Synthesis and Catalase Mimic Activity of MnO 2 Nano Powder Prepared by Hydrothermal Process

Manganese dioxide (MnO2) nanopowder has been synthesized by hydrothermal method. MnO2 was annealed at different temperatures (250, 400, 550, 700 ̊C). The crystal structure and surface morphology of these nanostructures were characterized by X-ray diffraction (XRD), Atomic Force Microscope (AFM) and Scanning Electron Microscopy (SEM). The catalase mimic activity (catalytic activity) of MnO2 against hydrogen peroxide (H2O2) was studied by using a new method and found that 400 ̊C is the best annealing temperature.


Introduction
For many years, manganese dioxide with diverse crystal morphologies are attracting a lot of attention, because of their outstanding structural flexibility combined with novel physical and chemical properties, which are of interest for the following applications, for example, molecular sieves, supercapacitors, catalysts and biosensors [1].It is n-type semiconductor material [2].Manganese dioxide exists in various polymorphic forms including α-, β-, γand δ-MnO2 which are different in the arrangement of basic octahedral [MnO6] units [3].The hydrothermal method is a powerful synthesis approach for synthesizing various forms of manganese oxides because of the choice of precursors that can be used and control of reaction time, pH, and temperature and it is a simple and inexpensive technique [4].
The catalytic (catalase) activity can be measured by determining the decrease of H2O2 absorption (at 240 nm) [5,6].The difficulties associated to this method, due to using high levels of substrate approximately (5-50 mM) to get acceptable absorbance [7].Moreover, the high levels of H2O2 lead to formation of bubbles in the test cell which cause mistake measurements [8].Catalase (catalytic) activity can be determined in other methods such as by titrimetric determination of H2O2 concentration, determination of oxygen production from decomposition of H2O2 by oxygen electrode [9,10].There are simple colorimetric methods such as by Goth [11] for catalase, by measuring of hydrogen peroxide (unreacted) spectrophotometrically by a complex reaction with ammonium molybdate.Sinha and Hadwan [12,13] use another simple method, in which the decomposition of hydrogen peroxide determined spectrophotometrically by a complex reaction with dichromate/acetic acid reagent.Another method for catalase activity measurement is the titration method, which is used when high (UV) absorption pigmentation or precipitation of the sample does not allow the use of the spectrophotometric method [8].
Our work is new modified method which use spectrophotometric assay to determination of H2O2 by potassium permanganate in acidic solution.

Theoretical Part
In the present work, we have prepared MnO2 nanopowder using KMnO4 and HCl as a precursor.The crystalline size for that peak alone calculated, using the Debye-Scherer formula [14]: D = kλ / βcosθ ……… (1) Where k is the constant (0.9), λ is the wave length of X-ray (1.54 nm), β is the full width half maximum (FWHM) of the peak and θ is the reflection angle.

Synthesis of MnO2 Nano powder
The hydrothermal reaction was done in a 100 mL Teflon-lined stainless steel (autoclave) under autogenous pressure.In this synthesis, 4.115 g (47.298 mmol) of KMnO4 was added into 70 mL of deionized water with vigorous stirring, and stirred for about 10 min.at room temperature.The solution filtered, then 3.405 ml concentrated HCl were added to the filtrated solution under stirring to form the precursor solution.Then the solution poured into a 80 Teflon-lined stainless steel autoclave.The autoclave was sealed and placed in an oven at 200 °C for 6 h. and hydrothermally treated at 200 °C for 12 h.After that, the autoclave was allowed to cool to room temperature naturally.The brown black precipitate (Mn(OH)4) was washed with distilled water (4-5 times), and collected by centrifugation, washed with ethanol (2 times) and lastly the washed precipitates were dried at 90˚C for 2 hours in air.

Catalase mimic activity (catalytic activity)
The concentration of KMnO4 was determined by titration with known concentration of sodium oxalate solution, then the concentration of H2O2 was determined by titration with known concentration of KMnO4.Standard curve consisted of (0, 1, 2, 3, 4 and 5) x10 -5 M of KMnO4 was prepared to find the concentration of color absorbed from KMnO4 (as shown in Fig. 1).Catalase mimic activity was determined by using the reaction with final concentration of manganese dioxide (MnO2) solution (2 mM), and hydrogen peroxide (750 μM), (as the following reaction) [13]: After five minutes that acidic solution consist from potassium permanganate (KMnO4) solution (300 μM as final concentration), acidity with some drops of sulphoric acid (H2SO4).The permanganate solution (purple color) will reacting with the excess of hydrogen peroxide (H2O2) (which no reacted with MnO2), and reduced to manganese sulfate (color less), as product reaction as following equation: Hydrogen peroxide concentration which used is directly proportional to the concentration of potassium permanganate that used in the reaction.The decreasing in permanganate concentration (color) is measured calorimetrically at 525 nm by using standard curve concentration.The procedure of Catalase mimic activity was done according following steps in describing in table 1.
Table -1 shows the X-ray diffraction patterns of prepared product (Mn(OH)2) at different annealing temperatures (250, 400, 550 and 700˚C) for 120 min.The increase of annealing temperature from 250 to 550˚C increased the intensity of diffraction and increase the lattice constant is in agreement with the reference [15].At annealing 700˚C the lattice constant is decrease because formation another new phase called Mn2O3 [16].For magnifications 5μm (Fig. 4a to d ) , the morphology of the MnO2 that prepared by hydrothermal method at different temperature (250-700)˚C was primarily investigated by SEM, according to the morphology of MnO2 there are smooth and high-quality nanowires with diameter of 17.33 to 42.89 nm and several micrometers in length for average.These nanowires aggregate into spherical shape with diameter of about 4.069 to 6.955 μm.

Fig. ( 3
Fig. (3-a to d) show the AFM images and the granularity accumulation distribution chart of MnO2 powders with annealing at (a-250, b-400, c-550, and d-700)˚C.The average grain size found to be (66.27-81.65 nm).AFM results show that the grain size increase by increasing temperature this is due to improving the crystalline of the powders.

Figure 3 :
Figure 3: Two and three dimensional AFM images and the morphology o for MnO2 with annealing temperatures at: (a) 250˚C, (b) 400˚C, (C) 550˚C and (d) 700˚C for 120 min.

Figure 4 :
Figure 4: SEM image for MnO2 at (a) 250˚C, (b) 400˚C, (C) 550˚C and (d) 700˚C for 120 min.The following equation was used to calculate the rate reaction of catalase mimic activity of MnO2 annealing at (As-prepared -700 o C) for 2 h: Rate of catalase activity (sec. - ) = (2.303/t)x (log (Co/C))…..(6) Where: t = time of reaction (seconds); Co and C are total concentration of hydrogen peroxide in cell reaction before and after reaction respectively.Our results show that the 400 o C is the best rate reaction of catalase mimic activity (2.59 x10 -2 S -1 ), these results are show in Fig.6and table 2.

Figure 6 -
Figure 6-The rate of reaction as catalase mimic activity (S -1 ) of MnO2 annealing at (as-prepared -700 o C for 2 h.

Table 2 : The rate of reaction as catalase mimic activity (S -1 ) of MnO2 annealing at (as-prepared -700 o C for 2 h.
MnO2 nanostructures were prepared by hydrothermal method and annealing at different temperatures (250, 400, 550 and 700 o C) for 2 h.The calculation rate of reaction (K) as catalase mimic activity against the low concentration of hydrogen peroxide (2 mM) has been done.The result found annealing at 400 o C were the highest activity (2.59 x10 -2 Sec.) among different annealing temperatures.