Effect of Reflux Time and Cow urine Concentration on Green synthesis

of Reduced Graphene Oxide (rGO)

Medha Gijare1 Sharmila Chaudhari2 Anil Garje3#
1
Department of physics, B.G. College, Sangvi, Pune, Maharashtra, India
2
Department of physics, Annasaheb Magar College, Hadapsar, Pune,Maharashtra, India
3#
Department of physics, S.P. College, Pune 411030, Maharashtra, India, adgarje@gmail.com

Abstract
In the present work, Graphene oxide (GO) was prepared using predefined improved modified
Hummer’s method. Reduced Graphene oxide (rGO) is synthesized by cow urine extract using
sonication and refluxing process. The influence of process time and concentration of cow urine
extract was optimized. Structural and optical characterization of GO and rGO was carried out
using XRD, UV-visible, FTIR, SEM. X-ray diffraction, FTIR, UV-visible spectroscopy detects
the formation of rGO. The surface morphology of synthesized rGO was studied by SEM
analysis. Preparation of reduced graphene oxide using cow urine extract is sustainable, cost
effective and more ecofriendly than other standard methods of reduction of nanoparticles. The
cow urine extract is the best alternative for the synthesis of rGOfor large scale production.
Keywords— graphene oxide, reduced graphene oxide, cow urine extract
I. INTRODUCTION
Graphene, a monolayer of sp2-bonded carbon atoms arranged in a honeycomb lattice, has
unique nanostructure and extraordinary properties [1, 2]. It has become a unique and extremely
promising material in the areas such as nanoelectronics, nanocomposites, optoelectronic
devices, electrochemical super capacitor devices, fabricated field-effect transistors, drug
delivery systems, solar cells, memory devices, and constructed ultrasensitive chemical sensors
such as pH sensors, gas sensors, biosensors, etc. [3-10]. Graphene is rapidly adopted due to its
diverse set of unusual properties as well as availability of techniques and processes. Graphene
shows large theoretical specific surface area (~ 2630 m 2 g-1, for single-layer graphene). It is a
unique bendable material with excellent mechanical flexibility and high crystallographic quality.
Graphene exhibits high optical transparency with absorption of ~ 2.3% toward visible light.
Graphene exhibits high values of Young’s modulus (~1.1 T pa). It is one of the thinnest known
materials. The carbon atoms are densely packed in a honeycomb crystal lattice with a bond

1
length of 0.141 nm. It exhibits excellent room temperature electrical conductivity (~ 7200 S.m-
1) [11]. Various methods are adopted for synthesis of graphene oxide.
Chemical reduction of graphite oxide colloidal suspensions has been considered as an
effective route to synthesize graphene sheets due to its simplicity, reliability, ability for large-
scale production and exceptionally low price. A number of reducing agents such as dimethyl
hydrazine, hydroquinone, aluminium powder, sodium borohydride, sulphur containing
compounds, hexamethylenetetramine, polyelectrolyte, ethylene diamine, sodium citrate , carbon
monoxide and norepinephrine have been experimented on and were found to have performed
under various conditions i.e., acid/alkali , thermal reflux and others refluxes such as laser,
plasmas , microwave, sonochemical , electrochemical , two-step reduction and so on. These
different reduction methods result in graphene with different properties [12-13]. In spite of the
distinct advantages of chemical route for reduction of GO, the reduced GO (rGO) tends to form
irreversible aggregation due to strong van der Waals attractive forces among the graphene planes
which creates bottle neck and confines its process ability[14-17]. However, this can be
eliminated by chemical modification of graphene using small organic molecules, biomolecules
etc. Another negative aspect of the chemical reduction method is the high toxic nature of the
reducing agents (hydrazine, dimethyl hydrazine, sodium borohydride, hydroquinone, etc). The
existence of such toxic agents could have harmful effect, particularly in cases of bio-related
applications such as catalysis and drug delivery. In the case of metal or hydrochloric acid
reduction of GO, particles traces of metals may stay behind as impurities. Here, the chemical
components that are associated in the bio-reduction process, formation and stabilization of
nanoparticles have been found to include biomolecules such as proteins, amino acids,
polysaccharides, alkaloids, alcoholic derivatives, polyphenolic compounds, enzymes, chelating
agents and vitamins[18].
Cow urine or “GOMUTRA”, has holy importance in Hindu mythology from ancient times in
India. The analysis of cow urine has shown that it contains nitrogen, sulphur, phosphate,
sodium, manganese, carbolic acid, iron, silicon, chlorine, magnesium, melci, citric, titric,
succenic, calcium salts, Vitamin A, B, C, D, E, minerals,lactose, enzymes, creatinine, hormones
and gold acids. It possesses high natural nitrogenous content. Cow urine contains total natural
N ranging from 6.8 to 21.6 g N per litre,consisting of urea (69%), allantoin (7.3%), hippuric acid
(5.8%), creatinine (3.7%), creatine (2.5%), uric acid (1.3%), hypoxanthine (0.5%), free amino
acid N (1.3%) and ammonia (2.8%). Cow urine has been found capable of curing several
diseases by urine therapy due to its excellent anti-oxidant and antibacterial activity [19]. In
addition, cow urine has also been used for the synthesis of silver nanoparticle (AgNPs) for

2
bacterial control and fungal pathogens [20-21], which proves cow urine to be a green alternative
to hazardous reducing agents for synthesis of nanomaterials.
In the present work, we report a simple green way to prepare reduced graphene oxide by
chemical reduction of GO by natural reducing agent, cow urine extract. The reduction was
carried out at variable concentrations of cow urine extract as well as variable time of refluxing
process. An approach for the preparation of reduced graphene oxide using cow urine extract is
sustainable, cost effective and more ecofriendly than other standard methods of reduction of
nanoparticles. The cow urine extract is the best alternative for the synthesis of rGOfor large
scale production.
II. EXPERIMENTAL:
Chemicals:
Graphite powder and H2SO4 (98 wt %), Potassium Permanganate (KMnO4), H3PO4 (85 wt %),
Hydrochloric acid (HCl 35 wt %), H 2O2 (30%), DI water, Cow urine extract
(GovidnyanSanstha). All reactants and solvents have been used without further purification.
Preparation of GO:
For a synthesis of GO, improved modified Hummer’s method [22-23] was used. 10 g graphite
powder and 5 g NaNo3 were mixed with 216 ml of concentrated H 2SO4 and 24 ml of
concentrated H3PO4 (9:1 volume ratio) . The mixture was kept in ice bath and stirred for 10 min
followed by addition of 30 g potassium permanganate very slowly for 1 hour. The reaction
mixture was kept in ice bath for 2 hrs followed by stirring for 1 hr, before increasing the
temperature. Thetemperature was increased to 40°C and the mixture was stirred for 1 hr
controlled by water bath. Finally the temperature was increased to 98 °C while stirring. DI water
was slowly added to the mixture till its volume becomes 800 ml to stop the reaction. Following
that 30 ml of H2O2 was added into the mixture. The addition of H 2O2 resulted in yellow color,
indicating high level of oxidation. The solution was then washed with 5% HCl and DI water
repeatedly using centrifuge (4000 rpm) until SO42- was removed completely and the pH of the
supernatant was neutral. Finally the material was dried for 24 h and a brown black sample was
obtained.
Mechanism of formation of graphene oxide:
The active component to oxidize graphite is diamanganese heptoxide (Mn2O7) which is obtained
via the reaction of monometallic tetra oxide and MnO3+ as shown in the reaction (1) below.
KMnO4 +3 H2SO4 K++MnO3++H3O++3HSO4- _____________ (1)
MnO3++MnO4- Mn2O7 ___________________ (2)

3
 C  O  C 
 
Mn2O7 +H2SO4+ + C (graphite
 C  OH  +MnO + H O+ SO
5 2 4
-

 COOH 
 
It is seen that the bimetallic form of manganese oxide gets destroyed when heated up to 55 °C
temperature or when reacted with organic compounds [24].
Reduction of GO using cow urine extract:
The graphene oxide solution (0.5 mg/ml) (yellow brown color) was mixed with 10 ml cow urine
extract in a ratio of (1:1) in 500 ml flask ; heated to 95°C using water bath to get homogeneous
heating under reflux condition for 15 hrs. The color of the mixture changes from yellow brown
to dark black after complete reduction. This indicates formation of rGO.
Effect of reflux time on reduction of GO:
The above process was repeated for various reflux time as 3,9,12 and 15 hrs. Each mixture was
centrifuged at 4000 rpm for 20 min. The precipitate obtained was washed repeatedly with DI
water and re-centrifuged to obtain rGO.
Effect of cow urine extract concentration on reduction of GO:
The graphene oxide solution (0.5 mg/ml) (yellow brown color) was mixed with different cow
urine extract concentration of 2, 5 and 10 ml in a ratio of (1:1 and heated to 95°C for 15 hrs.
Further process is repeated to obtain rGOas mentioned above.
III. Results and discussion:
The UV-visible spectra of the prepared graphene oxide are presented in (Figure1). It shows the
absorption peak at 228 nm, which correspond to the π→π*transition of C=C bonds [25]. It is
observed that with increasing cow urine extract concentration resulted in an increase in GO
reduction process. The shift in the absorption peak from 228 nm for GO to 265nm for rGO
indicates about ~37 nm red-shift relative to the absorption peak of the GO. This shift can be
assigned to deoxygenation of the GO sheets and the reconstruction of the sp 2 hybridized carbon
network due to the removal of the oxygen bearing functional groups. This similar shift was
observed for the reduction of GO by curcumin [26, 27]. This suggests that 10 ml cow urine
extract was the best extract concentration for GO reduction. After reduction of GO by refluxing
with cow urine extract , the absorption peak shifted from 228 nm to 265 nm (Figure 1) due to
removal of most of oxygen functional groups. The absorption peak for reduced GO had red

4
shifted to 265 nm. This phenomenon of red shift has been used as a monitoring tool for the
reduction of GO. During the reduction of GO, the colour of GO solution changes from yellow
brown to black after reduction. This is in agreement with the previous work where the black
colour is the first indication of the reduction of graphene oxideand the formation of reduced
graphene oxide (rGO).
The effect of various reflux time on GO reduction was studied and the results were
characterized by using UV-Visible Spectrophotometer. The graphene oxide (GO) was reduced
by reflux with 10 ml cow urine extract for (3, 6, 12, 15) hour(s). At reflux time of 3 hrs, the
UV-Visible spectra show the absorption peak shifted to 237 nm as shown in (Figure 2). At 6 hrs
reflux time the absorption peak shifted to 255 nm as show. At 12 hrs reflux time the absorption
peak shifted to 264 nm .At 15 hrs reflux time of the absorption peak changes slightly and
becomes 265 nm. So the best reflux time for GO reduction may be considered as 15 hr.
The XRD of the reduced graphene oxide (GO) using cow urine extract as green reducing agent
at best concentration 10 ml and at reflux time 15 hr is as shown in Figure 3. This broad peak
indicates the formation of few layers of reduced graphene oxide (rGO) sheets. The 2θ peak in
the range ~ 25° – 28° corresponds to interlayer distance of ~0.15 nm, which may be resulted
from some restacked reduced graphene oxide (rGO) layers and formation of a few layers rGO.
This interlayer spacing indicates the removal of oxygen functional groups [30].
The FTIR spectrum of graphite shows no significant peak for the functional groups, as shown
in (Figure 4). Graphene oxide shows several characteristic absorption bands of oxygen-
containing groups, demonstrating that the oxidation process was successful. A broad peak at
3395 cm−1 was observed for the O–H stretching vibrations from hydroxyl groups and water
molecules. The IR peaks corresponding to 2928 cm−1 and 2851 cm−1 are due the asymmetric
and symmetric CH2 stretching of GO. These bands can be seen in natural graphite due to the
presence of impurities or chemical defects in the sp2 graphitic plane. [30]. The intensity of
these bands is higher for GO show amount of C–H vibrations. Then, the vibrations for C=O
stretching and C–O stretching of –COOH groups generated at edges of the GO surface
appeared at 1741 cm−1 and 1217 cm−1, respectively. Some bands also appear around 1048 cm −1,
corresponding to the generation of C–O–C stretching vibrations of epoxy groups. It can be
observed that the intensities of absorption bands of oxygen-containing functional groups were
immensely reduced for the CrGO. The carbonyl C=O bands were disappeared, but the
spectrum retains a broad absorption band centred at 3395 cm −1 and presents a new band around
1048 cm−1was identified to the residual O–H groups in rGO.

5
The surface morphology of GO and CrGO have been analyzed by SEM as shown in (Fig.5).
SEM image of GO represents the agglomerated graphene nanosheets which consists of a large
number of randomly wrinkled thin sheets of oxygenated graphene layers as shown
(Fig.5a).The reflux treatment on GO with cow urine extract results in exfoliation of individual
layers of GO, and production of rGO. SEM image of CrGO is as shown in (Fig. 5b).
Conclusions
The cow urine extract was successfully used as green reducing agent for reduction of GO into
reduced graphene oxide (rGO). The main reward of the technique used over the traditional
chemical reduction is the ecofriendly approach, cost effectiveness, and simple process. The
cow urine extract is the best alternative for the synthesis of rGOfor large scale production.
Acknowledgment
The authors are thankful to Dept. of chemical engineering, AISSMS’s college of engineering
for providing all research facilities. Authors are also thankful to AISSMS’s college of
engineering, B.G. college and SPPU for facilitating characterization techniques.
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List of figures:

Figure 1: UV- visible spectrograph of rGO refluxed for 15 hrs at 95°C

Figure 2: UV- visible spectrograph of rGO (10 ml cow urine extract) refluxed at 95°C

Figure 3: XRD patteren for rGO refluxed for 15 hrs. at 95°C with 10 ml cow urine extract

Figure 4: FTIR graph for f Graphite, GO, CrGO

Figure 5: SEM images of synthesized GO and CrGO