Role of heat treatment on nickel oxide nanoparticles: energy band gap and their effectiveness as antibacterial agents

This paper discusses the critical role of thermal effect on the crystalline size, phase and energy band gap of nickel oxide nanoparticles synthesized utilizing the ASH SUPPORTED technique. These nanoparticles were baked from 300°C to 900°C. The phase of nickel oxide nanoparticles with isochronal annealing has been identified through structural characterization utilising X-ray diffraction of each sample. It was observed that nickel oxide nanoparticles have an average crystallographic size of about 26.2 nm. The pure phase of nickel oxide nanoparticles was obtained at 900°C, which was also supported by Raman spectra. Vibrational characteristics demonstrate the increase in intensity with temperature due to the improvement in the grain size. UV-Visible spectroscopy determines the direct variation of the band gap in the region of 2.14 eV - 3.37eV with varying crystallite size due to alteration in annealing temperature or may be due to surface & interface effect. It also exhibits excellent antibacterial activity against enteropathogenic bacteria Salmonella Sp, which shows zone of inhibition diameter of 10mm.


Introduction
Transition metal oxides at the nano-scale have drawn an adequate interest from researchers in recent years due to their remarkable performance in numerous applications.From a theoretical and practical perspective, nickel oxide (NiO) is an appealing material.NiO is the most extensively utilized metal oxide Because of it is inexpensive, stable, and improved physical and optoelectronics properties at the nanoscale [1][2][3].Due to quantum confine effect and increased aspect ratio, Nickel Oxide nanoparticles (NiO NPs) have dissimilar properties over 3D NiO particles.It is a p-type semiconductor, antiferromagnetic with a forbidden energy gap of 3.51 eV [4].NiO nanoparticles are used in numerous applications namely as a gas sensor, fuel cells, p-type transparent conducting films, electrochromic films, dye-sensitized photo-cathodes, anticancer, cytotoxicity and non-enzymatic glucose sensor etc. NiO NPs have antibacterial action against pathogens as well.[2][3].
Due to their natural abundance and capacity to catalyse processes via different routes, they are used as an inexpensive catalyst.Numerous biomedical uses exist for them, such as MRI, sorting of cells, delivery of drugs etc. NiO NPs have been found to have significant antibacterial, antifungal, anticancerous, and antiparasitic properties and are good antioxidants.Wastewater dye degradation, photocatalytic degradation is other field, where NiO NPs can be effectively used.NiO NPs have been shown to speed up seedling development, which means they can also aid in seed germination thus proving their capability to modernise agricultural process [5,6].
Present study emphasizes the thermal effect of temperature on size, phase and energy band gap of Nickel oxide nanoparticles based on structural, vibrational & optical characterization.Comparative study of crystalline size of NiO NPS has been done using XRD for two different molar concentrations.Relation between crystalline size & its band gap is also established.NiO Antibacterial action kills or inhibits the growth or reproduction of bacteria such as E. coli.Enterobacter species & Bacillus species, which causes various diseases, infections, illness, vomiting etc. [7].Consequently, it is the necessary requirement to control the growth of bacteria by using antibacterial/antimicrobial activity.NiO has also gained significant attention due to their major antibacterial activity [8].Thus NiO NPs of particular size synthesized using ASH SUPPORTED method have been used against some pathogens to check its applicability to restrain its growth.

Experimental
The ASH Supported technique was used to create nickel oxide nanoparticles with 0.5 M concentration [9].The obtained 'As Prepared' (ASP) sample was then annealed in air at 300 o C, 500 o C, and 900 o C, resulting in samples #1, #2, and #3 respectively.These samples were analysed using X-ray diffraction on Bruker Advance X-Ray Diffractometer at Cu K-alpha 0.154 nm.In order to conduct vibrational analysis, Raman spectroscopy was performed using a Jobin Yvon Horibra LABRAM-HR 800 visible (400 -1100 nm) device.In the current investigation, Raman spectra were obtained 50 cm -1 to 1500 cm - 1 .As an excitation Laser source, a He-Ne source with a wavelength of 632.8 nm was used.The optical characterization of NiO nanoparticles was done using a UV-Visible spectrometer from Perkin Elmer, model lambda -950, USA.This equipment is a double-beam spectrometer capable of recording spectra with a precision of 0.05mm from 170 nm to 3300 nm.All the above measurements were carried out at UGC-DAE-CSR, Indore, India.
Antimicrobial activity of all compounds was performed by Kirby bour method according to guidelines of CLSI [10].Methanolic and ethanolic extracts of the concentration of 100mg/ml compounds were prepared.All compounds were tested against three bacteria which is responsible for enteric diseases.All tests were executed in the microbiology lab at the Institute of Science and Research, IPS Academy, Indore.Pathogenic bacterial culture was inoculated in peptone water and turbidity of culture was measured in the form of optical density at 660 nm.The culture was spread on Muller Hinton agar plate.After that samples along with standard antibiotics were inoculated well in all plates and were kept for incubation at 37°C for 24-hrs.The diameter of the zone of inhibition was measured to investigate antibacterial activity.

Structural Characterization (XRD Analysis)
In the present work, 0.5 M concentration of Methanolic solution of Nickel Nitrate is used to synthesize the nanoparticles [9].The XRD results of NiO NPs with 0.25M concentration matches with our previous article [9].Spectra obtained for NiO nanoparticles displayed in figure 1    Figure 2 presents the comparative study of crystalline size of NiO nanoparticles prepared from two different molar concentration (0.25 M & 0.5 M) calculated using Debye sherrer formula.In both the cases, the behavior of crystalline size with enhancement in temperature is almost similar.Size increases with thermal effect.However, difference in crystallite size has been found.Obtained size is mention in table 2 .

Raman Spectroscopy
Raman spectra of Nickel Oxide nanoparticles consists of five vibrational bands at 400-440 cm -1 , 530-560 cm -1 , 740 cm -1 , 925 cm -1 and 1100 cm -1 due to one phonon (1P) TO and LO, two phonon (2P) 2TO, TO + LO, and 2LO respectively as well as two magnon excitation at 1500 cm -1 [11][12][13][14].The present study delineates the vibrational properties of NiO NPs at three temperatures namely 300 0 C, 500 0 C, and 900 0 C and of ASP sample shown in figure 3. Table 1 displays the observed peaks of samples.'ASP' sample and samples annealed at 300 0 C, and 500 0 C consists of peaks of NiO, O2 or both while at 900 o C it has peaks of only NiO.All of the samples have peaks at 358-385 cm −1 , 535-574 cm −1 , 685-702 cm −1 and 1064-1085 cm − 1 due to one-phonon (1P) transverse optical (TO), (1P) longitudinal optical (LO), two-phonon (2P) TO and 2P LO of Vibrational origin respectively.Extra peak is observed in the range 206-214cm -1 due to the zone-boundary phonon mode [14].Two phonon (2LO) peak is the most prominent peak (1064-1085 cm -1 ).A feeble two-magnon (2M) band linkedwith Ni 2+ -O 2− -Ni 2+ super exchange interaction is observed at 1528-1543 cm -1 in all the samples [11].While the 2-phonon band (TO + LO) is not detected.Table 1.Raman peaks of ASP sample and samples annealed at 300 0 C (sample #1), 500 0 C (sample#2) and at 900 0 C (sample#3).As the samples were annealed at different temperatures, the 2M peak broadens.It might be due to the change of phase from the mixed phase in the ASP sample to pure phase in sample#3 annealed at 900 0 C. Vibrational characteristics reveal an increase in intensity with increasing temperature due to an increase in crystallite size.

UV-VISIBLE Spectroscopy
Figure 4 illustrates Tauc plots for the direct band gap of nickel oxide nanoparticles for samples (ASP, #1, #2 & #3) estimated using absorption coefficient & incident photon energy [15].Tauc plots  The optical band gap is said to grow with heat treatment in a variety of publications on metal oxide nanoparticles [16].This demonstrates that as annealing temperature is raised, the crystalline structure changes.The effect of crystalline size on band gap is due to quantum size effect and surface effects.The relation of crystalline size and band gap of NiO nanoparticles is directly proportional to  each other because of larger lattice strain for smaller particles, which arises due to surface pressure enhancement [17].In our case, it is found that the crystal's size grows with temperature, increasing the band gap energy because of the crystal's transition from a mixed to a main phase, which is observed in figure 5.In addition, this figure suggests that the band gap energy and crystallite size is directly correlated with temperature [18].It is obvious that heat treatment and lattice strain have a significant impact on the phase shift of nanoparticles in the sample [19].
When samples are heated from 300 °C to 900 °C, it is shown that their crystal size grows from 22 nm to 33.3 nm (figure 5).Band gap for all of the samples have been increased, and this can be seen in table 2 from 2.14 eV to 3.37 eV.These band gap values are in good accordance with the published literature.Till 500 o C, sample is in mixed phase that it consists of both Ni and NiO and pure sample of NiO is obtained only at 900 o C which was confirmed by XRD well as Raman Spectra.
According to reported work [19], pure phase nanoparticles display smaller strain and a greater band gap at 900 o C than mixed phase nanoparticles, which show larger strain and a smaller band gap.Thus, it can be deduced that the influence of heat treatment is what causes nickel oxide nanoparticles to exhibit this kind of band gap behaviour.

Antimicrobial Activity
Figure 6 shows the results which were observed in the form of zone of inhibition.The compounds that are prepared to use their applicability against bacteria are mentioned in table 3. The compounds that illustrate antimicrobial property against bacteria shows zone of inhibition around the well.There zone inhibition diameter has been measured (shown in table 4).In the table given below, antimicrobial activity is shown for 800ºC to 1000ºC.As pure phase is observed above 500ºC (shown in figure 1), therefore antimicrobial activity is performed at 800ºC & above.
(a) Against Bacillus Sp.Anti-microbial activity of compounds shows that after 24 hrs growth Nickel oxides (900°C) inhibit the growth of Salmonella typhiiwhich is known as enteropthogenic bacteria and responsible for several food borne disease.Results suggested that Nickel Oxide nanoparticles annealed at 900ºC (pure NiO nanoparticles) shows inhibitory properties against all pathogens in ethanol.So these NiO nanoparticles are considered as good antimicrobial compound, which gives the zone of inhibition of 10 mm that confirm its antibacterial property.

Conclusion:
This research demonstrates that the direct modification in band gap with crystalline size is caused by the annealing effect, which alters the phase and size of nanoparticles.This band gap is proportional to the annealing temperature.As a result, phase is an important factor in the current investigation.Because of the modest lattice strain and high annealing temperature, pure NiO nanoparticles have a significant band gap at 900 o C. It is concluded from comparative study of crystalline size of NiO nanoparticles calculated from XRD data that smaller sized particles can be prepared by having higher molar concentration of the initial solution for synthesis of NiO NPs.Vibrational characteristics demonstrate an increase in intensity with increasing temperature due to an increase in grain size.Nickel oxide nanoparticles annealed at 900°C are found to inhibit the growth of Salmonella typhi which is known as enteropathogenic bacteria with a zone of inhibition of 10 mm that confirms its antibacterial efficiency.It is also concluded that present results are phase & size induced which is the effect of heat treatment.
with 0.5M concentration show the mixed phase for ASP, #1 & #2 samples.The one phase is of Nickel & other is of Nickel Oxide.NiO phase (presented as peak A) is prominent & Ni phase (presented as peak B) becomes feeble up to 500 o C. It is also observed that on heating the sample to 900 o C (sample #3) only the NiO phase becomes dominant with a cubic face centered structure (space group: Fm3m).

Figure 2 .
Figure 2.Variation of crystallite size with annealing temperature with two (0.25 M & 0.5M) different molar concentration.

Figure 6 :
Figure 6: Antimicrobial property of NiO nanoparticles against bacteria.

Table 3 .
Coding of the compounds used for antimicrobial propertyFigure 5.Variation of crystalline size & band gap of NiO nanoparticles

Table 4 .
Result of antimicrobial activity against different bacteria.