Carbohydrate Polymers 54 (2003) 343–351

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Studies on graft copolymerization of chitosan with synthetic monomers

K.V. Harish Prashanth, R.N. Tharanathan*
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570 013, India
Received 5 February 2003; revised 27 June 2003; accepted 30 June 2003

Abstract
Graft copolymerization of acrylonitrile and methylmethacrylate onto chitosan using potassium persulfate as an initiator was studied.
Evidence for graft-copolymerization was obtained by infrared spectroscopy and CP-MAS 13C-NMR data. The appearance of nitrile (– CxN)
at 2244 cm21 for chitosan-graft-polyacrylonitrile (C-g-PAN) and carbonyl (– CyO) at 1730 cm21 for chitosan-graft-polymethylmethacry-
late (C-g-PMMA) confirmed graft-copolymerization. CP-MAS 13C-NMR showed the appearance of a signal at 33 ppm assigned to the N –
CH group. With varying monomer concentration (40 – 180 mM), the percentage degree of substitution varied from 2 to 50. Maximum
grafting efficiency was obtained with 120 mM acrylonitrile and 0.74 mM potassium persulfate at 65 8C for 2 h under nitrogen atmosphere for
1% chitosan solution and for C-g-PMMA 140 mM methylmethacrylate at 75 8C gave maximum substitution. X-ray diffraction showed
changes in crystallinity pattern. Slightly different mechanisms in side chain substitution for these two copolymers were envisaged. DSC
thermogram showed a decomposition peak for C-g-PAN at around 255 8C and a melting peak for C-g-PMMA at around 400 8C. C-g-PMMA
could be thermopressed into films. Residual monomers were not found by HPLC in graft copolymers stored even for longer periods.
q 2003 Elsevier Ltd. All rights reserved.
Keywords: Chitosan; Polyacrylonitrile; Polymethymethacrylate; Copolymer; Grafting

1. Introduction tissue-anticalcification properties have been studied

(Shanthi & Panduranga Rao, 2001). However, modification
Chitin is one of the most abundant biopolymers found in of chitosan via grafting of vinyl monomers is one of the
the shells of crustacea, e.g. crab and shrimp, and cuticles of most effective methods to incorporate desirable properties
insects and also in the cell walls of some fungi and into chitosan without sacrificing its biodegradable nature.
microorganisms. Chitin consists of N-acetyl-D -glucosamine In the present work, the results of a study on grafting of
repeating units, linked by b-(1 ! 4) bonds. Due to its acrylonitrile (AN) and methylmethacrylate (MMA) onto
inherent intractability, it is often converted to chitosan, 2- chitosan in terms of different monomer concentrations and
amino-2-deoxy-(1 ! 4)-b-D -glucan by hot alkali treatment. using potassium persulfate as a free radical initiator, under
Chemical modification of chitosan is an important topic for innert conditions are described. Further, the effect of
the production of bifunctional materials. Work on graft grafting on the thermal behaviour and structural aspects of
copolymer synthesis based on chitin and chitosan and their chitosan have been examined. Attempts were made to
application has been reported. Chitosan-g-poly(glycidyl- prepare chitosan membranes out of graft copolymers and
methacrylate) copolymer was used for immobilization of also check for any residual monomers using HPLC.
urease (Chellapandian & Krishanan, 1998) and other
modifications such as the grafting of methylmethacrylate
onto chitin initiated by tributylborane (Kojima, Yoshikuni, 2. Experimental
& Suzuki, 1979), poly(3-hydroxyalkonate)-chitosan
conjugates (Yalpani, Marchessault, Morin, & Monosteries, 2.1. Preparation of graft copolymers of chitosan
1991), chitosan modified poly(glycidylmethacrylate –buty-
lacrylate) and chitosan grafted bovine pericardial Chitosan, as a solution in dilute acetic acid was graft
copolymerized under homogeneous conditions by the
* Corresponding author. Tel.: þ 91-821-514876; fax: þ 91-821-517233. method of Chellapandian and Krishanan (1998) with the
E-mail address: [email protected] (R.N. Tharanathan). following modifications.
0144-8617/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0144-8617(03)00191-7
344 K.V.H. Prashanth, R.N. Tharanathan / Carbohydrate Polymers 54 (2003) 343–351

2.1.1. Chitosan graft-polyacrylonitrile 2.5. High performance liquid chromatography

Chitosan solution (1.0%) was placed in a flat bottomed
three necked flask. Throughout the reaction time, nitrogen Separation of monomers was performed on a reverse
was purged at a constant temperature (60 8C) through the phase Shim-Pack C18 (ODS) column (Shimadzu, Japan, 15
stirred solution. Freshly prepared potassium persulfate cm £ 4.6 mm i.d.) in a Shimadzu HPLC system consisting
solution (0.74 mM) was added followed by dropwise of an LC 6A pump equipped with UV-Vis spectrophoto-
addition (40 – 140 mM) of acrylonitrile. The reaction was metric detector. Residual monomers of graft copolymers
conducted for 2 h with stirring continued for another 15 min were analyzed by HPLC according to the procedure of
at room temperature. Then the reaction product was Saroja, Gowda and Tharanathan (2000), which involves
precipitated out with 2 vol of isopropanol, filtered, washed separation using 0.05 M KH2PO4, pH 5.5 at 1 ml min21 at
with distilled water, to remove the unmodified soluble low 30 8C. A 10 ml volume of standards AN (acrylonitrile), AM
molecular weight chitosan (Harish Prashanth et al.— (acrylamide), AC (acrylic acid) and MMA (methylmetha-
unpublished data) and dried. It was again subjected to crylate, diluted in glass distilled water) were injected onto
Soxhlet refluxing for 8 – 12 h using N,N-dimethylformamide the column and detected at 220 nm. Their minimum
(DMF) to solubilize and remove the homopolymer, if any detection limit varied from 1 to 10 ng.
and finally lyophilized.
2.6. Differential scanning calorimetry
2.1.2. Chitosan graft-polymethylmethacrylate
To chitosan solution (1.0%) were added 0.74 mM The samples were analyzed using a Rheometric
potassium persulfate and varying concentration of methyl- Scientific (UK) equipment supported by thermal software
methacrylate (40 – 160 mM) and stirred continuously at S42 on a Compaq computer, which is precalibrated. The
75 8C. The reaction was continued as previously described equipment was provided with an autocool system.
for C-g-PAN. The copolymer was extracted with acetone Accurately weighed (5 mg) material was placed in an
and thoroughly washed with distilled water and lyophilized. aluminium cup and hermetically sealed. Empty sealed cup
The gain in weight of the copolymer is measured as percent was used as reference and runs were performed in
grafting ¼ Wg 2 W0 £ 100=W0 ; where Wg is the weight in duplicates. Analyses were done under continuous flow
grams of the grafted chitosan and W0 is the weight of native of dry nitrogen gas (10 ml min21) at a heating rate of
chitosan (Zhang & Chen, 2001). 20 8C min21 from 5 to 500 8C.

2.2. Infrared spectroscopy 2.7. X-ray diffractometry

IR spectra were recorded in KBr discs on a Perkin Powder X-ray diffraction patterns of chitosan and
Elmer Spectrum 2000 FTIR spectrometer under dry air at copolymers were obtained by using a EG-7G solid
room temperature. Approximately 6 mg of dried sample state germanium liquid nitrogen cooled detector Sintag
was blended with 200 mg of potassium bromide (IR XDS-2000 instrument equipped with a u – u goniometer,
grade) and about 40 mg of the mixture was used to under the following operating conditions; 30 kV and
prepare a pellet. 25 mA with Cu Ka1-radiation at l 1.54184 Å. The
relative intensity was recorded in the scattering range
13
2.3. Solid state CP-MAS C NMR ð2uÞ of 4– 608.

Approximately 300 mg of freeze dried sample were 2.8. Membrane preparation

inserted into a 7 mm ceramic rotor on a Bruker DSX 300
spectrometer. The spectra were recorded at 75.5 MHz. The The purified chitosan graft copolymers (1 g) were heat
crosspolarization pulse sequence was utilized for all pressed in between Teflon sheets at 115 ^ 2 8C at a
samples, which were spun at the magic angle at 4 kHz. A pressure of 150 kg cm22 for 20 min using polymer film
contact time of 1 ms and a pulse repetition time of 5 s were making machine (Techno Search (Pvt.) Ltd, India), which
used and more than 2500 scans were accumulated for each has both temperature/pressure control and autocooling
sample. systems.

2.4. Scanning electron microscopy

3. Results and discussion
The dry sample, spread on a double sided conducting
adhesive tape, pasted on a metallic stub, was coated (100 m) Polymer grafting reactions provide the potential for
with gold in a sputter coating unit for 2 min and observed in significantly altering the physical and mechanical proper-
a LEO-435-VP (LEO Electron Microscopy Ltd, Cambridge, ties of the starting materials. Some polymer grafts have
UK) electron microscope at 20 kV. tendency to form thin membranous films, which may be
 K.V.H. Prashanth, R.N. Tharanathan / Carbohydrate Polymers 54 (2003) 343–351 345

Fig. 1. Schematic representation of graft-copolymerization of chitosan.

useful for packaging applications. Polymer grafting is investigation, potassium persulfate was used as a free
usually carried out by radical initiation, which is done radical initiator to induce grafting.
either by photo induced free radical or by using free radical
initiators. The main disadvantage in the former is the 3.1. Characterization of the graft copolymers
formation of homopolymers, because the UV light
generates directly free radicals on vinyl monomers and From Table 1, it is obvious that the addition of side
thereby increasing homopolymerization, whereas with chains of PAN and PMMA to chitosan main chain (as
radical initiators the free radicals are produced on specific shown in Fig. 1) leads to increase in weight due to
sites, which also increases the grafting efficiency. The copolymerization. The percent grafting was considerably
reactive C-2 amino group in chitosan is important in more with C-g-PMMA than with C-g-PAN because of bulky
several of the structural modifications targeted because the methyl groups of PMMA in the former.
deprotonated amino group acts as a powerful nucleophile
ðpKa ø 6:3Þ readily reacting with electrophilic reagents 3.1.1. Solubility
(Chen, Kumar, Harris, Smith, & Payne, 2000). Even in free The solubility characteristic of the material is another
radical initiated copolymerization, NH2 groups of chitosan criterion for modification, since chitosan is soluble in water
involve in macroradical formation (Fig. 1). In the present in the presence of Hþ ions (acetic acid, lactic acid or HCl),
Table 1
Different monomer concentrations, their percent grafting and decomposition/melting temperatures

Acrylonitrile Decomposition peak Percent Methylmethacrylate Melting peak Percent

concentration (mM) temperature (8C)a grafting (%) concentration (mM) Temperature (8C)a grafting (%)

40 255.02 ^ 0.1 130 40 281.07 ^ 0.2 161

60 259.65 ^ 0.3 145 100 281.89 ^ 0.1 165
100 ND 142 120 391.49 ^ 0.3 273
120 302.10 ^ 0.2 249 140 393.81 ^ 0.5 276
140 255.00 ^ 0.3 170 180 390.01 ^ 0.1 271

ND, not determined.

a
The values presented are average of two separate runs with standard deviation.
346 K.V.H. Prashanth, R.N. Tharanathan / Carbohydrate Polymers 54 (2003) 343–351

whereas the grafted chitosans were insoluble even in and for C-g-PMMA with 140 mM methymethacrylate at
solvents like DMF and acetone, which are known to 75 8C.
solublize PAN and PMMA homopolymers, respectively.
In addition to the formation of graft copolymers, cross- 3.1.2. Infrared spectroscopy
linking between the chains of chitosan may also take place. Infrared spectroscopy is the best tool to confirm the
This was evident by the reaction between chitosan and the grafting reaction. The appearance in C-g-PAN of-CxN
initiator, in the absence of monomers, giving a product with (nitrile) absorption at around 2244 cm21 and the CH2
reduced solubility (unpublished data). Variations in initiator deformation vibration at around 1453 cm21 (Fig. 2a) and in
concentration, reaction temperature and time did not show C-g-PMMA of 1731 cm21 due to – CyO (carbonyl)
much effect on the yield and grafting efficiency, as absorption and methyl and methylene asymetric stretching
compared to that observed in changing monomer concen- vibrations at 2995 and 2951 cm21, respectively, (Fig. 2b)
tration (Table 1). Maximum grafting efficiency was confirmed grafting. With increase in percent grafting, the
obtained for C-g-PAN with 120 mM acrylonitrile at 65 8C, intensity of these absorption also increased (Fig. 3a and b).

Fig. 2. IR spectra of (a) C-g-PAN, (b) C-g-PMMA.

 K.V.H. Prashanth, R.N. Tharanathan / Carbohydrate Polymers 54 (2003) 343–351 347

Fig. 3. IR spectra of graft-copolymers with increase in percent grafting (a) C-g-PAN, (b) C-g-PMMA.

The absence of a strong (sharp) absorption around was shifted to a slightly higher frequency with a
3500 cm21 indicated the absence of free – OH groups of sharp band (1120 cm21), probably due to change in
chitosan, which probably are involved in some hydrogen crystallinity.
bonding. The region above 3000 cm21 was centered at
3396 cm21 for chitosan, which was shifted to a higher 3.1.3. CP-MAS 13C-NMR
frequency for graft copolymers, indicating an increase in Solid state NMR data indicated changes in the local
the ordered structure. The amide I and amide II order structure (Fig. 4). The line widths of chitosan in
absorptions were seen around 1630 and 1530 cm21, and graft copolymer were relatively broadened because of
with increase in percent grafting a progresssive weakening the introduction of side chains. The magnitude of –N – CH
of amide II band was observed. The – C – O –C – stretching signal (Capitani, Angelis, Crescenzi, Masci, & Segre,
absorption for chitosan was seen at 1080 cm21, which 2001; Rinaudo, Desbrieres, Le-Dung, Binh, & Dong,
348 K.V.H. Prashanth, R.N. Tharanathan / Carbohydrate Polymers 54 (2003) 343–351

3.1.4. Differential scanning calorimetry

In Fig. 5, thermograms of chitosan, C-g-PAN and C-g-
PMMA show the endotherms with a peak temperature at
around 150 8C, ascribable to the water holding capacity,
which is possible through the – OH and unsubstituted, free
– NH2 groups of chitosan (Sato et al., 1997). Additionally,
chitosan exhibits an exothermic peak temperature at 302 8C
(Fig. 5a). From our earlier observations it is likely that a
correlation between the decomposition peak temperature
and degree of polymerization of chitosan is possible (Harish
Prashanth, Kittur, & Tharanathan, 2002; Kittur, Harish
Prashanth, Udaya Sankar, & Tharanathan, 2002). In graft
copolymers, the decomposition peak temperature come
down drastically from around 300 to 255– 251 8C (Fig. 5b
and c) because of free radical induced depolymerization of
chitosan (Hsu, Don, & Chiu, 2002; Tharanathan & Harish
Prashanth, 2001). Prior to exothermic peak initiation a small
drift in the heat flow (indicated by an arrow in Fig. 5b and c)
was noticed which may correspond to the amorphous
chitosan region pendented with bulky side chains grafts. In
C-g-PAN, with increase in decomposition peak temperature

13
Fig. 4. CP-MAS C NMR spectra of (a) chitosan, (b) C-g-PAN, (c) C-g-
PMMA.

2001) at 33 ppm increased with increase in grafting in

C-g-PAN. Broad multiple splitting of C1 centered at
102.8 ppm, C3/C5 at 76.8 ppm and C2/C6 at 60.76 were
observed. The spectrum of C-g-PMMA showed – CyO
and CH3 signals at 180.5 and 18.9 ppm, respectively,
along with signals at 106 –100 and 77.8 ppm due to ring
carbons. Nevertheless a dominance of PMMA intensity
was ascribable to extended polymeric side chains. Based
on spectral peak intensities we envisage slightly different
mechanisms for these two copolymers. AN may undergo
regular substitution at most of the amino radicals of
chitosan, resulting in a frequent short PAN sidechain
appendages on the chitosan backbone; whereas in the case
of MMA the radical copolymerization may be taking
place at a few amino radicals, with simultaneous
elongation resulting in very long sidechains could be
due to difference in reactive ratios of AN and MMA
monomers. Fig. 5. DSC thermograms of (a) chitosan, (b) C-g-PAN and (c) C-g-PMMA.
 K.V.H. Prashanth, R.N. Tharanathan / Carbohydrate Polymers 54 (2003) 343–351 349

the percent grafting showed a slight decreasing trend, viz. 3.1.6. Scanning electron microscopy
145% C-g-PAN gave a decomposition peak temperature of Graft copolymerization considerably modifies chitosan
259.6 8C, whereas 170% graft copolymer gave 255 8C morphology, and also its physical, chemical and biode-
(Table 1). This may be attributed to the coinciding gradable characteristics, which varies with respect to the
decomposition peak temperatures of PAN and chitosan. nature of the synthetic side chains incorporated. SEM
The C-g-PAN indeed showed only decomposition peak but observations of native chitosan revealed its fibrous as well
not a melting peak, contrary to C-g-PMMA, which showed a as flaky nature (Fig. 7a), wherein innumerable thin fibre
the melting peak around 280 –400 8C. As the percent strands deposited over the surface are visible. The fibrous
grafting increased an increase in the melting peak nature of chitosan was totally modified in the grafted
temperature was observed, thus showing a good correlation materials, wherein distinct morphological differences were
ðr ¼ 0:996Þ: discernible in their surface topography. C-g-PAN showed a
soft porous structure (Fig. 7b), and C-g-PMMA showed
3.1.5. X-ray diffraction the appearance of clustered irregular beads (Fig. 7c, Don,
The powder X-ray diffractograms of the graft copoly- Hsu, & Chiu, 2001).
mers showed distinct crystalline peaks compared to the
native chitosan. The latter showed two major peaks at
around 10 and 208 due to 020 and 110 reflections, 3.2. Residual monomer detection
respectively (figure not shown). The highly grafted C-g-
PAN showed much variations in the peaks apart from the The residual monomers if any, in the graft copolymers
weak absorption at 21.048 and a strong absorption at were extracted with water and subjected to an isocratic
16.728, which were due to PAN crystalline regions (Fig. 6). reverse phase-HPLC separation (Saroja et al., 2000). No
On the otherhand, the C-g-PMMA showed a single broad detectable monomers were revealed even in C-g-PAN,
absorption at 13.58, appeared to be due to extensive side although acrylonitrile is known for degradation if kept for
chain coverage of PMMA, because the latter lacks long time, to acrylamide and acrylic acid (Zhang & Chen,
complete stereoregularity due to its bulky methyl side 2001). Even a sample of C-g-PAN stored for 6 months did
groups (Ren & Tokura, 1994). not show any residual monomers.

Fig. 6. X-ray diffraction patterns of (a) C-g-PAN, (b) C-g-PMMA.

350 K.V.H. Prashanth, R.N. Tharanathan / Carbohydrate Polymers 54 (2003) 343–351

Fig. 7. SEM photographs—low and high resolution of (a) chitosan, (b) C-g-PAN, (c) C-g-PMMA.

3.3. Copolymer membrane CP-MAS 13C NMR. Thermograms of C-g-PMMA showed

an increased peak temperature with increase in percent
The dry solid copolymers on heat pressing between grafting. X-ray diffraction revealed changes in crystallinity
Teflon sheets gave semi-transparent, pale brown coloured of grafted copolymers. In scanning electron micrographs
thin films with C-g-PMMA, whereas the C-g-PAN film was native chitosan appeared fibrous, whereas C-g-PAN and C-
too brittle for handling. The operating conditions to get C-g- g-PMMA appeared flaky and beads-like, respectively. No
PMMA film were 115 ^ 2 8C for 20 min. The brittleness of residual monomers were found in the graft copolymers,
the films can be overcome by incorporating plasticizers, and even after storage for long periods. The graft copolymers
such films may possibly be used as biodegradable packaging could be thermopressed to thin membranous films.
materials.

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