Modification of egg shell powder with in situ generated copper and cuprous oxide nanoparticles by hydrothermal method^*

In the present work, the waste egg shells were collected from the local restaurants and households. Copper sulfate pentahydrate supplied by M/s SDFCL, Mumbai, India was used as received. In all the present experiments, deionized water was used.

Initially, the procured chicken egg shells were thoroughly cleaned with deionized water and dried at ambient conditions in the open. Using a cleaned kitchen mixer, the dried egg shells were made into fine powder. The egg shell powder (ESP) was sieved and in the present work, ESP with a particle size <25 μm was used.

For modification of ESP, 3 g of sieved ESP was added to 100 ml of 250 mM aq.CuSO₄.5H₂O solution and the mixture was maintained at 80 °C for 24 h. The color of the ESP changed to greenish blue which preliminarily indicates the in situ generation of copper based nanoparticles in ESP. The obtained MESP was filtered, washed well with water and subsequently dried. The prepared MESP was stored in the desiccators till tested.

2.4.1. Visual observation

2.4.2. Scanning electron microscopy (SEM) analysis

2.4.3. Fourier transform infrared (FTIR) spectral analysis

2.4.4. x-ray analysis

2.4.5. Thermogravimetric analysis

2.4.6. Antibacterial activity analysis

In order to examine the appearance of the MESP with in situ generated copper based nanoparticles, the digital images of egg shells, the ESP and MESP were recorded and are presented In figure 1(a)–(c) respectively.

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From figure 1, it can be seen that the color of the ESP changed to greenish blue for the MESP. Though the source solution (aq.CuSO₄.5H₂O) was blue in color, the MESP was Greenish blue. The color change preliminarily indicates the in situ generation of copper based nanoparticles. Further, the color of the MESP remained unchanged even after several washings with water during preparation indicating the generation of permanent copper based nanoparticles in it.

In order to observe the in situ generated copper based nanoparticles on the surface of the MESP, the SEM analysis was carried out. The SEM image, EDX spectrum and the histogram indicating the particle size distribution of the generated copper based nanoparticles are presented in figures 2(a)–(c) respectively.

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From figure 2(a), it is evident that nanoparticles were generated on the surface of the calcite crystals of ESP. The element copper in the MESP was confirmed by the corresponding EDX spectrum (figure 2(b)) which indicates the peak corresponding to copper element. From figure 2(c), it is evident that the size of the generated copper based nanoparticles in the MESP varied between 50 nm and 120 nm with majority of them in the size range of 80 nm to 120 nm. The average size of these particles was found to be 90 nm. As ESP has 5 wt% organic matter in addition to 95 wt% of calcium carbonate (calcite), the functional groups present in the organic component might have reduced the copper salt source solution into copper based nanoparticles.

In order to probe the structural changes due to the in situ generation of copper based nanoparticles in ESP, the FTIR spectral analysis was carried out. The FTIR spectra of ESP and MESP are shown in figure 3.

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From figure 3, it is evident that the spectrum of ESP was broad while that of MESP with in situ generated CuNPs yielded sharp peaks. In the case of ESP, the broad peak observed at around 3400 cm⁻¹ corresponds to the moisture. The other peaks observed at 712 cm⁻¹, 874 cm⁻¹, 1460 cm⁻¹ and 2521 cm⁻¹ are characteristic peaks of CaCO₃. From figure 3, it can also be observed that the bands of MESP became sharp and shifted towards lower frequency. Of these, the broad and strong peak observed around 1460 cm⁻¹ corresponds to the vibrations of carbonate C–O bonds. The other peaks observed at 1803 cm⁻¹, 2874 cm⁻¹ and 2981 cm⁻¹ were attributed to the vibrations of C=O from the carbonate ion (${{{\rm{CO}}}_{3}}^{\mbox{--}2}$) [15, 16]. These observations confirm the presence of CaCO₃ in ESP. Further, as ESP contains 95 wt% CaCO₃ and 5 wt% X collagen as binding protein, the peaks at 1640 cm⁻¹, 1540 cm⁻¹ and 1250 cm⁻¹ corresponding to amide I, II and III groups [17] are expected. However, these bands could not be observed in figure 3 as the broad peak at 1460 cm⁻¹ (ranging from 1100 cm⁻¹ to 1700 cm⁻¹) obscured them. The observed shift of the peaks in MESP indicates the lowering of vibrational frequencies due to the electrostatic interactions between the CaCO₃ of ESP and the generated CuNPs and Cu₂ONPS. Further, the lowering of intensity in the case of MESP indicates the interactions between the generated nanoparticles and ESP.

In order to study the influence of the generated copper based nanoparticles on the morphology of MSEP, the x-ray analysis was carried out. The x-ray diffractograms of CuSO₄.5H₂O, ESP and MSEP are presented in figure 4(a).

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From figure 4(a), it is evident that in the case of the diffractogram of ESP, an intense peak at 2θ = 29.2° arose due to the reflections from (211) plane of the major component of CaCO₃ with calcite structure [18, 19]. In the case of CuSO₄.5H₂O, the peaks at 2θ = 15.9°, 1.9°, 25.4°, 27.2°, 38.6°, 49.9° and 51.3° arose due to the reflections from the (100), (011), (111), (130), (122),(202) and (123) planes respectively. This indicates the triclinic structure of CuSO₄.5H₂O used in the present work. As the intensity of the ESP is dominating, the additional peaks corresponding to the generated nanoparticles could not be seen in the overlapped diffractograms (figure 4(a)). In order to observe the peaks corresponding to the nanoparticles and also to establish their oxidative state, the diffractogram of MESP in the 2θ = 20° to 80° range in expanded form is presented in figure 4(b). From figure 4(b), additional peaks at 2θ = 32°, 36.7°, 42.4°, 43.6°, 50.4°, 60.4°, 61.2°, 73.6°, 74° and 77.4° can be observed. Of these, the peaks at 2θ = 43.6°, 50.4° and 74° arose due to the reflections from (111), (200) and (220) planes respectively of CuNPs whereas those observed at 2θ = 32°, 36.7°, 42.4°, 61.2°, 73.6° and 77.4° arose due to the reflections from (002), (111),(200), (220), (311) and (222) planes respectively of Cu₂O nanoparticles [20]. Thus x-ray analysis indicates the in situ generation of both CuNPs and Cu₂ONPs in MESP.

As the x-ray analysis indicated the generation of both CuNPs and Cu₂ONPs and as ESP has both CaCO₃ as major component and collagen protein as the binding material in MESP, there are two possibilities of the reactions. The first possibility is by the participation of CaCO₃ and CaO in the reaction by which the nanoparticles are generated. However such reactions are possible only at very high temperature [21]. The formation of CuNPs and Cu₂ONPs in aqueous phase is reported in the literature [22]. In the present work, the MESP was prepared by hydrothermal method and hence the other possibility is through the protein component route. Collagen protein in egg shell is composed of mainly three amino acids—Glycine, Proline and Hydroxy proline [23]. The hydroxyl proline can react with aqueous copper sulfate solution to undergo reduction of Cu⁺² to Cu° to form proline. The formed Cu metal undergoes complexation with amino group in glycine amino acid present in collagen [24]. The possible reactions involving the three amino acids of collagen component of ESP are presented in figure 5:

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Thus all the three amino acids of collagen component of ESP were involved in the formation and capping of both the CuNPs and Cu₂ONPs.

In order to study the influence of the in situ generated CuNPs and Cu₂ONPs on the thermal stability of the MESP under study, the thermogravimetric analysis was carried out. For comparison, the experiment was also conducted on the ESP. The primary thermograms of ESP and MESP are presented in figure 6.

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From figure 6, it is evident that the in situ generated copper based nanoparticles decreased the thermal stability of the MESP. It is understandable as the generated copper nanoparticles act as catalyst to increase the thermal degradation. However, even at 700 °C the MESP retained 70 wt% of its initial mass (for ESP, the weight at 700 °C was 87%) indicating that the thermal stability of the MESP is still good. Further, the weight variation for the MESP from initial weight in the 200 °C to 400 °C was 92% to 80%. This indicates that in the usage temperature (room temperature to 80 °C cleaning temperature range), the MESP did not thermally degrade.

In order to study the antibacterial activity of the MSEP, the well method was used. For comparison, the test was also conducted for ESP (Matrix), CuSO₄.5H₂O and Cu₂O powders. The zones of clearance formed in each case were photographed and the images are presented in figure 7(a).

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From figure 7(a), it is evident that both Cu₂O and CuSO₄.5H₂O exhibited significant antibacterial activity as evidenced by the formation of zones of clearance. Similar observations were made in the case of filter paper embedded with Cu₂O and CuSO₄.5H₂O [25] and CuSO₄.5H₂O [26]. To examine whether the MESP also exhibits the antibacterial activity or not against the bacteria, the antibacterial test for both ESP and MESP was carried out. The zones of clearance in each case were photographed and the images are shown in figure 7(b).

From figure7(b) it is evident that ESP did not form any zone of clearance and hence failed to exhibit antibacterial activity against both the Gram negative and Gram positive bacteria. However, the MSEP formed significant zones of clearance and hence exhibited excellent antibacterial activity. In order to quantify the antibacterial activity, the diameters of the zones of clearance were measured and are presented in table 1.

From table 1, it is clearly evident that in the case of both CuSO₄.5H₂O and Cu₂O powders, the diameter of the zone of clearance varied between 16 mm and 25 mm. On the otherhand, the MESP formed the clearance zones with diameters ranging from 36 mm to 40 mm. The higher zones of the clearance in the case of MSEP can be attributed to the higher surface energy of the CuNPs and CuONPs generated in it. Thus, the MESP exhibited excellent antibacterial activity and hence can find applications as antibacterial filler in the preparation of nanocomposites and also as antibacterial low cost cleaning powder for house wares.