Facile synthesis of hydroxyapatite nanoparticles from eggshell biowaste using Azadirachta indica extract as a green template

Punita Upadhyaya and Aman Ullah. Facile synthesis of hydroxyapatite nanoparticles from eggshell biowaste using Azadirachta indica extract as a green template. New J. Chem., 2024,48, 1424-1435

About

Hydroxyapatite (HAp) is an inorganic mineral which is chemically very similar to the naturally occurring main mineral component found in human bones and teeth. In recent years, hydroxyapatite nanoparticles (HANPs) have gained a lot of interest because of their use in various biomedical and industrial applications. Therefore, there is a great demand for developing simple, efficient, environmentally friendly, and rapid methods for the synthesis of HANPs. The materials used for synthesizing HANPs can be obtained from synthetic or natural sources. Among all the natural biomaterials, eggshells are an excellent source of calcium precursor for the synthesis of the HANPs. Eggshells are readily available, economical, and rich in calcium carbonate which make them an excellent calcium source for deriving HANPs. Use of eggshell waste to convert the valuable biomaterials promotes sustainable development by reducing environmental problems of eggshell disposal as well as decreasing carbon dioxide emission. The present work compared the synthesis of HANPs using microwave-assisted and conventional heating precipitation methods with and without green Azadirachta indica (AI) templates. Extensive characterization of the prepared HANPs in terms of structural, morphological, and thermal properties was carried out.

Approach

Eggshells were collected, washed several times with distilled water and heated at around 100C for 3 h to remove the surface impurities and thin inner layer membrane from the eggshell. After that, the eggshells were dried at 80C overnight in an oven and were ground into a fine powder and stored in a glass bottle. Then this pretreated eggshell powder was used for the synthesis of HANPs with the conventional heating method and microwave-assisted conversion method. The template was prepared with Azadirachta indica extract. The calcium precursor was obtained by slowly adding concentrated HNO3 into the eggshell powder (ES) with vigorous stirring for 30 min. The pH was adjusted, phosphate precursor and orthophosphoric acid were added to the resulting solution with continuous stirring. The white nano-hydroxyapatite powders were separated from the reaction mixture by two different methods. First, the solution mixture was subjected to microwave irradiation for 15 min at 40C. The reaction mixture was centrifuged and washed three times with distilled water and ethanol. Settled powder was dried in the oven at 100C for 24 hours. The final dried powder was calcinated using a furnace at 600C for 3 hours (herein named as HAp 01). In the second method, conventional heating was used. The reaction mixture was kept on stirring at 700 rpm on a hotplate at 40 C for 3 h and kept overnight to get white precipitation. The solution mixture was centrifuged, washed, dried, and calcined using the same approaches as described above for the microwave-assisted method (HAp 02). For the green synthesis of HANPs, the phosphate precursor (0.6 M H3PO4) was prepared using AI extract as a template. The aqueous solution of orthophosphoric acid was added to the calcium nitrate solution under vigorous stirring for 20 min; calcium ions formed a chelate complex with the presence of polyphenol of AI with the pH of the solution maintained at 9 by addition of sodium hydroxide. The synthesis was carried out using similar approaches as mentioned above for the synthesis of HAp 01 and HAp 02. The AI-templated nanoparticles were described as HAp 03 by the microwave method and HAp 04 using the conventional heating method.

Analysis of Results

It was observed that the HANPs had a pure hexagonal crystalline phase and no other impurities were detected. It was observed that the addition of the AI in both methods did not substantially impact the degree of crystallinity and crystallite size. It is shown that all the samples of HANPs have a pure single phase and HANPs synthesized using the microwave precipitation method give small particles compared to the conventional method with or without a template. The FTIR spectra obtained from all the methods clearly indicate the formation of pure HANPs without any impurities such as calcium carbonate and other calcium phosphate salts. No additional peaks in the AI-templated samples (HAp-03 and HAp-04) were noticed, indicating that AI acted only as a template and no AI residues were left after washing, purification and calcination. The surface morphology, shape, size, and distribution of HANPs synthesized by both microwave and conventional methods with and without a template were investigated. Addition of a template shows great change in uniformity and distribution of the nanoparticles. The use of AI template in both methods, resulted in more uniform distribution with less agglomeration in the micrograph of HAp-03 and HAp-04.

Application

Pure hydroxyapatite nanorods have been successfully synthesized from eggshell waste using two different methods, rapid microwave-assisted and conventional heating precipitation at a low temperature (40C) and with and without a green template (AI). The synthesized HANPs are biocompatible and biodegradable, and cheap starting materials (eggshells) make them economically viable. The MW synthesis is facile and could be easily scaled up, indicating that these biomaterials may be used for many applications, including biomedical applications such as bone repair, bone regeneration, and drug delivery and other applications like cosmetics, wastewater treatment and food packaging.

Abstract

In this study, hydroxyapatite nanoparticles (HANPs) were successfully synthesized from eggshell waste by microwave-assisted (MW) as well as conventional heating precipitation methods and a green template i.e., Azadirachta indica (AI) (neem) leaf extract. The synthesized HANPs were characterized by different analytical techniques such as Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA) for determining their functionality, shape, size, crystallinity, phase purity, and thermal stability. FTIR and Raman spectroscopy confirmed the pure hydroxyapatite phase with no other impurities. SEM and TEM analyses confirmed that HANPs displayed uniform distribution with less agglomeration by the MW method in the presence of AI when compared to the conventional heating method and without AI. TEM analyses revealed that the average size of HANPs from both methods without AI was 30–31 nm in width and 53–55 nm in length while in the presence of AI, it was 27–28 nm in width and 44–46 nm in length. XRD analysis indicated the formation of the pure crystalline structure of nanoparticles. The synthesized HANPs were thermally very stable and had a lower Ca/P ratio (1.81) from the MW method compared to the conventional heating method, which is 1.95. Thus, this study revealed that HANPs synthesized by the MW method in the presence of AI with a smaller crystallite size and less agglomerated particles have great potential to be used in the biomedical fields as well as different fields such as cosmetics, wastewater treatment, and food packaging applications.