Abstract

This is possibly the first report on the utilization of modified hummers method for the in situ
synthesis of a novel cellulose-graphene oxide nano bio-composite in a single reaction vessel,
with cellulose extracted from waste jute and graphite being the starting material. The synthesized
in situ nano biocomposite have been characterized by UV spectroscopy, FTIR, TEM, SEM,
DLS, XRD. The composite was employed as an excellent nano-adsorbent for the removal toxic
dye methylene blue. Process optimization with RSM studies revealed adsorption capacity of the
synthesized material to be as high as 300mg/g. Further investigation showed that the adsorption
followed a pseudo second order kinetics and also fitted well with the frendluich isotherm ,
thereby indicatinng that the surface functional groups of the composite are actively participating
in the sorption process. Owing to its efficient adsorption capacity and also simplicity of the
synthesis process as well as cost effectiveness, the composite has great potential for waste water
remedial application at a large scale.

Keywords: Modified Hummers method, Graphite, Graphene oxide, Cellulose, in-situ synthesis,
biocomposite, nanocomposite, Methylene blue, Adsorption, RSM, Optimization

1.0 Introduction
Nanomaterials based on carbon and its sister compound graphene has been enjoying a good deal
of attention from researchers for quite some time. Graphene is a planar sp-2 hybridized 2-
dimensional and single atomic layer thick material that acts as an excellent conductor. On the
other hand Graphene Oxide (GO) has an sp3 hybridized tetrahedral structure and contains high
amount of oxygenated functional moieties such as carboxyl group at its edge, hydroxyl and
epoxy group on its basal plane and is an insulator by nature. (Roy et al ,2016) Unlike graphene,
GO is readily dispersible in water and organic solvents and is also soluble in some solvents.
(Johnson and Coleman, 2015---paper in phn scrn shot ).Owing to its astonishing properties,
nontoxic nature, excellent chemical stability and environmental friendliness, GO and its
derivates have given rise to a never ending quest for extensive and countless applications in
various fields. GO nanoplatelets were successfully employed as potential adsorbent for the
removal of acutely toxic drug ibuprofen from aquesos medium. (Banerjee et al,2016 my paper).
Chitosan and GO based composite hydrogels were used as an effective tissue engeneering tool
to increase viability and proliferation of human embryonic stem cells (Jing et al ,2017). Hence,
we can see that in order to achieve potential candidates for highly specific end use, a large
number of organic and inorganic compounds, especially polymers are being explored for
developing very exclusive GO-based composites and hybrid products. In a very recent study GO
modified with Folic acid-PEG conjugate was implied to attenuate the migration of breast cancer
cell line providing insight to cancer treatment. (Basu et al, 2019).
 The first report on the synthesis of graphite oxide was made by Brodie in 1859 which was
improved by Staudenmaier in 1898. Much later in 1958, with the aim of improving
Staudenmaier’s approach for GO synthesis, Hummers and Offeman developed an alternate
oxidation technique, where NaNO3 and KMnO4 dissolved in con. H2SO4 was used to oxidize
graphite into graphite oxide (Hummers & Offeman, 1959). Since then, hummers method has
been popular amongst researchers and industrialists as the most promising route for synthesis of
graphene oxide from graphite. Innumerable scholars across the globe have followed the
hummers method as well as modified the protocol in several different ways to obtain improved
and high quality graphene oxide. Often a mixture of phosphoric acid and sulfuric acid is used
and temperature control is adjusted or even eliminated in order to simplify the process. (Huang
et.al, 2011). Depending on the desired properties and intended end use researchers tend to play
around with hummers synthesis process, such adapted versions of the hummers method are
mostly reported as modified hummers method or improved hummers method. (Sohail et al,
2017) (paper on mobile). However the application of hummers method have mostly been limited
to the synthesis of GO and precursors for rGO and graphene. Very few studies show the
utilization of this process for the oxidation or synthesis of any material other than GO. In a past
study a modified Hummers’ method was employed as an effective strategy to increase the
capacitance of commercially available carbon paper. (Qiu et al, 2014). In most of the studies
dealing with synthesis of GO based composites, GO is synthesized independently following
hummers or modified hummers method, and is then combined with the other ingredient/s mostly
polymers either by grafiting or crosslinking. Potato starch was cross linked with separately
synthesized GO using glutaraldehyde, a cross linking angent and the resultant composite was
used for adsorptive removal of Methylene blue. (Bhattacharya et al 2018)

Among the various biopolymers, cellulose is the most abundant on earth and is used for
the preparation of microcrystalline cellulose (MCC) which can be further used to obtain cellulose
nano crystals (CNC). Because of easy availability, low cost, biodegradibilty, and large surface
area (Jon paper) along with the presence of negetive surface functional groups (-OH), cellulose
has been selected as integral component for the in situ composite. In this study keeping in mind
the idea of minimal use of resources, jute waste (very short jute fibres) obtained from jute
carding machine has been used to extract MCC following a very simple and economical process
as previously reported (Jon paper)

This study for the first time makes use of modified hummers method for the direct one
pot in situ synthesis of a novel CNC-GO nano-bio-composite. Celulose extracted from jute waste
and procured graphite flakes were used as parent material and subjected to modified hummers
process in the same reaction vessel. Moreover there was absolutely no need of producing CNC
prior to the in situ synthesis, because the end product showed the presence of CNC indicating
that during the in situ synthesis, the MCC was converted to CNC in situ by broken down the
MCC into CNC. (refine the lang of this paragraph)
(One short para on charactrstn n successful applictn, metion use of RSM)
2.0 Materials and methods
Graphite flakes powder was obtained from Sigma-Aldrich Co., India. Sodium nitrate,
potassium permanganate, 98 % sulphuric acid and H2O2 were supplied by Merck Ltd., India.
Micro crystalline cellulose MCC MFC was synthesized from waste jute fibres. Methylene blue
(MB) dye was also obtained from Merck Ltd., India.

2.2 Methods
2.2.1 Extraction of micro crystalline cellulose from waste jute (cite Jonathan Da paper)
Jute waste fibres (JWF) were used to prepare pure cellulose by subjecting the JWF to
chemical disintegration process as described by jon et al. The process has been explained briefly
as following. At first JWF was treated with 25%v/v H2SO4 to produce micro crystalline jute
which was then thoroughly washed to neutral followed by delignification via treatment with 2%
H2O2 at slightly alkaline pH and 90 oC for an hour, washed to neutral and dried. The final
product was pure celullose in the form of MCC. This MCC was later used in the direct in situ
synthesis of GO-CNC composite.

2.2.2 Synthesis of the in situ nanocomposite

GO-CNC composites were prepared in-situ in a single reaction vessel via modified
hummers method. 3 gm of graphite powder and 1.5 gm of sodium nitrate were taken in a
flask and was completely dispersed in 100 ml of 98% H2SO4. The reaction mixture was
placed in an ice bath maintaining the temperature well below 10 degree. It was constantly
stirred for 20 mins using a magnetic stirrer at a moderate speed to allow adequate mixing.
12 grams of potassium permanganate was carefully added over 30 mins while the mixture
was still stirring. After 4 hours, the acid was diluted with slow addition of 200 ml of
distilled water. 3 gm of MCC was then added to the above mixture. After 15 mins the ice
bath was removed and the reaction was allowed to continue for 3 hours with constant
stirring at room temperature. After 3 hrs the reaction temperature was rapidly increased by
adding 100 ml of hot distiiled water. Temperature was then maintained at 90 0C for next 30
mins to achieve exfoliation. It was then cooled to room temperature and finally 30ml of 10%
H2O2 was added to remove the un-reacted MnO-4 and MnO-2 salts. The slurry which has then
turned dark yellow from blackish brown was washed to neutral with distilled water via
centrifugation at 13000 rpm .The residual mass was dried in a hot air oven at 60 0C to obtain
in-situ GO-CNC composite in powdered form.
For the purpose of better comparison, pure GO was separately synthesized via modified
Hummers method (Pablo da) from the same graphite source that was used for the composite.

2.3 Characterization of the GO-CNC composite

2.3.1 Scanning electron microscopy (SEM) analysis

 GO-CNC composite sample was dispersed in triple distilled water and sonicated for 2 hr.
A drop of the dispersed sample was casted on a silicon slide and dried. The cast was then used to
study the topography and structure of the sample using a ZEISS scanning electron microscope
(SEM).

2.3.2 Fourier Transform Infrared (FTIR) Spectroscopy

In order to determine the surface functional groups present on the GO-CNC insitu
composite, FTIR spectra of the sample was recorded using KBr pellet with Perkin Elmer
Spectrum Express Version 1.03.00 within the wave number range of 400–4000 cm-1.

2.3.3 X-ray Diffraction (XRD) Analysis

The crystalline properties of the synthesized composite was studied by recording XRD patterns
of the sample with X-PERT-PRO Pan Analytical Diffractometer using a Cu Kᾳ radiation
(λ=1.5406 nm) at 40kV voltage and 30mA current.

2.3.3 Dynamic light scattering (DLS) analysis

The measurements for the Composite particle size and zeta potential were made with a

Zetasizer Nano ZS (Malvern Instruments, Malvern UK, setting the detection angle to 174° at a

temperature of 25°C adopting water viscosity value for the measurement.

3. Adsorption Experiments

The synthesized GO-CNC composite was applied as an adsorbent for the removal of toxic dye
Methylene Blue (MB) (C16H18N3SCl; molecular weight 373.90 g/mol, absorption wavelength
664 nm). Adsorption experiments were conducted in a batch mode to study the effect of variation
in process parameters that greatly influence the adsorption capacity of the adsorbent material.
Critical parameters such as solution pH (2-10), reaction temperature (293K-323K), dose of
adsorbent (20-50 mg/L) and contact time (30-180 mins) were studied. For each experiment 70 ml
of freshly prepared 10mg/L MB solution were taken in Erlenmeyer flasks, each parameter was
varied one at a time by keeping the others fixed. A constant agitation speed of 120 RPM was
maintained using a shaker incubator. (Model ). Aliquot of samples were drawn at predetermined
intervals of time, and centrifuged to obtain adsorbent free residual MB solution. The
concentration of the residual MB solution, after adsorption was measured at 664 nm using UV-
Visible Spectrophotometer (Model: Perkin Elmer UV-VIS Spectrometer). Effect of initial dye
(MB) concentration (5-30 mg/L) was later studied separately, in the same manner. Each
experiment were repeated thrice and expressed as mean +_ SD. The percentacge of MB removed
from solution via adsorption (removal percentage = R %) and the adsorption capacity qt (mg g-1)
that is the amount of MB adsorbed onto the surface GO-CNC in time t, were calculted from the
following formulae:

R (%) = (C0 – Ct) /100 … … … … … (1)

Where C0 and Ct are the initial (t=0) and final (at any given time t) concentration of MB in
solution respectively.

qt = (C0-Ct) V/W ……………………...(2)

where V is the working volume of dye (MB) solution and W is the amount of adsorbent, GO-
CNC in ths case.

Process optimization by RSM

Adsorption Kinetics

The study of adsorption kinetics is very important for understanding the mechanism of the
adsorption process. With the help of different kinetic models, one can determine the adsorption
equilibrium time as well as the rate of adsorption which in turn reveals the efficiency of the
adsorbent. (Sromona, urban wood). 2.1mg of GO-CNC composite were added to 70 ml of MB
solution with an initial concentration of 10 mg/L and agitated at 120 rpm at 40o C and pH 10.
The final concentration that is the amount of residual MB in solution was measured and recorded
at a time interval of 30mins, starting from 0 mins to 180 mins. The data hence obtained were
investigated with three kinetic models namely the Lagergren pseudo-first-order, the pseudo-
second-order model and the intraparticle diffusion model.

The linear form of Lagergren pseudo-first-order is given as follows :

log (qe−qt) = log qe − (K1 /2.303) t …………………(3)

where, qe (mg g-1) and qt (mg g-1) represent the adsorption capacity at equilibrium and adsorption
capacity at any given time t, respectively while K1 (min -1) is Lagergren rate constant of
adsorption. The values of K1 and qe can be determined from the slope and intercept respectively,
obtained form a linear plot of log (qe−qt) against t.

The pseudo-2nd-order model equation is given as follows:

t/qt = (1/K2qe2) + (t/qe) …………… ……… (4)

In the above equation K2 (g mg-1 min-1) is the pseudo-second order rate constant which can be
calculated from a plot of (t/qt) versus t.

The inter-particle difusion model equation has been

given below

where qt vs. t1/2 graph was plotted and the intraparticle diffusion rate constant (Kdif) and constant (C) were
determined
from the slope and intercept of the plot, respectively

The activation energy (Ea) for adsorption of PAHs was

calculated using the Arrhenius equation (Banerjee et al.
2017a) given as follows:
where k was the exponential constant, A was the pre-exponential factor; Ea (J mol−1) was the activation energy, T (K)
was temperature and R (8.314 J mol−1 K−1) was the ideal
gas constant. Activation energy (Ea) was obtained from the
slope of the ln k vs. T−1/2 plot.
The inter-particle difusion model equation has been
given below:
where qt vs. t1/2 graph was plotted and the intraparticle diffusion rate constant (Kdif) and constant (C) were
determined
from the slope and intercept of the plot, respectively.
The Elovich model equation has been provided as follows:
where α and β represented initial rates of adsorption and
desorption, respectively. qt vs. ln(t) graph was plotted and
1/β and 1/β ln(αβ) was determined from the slope and intercept, respectively.
The R2 (correlation coefcients) calculated for all the
models indicated the most ftting model for this study.
Adsorption thermodynamic

Adsorption Isotherm

The adsorption isotherm can be defined as the relationship between the equilibrium
concentration of adsorbate and its mass adsorbed per unit of the adsorbent, at constant
temperature (Nur. H. Othman). Adsorption isotherm analysis is of utmost importance in
understanding the behavior of the adsorption molecules, their interaction and their distribution
between the solid and liquid phase in the equilibrium state (Yanhui Li).
 Several isotherm models are used for the evaluation of the process. Data obtained from
batch study and equilibrium adsorption study with varying initial adsorbate concentration at
equilibrium temperature, were subjected to three selected isotherm models namely Langmuir,
Freundlich and Temkin isotherms.

Langmuir Isotherm:
This isotherm model is based on the assumption that monolayer adsorption takes place on
a homogeneous surface which possess identical adsorption sites. The Langmuir equation is
expressed as:

𝐶𝑒 𝐶𝑒 1
= + (1)
𝑄𝑒 𝑄𝑚 𝐾𝐿 𝑄𝑚

RL Factor……

Freundlich Isotherm:
Freundlich isotherm model describes the multilayer adsorption of adsorbate molecules
from liquid phase onto a heterogeneous solid surface. This model assumes exponential
distribution of several adsorption sites with different adsorption energies that participate in
the sorption process. The linear for of the empirical equation is given below:

Temkin Isotherm:
This model not only explains the pattern of interaction between the adsorbate and the
adsorbent molecules but also specifically gives insight into the heat of sorption of the
adsorbate molecules. (Banerjee 2017)The model assumes that heat of sorption of molecules
present in the layers decreases linearly with coverage. (Barman 2018) From the Temkin’s
equation KT (mg/L) and BT (J/mol) that are coefficients of adsorption capacity and heat of
sorption respectively, can be obtained from a plot of qe vs ln Ce Banerjee 2016) Wherein T
represents the selected process temperature while R is the ideal gas constant (8.314J/mol/K)

Results and Discussion:

FTIR

The FTIR spectra of GO-CNC composite showed a strong peak at 1727 due to C=O stretching
vibrations of the carboxyl group (which is characteristically found in GO spectra)(Pablo da
proflavin) and –CO stretching vibrations at 1225, thereby confirming the successful formation of
GO in situ. To add to this confirmation, an intense broad peak at 3414 due to stretching vibrations
of Hydrogen bonded -OH was also found. A medium peak at 2918 which is present in cellulose
due to CH bending of methane(I&EC Research) has reduced in intensity and also shifted slightly
towards the right in the composite and appears at 2904 (alkane) (GO cnf xps new paper). This
change in the mode of CH vibrations indicate the incorporation of cellulose and its interaction with
GO in the in situ composite. The peak at 1628 ascribed to C=C vibrations in cellulose has shifted
and sharpened in the composite occurring at 1635. Another charactersictic absorption due to –CH
bending of Methyl group at 1373 (book) present in cellulose is found to have almost disappeared
in the composite, and appearance of an altogether new strong peak at 1053 that can be ascribed to
C-O of esters (book pg 28) validates the interaction between GO and cellulose during insitu
formation.