BIBECHANA 18 (1) (2021) 83-90 83 BIBECHANA ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher: Department of Physics, Mahendra Morang A.M. Campus, TU, Biratnagar, Nepal Facile methods of preparing pure hydroxyapatite nanoparticles in ordinary laboratories Gokarna Pandey1, Kedar Nath Dhakal1, Ajaya Kumar Singh2, Suresh Kumar Dhungel3, Rameshwar Adhikari1,4 * 1 Central Department of Chemistry, Tribhuvan University (TU), Kirtipur, Kathmandu, Nepal 2 Department of Chemistry, Govt. V. Y. T. PG Autonomous College, Durg, Chhattisgarh, India 3 Nepal Academy of Science and Technology (NAST), Lalitpur, Nepal 4 Research Centre for Applied Science and Technology (RECAST), TU, Kirtipur, Kathmandu, Nepal *Email: ram.adhikari.tu@gmail.com Article Information Received: June 1, 2020 Accepted: July 2, 2020 Keywords Hydroxyapatite Nanoparticles FTIR spectroscopy X-ray diffraction Bioceramic ABSTRACT The present work features some simple methods of preparing pure hydroxyapatite nanoparticles (nano-HAp), a useful biomaterial, via various wet chemical methods and using biogenic sources such as eggshells and animal bone. The nano-HAp hence prepared was subsequently characterized by FTIR spectroscopy and XRD technique. The FTIR spectra confirmed the presence of PO4 3- and OH- ions as major functional groups in the prepared material, with some additional peaks implying the presence of CO3- ions and adsorbed water molecules. The XRD patterns, in agreement with the JCPDS 09-432 data, demonstrated the crystalline nature of the nano-HAp and confirmed the phase as being apatite. The average grain diameter of the nanocrystallites was found in the range of 15-30 nm. The preparatory methods depicted herein can be easily employed in ordinary high school laboratories having basic facilities such as availability of distilled water, some handy glasswares, common laboratory chemicals and instruments such as balance, hot air oven and furnace. DOI: https://doi.org/10.3126/bibechana.v18i1.29600 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons.org/licenses/by-nc/4.0/ 1. Introduction Hydroxyapatite (HAp), with the chemical formula Ca10(PO4)6(OH)2, is one of the most attractive bioceramics for several biomedical applications due to its osteoconductive and antimicrobial properties and biocompatibility as well as lack of toxic and inflammatory responses [1-4]. HAp is the major mineral constituent of human bone accounting for approximately 65 weight fraction of the latter. Bone is, in fact, a nanocomposite material comprising HAp mineral and collagen proteins, its properties thus largely being dependent on the nanoscale morphology. The apatite crystals are the essential parts of tooth and bone of all the vertebrates. When used as implants, the synthetic HAp is able to provide a scaffold or template for new bone regeneration and growth, and supports the osteoblast adhesion and proliferation [3,4]. http://nepjol.info/index.php/BIBECHANA mailto:ram.adhikari.tu@gmail.com https://doi.org/10.3126/bibechana.v18i1.29600 https://creativecommons.org/licenses/by-nc/4.0/ Gokarna Pandey et al. / BIBECHANA 18 (1) (2021) 83-90 84 Such an important bio-ceramic can be synthesized either from natural sources such as bone, corals, eggshells, body fluids, etc. or via various chemical routes including sol-gel method, mechano-chemical precipitation, hydrothermal technique, flame spray and microwave assisted method, and others [2-16]. The major mineral constituents of human bone are calcium and phosphorous with traces of other substances [17,18]. However, a considerable amount of carbonate (CO3 2-) also appears to be present in the bone. The presence of CO3 2- ions in biological apatite is of great importance because it is the main source of lattice distortion, creating micro-stresses and crystalline defects in its surrounding area which, in turn, play a vital role in its solubility. Thus, synthetic apatite should exhibit small particle size and presence of CO3 2- [18]. It has been demonstrated that the nanocrystalline HAp (nano-HAp) exhibits enhanced biocompatibility, bioactivity and mechanical performance over microcrystalline or bulk HAp, which has been attributed to their unique quantum confinement effects and large surface area to volume ratio. Thus the nano-HAp can be used in designing superior biocompatible coatings for the implants and in developing high strength composite materials [4,12,17,18]. With the dawn of nanotechnological revolution during last few decades, and in context of unparalleled opportunities offered by these technological advances also in developing countries, the facile methods for nanomaterials synthesis are of tremendous scientific importance. There is overwhelming interest in the versatile routes for the synthesis of such materials among young scientists in their early career. Taking into account the vigor of scientists of new generation towards nanomaterials and their technological implications, the objective of present work has been to demonstrate simple methods of preparing nano-HAp in ordinary high school laboratories also using wet chemical methods by utilizing biogenic waste as resources. As evidence for the success of the methods used, the characterization of the materials by Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) are reported here. 2. Experimental Section 2.1 Materials Analytical grade calcium nitrate tetrahydrate, di- ammonium hydrogen phosphate, ammonium hydroxide (25%), ortho-phosphoric acid (85%), urea, calcium oxide, ethanol and ethylene-diamine- tetraacetic acid (EDTA) were purchased from Merck India Ltd. Egg shells and adult buffalo femoral bones were collected from local market in Kathmandu. All solutions were prepared in distilled water. 2.2 Preparation of nano-HAp Four different methods used for the preparation of nano-HAp were sol-gel method, precipitation method using calcium oxide and ortho-phosphoric acid as starting materials, precipitation method using aqueous slurry comprising of egg-shells powder and ortho-phosphoric acid solution, and calcination of animal bone. (a) Sol-gel Method This method was adopted from different research articles [11,12,19,20] published elsewhere. In this method, 10.8 g of calcium nitrate tetrahydrate, Ca(NO3)2.4H2O and 3.89 g of diammonium hydrogen phosphate, (NH4)2HPO4 were separately dissolved in 50 mL of water taken in a 100 mL conical flask for each. Phosphate solution was then added drop-wise to the calcium nitrate solution maintained at temperature of 75 °C. The pH was maintained at 11 throughout the experiment using ammonia solution. The resulting solution was continuously stirred for 2 hrs and the product was allowed to cool and precipitate for 24 hrs. The aged gel thus obtained was filtered and washed with doubly distilled water and ethanol several times till the filtrate was neutral. Finally, the resulting clear white powder was stored in a hot air oven Gokarna Pandey et al. / BIBECHANA 18 (1) (2021) 83-90 85 maintained at 85 °C for 12 hrs for further analyses. The reaction can be represented as: 10Ca(NO3)2 + 6(NH4)2 HPO4 + 8NH4OH Ca10(PO4)6(OH)2 + 20 NH4NO3 + 6H2O (b) Precipitation Method This method was adopted on the basis of works reported elsewhere [2,5,17,21,22]. Calcium oxide (CaO), ortho-phosphoric acid (H3PO4), and ammonium hydroxide (NH4OH) were used as starting substances for this method. First of all, 7.955 g of dry CaO powder was added to 50 mL of water taken in a 100 mL conical flask and vigorously stirred at 20 °C for 24 hrs to form aqueous suspension of Ca(OH)2. Then 9.732 g of 85% H3PO4 was slowly added to the solution at a rate of about 1.5 mL/min. The reactants were further stirred for 24 hrs to attain the maturation stage, under constant stirring. The pH of the solution was maintained at 10 using ammonia solution. The precipitate was filtered, washed and dried in hot air oven at 85 °C for 12 hrs. The chemical reaction may be written as: 3CaO+ 3H2O 3 Ca(OH)2 3Ca(OH)2+3H3PO4 3 CaHPO4+6 H2O 5Ca(OH)2+ 3H3PO4 ½ Ca10(PO4)6(OH)2+9H2O 2Ca(OH)2+3CaHPO4+6H2O ½ Ca10(PO)6(OH)2+9 H2O (c) Eggshells as Starting Material An eggshell that primarily comprises of calcium carbonate (CaCO3) can be converted into calcium oxide (CaO) via calcination, which can be then used as an inexpensive raw material for the HAp synthesis. Following the methods reported in elsewhere [6,23-25], the chicken eggs shells were collected and cleaned with tap water followed by washing with distilled water and acetic acid solution several times and dried and powdered using a grinder. The powder was hence subjected to thermal treatments in a furnace in three stages: firstly, at 150 ºC for 5 hrs to remove water and organic impurities, secondly, at 500 ºC for 3 hrs to remove any remaining organic residue, and finally at 1000 ºC for 2 hrs to transform the eggshell powder to CaO. The powder was then transformed into HAp in the phosphate solution using the following procedure. 5 g of calcium oxide was dissolved under constant stirring in 6 g of ortho-phosphoric acid. Then 0.4 g of urea was added along with 0.8 g of EDTA and 100 mL of water. The pH of the solution was adjusted to 10 using ammonia solution at 75 ºC. The reaction mixture was further stirred for half an hour and then kept relaxed for 24 hrs to allow nano-HAp to grow. The slurry was centrifuged at 5000 rpm for 2 min and the resulting sludge was thoroughly washed with distilled water until neutral pH. The precipitate was dispersed in 100 mL of ethanol and the mixture was sonicated for 1 hr. The residue was dried in air for 24 hrs and then at 85 ºC in an oven for 12 hrs. The reaction may be written as: CaCO3 CaO+CO2 10CaO+10H2O+6H3PO4 Ca10(PO4)6(OH)2+18 H2O (d) Animal Bone as Starting Material This method was adopted from different research papers [16,26-28]. Briefly, the femoral bones of buffalo was collected from butcher shop that were then cut into small pieces and boiled in water for 3 hrs. The bone was then washed with acetone for several times to remove fats and other impurities and hence dried at 160 °C for 48 hrs. The pieces were ground to powder with particle diameter less than 450 µm. The powder was treated with 4M NaOH (solid-liquid weight ratio of 1.40) for deproteinization. The content was heated at 250 °C for 5 hrs. The defatted and deproteinized powder was calcined at 700 °C for 6 hrs in a muffle furnace to obtain grayish white powder of HAp. The powder was further calcined at 1000 °C for another 6 hrs to study the effect of calcination temperature. 2.3 Methods for characterization Gokarna Pandey et al. / BIBECHANA 18 (1) (2021) 83-90 86 4000 3500 3000 2500 2000 1500 1000 500 0 20 40 60 80 100 T r a n s m it ta n c e ( % ) Wavenumber (cm -1 ) Adsorbed H2O O-H O-H CO3 2- CO3 2- PO4 3- P-O PO4 3- C=O (Carbon dioxide) Fourier Transform Infrared (FTIR) Spectroscopy The FTIR analysis was performed by using a Prestige-21 FTIR Spectrometer (Shimadzu Company, Japan). The spectra were collected in the range of 4700-400 cm−1 using KBr pellet method. X-Ray Diffraction (XRD) The crystal phase and structure of the samples were determined by Bruker D2 Phaser X-ray diffractometer with a monochromatic CuKα radiation source (λ = 0.15418 nm) with 2θ angles ranging from 20° to 80°. The accelerating voltage of 30 kV and emission current of 10 mA were used. 3. Results and Discussion The phase purity and presence of major functional groups of the HAp powder were attested by FTIR analyses. Fig. 1 shows the FTIR spectrum of the HAp powder chemically synthesized by sol-gel method. The spectrum is dominated by the typical PO4 3- bands of crystalline apatite phase characterized by the peaks representing triply degenerate υ3 (PO4 3-) asymmetric mode centered at 1021 cm-1 and 1087 cm-1 (shoulder), non-degenerate symmetric stretching mode υ1 (PO4 3-) at 962 cm-1 and components of the triplet of υ4 (PO4 3-) bending modes at 560 cm-1 and 470 cm-1 [2,6,12,21,23,29,30]. The broad band located between 3200-3600 cm−1, together with weak and broad band around 1627 cm−1 of H-O-H bending mode indicates the presence of absorbed water. The weak peak located at 3570 cm−1 corresponds to the vibrations of OH− ions in the HAP lattices [6,24,26]. The two peaks centered near 1490 and 1426 cm-1 are assigned to the ν3 vibration mode and the weak peak at about 870 cm-1 is due to the ν2 vibration mode of free, planar CO3 2- ions (group symmetry D3H) [12,20,23-25]. The weak band at 2360 cm-1 can be attributed to C=O bonds from adsorbed atmospheric carbon dioxide [25]. Fig. 1: FTIR spectrum of HAp powder synthesized by sol-gel method. In summary, the FTIR spectrum shown in Fig. 1 verifies, in consistence with several literatures, the success of the chemical synthesis of the HAp powder. Fig. 2 shows a closer look on structure of the raw eggshell and the HAp. Fig. 2: FTIR spectra of raw eggshells powder compared with the HAp from eggshells. The IR spectrum of the raw eggshell shows the strong carbonate peaks centered at 1490 cm-1 and 870 cm-1, and the sharp peak located at 710 cm-1 , which represents the absorbance by Ca-O bond [31]. The weak band at 2360 cm-1 is attributed to C=O bonds from carbonate [25,32] while the broad band at around 2863 cm-1 appears due to OH stretching vibration and that around 3600 cm-1 arises due to N-H bonds due to proteins present in the raw eggshells. 4000 3500 3000 2500 2000 1500 1000 500 T ra n s m it ta n c e ( a .u ) Wavenumber (cm -1 ) b) Raw eggshell a) HAp from eggshells O-H adsorbed H 2 O O-H CO3 2- PO 4 3- PO 4 3- P-O CO3 2- CO3 2- CO3 2- Ca-O C=O (carbonate) amide (proteins) PO 4 3- N-H Raw Powder HAp Powder Gokarna Pandey et al. / BIBECHANA 18 (1) (2021) 83-90 87 The IR spectrum of the HAp powder prepared from eggshells given towards the bottom of Fig. 2 is much different from the spectrum of raw eggshells as expected but contains the noteworthy peaks related to the HAp phosphates such as those centered around 470, 560, 1020, and 1090 cm-1 , as compared with Fig. 1. Fig. 3 shows the FTIR spectra of buffalo bone powder and the HAp prepared from it. Unlike in the eggshells, the spectrum of the buffalo bone powder, as shown in upper part of Fig. 3, clearly shows the presence of phosphate peaks centered at 1090, 1040, 603, 568 cm−1 [10,32,33] as well as - OH group at 3570 cm−1 [12,26,34] confirming the presence of hydroxyapatite as a major mineral component in the buffalo bone. The presence of sharp peaks in the aliphatic zone (2924 cm-1 and 2875 cm-1) due to the C-H elongation vibration together with the weak peaks at 1654 cm–1 due to the elongation vibration of C=O bonds and at 1541 cm–1 due to the deformation vibration of N-H bonds and elongation of C-N bonds in the bone powder results from the collagen protein [33-35]. Fig. 3: Comparison of the FTIR spectra of bone powder and HAp prepared there from. Likewise, the presence of broad bands in the range of 3200-3600 cm-1 result from adsorbed water and carbonate band located at 1450 cm-1 arises due to the presence of CaCO3 in the buffalo bone [35]. The spectrum is very much similar to that of HAp obtained from eggshells and sol-gel method except that it contains additional C-H peaks (2924 cm-1 and 2875 cm-1), which can be linked to the presence of residual organic impurities. The presence of carbonate peak (1450 cm-1 and 870 cm-1) [35] might imply that it still contains CaCO3 as impurity in the resulting HAp powder, which will be later confirmed by XRD spectra as shown in Fig. 5. Fig. 4 shows the XRD patterns of HAp powder synthesized by three different chemical methods: sol-gel method, precipitation method using eggshell and wet chemical precipitation method using CaO and H3PO4 as starting materials. The XRD patterns show that all the synthesized HAp materials are crystalline. The peaks located at 2θ values of 26°, 29°, 32°, 34°, 40°, 47°, 50°, 53° and 64°, correspond to (002), (102), (211), (112), (202), (310), (222), (213) and (004) Miller reflection planes of HAp, respectively (which comply with the JCPDS 09-432 data) [2,5,6,10,12,19, 23,27,30,36]. Fig. 4: XRD pattern of HAp powder synthesized by different methods as indicated. It is quite interesting to note that all the investigated samples have quite similar XRD patterns confirming the presence of the pure apatite phase in 4000 3500 3000 2500 2000 1500 1000 500 T ra n s m it ta n c e ( a .u .) Wavenumber (cm -1 ) O-H CO 3 2- CO 3 2- PO 4 3- PO 4 3- C-H (organic impurities) amide I (collagen matrix) CO 3 2- PO 4 3- C-H b) HAp from animal bone a) animal bone H 2 O O-H HAp Powder Bone Powder 20 30 40 50 60 70 80 In te n s it y ( a .u .) 2  (degrees) (002) (102) (211) (112) (202) (310) (222) (213) (004) (a) (b) (c) (a) HAp from Sol-gel method (b) HAp from egg shell (c) HAp from CaO and H3PO4 Gokarna Pandey et al. / BIBECHANA 18 (1) (2021) 83-90 88 the prepared samples. The absence of peaks corresponding to (024), (021) etc. planes also clearly hinted the absence of phases such as β- tricalcium phosphate [36]. The XRD patterns of the HAp from eggshell powder shows comparatively less impurities than from other methods which confirms that the impurities content in HAp can be reduced byheating the powder at higher temperatures, the same deduction as indicated by the FTIR results. Fig. 5 shows the XRD pattern of HAp samples obtained from bone powder calcined at two different temperatures, 700 °C and 1000 °C. XRD patterns of both samples are very similar to that obtained by chemical methods. However, the presence of a quite sharp peak located at 2θ value of 29° in the HAp prepared by calcination of the bone powder at 700 °C indicates the presence of considerable amount of CaCO3. Fig. 5: XRD patterns of HAp obtained from buffalo bone after calcination at 700 °C and 1000 °C. This peak, however, almost disappears when same HAp powder is heated at 1000 °C, indicating that the decomposition of CaCO3 takes place at higher temperature. Further, the HAp peaks were found to become sharper with increasing calcination temperature. The results imply the advantages of attaining better purity of the HAp by calcination of the bone powder at higher temperature. The diffraction peak observed at 2θ angle of 32o corresponding to the (211) plane, was chosen for the calculation of the crystallites size, as the former peak can be isolated from other peaks and is relatively sharper than the others. The average crystallite diameter was calculated by using Debye- Scherrer formula as reported elsewhere [12]:  = K/cosθ (i) where,‘ ’ is the average crystallite size (in nm), ‘K’ is the shape factor (K = 0.9 for most of the spherical crystallites), ‘λ’ is the wavelength of the X-rays (λ = 1.54056 Å for Cu K radiation), ‘’ is the full width at half maximum (FWHM) (in radian) and ‘θ’ is the Bragg’s diffraction angle. Table 1 compares the crystallite size of HAp particles prepared by various methods. It can be seen that size of the crystallites prepared by different methods lies in the range of 15-30 nm. The HAp synthesized by sol-gel method was found to have smallest crystallite size while the buffalo bone powder calcined at 1000 °C produced the largest crystallites. However, the size disparity is not too large. The bone calcined at 100 °C has produced the crystals slightly larger than that calcined at 700 °C. There might also be some other factors affecting the nano-crystal size during the synthesis. Table 1: Average crystallite size of HAp prepared by various methods calculated using equation (i). S. No. Methods 2θ (°)* Crystallite size (nm) 1 Sol-gel 32.668 14.80 2 Precipitation 32.796 28.11 3 Eggshell 32.647 17.51 4 Bone (powder calcined at 700 °C) 32.748 18.06 5 Bone (powder calcined at 1000 °C) 32.942 29.23 * 2θ values corresponding to (211) Miller planes at around 32° for each sample 20 30 40 50 60 70 80 In te n s it y ( a . u .) 2 (degrees) (211) (002) (102) (112) (202) (310) (222) (213) (004) Bone calcination (700 °C) Bone calcination (1000 °C) 2 q (degrees) Gokarna Pandeyet al. / BIBECHANA 18 (2021) 96-103 89 4. Conclusions A comparative study on simple, inexpensive, and facile approaches for the synthesis of nano-HAp has been presented. It has been demonstrated that the nano-HAp with reasonably high crystallinity and purity can be prepared in an ordinary senior high school chemistry laboratory. Among the methods presented, the procedure employed on the animal bone offers reliable, most cost effective and greener way of preparing the nano-HAp with high degree of purity and yield. The FTIR spectra of all HAp samples showed typical peaks centered around 470, 560, 960, 1020 and 1090 cm-1 corresponding to phosphate groups of apatite phase. All the specimens showed the presence of adsorbed water (band 3200-3600 cm-1) and vibration of OH- ions (3570 and 1027 cm-1) in HAp lattices. The presence of peak corresponding to carbonate ions (1426-1490 and 870 cm-1) revealed the presence of impurities that might not appear on performing the experiment under inert atmosphere. The XRD results showed that all the synthesized HAp samples were fairly nano-crystalline which also matched the corresponding (JCPDS 09-432) files for apatite crystals with corresponding (002), (102), (211), (112), (202), (310), (222), (213) and (004) lattice planes. 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