMRE), A*STAR (Agency for Science,
Technology and Research), Singapore 138634, Singapore *Corresponding author: M Tarik Arafat. Department of Biomedical Engineering, Bangladesh University of Engineering and Technology (BUET), Dhaka 1205, Bangladesh Email: [email protected] Phone: +880255167100 Ext. 6133
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Abstract Carbonated hydroxyapatite (CHA) was synthesized through a precipitation method using different organic modifiers namely citric acid, acetic acid, glutamic acid and gallic acid. These organic modifiers were used in order to control the nucleation of the synthesis process and crystallinity of the particles. Field Emission Scanning Electron Microscopy (FESEM) and Transmission Emission Microscopy (TEM) data showed that among the samples, CHA produced in the presence of citric acid was the smallest with rod shape (1925 nm) whereas, in the presence of gallic acid CHA particles became largest with flake shape (127-143 nm). Citric acid controlled nucleation by inhibiting crystal growth along a axis whereas, glutamic acid influenced nucleation through inhibition along c axis both resulting in rod shaped particles. Acetic acid also hindered crystal growth, however, to a lesser extent and the particles were flake shaped. CHA particles synthesized with gallic acid connected with each other due to π-π stacking interaction producing the largest particles. The synthesized particles were poorly crystallized with crystallinity ranging from 18.5% to 30%. Fourier Transform Infrared Spectroscopy (FTIR) results confirmed that all the samples were carbonated hydroxyapatite and from Energy Dispersive X-Ray (EDX) it was confirmed that the Ca/P ratio of the samples was around 1.7 indicating non-stoichiometric CHA. It was also found that with the increase in temperature, both the particle size and crystallinity increased. Keywords: Chemical precipitation; Carbonated hydroxyapatite; Organic modifiers; Biomedical applications.
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1. Introduction Hydroxyapatite (Ca10(PO4)6(OH)2) (HA) is the main inorganic compound of natural bone and can be considered as an excellent bioceramic material [1,2]. However, in a biological environment, the natural bone mineral is always carbonated and poorly crystallized [3]. Hence, efforts have been made to synthesize carbonated hydroxyapatite (CHA) using various methods such as hydrothermal treatment [4], nano-emulsion [5], solgel [6], mechanochemical [7,8] and chemical precipitation [9–14]. Among these methods, as hydrothermal method involves heat treatment, only highly crystallized HA can be synthesized [4]. On the other hand, sol-gel method is cost effective and simple. However, it requires a long time to synthesize CHA [6]. Chemical precipitation is an effective method to synthesize CHA. However, it is always challenging to control the size distribution and morphology of CHA through precipitation method which is very important for drug delivery, biomineralization process and dental applications [15–18]. Although CHA bioceramic has been synthesized through biomimetic precipitation [9,10,19], controlling the synthesis mechanism to yield CHA particles of preferential shape, morphology and crystallinity through the precipitation process has not been studied much. In precipitation process calcium and phosphate salts with a specific Ca/P molar ratio are used as precursors in an aqueous medium which undergoes nucleation. The HA particles thus grown form a solution whose solubility is low and therefore, HA particles precipitate. In the chemical precipitation process, organic modifiers can also be used to control the nucleation of the precipitation process. Organic modifiers work as inhibitors in the growth of HA particles and can influence the nucleation according to their chemical structures. For example, citric acid has been used as organic modifier to mediate the 3
nucleation process and crystallinity of HA [20]. It was found out that the particle size and morphology were dependent on the supersaturated solution containing citric acid. Citric acid mobilized calcium ions as calcium-citrate complex which inhibited the crystal growth process and produced crystallites of ~20 nm size [20]. Initially, citric acid surrounds the calcium ions and forms a supersaturated solution. This inhibits the spontaneous precipitation reaction of calcium phosphate and results in highly amorphous HA particles [21]. These results showed that using organic modifiers could be an effective way to control the synthesis process of HA. Gallic acid is a polyphenol acid and glutamic acid is a biomolecule and both of them can be used as organic modifiers. Acetic acid is another modifier that serves the purpose of comparing its effect on the formation of apatite particles with others. In this respect, a comparative study using these modifiers to synthesize CHA through precipitation process to better understand and control the nucleation process could be useful. This study aims to control the precipitation mechanisms by using different organic modifiers which are easily available such as citric acid, acetic acid, glutamic acid and gallic acid to synthesize CHA of different size distribution and morphology. Along with the organic modifiers, temperature of synthesis process has also been varied to observe the effect of temperature on the synthesized apatite. The varied morphology, size distribution, crystallinity, composition and other properties were investigated through a series of characterization techniques such as Field Emission Scanning Electron Microscopy (FESEM), Transmission Emission Microscopy (TEM), X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-Ray Analysis (EDX) and
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Thermogravimetric Analysis (TGA). Detailed mechanism of the apatite nucleation while using different modifiers has also been proposed. 2. Materials and Methods 2.1. Materials Calcium chloride (CaCl2.2H2O), ortho-phosphoric acid (85% H3PO4), sodium carbonate (Na2CO3), sodium hydroxide (NaOH), acetic acid (100% CH3COOH), citric acid, (C6H8O7), gallic acid (C7H6O5) and glutamic acid (C5H9NO4) were used to synthesize CHA. All of these chemicals were analytical grade and purchased from Merck. 2.2. Methods CHA was synthesized by adopting a previously developed method [10,19]. In brief, 20 ml of 0.1 M CH3COOH was stirred at 80 °C where 10 ml of 0.1 M CaCl2 and 6 ml 0.1 M H3PO4 solution were added drop by drop to maintain a Ca/P ratio of 10:6. Here CH3COOH was used as organic modifier. The solution was kept stirring for 30 minutes. Then, 18 ml of 0.1 M Na2CO3 was added slowly. After stirring for 30 minutes, NaOH solution was added quantitatively to get the pH of the solution 9. The solution was kept stirring for 4 hours during which the synthesized particles precipitated and then the samples were collected. The synthesized apatites were rinsed with ethanol and collected through centrifugation. Subsequently, the collected sample was dried at 50 °C overnight. The synthesized sample was named as CHA-Ace. Similarly, the whole procedure was repeated for other modifiers, namely citric acid, glutamic acid and gallic acid and the synthesized samples were named as CHA-CA, CHA-Glu and CHA-GA, respectively.
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To understand the effect of synthesis temperature, 60 °C and room temperature were also used along with the different organic modifiers. However, glutamic and gallic acid do not dissolve in water in room temperature and glutamic acid does not dissolve in water even at 60 °C [22–24]. Hence for acetic acid, citric acid and gallic acid, samples were prepared at 60 °C and were named as CHA-Ace', CHA-CA' and CHA-GA', respectively. On the other hand, for acetic acid and citric acid two more samples were prepared at room temperature and were named as CHA-Ace-RT, and CHA-CA-RT, respectively. The images of the final solution are given in Fig. S1. 3. Characterization The morphology of the synthesized CHA was observed using FESEM (JEOL-JSM7600F) with an acceleration voltage of 5 kV and a spot size of 8 mm. Before that, the samples were coated with platinum using ion sputtering method by JEOL-JFC 1600 auto fine coater. EDX was performed along with FESEM to obtain the Ca /P ratio of the synthesized CHA. In preparing samples for TEM (JEOL 2100), the samples were dispersed in ethanol and sonicated. Subsequently, they were dripped onto the TEM grid for observation. The lattice parameters and crystallinity of the synthesized CHA particles were analyzed using XRD which was performed by Rigaku Ultima IV, X-Ray diffractometer, Japan. Ni filtered CuKα radiation was used in the range of 2θ = 5–70° with the wavelength of 1.5418 Å. The percentage crystallinities of the samples were calculated byCrystallinity(%) = (1-
υ(112/300) I300
)* 100%
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where υ(112/300) is the intensity of the hollow between the diffraction peaks at (112) and (300) and I300 is the intensity of the diffraction peak at (300) of the samples. The Full Width at Half Maximum (FWHM) was calculated from each of the spectra and that was used to calculate the average crystallite size using the Scherrer equation with a shape factor of 0.94 for rod shaped particles [25]. D=
0.94λ β cos θ
where λ is the X-ray wavelength of the CuKα radiation (=1.5418 Å), β is the FWHM of the major XRD peak and θ is the angle of diffraction for characteristic peak (002) and 0.94 is the shape factor. The lattice parameters were calculated by using Bragg’s law to determine the inter-planar distance for characteristics planes such as (112) and (300). Bragg’s law is formulated asd(hkl) =
λ 2sinθ
where d(hkl) is the inter-planar distance at Miller indices (hkl), λ is the X-ray wavelength of the CuKα radiation (=1.5418 Å) and θ is the angle of diffraction for peaks at (hkl). The d(hkl) values of peak (112) and (300) were used to determine lattice parameters a and c from the relation of hexagonal unit cells1 d
2
4 h2 +hk+k2
= ( 3
a2
)+
l2 c2
where h, k and l are the Miller indices; a and c are lattice constants for a and c axis. To investigate the compositional analysis and presence of some of the specific groups, fourier transform infrared (Schimadzu IR Prestige21, Japan) spectroscopy (FTIR)
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was conducted in the range of 4000–400 cm-1. To prepare FTIR samples, around 200 mg of potassium bromide (KBr) was pressed with 2 mg of dried samples into pellets. TG Analysis was used to analyze thermal properties of the apatites. TGA Q500 (TA Instruments) was used and the range of heating was selected from room temperature upto 900 °C, with a heating rate of 10 °C /min. Nitrogenous environment with a flow rate of 40 ml/min was ensured during the analysis procedure. 4. Results 4.1. Morphology The FESEM and TEM images of the synthesized CHA samples are shown in Fig. 1 and 2, respectively. From these images it can be observed that citric acid and glutamic acid produced CHA particles that resembled the shape of rod with different size distribution by controlling the nucleation mechanism. Both acetic acid and gallic acid produced particles that were connected and thereby resulted in a flake-like morphology and their sizes were observed to be larger than CHA-CA and CHA-Glu. The length, width, equivalent circle area diameter, equivalent circle perimeter diameter and shape of the samples are summarized in Table 1. CHA-GA particles were the largest among all the samples whereas, CHA-CA particles were the lowest. The particle size distribution presented in Fig. 3 shows that the particle sizes are significantly different from each other. Fig. 4 shows the particle size distribution and TEM images of CHA-Ace-RT and CHA-CA-RT samples which signifies that relatively lower synthesis temperature yields smaller particle size. CHA-Ace-RT had aggregated rod-shaped morphology which is different from flake morphology of CHA-Ace synthesized at 80 °C. To further clarify the
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effect of temperature on morphology and size of the particles, TEM images of CHA-Ace', CHA-CA' and CHA-GA' have been given on Fig. S2. From the TEM images it can be observed that CHA-Ace' had flake shaped morphology which was smaller than CHA-Ace and larger than the aggregated nano-rod shape of CHA-Ace-RT. On the other hand, CHACA-RT had almost spherical shaped morphology different from rod shaped morphology of CHA-CA. CHA-CA' had rod shaped morphology smaller than the rod shape of CHA-CA. CHA-GA' had flake shaped morphology similar to CHA-GA however smaller than CHAGA. It can be concluded that lower synthesis temperature produced apatites with smaller particles and mostly similar morphology. The detailed mechanism of the formation of different morphology with different organic modifier and effect of temperature has been discussed thoroughly in ‘discussion’ section. 4.2. XRD The XRD spectra of the as prepared samples with the four different organic modifiers are shown in Fig. 5. All the diffraction peaks of the samples correspond to CHA peaks. The main peaks indexed to (002) at about 26°, broad and overlapped peaks of (211), (112), (300), (202) at approximately 32°, (310) at 40°, (222) at about 47°, (213) at 50° and (004) at 54° indicate that the synthesized materials are CHA [9]. After comparing with biological apatites it was concluded that the synthesized apatites resembled bone like apatite [26]. The JCPDS reference spectra for HA is also given in Fig. S3 which shows that the peaks of the synthesized apatites have broadened. This indicates carbonate incorporation in the apatite crystal and low crystallinity of the apatites [27]. To find the effect of organic modifier, the XRD of a sample synthesized without the modifiers, is given
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in Fig. S3. The highest intensity peak was for CHA-CA indicating preferred orientation of this sample. For CHA-GA, the intensity of the characteristic (002) and (211) plane was lower compared to CHA-Ace and CHA-Glu, indicating relatively lowered preferred orientation of this sample. The crystallinity, average crystallite size, lattice strain values and values of lattice parameters are tabulated in Table 2. The crystallinity of the samples ranged from 18.5% to 30%, which implies that the synthesized samples were poorly crystallized. The crystallinity of CHA-Ace was the highest and that of CHA-GA was the lowest among the samples. CHA-Ace exhibited the sharpest peak among the samples. Sharp peaks result in larger crystals and hence, CHA-Ace had the largest crystallite size and the lowest one was for CHA-CA and then CHA-GA and CHA-Glu, respectively. Broadening of peaks may also occur from carbonate substitution and strain of the unit cells as carbonate substitution hinders crystal growth to some extent and it introduces lattice strain in the crystallites [28]. The value of a axis was the largest for CHA-Glu and that of c axis was the largest for CHA-CA and least for CHA-Ace. CHA-GA hindered crystal growth along a axis as well and crystals grew along c axis. This indicates that glutamic acid had the effect of hindering the growth along c axis and assisting along a axis whereas, citric acid and gallic acid had the opposite effect. Acetic acid also assisted in growth along c axis, however, the effect was less prominent than citric acid and gallic acid as can be seen from its lattice parameters and c/a ratio. The change of growth along c axis for the samples can be determined by the combined effect of organic modifiers used and the substitution of carbonate atoms for phosphate ions. After comparing with the XRD of CHA-without modifiers, it was found out that without modifiers, the synthesized CHA had substantial growth along both the a and c axis which proves that organic modifiers inhibited growth along these axes. Hence, 10
the crystallite size of this particle was also the greatest among all the particles. CHAwithout modifiers also had the highest crystallinity among the samples and by using the organic modifiers, the crystallinity of the particles decreased. The XRD spectra of CHA-Ace', CHA-CA', CHA-GA', CHA-Ace-RT and CHACA-RT are given in Fig. S3. The crystallinity, lattice parameter values, average crystallite size and lattice strain of CHA-Ace', CHA-CA', CHA-GA' and CHA-without modifiers are given in Table S1. The XRD spectra of CHA-CA-RT has very low intensity and broad peaks unlike the other samples which means that this sample was highly amorphous. XRD of CHA-CA' is not as broad as CHA-CA-RT and this apatite had higher crystallinity than CHA-CA-RT and lower than CHA-CA. Thus it can be observed that lower synthesis temperature produced relatively poor crystallized particles. The variation of crystallinity with the change in temperature for acetic and citric acid as modifier is given in Fig. S4. CHA-CA-RT had smallest crystallite size and CHA-CA had the largest. Similar effect of temperature on crystallite size was observed for other organic modifiers as well. 4.3. FTIR Fig. 6 demonstrates the FTIR spectra of the characteristic peak of CHA for the synthesized apatite. The intense peaks noted around 561 and 601 cm−1 were observed due to the bending modes of PO43− ions in CHA [29,30]. The bands at 1036 and 1110 cm−1 are the bands which represent the basic characteristic of asymmetric (P-O) stretching vibration of PO43− ion in CHA [30]. The peak at 1411 cm−1 represents the CO32- ions [31]. The carbonate bands of the samples confirm that the synthesized apatites were carbonated HA. In addition to that the carbonate bands of the samples are around 1410 cm-1 which indicate
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the synthesized samples to be CHA B-type [32]. The FTIR spectra of CHA-Ace, CHAAce', CHA-CA, CHA-CA' and CHA-GA' are given in Fig. S5.
4.4. EDX From the elemental analysis by EDX, Ca/P ratio was obtained and the results are summarized in the Table 2 and Fig. 7. As biological apatite has Ca/P ratio between 1.6-1.87 [33], all the samples are biological in nature, although non-stoichiometry can be observed. CHA-CA, CHA-GA has higher Ca/P ratio which points out substantial phosphate substitution by carbonate ions [34]. These samples also have higher lattice strain indicating greater amount of carbonate substitution. The EDX of CHA-Ace-RT and CHA-CA-RT are given in Fig S6. The Ca/P ratio of CHA-CA-RT is lower than CHA-CA and thereby had lower crystallite size. CHA-Ace-RT has Ca/P ratio of 1.5 which is lower than that of CHA-Ace, however, its crystallite size was not larger and its lattice strain was not lower. This could be due to the effect of low temperature that was more dominating than carbonate substitution and the crystal growth was hindered. This resulted in smaller crystallite size of CHA-Ace-RT than CHA-Ace. 4.5. TGA Thermal analysis of the CHA samples are presented in Fig. 8. The TGA curves for all the samples showed gradual mass loss due to the removal of adsorbed water and decomposition of carbonate components. At around 300 °C mass loss was observed due to the dehydration of the synthesized samples and loss of physically adsorbed water molecules [35–37]. At around 400 °C, only the mineral phase of CHA was left. With the increase in
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temperature from 400 to 800 °C, the rest of the mass was lost because of the decomposition of carbonate [35–37]. The carbonate content of the apatites is given in Fig. S8 which shows that with the increase in synthesis temperature the carbonate content decreases for a specific organic modifier. The carbonate contents of the samples are given in Table S2. 5. Discussion 5.1. Effects of organic modifiers on CHA particles Fig. 9 demonstrates the schematic of the possible formation of CHA using citric acid, acetic acid, glutamic acid and gallic acid as organic modifiers. Citrate ion plays an inhibiting role on the nucleation of CHA by chelating with three calcium ions and limiting their availability on the solution [18,38,39]. After the addition of precursor ions on the citric acid solution, calcium citrate complex functioned as the nucleation sites for the growth of CHA crystals. The normal precipitation reaction between the precursors was inhibited and calcium phosphate precipitation was limited [18,38,39]. There was a decrease in the supersaturation of the solution which resulted in the low precipitation of calcium phosphate [38] This slowed the nucleation process of CHA to a great extent. As the solution was kept stirring, calcium ions were slowly released from the calcium citrate chelation complex and CHA crystals started to form. Besides this effect on CHA particles, citrate ions also affected the crystal growth of CHA [40]. They adsorbed preferentially to its hexagonal unit cell surface by its COO- group mainly on the rectangular facet ac or bc of the unit cell. These two faces are rich in calcium ions than the ab facet. The adsorption process made the surface of the unit cell negatively charged because of the two free COOgroups of the citrate ion which repelled other crystals inhibiting facet to facet crystal
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aggregation [40]. Thus the crystal growth at the a and b axis was hindered and crystals grew along the c axis (Fig. 9(b.i)) [41,42]. From the XRD analysis it is apparent that the c/a ratio for CHA-CA is higher than other samples indicating preferential growth along the c axis. This made the particles rod-shaped and it was the smallest out of the samples. As the synthesized CHA was B type in nature, phosphate ions were substituted by carbonate ions and this exchange process also resulted in small particles by inhibiting growth of crystals along all the axis except c. Thus crystals grew along this direction due to the added effect of carbonate substitution. TGA result also shows that CHA-CA had phosphate ions substituted by carbonate ions. Carbonate substitution also creates strain on lattice unit cells and it is clear from Table 2 that strain of CHA-CA was the second highest among all the samples. The XRD peaks of CHA-CA were also broader due to the combined effect of citric acid as organic modifier and carbonate substitution that resulted in small crystals. The effect of acetic acid on the growth of CHA is similar to the effect of citric acid. As acetic acid is a monoprotic acid, it chelated to only one calcium ion per acetate ion. This limits calcium availability for CHA particle growth to a lesser extent and nucleation was less controlled by acetic acid than citric acid. The calcium acetate compound acted as nucleation sites and growth of CHA crystals occurred in these sites. The inhibiting effect of acetate ion was mainly on the nucleation rather than the crystal growth and the particles were larger than CHA-CA. The c/a ratio of CHA-Ace is smaller than CHA-CA. This indicates that acetic acid did not inhibit growth along a or b axis to the same extent as citric acid did (Fig. 9(b.ii)). Crystal agglomeration was not hindered by negatively charged crystals like CHA-CA because there were no leftover COO- ions after the adsorption of acetate ions. That is why the resulting particles agglomerated and resembled flake like 14
CHA particle which is apparent from FESEM and TEM images of Fig. 1 and 2, respectively. The carbonate substitution was less as can be found from TGA result and crystal growth inhibition due to this effect was also less than CHA-CA which is evident from XRD by sharp peaks of CHA-Ace. Unlike citric acid and acetic acid, glutamic acid mainly played role on the CHA crystal growth rather than on the nucleation of CHA in the solution. Glutamic acid has two COO- sides and one NH3+ side. It made a polydentate complex with calcium ions through its NH3+ ions to a lesser amount than acetic acid and citric acid (Fig. 9(b.iii.1). This phenomenon did not inhibit the nucleation of CHA to a great degree and thus nucleation occurred spontaneously [43]. Glutamic acid adsorbed on both the (001) and (100) surface of the CHA crystal. The adsorption on the (001) face was due to NH3+ positioning on the vacant calcium sites mainly. Additionally, the adsorption was due to the electrostatic attraction between the NH3+ group and carbonate, phosphate and hydroxyl groups. However, the majority of adsorption occurred due to the substitution of NH3+ in the vacant calcium sites of the crystal. Glutamic acid also adsorbed on the (100) crystal face by electrostatically attracting the calcium ions of the crystal by its COO- group. However, the adsorption amount was greater in (001) surface compared to (100) because of the presence of greater number of calcium ions in the (001) [43–45]. Glutamic acid being an acidic amino acid, adsorbed preferentially on the positively charged surface of CHA as shown by previous works [45,46]. To determine the inhibitory effect of glutamic acid, geometrical factor must also be considered. Adsorbed glutamic acid has an axis of free rotation perpendicular to the adsorbed surface of the crystal. The volume resulted due to the rotation
15
can be described as a cone that has a radius of 5.23 Å as found previously [43]. The projected area due to the rotation covered up a large portion of the crystal structure and hence the crystal could not further accommodate calcium ions and thus development of crystals stopped. The (001) surface accommodated higher amount of glutamate ions than the (100) surface and hence, crystal structure could not complete in this direction (Fig. 9(b.iii.2)) [43–45]. This resulted in an inhibition of crystal growth along the c axis unlike the effect of citric acid [43]. The crystals grew in a or b direction resulting in rod shaped particles and they were observed in the FESEM and TEM images in Fig. 1 and 2, respectively. XRD data show that the value of a axis is the greatest for CHA-Glu and c/a ratio is the lowest which also indicates the crystal growth along a axis being more profound. The c axis value should have been smaller compared to other samples. However, Table 2 points out that CHA-Glu had some growth along c axis as well. This took place because of carbonate substitution which promoted growth along c axis as oppose to glutamic acid. The final value of a and c axis resulted due to the combined and opposing effect of glutamic acid adsorption and carbonate substitution. Between them, the effect of glutamic acid dominated and determined the size of CHA-Glu particles. CHA-Glu particles were larger than CHA-CA because citric acid had more inhibition on solution chemistry controlling the nucleation rate unlike Glu. CHA-GA particles had flake like morphology although particles were larger than CHA-Ace. As gallic acid has COO- group, it bonded to the calcium ions of the solutions by electrostatic attraction and thus acted as a nucleation site. It inhibited crystal growth in a or b axis to some extent and crystals grew along c axis. Table 2 points out that c axis of CHA-
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GA grew more than CHA-Ace and CHA-Glu. Thus particles started to grow and after the growth of CHA particles, they might have bonded with the gallic acid molecules by the hydroxyl groups of each through hydrogen bonding between the hydroxyl groups. Each of the CHA-GA units might have connected to each other via the intermolecular force or the π-π stacking interaction between its aryl rings and CHA-GA particles assembled (Fig. 9(b.iv)) [47,48]. The interconnection between the CHA-GA molecules resulted in a flake like morphological shape evidenced by the FESEM and TEM images (Fig. 1 and Fig. 2). All the synthesized samples were poorly crystallized which is similar to the nature of biological apatites. The crystallization activation temperature plays a key role in developing crystallinity of CHA particles at different synthesis temperature which was affected by the organic modifiers. CHA-Ace showed the highest crystallinity among the samples because at 80 °C, the synthesis temperature overcame the activation temperature or was closer to it. CHA-CA had low crystallinity as citric acid chelated to Ca2+ to a great amount resulting in random growth of CHA particles. CHA-GA had the lowest crystallinity among the samples. Because of the presence of gallic acid, the crystallization activation temperature could not be reached easily as the heat was used by the system to make the gallic acid particles soluble in the water. The remaining heat was not enough to reach the crystallization activation temperature which resulted in the lowest crystallinity of 25% among the samples. CHA-Glu also exhibited similar effects, however, the solubility of glutamic acid is greater than gallic acid at the same temperature thus less temperature was needed to maintain the solubility of glutamic acid [22–24]. Hence, the temperature was closer to the crystallization activation temperature and crystallinity of 27.7% resulted. Low
17
crystalline apatites improve the surface activity of the implant [49]. It can also dissolve more easily than highly crystallized ones which is beneficial to drug delivery system and in clinical applications where absorption-recrystallization is wanted. 5.2. Effect of temperature on CHA particles Synthesis temperature had important effects on the size and morphology of the CHA samples. With the increase in temperature, the rate of collision between two or more molecules increased as the intermolecular distance reduced because of the increased mobility of the particles. Thus, effective collisions of the reactant ions increased the size of the nuclei with increasing temperature. Hence, the particle size increased with temperature as can be seen from TEM and particle size distribution analysis (Fig. 4) of CHA-Ace, CHA-Ace-RT, CHA-CA and CHA-CA-RT and also from Table 1. The size of CHA-Ace was greater than CHA-Ace' and almost double than CHA-Ace-RT showing the effect of synthesis temperature. CHA-Ace' had flake shaped morphology with similar particle forming mechanism as CHA-Ace and it was smaller than CHA-Ace because of the decreased amount of effective collision. CHA-Ace-RT had aggregated nano rod shaped morphology. This indicates that as the synthesis temperature increased, the morphology changed from aggregated nano rod shape to flake shape and particle size increased due to more collision among the precursors. CHA-CA, on the other hand, exhibited slight increase in size than CHA-CA' and CHA-CA-RT as citric acid chelated to calcium ion more efficiently than acetic acid. This resulted in a decreased mobility of calcium ion. CHA-CA' and CHA-CA-RT had somewhat rod shaped particle and almost spherical particles, respectively. Due to lower synthesis temperature and high chelation effect of citric acid, the collision rate of the reactants was the lowest. Thus the growth of CHA-CA-RT particles 18
was almost equal in every direction which resulted in particles resembling spheres. CHACA' particles were of nano rod shaped similar to CHA-Ace. However, the particle size was smaller than CHA-CA indicating the effect of temperature. Also, the crystallite size decreased with the increase in carbonate content and CHA-CA-RT had the smallest crystallite size among CHA-CA, CHA-CA' and CHA-CA-RT because it had the highest carbonate content. Similar effect of temperature was also found on the morphology and shape of CHA-GA and CHA-GA'. The particle size increased with the increase in temperature with similar particle forming mechanism. Lower synthesis temperature also yielded lower crystallinity of the samples. Crystallinity of CHA-CA-RT is even lower than CHA-CA' and CHA-CA because of lower synthesis temperature and high chelation effect of citric acid. Lower temperature had similar effects on the crystallinity of CHA-Ace-RT, CHA-Ace' and CHA-GA' as well. 6. Conclusion A precipitation process has been developed to synthesize CHA with controlled size, morphological shape and crystallinity. Organic modifiers which controls the nucleation process of the synthesis procedure were used in this regard. The largest particle size obtained was CHA-GA (127-143 nm) and CHA-CA was the lowest particle (19-25 nm) in size. Significant changes in morphology were also obtained. CHA-CA and CHA-Glu showed rod like morphology whereas, CHA-Ace and CHA-GA exhibited flake morphology. All the samples were poorly crystallized with crystallinity values ranging from 18.5% to 30% resembling amorphous structure of natural bone. The samples were also B-type carbonated apatite which was confirmed by FTIR. The Ca/P ratio was around
19
1.7 obtained from EDX indicating non-stoichiometric CHA. Furthermore, different synthesis temperatures were used to understand the effect of temperature on the synthesis and CHA crystallization process. Samples synthesized at room temperature were smaller than the samples synthesized at higher temperature with the same modifier. This indicates that increased temperature results in larger particles. Morphologically, CHA-Ace-RT had aggregated rod-shaped particles whereas, CHA-CA-RT particles were almost spherical shaped. It was also found out that crystallinity also increased with increased synthesis temperature. Thus, in this study, using inexpensive and readily available organic modifiers, CHA nanocrystals of different size, morphology and crystallinity were obtained with a detailed understanding of the precipitation mechanism. The synthesized CHA particles could be useful in preventive, restorative and regenerative dentistry, structural biomaterial for prosthetic applications, bone-tissue engineering and drug delivery systems. 7. Acknowledgement The project was funded by University Grants Commission (UGC), Bangladesh under the grant no. 6(76) UGC/S&T/CHEMICAL-25/2018/3282. 8. References [1]
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Fig. Captions Fig. 1. FESEM images of samples (a) CHA-Ace particles with flake like morphology, (b) CHA-CA particles resembling somewhat rod shaped morphology, (c) CHA-GA particles with flake shaped morphology and (d) CHA-Glu particles having rod shaped morphology Fig. 2. TEM images of samples (a) CHA-Ace with flake-like morphology, (b) CHA-CA particles having somewhat rod shape, (c) CHA-GA particles with flake-like morphology and (d) CHA-Glu particles with rod shaped morphology Fig. 3. Particle size distribution of all the synthesized apatite. CHA-GA with the highest particle size distribution with 127-144 nm and CHA-CA with the lowest particle size distribution with 19-25 nm. All samples are significantly different compared to CHA-CA and the differences are denoted by * (p<0.05) Fig. 4. Particle size distribution and respective TEM images of the samples CHA-Ace, CHA-Ace-RT, CHA-CA, CHA-CA-RT showing that higher synthesis temperature yields larger particles. Samples with significant size differences are denoted by * (p<0.05)
28
Fig. 5. XRD patterns of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid at synthesis temperature of 80 °C. All the samples exhibited characteristics HA at about 26°, 32°, 40°, 47°, 50° and 54°. Fig. 6. FTIR spectra of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid and at synthesis temperature of 80 °C. All the samples have the characteristics CHA peaks. Fig. 7. EDX data of samples (a) CHA-Ace with Ca/P of 1.66, (b) CHA-CA with Ca/P=1.88, (c) CHA-GA with Ca/P=1.71, (d) CHA-Glu with Ca/P=1.68; Higher values of Ca/P indicate that PO43− had been substantially substituted by CO32- ions. Fig. 8. TGA curves of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid. All the samples have a mass loss due to water removal upto 200-300 °C. The next portion of the curve upto 800 °C shows decomposition of HPO42- and CO32-. Further increase in temperature shows more weight loss in CHA-CA and CHA-Glu whereas, slight mass gain in CHA-Ace and CHA-GA. Fig. 9. (a) CHA synthesis process, (b) (i) Citric acid inhibiting particle growth by chelating to Ca2+ ions and working as nucleation sites by slowly releasing Ca2+; then adsorbing onto (001) face and producing negatively charged crystal hindering crystal aggregation along a and b axis. (ii) Acetic acid producing similar effect but to a lesser extent and no adsorption effect like citric acid. (iii.a) Glutamic acid making polydentate chelation with Ca2+ by two NH3+ groups limiting its availability. (iii.b) Glutamic acid mostly inhibiting crystal growth along c axis by preferentially adsorbing to this face in the vacant Ca2+ sites by NH3+ groups
29
and preventing Ca2+ ions to fill the vacant sites due to effective volume of rotation. (iv) Gallic acid hindering crystal growth by binding to Ca2+ and aggregating to large grains by π-π stacking interaction between its aryl rings after forming CHA-GA crystal. Table Captions Table 1. The size distribution (nm) and Morphological shape and Ca/P ratio of the synthesized CHA samples Table 2. The Crystallinity (%), Lattice parameters (nm), c/a ratio, Strain (%) and Ca/P ratio of the synthesized CHA samples
30
(a)
CHA-Ace
(b)
CHA-CA
(c)
CHA-GA
(d)
CHA-Glu
Fig. 1. FESEM images of samples (a) CHA-Ace particles with flake like morphology, (b) CHACA particles resembling somewhat rod shaped morphology, (c) CHA-GA particles with flake shaped morphology and (d) CHA-Glu particles having rod shaped morphology
31
(a)
CHA-Ace
50 nm
CHA-CA
50 nm
50 nm
(c)
(b)
CHA-GA
(d)
CHA-Glu
50 nm
Fig. 2. TEM images of samples (a) CHA-Ace with flake-like morphology, (b) CHA-CA particles having somewhat rod shape, (c) CHA-GA particles with flake-like morphology and (d) CHA-Glu particles with rod shaped morphology
32
*
CHA-GA
* * CHA-Ace CHA-Glu CHA-CA
Fig. 3. Particle size distribution of all the synthesized apatite. CHA-GA with the highest particle size distribution with 127-144 nm and CHA-CA with the lowest particle size distribution with 1925 nm. All samples are significantly different compared to CHA-CA and the differences are denoted by * (p<0.05)
33
*
* 100 nm
CHA-Ace
50 nm 50 nm
CHA-Ace-RT
CHA-CA
100 nm
CHA-CA-RT
Fig. 4. Particle size distribution and respective TEM images of the samples CHA-Ace, CHA-AceRT, CHA-CA, CHA-CA-RT showing that higher synthesis temperature yields larger particles. Samples with significant size differences are denoted by * (p<0.05)
34
(211) (112) (300) (202)
(002)
(310)
(222) (213) (004)
CHA-Glu
CHA-GA
CHA-CA CHA-Ace 5
10
15
20
25
30
35
40
45
50
55
60
65
70
2theta (deg)
Fig. 5. XRD patterns of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid at synthesis temperature of 80 °C. All the samples exhibited characteristics HA peaks at about 26°, 32°, 40°, 47°, 50° and 54°.
35
CO32CHA-Glu PO4 3− PO4 3− CHA-GA
CHA-CA
CHA-Ace
2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (1/cm)
Fig. 6. FTIR spectra of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid, and glutamic acid at synthesis temperature of 80 °C. All the samples have the characteristics CHA peaks.
36
(a)
CHA-Ace
(b)
CHA-CA
(c)
CHA-GA
(d)
CHA-Glu
Fig. 7. EDX data of samples (a) CHA-Ace with Ca/P of 1.66, (b) CHA-CA with Ca/P=1.88, (c) CHA- GA with Ca/P=1.71, (d) CHA-Glu with Ca/P=1.68; Higher values of Ca/P indicate that PO43− had been substantially substituted by CO32- ions.
37
100
CHA-Ace CHA-CA
95
CHA-GA
Weight loss (%)
CHA-Glu 90
85
80
75 0
100
200
300
400
500
600
700
800
900
Temperature (℃)
Fig. 8. TGA curves of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid.
38
(a) CHA synthesis process
(b) Crystal forming mechanism in the presence of organic modifiers Citrate
i. Precursor Acetate
ii. Precursor
Glutamate (iii.1)
iii. Precursor
(iii.2)
Gallic
iv. Precursors
Fig. 9. (a) CHA synthesis process, (b) (i) Citric acid inhibiting particle growth by chelating to Ca2+ ions and working as nucleation sites by slowly releasing Ca2+; then adsorbing onto (001) face and producing negatively charged crystal hindering crystal aggregation along a and b axis. (ii) Acetic
39
acid producing similar effect but to a lesser extent and no adsorption effect like citric acid. (iii.a) Glutamic acid making polydentate chelation with Ca2+ by two NH3+ groups limiting its availability. (iii.b) Glutamic acid mostly inhibiting crystal growth along c axis by preferentially adsorbing to this face in the vacant Ca2+ sites by NH3+ groups and preventing Ca2+ ions to fill the vacant sites due to effective volume of rotation. (iv) Gallic acid hindering crystal growth by binding to Ca2+ and aggregating to large grains by π-π stacking interaction between its aryl rings after forming CHA-GA crystal. Table 1. The Length (nm), Width (nm), Equivalent circle area diameter (nm), Equivalent circle perimeter diameter (nm) and Morphological shape of the synthesized CHA samples Sample
Length (nm)
Width (nm)
Equivalent circle area diameter (nm)
Equivalent circle perimeter diameter (nm)
Morphological shape
CHAAce CHA-CA
55-68
22-50
14-20
19-27
Flake shaped morphology
19-25
13-20
9-14
14-19
CHA-GA
127-143
56-122
40-47
88-101
Somewhat rod shape with elongation along c axis Flake shaped morphology
CHAGlu CHAAce-60 CHACA-60 CHAGA-60 CHAAce-RT
33-43
16-31
13-18
23-32
29-40
20-33
11-16
21-29
14-23
9-17
7-12
11-17
42-68
23-55
18-26
31-48
23-32
18-26
14-20
19-27
Somewhat rod shape with elongation along c axis Somewhat rod shaped with elongation along c axis Aggregated nano rod shape
CHACA-RT
15-19
10-14
10-13
12-16
Almost spherical shape
Rod shaped with elongation along a or b axis Flake shaped morphology
40
Table 2. The Crystallinity (%), Lattice parameters (nm), c/a ratio, Strain (%) and Ca/P ratio of the synthesized CHA samples Sample
Crystallinity (%)
CHA-Ace
30
CHA-CA CHAGA CHA-Glu CHAAce-RT CHACA-RT
Lattice parameters (nm) a=b axis c axis 0.909 0.651
c/a
Crystallite Size(nm)
Strain (%)
Ca/P ratio
0.716
26
0.62
1.66
28.6
0.917
0.677
0.738
16
1.02
1.88
25
0.926
0.673
0.727
21
1.78
1.71
27.7
0.93
0.66
0.709
25
0.64
1.68
25
0.91
0.65
0.714
20
0.79
1.5
18.5
0.92
0.677
0.735
12
1
1.82
41
S0272-8842(19)32408-3
DOI:
https://doi.org/10.1016/j.ceramint.2019.08.211
Reference:
CERI 22684
To appear in:
Ceramics International
Received Date: 27 May 2019 Revised Date:
10 August 2019
Accepted Date: 22 August 2019
Please cite this article as: T.H. Aneem, S.K. Saha, R.A. Jahan, S.Y. Wong, X. Li, M.T. Arafat, Effects of organic modifiers and temperature on the synthesis of biomimetic carbonated hydroxyapatite, Ceramics International (2019), doi: https://doi.org/10.1016/j.ceramint.2019.08.211. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
Effects of organic modifiers and temperature on the synthesis of biomimetic carbonated hydroxyapatite Taufiq Hasan Aneema, Sushanto Kumar Sahaa, Rumana A Jahanb, Siew Yee Wongc, Xu Lic, M Tarik Arafata,* a
Department of Biomedical Engineering, Bangladesh University of Engineering and
Technology (BUET), Dhaka 1205, Bangladesh b
Center for Advanced Research in Science (CARS), University of Dhaka, Dhaka 1000,
Bangladesh c
Institute of Materials Research and Engineering (IMRE), A*STAR (Agency for Science,
Technology and Research), Singapore 138634, Singapore *Corresponding author: M Tarik Arafat. Department of Biomedical Engineering, Bangladesh University of Engineering and Technology (BUET), Dhaka 1205, Bangladesh Email: [email protected] Phone: +880255167100 Ext. 6133
1
Abstract Carbonated hydroxyapatite (CHA) was synthesized through a precipitation method using different organic modifiers namely citric acid, acetic acid, glutamic acid and gallic acid. These organic modifiers were used in order to control the nucleation of the synthesis process and crystallinity of the particles. Field Emission Scanning Electron Microscopy (FESEM) and Transmission Emission Microscopy (TEM) data showed that among the samples, CHA produced in the presence of citric acid was the smallest with rod shape (1925 nm) whereas, in the presence of gallic acid CHA particles became largest with flake shape (127-143 nm). Citric acid controlled nucleation by inhibiting crystal growth along a axis whereas, glutamic acid influenced nucleation through inhibition along c axis both resulting in rod shaped particles. Acetic acid also hindered crystal growth, however, to a lesser extent and the particles were flake shaped. CHA particles synthesized with gallic acid connected with each other due to π-π stacking interaction producing the largest particles. The synthesized particles were poorly crystallized with crystallinity ranging from 18.5% to 30%. Fourier Transform Infrared Spectroscopy (FTIR) results confirmed that all the samples were carbonated hydroxyapatite and from Energy Dispersive X-Ray (EDX) it was confirmed that the Ca/P ratio of the samples was around 1.7 indicating non-stoichiometric CHA. It was also found that with the increase in temperature, both the particle size and crystallinity increased. Keywords: Chemical precipitation; Carbonated hydroxyapatite; Organic modifiers; Biomedical applications.
2
1. Introduction Hydroxyapatite (Ca10(PO4)6(OH)2) (HA) is the main inorganic compound of natural bone and can be considered as an excellent bioceramic material [1,2]. However, in a biological environment, the natural bone mineral is always carbonated and poorly crystallized [3]. Hence, efforts have been made to synthesize carbonated hydroxyapatite (CHA) using various methods such as hydrothermal treatment [4], nano-emulsion [5], solgel [6], mechanochemical [7,8] and chemical precipitation [9–14]. Among these methods, as hydrothermal method involves heat treatment, only highly crystallized HA can be synthesized [4]. On the other hand, sol-gel method is cost effective and simple. However, it requires a long time to synthesize CHA [6]. Chemical precipitation is an effective method to synthesize CHA. However, it is always challenging to control the size distribution and morphology of CHA through precipitation method which is very important for drug delivery, biomineralization process and dental applications [15–18]. Although CHA bioceramic has been synthesized through biomimetic precipitation [9,10,19], controlling the synthesis mechanism to yield CHA particles of preferential shape, morphology and crystallinity through the precipitation process has not been studied much. In precipitation process calcium and phosphate salts with a specific Ca/P molar ratio are used as precursors in an aqueous medium which undergoes nucleation. The HA particles thus grown form a solution whose solubility is low and therefore, HA particles precipitate. In the chemical precipitation process, organic modifiers can also be used to control the nucleation of the precipitation process. Organic modifiers work as inhibitors in the growth of HA particles and can influence the nucleation according to their chemical structures. For example, citric acid has been used as organic modifier to mediate the 3
nucleation process and crystallinity of HA [20]. It was found out that the particle size and morphology were dependent on the supersaturated solution containing citric acid. Citric acid mobilized calcium ions as calcium-citrate complex which inhibited the crystal growth process and produced crystallites of ~20 nm size [20]. Initially, citric acid surrounds the calcium ions and forms a supersaturated solution. This inhibits the spontaneous precipitation reaction of calcium phosphate and results in highly amorphous HA particles [21]. These results showed that using organic modifiers could be an effective way to control the synthesis process of HA. Gallic acid is a polyphenol acid and glutamic acid is a biomolecule and both of them can be used as organic modifiers. Acetic acid is another modifier that serves the purpose of comparing its effect on the formation of apatite particles with others. In this respect, a comparative study using these modifiers to synthesize CHA through precipitation process to better understand and control the nucleation process could be useful. This study aims to control the precipitation mechanisms by using different organic modifiers which are easily available such as citric acid, acetic acid, glutamic acid and gallic acid to synthesize CHA of different size distribution and morphology. Along with the organic modifiers, temperature of synthesis process has also been varied to observe the effect of temperature on the synthesized apatite. The varied morphology, size distribution, crystallinity, composition and other properties were investigated through a series of characterization techniques such as Field Emission Scanning Electron Microscopy (FESEM), Transmission Emission Microscopy (TEM), X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-Ray Analysis (EDX) and
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Thermogravimetric Analysis (TGA). Detailed mechanism of the apatite nucleation while using different modifiers has also been proposed. 2. Materials and Methods 2.1. Materials Calcium chloride (CaCl2.2H2O), ortho-phosphoric acid (85% H3PO4), sodium carbonate (Na2CO3), sodium hydroxide (NaOH), acetic acid (100% CH3COOH), citric acid, (C6H8O7), gallic acid (C7H6O5) and glutamic acid (C5H9NO4) were used to synthesize CHA. All of these chemicals were analytical grade and purchased from Merck. 2.2. Methods CHA was synthesized by adopting a previously developed method [10,19]. In brief, 20 ml of 0.1 M CH3COOH was stirred at 80 °C where 10 ml of 0.1 M CaCl2 and 6 ml 0.1 M H3PO4 solution were added drop by drop to maintain a Ca/P ratio of 10:6. Here CH3COOH was used as organic modifier. The solution was kept stirring for 30 minutes. Then, 18 ml of 0.1 M Na2CO3 was added slowly. After stirring for 30 minutes, NaOH solution was added quantitatively to get the pH of the solution 9. The solution was kept stirring for 4 hours during which the synthesized particles precipitated and then the samples were collected. The synthesized apatites were rinsed with ethanol and collected through centrifugation. Subsequently, the collected sample was dried at 50 °C overnight. The synthesized sample was named as CHA-Ace. Similarly, the whole procedure was repeated for other modifiers, namely citric acid, glutamic acid and gallic acid and the synthesized samples were named as CHA-CA, CHA-Glu and CHA-GA, respectively.
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To understand the effect of synthesis temperature, 60 °C and room temperature were also used along with the different organic modifiers. However, glutamic and gallic acid do not dissolve in water in room temperature and glutamic acid does not dissolve in water even at 60 °C [22–24]. Hence for acetic acid, citric acid and gallic acid, samples were prepared at 60 °C and were named as CHA-Ace', CHA-CA' and CHA-GA', respectively. On the other hand, for acetic acid and citric acid two more samples were prepared at room temperature and were named as CHA-Ace-RT, and CHA-CA-RT, respectively. The images of the final solution are given in Fig. S1. 3. Characterization The morphology of the synthesized CHA was observed using FESEM (JEOL-JSM7600F) with an acceleration voltage of 5 kV and a spot size of 8 mm. Before that, the samples were coated with platinum using ion sputtering method by JEOL-JFC 1600 auto fine coater. EDX was performed along with FESEM to obtain the Ca /P ratio of the synthesized CHA. In preparing samples for TEM (JEOL 2100), the samples were dispersed in ethanol and sonicated. Subsequently, they were dripped onto the TEM grid for observation. The lattice parameters and crystallinity of the synthesized CHA particles were analyzed using XRD which was performed by Rigaku Ultima IV, X-Ray diffractometer, Japan. Ni filtered CuKα radiation was used in the range of 2θ = 5–70° with the wavelength of 1.5418 Å. The percentage crystallinities of the samples were calculated byCrystallinity(%) = (1-
υ(112/300) I300
)* 100%
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where υ(112/300) is the intensity of the hollow between the diffraction peaks at (112) and (300) and I300 is the intensity of the diffraction peak at (300) of the samples. The Full Width at Half Maximum (FWHM) was calculated from each of the spectra and that was used to calculate the average crystallite size using the Scherrer equation with a shape factor of 0.94 for rod shaped particles [25]. D=
0.94λ β cos θ
where λ is the X-ray wavelength of the CuKα radiation (=1.5418 Å), β is the FWHM of the major XRD peak and θ is the angle of diffraction for characteristic peak (002) and 0.94 is the shape factor. The lattice parameters were calculated by using Bragg’s law to determine the inter-planar distance for characteristics planes such as (112) and (300). Bragg’s law is formulated asd(hkl) =
λ 2sinθ
where d(hkl) is the inter-planar distance at Miller indices (hkl), λ is the X-ray wavelength of the CuKα radiation (=1.5418 Å) and θ is the angle of diffraction for peaks at (hkl). The d(hkl) values of peak (112) and (300) were used to determine lattice parameters a and c from the relation of hexagonal unit cells1 d
2
4 h2 +hk+k2
= ( 3
a2
)+
l2 c2
where h, k and l are the Miller indices; a and c are lattice constants for a and c axis. To investigate the compositional analysis and presence of some of the specific groups, fourier transform infrared (Schimadzu IR Prestige21, Japan) spectroscopy (FTIR)
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was conducted in the range of 4000–400 cm-1. To prepare FTIR samples, around 200 mg of potassium bromide (KBr) was pressed with 2 mg of dried samples into pellets. TG Analysis was used to analyze thermal properties of the apatites. TGA Q500 (TA Instruments) was used and the range of heating was selected from room temperature upto 900 °C, with a heating rate of 10 °C /min. Nitrogenous environment with a flow rate of 40 ml/min was ensured during the analysis procedure. 4. Results 4.1. Morphology The FESEM and TEM images of the synthesized CHA samples are shown in Fig. 1 and 2, respectively. From these images it can be observed that citric acid and glutamic acid produced CHA particles that resembled the shape of rod with different size distribution by controlling the nucleation mechanism. Both acetic acid and gallic acid produced particles that were connected and thereby resulted in a flake-like morphology and their sizes were observed to be larger than CHA-CA and CHA-Glu. The length, width, equivalent circle area diameter, equivalent circle perimeter diameter and shape of the samples are summarized in Table 1. CHA-GA particles were the largest among all the samples whereas, CHA-CA particles were the lowest. The particle size distribution presented in Fig. 3 shows that the particle sizes are significantly different from each other. Fig. 4 shows the particle size distribution and TEM images of CHA-Ace-RT and CHA-CA-RT samples which signifies that relatively lower synthesis temperature yields smaller particle size. CHA-Ace-RT had aggregated rod-shaped morphology which is different from flake morphology of CHA-Ace synthesized at 80 °C. To further clarify the
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effect of temperature on morphology and size of the particles, TEM images of CHA-Ace', CHA-CA' and CHA-GA' have been given on Fig. S2. From the TEM images it can be observed that CHA-Ace' had flake shaped morphology which was smaller than CHA-Ace and larger than the aggregated nano-rod shape of CHA-Ace-RT. On the other hand, CHACA-RT had almost spherical shaped morphology different from rod shaped morphology of CHA-CA. CHA-CA' had rod shaped morphology smaller than the rod shape of CHA-CA. CHA-GA' had flake shaped morphology similar to CHA-GA however smaller than CHAGA. It can be concluded that lower synthesis temperature produced apatites with smaller particles and mostly similar morphology. The detailed mechanism of the formation of different morphology with different organic modifier and effect of temperature has been discussed thoroughly in ‘discussion’ section. 4.2. XRD The XRD spectra of the as prepared samples with the four different organic modifiers are shown in Fig. 5. All the diffraction peaks of the samples correspond to CHA peaks. The main peaks indexed to (002) at about 26°, broad and overlapped peaks of (211), (112), (300), (202) at approximately 32°, (310) at 40°, (222) at about 47°, (213) at 50° and (004) at 54° indicate that the synthesized materials are CHA [9]. After comparing with biological apatites it was concluded that the synthesized apatites resembled bone like apatite [26]. The JCPDS reference spectra for HA is also given in Fig. S3 which shows that the peaks of the synthesized apatites have broadened. This indicates carbonate incorporation in the apatite crystal and low crystallinity of the apatites [27]. To find the effect of organic modifier, the XRD of a sample synthesized without the modifiers, is given
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in Fig. S3. The highest intensity peak was for CHA-CA indicating preferred orientation of this sample. For CHA-GA, the intensity of the characteristic (002) and (211) plane was lower compared to CHA-Ace and CHA-Glu, indicating relatively lowered preferred orientation of this sample. The crystallinity, average crystallite size, lattice strain values and values of lattice parameters are tabulated in Table 2. The crystallinity of the samples ranged from 18.5% to 30%, which implies that the synthesized samples were poorly crystallized. The crystallinity of CHA-Ace was the highest and that of CHA-GA was the lowest among the samples. CHA-Ace exhibited the sharpest peak among the samples. Sharp peaks result in larger crystals and hence, CHA-Ace had the largest crystallite size and the lowest one was for CHA-CA and then CHA-GA and CHA-Glu, respectively. Broadening of peaks may also occur from carbonate substitution and strain of the unit cells as carbonate substitution hinders crystal growth to some extent and it introduces lattice strain in the crystallites [28]. The value of a axis was the largest for CHA-Glu and that of c axis was the largest for CHA-CA and least for CHA-Ace. CHA-GA hindered crystal growth along a axis as well and crystals grew along c axis. This indicates that glutamic acid had the effect of hindering the growth along c axis and assisting along a axis whereas, citric acid and gallic acid had the opposite effect. Acetic acid also assisted in growth along c axis, however, the effect was less prominent than citric acid and gallic acid as can be seen from its lattice parameters and c/a ratio. The change of growth along c axis for the samples can be determined by the combined effect of organic modifiers used and the substitution of carbonate atoms for phosphate ions. After comparing with the XRD of CHA-without modifiers, it was found out that without modifiers, the synthesized CHA had substantial growth along both the a and c axis which proves that organic modifiers inhibited growth along these axes. Hence, 10
the crystallite size of this particle was also the greatest among all the particles. CHAwithout modifiers also had the highest crystallinity among the samples and by using the organic modifiers, the crystallinity of the particles decreased. The XRD spectra of CHA-Ace', CHA-CA', CHA-GA', CHA-Ace-RT and CHACA-RT are given in Fig. S3. The crystallinity, lattice parameter values, average crystallite size and lattice strain of CHA-Ace', CHA-CA', CHA-GA' and CHA-without modifiers are given in Table S1. The XRD spectra of CHA-CA-RT has very low intensity and broad peaks unlike the other samples which means that this sample was highly amorphous. XRD of CHA-CA' is not as broad as CHA-CA-RT and this apatite had higher crystallinity than CHA-CA-RT and lower than CHA-CA. Thus it can be observed that lower synthesis temperature produced relatively poor crystallized particles. The variation of crystallinity with the change in temperature for acetic and citric acid as modifier is given in Fig. S4. CHA-CA-RT had smallest crystallite size and CHA-CA had the largest. Similar effect of temperature on crystallite size was observed for other organic modifiers as well. 4.3. FTIR Fig. 6 demonstrates the FTIR spectra of the characteristic peak of CHA for the synthesized apatite. The intense peaks noted around 561 and 601 cm−1 were observed due to the bending modes of PO43− ions in CHA [29,30]. The bands at 1036 and 1110 cm−1 are the bands which represent the basic characteristic of asymmetric (P-O) stretching vibration of PO43− ion in CHA [30]. The peak at 1411 cm−1 represents the CO32- ions [31]. The carbonate bands of the samples confirm that the synthesized apatites were carbonated HA. In addition to that the carbonate bands of the samples are around 1410 cm-1 which indicate
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the synthesized samples to be CHA B-type [32]. The FTIR spectra of CHA-Ace, CHAAce', CHA-CA, CHA-CA' and CHA-GA' are given in Fig. S5.
4.4. EDX From the elemental analysis by EDX, Ca/P ratio was obtained and the results are summarized in the Table 2 and Fig. 7. As biological apatite has Ca/P ratio between 1.6-1.87 [33], all the samples are biological in nature, although non-stoichiometry can be observed. CHA-CA, CHA-GA has higher Ca/P ratio which points out substantial phosphate substitution by carbonate ions [34]. These samples also have higher lattice strain indicating greater amount of carbonate substitution. The EDX of CHA-Ace-RT and CHA-CA-RT are given in Fig S6. The Ca/P ratio of CHA-CA-RT is lower than CHA-CA and thereby had lower crystallite size. CHA-Ace-RT has Ca/P ratio of 1.5 which is lower than that of CHA-Ace, however, its crystallite size was not larger and its lattice strain was not lower. This could be due to the effect of low temperature that was more dominating than carbonate substitution and the crystal growth was hindered. This resulted in smaller crystallite size of CHA-Ace-RT than CHA-Ace. 4.5. TGA Thermal analysis of the CHA samples are presented in Fig. 8. The TGA curves for all the samples showed gradual mass loss due to the removal of adsorbed water and decomposition of carbonate components. At around 300 °C mass loss was observed due to the dehydration of the synthesized samples and loss of physically adsorbed water molecules [35–37]. At around 400 °C, only the mineral phase of CHA was left. With the increase in
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temperature from 400 to 800 °C, the rest of the mass was lost because of the decomposition of carbonate [35–37]. The carbonate content of the apatites is given in Fig. S8 which shows that with the increase in synthesis temperature the carbonate content decreases for a specific organic modifier. The carbonate contents of the samples are given in Table S2. 5. Discussion 5.1. Effects of organic modifiers on CHA particles Fig. 9 demonstrates the schematic of the possible formation of CHA using citric acid, acetic acid, glutamic acid and gallic acid as organic modifiers. Citrate ion plays an inhibiting role on the nucleation of CHA by chelating with three calcium ions and limiting their availability on the solution [18,38,39]. After the addition of precursor ions on the citric acid solution, calcium citrate complex functioned as the nucleation sites for the growth of CHA crystals. The normal precipitation reaction between the precursors was inhibited and calcium phosphate precipitation was limited [18,38,39]. There was a decrease in the supersaturation of the solution which resulted in the low precipitation of calcium phosphate [38] This slowed the nucleation process of CHA to a great extent. As the solution was kept stirring, calcium ions were slowly released from the calcium citrate chelation complex and CHA crystals started to form. Besides this effect on CHA particles, citrate ions also affected the crystal growth of CHA [40]. They adsorbed preferentially to its hexagonal unit cell surface by its COO- group mainly on the rectangular facet ac or bc of the unit cell. These two faces are rich in calcium ions than the ab facet. The adsorption process made the surface of the unit cell negatively charged because of the two free COOgroups of the citrate ion which repelled other crystals inhibiting facet to facet crystal
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aggregation [40]. Thus the crystal growth at the a and b axis was hindered and crystals grew along the c axis (Fig. 9(b.i)) [41,42]. From the XRD analysis it is apparent that the c/a ratio for CHA-CA is higher than other samples indicating preferential growth along the c axis. This made the particles rod-shaped and it was the smallest out of the samples. As the synthesized CHA was B type in nature, phosphate ions were substituted by carbonate ions and this exchange process also resulted in small particles by inhibiting growth of crystals along all the axis except c. Thus crystals grew along this direction due to the added effect of carbonate substitution. TGA result also shows that CHA-CA had phosphate ions substituted by carbonate ions. Carbonate substitution also creates strain on lattice unit cells and it is clear from Table 2 that strain of CHA-CA was the second highest among all the samples. The XRD peaks of CHA-CA were also broader due to the combined effect of citric acid as organic modifier and carbonate substitution that resulted in small crystals. The effect of acetic acid on the growth of CHA is similar to the effect of citric acid. As acetic acid is a monoprotic acid, it chelated to only one calcium ion per acetate ion. This limits calcium availability for CHA particle growth to a lesser extent and nucleation was less controlled by acetic acid than citric acid. The calcium acetate compound acted as nucleation sites and growth of CHA crystals occurred in these sites. The inhibiting effect of acetate ion was mainly on the nucleation rather than the crystal growth and the particles were larger than CHA-CA. The c/a ratio of CHA-Ace is smaller than CHA-CA. This indicates that acetic acid did not inhibit growth along a or b axis to the same extent as citric acid did (Fig. 9(b.ii)). Crystal agglomeration was not hindered by negatively charged crystals like CHA-CA because there were no leftover COO- ions after the adsorption of acetate ions. That is why the resulting particles agglomerated and resembled flake like 14
CHA particle which is apparent from FESEM and TEM images of Fig. 1 and 2, respectively. The carbonate substitution was less as can be found from TGA result and crystal growth inhibition due to this effect was also less than CHA-CA which is evident from XRD by sharp peaks of CHA-Ace. Unlike citric acid and acetic acid, glutamic acid mainly played role on the CHA crystal growth rather than on the nucleation of CHA in the solution. Glutamic acid has two COO- sides and one NH3+ side. It made a polydentate complex with calcium ions through its NH3+ ions to a lesser amount than acetic acid and citric acid (Fig. 9(b.iii.1). This phenomenon did not inhibit the nucleation of CHA to a great degree and thus nucleation occurred spontaneously [43]. Glutamic acid adsorbed on both the (001) and (100) surface of the CHA crystal. The adsorption on the (001) face was due to NH3+ positioning on the vacant calcium sites mainly. Additionally, the adsorption was due to the electrostatic attraction between the NH3+ group and carbonate, phosphate and hydroxyl groups. However, the majority of adsorption occurred due to the substitution of NH3+ in the vacant calcium sites of the crystal. Glutamic acid also adsorbed on the (100) crystal face by electrostatically attracting the calcium ions of the crystal by its COO- group. However, the adsorption amount was greater in (001) surface compared to (100) because of the presence of greater number of calcium ions in the (001) [43–45]. Glutamic acid being an acidic amino acid, adsorbed preferentially on the positively charged surface of CHA as shown by previous works [45,46]. To determine the inhibitory effect of glutamic acid, geometrical factor must also be considered. Adsorbed glutamic acid has an axis of free rotation perpendicular to the adsorbed surface of the crystal. The volume resulted due to the rotation
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can be described as a cone that has a radius of 5.23 Å as found previously [43]. The projected area due to the rotation covered up a large portion of the crystal structure and hence the crystal could not further accommodate calcium ions and thus development of crystals stopped. The (001) surface accommodated higher amount of glutamate ions than the (100) surface and hence, crystal structure could not complete in this direction (Fig. 9(b.iii.2)) [43–45]. This resulted in an inhibition of crystal growth along the c axis unlike the effect of citric acid [43]. The crystals grew in a or b direction resulting in rod shaped particles and they were observed in the FESEM and TEM images in Fig. 1 and 2, respectively. XRD data show that the value of a axis is the greatest for CHA-Glu and c/a ratio is the lowest which also indicates the crystal growth along a axis being more profound. The c axis value should have been smaller compared to other samples. However, Table 2 points out that CHA-Glu had some growth along c axis as well. This took place because of carbonate substitution which promoted growth along c axis as oppose to glutamic acid. The final value of a and c axis resulted due to the combined and opposing effect of glutamic acid adsorption and carbonate substitution. Between them, the effect of glutamic acid dominated and determined the size of CHA-Glu particles. CHA-Glu particles were larger than CHA-CA because citric acid had more inhibition on solution chemistry controlling the nucleation rate unlike Glu. CHA-GA particles had flake like morphology although particles were larger than CHA-Ace. As gallic acid has COO- group, it bonded to the calcium ions of the solutions by electrostatic attraction and thus acted as a nucleation site. It inhibited crystal growth in a or b axis to some extent and crystals grew along c axis. Table 2 points out that c axis of CHA-
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GA grew more than CHA-Ace and CHA-Glu. Thus particles started to grow and after the growth of CHA particles, they might have bonded with the gallic acid molecules by the hydroxyl groups of each through hydrogen bonding between the hydroxyl groups. Each of the CHA-GA units might have connected to each other via the intermolecular force or the π-π stacking interaction between its aryl rings and CHA-GA particles assembled (Fig. 9(b.iv)) [47,48]. The interconnection between the CHA-GA molecules resulted in a flake like morphological shape evidenced by the FESEM and TEM images (Fig. 1 and Fig. 2). All the synthesized samples were poorly crystallized which is similar to the nature of biological apatites. The crystallization activation temperature plays a key role in developing crystallinity of CHA particles at different synthesis temperature which was affected by the organic modifiers. CHA-Ace showed the highest crystallinity among the samples because at 80 °C, the synthesis temperature overcame the activation temperature or was closer to it. CHA-CA had low crystallinity as citric acid chelated to Ca2+ to a great amount resulting in random growth of CHA particles. CHA-GA had the lowest crystallinity among the samples. Because of the presence of gallic acid, the crystallization activation temperature could not be reached easily as the heat was used by the system to make the gallic acid particles soluble in the water. The remaining heat was not enough to reach the crystallization activation temperature which resulted in the lowest crystallinity of 25% among the samples. CHA-Glu also exhibited similar effects, however, the solubility of glutamic acid is greater than gallic acid at the same temperature thus less temperature was needed to maintain the solubility of glutamic acid [22–24]. Hence, the temperature was closer to the crystallization activation temperature and crystallinity of 27.7% resulted. Low
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crystalline apatites improve the surface activity of the implant [49]. It can also dissolve more easily than highly crystallized ones which is beneficial to drug delivery system and in clinical applications where absorption-recrystallization is wanted. 5.2. Effect of temperature on CHA particles Synthesis temperature had important effects on the size and morphology of the CHA samples. With the increase in temperature, the rate of collision between two or more molecules increased as the intermolecular distance reduced because of the increased mobility of the particles. Thus, effective collisions of the reactant ions increased the size of the nuclei with increasing temperature. Hence, the particle size increased with temperature as can be seen from TEM and particle size distribution analysis (Fig. 4) of CHA-Ace, CHA-Ace-RT, CHA-CA and CHA-CA-RT and also from Table 1. The size of CHA-Ace was greater than CHA-Ace' and almost double than CHA-Ace-RT showing the effect of synthesis temperature. CHA-Ace' had flake shaped morphology with similar particle forming mechanism as CHA-Ace and it was smaller than CHA-Ace because of the decreased amount of effective collision. CHA-Ace-RT had aggregated nano rod shaped morphology. This indicates that as the synthesis temperature increased, the morphology changed from aggregated nano rod shape to flake shape and particle size increased due to more collision among the precursors. CHA-CA, on the other hand, exhibited slight increase in size than CHA-CA' and CHA-CA-RT as citric acid chelated to calcium ion more efficiently than acetic acid. This resulted in a decreased mobility of calcium ion. CHA-CA' and CHA-CA-RT had somewhat rod shaped particle and almost spherical particles, respectively. Due to lower synthesis temperature and high chelation effect of citric acid, the collision rate of the reactants was the lowest. Thus the growth of CHA-CA-RT particles 18
was almost equal in every direction which resulted in particles resembling spheres. CHACA' particles were of nano rod shaped similar to CHA-Ace. However, the particle size was smaller than CHA-CA indicating the effect of temperature. Also, the crystallite size decreased with the increase in carbonate content and CHA-CA-RT had the smallest crystallite size among CHA-CA, CHA-CA' and CHA-CA-RT because it had the highest carbonate content. Similar effect of temperature was also found on the morphology and shape of CHA-GA and CHA-GA'. The particle size increased with the increase in temperature with similar particle forming mechanism. Lower synthesis temperature also yielded lower crystallinity of the samples. Crystallinity of CHA-CA-RT is even lower than CHA-CA' and CHA-CA because of lower synthesis temperature and high chelation effect of citric acid. Lower temperature had similar effects on the crystallinity of CHA-Ace-RT, CHA-Ace' and CHA-GA' as well. 6. Conclusion A precipitation process has been developed to synthesize CHA with controlled size, morphological shape and crystallinity. Organic modifiers which controls the nucleation process of the synthesis procedure were used in this regard. The largest particle size obtained was CHA-GA (127-143 nm) and CHA-CA was the lowest particle (19-25 nm) in size. Significant changes in morphology were also obtained. CHA-CA and CHA-Glu showed rod like morphology whereas, CHA-Ace and CHA-GA exhibited flake morphology. All the samples were poorly crystallized with crystallinity values ranging from 18.5% to 30% resembling amorphous structure of natural bone. The samples were also B-type carbonated apatite which was confirmed by FTIR. The Ca/P ratio was around
19
1.7 obtained from EDX indicating non-stoichiometric CHA. Furthermore, different synthesis temperatures were used to understand the effect of temperature on the synthesis and CHA crystallization process. Samples synthesized at room temperature were smaller than the samples synthesized at higher temperature with the same modifier. This indicates that increased temperature results in larger particles. Morphologically, CHA-Ace-RT had aggregated rod-shaped particles whereas, CHA-CA-RT particles were almost spherical shaped. It was also found out that crystallinity also increased with increased synthesis temperature. Thus, in this study, using inexpensive and readily available organic modifiers, CHA nanocrystals of different size, morphology and crystallinity were obtained with a detailed understanding of the precipitation mechanism. The synthesized CHA particles could be useful in preventive, restorative and regenerative dentistry, structural biomaterial for prosthetic applications, bone-tissue engineering and drug delivery systems. 7. Acknowledgement The project was funded by University Grants Commission (UGC), Bangladesh under the grant no. 6(76) UGC/S&T/CHEMICAL-25/2018/3282. 8. References [1]
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Fig. Captions Fig. 1. FESEM images of samples (a) CHA-Ace particles with flake like morphology, (b) CHA-CA particles resembling somewhat rod shaped morphology, (c) CHA-GA particles with flake shaped morphology and (d) CHA-Glu particles having rod shaped morphology Fig. 2. TEM images of samples (a) CHA-Ace with flake-like morphology, (b) CHA-CA particles having somewhat rod shape, (c) CHA-GA particles with flake-like morphology and (d) CHA-Glu particles with rod shaped morphology Fig. 3. Particle size distribution of all the synthesized apatite. CHA-GA with the highest particle size distribution with 127-144 nm and CHA-CA with the lowest particle size distribution with 19-25 nm. All samples are significantly different compared to CHA-CA and the differences are denoted by * (p<0.05) Fig. 4. Particle size distribution and respective TEM images of the samples CHA-Ace, CHA-Ace-RT, CHA-CA, CHA-CA-RT showing that higher synthesis temperature yields larger particles. Samples with significant size differences are denoted by * (p<0.05)
28
Fig. 5. XRD patterns of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid at synthesis temperature of 80 °C. All the samples exhibited characteristics HA at about 26°, 32°, 40°, 47°, 50° and 54°. Fig. 6. FTIR spectra of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid and at synthesis temperature of 80 °C. All the samples have the characteristics CHA peaks. Fig. 7. EDX data of samples (a) CHA-Ace with Ca/P of 1.66, (b) CHA-CA with Ca/P=1.88, (c) CHA-GA with Ca/P=1.71, (d) CHA-Glu with Ca/P=1.68; Higher values of Ca/P indicate that PO43− had been substantially substituted by CO32- ions. Fig. 8. TGA curves of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid. All the samples have a mass loss due to water removal upto 200-300 °C. The next portion of the curve upto 800 °C shows decomposition of HPO42- and CO32-. Further increase in temperature shows more weight loss in CHA-CA and CHA-Glu whereas, slight mass gain in CHA-Ace and CHA-GA. Fig. 9. (a) CHA synthesis process, (b) (i) Citric acid inhibiting particle growth by chelating to Ca2+ ions and working as nucleation sites by slowly releasing Ca2+; then adsorbing onto (001) face and producing negatively charged crystal hindering crystal aggregation along a and b axis. (ii) Acetic acid producing similar effect but to a lesser extent and no adsorption effect like citric acid. (iii.a) Glutamic acid making polydentate chelation with Ca2+ by two NH3+ groups limiting its availability. (iii.b) Glutamic acid mostly inhibiting crystal growth along c axis by preferentially adsorbing to this face in the vacant Ca2+ sites by NH3+ groups
29
and preventing Ca2+ ions to fill the vacant sites due to effective volume of rotation. (iv) Gallic acid hindering crystal growth by binding to Ca2+ and aggregating to large grains by π-π stacking interaction between its aryl rings after forming CHA-GA crystal. Table Captions Table 1. The size distribution (nm) and Morphological shape and Ca/P ratio of the synthesized CHA samples Table 2. The Crystallinity (%), Lattice parameters (nm), c/a ratio, Strain (%) and Ca/P ratio of the synthesized CHA samples
30
(a)
CHA-Ace
(b)
CHA-CA
(c)
CHA-GA
(d)
CHA-Glu
Fig. 1. FESEM images of samples (a) CHA-Ace particles with flake like morphology, (b) CHACA particles resembling somewhat rod shaped morphology, (c) CHA-GA particles with flake shaped morphology and (d) CHA-Glu particles having rod shaped morphology
31
(a)
CHA-Ace
50 nm
CHA-CA
50 nm
50 nm
(c)
(b)
CHA-GA
(d)
CHA-Glu
50 nm
Fig. 2. TEM images of samples (a) CHA-Ace with flake-like morphology, (b) CHA-CA particles having somewhat rod shape, (c) CHA-GA particles with flake-like morphology and (d) CHA-Glu particles with rod shaped morphology
32
*
CHA-GA
* * CHA-Ace CHA-Glu CHA-CA
Fig. 3. Particle size distribution of all the synthesized apatite. CHA-GA with the highest particle size distribution with 127-144 nm and CHA-CA with the lowest particle size distribution with 1925 nm. All samples are significantly different compared to CHA-CA and the differences are denoted by * (p<0.05)
33
*
* 100 nm
CHA-Ace
50 nm 50 nm
CHA-Ace-RT
CHA-CA
100 nm
CHA-CA-RT
Fig. 4. Particle size distribution and respective TEM images of the samples CHA-Ace, CHA-AceRT, CHA-CA, CHA-CA-RT showing that higher synthesis temperature yields larger particles. Samples with significant size differences are denoted by * (p<0.05)
34
(211) (112) (300) (202)
(002)
(310)
(222) (213) (004)
CHA-Glu
CHA-GA
CHA-CA CHA-Ace 5
10
15
20
25
30
35
40
45
50
55
60
65
70
2theta (deg)
Fig. 5. XRD patterns of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid at synthesis temperature of 80 °C. All the samples exhibited characteristics HA peaks at about 26°, 32°, 40°, 47°, 50° and 54°.
35
CO32CHA-Glu PO4 3− PO4 3− CHA-GA
CHA-CA
CHA-Ace
2000
1800
1600
1400
1200
1000
800
600
400
Wavenumber (1/cm)
Fig. 6. FTIR spectra of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid, and glutamic acid at synthesis temperature of 80 °C. All the samples have the characteristics CHA peaks.
36
(a)
CHA-Ace
(b)
CHA-CA
(c)
CHA-GA
(d)
CHA-Glu
Fig. 7. EDX data of samples (a) CHA-Ace with Ca/P of 1.66, (b) CHA-CA with Ca/P=1.88, (c) CHA- GA with Ca/P=1.71, (d) CHA-Glu with Ca/P=1.68; Higher values of Ca/P indicate that PO43− had been substantially substituted by CO32- ions.
37
100
CHA-Ace CHA-CA
95
CHA-GA
Weight loss (%)
CHA-Glu 90
85
80
75 0
100
200
300
400
500
600
700
800
900
Temperature (℃)
Fig. 8. TGA curves of the synthesized CHA samples with organic modifiers acetic acid, citric acid, gallic acid and glutamic acid.
38
(a) CHA synthesis process
(b) Crystal forming mechanism in the presence of organic modifiers Citrate
i. Precursor Acetate
ii. Precursor
Glutamate (iii.1)
iii. Precursor
(iii.2)
Gallic
iv. Precursors
Fig. 9. (a) CHA synthesis process, (b) (i) Citric acid inhibiting particle growth by chelating to Ca2+ ions and working as nucleation sites by slowly releasing Ca2+; then adsorbing onto (001) face and producing negatively charged crystal hindering crystal aggregation along a and b axis. (ii) Acetic
39
acid producing similar effect but to a lesser extent and no adsorption effect like citric acid. (iii.a) Glutamic acid making polydentate chelation with Ca2+ by two NH3+ groups limiting its availability. (iii.b) Glutamic acid mostly inhibiting crystal growth along c axis by preferentially adsorbing to this face in the vacant Ca2+ sites by NH3+ groups and preventing Ca2+ ions to fill the vacant sites due to effective volume of rotation. (iv) Gallic acid hindering crystal growth by binding to Ca2+ and aggregating to large grains by π-π stacking interaction between its aryl rings after forming CHA-GA crystal. Table 1. The Length (nm), Width (nm), Equivalent circle area diameter (nm), Equivalent circle perimeter diameter (nm) and Morphological shape of the synthesized CHA samples Sample
Length (nm)
Width (nm)
Equivalent circle area diameter (nm)
Equivalent circle perimeter diameter (nm)
Morphological shape
CHAAce CHA-CA
55-68
22-50
14-20
19-27
Flake shaped morphology
19-25
13-20
9-14
14-19
CHA-GA
127-143
56-122
40-47
88-101
Somewhat rod shape with elongation along c axis Flake shaped morphology
CHAGlu CHAAce-60 CHACA-60 CHAGA-60 CHAAce-RT
33-43
16-31
13-18
23-32
29-40
20-33
11-16
21-29
14-23
9-17
7-12
11-17
42-68
23-55
18-26
31-48
23-32
18-26
14-20
19-27
Somewhat rod shape with elongation along c axis Somewhat rod shaped with elongation along c axis Aggregated nano rod shape
CHACA-RT
15-19
10-14
10-13
12-16
Almost spherical shape
Rod shaped with elongation along a or b axis Flake shaped morphology
40
Table 2. The Crystallinity (%), Lattice parameters (nm), c/a ratio, Strain (%) and Ca/P ratio of the synthesized CHA samples Sample
Crystallinity (%)
CHA-Ace
30
CHA-CA CHAGA CHA-Glu CHAAce-RT CHACA-RT
Lattice parameters (nm) a=b axis c axis 0.909 0.651
c/a
Crystallite Size(nm)
Strain (%)
Ca/P ratio
0.716
26
0.62
1.66
28.6
0.917
0.677
0.738
16
1.02
1.88
25
0.926
0.673
0.727
21
1.78
1.71
27.7
0.93
0.66
0.709
25
0.64
1.68
25
0.91
0.65
0.714
20
0.79
1.5
18.5
0.92
0.677
0.735
12
1
1.82
41