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A facile
strategy to synthesis GO-ECH-DETA nanocomposite by the reaction of graphite
oxide (GO) with epichlorohydrin (ECH) as a coupling agent and allylamine
hydrochloride (AAH) as a cationic ligand (GO-ECH-AAH), were prepared and
employed for endotoxin accumulation and removal from aqueous solution. In this
work, the synthesis and characterization of GO-ECH-DETA nanocomposites through
seeded emulsion polymerization and the selective light reflection properties of
dry films have been reported. Amphiphilic molecule sulfonated 3‐pentadecyl
phenol was used as a co‐surfactant to stabilize GO dispersions during the
emulsion polymerization process. The synthesis mechanism of the nanocomposite
was studied and characterized with Scanning Electron Microscopy (SEM), X-ray
diffraction patterns, Transmission Electron Microscopy (TEM), Fourier Transform
Infrared Spectroscopy (FTIR), X-ray Photoelectron Spectroscopy (XPS), Raman
spectroscopy, UV-Vis spectra and Atomic Force Microscopy (AFM) were used to
study the morphology and structure of the composite particles on drying. The
AFM study confirms the non‐spherical shape of the particles. This kind of
nanocomposite undoubtedly shows promising applications in the fabrication of
multi-functional materials and can be used as a potential candidate for removal
of PLS from aqueous solution. Batch adsorption studies showed that the product
possesses superior adsorption capacity of endotoxin from aqueous solution. This
is a simple and cheap procedure, has proven to remove endotoxins without
affecting any significant losses in protein yields and biological activities.
The physicochemical properties of the nanocomposite were fully characterized,
adsorption equilibrium and kinetic analysis indicated that the adsorption
isotherm was well fitted by Langmuir isothermal model with the maximum
adsorption capacity of 138.63 mg g⁻¹, the kinetics of the endotoxin adsorption
process was shown to follow the pseudo-second-order model. The results showed
that the optimum condition for endotoxin removal was obtained at pH of 6.05,
GO-ECH-AAH dosage of 500 mg L⁻¹, contact time of 60 min and endotoxin
concentration of 200.0 endotoxin units per milliliter (EU mL⁻¹).
Keywords: Graphene oxide, Epichlorohydrin, Allylamine hydrochloride,
Nanocomposites, Endotoxin, Adsorption
INTRODUCTION
Lipopolysaccharides (LPS), also called endotoxins, are one
of the main pollutants found in commercially produced proteins and biologically
active substances, which often interfere with biological effects of the main
ingredient. The presence of small amounts of endotoxin in recombinant protein
preparations causes tissue injury, endotoxin shock and even death. Thus, it is
often essential to remove endotoxins from drugs, injectable and other
biological and pharmaceutical products. LPS are derived from cell membrane of
Gram-negative bacteria and are responsible for its organization and stability.
In pharmaceutical industries endotoxins always found in the final product.
Endotoxins release does not happen only with cell death but also liberated
during growth and division into the surrounding environment. Even if the media
is poor in nutrients, such as clean or saline water, endotoxins are found
almost everywhere. However, upon cell death LPS are shed in large amount than
during growth and division. They are highly heat-stable and are not destroyed
under regular sterilizing conditions. However, LPS can be inactivated when
exposed
The most biological activate part of endotoxin is lipid A
and indeed is responsible for its toxicity. Endotoxin is composed of
b-1,6-linked D-glucosamine residues, covalently linked to 3-hydroxy-acyl
substituents with 12-16 carbon atoms via amide and ester bonds. These further
esterified with saturated fatty acids. This hydrophobic part of endotoxin adopts
an ordered hexagonal arrangement, resulting in a more rigid structure compared
to the rest of the molecule [12,14]. The core oligosaccharide has an inner
3-deoxy-D-manno-2-octulosonic acid (KDO) - heptose structure region and an
outer hexose region. In E. coli species, five different core types are
known and Salmonella species
share only one core structure. The core region close to lipid A and lipid A
itself are partially phosphorylated (pK1=1.3, pK2=8.2 of phosphate groups at
lipid A). Thus endotoxin molecules exhibit a net negative charge in common
protein solutions [10]. The O-antigen is generally composed of a sequence of
identical oligosaccharides (with three to eight monosaccharides each), which
are strain specific and determinative for the serological identity of the
respective bacterium [8]. The endotoxin monomer molar mass range from 10 to 20
kDa, owing to the variability of the oligosaccharide chain; even extreme masses
of 2.5 (O-antigen-deficient) and 70 (very long O-antigen) kDa can be found. There
are various forms of endotoxins supra-molecular aggregates in aqueous solutions
due to their amphipathic structures. Based on molecular dynamics, the
three-dimensional structure of endotoxin, especially the long surface antigen,
is much more flexible than the globular structure of proteins [13]. These
aggregates result from non-polar interactions between lipid chains as well as
of bridges generated among phosphate groups by divalent cations [1]. The
aggregate structures have been studied by different techniques such as electron
microscopy, X-ray diffraction, FT-IR spectroscopy and NMR. Results from these
studies have shown that, in aqueous solutions, endotoxins can self assemble in
a variety of shapes, such as lamella, cubic and hexagonal inverted arrangements,
with diameters up to 0.1 mm and 1000 kDa and high stability depending on the
solution characteristics (pH, ions, surfactants, etc.) [1,2].
The old approved techniques used by FDA for endotoxin
detection are the rabbit pyrogen test and Limulus Amoebocyte Lysate (LAL) assay
[15,16]. The rabbit pyrogen
test, is an old technique used in 1920s, involves measuring the rise in
temperature of rabbits after intravenous injection of a test solution. Due to
its high cost and long turnaround time, the use of the rabbit pyrogen test has
diminished and is now only applied in combination with the LAL test to analyze
biological compounds in the earlier development phase of parenteral devices.
Today the most popular endotoxin detection systems are based on LAL, which is
derived from the blood of horseshoe crab, Limulus polyphemus and clots
upon exposure to endotoxin. The simplest form of LAL assay is the LAL gel-clot
assay. When LAL assay is combined with a dilution of the sample containing
endotoxin, a gel will be formed proportionally to the endotoxin sensitivity of
the given assay. The endotoxin concentration is approximated by continuing to
use an assay of less sensitivity until a negative reaction (no observable clot)
is obtained. This procedure can require several hours [16]. The concentration
of 0.5 EU/mL was defined as the threshold between pyrogenic and non-pyrogenic
samples [16].
In addition to the gel-clot technique, scientists have also
developed two other techniques: turbidimetric LAL technique and the chromogenic
LAL technique. These newer techniques are kinetic based, which means they can
provide the concentration of endotoxin by extracting the real-time responses of
the LAL assay. Turbidimetric LAL assay contains enough coagulogen to form
turbidity when cleaved by the clotting enzyme, but not enough to form a clot
[17]. The LAL turbidimetric assay, when compared to the LAL gel-clot assay,
gives a more quantitative measurement of endotoxin over a range of
concentrations (0.01 EU/mL to 100.0 EU/mL). This assay is based on the
turbidity increase due to protein coagulation related to endotoxin
concentration in the sample. The optical densities of various test-sample
dilutions are measured and correlated to endotoxin concentration helped by a
standard curve obtained from samples with known amounts of endotoxin [18]
kinetic chromogenic substrate assay differs from gel-clot and turbidimetric
reactions because the coagulogen is partially or completely replaced by a
chromogenic substrate [19]. When hydrolyzed by the pre-clotting enzyme, the
chromogenic substrate releases a yellow-colored substance known as p-nitroaniline.
The time required to attain the yellow substance is related to the endotoxin
concentration [18]. However, kinetic turbidimetric and chromogenic tests,
although more accurate and faster than the gel-clot, cannot be used for fluids
with inherent turbidity such as blood and yellow-tinted liquids, e.g. urine and
their performance may be compromised by any precipitation from solution [19].
Therefore, different new methods for detection of endotoxin in different
samples have been studied and approved [20,21].
Carbonaceous nanomaterials, including carbon nanotubes
(CNTs) and graphene based materials (GBMs) such as graphene oxide (GO), hold
significant promise in engineering and medicine due to their intrinsic
electro-mechanical properties. Graphene and graphine
oxide are the most basic form of carbon, it is composed of sp2 bonded carbon
atoms arranged in a hexagonal arrangement in a 2D plane [22]. The lattice of
graphene consists of two interleaved triangular shaped carbon sub lattices. The
sub lattices overlap in such a way that carbon atom from one sub lattice is at
the centroid of the other sub lattice. Graphene has been utilized in many
engineering and industrial applications and graphene-based polymer
nanocomposites exhibit superior promising properties. For example,
graphene-based polymer composites show better thermal, mechanical and
electrical properties than the normal polymer [23,24]. It has been shown that
the mechanical and electrical properties of graphene-based polymer composites
are much better in comparison to clay or other carbon filler-based polymer
composites [25-29]. One of the main applications of graphene sheets is use as
reinforcement agents for the preparation of nanocomposites with different types
of polymers. Other than mechanical properties, electrical and thermal
properties of the polymeric matrix can also be enhanced. It is a fact that the
graphene-based nanocomposites present improved properties compared to the
original raw form of graphene. Graphene nanocomposites with polysaccharides
such as chitosan have many diverse new applications. Polysaccharide exist both
as linear or branched polymers, since their repeating monosaccharide units are
connected via O-glycosidic bonds [30]. Their properties, including gelation,
water solubility and other surface properties depend on the type of
monosaccharide composition. Advantages such as abundance in nature,
biocompatibility, biodegradability, easy functionalization and relatively easy
isolation from their natural sources have led to their study and use in several
applications, especially in the field of drug delivery and biomaterials [31].
Another application of graphene derivatives with polysaccharide is the use in
accumulation and removal of various types of pollutants from wastewater
effluents [32-34]. Graphene nanocomposites with chitosan have been used for the
removal of dyes [35,36], heavy metal ions [32-34] and pharmaceutical compounds
[32-34] from aqueous solutions. Despite the intriguing properties of
polysaccharides, their poor mechanical properties limit their applications.
Nanofillers such as graphene are known to improve the properties of raw
polymers, not only the mechanical but also the thermal and electrical
properties [37,38]. Moreover, the effects of the incorporation of graphene and
graphene oxide together in raw polymers have been extensively studied, mostly
synthetic polymers reinforced by graphene and graphene oxide find several
improvement in properties such as mechanical strength, thermal stability, gas
barrier properties, electrical and thermal conductivity, etc. [39-46].
Cationic
polymers which are useful as flocculants are prepared by condensation reaction
of a dialkylamine, dicyandiamide and a polyalkylenepolyamine, with a
difunctional epoxide. Functionalization of graphene oxide (GO) by crown ether
moiety to attach Li+ in the cage of five oxygen is a useful tool to
achieve 2D material for Li ion battery. The attachment of crown ether occurs
only through the reaction with epoxy groups of GO. Epichlorohydrin is used to
increase the number of the epoxy group to enhance and precise control of the Li+
content for tuning the activation energy of Li+ migration [47].
The final properties
of nanocomposites depend on various factors; the most important is the
interfacial bonding between the filler and the matrix. Poor adhesion can lead
to aggregates of the nanofillers or gaps between the surface of the composites
components, acting as stress concentration points and therefore causing
premature failure of the materials. Besides, the compatibility between
nanofiller and matrix, the geometrical and the aspect ratio of the fillers play
a similarly important role. Graphene possesses a high surface area, high aspect
ratio and high strength which are reasons for the enhanced performance of its
nanocomposites. Large graphene or GO flakes with high surface areas have proved
to be more efficient reinforcing agents than similar structures with smaller
aspect ratio.
Some commonly used techniques have been extensively used
for removing endotoxin contaminants are ultrafiltration [48] and ion exchange
chromatography. Ultrafiltration has been successfully effective in removing
endotoxins from water. Nevertheless, universal adoption of this technology is
limited by the presence of proteins, which can be damaged by physical forces [49].
Anion exchangers, which take advantage of the negative net charge of
endotoxins, have been used for endotoxin adsorption. However, when negatively
charged proteins need to be decontaminated, they may co-adsorb onto the matrix
and cause a significant loss of biological material. Also, net-positively
charged proteins form complexes with endotoxins, causing the proteins to drag
endotoxin along the column and consequently minimizing the endotoxin removal
efficiency [50]. LPS removal is more efficient on cationic exchangers than on
anionic exchangers. In recent years, alkane diols were shown to be effective
agents for the separation of LPS from LPS-protein complexes during
chromatography with ionic supports. Their effectiveness in reducing the protein
complexation with LPS is dependent on (I) the size of the alkanediol, (II) the
isomeric form of the alkanediol, (III) the length of the alkanediol wash, (IV)
the concentration of alkanediol and (V) the type of ionic support used,
cationic or anionic. Membrane-based chromatography has been successfully used
for PLS separations form protein, universal adoption of this technology has not
taken place because membrane chromatography is limited by the binding capacity,
which is small when compared to that of bead-based columns, even though the
high flux advantages provided by membrane adsorbers would lead to higher
productivity [51-60].
Jann et al. [61] tested that slab-polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) can be
used for the separation of LPS. Several methods have been used to separate the
different subclasses of LPS from individual strains, with sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration being
perhaps the most successful Tsai and Frasch [62],
McIntire et al. [63], Oroszlan and Mora [64], Weiser and Rothfield [65], Ribi
et al. [66], Hannecart-Pokorni et al. [67], McIntire et al. [68], Kim et al. [69],
Morrison and Leive [51], Maccari and Volpi [70] and Dietrich and Dietrich [71].
These methods are hampered by the tendency of LPS to
aggregate and by the difficulty in detecting and identifying each distinct
subclass [72]. Reichelt et al. [73] reported that the removal of endotoxin
could be achieved during chromatography purification with the use of Triton
X-114 in the washing steps. The application of 0.1% Triton X-114 in the washing
steps was successful at reducing endotoxins during histidine and GST (resin GST
sepharose) fusion protein purification, whereas washing steps lacking
surfactant were ineffective in eliminating endotoxins. In contrast to purified
materials employing the standard protocol which contained from 2500 to 34000 EU
mg-1, purified recombinant proteins treated with Triton X-114
contained concentrations as low as 0.2 to 4 EU mg-1 (less than 1% of
initial endotoxin content). Reichelt et al. [73] studied whether the use of
Triton X-114 in washing steps could eliminate endotoxins from proteins with a pI above 8.5. They found that washing
with Triton X-114 coupled with affinity chromatography effectively removed
endotoxins from negatively-charged proteins (SyCRP and NdhR). The minimal endotoxin
concentration achieved was lower than 0.2 EU mg-1; protein recovery
and yield were close to 100% [73]. The use of two-phase aqueous micellar
systems for the purification or concentration of biological molecules, such as
proteins and viruses has been growing [74-76]. In these systems an aqueous
surfactant solution, under the appropriate solution conditions, spontaneously
separates into two predominantly aqueous, yet immiscible, liquid phases, one of
which has a greater concentration of micelles than the other [77]. The
difference between the physicochemical environments in the micelle-rich phase
and in the micelle-poor phase forms the basis of an effective separation and
makes two-phase aqueous micellar systems a convenient and potentially useful
method for the separation, purification, and concentration of biomaterials [78].
Particularly for endotoxin removal, above the critical micelle concentration
(CMC) of surfactants, endotoxins are accommodated in the micellar structure by
non-polar interactions of alkyl chains of lipid A and the surfactant tail
groups and are consequently separated from the water phase (micelle-poor
phase). Surfactants of the Triton series show a miscibility gap in aqueous
solutions. Above a critical temperature, the so-called cloud point, micelles
aggregate to droplets with very low water content, by that forming a new phase.
Endotoxins remain in the surfactant-rich phase. Through centrifugation or
further increase in temperature the two-phases separate with the
surfactant-rich phase being the bottom phase [79,80]. If necessary, this process is repeated until the
remaining endotoxin concentration is below the threshold limit. The cloud point
of Triton X-114 is at 22°C, which is advantageous when purifying proteins [81]
used Triton X-114, showed that a 100-fold endotoxin reduction in two steps with
a final endotoxin content of 30 EU mg-1 and 50% loss in bioactivity
of the exopolysaccharide. In addition, about 100-fold endotoxin reduction was
shown by Cotten et al. [82] from plasmid DNA preparation with a final endotoxin
content of 0.1 EU in 6 μg DNA. The detergents, even though they were also very
effective at reducing the LPS levels, are relatively expensive, would add
significant cost to a manufacturing process, and may affect the bioactivity of
the protein of interest. Alternative chemicals are desired that could safely
and cost effectively be used in place of the alcohols or detergents as washing
agents for the separation of LPS from proteins during chromatographic unit
operations [83]. Indeed, these chemicals would be relatively inexpensive,
chemically well-defined, present minimal safety issues and ideally have minimal
impact on the bioactivity of the protein in question when implemented into a
process.
The objective of this research is to test the
synthesis and applications of a new nanocomposite polymer formed by mixing and
cross-linking networks graphite oxide (GO) with epichlorohydrin (ECH) as a
coupling agent and allylamine hydrochloride (AAH) as a cationic ligand to form
(GO-ECH-AAH). Then, we will examine the mechanical properties of the
nanocomposite polymer as well as its various applications in the accumulation
of PLS. The fabricated GO nanocomposites polymers have better mechanical and
thermal properties and stabilities than GO nanocomposites alone. The results
has shown strong interactions between the functional groups of the three
components, confirmed by FTIR spectra, led to a series of improved properties,
including mechanical strength in both wet and dry conditions. The results of
this research has shown that the novel graphene oxide (GO)-based adsorbent
embedded with epichlorohydrin (ECH) as a coupling agent and allylamine
hydrochloride (AAH) as a cationic ligand (GO-ECH-AAH), has excellent endotoxin removal from aqueous
solutions and biotechnological preparations. The results of our studies showed
that endotoxin strongly loads on the GO-ECH-AAH nanocomposite derivative via
σ–σ, π–π and n–π interaction and bonding between the nanocmposite and the LPS.
The interaction and adsorption behavior of the prepared composite was
elucidated with a series of experiments. The results revealed that the
adsorption mechanism dominated between endotoxin molecules and the GO-ECH- AAH
matrix favor acidic conditions were the optimum for the adsorption process at
pH 5.5. The Langmuir-Hinshelwood kinetic model adequately describes the
experimental results; both the pseudo-first order kinetic constants of the
reactions and the adsorption constants were calculated. GO-ECH- AAH was more
active than GO alone for the endotoxin accumulation. The reduction of 90% of
the endotoxin was observed after 1hr. Thus, graphene polymer nanocomposites
(GO-ECH-AAH) offer a green alternative to synthetic polymers in the preparation
of soft nanomaterials, results indicated that a significant interaction of the
allylamine hydrochloride of the AAH with both GO and ECH and ECH-AAH polymer
were inserted between the GO layers and (ii) ECH reacted with carboxyl and epoxy
groups of GO, leading to its reduction and hence the destruction of the layered
structure.
MATERIALS AND METHODS
Go synthesis
GO was synthesized through a modified Hummer's method
[84]: 6 g of natural graphite powder (Graphene
Laboratories Inc.), 4.5 g sodium nitrate and 207 mL sulphuric acid were added
in a reaction flask, kept at 10°C and stirred for 30 min, followed by the
addition of 27 g potassium permanganate. The solution was stirred for 45 min
and then 414 mL of water was added. After 12 h, 1260 mL of warm water and 45 mL
oxygen peroxide (30%) were added. The suspension was filtered, washed several
times and finally dried at 60°C in a vacuum oven.
Nanocomposite fabrication
The epoxy resin
used in this study was epichlorohydrin (ECH) as a coupling agent and Allyl
Amine Hydrochloride (AAH) a ligand and the hardener, both supplied by Huntsman.
Nanocomposite samples were prepared without nanomaterials (i.e., neat epoxy
resin), with GO and with AAH. They all received the same amount of filling
material, 0.25% wt. and the same amount of hardener, 27% wt. To improve GO
dispersion, these fillers were each mixed with acetone through bath sonication
(25 kHz) for 30 min. An aqueous solution of epichlorohydrin (ECH) (5 wt%) was
prepared at 100°C upon stirring for 1 h and subsequently cooled to room
temperature. An aqueous solution of Allyl Amine Hydrochloride (AAH) (5 wt%) was
prepared at 90°C upon stirring for 2 h and subsequently cooled to room
temperature. The Allyl Amine Hydrochloride (AAH) a ligand hardener was added to
each mixture and heated to 90°C to melt down. The chemically reduced GO
dispersion was then mixed with epichlorohydrin (ECH) epoxy resin solution at
65°C and ultrasonicated (42 kHz) for another additional 1 h. The resulting
mixtures were degasified at 80°C for 24 h to eliminate volatiles such as
acetone and avoid bubbles in the final nanocomposites. Curing process was held
in two stages: at 80°C for 1 h and at 120°C for 2 h, according to the
manufacturer's instructions.
Endotoxin
detection methods
We used Sun et
al. [85] applied cysteamine-modified gold nanoparticles to detect LPS by UV-Vis
spectrum and the detection limit was decreased to 3.3 × 10−10 mol/L.
More facilely, a colorimetric biosensor fabricated with gold nanorods was
developed and can detect LPS in the concentration range of 0.01 to 0.6 µM.
Nanorods of a high aspect ratio were also demonstrated to show superiority in
sensing. The facile assay for the rapid visual detection of lipopolysaccharide
(LPS) molecules down to the low nanomolar level by taking advantage of the
electrostatic interaction between GO and Epichlorohydrin (ECH) and Allyl Amine
Hydrochloride (AAH). The large amount of negatively charged groups on the LPS
molecules make LPS highly negatively charged. Thus, when modified with
cysteamine, the positively charged gold nanoparticles can aggregate in the
presence of trace amounts of LPS. The probe is simple, does not require any advanced
instrumentation, and the limit of detection (LOD) was determined to be as low
as 3.3 × 10−10 mol/L. To the best of our knowledge, it is the most
sensitive synthetic LPS sensor reported so far.
Characterization
techniques
Raman spectrum
was used to show the graphitic ordering before and after functionalization
treatments on GO and GO-ECH-AAH samples. It was acquired on a Renishaw 2000
Micro-Raman, with Ar laser (λ=514.5 nm) and range of 500-3500 cm-1
(only first order spectrum is shown in results). XPS high-resolution spectra
were obtained to determine atomic composition of GO in a UNI-SPECS UHV
spectrometer (5 × 10-7 Pa, hν=1253.6 eV). FT-IR was used to
characterize the presence of chemical groups on GO surfaces. Infrared spectra
were recorded on a Perkin-Elmer Spectrum GX, in the range of 4000-400 cm-1
with 4 cm-1 resolution, 12 scans and KBr pellet method. X-ray
photoelectron spectroscopy (XPS) was employed for the analysis of the surface
chemistry of GO and GO-ECH-DETA, using a SPECS system equipped with a Phoibos
150 1D-DLD analyser (Berlin, Germany) and monochromatic Al Kα X-ray source
(1486.6 eV). The XPS survey-scan spectra were recorded with pass energy of 80
eV, step energy 1 eV and dwell time 0.1 s; whereas the individual
high-resolution spectra were collected with pass energy of 30 eV, step energy
0.1 eV and dwell time 0.1 s, at an electron take-off angle of 90°. A Renishaw
Invia microscope (Gloucestershire, UK) with laser frequency of 514 nm was used
to obtain the Raman spectra of the graphenic materials from 500 to 3500 cm−1.
The information about the methods for the structural, morphological,
microstructural and thermal characterization of GO and GS is displayed in the Supplementary Material. The XRD
patterns of graphenic materials, GO and GO-ECH- AAH nanocomposites were
performed on a Malvern Panalytical (Almelo, Netherlands) X’PERT PRO automatic
diffractometer operating at 40 kV and 40 mA, in theta-theta configuration,
secondary monochromator with Cu-Kα radiation (λ=0.154 nm) and a PIXcel solid
state detector (active length in 2θ 3.347°). Data were collected in the
range of 2θ =1-50° (step size of 0.026° and time per step of 80 s, total
time 20 min) at room temperature. A variable divergence slit giving a constant
5 mm area of sample illumination was used. The Bragg equation (λ=2d sinθ)
was used to determine the interlayer distance in the graphenic materials. A
Hitachi S-4800 scanning electron microscope (Tokyo, Japan) operating at an
accelerating voltage of 15 kV was used to obtain SEM images of the neat
GO-ECH-DETA nanocomposite films, after being freeze fractured by liquid
nitrogen and sputtered with gold. TEM micrographs of nanocomposites were
obtained with a Philips Tecnai G2 20 TWIN TEM (Eindhoven, Netherlands) at 200
kV accelerated voltage after cutting the GO-ECH-DETA films into thin sections
with a Leica EM UC6 ultramicrotome apparatus, at room temperature and placing
the sliced specimens in copper grids. Differential scanning calorimetry
analyses were performed by a Mettler Toledo DSC 3+ unit (Greifensee,
Switzerland). The samples were heated from −30°C to 250°C at a heating rate of
10°C/min under a nitrogen gas flow of 20 mL/min. Values were obtained from the
first cooling and second heating scans. Thermogravimetric analysis was performed
on a TA instruments TG-Q-500 (New Castle, DE, USA) at a heating rate of
10°C/min from 40°C to 800°C in nitrogen or air-flow. An electromechanical
testing machine (Instron 5967, Norwood, MA, USA) operating at room temperature
with a load cell of 500 N, a gauge length of 10 mm and a cross head speed of 5
mm/min was used to performed tensile tests. Films were cut into a dog-bone
shape before testing and kept at a relative humidity of 58% at room temperature
for more than one week to ensure equilibration of the moisture uptake in the
films. Testing was carried out on at least ten identical composite films of
each composition and the average values were reported.
RESULTS AND DISCUSSION
X-ray
diffraction (XRD) analysis
SEM images
TEM images
Figure 4 shows the TEM
images of GO before functionalization treatments; (b) after functionalization
treatments of GO with ECH-DETA to form GO-ECH- AAH nanocomposite treatment. It indicates a brittle wrinkled fracture of GO. After formulation
with ECH-AAH, by solution blending, the GO could be homogenously dispersed in
the ECH-AAH matrix, becomes cloud-like and rough. The cracks become more
randomly dispersed, indicating that the GO network acts as an obstacle to crack
propagation. In this image the TEM of Figure
3b have a display surface in some areas of irregular morphology of fibers
and various forms of holes evenly along the fiber, are structures are similar
to the rod. But, there are 2 wt.% graphene oxide inclusions longer the polymer,
the fibers presented in the fracture morphology is connected; indicating a high
physical interaction existing between ECH-AAH and GO nanocomposite. Overall,
graphene has carboxylic acid functional group provides an intermolecular force
calling itself bridge effect. Providing a cohesive bond on the GO with ECH-AAH
system provides a more efficient load transfer to the polymer matrix. Thus,
ECH-AAH addition enhances the strength of the composite. However, further
addition of ECH-AAH (beyond 1.5 vol%) leads to the formation of clusters within
the GO network that are in the scale of microns. This inhibits the stress
transfer from the ECH-DETA matrix to the GO network, thus deteriorating the
strength of the composite. It is clear from the SEM image that GO is generally
dispersed properly in the matrix. Upon impact, the crack propagates in the
direction of the tension, and then proceeds to the weak interfaces, finally
damaging the material. At 4.5 vol% ECH-AAH, the fracture surface is
non-uniform. The ECH-AAH flow is hindered by the GO agglomerates. The composite
surface shows signs of brittleness, with a rougher surface than neat ECH-AAH.
The pull-out of the GO in the ECH-AAH matrix is also seen to decrease.
Polarized optical microscopy (POM) images
Composites
crystallization behaviors via POM are shown in Figures 5 and 6. The POM photographs of pure GO and GO-ECH- AAH
nanocomposite are shown in Figure 5.
The thin nanosheet, wrinkled and fine morphology of pure GO is seen in Figure 5a. Compared to AAH
polymerization crystalization, the GO-ECH-AAH nanocomposite shows faultiness in
crystallization after the reaction process was complete. The phenomenon may be
due to the intrinsic slow rate of crystallization of GO-ECH-AAH. Figure 6 shows polarized optical
microscopy (POM) images of (a) pure poly AAL and (b) GO-ECH-AAH at 65°C during
non-isothermal crystallization from their melts at a cooling rate of 2°C/min.
Nevertheless, the results suggest that GO can be an efficiency nucleation
accelerator for AAH, it is heterogeneous nucleating agent during the
non-isothermal crystallization process of AAH and can accelerate the
polymerization process of ECH-AAH. Moreover, the low MW of ECH and AAH compared
to the hybrid GO-ECH-AAH nanocomposite, POM photographs of the high MW of
GO-ECH-AAH shows more black flakes, the result should contribute to the
inferior disperse ability of hybrid ECH-AAH within the GO phases.
XPS scanning
spectrum
The presence of functional groups GO surface
was confirmed by XPS analysis. As expected, oxygen and nitrogen were found in
GO exhibits high oxygen content from its oxidation. Also, exploratory scans
have indicated the residual presence of sulfur from growth and
functionalization processes in. In our studies, ECH-AAH was
grafted onto the GO sheets by in situ
ring opening polymerization of GO. The grafted GO-ECH-AAH dissolved well in
dichloromethane, chloroform, DMF, THF, toluene and ethylene acetate. The
homogeneous dispersion of GO in the polymer matrix improved the mechanical
properties of ECH-AAH. The aggregation and stacking of GO nanosheets were also
supported by tethering GO sheets on the ECH-AAH chains. We verified this morphology
the presence of well-dispersed layers, indicating that after chemical reduction
the material has not organized its crystal structure. Consequently, an increase
in surface area of these nanoparticles in the nanocomposite, allowing the
modification of the polymeric matrix structure, and this may result in
increased elastic modulus and hardness of the sample; possibly change in the
degree of crystalline because smaller nanoparticles can act as nucleation
sites.
The
presence of functional groups on GO
surface before and after functionalization treatments
of GO with ECH-AAH to form GO-ECH-AAH nanocomposite polymer surface
was confirmed by XPS analysis (Table 1).
As expected, oxygen and nitrogen were found in GO and
GO-ECH-AAH exhibits high oxygen content from its
oxidation. Also, exploratory scans have indicated the residual presence of
sulphur and from growth and functionalization processes in GO-ECH-AAH, as shown in Figure 7, as well as residual presence of sulphur in GO from its
synthesis process as shown in Figure 8.
Raman
spectrum of GO and GO-ECH-AAH nanocomposite
Figure 9 shows the Raman
spectrum of GO and GO-ECH-AAH nanocomposite at different temperature as grown have clear bands at 1344.1 cm-1
(D band) and 1575.99 cm-1 (G band) and a shoulder near 1586.1 cm-1
(D' band). After functionalization, GO-ECH-AAH nanocomposite presents two bands
at 1353.7 cm-1 (D band) and 1590.9 cm-1 (G band) and a
shoulder near 1614.64 cm-1 (D' band). These bands are characteristic
of multi-walled GO. The higher intensity of the G band for CNTs as grown
indicates a higher degree of graphitisation/crystallinity, while D band is
typically attributed to disordered structures (defective GO and non-crystalline
carbon. The change in intensities of D and G band could be observed on the
Raman spectrum of GO-ECH-AAH due to the acid and amino functionalization
process. It is known that during oxidizing treatments of graphitic structures
two concurring phenomena take place: the removal of amorphous carbon from the
GO and the formation of oxygenated functional groups, changing the atomic
structure from C-C sp2 to C-C sp3. Due to this change, a displacement in the
position of G band and a higher intensity of D band can be observed.
UV-Vis
absorption spectra
Figure 10 shows the UV-vis
absorption spectra of the GO, the ECH-AAH and GO-ECH-AAH nanocomposite. GO
presented by characteristic peak at 229 nm corresponding to π–π* transitions of
aromatic C–C bonds. Red shift peak of the graphene presented at 260 nm because
the electronic conjugation in the graphene. While for GO-ECH-AAH nanocomposite,
the absorption peak was shifted to 280 nm, suggesting that the covalent
attachment of ECH-AAH on to GO surface. As shown in Figure 10, there is a recorded blue shift in absorption maxima for
the pure Allyl Amine Hydrochloride (AAH) polymer complex with ECH-AAH when
complexed with GO sheet. I guess it is caused by complexation and an electron
transfer between GO and ECH-AAH. So the active sites of GO sheet were blocked
by complex formation with ECH-AAH polymer NPs.
Crystallinity of carbonaceous materials can
be evaluated by the ratio between D and G band intensity (ID/IG), as well as
full width at half height (FWHM) of G band. Table 2 shows the information from the deconvoluted spectra.
ID/IG ratio
changed from 0.45 for GO as grown to 1.25 for functionalized GO-ECH-DETA
nanocomposite. The increase in ID/IG ratio suggests that formation of
oxygenated functional groups was more intense than removal of amorphous carbon.
FT-IR
analysis
The FTIR
spectra were obtained between 4000 and 350 cm-1. Figure 11 shows the FT-IR spectrum of
(a) GO and (b) GO-ECH-AAH nanocomposite. The technique was used to investigate
the structure and functional groups of both samples. Figure 11b shows FT-IR spectrum of GO, shows adsorption bands at
1723 cm-1, due to the C=O stretch of COOH group; at 1621 cm-1,
for stretch of C=C groups; at 1220 cm-1, for C=C skeleton vibration;
and at 1043 cm-1for alkoxy C-O groups. Although graphite had been
oxidized into GO, C=C groups led to the conclusion that the main structure of
graphite layer was retained. The presence of oxygen-containing functional
groups confirmed that the graphite was greatly oxidized into GO and was in
agreement with the literature. FT-IR spectrum for GO-ECH-AAH nanocomposite
sample (Figures 7c and 7d) shows the
following bands and peaks of interest: at 1670 cm-1, corresponding
to the amide carbonyl (C=O) stretching; at 3728 cm-1, due to -NH
stretching; at 1587 cm-1, because of N-H in-plane bending; and at
1220 and 1047 cm-1, assigned to C-N stretching. These bands prove
the presence of amide groups and lead to the conclusion that carboxylic groups
on GOs surface were modified by amine from AAH. The band in the range of
3445.15 cm-1 showed a relatively broad bandwidth that is probably
related to the axial deformation of the O-H bond due to the reaction with ECH.
The other bands in the range of 3026.35 cm-1 C-H show that the
nanocomposite formation process between the ECH, AAH and GO on the surface was
successful. However, the characteristics and similar bands have the
predominance of GO. The oxygen atoms tend to combine with carbon atoms thereby
forming an array of functionality, among which can be mentioned: ketones,
esters, carboxylic acids and others. The three faint bands were observed in the
region between 1651 and 1452-1366 cm-1 due to bending vibrations and
axial deformation of the C=C bonds is low because of GO with respect to the
nanocomposite polymer matrix. The two most intense peaks has its stretching
vibration ascribed to C=O appeared in the range 1493-1601 cm-1 are
due to the formation of hydroxyl and carboxyl groups, resulting from the
chemical reaction. The band located at 748 cm-1 is related to the
C-axial deformation the primary alcohols and other of band and 540 cm-1
are due to the angular deflection of C-H with H out of plane. Therefore, GOs
were indeed functionalized through ECH and AAH treatments.
LPS removal
by GO-ECH-AAH nanocomposite
CONCLUSION
In summary, we
developed multi-functional GO-based nanocomposites, GO-ECH-AAH, by the reaction
of graphite oxide (GO) with epichlorohydrin (ECH) as a coupling agent and
allylamine hydrochloride (AAH) a ligand. The instrumental analysis of the
nanocomposite prove that there is chemical interaction between ECH, AAH and GO
on the surface of GO. For example, the presence of amide groups and lead to the
conclusion that carboxylic groups on GOs surface were modified by amine from
AAH. The band in the range of 3445.27 cm-1 showed a relatively broad
bandwidth that is probably related to the axial deformation of the O-H bond due
to the reaction with ECH. The other bands in the range of 3026.55 cm-1
C-H show that the nanocomposite formation process between the ECH, AAH and GO
on the surface was successful. The polymeric chains on the surface of GO
endowed GO-ECH-AAH nanocomposite with excellent chain stability. Additionally,
from the SEM images we observed the interface between the GO and the epoxy
composite ECH-AAH suggests chemical interaction. As can be seen from this
research, the GO-ECH-AAH nanocomposite can be used as a potential candidate for
removal of PLS from aqueous solution.
CONFLICTS OF
INTEREST
The authors
declare no conflict of interest regarding the publication of this paper.
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