Page | 880

Synthesis and characterization of methyl acrylamide cellulose nanowhiskers for

environmental applications

Swati Sharma 1

, Ch. Shiney Rashmitha 2

, Lalit M. Pandey 1, * 1Bio-interface & Environmental Engineering Lab Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam, 781039, India 2Department of Biotechnology Motilal Nehru National Institute of Technology, Allahabad, Prayagraj, Uttar Pradesh, 211004, India

*corresponding author e-mail address: swatisharma7503@iitg.ac.in, chsrashmitha29@gmail.com, lalitpandey@iitg.ac.in | Scopus ID 36984082800

ABSTRACT

Cellulose is the most abundant biopolymer on earth with excellent mechanical strength, biocompatibility, and biodegradability. Yet, its

poor chemical reactivity deceives its application in various biotechnological sectors. In this study, the microcrystalline cellulose was

used as low cost substrate for the synthesis of cellulose nanowhiskers (CNWs) using controlled acid hydrolysis. In order to improve their

chemical reactivity, synthesized CNWs were modified with carboxyl functional groups using 2,2,6,6-Tetramethylpiperidinyloxyl

(TEMPO) mediated oxidation reaction. These carboxylated cellulose nanowhiskers (CCNWs) were functionalized with amine functional

groups using carbodiimide reaction. The as-synthesized amine terminated amido-cellulose nanowhiskers (ACNWs) were modified with

methyl acryl functional groups using Michael addition resulting methyl acryl functionalized amido-cellulose nanowhiskers

(MAACNWs). The synthesized MAACNWs were characterized using Fourier-transform infrared spectroscopy, UV-visible spectroscopy

and nuclear magnetic resonance spectroscopy. The physical morphology was studied using Atomic force microscopy, X-Ray Diffraction

and zeta analyser. The modified cellulose nanowhiskers were found to exhibit 4 folds increased surface roughness confirming fabrication

of negatively charged chemical groups on CNWs yielding 7 folds higher surface charge than pristine nanowhiskers. Hence, the modified

CNWs could be acknowledged as potential candidate for effective heavy metal biosorption and remediation studies.

Keywords: Cellulose nanowhiskers; Michael addition; Methyl acrylamide; Carboxylation.

1. INTRODUCTION

Nanomaterials are the major focus of present material

science studies due to their magnificent ability to be custom

tailored as per the synthesis conditions with desired morphological

and chemical characteristics for the diverse applications [1, 2].

They have been explored in various area of research

ranging from drug delivery, drug design to various photocatalytic

and environmental applications [3-7]. Cellulose is the most

abundant renewable biopolymer existing on the earth with distinct

characteristic properties of high mechanical strength,

biocompatibility, biodegradability and economic feasibility.

Owing to such features, makes cellulose a suitable substrate for

the nanomaterial synthesis [8].

Recent studies have explored various forms of cellulose

nanostructures such as cellulose nanocrystals and cellulose

nanotubes for various biotechnological and environmental

applications. In contrast to carbon nanotubes, the presence of

surface active hydroxyl groups on their surfaces boosts cellulose

nano-whiskers exploration in the field of biotechnology due to

their improved solvent dispersity [8, 9]. Apart from surface active

groups, its high surface to volume ratio encourages towards

surface functionalization studies. However, very few reports are

available based on the surface modification of cellulose nano-

whiskers [10].

Branched chain of polymers of ethylene diamine

conjugated methyl meth acrylate have been explored for

improving the surface functionality, overall surface area and

charge. Their chemically reactive structure gains the advantage in

the synthesis of a vigorously branched structure known as

dendrimers [10, 11].

In this study we have fabricated novel acrylamide capping

on the surface of cellulose nano-whiskers using a convergent

approach. The successful functionalization is monitored using

FTIR, UV and NMR spectroscopy. The physical properties of the

modified nano-whiskers like surface charge and surface

morphology are measured. This modified nanomaterial with

featured characteristics could be further explored in the field of

biological and various environmental applications.

2. MATERIALS AND METHODS

2.1. Chemicals and reagents.

Microcrystalline cellulose powder (MCC) (GRM333) and

Sulphuric acid (H2SO4) (RM6244) were used for synthesis of

cellulose nano-whiskers (CNWs). Sodium hypochlorite (NaClO)

(PCT1311-50ML), Sodium bromide (NaBr) (GRM7496), 2,2,6,6-

Tetramethyl-1-piperidinyloxy radical (TEMPO)( RM4807), and

absolute ethanol (MB106) were used for the preparation of

carboxylated CNWs (CCNWs). Ethylenediamine (EDA), 1-Ethyl-

3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)

(RM1817), N-Hydroxysuccinimide (NHS) (RM1120), Methyl

methacrylate (MMA) (GRM8641), (4-(2-Hydroxyethyl)-1-

piperazineethanesulfonic acid) HEPES buffer (TCL021), and

Dimethyl formamide (DMF) (MB036) were required for the

acrylamide functionalization of CCNWs. Double distilled water

(Mili Q, 18mΩ, Millipore systems) was used in all the

experiments.

Volume 9, Issue 1, 2020, 880 - 884 ISSN 2284-6808

Open Access Journal Received: 19.02.2020 / Revised: 04.03.2020 / Accepted: 06.03.2020 / Published on-line: 10.03.2020

Original Research Article

Letters in Applied NanoBioScience https://nanobioletters.com/

https://doi.org/10.33263/LIANBS91.880884

https://www.scopus.com/authid/detail.uri?authorId=36984082800https://orcid.org/0000-0002-1664-5730https://nanobioletters.com/https://doi.org/10.33263/LIANBS91.880884

Synthesis and characterization of methyl acrylamide cellulose nanowhiskers for environmental applications

Page | 881

2.2. Preparation of cellulose nanowhiskers.

In this study we prepared cellulose nanowhiskers using

sulfuric acid hydrolysis as reported elsewhere by our group [9].

Briefly, microcrystalline cellulose powder was treated with 65%

(v/v) sulfuric acid in 1:10 (w/v) ratio at 50°C under constant

stirring for 2 h. The reaction was stopped by adding two folds of

ice cold water to the solution. The obtained solution was further

centrifuged at 10,000 rpm for 20 min at 4°C and washed several

times to neutralize the pH. Finally, the pellet was lyophilized to

obtain CNWs.

2.3. Preparation of carboxylated CNWs.

The hydroxyl (-OH) surface groups of CNWs was initially

oxidized to carboxylic acid (-COOH) functional groups using

TEMPO-mediated oxidation system [9]. For this, 1% (w/v) of

prepared CNWs were mixed with TEMPO (70 mg/g) and NaBr

(180 mg/g) under constant stirring at room temperature. To this

solution, 9% (v/v) of NaClO was added slowly to maintain the pH

of the solution at 10.5 for 10 h. Finally, to arrest the reaction 20

mL of ethanol was added. Further, the obtained mixture washed

and centrifuged at 10000 rpm for 10 min at 4°C till the pH

neutralized and later lyophilized to obtain CCNWs.

2.4. Preparation of acrylamide coated CCNWs.

The acrylamide coated CCNWs were synthesized using

divergent mode of synthesis (Figure 1). In this, the core CNWs

were oxidized to obtain surface active –COOH functional groups

which acted as binder for the acrylamide functionalization. The

prepared CCNWs were used as monomer units for the further

modification. Initially, for the coupling of EDA on the CCNWs,

carbodiimide crosslinking technique was employed [10]. EDC-

NHS were used in HEPES buffer for the formation of amide

bonding between –COOH group of CCNWs and NH2 group of

EDA. To this, methyl methacrylate coating was done using

Michael addition reaction resulting the formation of methyl acryl

amido CCNWs (MAACNWs) [11].

Figure 1. Schematic representation of acrylamide coated cellulose

nanowhiskers.

2.5. Characterization of the functionalized CNWs.

The synthesized modified CNWs were analyzed both

chemically and physically to explore its surface characteristics.

The morphology of MAACNWs was compared to pristine CNWs

using Atomic force microscopy (AFM). Also. X-ray Diffraction

(XRD) was performed to analyse any change in the crystal

structure due to modification conditions. In addition, the prepared

MAACNWs were characterized for the presence of surface active

functional groups using Fourier Transform Infrared spectroscopy

(FTIR). Proton nuclear magnetic resonance (1H NMR) was also

recorded for the prepared material. The surface charge of the

prepared MAACNWs was analysed using zeta potential analyser.

2.5.1. Chemical characterization.

The functional groups analysis of cellulose nano-whiskers

and modified MAACNWs was characterized using FTIR (Perkin

Elmer, Spectrum Two) equipped with ZnSe crystal in attenuated

total reflectance (ATR) mode. Initially, the samples were dried

overnight and later placed directly over the crystal using pressure

clamp. All the spectra were recorded in the range of 400-4000 cm-

1 with an average 32 scans and 4 cm-1 resolution. FTIR spectra

were analysed at each step of modification as qualitative

conformation for proceeding further steps. The synthesized

MAACNWs were also analysed using 400 MHz Nuclear Magnetic

Resonance Spectrometer (Make: VARIAN, Model: MERCURY

PLUS) using D2O as solvent.

The synthesized MAACNWs were also examined using

UV-spectroscopic technique (Carry 100 Bio UV-Vis

spectrophotometer). For this study, 10 mg of synthesized material

was dissolved in MiliQ and scanned in the range of 200-350 nm.

Only cellulose nanowhiskers with no surface modification were

used as control. The presence of additional peak suggested the

change in surface chemistry of material due to modifications.

2.5.2. Physical characterization.

The surface morphology of synthesized CNWs and

MAACNWs were analysed using AFM (Oxford, Model: Cypher)

equipped with silicon nitride tip (Radii

Swati Sharma, Ch. Shiney Rashmitha, Lalit M. Pandey

Page | 882

sulfonate groups was seen at 1205 cm-1 confirming the formation

of nano-whiskers due to sulfate esterification. Various literatures

have reported sulfate peak as conformation of hydrolysis of

cellulose [9]. Further, the prepared CNWs were carboxylated

using TEMPO mediated oxidation reaction. In this, the –OH

groups of CNWs were oxidized to –COOH groups which were

observed as a new peak at 1740 cm-1 (Figure 2B) yielding

carboxylated cellulose nanowhiskers (CCNWs) [13].

These CCNWs were further modified using carbodiimide

reaction between EDA and CCNWs. In agreement with that,

appearance of peak at 1545 cm-1 demonstrated the amide II

linkage on the surface of CNWs (Figure 2C) [14, 15].

Furthermore, the MMA coated amido CNWs were explored for

the grafting of esterified amide groups. The presence of peaks at

1450 cm-1 and 1260 cm-1 showed the presence of functional groups

for CH2 groups and CN groups explained the functionalization

MMA on the surface of this synthesized amido-cellulose

nanowhiskers (ACNWs) (Figure 2D) [11, 16]. Hence, FTIR

spectra revealed the successful grafting of methyl acrylate groups

on the surface of CNWs yielding a material with highly enriched

negatively charged functional groups on its surface.

Figure 2. FTIR spectra for (A) CNWs, (B) CCNWs, (C) ACNWs, and

(D) MAACNWs.

Figure 3. UV spectra for pristine CNWs and functionalized MAACNWs.

Thus synthesized material was further explored using UV-

spectroscopy. UV-spectra revealed the presence of a new peak at

255 nm in MAACNWs, which was absent in CNWs (Figure 3).

Literature reports this peak of the presence of amide bonds as in

present in MAACNWs. A similar peak has been reported in

literature for methyl acryl amide surface modifications [17, 18].

NMR spectra of synthesized MACNWs depicted the

presence of peaks at 3.36 and 3.07 ppm signifying the -OCH3

protons and -N-CH3 protons, respectively on the surface of CNWs

after grafting of methyl meth acrylate [19] [20]. The peaks at 4.4

to 4.8 ppm stand for the D2O solvent peaks (Figure 4)

Figure 4. 1H NMR spectra of the synthesized MAACNWs

3.2. Physical characterization.

Due to strong acid based hydrolysis the precursor

microcrystalline cellulose gets deconstructed to smaller fragments

taking various shape based on the reaction conditions used for the

hydrolysis. In the present study, microcrystalline cellulose got

transformed into nanowhiskers, as analysed using AFM. It was

observed that CCNWs were rod shape in morphology with a

length of ~ 144 ± 30 nm and width of about ~25 ± 5 nm (Figure

5A). However, after conjugation with acrylamide, various

extensions in the form of branched projection were observed

(Figure 5B). MAACNWs were found to be ~ 215 ± 20 nm in

length and 30 ± 7 nm in width, deciphering the formation of

branched morphology. Figure 5 showed the length and thickness

of whiskers increased upon modification due to the formation of

branched structures. Prior to functionalization, the surface of

CNWs were found to be comparatively smooth.

Figure 5. AFM images of (A) CNWs and (B) MAACNWs.

The effect of surface functionalization on the morphology

of CNWs was estimated by analyzing the surface roughness in

terms of change in average height (Ra). The Ra value for bare

CNWs was 0.67 nm, which increased to 2.55 nm after surface

functionalization indicated successful modification. Few

Synthesis and characterization of methyl acrylamide cellulose nanowhiskers for environmental applications

Page | 883

unbranched whiskers were also present suggesting all the reactive

sites were not occupied. Hence, the present base material can

accommodate more branched structures on its surface and could

be further optimized for effective functionalization.

To see the effect of chemical modification on crystal

structure, XRD was performed for pristine and modified CNWs.

Figure 6A illustrates the diffraction peaks at 15.9, 22.8 and 33.8

angles which corresponded to diffraction planes of Cellulose-Iß

(110), (200) and (211), respectively (JCPDS 00-060-1502). Figure

6(B and C) showed that even after strong acid hydrolysis and

carboxylation reaction, no damage was done to the crystal

structure for prepared pristine and modified CNWs. However

there was a slight blue shift observed in the case of modified

(MAACNWs) (2θ = 22.5) from pristine CNWs (2θ = 23.05) which

could be due to change in chemical composition of modified

CNWs. Yet there was no significant change in crystal size (i.e.

11.02 nm), as calculated from Scherrer equation [4].

The prepared MAACNWs were further studied for their

overall surface charge using zeta potential analysis. It was

observed that obtained pristine CCNWs exhibited a surface

potential of -2.5 ± 1 mV which transformed to a more negative

surface potential of -17.7 ± 1.2 mV after modification. This

explained the presence of a higher amount of acrylate groups on

the surface of functionalized CCNWs. Such improved surface

charge by ~7 folds, signified the successful modification of

cellulose nanowhiskers. This was due to the availability of high

surface area to volume ratio nanowhiskers that encouraged higher

modification sites. Hence, the synthesized methyl acrylamide

cellulose nanowhiskers are found to be highly surface active with

suitable surface area and charge distribution that could be further

optimized for biosorption and other environmental applications.

Figure 6. XRD diffractogram for standard Cellulose 1β (A), modified

CNWs (B) and pristine CNWs.

4. CONCLUSIONS

In this study, cellulose nanowhiskers were prepared and

carboxylated using TEMPO mediated oxidation reaction. These

CCNWs were modified using carbodiimide reaction to obtain

acryl amine coated CNWs with highly improved surface

reactivity. The synthesized nanostructure was found to be whisker

shaped in morphology increased surface roughness after surface

modification indicating the successful functionalization on CNWs.

The crystallinity of cellulose nanowhiskers was retained after

surface modification explaining its high stability. Functional group

analysis showed the presence of acrylate groups on the surface

causing increased negative surface charge by 7 folds after

modification. Conclusively, the synthesized modified

nanowhiskers were found to exhibit excellent surface

functionalization and charge and hence could be further optimized

to improve the overall selectivity and sorption capacity for various

environmental applications.

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6. ACKNOWLEDGEMENTS

The authors would like to express gratitude to the Department of Science and Technology, Government of India for financial

assistance (Sanction Nos: DST/INSPIRE/04/2014/002020, ECR/2016/001027, and DST/INT/UK/P-155/2017) for this work. The authors

would like to acknowledge Central Instrument Facility (CIF), IIT Guwahati for allowing the access of various facilities required for the

present study.

© 2020 by the authors. This article is an open access article distributed under the terms and conditions of the

Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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