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Vol. 9. Issue 1.
Pages 994-1004 (January - February 2020)
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Vol. 9. Issue 1.
Pages 994-1004 (January - February 2020)
Original Article
DOI: 10.1016/j.jmrt.2019.11.039
Open Access
A facile method for synthesis of nanostructured copper (II) oxide by coprecipitation
Wellington Marques Rangel
Corresponding author
, Rozineide A. Antunes Boca Santa, Humberto Gracher Riella
Chemical Engineering Graduate Program (PósENQ), Department of Chemical and Food Engineering (EQA), Laboratory of Materials and Corrosion (LabMAC), Federal University of Santa Catarina (UFSC), Florianópolis, SC 88040-900, Brazil
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Tables (5)
Table 1. Experiments prepared for CuO nanoparticles synthesis.
Table 2. Average Crystallite size (nm) of CuO, calculated by the Scherrer equation, according to the type of precursor and reaction conditions.
Table 3. Average crystallite size (nm) obtained by the Scherrer method and TEM microphotographs.
Table 4. Results obtained experimentally in the thermal analysis of sample B: precursor CuSO4. Synthesis conditions: temperature 50°C and precursor salt concentration 0.2M.
Table 5. Results obtained experimentally in the thermal analysis of sample F: precursor CuCl2. Synthesis conditions: temperature 50°C and precursor salt concentration 0.2M.
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Due to their versatility e wide range of physical and chemical properties, transition metal oxides are very promising as nanostructured materials. Copper oxides are indicated for applications in increasingly wider areas as gas sensing, catalysts, solar cells, electrochromic devices, and antimicrobial materials. In this study, copper (II) oxide nanoparticles were synthesized by the coprecipitation method from two precursors: copper sulfate and copper chloride. The evaluated precursor concentrations were 0.1 M and 0.2 M and temperatures were 50 °C and 75 °C. Different results were found, according to synthesis parameters and precursor used. Using copper sulfate as a precursor, a single phase of polycrystalline CuO could be obtained, and only a mixture of crystalline phases of CuO and cllinoatacamite (Cu2(OH)3Cl) were found using copper chloride as the precursor. A known polymorphism phenomenon was observed in clinoatacamite according to synthesis reaction parameters, and all three polymorphs were found mixed with CuO nanoparticles. Crystallite sizes of about 10 nm were found in this study, with leaf and rod-like nanostructured particles with dimensions range from 190 to 700 nm, and very good thermal stability. Under the synthesis conditions selected in this study, nanostructured materials made with copper sulfate precursor exhibited smaller crystallite sizes and better thermal stability. The coprecipitation method used was considered adequate to obtain nanostructured materials, with the advantages of being simple, fast and inexpensive.

Copper (II) oxide
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The broad properties spectrum of nanostructured transition metal oxides has received great attention from researchers. However, their size and morphology control is still in development [1]. And, depends on the reaction conditions and several interdependent parameters such as temperature, electrostatic interactions and sterical confinements [2].

Copper Oxide (CuO) due to its excellent physical and chemical properties [3] has numerous potential applications, including research fields as catalysis [4,28] and gas sensors [5,31]. More advanced applications as solar cells, light-emitting diodes, photocatalyst, electrochemical, electrochromic devices are possible research fields for copper oxide nanostructured materials [6]. Furthermore, nanostructured CuO is used for several applications in the removal of inorganic pollutants and could be used as potential components for the fabrication of nanodevices [7]. Nanoparticles specially designed for antimicrobial applications in biotechnology and pharmaceuticals have great demand and its market was valued at US$ 17.5 billion in 2011 [8].

Copper is widely used as a potent biocidal for centuries and both gram-positive and gram-negative bacteria are killed when exposed to high copper concentrations [32]. The same biocidal effect is reported in the literature for copper oxide nanoparticles as a promising material to prepare antimicrobial textiles, with very effective results in cotton fabrics [9]. Recent developments have also reported the antimicrobial properties of copper oxide nanoparticles in textiles for biomedical applications [10]. The copper oxide impregnated into cotton or polyester fabrics has shown good biocidal results against various microorganisms and viruses. The low risk of causing unfavorable effects on the skin is an advantage of copper oxide nanoparticles application [11].

Nanostructured materials have a high antimicrobial effect due to its high surface area to volume ratio and the dependence of its cytotoxicity to particle size is reported in the literature. It was also observed higher levels of cytotoxicity in copper oxide nanoparticles than other transition metal oxides such as iron oxide, titanium dioxide, and zinc oxide [12,29].

Their promising properties and low cost compared to other metal oxides have increased the interest in the development of nanostructured materials of copper (II) oxide. Its structure is considered more efficient and stable for antimicrobial applications than copper (I) oxide at room temperature [3].

Many synthesis routes were reported to prepare nanostructured CuO particles, each one with advantages and drawbacks, including sol-gel [13], hydrothermal [14], thermal decomposition [15,16], microwave irradiation [17], alcothermal [18] and sonochemical processes [19,30]. In this context, this study proposed the synthesis and characterization of copper (II) oxide nanoparticles using two different precursors. The coprecipitation synthesis method was chosen for its simplicity, reaction rates at low temperatures and low cost.

Copper oxide nanoparticles were produced in two reaction conditions, in an alkaline medium using sodium hydroxide (NaOH). The precursors were copper sulfate (CuSO4.5H2O) and copper chloride (CuCl2.2H2O). The synthesis was conducted at temperatures of 50°C and 75°C and the concentrations of metal salts were 0.1M and 0.2M. After preparing, copper (II) nanoparticles made with copper sulfate as precursor showed better results (minor crystallite sizes). The coprecipitation method was considered suitable for obtaining nanostructured materials, with the advantages of being simple, fast and inexpensive.


Two copper salts were used to prepare CuO nanostructured particles, copper sulfate (CuSO4.5H2O) 98 % PA (SYNTH) and copper chloride (CuCl2.2H2O) 99 % PA (VETEC); sodium hydroxide (NaOH) 97 % PA (NAFAN) was used as an alkaline reagent. All reagents were used without purification and solutions prepared with distilled and deionized water.

2.2Synthesis method

Copper oxide nanoparticles were prepared using a chemical coprecipitation method and NaOH as an alkaline reagent. First, 100mL precursor solutions were prepared using distilled and deionized water (0.1 and 0.2M) and kept in constant magnetic stirring in a rounded bottom three-neck flask. The temperature was raised and maintained at 50 or 75°C as summarized in Table 1.

Table 1.

Experiments prepared for CuO nanoparticles synthesis.

samples  precursors  Temperatures (°C)  Concentration (M) 
CuSO4.5H250  0.1 
CuSO4.5H250  0.2 
CuSO4.5H275  0.1 
CuSO4.5H275  0.2 
CuCl2.2H250  0.1 
CuCl2.2H250  0.2 
CuCl2.2H275  0.1 
CuCl2.2H275  0.2 

After, an alkali (NaOH 1,0M) solution previously prepared, was added drop-wise using a burette, until a volume ratio of 1:1 was obtained. Constant stirring (500rpm) was maintained in the whole experiment and alkali solution was added at a flow rate of 5ml/min obtaining a pH 14 at the end of alkali addition. The solution color gradually changed from blue to black, and a considerable amount of black precipitate was formed. The reaction conditions were maintained for an additional 30min at the same temperature and constant stirring. The precipitates obtained were filtered under vacuum and washed with deionized water at room temperature. After washing, the powder was dried in a vacuum drying oven for 5h at 80°C, and characterization was performed.

2.3Characterization of CuO nanoparticles

The samples were characterized by X-ray diffraction (XRD) analysis to evaluate the existing crystalline phases. The equipment used was a Philips diffractometer X'pert model with copper Kα radiation (λ=1.5418Å), 40kV and 30mA, 2θ ranging from 5 to 110 and an angular step of 0.05°.

Thermogravimetric analysis was applied (TGA) for measuring the mass variation according to temperature. The exothermic and endothermic peaks were also analyzed by differential scanning calorimetry techniques (DSC). A thermal analyzer Netzsch Simultaneous Thermal Analyzer STA 449 F3 was used and samples were evaluated in the range 22−1000°C at a heating rate of 10°C / min, platinum sample holder and with flowing nitrogen atmosphere of 20mL/min.

The morphology of the material was analyzed at the Electronic Microscopy Laboratory (LCME) at UFSC, through scanning electron microscopy techniques (SEM) and transmission electron microscopy (TEM). For SEM a JEOL model JSM-6390LV was used, coupled to EDX (Energy Dispersive X-ray Spectroscopy), which was used for qualitative elemental analysis. Samples were pre-coated with gold and the images were obtained at 15kV accelerating voltage. The TEM analyses were performed in a transmission electron microscope JEOL JEM-1011 model, with 100kV voltage.

3Results and discussion3.1X-ray diffraction of nanoparticles synthesized from copper sulfate

The XRD patterns of prepared precipitates with copper sulfate as a precursor are presented in Fig. 1. The characteristic peaks in the XRD patterns are shown 2θ=35,6°; 2θ=38.7° e 2θ=48,9°; corresponding to planes (002) and (−111); (200) and (111); and (−202) of CuO. With monoclinic crystal system, space group C2/c and lattice parameters: a=4.668Å, b=3.423Å, c=5.132Å, α=γ=90° and β=99.54° [20]. Similar results were found by Lange et al. [33], using the coprecipitation method with a 0.02M solution of copper acetate as a precursor. Comparable XRD results were found using copper sulfate as a precursor and the coprecipitation method for coating cotton fibers [9]; and using coprecipitation method with subsequent application of ultrasound [21].

Fig. 1.

XRD patterns of the samples prepared with the precursor CuSO4.5H2O: A - 50°C / 0.1M; B - 50°C / 0.2M; C - 75°C / 0.1M and D - 75°C / 0.2M.


In sample C, additional peaks observed are due to the formation of brochantite (Cu4(SO4)(OH)6) e de posnjakite (Cu4(SO4)(OH)6.H2O), confirmed by the XRD patterns presented in Fig. 2. In the work of Darezereshki and Bakhtiari [22] similar results were obtained using CuSO4 as precursor salt.

Fig. 2.

XRD patterns of the samples prepared with the precursor CuCl2.2H2O, under the conditions: E - 50°C / 0.1M; F - 50°C / 0.2M; G - 75°C / 0.1M; H – 75°C / 0.2M.


According to Frost [23], in highly alkaline medium, copper (II) oxide is the most stable form and brochantite becomes the more stable phase in case of pH in 4–6 range.

All samples exhibited similar diffraction patterns with small variations in intensity and width of the peaks. Only when prepared at 75°C and at 0.1M CuSO4 the presence of compounds like brochantite and posnjakite were detected indicating that the increase in temperature and the use of lower concentrations difficult the removal of sulfate ions (SO4)2- during the formation of CuO crystals. The absence of impurity peaks, like Cu(OH)2 and Cu2O, demonstrated the high purity of the nanostructured material produced.

3.2X-ray diffraction of nanoparticles synthesized from copper chloride

In the diffraction data obtained for the precursor copper chloride, there was the formation of different crystalline phases (Fig. 2). Copper oxide characteristic peaks were found at 2θ=35,5°; 2θ=38,7° and 2θ=48,9°, confirming the formation of copper oxide in the nanostructured particles.

However, other peaks were found, suggesting a mixture of crystalline phases. Peaks at: 2θ=16,3°; 2θ=32,5°; and 2θ=39,7° e 2θ=53,8°; indicated the formation of chlorinated compounds such as clinoatacamite (Cu2(OH)3Cl) and its polymorphs.

According to Frost [23], the mineral clinoatacamite (monoclinic) has polymorphism and also is shown in the form of botallackite (monoclinic) and atacamite (orthorhombic). The stability order for these phases is clinoatacamite > atacamite > botallackite and, according to the Ostwald rule in a chemical reaction where different products are formed, the first being formed is preferably the lowest Gibbs free energy chemical compound, botallackite, in this case.

It was observed in the results, that increasing temperature or the precursor salt concentration favored the formation of chlorinated compounds. Under the synthesis conditions in this study, the formation of a single phase of copper oxide was not observed.

As prepared, CuO nanostructured particles made with copper sulfate precursor demonstrated better purity and could be used in applications like antimicrobial textile materials without further purification. Using copper chloride as precursor salt, chlorinated impurities were found.

3.3Average crystallite size

The crystallite size, D, was estimated according to the Scherrer equation (Eq. 1), using as reference the highest intensity peak at 2θ=35.6°. The angle parameter (θ) and full width at half maximum (FWHM) were calculated using the OriginPro 8 software, λ is the copper Kα wavelength 1.5418Å) and the results are shown in Table 2.

Table 2.

Average Crystallite size (nm) of CuO, calculated by the Scherrer equation, according to the type of precursor and reaction conditions.

Precursor  Temperature (°C)  Concentration (M)  Cryst. Size (nm) 
CuSO4.5H2O500.1  10.7 
0.2  10.0 
750.1  13.8 
0.2  14.1 
CuCl2.2H2O500.1  14.5 
0.2  12.0 
750.1  14.5 
0.2  13.2 

Smaller nanoparticles were obtained using CuSO4 as precursor salt. The reaction temperature and concentration of precursor salt also influenced the crystallite size, where an increase of temperature generates an increase of crystallite size, and for most cases, an increase of salt concentration causes a decrease of crystallite size.

Rodriguez and Fernandez-Garcia [24] reported crystallite sizes considerably smaller in the range of 7−9nm for copper (II) oxide. The crystallite size of the CuO nanoparticles depends on the preparation method used. According to Aparna et al. [25], the average particle diameter is: (i) about 10nm when prepared by sonochemical; (ii) 1–10nm by the sol-gel method; (iii) 3–9nm in the decomposition of copper acetate; (iv) 15–20nm in solid-state reactions; and (v) 4nm when using the electrochemical method.

It is observed that any material which contains particles or agglomerates of smaller sizes than 100nm, or films, and filaments of this range are considered nanostructured materials [26]. For Rodriguez and Fernandez-Garcia [24] nanoparticle sizes definition includes the range from 1 to 1000nm, which can be amorphous, fully crystalline or small aggregates of crystallites. A nanocrystal is formed by only one crystalline phase and should have dimensions in the nanometric order, but for nanoparticles, a crystalline phase is not strictly necessary.

3.4Transmission electron microscopy (TEM), nanoparticles prepared with copper sulfate

In Figs. 3 and 4, TEM microphotographs for nanoparticles obtained from CuSO4.5H2O precursor are presented. The nanostructured particles synthesized at 50°C at both concentrations have shown the same morphology. Agglomerates were built with an average crystallite size between 10 and 11nm. The rod-like crystals formed agglomerates with 190nm width in the center and thinner on the ends. Larger star-like agglomerates formed three-dimensional clusters with diameters ranging from 400 to 700nm. In Fig. 3c, the image was carried out by electron diffraction and indicated a nanostructured and polycrystalline material.

Fig. 3.

Nanoparticles obtained with CuSO4.5H2O, 0.1M concentration and temperature 50°C: a) and b) bright field image; c) diffraction pattern; d) crystallite details.

Fig. 4.

Nanoparticles obtained with CuSO4.5H2O, concentration 0.2M and temperature 50°C: a) and b) bright field images; c) detail of the formed particle; d) dark field image.


With temperature increasing to 75°C (Figs. 5 and 6), larger crystallites with an average size of 14.5nm were formed (0.2M precursor concentration). The sample prepared with 0.1M precursor (Fig. 5) showed irregular structure, probably caused by the presence of those chemical species: brochantite (Cu4(SO4)(OH)6) and posnjakite (Cu4(SO4)(OH)6.H2O), detected in XRD analysis.

Fig. 5.

Nanoparticles obtained with CuSO4.5H2O, concentration 0.1M and temperature 75°C: a) and b) bright field images of formed particles; c) and d) bright and dark field images of a nanostructured particle.

Fig. 6.

Nanoparticles obtained with CuSO4.5H2O, concentration 0.2M and temperature 75°C: a) bright field image; b) and d) bright and dark field images of a nanostructured particle prepared; c) crystallite details.


The rods formed in sample D (Fig. 6) had an average width of 220nm and an average length of 800nm, forming larger structures than the synthesis carried out at 50°C.

The reaction temperature also has influenced on the preferred orientation of crystal growth. At 50°C, the crystallites agglomerated and built a three-dimensional structure with 6 edges, and at 75°C, the crystal growth followed a linear direction, forming small particles similar to a rod.

3.5Transmission electron microscopy (TEM), nanoparticles prepared with copper chloride

Sample E was selected for TEM analysis and results obtained with the precursor CuCl2.2H2O, are shown in Fig. 7. Agglomerates were visualized in this sample also, and crystallite size was greater than those obtained with copper sulfate as a precursor (considering the same conditions: 50°C and 0.1M). In this case, the crystallites obtained had an average size of 21nm and formed rod-like structures with an average size of 220nm width and 830nm in length, with subsequent cluster formation in three-dimensional structures.

Fig. 7.

Nanoparticles obtained with CuCl2.2H2O, concentration 0.1M and temperature 50°C: a) and b) bright field image of particles; c) and d) bright and dark field image of one nanostructured particle prepared.


The crystallite size calculated from TEM micrographs and the values obtained by the Scherrer method revealed good correlation as presented in Table 3. The major differences observed in samples C (Fig. 5) and E (Fig. 7) represents synthesis conditions where the nanoparticles were a mixture of copper oxide and different contaminants.

Table 3.

Average crystallite size (nm) obtained by the Scherrer method and TEM microphotographs.

Precursor  Sample  Scherrer (nm)  TEM (nm) 
CuSO4.5H2O10.7  10.7 
10.0  10.9 
13.8  36.9 
14.1  14.6 
CuCl2.2H214.5  20.8 
3.6Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDX)3.6.1Copper sulfate as a precursor

Samples A, B, C and D (CuSO4 precursor) are presented in Figs. 8, 9, 10, and 11, respectively. The results of analysis by energy dispersive spectroscopy indicate the presence of only copper and oxygen in the samples developed with this precursor. The other two peaks appearing in Figs. 8 (b), 9 (b), 10 (b) and 11 (b) correspond to the presence of carbon and gold used, respectively, in the carrier tape and covering the samples.

Fig. 8.

CuO nanoparticles obtained with the precursor CuSO4, conc. 0.1M and temperature 50°C: a) micrograph; b) elemental analysis by EDX.

Fig. 9.

CuO nanoparticles obtained with the precursor CuSO4, conc. 0.2M and temperature 50°C: a) micrograph; b) elemental analysis by EDX.

Fig. 10.

CuO nanoparticles obtained with the precursor CuSO4, conc. 0.1M and temperature 75°C: a) micrograph; b) elemental analysis by EDX.

Fig. 11.

CuO nanoparticles obtained with the precursor CuSO4, conc. 0.2M and temperature 75°C: a) micrograph; b) elemental analysis by EDX.

3.6.2Copper chloride as a precursor

Sample E (Fig. 12a) prepared with precursor copper chloride (CuCl2) was selected for this analysis (synthesis conditions: 50°C and 0.1M). In EDX results, Fig. 12b, a presence of the atom Chlorine is confirmed. This is due to the formation of clinoatacamite and its polymorphs in the sample, as confirmed by XRD results. The same peaks of carbon and gold are also observed because of experimental conditions.

Fig. 12.

CuO nanoparticles obtained with the precursor CuCl2, conc. 0.1M and temperature 50°C: a) micrograph; b) elemental analysis by EDX.

3.7Thermal analysis (TGA and DSC)

Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) was performed on samples synthesized at 50°C and with 0.2M of precursor salt concentration (Samples B and F) and the results are exhibited, respectively, in Fig. 13a and b.

Fig. 13.

Thermal analysis (TGA and DSC) of CuO nanostructured particles: a) sample B, precursor CuSO4; b) sample F, precursor CuCl2. Synthesis conditions: temperature 50°C and precursor salt concentration 0.2M.


According to Patnaik [27], the heating when the temperature is above 800°C converts the copper (II) oxide into copper (I) oxide. Above this temperature, copper (I) oxide becomes the more stable phase. For this reason, the samples were tested until 1000°C to observe the thermal stability of the obtained CuO samples.

Sample B (Fig. 13a) produced with the precursor copper sulfate, showed a weight loss of about 3.4 %; in the temperature range between 25 and 136°C, probably due to the vaporization of water contained in the sample. There is a small signal at 283°C associated with a weight loss of 1.15 % due to crystallization water removal; these events are summarized in Table 4 (peaks values estimated in OriginPro 8).

Table 4.

Results obtained experimentally in the thermal analysis of sample B: precursor CuSO4. Synthesis conditions: temperature 50°C and precursor salt concentration 0.2M.

Event  Temp. Range (oC)  Central Peak (°C)  Weight loss (%) 
25.6–136.4  58.2  3.38 
230.8–349.0  283.1  1.15 
909.2–995.6  943.8  5.98 

Sample B exhibited very good thermal stability, losing only 5.6 % of its mass until the decomposition of CuO started at about 900°C. The difference in CuO decomposition temperature, when compared with the literature, was probably an effect of the resolution used (10°C / min).

The thermal behavior of sample F (Fig. 13b) which is a mixture of copper (II) oxide and chlorinated compounds of chemical formula Cu2(OH)3Cl, showed less thermal stability and larger weight loss in the temperature range observed. The different weight loss results observed in Figs. 13a and b are probably due to chlorine gas, eliminated by heat in sample F.

Performing the analysis by the DSC curve, three endothermic peaks were observed until 250°C, probably induced by dehydration of the nanostructured crystals. The five events exhibited by this sample are displayed in Table 5 (data estimated in OriginPro 8). Another endothermic peak appeared at about 800°C, associated with a small weight loss (0.36 %), indicating a probable crystalline transition, followed by a massive loss indicating material decomposition.

Table 5.

Results obtained experimentally in the thermal analysis of sample F: precursor CuCl2. Synthesis conditions: temperature 50°C and precursor salt concentration 0.2M.

Event  Temp. Range (oC)  Central Peak (°C)  Weight loss (%) 
25.9–107.3  66.1  15.84 
141.4–154.1  147.8  0.63 
201.3–252.0  218.5  3.25 
782.7–800.1  796.1  0.36 
943.7–985.7  970.8  11.20 

Nanoparticles of copper oxide nanoparticles (II) were made from two precursors: copper sulfate (CuSO4.5H2O) and copper chloride (CuCl2.2H2O) using concentrations of 0.1M and 0.2M; and temperatures of 50°C and 75°C.

The characterization by X-ray diffraction (XRD) showed the presence of a crystalline phase of copper (II) oxide in the samples, using copper sulfate as a precursor. However, with the same precursor, increasing the temperature to 75°C caused the appearance of compounds like brochantite (Cu4(SO4)(OH)6) and Posnjakite (Cu4(SO4)(OH)6.H2O) together with copper (II) oxide phase, only with precursor salt concentration of 0.1M.

All samples prepared with the copper chloride precursor has been identified as a mixture of crystalline phases, with the presence of chlorinated compounds with chemical formula Cu2(OH)3Cl, and a copper (II) oxide phase. These chlorinated chemical compounds exhibited a polymorphism phenomenon and its most stable form is called clinoatacamite.

The crystallite size was calculated by Scherrer formula and lower average crystallite sizes were obtained using the copper sulfate solution as a precursor. It was observed that the increase of reaction temperature promotes the development of the chlorinated crystalline phases during the nanostructured material formation.

Thermal analysis revealed the different behavior of the nanostructured materials obtained. When prepared with copper sulfate salt the material exhibited only 5.6 % weight loss until decomposition temperature of CuO. As a comparison, the sample made from copper chloride has lost more than 30 % of the mass, considering 800°C as the decomposition temperature, probably due to chlorine gas elimination.

The analysis of its elemental composition by EDX evidenced the presence of chlorine, copper, and oxygen in the samples prepared with the precursor copper chloride, and, only copper and oxygen in samples prepared from copper sulfate.

The morphology of the nanoparticles and their dimensions were observed in TEM photomicrographs, in which the nanoparticles were presented as aggregates of crystallites in the form of a small rod, organized into three-dimensional structures with average dimensions differing according to synthesis conditions. The samples synthesized at 50°C precursor with the copper sulfate had structures with smaller dimensions than using 75°C as reaction temperature, thereby forming a more interesting material for future applications.

With the use of copper chloride as a precursor, leaf-like nanoparticles were prepared, which then formed larger three-dimensional structures with about 830nm in length, similar to those found with the other precursor.

The average crystallite size showed a good correlation between the values obtained by the Scherrer method and TEM photomicrographs. The formation of a single-phase copper oxide (II) in the synthesized material from the precursor copper sulfate, using appropriate reaction conditions allows the preparation of copper oxide nanocrystals for numerous applications.

The coprecipitation method used revealed to be fast and simple, using only common materials of a chemical laboratory and with low reaction temperatures.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The authors wish to thank the National Council for Scientific and Technological Development (CNPq) - Grant 134522/2013-3), the Central Laboratory of Electron Microscopy (LCME), the Federal Institute of Santa Catarina (IFSC) and the Federal University of Santa Catarina (UFSC).

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