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Green Synthesis of N-Doped Graphene

Nanosheets by Cow Urine

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Current Graphene Science, 2017, 1, 000-000 1

RESEARCH ARTICLE

Green Synthesis of N-Doped Graphene Nanosheets by Cow Urine

Pankaj Chamoli1, Malay K. Das2 and Kamal K. Kar1,2*

1
Advanced Nanoengineering Materials Laboratory, Materials Science Programme, Indian Institute of Technology
Kanpur, Kanpur-208016, India; 2Advanced Nanoengineering Materials Laboratory, Department of Mechanical
Engineering, Indian Institute of Technology Kanpur, Kanpur-208016, India

ARTICLE HISTORY
Received: November 10, 2016
Revised: February 14, 2017
Accepted: February 14, 2017
DOI:
10.2174/2452273201666170321163529
Keywords: Graphene, nitrogen doping, N-doped graphene, cow urine, green dopant, solvothermal method.
Abstract: Background: Nitrogen (N)-doping in the graphene lattice is a process to tailor the physico-
chemical properties of graphene nanosheets, and explored for potential use in the various modern appli-
cations.
Objective: Synthesis of N-doped graphene nanosheets via green route in contrast to the post traditional
harmful methods.
Method: N-doped graphene nanosheets have been synthesized by simple solvothermal method at 180 oC
using cow urine as a natural dopant of nitrogen.
Results: Experimental results confirm that the cow urine has deoxygenated the graphene oxide to pro-
duce graphene nanosheets, and simultaneously nitrogen atoms are successfully doped into graphene
lattice from cow urine. The current approach produces high yield N-doped graphene nanosheets with N
content ~ 3.2 at.% into the graphene lattice.
Conclusion: A strikingly simple, cost effective and environmental friendly approach has been demon-
strated for the synthesis of N-doped graphene nanosheets by simple solvothermal method using cow
urine as a natural dopant of N atoms. The proposed approach produces N-doped graphene nanosheets at
bulk scale.

1. INTRODUCTION two steps: first direct synthesis of graphene by arc-discharge,

Graphene emerges as a wonder material worldwide by
owning remarkable properties such as high mobility of
charge carriers (~ 200,000 cm2 V
1 s
1), high optical trans-
parency (~ 97.7%), high thermal conductivity (
5000 W
m
1 K
1), excellent Youngs modulus (
1 TPa), high
strength (
130 GPa), and high specific surface area (2630
m2 g
1); and is used for various applications such as displays,
energy storage and conversion devices, water treatment, and
electrochemical sensor [1-8]. Doping of heteroatoms such as
boron, sulphur, phosphorus, nitrogen, etc. into graphene lat-
tice have been explored to tailor the graphenes optoelectri-
cal, magnetic and electrochemical signatures [9-10]. Particu-
larly, nitrogen (N)-doped graphene nanosheets (NGns) have
been potentially used in various applications such as fuel
cells, water splitting, batteries, electrochemical bio-sensing,
electrocatalytic performances, and super capacitor [11].
Various approaches have been adopted to produce NGns in
*Address correspondence to this author at the Indian Institute of
Technology, Advanced Nanoengineering Materials Laboratory, Department
of Mechanical Engineering and Materials Science Programme Kanpur-
208016, India; Tel: +91-512-2597687; Fax: +91-512-2597408;
Email: kamalkk@iitk.ac.in
chemical vapor deposition (CVD), and wet chemical
method; then plasma, thermal, or NH3/N2H4 treatment to the
graphene lattice. Apart from these methods, wet chemical
method is more versatile and widely used for the synthesis of
graphene nanosheets (Gns) followed by chemical reduction
of graphene oxide (GO) at large scale. Mostly, N doping into
the graphene lattice is done by NH3 and N2H4 [12]. These
materials are highly toxic and have serious environmental
impacts. As the consequence, development of simple eco-
friendly and cost effective approach for synthesis of N-doped
graphene remains an important challenge.
Cow urine has a unique importance in Ayurveda and is
considered to be the most effective animal originated secre-
tions with innumerable therapeutic values. In Hindu mythol-
ogy, it has great sacred importance and drinking of cows
urine has been practiced for thousands of years [13]. Cow
urine inherently owns excellent anti-oxidant and antibacterial
activity by virtue of its high natural nitrogenous content and
is used singly or in combination with some other herbs
against many diseases. Total natural N content is found ~ 6.8
to 21.6 g N litre
1 in the cow urine, consisting of ~ 69%
(urea), 7.3% (allantoin), ~ 5.8% (hippuric acid), 3.7%
(creatinine), 2.5% (creatine), 1.3% (uric acid), 0.5% (hy-

2452-2732/17 $58.00+.00 2017 Bentham Science Publishers

2 Current Graphene Science, 2017, Vol. 1, No. 1
poxanthine), 1.3% (free amino acid N) and 2.8% (ammonia)
[14]. Due to its excellent anti-oxidant and antibacterial activ-
ity, Ayurveda practitioners use cow urine as a medicine to
cure several diseases such as cancer, allergies, bacterial/viral
infections, tuberculosis, hepatitis, ulcer, heart disease, skin
infections, aging, chemical intoxication, etc. [15]. Several
patents have been awarded on the benefits of cow urine such
as: (i) pharmaceutical composition of cow urine distillate and
antibiotic to enhance antimicrobial effect, (ii) composition of
cow urine useful for protecting and repairing DNA from
oxidative damages, and (iii) pharmaceutical composition of
cow urine distillate and antibiotic as anti-infective and anti-
cancer agents [16-17]. In addition, cow urine has been used
in the field of nanoscience and technology as a biological
material, which has the potential to reduce the metal ions
into metal nanoparticles (NPs). For example, Prabhu et al.
have used fermented cow urine as a reducing medium to the
synthesis of silver nanoparticles (Ag NPs) for antibacterial
activity [18]. Meanwhile, Govarthanan, et al. have synthe-
sized Ag NPs using panchakavya (Indian traditional farming
formulating agent, a mixture of cow products such as urine,
milk, dung, cured and ghee) as a reducing agent [19]. Re-
cently, high quality graphene nanosheets have been synthe-
sized by Chamoli, et al. using cow urine as a reducing agent
for GO [20]. Therefore, high natural nitrogenous content
makes cow urine as a suitable natural dopant material for N
doping into the graphene lattice.
Herein, simple green approach has been demonstrated for
the synthesis of NGns via solvothermal method using natural
nitrogen dopant, cow urine. The schematic of various steps
in the synthesis process is shown in Fig. (1). This synthesis
process has several distinctive features such as low-cost and
Fig. (1). Schematic of various steps in the synthesis process of NGns.
Chamoli et al.
natural dopant, a single step reaction, mild synthesis condi-
tions, mass-scale production of NGns with high N content 

3.2 at.% into the graphene lattice.
2. MATERIALS AND METHODS
2.1. Materials
Natural flake graphite (NFG) (Sigma Aldrich, 99.99%)
was used as carbon source. Potassium permanganate
(KMnO4), hydrogen peroxide (H2O2, 30%), sulfuric acid
(H2SO4, 98%), sodium nitrate (NaNO3) from Qualigens Fine
Chemicals, India and cow urine (Divya Godhan Ark, Patan-
jali Ayurved Ltd., Haridwar, Uttarakhand, India) were used.
All chemicals were used without further purification.
2.2. Preparation of GO
GO was prepared by modified Hummers and Offeman
method [21]. In brief, 2 g NFG was added into the mixture of
96 ml H2SO4 and 2 g NaNO3 under ice bath. 12 g KMnO4
was added slowly to the mixture stirred below 20 o C, mixture
changes to green color. Then, the mixture was transferred to
water bath maintaining at 35 oC for 1 h, color of solution was
changed from green to purple brown paste. 80 ml of deion-
ized (DI) water was added drop wise to the mixture and tem-
perature was raised to 98 oC. Further, 200 ml DI containing
10 ml of H2O2 aqueous solution was added until bubble for-
mation stopped, and stirred for 0.5 h. The color of mixture
was changed to yellowish, which indicates the oxidation of
NFG. Finally, the solution mixture was allowed to cool at
room temperature and filtered, washed several times with DI
water to remove the remaining impurities. The resultant GO
was dried at 60 oC for 24 h in ambient condition.
N-Doped Graphene by Cow Urine Current Graphene Science, 2017, Vol. 1, No. 1 3

2.3. Synthesis of NGns

GO powder (0.5 mg/ml) as prepared by modified Hum-
mers method was dispersed in 100 ml cow urine and ultra-
sonicated at 100 W for 2 h. Once, GO was fully dispersed in
cow urine then dispersed solution was directly placed into
200 ml Teflon-vessel stainless steel autoclave at 180 oC for
12 h. Resultant i.e., NGns were filtered (0.2
m membrane
filter), washed several times with DI water and dried for 3 h
in vacuum oven at 60 oC.

2.4. Characterizations
X-ray diffraction patterns of NFG, GO and NGns were
recorded with XPert Powder PANalytical, Advanced X-Ray
Diffractometer with a Cu K adsorption spectrometer. Ra-
man analysis was carried out by LabRam Micro-Raman
spectrometer (Jobin-Yuon HR 800 UV) using a HeNe (-
633nm) laser excitation source. The morphology of GO and Fig. (2). XRD pattern of (a) NFG, (b) GO and (c) NGns.
NGns were examined by field scanning electron microscopy
(FESEM) JEOL JSM-7100F. Fourier transform infrared
spectroscopy (Bruker-FTIR) of GO and NGns were con-
ducted by using KBr pallet in the range of 4000-400 cm-1 .
The characteristic absorption of GO and NGns were acquired
by the UV-vis diffuse reflectance spectra (UV-vis DRS) with
a UV-vis-NIR spectrophotometer (Perkin-Elmer Lambda
1050). The surface compositional analysis of GO and NGns
were carried out by Multifunctional XPS (ULVAC, PHI500
VersaProbe II with 1486.6eV K Al X-ray source).

3. RESULTS AND DISCUSSION

Figure 2 shows the XRD patterns of NFG, GO, and
NGns. XRD pattern of NFG exhibits two sharp peaks at 2
of 26.56 (002) and 54.66 (004), respectively and d-spacing
is found ~ 3.33 as shown in Fig. (2a). In case of GO in the
Fig. (2b), the main characteristic peak of graphite at 2 of
26.56 is completely suppressed, reappeared at 2 of 14.10
(001), and confirmed the intercalation of water molecule, as
well as generation of oxidation groups such as hydroxyl,
epoxy between the graphite galleries and d-spacing is in-
creased to ~ 6.24 from 3.33 (NFG). It is also observed that
NGns exhibits a sharp broad peak centered at 2 of 26.10
(002), having d-spacing ~ 3.40 , indicates the existence of
 stacking between graphene sheets and the exfoliation of
graphene sheets from GO as shown in Fig. (2c) [22].
Figure 3 shows the Raman spectra of GO and NGns.
Raman spectra of GO exhibits two sharp peaks. D band at
1337 cm1 is due to the disorder resulting from defects in the
GO, and G band at 1603 cm1, which is a main spectral fea-
ture of graphite, arising due to the in-plane vibration of car-
bon atoms as shown in Fig. (3a) [23]. Meanwhile, Raman
spectra of NGns show two sharp peaks at 1342 and 1608 cm-
1
, corresponds to D band (disordered sp2 microdomains by
introduction of N atoms into the graphene lattice) and G
band (symmetric E2g vibrational mode of sp2 carbon atoms),
respectively as shown in Fig. (3b). It is observed that the
ID/IG ratio increases from 1.10 to 1.22, indicating the reduc-
tion of oxygen functional groups from GO. In addition, G
band (1603 cm-1) has been shifted to higher frequency (1608
cm-1), which strongly indicates structural distortion induced
by N atoms. The crystalline domain size (La) is calculated
Fig. (3). Raman spectra of (a) GO and (b) NGns; Inset: 2D peak.
using Tuinstra-Koenig (TK) relation and retrieved by ID/IG
[24]. The La value is decreased to 35.02 and 31.58 nm for
GO and NGns respectively, suggesting the more disordered
arrangement in honeycomb lattice by N-doping. In addition,
the intensity ratio of Raman 2D and G band (I2D/I G) gives the
estimation of number of layers of graphene sheets; and typi-
cally found for single ~1.6, double ~ 0.8, triple ~ 0.30 and
multi (>4) ~ 0.07 layers [25]. Less intense 2D band has been
observed at 2675, 2679 cm-1 with I2D/IG ratio ~ 0.10, 0.07 for
GO and NGns, respectively as shown in Fig. (3) (Inset). The
I2D/IG ratio ~ 0.07 indicates that cow urine reduced GO into
multi (>4) layer NGns. Cow urine contains high amount of
natural nitrogenous compound (i.e. urea) and possible reduc-
tion mechanism may be predicted on the basis of urea. Urea
produces redox reactions between electrochemically active
functional groups in the carbon skeleton and H2O, and re-
sponsible for the removal of oxygen functionality from GO,
yields graphene nanosheets at large scale [26].
4 Current Graphene Science, 2017, Vol. 1, No. 1 Chamoli et al.

FTIR spectroscopy is used to analyze the types of chemi- red-shifted absorption signature is observed at 272 nm for
cal bonding in GO and NGns as shown in Fig. (4). The FTIR NGns shown in Fig. (5b), indicates the restoration of elec-
spectra of GO shows bond stretching at 3434 cm-1, which tronic conjugation within the graphene sheets [27].
indicates OH stretching vibrations, 1716 cm-1 indicates the
C=O stretching vibrations, 1632 cm-1 indicates the C=C
stretching vibrations and 1198, 1061, 843, 848 and 585 cm-1
indicate the C-O stretching vibration as shown in Fig. (4a)
[27]. Figure 4b shows FTIR of NGns. It exhibits two peaks
at 1564 and 1195 cm-1. These are due to the introduction of
heteroatoms into graphene lattice and can be attributed as
C=C or C-N, and C-N, respectively. Moreover, C-O stretch-
ing vibration peaks at 1061, 843, 848 and 585 cm-1 have
suppressed, and suggested that the cow urine deoxygenates
the GO, and the less oxygen contenting NGns are success-
fully synthesized.

Fig. (5). UV-Vis spectra of (a) GO and (b) NGns.

Figure 6 shows FESEM image of GO and NGns. FE-

Fig. (4). FTIR spectra of (a) GO and (b) NGns.
Figure 5 represents the UVvis studies to understand the
characteristic absorption of GO and NGns. GO exhibits a
broad absorption peak around 234 nm, assign to the 
*
transition of aromatic CC bonds as shown in Fig. (5a). A
SEM morphology of GO depicts the thin oxidized graphene
sheets, ranging from several nanometer to micrometer, are
closely associated with each other as shown in Fig. (6a).
Moreover, the cow urine deoxygenates GO, and simultane-
ously N atoms are doped in individual layers graphene
sheets to produced NGns. Sheet like morphology with
wrinkled structure has been observed in NGns as shown in
Fig. (6b).
Figure 7 shows the XPS spectrum of GO and NGns, and
analyses the level of N atoms introduced into the graphene
lattice. XPS survey of NGns has confirmed that ~ 3.2 at.%
N content is successfully doped into the graphene lattice by
cow urine and C/O ratio has increased from 2.07 to 5.27 as
shown in Fig. (7a). The comparison of N-doped content in

Table 1. Comparison of nitrogen content doped into the graphene lattice by different dopants.

Method N-dopant N Content (at. %) Reference

CVD NH3  8.9 Wei et al. [28]

CVD NH3 4 Qu et al. [29]

Arc-discharge NH3 1 Li et al. [30]

Thermal treatment NH3  1.1 Guo et al. [31]

Plasma treatment N2 3 Jafri et al. [32]

Solvothermal NH3 3-5 Li et al. [33]

Solvothermal N2  2.66 Jung et al. [34]

Solvothermal Cow urine 
 Present work

N-Doped Graphene by Cow Urine Current Graphene Science, 2017, Vol. 1, No. 1 5

Fig. (6). FESEM image of (a) GO and (b) NGns.

the graphene lattice by different methods has been tabu-

lated in Table 1 and the results are within the reported val-
ues [28-34]. The high resolution C1s spectra with Shirley
background of GO ascribed an intense peak at 284.5 eV
(C-C bonds), is corresponded to conjugated honey-comb
lattice of carbon atoms. During synthesis of GO, strong
oxidation as well as destruction are occurred in the sp 2
atomic structure of graphite, and C-O bonding configura-
tion peaks at 286.5, 287.1, 288.3 eV could be attributed to
the C-O, C=O and O-C=O, respectively as shown in Fig.
(7b) [35]. After processed by cow urine, substantial decre-
ment is observed in all oxygen related peaks, confirms the
removal of oxygen spices from GO and delocalized
-
conjugation restored in NGns as shown in Fig. (7c). The
high resolution XPS spectrum of N1s peak in NGns can be
deconvoluted into three broad peaks with binding energies
of 398.8, 399.8 and 401.1eV, which correspond to pyridinic
N, pyrrolic N and graphitic N, respectively as shown in Fig.
(7d) [36].

CONCLUSION
In summary, we have successfully synthesized NGns by
simple solvothermal reaction using cow urine as a natural
dopant of N atoms. The XPS analysis reveals that N atoms
are successfully doped in graphene lattice from cow urine.
The proposed approach has several advantages over tradi-
tional methods for the synthesis of NGns: (1) cow urine is
natural dopant material and easily available, and highly toxic
dopant material such as NH3 is avoided. (2) This approach
produces NGns with high value of N content ~ 3.2 at. % in
graphene lattice. In addition, the process is a unique, strik-
Fig. (7). XPS analysis (a) XPS survey of GO and NGns, (b) high
ingly simple, cost effective, environmental friendly and pro- resolution spectra C1s of GO, (c) high resolution spectra C1s of
duced NGns at large scale. NGns and (d) high resolution spectra N1s of NGns.
 6 Current Graphene Science, 2017, Vol. 1, No. 1
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The authors acknowledge the financial support provided
by Imprint, Proposal-6134 (Ministry of Textiles and Ministry
of Human Resource Development) and Indian Institute of
Technology Kanpur, India for carrying out this research
work.
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