Calcium carbonate nanoparticles of quail’s egg shells: Synthesis and characterizations


 Avian eggshell is a natural biomaterial that has been used as an alternative natural source of CaCO3 and is accessible in big amounts from egg manufacturing. This study was planned to estimate CaCO3 in quail’s eggshell because it has a probable use in the progress of a novel choice of many applications. Physical properties: mineralogical documentation of the natural eggshell nanoparticles were approved using XRD and FTIR to explore the chemical bond or molecular structure of the materials. Micrographs were obtained using FESEM/EDX and TEM to identify the morphology and size of nanoparticles. The results showed that quail eggshell was soft, with white to light sand color, and a smooth texture which allows good deposition of different color spots, from black to brown spots. The resulted of eggshells signifies almost 8.4% w/w of the overall weight (12.2) gram of quail egg and 91.60% w/w of the micropowder to the full weight of (0.94) gram of quail eggshell. The results presented that calcium is the main element in an eggshell; frequently occurs in a formula of CaCO3 and the crystal construction was almost pure calcite. FTIR spectra for quail eggshell demonstrated the existence of the out of plane bending, the asymmetric stretching, and the plane bending styles of the carbonate groups, specific of normal dolomite, situated at 873 cm–1, 1405 cm–1, and 710 cm–1, respectively. The FESEM and TEM for nanoparticles were shown calcite CaCO3 nanoparticles with an ordinary size of ≤ 100 nm for FESEM and with a variety size of ≤ 50 nm for TEM. Unfortunately, eggshell is an egg product manufacturing deposit. These incomes will let fast developments in proportional studies of the organic elements of avian eggshell and their purposeful consequences by usages of eggshell in nourishment and medicine which can be applied for many resolutions that diminish their consequence on environmental contamination.


Introduction
The egg and egg derived intake harvest a big quantity of remaining shells which carriage an ecological contamination as an outcome of microbial accomplishment as it is hard to be despoiled by soil microorganisms [1][2][3]. Eggshell are discarded materials from hatcheries, homes and fastfood manufacturing and can be freely composed in sufficiently [4,5]. Most of the discarded is generally predisposed of in a landfill without any pretreatment, producing smells from biodegradation, terminating microbial action, and exchanging the worth of soil [6]. Challenges related to eggshell discarding need cost, obtainability of discarding sites, odor, flies and insensitivity [5]. Calcium carbonate (CaCO 3 ) itself can be initiate in big amounts in nature, with submissions frequently as fresh material for the ceramic calcium carbonate progress in quickly growing technology and research, for example hydroxyl apatite (HA) material production as a substitute to the teeth and bones of human [7,8]. Quick technological expansions have mandatory research in all fields of science and technology is no exclusion to remain to renovate in the technology of discarded exploitation and waste [9]. It is chiefly motivating to study the discarded bin of numerous types of poultry eggshells, with a statement that a lot of eggshells have calcium carbonate. Application of discarded eggshells as a source material for manufacturing other combinations those are able for association to harvest a new compound. Eggshells are a well basis of calcium for calcium oxide (CaO), calcium carbonate (CaCO 3 ), calcium hydroxide (Ca(OH) 2 ), calcium phosphate, or hydroxyl apatite (HA), in comparison to other sources for example carbonaceous rock, occasioned soil, teeth and bone, and crab or shrimp shells and the structure of an eggshell is actually like to that of our bones and teeth [6,10,11].
Chicken eggs are the maximum normally spent eggs; they are also a cheap protein source [12][13][14]. Paralleled to chicken egg duck egg is 30% larger and its nutritive profile is actually high [15]. Ingesting of quail eggs has increased in current years, due to their dietary qualities, as a good source of proteins, antioxidants, lysozyme, vitamins, and minerals, further than their dietetic importance which is 4-5 times greater than that of chicken eggs [16].
The benefits of eggshell remaining applications are to provide the normal sources of calcium (CaCO 3 , CaO, Ca(OH) 2 ), to decrease the discarded problematic in household, to preserve ordinary resources from rock and soil, to moderate the global environment warming, and to improve originality of green ceramic materials and products: dielectrics, catalysts, biomaterials, fuel cell, and fillers [6,[17][18][19][20][21][22]; and these incomes will permit quick improvements in proportional studies of the organic elements of quail eggshells and their serviceable effects [23,24].
This research aims to product of CaCo 3 nanopowder from uncooked quail eggshells and examinations of the nanostructure and particle size, chemical composition, organics (C/H/N) substances and practical groups, and provide the endorsements for uses of this type of nanoparticles in the future.

Materials and methods
Fifteen quail eggs were collected from animal house in the College of Veterinary Medicine which took from adult quail (Coturnix coturnix) aged 12 weeks that were fed on a standard ration. Quail eggs transported to the laboratory of Anatomy in the College of Veterinary Medicine, University of Mosul. In the laboratory, egg sample was weighed for whole on a weighing balance (Sartorius AG Gottikàen, GP5202, d = 0.01 g, Germany), and detached the eatable part of eggs. Natural and raw eggshells were carefully washed numerous times only with warm water and a piece of gauze till lost their natural spots pattern, and eggshells white was totally removed and skin off all of the shell's membranes from inside of the shells. The shells were washed again with deionized water then rap into a toilet paper to dry the water contented totally. The remainder of eggshells were weighed and crushed into small pieces in a porcelain mortar and grinded into a fine powder using blender (Good and Well ® , Taiwan) 25 , then weighed, sieved through ≤ 75 µm sieve (Endecotts Ltd, London, England). The micropowder was more dried in the oven (Memmert, UM 500, Germany) at 50 ∘ C for 5 days and converted into nanoparticles using a mechanical method in the existence of Ball mill (Wisd ® Ball Mill, Korea) for 7 days (Figure 1) then kept at 50 ∘ C in a sterile container before use [26,27]. Physical properties of the samples like color, texture and hardness were considered. The mineralogical documentation of the natural eggshells was approved out using Powder X-ray diffraction (PXRD) on a diffractometer (Angstrom Advanced Inc. ADX-2700, X-Ray Powder Diffraction Instrument) in a tube CuKα operating at a voltage of 40kV and a current 40mA to identify the crystalline phases and the crystallite size of eggshells [19,28,29]. To complete the analysis of the powders were located in the cavity of a support used as a sample container. The crystalline phases achieved matched by Joint Committee on Powder Diffraction Standards (JCPDS). Fourier Transform Infrared (FTIR) which was run in the variety of 4000-400 wavenumber/cm −1 to explore the chemical bond or molecular structure of the materials [19]. Micrographs were gotten using a Field Emission Scanning Electron Microscope, (FESEM) tracked by image analysis using their particular software's correspondingly and a Transmission Electron Microscope (TEM) (Hitachi H-7100, Japan) to identify the morphology and size of nanoparticles. The preparation of SEM and TEM samples of the raw materials were conceded out on a grid. The samples were firstly enclosed with a thin layer of gold (10 nm) using a sputter coater and detected using (FESEM/EDX) (20 kV) under a vacuum of 1.33 × 10 −6 mbar (Joel, Japan) [19,30,31].

Results
The results involved physical description data showed that raw quail eggshells had smooth texture, soft shell and white to light color with different black to brown spots. The proportional analysis of calcium carbonate (CaCO 3 ) content in them, which include percentage method of analysis by using 15 eggs presented in Table 1.
The ultrastructure and crystal construction of quail eggshells was considered in laboratory. The eggshell crystal construction was distinguished using an X-ray diffraction spectra (XRD) of normal (not boiling or exposure to any treatment) eggshells sample which achieved with CuKα radiation (λ = 0.15406 nm) at 30 kV, 16 mA, scan speed of 8.0 θ/min and scan range 5-60 θ. Figure 2 appearances a Xray diffraction spectrum of normal eggshells. Four obvious peaks were start individually in CaCO 3 calcite nanoparticles powder bands. The 1 st conventional peaks were 2θ = 23.057 ∘ to 29.408 ∘ , while the 2 nd conventional peaks were 2θ = 31.46 ∘ to 39.456 ∘ and the 3 rd conventional peaks were 2θ = 43.209 ∘ to 48.601 ∘ and the 4 th conventional peaks were 2θ = 56.755 ∘ to 58.148 ∘ . Chief peak performed at 2θ = 29.4. Equaling the XRD peak data of (Figure 2 and Table 2) with the classic CaCO 3 diffractogram from the shell powders seen to match systematically to calcite phase of a typical CaCO 3 structure ICDD-card number 01-083-1762. The deflection peaks were sharp and deep, signifying that the nanoparticles are extremely crystalline and equal very     well with the typical data card. There is no signal of any contamination phase current in the sample. The results presented that calcium is the main element in an eggshell; frequently occurs in a formula of CaCO 3 and the crystal construction was almost pure calcite.
The quail eggshells expressions a particle size of ≤ 50 nm (CaCO 3 ). The reduction of the crystal size can be recognized to the mechanical method using roller mill method. The lower strength peaks for quail eggshells could be associated to the decrease in the crystallite size. Figure 3 displays the FTIR spectra for mixed of all samples of quail eggshells. The spectrum of quail eggshells in Figure 3 demonstrations the existence of the out of plane bending, the asymmetric stretching, and the in plane bending styles of the carbonate groups, specific of normal dolomite, situated at 873 cm −1 , 1405 cm −1 , and 710 cm −1 , respectively. In addition, the internal modes, the grouping of the prior bending modes has also been detected at 1795 cm −1 and 2515 cm −1 . Lastly, the band positioned at 3668 cm −1 has been accredited to H-bonded water of the humidity. Moreover, a band about 2902 cm −1 performs apportioned to alkyl C-H stretch because of the organic substance of the quail eggshells.
The FESEM and TEM for nanoparticles shown calcite calcium carbonate nanoparticles with an ordinary size of ≤ 100 nm for FESEM and with variety size of ≤ 50 nm for TEM (Figures 4(A) and (B)). There was no conversion in the elemental structures of the gotten CaCO 3 calcite nanoparticles powder after production using of roller mill method. This reveals the roll milling in the breakdown of the larger sized calcite rods into very smaller spherical ones. Figure 4(A) appearances the SEM micrographs of the organized eggshells NPs. The particles were spherical in shape with constant size spreading. The eggshell ultrastructure was detected using SEM gotten from standard eggshells under high magnification (50000X). TEM images of the raw quail eggshells samples are presented in Figure 4(B) at a magnification of 50 nm. The raw quail eggshells had a normally regular crystal construction.

Discussion
The results showed that quail eggshells were soft, with color between white to light sand color, and a smooth texture which allows good deposition of color spots, with different color levels from black to brown spots. These results agreed with the studies by Sezer & Tekelioglu [32], Duval et al. [33]; Stolić et al. [34] and Drabik et al. [35].
The resulted of eggshells signifies almost 8.4% w/w of the overall weight (12.2) gram of quail egg and 91.60% w/w of the micropowder to the full weight of (0.94) gram of quail eggshell. The study of the quail eggshells exposed a high quantity of calcium in formula CaCO 3 . These results confirm the results of studies by Thapon & Bourgeois [1]; Romanoff & Romanoff [36] and Nys et al. [37].
The eggshells contain chiefly of CaCO 3 , therefore calcium shows an essential part in the eggshells construction [38]. The previous studies determined that the chief structure material of the shell represented by CaCO 3 (96%), and the residual modules are magnesium, phosphorus, but also copper, zinc, iron [39] and numerous trace elements, amongst them lithium, strontium and bar [40]. They described that all eggshells had parallel chemical substances which mostly composed of CaCO 3 and a little of other elements, i.e. S, Mg, P, Al, K and Sr.
The quail eggshells comprise more calcium levels [24,41,42]. They are spent often, but till now the shells are unused and static infrequently used particularly for HA material [43,44].
The ultrastructure and crystal construction of quail eggshells was considered in laboratory [45]. The eggshell crystal construction was distinguished using an X-ray diffractometer. Results presented that the eggshells crystal construction was almost pure calcite (CaCO 3 ) in quail eggs. These results agreed with the results of studies by Cahya & Marfuah [7] and Murakami et al. [28].
The FTIR spectra for quail eggshells displayed in Figure 3 demonstrates the existence of the outer plane bending, the asymmetric stretching and the in plane bending styles of the carbonate groups, specific of normal dolomite, situated at 873 cm −1 , 1405 cm −1 , and 710 cm −1 , respectively. In addition, the internal modes, the grouping of the prior bending modes has also been detected at 1795 cm −1 and 2515 cm −1 . Lastly, the band positioned at 3668 cm −1 has been accredited to H-bonded water of the humidity. Moreover, a band about 2902 cm −1 performs apportioned to alkyl C-H stretch because of the organic substance of the quail eggshells. These results confirm the results of studies by Cahya & Marfuah [7]; Wei et al. [45]; Viriya-empikul et al. [46] and Correia et al. [47].
The eggshells ultrastructure was detected using SEM presented in Figure 4, gotten from standard eggs [42]. The quail eggshells expressions a particle size of ≤ 50 nm (CaCO 3 ). The reduction of the crystal size can be recognized to the exothermic circumstances of the mechanical method using roll mill machine. The lower strength peaks for quail eggshells could be associated with decrease in the crystallite size. These results agreed with the results of studies by Park et al. [10]; Marques et al. [25] and Mahmood et al. [28].
The persistence of SEM and TEM description is to conclude the morphology of the quail shell nanopowder which are presented in Figure 4. This image indicates that the typical nanoparticles of quail eggshells nanopowder is the spherical nanoparticles with diameter about 50 nm. The image also designates the existence of agglomeration which occurred because of the existence of other phosphate compounds [48]. This remark is agreed with the results described by Park et al. [10]; Marques et al. [25]; Mahmood et al. [28] and Kalita & Verma [49].
Consequently, quail eggshell is a rich basis of mineral salts, principally calcium carbonate that is possibly the superlative normal source of Ca +2 .

Conclusion
Shells of quail eggs are a remarkable alternate to the presently used products in supplementation of additional normal sources of Ca +2 to humans and animals. Greater solubility of CaCO 3 from the shells of quail eggs, matched to synthesized CaCO 3 mark them a brilliant biomaterial for the manufacture of new nutritional supplements. Furthermore, the alteration of CaCO 3 micropowder, consequences in Ca +2 salt nanoparticles with enhanced properties matched to CaCO 3 . The CaCO 3 got from quail eggshells is categorized by an appropriate purity and comprises valued minerals in its structure. These incomes will let fast developments in proportional studies of the organic elements of avian eggshells and their purposeful consequences by using of eggshells in nourishment and medicine which can be applied for many resolutions that diminish their consequence on environmental contamination.