Accepted Manuscript Title: Surface-initiated atom transfer radical polymerization from chitin nanofiber macroinitiator film Author: Kazuya Yamamoto Sho Yoshida Jun-ichi Kadokawa PII: DOI: Reference:
S0144-8617(14)00557-8 http://dx.doi.org/doi:10.1016/j.carbpol.2014.05.079 CARP 8946
To appear in: Received date: Revised date: Accepted date:
19-3-2014 21-5-2014 29-5-2014
Please cite this article as: Yamamoto, K., Yoshida, S., and Kadokawa, J.-i.,Surfaceinitiated atom transfer radical polymerization from chitin nanofiber macroinitiator film, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.05.079 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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Chitin nanofiber (CNF) film was used for surface-initiated graft polymerization.
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It derived to macroinitiator for atom transfer radical polymerization (ATRP).
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Surface-initiated graft ATRP of acrylate monomer from the product was performed.
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The analytical results showed the production of CNF-graft-polyacrylate film.
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Surface-initiated atom transfer radical polymerization
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from chitin nanofiber macroinitiator film
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Kazuya Yamamoto a, Sho Yoshida a, Jun-ichi Kadokawa a,b,*
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a
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890-0065, Japan
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b
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Technology, 27-1 Mizumoto-cho, Muroran, Hokkaido 050-8585, Japan
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Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima
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Research Center for Environmentally Friendly Materials Engineering, Muroran Institute of
19 ABSTRACT
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This paper reports the preparation of chitin nanofiber-graft-poly(2-hydroxyethyl acrylate) (CNF-g-
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polyHEA) films by surface-initiated atom transfer radical polymerization (ATRP) of HEA monomer
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from a CNF macroinitiator film. First, a CNF film was prepared by regeneration from a chitin ion gel
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with an ionic liquid. Then, acylation of the CNF surface with α-bromoisobutyryl bromide was carried
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out to obtain the CNF macroinitiator film having the initiating moieties (α-bromoisobutyrate group). The
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surface-initiated graft polymerization of HEA from the CNF macroinitiator film by ATRP was
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performed to produce the CNF-g-polyHEA film. The IR, XRD, and SEM measurements of the resulting
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film indicated the progress of the graft polymerization of HEA on surface of CNFs. The molecular
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weights of the grafted polyHEAs increased with prolonged polymerization times, which affected the
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mechanical properties of the films under tensile mode.
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Keywords: Chitin nanofiber film; Atom transfer radical polymerization; Surface-initiated graft
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polymerization; Macroinitiator
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*Corresponding author. Tel.: +81 99 285 7743; fax: +81 99 285 3253
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E-mail address: [email protected] (J. Kadokawa).
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1. Introduction Polysaccharides are widely distributed in nature and have been regarded as structural materials and as suppliers of energy (Shuerch, 1986). Because of better recent understanding of their possessing unique
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structures and properties, much attention has been paid to them as material sources. Of the many kinds
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of polysaccharides, chitin, which is an aminopolysaccharide composed of N-acetyl-D-glucosamine
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residues linked through -(14)-glycosidic bonds, is one of the most abundant polysaccharides in
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nature and occurs mainly in exoskeletons of crustaceans, shellfish, and insects (Kurita, 2006; Muzzarelli,
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2011; Pillai, Paul, & Sharma, 2009; Rinaudo, 2006). However, it still remains mostly as an unutilized
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biomass resource primary because of its intractable bulk structure and insolubility in water and common
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organic solvents. Therefore, research concerning conversion of chitin into various useful bio-based
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materials after proper dissolution of it in suitable solvents has attracted much attention even in recent
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years.
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To efficiently provide new chitin-based materials, we have focused on ionic liquids, which are low-
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melting point salts that form liquids at temperatures below the boiling point of water. Since it was
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reported that 1-butyl-3-methylimidazolium chloride of an ionic liquid dissolved cellulose in relatively
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high concentrations (Swatloski, Spear, Holbrey, & Rogers, 2002), various ionic liquids have been used
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as good solvents for the derivatization and material processing of cellulose (Feng, & Chen, 2008;
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Liebert, & Heinze, 2008; Pinkert, Marsh, Pang, & Staiger, 2009; Zakrzewska, Lukasik, & Lukasik,
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2010). However, only the limited investigations have been reported regarding the dissolution of chitin
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with ionic liquids (Bochek, Murav'ev, Novoselov, Zaborski, Zabivalova, Petrova, Vlasova, Volchek, &
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Lavrent'ev, 2012; Jaworska, Kozlecki, & Gorak, 2012; Muzzarelli, 2012; Qin, Lu, Sun, & Rogers, 2010;
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Wang, Zhu, Wang, Huang, & Wang, 2010; Wu, Sasaki, Irie, & Sakurai, 2008). We also found that an
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ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), dissolved or swelled chitin to form weak
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gel-like materials (ion gels) (Prasad, Murakami, Kaneko, Takada, Nakamura, & Kadokawa, 2009;
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Yamazaki, Takegawa, Kaneko, Kadokawa, Yamagata, & Ishikawa, 2009). By using AMIMBr as a
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solvent, acetylation of chitin using acetic anhydride produced an acetylated chitin with a high degree of
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substitution (DS) under selected conditions (Mine, Izawa, Kaneko, & Kadokawa, 2009). Furthermore,
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this reaction manner, i.e., the esterification of hydroxy groups in chitin in the AMIMBr solvent was
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employed to introduce initiating moieties for atom transfer radical polymerization (ATRP) attached
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through ester linkages on chitin. Then, grafting of styrene from the resulting macroinitiator via ATRP
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was performed to produce a chitin-graft-polystyrene (Yamamoto, Yoshida, Mine, & Kadokawa, 2013).
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ATRP is a robust and versatile technique to accurately control chain length and polydispersity of the
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resulting polymers, and thus has been used to synthesize a wide range of copolymers with the controlled
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structure, unit length, and block sequence (Kamigaito, Ando, & Sawamoto, 2001; Matyjaszewski,
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2012).
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On the other hand, graft copolymers based on a chitin nanofiber (CNF) film have been prepared by us. In the previous study, we reported that CNF film was facilely obtained by regeneration from the
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aforementioned chitin ion gel with AMIMBr in methanol, followed by filtration (Fig. 1a) (Kadokawa,
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Takegawa, Mine, & Prasad, 2011; Tajiri, Setoguchi, Wakizono, Yamamoto, & Kadokawa, 2013).
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Approaches to the production of nanofibers from chitin have often been performed upon top-down
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procedures that break down the starting bulk materials from native chitin resources (Fan, Saito, & Isogai,
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2008; Ifuku, & Saimoto, 2012; Zeng, He, Li, & Wang, 2012). Our method applies a completely different
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approach from the above to produce CNFs, that accords to a self-assembling generative (bottom-up)
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route. We have continuously studied surface-initiated graft polymerization approach from the CNF film
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to prepare new chitin-based materials. For example, we performed the surface-initiated ring-opening
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graft copolymerization of L-lactide (LA)/ε-caprolactone (CL) from the CNF film to produce a
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environmentally benign composite material because the ring-opening copolymerization is initiated from
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a hydroxy group and has been known to efficiently produce a biodegradable and biocompatible
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poly(LA/CL) (Setoguchi, Yamamoto, & Kadokawa, 2012). Recently, the surface-initiated grafting
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technique from the CNF film was extended to use a synthetic polypeptide as another biocompatible
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polymer. Because it has been well-known that synthetic polypeptides with well-defined structures are
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synthesized by ring-opening polymerization of α-amino acid N-carboxyanhydride (NCA) accompanied
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with decarboxylation initiated from amino groups, we reported the surface-initiated graft polymerization
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of NCA from amino initiating groups on surface of a partially deacetylated CNF film to give a CNF-
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graft-polypeptide film (Kadokawa, Setoguchi, & Yamamoto, 2013). By free radical polymerization
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approach, grafting of acrylic acid on CNFs, which were prepared by the aforementioned top-down
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procedure, was also carried out with potassium persulfate as a initiator, where radicals are formed along
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the chitin back-bone, followed by the progress of free radical propagation (Ifuku, Iwasaki, Morimoto, &
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Saimoto, 2012).
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In this paper, on the basis of the above previous investigations, we report the preparation of CNF-
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graft-poly(2-hydroxyethyl acrylate) (CNF-g-polyHEA) films by the surface-initiated grafting technique
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via ATRP using a CNF macroinitiator film. The CNF macroinitiator film for ATRP was first prepared
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by introduction of the initiating moieties attached through ester linkages on CNF surfaces. Then, the
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surface-initiated graft polymerization of HEA from the CNF macroinitiator film by ATRP was
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performed under the appropriate conditions to produce the CNF-g-polyHEA films. To the best of our
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knowledge, this is the first example of the surface-initiated ATRP from CNF materials. Because the
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present surface-initiated ATRP technique using the CNF macroinitiator film has a potential to employ
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various monomers, we are convinced that the present approach will contribute to the development of
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practical chitin-based composite materials in the future.
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2. Experimental Part
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2.1.
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Chitin powder from crab shells was purchased from Wako Pure Chemical Industries, Ltd., Japan. The
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weight-average molecular weight value of the chitin sample was estimated by viscometric analysis to be
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7 x 105 (Poirier & Charlet, 2002). An ionic liquid, AMIMBr, was prepared by reaction of 1-
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methylimidazole with 3-bromo-1-propene according to the method adapted from the literature procedure
Materials and methods
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(Zhao, Fei, Geldbach, Scopelliti, Laurenczy, & Dyson, 2005). All other reagents and solvents were
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used as received from commercial sources. IR spectra were recorded with samples in KBr using a
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SHIMADZU FTIR-8400 spectrometer. The powder X-ray diffraction (XRD) measurements were
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conducted using a PANalytical X’Pert Pro MPD with Ni-filtered CuKα radiation (λ = 0.15418 nm). The
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SEM images were obtained using Hitachi S-4100H electron microscope. The energy-dispersive X-ray
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spectroscopy was performed using a Philips XL30 CP scanning electron microscope (SEM-EDX). 1H
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NMR spectrum was recorded on a JEOL ECX 400 spectrometer. GPC measurement was performed with
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two Shodex GPC KF-804L and KF-803L columns and equipped with HITACHI pump L-2130
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connected to HITACHI RI detector L-2490. Chloroform was used as the eluent at a flow rate of 1.0 mL
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min-1. The calibration was established with polystyrene standards. The stress-strain curves were
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measured using a tensile tester (Little Senster LSC-1/30, Tokyo Testing Machine).
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2.2.
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First, the preparation of CNF film was conducted according to the method reported in our previous
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publication (Kadokawa, Takegawa, Mine, & Prasad, 2011). Chitin powder (0.125 g, 0.61 mmol) was
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immersed in AMIMBr (1.0 g, 4.9 mmol) at room temperature for 24 h, followed by heating the mixture
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at 100 °C for 24 h to give a chitin/AMIMBr ion gel. After methanol (40 mL) was added to the gel and
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the system was left standing at room temperature for 24 h, the mixture was sonicated for 5 min to give a
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chitin dispersion. Then, the dispersion was subjected to filtration and a residue was dried under reduced
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pressure to give a film. The film was subjected to Soxhlet extraction with methanol (70 mL) for 5 h,
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followed by drying under reduced pressure to give a CNF film. A typical procedure for the synthesis of
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the CNF macroinitiator film was as follows (entries 1-4, Table 1; DS=0.61). For the pre-treatment of
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the resulting CNF film (49 mg, 0.24 mmol/unit), it was immersed in 3.2 wt% LiCl/N,N’-
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dimethylacetamide (DMAc) solution (5 g) and the mixture was heated at 80 °C for 14 h. Then, the
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solution was removed and the film was washed with DMAc. After the pre-treated CNF film was
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immersed in DMAc (1.0 mL) and α-bromoisobutyryl bromide (1.73 g, 7.6 mmol; 30 equiv. for a unit of
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chitin) and pyridine (96 mg, 1.2 mmol; 5 equiv. for a unit of chitin) were added to the mixture, it was
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heated at 60 °C for 24 h. The film was taken out, subjected to Soxhlet extraction with methanol for 6 h,
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and dried under reduced pressure at 60 °C for 24 h to produce a CNF macroinitiator film (44 mg,
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DS=0.61 by SEM-EDX analysis). By the same acylation procedure using 10 and 20 equiv. of α-
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bromoisobutyryl bromide, CNF macroinitiator films with DSs=0.20 and 0.30 were prepared.
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2.3.
Surface-initiated ATRP of HEA from CNF macroinitiator film.
A typical experimental procedure for the surface-initiated ATRP was as follows (entry 4, Table 1). For
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the pre-treatment of the CNF macroinitiator film (34 mg, 0.17 mmol, DS=0.61), it was immersed in 3.0
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wt% LiCl/DMAc solution (5 g) and the mixture was heated at 80 °C for 48 h. Then, the film was taken
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out and subjected to Soxhlet extraction with methanol for 15 h. Then, HEA (254 mg, 2.20 mmol; 20
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equiv. for an initiate site), 2,2’-bipyridine (Bpy) (70 mg, 0.45 mmol; 4 equiv. for an initiate site) and
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copper(I) bromide (CuBr) (33 mg, 0.23 mmol; 2 equiv. for an initiate site) were added to the pre-treated
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mixture, it was heated at 60 °C for 24 h under argon. After water was added to the reaction mixture and
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the solution was filtered off, the film was washed successively with methanol and acetone, and dried
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under reduced pressure to produce a CNF-g-polyHEA film (216 mg) in 71 % yield.
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2.4.
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Separation and methyl esterification of grafted polyHEAs on CNF Films.
A mixture of the CNF-g-polyHEA film (46 mg, entry 4, Table1) with 5.0 mol/L NaOH aqueous
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solution (5.0 mL) was heated at 60 °C for 18 h. After the insoluble residue was filtered off, the filtrate
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was diluted with water and treated with cation-exchange resin (DowexTM 50 W H+ form) at room
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temperature. The resin was removed by filtration and the filtrate was concentrated and dried under
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reduced pressured at 60 °C for 8 h. The obtained product (24 mg) was dispersed in a mixed solvent of
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THF/methanol (1/3 v/v). After trimethylsilyldiazomethane (0.5 mL, 0.3 mmol) was added to the
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dispersion, the mixture was stirred at room temperature for 15 h under nitrogen. The reaction mixture
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was diluted with water and extracted with chloroform. After the insoluble material present in the 8
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chloroform layer was removed by filtration, the filtrate was dried over anhydrous Na2SO4 and
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concentrated. Then, the concentrated solution was poured into a large amount of hexane to precipitate
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the product. The precipitate was isolated by filtration, and dried under reduced pressure at 60 °C for 8 h
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to give poly(methyl acrylate) (6 mg); 1H NMR (CDCl3)δ1.31-2.46 (br, -CH2-CH-, 3H),δ3.60 (br, –
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CH3, 3H). The number-average molecular weight (Mn) and molecular weight distribution (Mw / Mn) of
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the product were estimated by the GPC measurement with chloroform as the eluent.
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3. Results and discussion
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As previously reported by us, the CNF film was obtained through the gelation of chitin with AMIMBr, followed by regeneration in methanol and filtration according to Fig. 1a. The CNF macroinitiator film
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having the initiating moieties (α-bromoisobutyrate group) was prepared by acylation on the CNF surface
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with α-bromoisobutyryl bromide as shown in Fig. 1b. To promote the solid-liquid reaction, the CNF
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film was first pre-treated in 3.2 wt% LiCl/DMAc solution. Then, the pre-treated CNF film was reacted
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with 10, 20, and 30 equiv. of α-bromoisobutyryl bromide for a repeating unit of chitin in the presence of
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pyridine in DMAc. Then, the structure and the presence of the initiating moieties of the obtained film
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were evaluated by the IR, XRD, SEM, and SEM-EDX analyses. Fig. 2b shows the IR spectrum of the
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product obtained using 30 equiv. of α-bromoisobutyryl bromide for a repeating unit of chitin in
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comparison with that of the CNF film (Fig. 2a). In addition to the carbonyl absorption at 1665 cm-1 due
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to acetamide, the detection of that at 1745 cm-1 due to ester suggested the presence of the initiating
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moieties in the product. The XRD profile of the product (Fig. 3b) showed typical four diffraction peaks
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at around 9.5, 19.5, 20.5, and 23.4° assignable to 020, 110, 120, and 130 planes, respectively, of the
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antiparallel α-chitin arrangement and the pattern was same as that of the original CNF film (Fig 3a).
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These results indicated the crystalline structure of α-chitin in CNFs was still maintained even after the
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acylation, indicating the introduction of the initiating moieties on surface of CNFs with remaining the
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nanofiber morphology. The SEM images of the produced films further revealed remaining the nanofiber
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morphology as seen in Fig. 5a (30 equiv. of α-bromoisobutyryl bromide). The SEM-EDX spectra of the
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products also suggested the presence of the initiating moieties (α-bromoisobutyrate group) on the film
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surface, because signals assignable to C, O, and Br elements were observed. By comparison of the signal
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intensities of the O and Br elements, the DS values on the film surfaces of the products obtained using
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10, 20, and 30 equiv. of α-bromoisobutyryl bromide were calculated to be 0.20, 0.30, and 0.61,
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respectively.
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Then, the surface-initiated graft polymerization of HEA from the CNF macroinitiator film via ATRP
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was performed to produce the CNF-g-polyHEA film as shown in Fig. 1b. The CNF macroinitiator films
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with the different DSs (0.20, 0.30, and 0.61) were pre-treated in 3.0 wt% LiCl/DMAc solution. Then,
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the surface-initiated ATRP of HEA (20 equiv. for an initiate site) from the pre-treated films was
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performed in the presence of Bpy (4 equiv. for an initiate site) and CuBr (2 equiv. for an initiate site) at
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60 °C for desired times under argon. After water was added to the mixtures, the films were taken out,
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washed successively with methanol and acetone, and dried under reduced pressure. Table 1 shows the
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results of the surface-initiated graft polymerization of HEA from the CNF macroinitiator films under
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conditions of different reaction times (3, 6, 12, and 24 h). The conversions of HEA were estimated by
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the weight increases of the products from the CNF macroinitiator film. The yields/conversions of the
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products obtained using the CNF macroinitiator films with DS=0.61 increased with increasing
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polymerization times (entries 1-4, Table 1). When the surface-initiated graft polymerization of HEA
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from the CNF macroinitiator films with the lower DSs (0.20 and 0.30) was carried out for 24 h,
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similarly, the polymerization proceeded (entries 5 and 6, Table 1, conversion 68 and 71%). Fig. 2c
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shows the IR spectrum of product (entry 4, Table 1, conversion 71%). The intensity ratio of the ester
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absorption (1745 cm-1) to the amide I absorption (1665 cm-1) of acetamide group increased in
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comparison with the CNF macroinitiator film (Fig. 2b), suggesting the progress of the graft
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polymerization of HEA. The XRD pattern (Fig. 3c) of the product was almost the same as that of the
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original CNF and CNF macroinitiator film (Fig. 3a and b), indicating the crystalline structure of the
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chitin chains was not disrupted owing to the occurrence of the graft polymerization only on surface of
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CNFs. To estimate the Mn values of the grafted polyHEA chains, their separation from the CNFs was
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conducted by alkaline hydrolysis of the products in 5 mol/L NaOH aq. (Scheme 1). For the GPC
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measurement, the hydrolysate, i.e., poly(acrylic acid) produced by alkaline hydrolysis of polyHEA, was
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modified by methylation of the carboxylic acid groups using trimethylsilyldiazomethane. The water-
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insoluble fraction in the methylation experiment was characterized by the 1H NMR analysis in CDCl3 to
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be the structure of poly(methyl acrylate). Fig. 4 shows the GPC traces of poly(methyl acrylate)s obtained
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from the CNF-g-polyHEA films of entries 3 and 4 in Table 1, respectively. When the polymerization
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time increases, the GPC peaks shifted to higher molecular weight region (Mn; 1490 and 4000, estimated
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using polystyrene standards) and their Mw/Mn values were relatively low (1.64 and 1.22). The GPC
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analysis supported that the longer polyHEA chains were grafted on the CNF films by the further
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progress of ATRP with prolonged reaction time. The surface morphologies of the CNF-g-polyHEA
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films were investigated by SEM measurement. At lower conversion of HEA in the graft polymerization
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(entry 1, Table 1), the SEM image (Fig. 5b) indicated that the morphology of the nanofibers on the films
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still remained, but the fiber widths (ca. 40 nm) increased compared with those of the CNF macroinitiator
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film (ca. 30 nm, Fig. 5a). On the other hand, the nanofiber morphologies were not seen in the SEM
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images of the films produced at higher conversions of HEA (Fig. 5c-e), indicating that the nanofibers
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were covered by the grafted longer polyHEA chains. When the CNF macroinitiator films with the lower
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DSs (0.20 and 0.30, entries 4 and 5, Table 1) were used for the graft polymerization of HEA, the SEM
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images showed the morphology of the nanofibers on the films (ca. 40 nm, Fig. 5f and ca. 51 nm, Fig.
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5g). Because the conversions of HEA in the graft polymerization from the three CNF macroinitiator
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films were comparable, which in turn suggested the similar molecular weights of the grafted polyHEAs
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(entries 4-6, Table 1), the functionality of the grafted chains on CNF surfaces was also shown to affect
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the nanofiber morphologies. The above SEM results supported the presence of the grafted polyHEA
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chains on surface of the CNF films and suggested that the nanofiber morphologies of the films were
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affected by both the molecular weight and functionality of the grafted polyHEAs.
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The mechanical properties of the CNF-g-polyHEA films were evaluated by tensile testing (Fig. 6). The stress-strain curves of the films obviously showed the larger fracture strain values (3.9 – 12.6 %)
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compared with those of the original films (1.1 %). Furthermore, the fracture strain values increased with
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increasing the conversions of HEA, whereas the fracture stress values decreased in this order and the
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film with the longer polyHEA graft chains showed the much larger fracture strain value than that of
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CNF-graft-poly(LA/CL) and CNF-graft-polypeptide films, which were provided in our previous studies
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(Setoguchi, Yamamoto, & Kadokawa, 2012; Kadokawa, Setoguchi, & Yamamoto, 2013). These results
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suggested the effect on enhancement of the flexibility by grafting the longer polyHEA chains on the
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CNFs films.
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4. Conclusions
In this study, we prepared the CNF macroinitiator film for ATRP by introduction of the initiating
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moieties on surface of the CNF films. The surface-initiated graft polymerization of HEA from the CNF
255
macroinitiator film by ATRP was performed to produce the CNF-g-polyHEA films. The IR, XRD, and
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SEM measurements of the resulting films as well as the GPC analysis after separation of the graft chains
257
indicated the progress of the graft polymerization of HEA on surface of CNFs. Furthermore, the CNF-g-
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polyHEA films showed more flexible nature than the original CNF film. Because the present films are
259
efficiently obtained by the surface-initiated ATRP, this approach has a potential to produce the CNF
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composite materials with various synthetic polymers, which will be used as practical bio-based materials
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in the future.
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References
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Bochek A. M, Murav'ev, A. A., Novoselov, N. P., Zaborski, M., Zabivalova, N. M., Petrova, V. A.,
266
Vlasova, E. N., Volchek, B. Z., & Lavrent'ev V. K. (2012). Specific features of cellulose and chitin
267
dissolution in ionic liquids of varied structure and the structural organization of regenerated
268
polysaccharides. Russian Journal of Applied Chemistry, 85, 1718-1725.
269
Fan, Y., Saito, T., & Isogai, A. (2008). Chitin nanocrystals prepared by tempo-mediated oxidation of -
270
chitin. Biomacromolecules, 9, 192-198.
271
Feng, L., & Chen, Z.-I. (2008). Research progress on dissolution and functional modification of
272
cellulose in ionic liquids. Journal of Molecular Liquids, 142, 1-5.
273
Ifuku, S., & Saimoto, H. (2012). Chitin nanofibers: Preparations, modifications, and applications.
274
Nanoscale, 4, 3308-3318.
275
Ifuku, S., Iwasaki, M., Morimoto, M., & Saimoto, H. (2012). Graft polymerization of acrylic acid onto
276
chitin nanofiber to improve dispersibility in basic water. Carbohydrate Polymers, 90, 623-627.
277
Jaworska, M. M., Kozlecki, T., & Gorak, A. (2012). Review of the application of ionic liquids as
278
solvents for chitin. Journal of Polymer Engineering, 32, 67-69.
279
Kadokawa, J., Setoguchi, T., & Yamamoto, K. (2013). Preparation of highly flexible chitin nanofiber-
280
graft-poly(gamma-L-glutamic acid) network film. Polymer Bulletin, 70, 3279-3289.
281
Kadokawa, J., Takegawa, A., Mine, S., & Prasad, K. (2011). Preparation of chitin nanowhiskers using
282
an ionic liquid and their composite materials with poly(vinyl alcohol). Carbohydrate Polymers, 84,
283
1408-1412.
284
Kamigaito, M., Ando, T., & Sawamoto, M. (2001). Metal-Catalyzed Living Radical Polymerization.
285
Chemical Reviews, 101, 3689-3745.
Ac ce p
te
d
M
an
us
cr
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264
13
Page 13 of 22
Kurita, K. (2006). Chitin and chitosan: Functional biopolymers from marine crustaceans. Marine
287
Biotechnology, 8, 203-226.
288
Liebert, T., & Heinze, T. (2008). Interaction of ionic liquids with polysaccharides. 5. Solvents and
289
reaction media for the modification of cellulose. BioResources, 3,576-601.
290
Matyjaszewski, K. (2012). Atom Transfer Radical Polymerization (ATRP): Current Status and Future
291
Perspectives, Macromolecules, 45, 4015-4039.
292
Mine, S., Izawa, H., Kaneko, Y., & Kadokawa, J. (2009). Acetylation of a-chitin in ionic liquids.
293
Carbohydrate Research, 344, 2263-2265.
294
Muzzarelli, R.A.A. (2011). Biomedical exploitation of chitin and chitosan via mechano-chemical
295
disassembly, electrospinning, dissolution in imidazolium ionic liquids, and supercritical drying. Marine
296
Drugs, 9, 1510-1533.
297
Muzzarelli, R.A.A. (2012). Nanochitins and nanochitosans, paving the way to eco-friendly and energy-
298
saving exploitation of marine resources. Polymer Science: A Comprehensive Reference, 10, 153-164.
299
Pillai, C. K. S., Paul, W., & Sharma, C. P. (2009). Chitin and chitosan polymers: Chemistry, solubility
300
and fiber formation. Progress in Polymer Science, 34, 641-678.
301
Pinkert, A., Marsh, K. N., Pang, S., & Staiger, M. P. (2009). Ionic liquids and their interaction with
302
cellulose. Chemical Reviews, 109, 6712-6828.
303
Poirier, M., & Charlet, G. (2002). Chitin fractionation and characterization in N,N-
304
dimethylacetamide/lithium chloride solvent system. Carbohydrate Polymers, 50, 363-370.
305
Prasad, K., Murakami, M., Kaneko, Y., Takada, A., Nakamura, Y., & Kadokawa, J. (2009). Weak gel of
306
chitin with an ionic liquid, 1-allyl-3-methylimidazolium bromide. International Journal of Biological
307
Macromolecules, 45, 221-225.
Ac ce p
te
d
M
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286
14
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Qin Y., Lu X., Sun N., & Rogers R. D. (2010). Dissolution or extraction of crustacean shells using ionic
309
liquids to obtain high molecular weight purified chitin and direct production of chitin films and fibers.
310
Green Chemistry, 12, 968-971
311
Rinaudo, M. (2006). Chitin and chitosan: Properties and applications. Progress in Polymer Science, 31,
312
603-632.
313
Schuerch, C. (1986). Polysaccharides. In H. F. Mark, N. Bilkales, & C. G. Overberger (Eds.),
314
Encyclopedia of polymer science and engineering (2nd edition, 13, pp. 87–162). New York: John Wiley
315
& Sons.
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Setoguchi, T., Yamamoto, K., & Kadokawa, J. (2012). Preparation of chitin nanofiber-graft-poly(L-
317
lactide-co-ε-caprolactone) films by surface-initiated ring-opening graft copolymerization. Polymer, 53,
318
4977-4982.
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Swatloski, R. P., Spear, S. K., Holbrey, J. D., & Rogers, R. D. (2002). Dissolution of cellulose with
320
ionic liquids. Journal of the American Chemical Society, 124, 4974-4975.
321
Tajiri, R., Setoguchi, T., Wakizono, S.,Yamamoto, K., & Kadokawa, J. (2013). Preparation of Self-
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assembled Chitin Nanofibers by Regeneration from Ion Gels Using Calcium Halide･dihydrate/methanol
323
Solutions. Journal of Biobased Materials and Bioenergy, 7, 655-659.
324
Wang, W. T., Zhu, J., Wang, X. L., Huang, Y, & Wang, Y. Z. (2010). Dissolution behavior of chitin in
325
ionic liquids. Journal of Macromolecular Science, Part B: Physics, 49, 528-541.
326
Wu, Y., Sasaki, T., Irie, S., & Sakurai, K. (2008). A novel biomass-ionic liquid platform for the
327
utilization of native chitin. Polymer, 49, 2321-2327.
328
Yamazaki, S., Takegawa, A., Kaneko, Y., Kadokawa, J., Yamagata, M., & Ishikawa, M. (2009). An
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acidic cellulose-chitin hybrid gel as novel electrolyte for an electric double Layer capacitor.
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Electrochemistry Communications, 11, 68-70.
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Yamamoto, K., Yoshida, S., Mine, S., & Kadokawa, J. (2013). Synthesis of chitin-graft-polystyrene via
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atom transfer radical polymerization initiated from chitin macroinitiator. Polymer Chemistry, 4, 3384-
333
3389.
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Zakrzewska, M. E., Lukasik, E. B., & Lukasik, R. B. (2010). Solubility of carbohydrates in ionic liquids.
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Energy Fuels, 24, 737-745.
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Zeng, J. B., He, Y. S., Li, S. L., & Wang, Y. Z. (2012). Chitin whiskers: A overview.
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Biomacromolecules, 13, 1-11.
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Zhao, D., Fei, Z., Geldbach, T. J., Scopelliti, R., Laurenczy, G., & Dyson, P. J. (2005) Allyl-
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functionalised ionic liquids: synthesis, characterisation, and reactivity. Helvetica Chimica Acta, 88, 665-
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675.
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Figure and Scheme Captions
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Fig. 1. Procedures for preparations of (a) CNF film through chitin ion gel with AMIMBr, (b) chitin
345
macroinitiator film, and (c) CNF-graft-polyHEA film via surface-initiated ATRP.
346
Fig. 2. IR spectra of (a) CNF film, (b) CNF macroinitiator film (30 equiv. of α-bromoisobutyryl
347
bromide), and (c) CNF-g-polyHEA film (entry 4, Table 1).
348
Fig. 3. XRD patterns of (a) CNF film, (b) CNF macroinitiator film and, (c) CNF-g-polyHEA film (entry
349
4, Table 1).
350
Fig. 4. GPC traces of poly(methyl acrylate)s from CNF-g-polyHEA films of (a) entry 3 and (b) entry 4
351
in Table 1.
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Fig. 5. SEM images of (a) chitin macroinitiator film (30 equiv. of α-bromoisobutyryl bromide) and (b)–
353
(g) CNF-g-polyHEA films of entries 1-6 in Table 1, respectively.
354
Fig. 6. Stress-strain curves of (a) chitin macroinitiator film (30 equiv. of α-bromoisobutyryl bromide)
355
and (b)-(e) CNF-g-polyHEA films for of entries 1-4 in Table 1, respectively; the fracture strain and
356
stress values are shown in parentheses.
357
Scheme 1. Alkaline hydrolysis of grafted polyHEA on CNF film and methylation of carboxylic acid
358
groups.
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Table 1
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Surface-initiated ATRP of HEA from CNF macroinitiator films with different DSs under conditions of
361
different reaction timesa
362
Entry
363
1
364
Yield / mg
0.61
3
52
2
0.61
6
65
365
3
0.61
12
161
366
4
0.61
24
216
367
5
0.20
24
368
6
0.30
24
Conversion of HEA / % b
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68
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DS
71
143
369
a
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Reaction conditions; [Initiating site]/[HEA]/[Bpy]/[CuBr] =1 / 20 / 4 / 2, Temp.; 60 °C
371
b
373
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Estimated by the weight increases of the products from the CNF macroinitiator film.
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A feed amount of chitin macroinitiator film was 34 mg (0.17 mmol, DS; 0.20, 0.30, and 0.61).
18
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373 374 375 376
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Fig. 1. Procedures for preparations of (a) CNF film through chitin ion gel with AMIMBr, (b) chitin
389
macroinitiator film, and (c) CNF-graft-polyHEA film via surface-initiated ATRP.
391 392 393
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Fig. 2. IR spectra of (a) CNF film, (b) CNF macroinitiator film (30 equiv. of α-bromoisobutyryl
398
bromide), and (c) CNF-g-polyHEA film (entry 4, Table 1). 19
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399 400 401
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Fig. 3. XRD patterns of (a) CNF film, (b) CNF macroinitiator film and, (c) CNF-g-polyHEA film (entry
408
4, Table 1).
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Fig. 4. GPC traces of poly(methyl acrylate)s from CNF-g-polyHEA films of (a) entry 3 and (b) entry 4
421
in Table 1.
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Fig. 5. SEM images of (a) chitin macroinitiator film (30 equiv. of α-bromoisobutyryl bromide) and (b)–
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(g) CNF-g-polyHEA films of entries 1-6 in Table 1, respectively.
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436 437 438
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Fig. 6. Stress-strain curves of (a) chitin macroinitiator film (30 equiv. of α-bromoisobutyryl bromide)
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and (b)-(e) CNF-g-polyHEA films for of entries 1-4 in Table 1, respectively; the fracture strain and
446
stress values are shown in parentheses.
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Scheme 1. Alkaline hydrolysis of grafted polyHEA on CNF film and methylation of carboxylic acid
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groups. 22
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S0144-8617(14)00557-8 http://dx.doi.org/doi:10.1016/j.carbpol.2014.05.079 CARP 8946
To appear in: Received date: Revised date: Accepted date:
19-3-2014 21-5-2014 29-5-2014
Please cite this article as: Yamamoto, K., Yoshida, S., and Kadokawa, J.-i.,Surfaceinitiated atom transfer radical polymerization from chitin nanofiber macroinitiator film, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.05.079 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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Chitin nanofiber (CNF) film was used for surface-initiated graft polymerization.
3
It derived to macroinitiator for atom transfer radical polymerization (ATRP).
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Surface-initiated graft ATRP of acrylate monomer from the product was performed.
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The analytical results showed the production of CNF-graft-polyacrylate film.
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Surface-initiated atom transfer radical polymerization
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from chitin nanofiber macroinitiator film
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Kazuya Yamamoto a, Sho Yoshida a, Jun-ichi Kadokawa a,b,*
15
a
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890-0065, Japan
17
b
18
Technology, 27-1 Mizumoto-cho, Muroran, Hokkaido 050-8585, Japan
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us
Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima
M
an
Research Center for Environmentally Friendly Materials Engineering, Muroran Institute of
19 ABSTRACT
21
This paper reports the preparation of chitin nanofiber-graft-poly(2-hydroxyethyl acrylate) (CNF-g-
22
polyHEA) films by surface-initiated atom transfer radical polymerization (ATRP) of HEA monomer
23
from a CNF macroinitiator film. First, a CNF film was prepared by regeneration from a chitin ion gel
24
with an ionic liquid. Then, acylation of the CNF surface with α-bromoisobutyryl bromide was carried
25
out to obtain the CNF macroinitiator film having the initiating moieties (α-bromoisobutyrate group). The
26
surface-initiated graft polymerization of HEA from the CNF macroinitiator film by ATRP was
27
performed to produce the CNF-g-polyHEA film. The IR, XRD, and SEM measurements of the resulting
28
film indicated the progress of the graft polymerization of HEA on surface of CNFs. The molecular
29
weights of the grafted polyHEAs increased with prolonged polymerization times, which affected the
30
mechanical properties of the films under tensile mode.
31
Keywords: Chitin nanofiber film; Atom transfer radical polymerization; Surface-initiated graft
32
polymerization; Macroinitiator
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*Corresponding author. Tel.: +81 99 285 7743; fax: +81 99 285 3253
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E-mail address: [email protected] (J. Kadokawa).
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37 38
1. Introduction Polysaccharides are widely distributed in nature and have been regarded as structural materials and as suppliers of energy (Shuerch, 1986). Because of better recent understanding of their possessing unique
40
structures and properties, much attention has been paid to them as material sources. Of the many kinds
41
of polysaccharides, chitin, which is an aminopolysaccharide composed of N-acetyl-D-glucosamine
42
residues linked through -(14)-glycosidic bonds, is one of the most abundant polysaccharides in
43
nature and occurs mainly in exoskeletons of crustaceans, shellfish, and insects (Kurita, 2006; Muzzarelli,
44
2011; Pillai, Paul, & Sharma, 2009; Rinaudo, 2006). However, it still remains mostly as an unutilized
45
biomass resource primary because of its intractable bulk structure and insolubility in water and common
46
organic solvents. Therefore, research concerning conversion of chitin into various useful bio-based
47
materials after proper dissolution of it in suitable solvents has attracted much attention even in recent
48
years.
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To efficiently provide new chitin-based materials, we have focused on ionic liquids, which are low-
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melting point salts that form liquids at temperatures below the boiling point of water. Since it was
51
reported that 1-butyl-3-methylimidazolium chloride of an ionic liquid dissolved cellulose in relatively
52
high concentrations (Swatloski, Spear, Holbrey, & Rogers, 2002), various ionic liquids have been used
53
as good solvents for the derivatization and material processing of cellulose (Feng, & Chen, 2008;
54
Liebert, & Heinze, 2008; Pinkert, Marsh, Pang, & Staiger, 2009; Zakrzewska, Lukasik, & Lukasik,
55
2010). However, only the limited investigations have been reported regarding the dissolution of chitin
56
with ionic liquids (Bochek, Murav'ev, Novoselov, Zaborski, Zabivalova, Petrova, Vlasova, Volchek, &
57
Lavrent'ev, 2012; Jaworska, Kozlecki, & Gorak, 2012; Muzzarelli, 2012; Qin, Lu, Sun, & Rogers, 2010;
58
Wang, Zhu, Wang, Huang, & Wang, 2010; Wu, Sasaki, Irie, & Sakurai, 2008). We also found that an
59
ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), dissolved or swelled chitin to form weak
60
gel-like materials (ion gels) (Prasad, Murakami, Kaneko, Takada, Nakamura, & Kadokawa, 2009;
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Yamazaki, Takegawa, Kaneko, Kadokawa, Yamagata, & Ishikawa, 2009). By using AMIMBr as a
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solvent, acetylation of chitin using acetic anhydride produced an acetylated chitin with a high degree of
63
substitution (DS) under selected conditions (Mine, Izawa, Kaneko, & Kadokawa, 2009). Furthermore,
64
this reaction manner, i.e., the esterification of hydroxy groups in chitin in the AMIMBr solvent was
65
employed to introduce initiating moieties for atom transfer radical polymerization (ATRP) attached
66
through ester linkages on chitin. Then, grafting of styrene from the resulting macroinitiator via ATRP
67
was performed to produce a chitin-graft-polystyrene (Yamamoto, Yoshida, Mine, & Kadokawa, 2013).
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ATRP is a robust and versatile technique to accurately control chain length and polydispersity of the
69
resulting polymers, and thus has been used to synthesize a wide range of copolymers with the controlled
70
structure, unit length, and block sequence (Kamigaito, Ando, & Sawamoto, 2001; Matyjaszewski,
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2012).
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On the other hand, graft copolymers based on a chitin nanofiber (CNF) film have been prepared by us. In the previous study, we reported that CNF film was facilely obtained by regeneration from the
74
aforementioned chitin ion gel with AMIMBr in methanol, followed by filtration (Fig. 1a) (Kadokawa,
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Takegawa, Mine, & Prasad, 2011; Tajiri, Setoguchi, Wakizono, Yamamoto, & Kadokawa, 2013).
76
Approaches to the production of nanofibers from chitin have often been performed upon top-down
77
procedures that break down the starting bulk materials from native chitin resources (Fan, Saito, & Isogai,
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2008; Ifuku, & Saimoto, 2012; Zeng, He, Li, & Wang, 2012). Our method applies a completely different
79
approach from the above to produce CNFs, that accords to a self-assembling generative (bottom-up)
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route. We have continuously studied surface-initiated graft polymerization approach from the CNF film
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to prepare new chitin-based materials. For example, we performed the surface-initiated ring-opening
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graft copolymerization of L-lactide (LA)/ε-caprolactone (CL) from the CNF film to produce a
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environmentally benign composite material because the ring-opening copolymerization is initiated from
84
a hydroxy group and has been known to efficiently produce a biodegradable and biocompatible
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poly(LA/CL) (Setoguchi, Yamamoto, & Kadokawa, 2012). Recently, the surface-initiated grafting
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technique from the CNF film was extended to use a synthetic polypeptide as another biocompatible
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polymer. Because it has been well-known that synthetic polypeptides with well-defined structures are
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synthesized by ring-opening polymerization of α-amino acid N-carboxyanhydride (NCA) accompanied
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with decarboxylation initiated from amino groups, we reported the surface-initiated graft polymerization
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of NCA from amino initiating groups on surface of a partially deacetylated CNF film to give a CNF-
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graft-polypeptide film (Kadokawa, Setoguchi, & Yamamoto, 2013). By free radical polymerization
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approach, grafting of acrylic acid on CNFs, which were prepared by the aforementioned top-down
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procedure, was also carried out with potassium persulfate as a initiator, where radicals are formed along
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the chitin back-bone, followed by the progress of free radical propagation (Ifuku, Iwasaki, Morimoto, &
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Saimoto, 2012).
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In this paper, on the basis of the above previous investigations, we report the preparation of CNF-
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graft-poly(2-hydroxyethyl acrylate) (CNF-g-polyHEA) films by the surface-initiated grafting technique
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via ATRP using a CNF macroinitiator film. The CNF macroinitiator film for ATRP was first prepared
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by introduction of the initiating moieties attached through ester linkages on CNF surfaces. Then, the
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surface-initiated graft polymerization of HEA from the CNF macroinitiator film by ATRP was
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performed under the appropriate conditions to produce the CNF-g-polyHEA films. To the best of our
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knowledge, this is the first example of the surface-initiated ATRP from CNF materials. Because the
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present surface-initiated ATRP technique using the CNF macroinitiator film has a potential to employ
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various monomers, we are convinced that the present approach will contribute to the development of
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practical chitin-based composite materials in the future.
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2. Experimental Part
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2.1.
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Chitin powder from crab shells was purchased from Wako Pure Chemical Industries, Ltd., Japan. The
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weight-average molecular weight value of the chitin sample was estimated by viscometric analysis to be
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7 x 105 (Poirier & Charlet, 2002). An ionic liquid, AMIMBr, was prepared by reaction of 1-
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methylimidazole with 3-bromo-1-propene according to the method adapted from the literature procedure
Materials and methods
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(Zhao, Fei, Geldbach, Scopelliti, Laurenczy, & Dyson, 2005). All other reagents and solvents were
114
used as received from commercial sources. IR spectra were recorded with samples in KBr using a
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SHIMADZU FTIR-8400 spectrometer. The powder X-ray diffraction (XRD) measurements were
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conducted using a PANalytical X’Pert Pro MPD with Ni-filtered CuKα radiation (λ = 0.15418 nm). The
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SEM images were obtained using Hitachi S-4100H electron microscope. The energy-dispersive X-ray
118
spectroscopy was performed using a Philips XL30 CP scanning electron microscope (SEM-EDX). 1H
119
NMR spectrum was recorded on a JEOL ECX 400 spectrometer. GPC measurement was performed with
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two Shodex GPC KF-804L and KF-803L columns and equipped with HITACHI pump L-2130
121
connected to HITACHI RI detector L-2490. Chloroform was used as the eluent at a flow rate of 1.0 mL
122
min-1. The calibration was established with polystyrene standards. The stress-strain curves were
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measured using a tensile tester (Little Senster LSC-1/30, Tokyo Testing Machine).
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2.2.
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First, the preparation of CNF film was conducted according to the method reported in our previous
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publication (Kadokawa, Takegawa, Mine, & Prasad, 2011). Chitin powder (0.125 g, 0.61 mmol) was
128
immersed in AMIMBr (1.0 g, 4.9 mmol) at room temperature for 24 h, followed by heating the mixture
129
at 100 °C for 24 h to give a chitin/AMIMBr ion gel. After methanol (40 mL) was added to the gel and
130
the system was left standing at room temperature for 24 h, the mixture was sonicated for 5 min to give a
131
chitin dispersion. Then, the dispersion was subjected to filtration and a residue was dried under reduced
132
pressure to give a film. The film was subjected to Soxhlet extraction with methanol (70 mL) for 5 h,
133
followed by drying under reduced pressure to give a CNF film. A typical procedure for the synthesis of
134
the CNF macroinitiator film was as follows (entries 1-4, Table 1; DS=0.61). For the pre-treatment of
135
the resulting CNF film (49 mg, 0.24 mmol/unit), it was immersed in 3.2 wt% LiCl/N,N’-
136
dimethylacetamide (DMAc) solution (5 g) and the mixture was heated at 80 °C for 14 h. Then, the
137
solution was removed and the film was washed with DMAc. After the pre-treated CNF film was
138
immersed in DMAc (1.0 mL) and α-bromoisobutyryl bromide (1.73 g, 7.6 mmol; 30 equiv. for a unit of
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chitin) and pyridine (96 mg, 1.2 mmol; 5 equiv. for a unit of chitin) were added to the mixture, it was
140
heated at 60 °C for 24 h. The film was taken out, subjected to Soxhlet extraction with methanol for 6 h,
141
and dried under reduced pressure at 60 °C for 24 h to produce a CNF macroinitiator film (44 mg,
142
DS=0.61 by SEM-EDX analysis). By the same acylation procedure using 10 and 20 equiv. of α-
143
bromoisobutyryl bromide, CNF macroinitiator films with DSs=0.20 and 0.30 were prepared.
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2.3.
Surface-initiated ATRP of HEA from CNF macroinitiator film.
A typical experimental procedure for the surface-initiated ATRP was as follows (entry 4, Table 1). For
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the pre-treatment of the CNF macroinitiator film (34 mg, 0.17 mmol, DS=0.61), it was immersed in 3.0
148
wt% LiCl/DMAc solution (5 g) and the mixture was heated at 80 °C for 48 h. Then, the film was taken
149
out and subjected to Soxhlet extraction with methanol for 15 h. Then, HEA (254 mg, 2.20 mmol; 20
150
equiv. for an initiate site), 2,2’-bipyridine (Bpy) (70 mg, 0.45 mmol; 4 equiv. for an initiate site) and
151
copper(I) bromide (CuBr) (33 mg, 0.23 mmol; 2 equiv. for an initiate site) were added to the pre-treated
152
mixture, it was heated at 60 °C for 24 h under argon. After water was added to the reaction mixture and
153
the solution was filtered off, the film was washed successively with methanol and acetone, and dried
154
under reduced pressure to produce a CNF-g-polyHEA film (216 mg) in 71 % yield.
155 156 157
2.4.
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Separation and methyl esterification of grafted polyHEAs on CNF Films.
A mixture of the CNF-g-polyHEA film (46 mg, entry 4, Table1) with 5.0 mol/L NaOH aqueous
158
solution (5.0 mL) was heated at 60 °C for 18 h. After the insoluble residue was filtered off, the filtrate
159
was diluted with water and treated with cation-exchange resin (DowexTM 50 W H+ form) at room
160
temperature. The resin was removed by filtration and the filtrate was concentrated and dried under
161
reduced pressured at 60 °C for 8 h. The obtained product (24 mg) was dispersed in a mixed solvent of
162
THF/methanol (1/3 v/v). After trimethylsilyldiazomethane (0.5 mL, 0.3 mmol) was added to the
163
dispersion, the mixture was stirred at room temperature for 15 h under nitrogen. The reaction mixture
164
was diluted with water and extracted with chloroform. After the insoluble material present in the 8
Page 8 of 22
chloroform layer was removed by filtration, the filtrate was dried over anhydrous Na2SO4 and
166
concentrated. Then, the concentrated solution was poured into a large amount of hexane to precipitate
167
the product. The precipitate was isolated by filtration, and dried under reduced pressure at 60 °C for 8 h
168
to give poly(methyl acrylate) (6 mg); 1H NMR (CDCl3)δ1.31-2.46 (br, -CH2-CH-, 3H),δ3.60 (br, –
169
CH3, 3H). The number-average molecular weight (Mn) and molecular weight distribution (Mw / Mn) of
170
the product were estimated by the GPC measurement with chloroform as the eluent.
ip t
165
173
3. Results and discussion
us
172
cr
171
As previously reported by us, the CNF film was obtained through the gelation of chitin with AMIMBr, followed by regeneration in methanol and filtration according to Fig. 1a. The CNF macroinitiator film
175
having the initiating moieties (α-bromoisobutyrate group) was prepared by acylation on the CNF surface
176
with α-bromoisobutyryl bromide as shown in Fig. 1b. To promote the solid-liquid reaction, the CNF
177
film was first pre-treated in 3.2 wt% LiCl/DMAc solution. Then, the pre-treated CNF film was reacted
178
with 10, 20, and 30 equiv. of α-bromoisobutyryl bromide for a repeating unit of chitin in the presence of
179
pyridine in DMAc. Then, the structure and the presence of the initiating moieties of the obtained film
180
were evaluated by the IR, XRD, SEM, and SEM-EDX analyses. Fig. 2b shows the IR spectrum of the
181
product obtained using 30 equiv. of α-bromoisobutyryl bromide for a repeating unit of chitin in
182
comparison with that of the CNF film (Fig. 2a). In addition to the carbonyl absorption at 1665 cm-1 due
183
to acetamide, the detection of that at 1745 cm-1 due to ester suggested the presence of the initiating
184
moieties in the product. The XRD profile of the product (Fig. 3b) showed typical four diffraction peaks
185
at around 9.5, 19.5, 20.5, and 23.4° assignable to 020, 110, 120, and 130 planes, respectively, of the
186
antiparallel α-chitin arrangement and the pattern was same as that of the original CNF film (Fig 3a).
187
These results indicated the crystalline structure of α-chitin in CNFs was still maintained even after the
188
acylation, indicating the introduction of the initiating moieties on surface of CNFs with remaining the
189
nanofiber morphology. The SEM images of the produced films further revealed remaining the nanofiber
Ac ce p
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9
Page 9 of 22
morphology as seen in Fig. 5a (30 equiv. of α-bromoisobutyryl bromide). The SEM-EDX spectra of the
191
products also suggested the presence of the initiating moieties (α-bromoisobutyrate group) on the film
192
surface, because signals assignable to C, O, and Br elements were observed. By comparison of the signal
193
intensities of the O and Br elements, the DS values on the film surfaces of the products obtained using
194
10, 20, and 30 equiv. of α-bromoisobutyryl bromide were calculated to be 0.20, 0.30, and 0.61,
195
respectively.
ip t
190
Then, the surface-initiated graft polymerization of HEA from the CNF macroinitiator film via ATRP
197
was performed to produce the CNF-g-polyHEA film as shown in Fig. 1b. The CNF macroinitiator films
198
with the different DSs (0.20, 0.30, and 0.61) were pre-treated in 3.0 wt% LiCl/DMAc solution. Then,
199
the surface-initiated ATRP of HEA (20 equiv. for an initiate site) from the pre-treated films was
200
performed in the presence of Bpy (4 equiv. for an initiate site) and CuBr (2 equiv. for an initiate site) at
201
60 °C for desired times under argon. After water was added to the mixtures, the films were taken out,
202
washed successively with methanol and acetone, and dried under reduced pressure. Table 1 shows the
203
results of the surface-initiated graft polymerization of HEA from the CNF macroinitiator films under
204
conditions of different reaction times (3, 6, 12, and 24 h). The conversions of HEA were estimated by
205
the weight increases of the products from the CNF macroinitiator film. The yields/conversions of the
206
products obtained using the CNF macroinitiator films with DS=0.61 increased with increasing
207
polymerization times (entries 1-4, Table 1). When the surface-initiated graft polymerization of HEA
208
from the CNF macroinitiator films with the lower DSs (0.20 and 0.30) was carried out for 24 h,
209
similarly, the polymerization proceeded (entries 5 and 6, Table 1, conversion 68 and 71%). Fig. 2c
210
shows the IR spectrum of product (entry 4, Table 1, conversion 71%). The intensity ratio of the ester
211
absorption (1745 cm-1) to the amide I absorption (1665 cm-1) of acetamide group increased in
212
comparison with the CNF macroinitiator film (Fig. 2b), suggesting the progress of the graft
213
polymerization of HEA. The XRD pattern (Fig. 3c) of the product was almost the same as that of the
214
original CNF and CNF macroinitiator film (Fig. 3a and b), indicating the crystalline structure of the
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10
Page 10 of 22
215
chitin chains was not disrupted owing to the occurrence of the graft polymerization only on surface of
216
CNFs. To estimate the Mn values of the grafted polyHEA chains, their separation from the CNFs was
218
conducted by alkaline hydrolysis of the products in 5 mol/L NaOH aq. (Scheme 1). For the GPC
219
measurement, the hydrolysate, i.e., poly(acrylic acid) produced by alkaline hydrolysis of polyHEA, was
220
modified by methylation of the carboxylic acid groups using trimethylsilyldiazomethane. The water-
221
insoluble fraction in the methylation experiment was characterized by the 1H NMR analysis in CDCl3 to
222
be the structure of poly(methyl acrylate). Fig. 4 shows the GPC traces of poly(methyl acrylate)s obtained
223
from the CNF-g-polyHEA films of entries 3 and 4 in Table 1, respectively. When the polymerization
224
time increases, the GPC peaks shifted to higher molecular weight region (Mn; 1490 and 4000, estimated
225
using polystyrene standards) and their Mw/Mn values were relatively low (1.64 and 1.22). The GPC
226
analysis supported that the longer polyHEA chains were grafted on the CNF films by the further
227
progress of ATRP with prolonged reaction time. The surface morphologies of the CNF-g-polyHEA
228
films were investigated by SEM measurement. At lower conversion of HEA in the graft polymerization
229
(entry 1, Table 1), the SEM image (Fig. 5b) indicated that the morphology of the nanofibers on the films
230
still remained, but the fiber widths (ca. 40 nm) increased compared with those of the CNF macroinitiator
231
film (ca. 30 nm, Fig. 5a). On the other hand, the nanofiber morphologies were not seen in the SEM
232
images of the films produced at higher conversions of HEA (Fig. 5c-e), indicating that the nanofibers
233
were covered by the grafted longer polyHEA chains. When the CNF macroinitiator films with the lower
234
DSs (0.20 and 0.30, entries 4 and 5, Table 1) were used for the graft polymerization of HEA, the SEM
235
images showed the morphology of the nanofibers on the films (ca. 40 nm, Fig. 5f and ca. 51 nm, Fig.
236
5g). Because the conversions of HEA in the graft polymerization from the three CNF macroinitiator
237
films were comparable, which in turn suggested the similar molecular weights of the grafted polyHEAs
238
(entries 4-6, Table 1), the functionality of the grafted chains on CNF surfaces was also shown to affect
239
the nanofiber morphologies. The above SEM results supported the presence of the grafted polyHEA
Ac ce p
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Page 11 of 22
240
chains on surface of the CNF films and suggested that the nanofiber morphologies of the films were
241
affected by both the molecular weight and functionality of the grafted polyHEAs.
242
The mechanical properties of the CNF-g-polyHEA films were evaluated by tensile testing (Fig. 6). The stress-strain curves of the films obviously showed the larger fracture strain values (3.9 – 12.6 %)
244
compared with those of the original films (1.1 %). Furthermore, the fracture strain values increased with
245
increasing the conversions of HEA, whereas the fracture stress values decreased in this order and the
246
film with the longer polyHEA graft chains showed the much larger fracture strain value than that of
247
CNF-graft-poly(LA/CL) and CNF-graft-polypeptide films, which were provided in our previous studies
248
(Setoguchi, Yamamoto, & Kadokawa, 2012; Kadokawa, Setoguchi, & Yamamoto, 2013). These results
249
suggested the effect on enhancement of the flexibility by grafting the longer polyHEA chains on the
250
CNFs films.
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d
253
4. Conclusions
In this study, we prepared the CNF macroinitiator film for ATRP by introduction of the initiating
te
252
M
251
moieties on surface of the CNF films. The surface-initiated graft polymerization of HEA from the CNF
255
macroinitiator film by ATRP was performed to produce the CNF-g-polyHEA films. The IR, XRD, and
256
SEM measurements of the resulting films as well as the GPC analysis after separation of the graft chains
257
indicated the progress of the graft polymerization of HEA on surface of CNFs. Furthermore, the CNF-g-
258
polyHEA films showed more flexible nature than the original CNF film. Because the present films are
259
efficiently obtained by the surface-initiated ATRP, this approach has a potential to produce the CNF
260
composite materials with various synthetic polymers, which will be used as practical bio-based materials
261
in the future.
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References
265
Bochek A. M, Murav'ev, A. A., Novoselov, N. P., Zaborski, M., Zabivalova, N. M., Petrova, V. A.,
266
Vlasova, E. N., Volchek, B. Z., & Lavrent'ev V. K. (2012). Specific features of cellulose and chitin
267
dissolution in ionic liquids of varied structure and the structural organization of regenerated
268
polysaccharides. Russian Journal of Applied Chemistry, 85, 1718-1725.
269
Fan, Y., Saito, T., & Isogai, A. (2008). Chitin nanocrystals prepared by tempo-mediated oxidation of -
270
chitin. Biomacromolecules, 9, 192-198.
271
Feng, L., & Chen, Z.-I. (2008). Research progress on dissolution and functional modification of
272
cellulose in ionic liquids. Journal of Molecular Liquids, 142, 1-5.
273
Ifuku, S., & Saimoto, H. (2012). Chitin nanofibers: Preparations, modifications, and applications.
274
Nanoscale, 4, 3308-3318.
275
Ifuku, S., Iwasaki, M., Morimoto, M., & Saimoto, H. (2012). Graft polymerization of acrylic acid onto
276
chitin nanofiber to improve dispersibility in basic water. Carbohydrate Polymers, 90, 623-627.
277
Jaworska, M. M., Kozlecki, T., & Gorak, A. (2012). Review of the application of ionic liquids as
278
solvents for chitin. Journal of Polymer Engineering, 32, 67-69.
279
Kadokawa, J., Setoguchi, T., & Yamamoto, K. (2013). Preparation of highly flexible chitin nanofiber-
280
graft-poly(gamma-L-glutamic acid) network film. Polymer Bulletin, 70, 3279-3289.
281
Kadokawa, J., Takegawa, A., Mine, S., & Prasad, K. (2011). Preparation of chitin nanowhiskers using
282
an ionic liquid and their composite materials with poly(vinyl alcohol). Carbohydrate Polymers, 84,
283
1408-1412.
284
Kamigaito, M., Ando, T., & Sawamoto, M. (2001). Metal-Catalyzed Living Radical Polymerization.
285
Chemical Reviews, 101, 3689-3745.
Ac ce p
te
d
M
an
us
cr
ip t
264
13
Page 13 of 22
Kurita, K. (2006). Chitin and chitosan: Functional biopolymers from marine crustaceans. Marine
287
Biotechnology, 8, 203-226.
288
Liebert, T., & Heinze, T. (2008). Interaction of ionic liquids with polysaccharides. 5. Solvents and
289
reaction media for the modification of cellulose. BioResources, 3,576-601.
290
Matyjaszewski, K. (2012). Atom Transfer Radical Polymerization (ATRP): Current Status and Future
291
Perspectives, Macromolecules, 45, 4015-4039.
292
Mine, S., Izawa, H., Kaneko, Y., & Kadokawa, J. (2009). Acetylation of a-chitin in ionic liquids.
293
Carbohydrate Research, 344, 2263-2265.
294
Muzzarelli, R.A.A. (2011). Biomedical exploitation of chitin and chitosan via mechano-chemical
295
disassembly, electrospinning, dissolution in imidazolium ionic liquids, and supercritical drying. Marine
296
Drugs, 9, 1510-1533.
297
Muzzarelli, R.A.A. (2012). Nanochitins and nanochitosans, paving the way to eco-friendly and energy-
298
saving exploitation of marine resources. Polymer Science: A Comprehensive Reference, 10, 153-164.
299
Pillai, C. K. S., Paul, W., & Sharma, C. P. (2009). Chitin and chitosan polymers: Chemistry, solubility
300
and fiber formation. Progress in Polymer Science, 34, 641-678.
301
Pinkert, A., Marsh, K. N., Pang, S., & Staiger, M. P. (2009). Ionic liquids and their interaction with
302
cellulose. Chemical Reviews, 109, 6712-6828.
303
Poirier, M., & Charlet, G. (2002). Chitin fractionation and characterization in N,N-
304
dimethylacetamide/lithium chloride solvent system. Carbohydrate Polymers, 50, 363-370.
305
Prasad, K., Murakami, M., Kaneko, Y., Takada, A., Nakamura, Y., & Kadokawa, J. (2009). Weak gel of
306
chitin with an ionic liquid, 1-allyl-3-methylimidazolium bromide. International Journal of Biological
307
Macromolecules, 45, 221-225.
Ac ce p
te
d
M
an
us
cr
ip t
286
14
Page 14 of 22
Qin Y., Lu X., Sun N., & Rogers R. D. (2010). Dissolution or extraction of crustacean shells using ionic
309
liquids to obtain high molecular weight purified chitin and direct production of chitin films and fibers.
310
Green Chemistry, 12, 968-971
311
Rinaudo, M. (2006). Chitin and chitosan: Properties and applications. Progress in Polymer Science, 31,
312
603-632.
313
Schuerch, C. (1986). Polysaccharides. In H. F. Mark, N. Bilkales, & C. G. Overberger (Eds.),
314
Encyclopedia of polymer science and engineering (2nd edition, 13, pp. 87–162). New York: John Wiley
315
& Sons.
316
Setoguchi, T., Yamamoto, K., & Kadokawa, J. (2012). Preparation of chitin nanofiber-graft-poly(L-
317
lactide-co-ε-caprolactone) films by surface-initiated ring-opening graft copolymerization. Polymer, 53,
318
4977-4982.
319
Swatloski, R. P., Spear, S. K., Holbrey, J. D., & Rogers, R. D. (2002). Dissolution of cellulose with
320
ionic liquids. Journal of the American Chemical Society, 124, 4974-4975.
321
Tajiri, R., Setoguchi, T., Wakizono, S.,Yamamoto, K., & Kadokawa, J. (2013). Preparation of Self-
322
assembled Chitin Nanofibers by Regeneration from Ion Gels Using Calcium Halide･dihydrate/methanol
323
Solutions. Journal of Biobased Materials and Bioenergy, 7, 655-659.
324
Wang, W. T., Zhu, J., Wang, X. L., Huang, Y, & Wang, Y. Z. (2010). Dissolution behavior of chitin in
325
ionic liquids. Journal of Macromolecular Science, Part B: Physics, 49, 528-541.
326
Wu, Y., Sasaki, T., Irie, S., & Sakurai, K. (2008). A novel biomass-ionic liquid platform for the
327
utilization of native chitin. Polymer, 49, 2321-2327.
328
Yamazaki, S., Takegawa, A., Kaneko, Y., Kadokawa, J., Yamagata, M., & Ishikawa, M. (2009). An
329
acidic cellulose-chitin hybrid gel as novel electrolyte for an electric double Layer capacitor.
330
Electrochemistry Communications, 11, 68-70.
Ac ce p
te
d
M
an
us
cr
ip t
308
15
Page 15 of 22
Yamamoto, K., Yoshida, S., Mine, S., & Kadokawa, J. (2013). Synthesis of chitin-graft-polystyrene via
332
atom transfer radical polymerization initiated from chitin macroinitiator. Polymer Chemistry, 4, 3384-
333
3389.
334
Zakrzewska, M. E., Lukasik, E. B., & Lukasik, R. B. (2010). Solubility of carbohydrates in ionic liquids.
335
Energy Fuels, 24, 737-745.
336
Zeng, J. B., He, Y. S., Li, S. L., & Wang, Y. Z. (2012). Chitin whiskers: A overview.
337
Biomacromolecules, 13, 1-11.
338
Zhao, D., Fei, Z., Geldbach, T. J., Scopelliti, R., Laurenczy, G., & Dyson, P. J. (2005) Allyl-
339
functionalised ionic liquids: synthesis, characterisation, and reactivity. Helvetica Chimica Acta, 88, 665-
340
675.
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Figure and Scheme Captions
344
Fig. 1. Procedures for preparations of (a) CNF film through chitin ion gel with AMIMBr, (b) chitin
345
macroinitiator film, and (c) CNF-graft-polyHEA film via surface-initiated ATRP.
346
Fig. 2. IR spectra of (a) CNF film, (b) CNF macroinitiator film (30 equiv. of α-bromoisobutyryl
347
bromide), and (c) CNF-g-polyHEA film (entry 4, Table 1).
348
Fig. 3. XRD patterns of (a) CNF film, (b) CNF macroinitiator film and, (c) CNF-g-polyHEA film (entry
349
4, Table 1).
350
Fig. 4. GPC traces of poly(methyl acrylate)s from CNF-g-polyHEA films of (a) entry 3 and (b) entry 4
351
in Table 1.
352
Fig. 5. SEM images of (a) chitin macroinitiator film (30 equiv. of α-bromoisobutyryl bromide) and (b)–
353
(g) CNF-g-polyHEA films of entries 1-6 in Table 1, respectively.
354
Fig. 6. Stress-strain curves of (a) chitin macroinitiator film (30 equiv. of α-bromoisobutyryl bromide)
355
and (b)-(e) CNF-g-polyHEA films for of entries 1-4 in Table 1, respectively; the fracture strain and
356
stress values are shown in parentheses.
357
Scheme 1. Alkaline hydrolysis of grafted polyHEA on CNF film and methylation of carboxylic acid
358
groups.
cr
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Page 17 of 22
359
Table 1
360
Surface-initiated ATRP of HEA from CNF macroinitiator films with different DSs under conditions of
361
different reaction timesa
362
Entry
363
1
364
Yield / mg
0.61
3
52
2
0.61
6
65
365
3
0.61
12
161
366
4
0.61
24
216
367
5
0.20
24
368
6
0.30
24
Conversion of HEA / % b
ip t
Time / h
6
an
us
cr
13 51 71
68
68
M
DS
71
143
369
a
370
Reaction conditions; [Initiating site]/[HEA]/[Bpy]/[CuBr] =1 / 20 / 4 / 2, Temp.; 60 °C
371
b
373
d
te
Estimated by the weight increases of the products from the CNF macroinitiator film.
Ac ce p
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A feed amount of chitin macroinitiator film was 34 mg (0.17 mmol, DS; 0.20, 0.30, and 0.61).
18
Page 18 of 22
373 374 375 376
ip t
377 378
cr
379 380
us
381 382
an
383 384
387
M
385
388
Fig. 1. Procedures for preparations of (a) CNF film through chitin ion gel with AMIMBr, (b) chitin
389
macroinitiator film, and (c) CNF-graft-polyHEA film via surface-initiated ATRP.
391 392 393
te
Ac ce p
390
d
386
394 395 396 397
Fig. 2. IR spectra of (a) CNF film, (b) CNF macroinitiator film (30 equiv. of α-bromoisobutyryl
398
bromide), and (c) CNF-g-polyHEA film (entry 4, Table 1). 19
Page 19 of 22
399 400 401
ip t
402
cr
403
us
404 405
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Fig. 3. XRD patterns of (a) CNF film, (b) CNF macroinitiator film and, (c) CNF-g-polyHEA film (entry
408
4, Table 1).
d
409
M
407
te
410
Ac ce p
411 412 413 414 415 416 417 418 419 420
Fig. 4. GPC traces of poly(methyl acrylate)s from CNF-g-polyHEA films of (a) entry 3 and (b) entry 4
421
in Table 1.
422 20
Page 20 of 22
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423
Fig. 5. SEM images of (a) chitin macroinitiator film (30 equiv. of α-bromoisobutyryl bromide) and (b)–
425
(g) CNF-g-polyHEA films of entries 1-6 in Table 1, respectively.
426 427 428 429
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424
430 431 432 433 434 435 21
Page 21 of 22
436 437 438
ip t
439
cr
440
us
441 442
an
443
Fig. 6. Stress-strain curves of (a) chitin macroinitiator film (30 equiv. of α-bromoisobutyryl bromide)
445
and (b)-(e) CNF-g-polyHEA films for of entries 1-4 in Table 1, respectively; the fracture strain and
446
stress values are shown in parentheses.
te
447
d
M
444
Ac ce p
448 449 450 451 452 453 454
Scheme 1. Alkaline hydrolysis of grafted polyHEA on CNF film and methylation of carboxylic acid
455
groups. 22
Page 22 of 22
