Immobilization of quaternized polymers on bacterial cellulose by different grafting techniques

Article abstract of DOI:10.1039/c9nj02199j

Since the discovery of the extraordinary mechanical properties of pristine bacterial cellulose (BC) few decades ago, its potential applications as a textile material and leather-like material have been realized. Immobilization of suitable polymers on the surface of BC can provide two of the most desirable characteristics in the current scenario: improved mechanical strength and variable hydrophobicity. Here, we report the immobilization of suitable polymers on BC surfaces in convenient ways. Different polymers (phosphonium-based or pyridinium-based), different grafting techniques (graft to or direct graft) and different surfaces (bacterial cellulose (BC) or bacterial cellulose-cotton composites (BCC)) were particularly used for comparison. These polymer-functionalized surfaces were characterized by standard instrumentation techniques. Significant improvements in mechanical properties and hydrophobicity were observed for some of the materials after functionalization with the polymers.

Full text of DOI:10.1039/c9nj02199j

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Immobilization Of Quaternized Polymers on Bacterial Cellulose by Different
Grafting Techniques†
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P. Ramar,a,b Sourita Jana,a,c Sandipan Chatterjee,d S. N. Jaisankar,*a and Debasis Samanta*a,b
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aPolymer Science & Technology Department, CSIR-Central Leather Research Institute (CSIR-CLRI), Adyar, Chennai-600020, India. Fax: 91-
44-24911589, Tel: 91 -44 24422059 E-mail: debasis@clri.res.in (DS), snjsankar@clri.res.in (SNJ)
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bAcademy of Scientific and Innovative Research (AcSIR), India
cUniversity of Madras, Chennai-600005, India
dRCED, CLRI, Kolkata, India
10 Key words: surface, polymer immobilization, graft to, direct grafting, Bacterial cellulose,
Abstract
Following the discovery of extraordinary mechanical properties of pristine bacterial cellulose (BC)
in a couple of decades ago, its potential applications as textile material and leather like material was
realized. Immobilization of suitable polymers to the surface of BC can provide two of the most desirable
15 characteristics in current scenario; improved mechanical strength and variable hydrophobicity. Here we
report the immobilization of suitable polymers to BC surfaces in convenient ways. Different polymers
(phosphonium based or pyridinium based), different grafting techniques (graft to or direct graft) and
different surfaces (bacterial cellulose (BC) or bacterial cellulose-cotton composites (BCC)) were
particularly used for comparison. These polymer functionalized surfaces were characterized by standard
20 instrumentation techniques. Significant improvements in mechanical properties and hydrophobicity
were observed for some of the materials after the functionalization with polymers.
1. Introduction
Unlike the most common plant-cellulose, bacterial
35 also necessary to modify the BC surfaces to improve certain
properties, like hydrophilicity, texture, mechanical properties etc.
The presence of robust β-1,4-glycosidic linkages (C–O–C) between
anhydro glucose units with triclinic (Iα) form of crystalline
structure and large number of active hydroxyl groups, facilitates
40 to making of different chemically functionalized BC materials.10
The bacterial cellulose surfaces can be modified by composites
formation with nanoparticles11 and polymers12 etc. Ul-Islam et al.
has recently reviewed the preparation strategies and applications
of BC composites with polymeric, non-polymeric compounds and
45 nanoparticles.13 Enhancement of the properties of BC materials,
by making composites or conjugates with foreign material on the
surfaces were reported by several researchers.14-16 Tercjak et al.
25 cellulose (BC) is produced by fermentation process induced by
bacteria and fungus. It has usually high degree of natural purity
without lignin and hemicellulose. Due to their fibrous nature, it
has high tensile strength and crystallinity, biodegradability and
biocompatibility.1 The BC and modified BC materials find different
30 applications, particularly as food packaging materials, transparent
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surfaces,2 supercapacitors,3,
metal ions adsorbent surfaces,
chemosensory materials,5 controlled drug releasing gels,6 wound
healing materials,7 electrical and medical devices8, 9 etc. For many
of the applications, chemical modification of BC is necessary. It is
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investigated the improvement of the mechanical properties of
bacterial cellulose by in situ formation of the bio composites with
block copolymers like PEO ‑ b ‑ PPO ‑ b ‑ PEO.12 Biocompatible
polymers like polyethylene glycol,17 or acrylate polymers18 and
(XRD) after and before grafting reaction. The change in
hydrophobicity was studied by water contact angle
measurements. This is particularly important since this type of BC-
45 polymer conjugates can be useful as leather-like materials (ESI
video), which require better protection against water in different
climatic conditions.
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conjugated polymers4, were used for the functionalization of
cellulosic fibres. However, to the best of our knowledge
zwitterionic polymers remained unexplored for the modification
of bacterial cellulose, although they have interesting properties
like pH-responsive hydrophilicity.20 In this paper, we report
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2. Results and Discussions
2.1 Synthesis and characterization of monomers and
50 quaternized P4VP polymers
10 covalent functionalization of bacterial cellulose by both cationic
and zwitterionic polymers.
First, we prepared compound 1(1,4-dibromo-2-
(bromomethyl)-5-methylbenzene) and
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(1,4-dibromo-2,5-
In this context, method of functionalization is also important for
better convenience and reliability. In most cases, “graft from”
technique or “graft through” technique21 has been chosen to
15 grow polymers from the surface of the cellulose materials, where
initiator or catalysts are already immobilized. For example, Roy et.
al. described the growing of methacrylate polymeric chains from
cellulosic fibres by using RAFT polymerization.22 In this case, S-
methoxycarbonylphenylmethyl dithiobenzoate was immobilized
20 first on cellulosic fibres, which was then used in presence of
monomer, free chain transfer agent and AIBN to grow the
polymeric chains. Atom transfer radical polymerization was used
by Mortis et. al. after the deposition of initiator to the cellulosic
surface.23 The graft from methods require prior immobilization of
25 initiator to the surface followed by the growing of polymeric
chains from the surface. The alternative approaches are “graft to”
or “direct” immobilization method, where polymer is first formed
in solution followed by immobilization to surface with surface-
active groups.24
bis(bromomethyl)benzene) from 2,5-dibromo-p-xylene by
reaction with N-bromosuccinimide using a modified literature
55 procedure25 (ESI scheme S1 and S2). The Nuclear Magnetic
Resonance spectroscopy (NMR) data related to compound 1 and
2 were provided in ESI figure S1-S6. The compound 3 ((2,5-
dibromo-4-(bromomethyl)benzyl)triphenyl
phosphonium
bromide) was synthesized26 from compound 2 (Scheme 1) by
60 reaction with triphenylphosphine and the formation of the
compound was confirmed by 1H, 13C and 31P NMR analysis (ESI
1
figure S7-S8). In H NMR, characteristic peaks were observed at
5.81 and 4.45 ppm due to benzylic protons attached to bromide
and phosphonium group respectively. The aromatic protons were
65 observed at 7.82-7.64 ppm. In 31P NMR (figure 2 (B)) the single
peak was observed at 24.66 ppm corresponds to the phosphorous
nucleus of phosphonium group. Initially, as depicted in scheme 1,
we performed a model quaternization reaction to prepare
quaternized poly-4-vinyl pyridinium polymer (compound 4,
70 QP4VP-Bz) by reaction of poly-4-vinyl pyridine with 1.1 equivalent
of compound 1 (1,4-dibromo-2-(bromomethyl)-5-methylbenzene)
The spectral data of compound 4 confirmed the success of
quaternization (in ESI figure S10). Then we prepared quaternized
polymer containing phosphonium group and trimethoxysilane
75 group (Compound 5, QP4VP-PBz), by reaction of P4VP with 3-
iodopropyl trimethoxysilane followed by reaction with compound
3 (Scheme 1). The unreacted compound 3 was removed by
washing with chloroform repeatedly. The formation of the
compound was confirmed by NMR and FT-IR spectroscopy
80 (described below). This compound 5 is useful for direct grafting to
BC surfaces (vide infra).
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In this work, we demonstrated that while pyridinium
polymers can be conveniently immobilized by “graft to”
technique, for phosphonium polymers direct grafting method can
be used. Various thermal, mechanical and surface properties were
studied to understand the effect of binding of those polymers to
35 bacterial cellulose, which may facilitate the cross linking among
fibres leading to improvement in different properties. We
analysed the chemical structures by Fourier Transform Infra-red
spectroscopy (FTIR) and Nuclear Magnetic Resonance
spectroscopy (NMR), structural patterns by Small Angle X-Ray
40 Scattering spectroscopy (SAXS), Atomic Force Microscopy (AFM),
Scanning Electron Microscopy (SEM), X-Ray Diffraction analysis
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maximum amount of quaternization was obtained at around 85%
after 24 hours of refluxing.
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Figure 1. (A) FT-IR spectra of various pyridinium and phosphonium
polymers (B) ATR-IR spectra measured at different time interval of
quaternization reaction with alkylhalides. The percentage of
35 quaternization is mentioned on the respective spectra. The peak marked
by blue arrow represents the product peak while the peak marked red
arrow corresponds to reactant peak.
Scheme 1. Synthesis of different phosphonium and pyridinium polymers
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Further, the polymers were studied by 1H NMR
spectroscopy in dimethyl sulfoxide-d6 solvent. In 1H NMR
40 spectrum of compound 4 (QP4VP-Bz), (showed in ESI figure S10)
after quaternization the aromatic ortho and meta (to the N atom)
protons signal of pyridine/pyridinium ring shifted slightly from
6.21-6.58 ppm to 7.6-7.9 and 8.16-8.49 ppm to 8.74-9.23 ppm
respectively. The chemical shift value of phenyl ring aromatic C-H
45 protons was in the range of 7.8-8.3 ppm; benzyl -CH2 proton
signal (near to quaternary N) appeared at 5.6-6.3 ppm and CH3
proton signal appeared at 2.35-2.47 ppm. Similarly, in the final
quaternized polymer 5 (QP4VP-PBz) the 1H NMR signal of pyridine
ring C-H protons appeared in 7.12-7.53 ppm (ortho protons) and
50 8.74-9.28 ppm (meta protons) as shown in figure 2 (A). The
chemical shift was obtained at 7.45-7.64 ppm and 8.32-8.65 ppm
corresponds to the phenyl ring aromatic C-H protons of ortho and
meta to the C-CH2N+ respectively. The peaks at 7.67-8.12 ppm
value indicate the presence of all other aromatic C-H protons of
55 triphenyl phosphonium rings. In addition, the benzyl CH2 protons
(near to the quaternary N atom) signal was shifted from 4.56 ppm
to 5.02-5.41 ppm due to the decrease of the electron density
after quaternization and the chemical shift value of benzyl CH2
proton (near to the quaternary phosphorus atom) was also
60 shifted slightly after reaction from 5.67 ppm to 5.83-6.27 ppm.
Further in 31P NMR spectrum of QP4VP-PBz polymer, the
quaternary phosphorus nucleus signal was observed at 24.44 ppm
for immobilization to bacterial cellulose surfaces by “Graft to and “Direct”
method of immobilization
5
As observed in FT-IR spectra (figure 1 (A)), in both the
cases of quaternized polymers (QP4VP-Bz and QP4VP-PBz), the
new peak appeared at 1640 cm-1 corresponds to the C=N+ bond
stretching frequency of pyridinium moiety. The disappearance of
peak at 1595 cm-1 corresponds to C=N bond stretching frequency
10 of pyridine ring was also observed. Moreover, we estimated the
amount of quaternization in terms of percentage during the
synthesis of QP4VP-PBz polymer at different time intervals of
reaction (like 1h, 2h etc till 24 hours) by attenuated total
reflection-Infrared (ATR-IR) analysis27 from the relative peak
15 intensity values of two peaks at 1595 and 1640 cm-1 (showed in
figure 1 (B)). Initially, we added 10 mol% of 3-iodopropyl
trimethoxysilane to the solution of P4VP (Scheme 1) and refluxed
for 3 h. The small peak appeared at 1640 cm-1 (blue arrow, Fig.
1(B), red curve) next to the peak at 1595 cm-1 (red arrow, Fig.
20 1(B), red curve). Hence the corresponding amount of
quaternization was calculated to be around 6%, indicating that
around 6% of pyridine ring of P4VP was quaternized by 3-
iodopropyl trimethoxy silane moieties. Then we added compound
3 in the reaction mixture all at a time and the relative intensities
25 of those characteristic peaks at 1595 and 1640 cm-1 were
measured in 1 h interval, up to 24 h; to calculate the amount of
quaternization, mentioned in the figure 1 (B). Finally, the
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showing that phosphonium moieties were successfully 35 evidence of the disappearance of peak at (2Ɵ = 10°) and
incorporated with backbone of P4VP polymer during
quaternization (figure 2 (B)).
appearance of new broad peak at 28° (adjacent to 2Ɵ = 21°) with
different intensities for both cases of QP4VP-Bz and QP4VP-PBz.
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5 Figure 2. (A) 1H NMR spectra of QP4VP-PBz polymer and (B) 31P NMR
spectra of compound 3 (PBz) and compound 5 (QP4VP-PBz).
The thermal stabilities of polymers were studied by the
thermal analysis like thermogravimetric analysis (TGA) and
differential scanning calorimetry (DSC) techniques as seen in
10 figure 3 (A) and 3 (B). As observed from TGA thermogram, the
first decomposition temperature onset value for P4VP is 290°C,
for QP4VP-Bz, it is 250°C and for QP4VP-PBz, it is 220°C indicating
the lowering of thermal stability after quaternization.20 The glass
transition temperature of poly-4-vinyl pyridine polymer is 156-
15 160°C, as observed in DSC. After reaction, the quaternized
polymers QP4VP-Bz and QP4VP-PBz did not show any glass
transition in the similar temperature range, possibly due to
restrictions in polymer chain movement by electrostatic
interactions.
Figure 3. (A) TGA, (B) DSC, (C) UV-Vis absorption spectra and (D) XRD
40 pattern of poly-4-vinyl pyridine (P4VP), pyridinium (QP4VP-Bz) and
phosphonium (QP4VP-PBz) polymers.
2.2 “graft to” and “direct graft” method of immobilization of
polymers on BC and BCC surfaces and characterization
For “graft to” method (scheme 2 (A)) first the surfaces were
45 reacted with 10% (w/w) solution of 3-iodopropyltrimethoxysilane
in methanol under optimum condition to prepare BCTMS (3-
iodopropyl trimethoxy silane grafted bacterial cellulose) and
BCCTMS (3-iodopropyl trimethoxy silane grafted cotton-bacterial
cellulose) containing the self-assembled monolayer (SAM) of
50 reactive iodide. Then the SAM layer of BCTMS and BCCTMS
surfaces were reacted with poly-4-vinyl pyridine in methanol
under N2 atmosphere for 12 hours to facilitate the quaternization.
After completion of reaction, we obtained pale yellow colour
P4VP polymer functionalized QBC (poly-4-vinylpyridinium polymer
55 grafted bacterial cellulose) and QBCC (poly-4-vinylpyridinium
polymer grafted cotton-bacterial cellulose) surfaces. On the other
hand, “direct graft” method (Scheme 2(B)) is a one step process
on BC and BCC surfaces with pre-synthesised QP4VP-PBz
polymers containing surface anchoring groups to form a QBCP
60 (phosphonium salt-based poly-4-vinylpyridinium polymer grafted
bacterial cellulose) and QBCCP (phosphonium salt-based poly-4-
vinylpyridinium polymer grafted cotton-bacterial cellulose)
surfaces. In this case, the pre-synthesized polymer (QP4VP-PBz)
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Optical properties of P4VP and quaternized polymers were
studied by UV-vis absorption spectroscopy (Figure 3C). For P4VP
polymer the two broad peaks appeared at 217 nm and 255 nm
corresponding to the π→π* and n→π* electronic transitions of
aromatic pyridine rings. After reaction the π→π* peak at 217 nm
25 was shifted to 210 nm and 216 nm for QP4VP-Bz and QP4VP-PBz
polymer respectively. The n→π* peak (255 nm) was also shifted
to 231 nm for QP4VP-Bz and 224 nm for QP4VP-PBz due to the
reduced availability of lone pair of electrons on N of pyridine after
the quaternization reaction. Finally, the structure arrangement of
30 polymers was studied by X-ray diffraction in 2Ɵ angle range
between 5 to 80° (figure 3 (D)). The poly-4-vinyl pyridine polymer
has amorphous nature with two broad peaks at 2Ɵ value of 10°
and 21° in XRD pattern. After quaternization due to the salt
formation the polymer chain arrangement was changed with
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Scheme 2. Immobilization of different polymers on bacterial cellulose (BC and BCC) surfaces by various grafting methods. (A) “graft to” method of
immobilization of poly-4-vinylpyridinium polymer, and (B) “direct graft” method of immobilization of phosphonium polymers on bacterial cellulose.
25 line with observations by Fernandas et al., who reported that
was reacted with BC or BCC surfaces in methanol under optimized
after sillylation the -Si-O-CH2- bond vibrations are typically
5
condition at 65°C for 12 hours (depicted in reaction scheme 2).
observed at around 1150 cm-1 and these peaks are not observed
Finally, the pristine surfaces (BC and BCC), silyllated surfaces
sometimes due to masking of peaks by cellulose -O-C-O- glyosidic
(BCTMS and BCCTMS) and polymer functionalized surfaces (QBC,
vibration peaks. Further, we observed small sharp peaks at 2890
QBCP, QBCC, and QBCCP) were characterized by ATR-IR and solid
30 cm-1 corresponding to the CH2 stretching frequency of propyl
state 13C NMR spectroscopy, elemental analysis, XRD, TGA, DSC,
groups. In “graft to” functionalized surfaces (QBC and QBCC) the
10 SAXS, SEM, Fluorescence microscope, contact angle meter, tensile
strength measurement and AFM. Quantification of the density of
quaternary ammonium groups on surfaces was done with a
fluorescent dye by optical spectroscopy.
two new peaks appeared at 1600 and 1640 cm-1 indicating the
characteristic stretching frequency of C=N and C=N+ bonds of
pyridine ring respectively which indicates that partial
35 quaternization was happened on the surfaces and that QBC
surfaces contain quaternized pyridine ring as well as free pyridine
ring.27 Similarly, for the surfaces functionalized by direct grafting,
the characteristic peaks for QBCP and QBCCP were observed at
1640, 2850 and 2920 cm-1 corresponds to the stretching
40 frequency of C=N+ (pyridine ring), aliphatic CH2 and aromatic C-H
bonds respectively, confirming the success of polymerization.
2.2.1. ATR-IR and Elemental analysis
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The ATR-IR spectral analysis of BC materials before and
after functionalization is shown in figures 4. The ATR-IR spectrum
of our prepared dry BC material shows the characteristic peaks at
around 3340 cm-1, 2894 cm-1 and 1045-1150 cm-1 corresponding
to the O-H stretching frequencies, aliphatic CH2 stretching
20 frequency, and -O-C-O- glycosidic linkages vibration frequencies
respectively. In BCC material, other than bacterial cellulose peak
cotton cellulose characteristic peaks were observed at around
3254 cm-1, 2852 cm-1 and 890 cm-1. After sillylation reaction, the
BC-TMS and BCC-TMS surfaces shows little changes only. This is in
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ppm corresponding to methylene carbon of basic cellulose unit,
assigned in figure 5 (black colour spectrum). After “graft to”
reaction, the new carbon signals were appeared at 28-42 ppm
30 and 130-165 ppm (3 peaks) corresponding to the methylene
carbon of P4VP backbone and aromatic carbon of pyridine ring
respectively. In addition, the 13C signal of methylene group in silyl
moieties were observed at around 10, 25 and 58 ppm with line
broadening.28 These results clearly show the successful
35 immobilization of poly-4-vinyl pyridinium polymer on BC
surfaces.
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Figure 4 ATR-IR spectra of (A) BC and (B) BCC materials before and after
functionalization. XRD pattern of (C) BC and (D) BCC materials before and
after functionalization
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The elemental composition of pristine and functionalized
bacterial cellulose surfaces was studied by CHNS analysis. In
pristine BC it was found that 43.05 % of carbon and 7.19 % of
hydrogen elements were present (showed in ESI table S1). After
silyllation the BCTMS surfaces show that the amount of C and H
Figure 5. CPMAS-13C solid state NMR of BC and QBC materials
2.2.3. XRD analysis
40
The effect of grafting on crystallinity of BC and BCC
surfaces was studied by X-ray diffractometer at 2Ɵ ranges
between 5-80 degrees and percentage of crystallinity index was
10 was reduced to 36.68 % and 6.14 % respectively due to the
incorporation of the siloxane moiety on the cellulose surfaces.
The elemental composition of polymer functionalized BC surfaces
further indicated the success of immobilization of pyridinium
polymer on BC surfaces by showing the presence of N (pyridinium
15 group) contents with 2.18 % and 1.12 % for QBC and QBCP
surfaces respectively. In addition, the changes in carbon and
hydrogen elements were also observed in both cases.
calculated by peak height method29 using the formula CI = (I200
Iam) / I200 as shown in figure 4 (C) and 4 (D). By this method,
45 Toakaew et al.30 found that the crystallinity of BC films was
around 91.8%. Generally, the bacterial cellulose exhibit
-
a
diffraction pattern of cellulose I type.31 After grafting, the changes
in peak intensity were observed in all the cases. The results show
that the crystallinity of pristine bacterial cellulose (BC) was around
50 97% by peak height method calculation. In silyllated BC surfaces
(BCTMS) the crystallinity was slightly decreased to 90% due to the
restriction of hydrogen bonding of cellulose units during silyl
addition. The crystallinity of QBC surfaces significantly decreased
to 68% when the P4VP polymers were grafted on BCTMS surface
55 by quaternization. The change in crystallinity may be attributed
to the immobilisation of different polymers leading to cross linking
of fibres. The decrease in crystallinity of cellulose materials by
2.2.2. Solid state 13C NMR spectroscopy analysis
Grafting on bacterial cellulose surfaces (BC and BCC)
20 was further analysed by CP-MAS solid state NMR spectroscopy
technique with 5000 scans (showed in figure 5). Since the BC and
QBC samples were not soluble in any organic solvent, the samples
were submitted in powder form (using lyophilization followed by
grinding) to perform the solid-state NMR. In the pristine BC
25 surface, all characteristic carbon nuclei peaks of bacterial
cellulose were observed at 65 ppm, 69-76 ppm, 89 ppm and 105
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polymer grafting was also reported by Ikkala, and others.23 The
restriction of movements in crystalline phase of cellulose chains
during polymer functionalization with crosslinking.
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pristine BCC material also exhibits a cellulose type I crystallinity,
similar to bacterial cellulose XRD pattern. However, due to
overlapping of different cellulose forms, the crystallinity of BCC
materials was not determined.
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Thermogravimetric analysis of bacterial cellulose surfaces
was studied with the aim of determining the changes of thermal
stability by influence of polymer grafting (shown in figure. 6 (A)
10 and 6 (B)). The literature report suggested that the typical range
of onset decomposition temperature (Td) of neat BC surfaces is
around 280-389°C.32 Along the same line, we observed that the
first onset decomposition temperature (Td) of neat BC is 326°C
and neat BCC is 309°C which is attributed to cellulose pyrolysis.
15 After sillylation, the residue amount was increased to around 44.2
% and 46.3% and the Td value was decreased to 260°C and 280°C
for BCTMS and BCCTMS surfaces respectively, possibly due to the
elimination of iodine groups and cleavage of cellulose-O-Si bonds
at higher temperature. Generally, the polymer grafted bacterial
20 cellulose has low thermal stability compared to pristine BC
because of more chances to decomposing with different factors
like depolymerization, pyrolysis etc. In our case, we observed two
decomposition stages for QBC and QBCC surfaces at 251 and
337°C and 256 and 348°C respectively, which was functionalized
25 by “graft to” method. It may be attributed to the decomposition
of silane moiety and polymer backbone. But in this case of QBCP
and QBCCP surfaces which was functionalized by “direct graft”
method we observed only one decomposition stage at 315°C (for
Figure 6. TGA thermogram of (A) BC and (B) BCC materials before and
45 after immobilization of polymer. DSC thermogram of (C) BC and (D) BCC
materials before and after polymer immobilization
Table 1. Physical properties of different materials before and after
functionalization from TGA, DSC and XRD
Surface
BC
Td °C
Tm°C
CI (%)
Surface
BCC
Td °C
Tm°C
(Residue)
(Residue)
326 (29.3)
260 (44.2)
71.1
97.8
309 (33.9)
280 (46.3)
89.6
BCTMS
QBC
44.1
58.1
90.1
68.5
BCCTMS
QBCC
62.1
83.4
251, 337
(38.9)
256, 348
(38.3)
QBCP
315 (30.2)
68.2
95.0
QBCCP
299 (40.6)
66.1
QBCP) and 299°C (for QBCCP) (Table 1) and it may be due to
30 the decompositions of polymer chains during higher temperature.
DSC analysis of surfaces were studied at -50 to 175°C with heating
rate of 10°C/ min (showed in figure 6 (C) and 6 (D)). In DSC, we
observed the broad endo thermic peak in the range of 60-90 OC
which is attributed to the melting of crystalline phase of
35 cellulose33 (Tm) (table 1). In pristine BC and BCC surfaces, we
observed the Tm values at 71.1 and 89.6°C. After sillylation the Tm
transition temperature was decreased in BC-TMS and BCC-TMS
surfaces to 44.1 and 62.1°C respectively. The decrease in values of
Tm compared with pristine surfaces was also observed after
40 polymer functionalization which may be attributed to the
50
2.2.5. Small angle X-ray Scattering analysis
The nanofiber structures of bacterial cellulose surfaces (BC,
BCTMS and QBC) were analysed by small angle X-ray scattering
technique (SAXS).34 The SAXS data is obtained as a plot between
55 scattering intensity and scattering vector q (q =4π sinɵ/λ) to
understand the dimensions of BC fibers (ESI Fig. S16). Our AFM
analysis and previous reports of bacterial cellulose reveals that
the cellulose nanofibers have long rod like structure with
rectangular cross section shapes due to aggregation occurring
60 between several numbers of fibres. In this case, the SAXS data can
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provide information about cross section dimensions of the
scattering elements belonging to the rectangular cross section of
long rod like structure. We obtained experimentally the
(BC, BCTMS and QBC) from guinier plot fitting and provided in
table 2. This result show that the rc values were decreased and
scattering intensity also varied from BC to QBC (ESI figure S16).
dimensions of single BC nanofiber is around 31 nm and 18 nm 45 Finally, we obtained the dimension values a and b for BC is 21.1
5
from AFM and SEM analysis (in ESI figure S15). Khandelwal et al.
estimated the dimensions of cross section of bacterial cellulose
fibers was 32 nm by 16 nm from SAXS analysis and this was more
which is comparable with our experimental results. So, we have
nm by 6.9 nm, for BCTMS is 19.3 nm by 3.7 nm and for QBC is 8.0
nm by 7.0 nm. In BCTMS surfaces the dimension were decreased
slightly which may be attributed to the breaking of the hydrogen
bonds of cellulose units with separation of fibres during the sillyl
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tried to calculate the value of cross sectional dimension (a and b) 50 addition. In the case of P4VP polymer grafted BC (QBC) surfaces,
10 by SAXS analysis with Guinier approximation theory (from low q
the dimension values were dramatically changed compared to BC
and BCTMS surfaces. This observation was corroborated with
AFM analysis of QBC surfaces (figure 9). This is plausibly due to
the crosslinking of fibres during the polymer grafting by “graft to”
region of SAXS scattering element) using the formula qI(q) = G
2
exp (-q2rc /2)). Where G is scaling constant, q is scattering vector
and rc is radius of homogeneous cross section. Moreover, this
theory is only valid for very dilute system, so we have chosen very 55 methods of immobilization to facilitate the separation of
15 thin layer of bacterial cellulose film for all reactions. Since the
cellulose fibre structure has rectangular cross section, so the
relation between the dimensions and radius of cross section
values can be given as a2+b2 = 3 rc2 and another relation between
the dimensions and cross-sectional areas (S) can be given as
20 S=4ab. The value of S can be estimated from the equation:
S=2πG/Q when q→0, the value of qI(q) is equal to scaling
constant G, where Q is invariant of the cross-sectional area of
both region of guinier and porod range. Finally, we obtained the
values of rc, G and Q from extrapolating the guinier and porod
25 plots (ESI, Fig S16), followed by the calculation of S, to obtain the
cross-section dimension value a and b by simple arithmetic
equation.
nanofiber.
Table 2. Result from SAXS analysis (Figure S16 in ESI) of different
materials
Radius
of cross
section
(rc)
Cross
Dimension of
Rectangular
cross section
surfaces
G
Q
sectional
area (S)
invariant
a
B
BC
13.06
1.87×106
1.005×106
43119
19075
22536
1152
615
280
235
21.1
nm
19.3
nm
8.0
6.9
nm
3.7
nm
7.0
nm
BCTMS
QBC
11.59
5.00
nm
2.2.6. Density of quaternary ammonium groups Quantification
The polymer grafted surfaces (QBC, QBCC, QBCP, and
QBCCP) contain more amounts of quaternary ammonium groups
and the density of quaternary ammonium groups on the surfaces
was calculated after counter ion exchange by fluorescein anion
(slightly modified from reported procedure36 was described in
60
After completing the SAXS experiment, the sample
scattering data was subtracted from the scattering data of blank.
30 Then with desmear scattering data, we can draw the guinier plot
(plot between ln qI(q) and q2 according to the guinier equation)
and porod plot (figure S16). In guinier plot, the first few points of
low q region were fitted with SAXS quant software. For knowing
the differences in samples scattering, the guinier fitting q2 range
35 was kept same for all three samples typically at 0.018 to 0.028.
The radius of homogeneous cross section (rc) and scaling constant
(G) can be determined by extrapolating the guinier plot. Tischer et
al. investigated the rc values of bacterial cellulose with different
time of ultrasonication treatment and they have found increasing
40 the rc values with increasing the time of ultrasonication due to the
aggregation of BC fibers.35 We obtained the rc values of surfaces
65 ESI). For this, first the surface quaternary ammonium groups were
treated with fluorescein anion to replace halide ions from
polymer grafted bacterial cellulose surfaces. After that, desorbing
the
exchanged
fluorescein
molecules
was
done
by hexadecyltrimethylammonium bromide solutions from the
70 surfaces. Then, the UV absorption values at 501 nm
(corresponding to the fluorescent molecule) of final solution (ESI
figure S14) was measured to calculate the density of quaternary
ammonium groups present in a given area by using Beer-Lambert
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law. The results show, in control surfaces (BC and BCC) there is no 40 cellulose units by 3-iodo propyl trimethoxy silane during grafting
2
quaternary ammonium group (shown in ESI table S2). It was thus
observed that both the polymer functionalized surfaces obtained
by “graft to” and “direct graft” method has an appreciable
amount of quaternary ammonium group. Further, surfaces by the
“direct graft” methods have more density (QBCP is 0.047 × 1015
and QBCCP is 0.16 × 1015) compared to “graft to” surfaces (QBC is
0.050 × 1015 and QBCC is 0.035 × 1015). This may be attributed to
the reason that in “graft to” method the P4VP polymer chains
reaction.
3
4
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10 were directly reacted on sillylated surfaces to form quaternary
ammonium groups and due to the steric hindrance, the entire
pyridine ring N was not able to participate in quaternization. So,
these surfaces have free pyridine ring of polymer chains. But in
the case of “direct graft” surfaces the density is more because,
15 the quaternization was performed before grafting.
Figure 7. Fluorescence images of bacterial cellulose (A) BC and (B) QBC, (C)
QBCP, (D) BCC, (E) QBCC, and (F) QBCCP surfaces before and after
45 reaction.
2.2.7. Surface morphology analysis by Fluorescence microscope,
SEM and AFM analysis
Surface morphology was studied by fluorescence
microscope (with 365 nm wavelength excitation), scanning
20 electron microscope (SEM) and atomic force microscope (AFM). In
line with the observations by others we did not observe
fluorescence emission in pristine bacterial cellulose (BC and BCC)
surfaces due to absence of fluorescent chromophore in cellulose
unit.37 (figure 7). But in the case of polymer functionalized
25 surfaces greenish red emission was observed. It indicates the
polymers were successfully immobilized on the bacterial cellulose
surfaces. Functionalized surfaces with quaternized polymer
(QP4VP-PBz) show fluorescent emission at 400 nm. Then, the
polymer was drop casted on the glass plate and analysed with the
30 fluorescence microscope. It shows pale red emission (ESI figure.
S12). The SEM images showed that dried BC material has clear
nanofiber with fibre size less than 100 nm (SEM images of BC and
BCC surfaces are provided in ESI figure S13). In BCC surfaces we
obtained two different types of fibres: one is the BC nanofiber
35 (<100 nm) and another cotton micro fibre (≈18 µm). It reveals
that in BCC materials, during culture process, the bacterial
cellulose was growing on the cotton fibres as observed from
figure 8, after functionalization the BC nanofiber was embedded
with polymer chains due the crosslinking between the adjacent
Figure 8. SEM images of different surfaces after polymer functionalization:
(A) QBC, (B) QBCC, (C) QBCP and (D) QBCCP surfaces
Further, atomic force microscopic (AFM) study of pristine
50 BC and polymer functionalized surfaces (QBC, QBCP, QBCC and
QBCCP) showed the changes of fibers morphology while
incorporating the polymers through grafting method (figure 9). In
AFM topography, image of pristine BC fibers clearly shows endless
long rod like structure with rectangular cross section shapes due
55 to aggregation (several number of cellulose fibers were
combined). After polymer functionalization the topography of
QBC and QBCC surfaces was completely changed from fibrillar
structure to a crosslinked structure (Fig. 9(B), 9(C), 9(D) and 9(E))
with increasing the surface roughness values of BC, QBC, QBCC,
60 QBCP and QBCCP to 15.42 nm, 21.92 nm, 137 nm, 18.52 nm and
59.07 nm respectively.
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(ɛ) after reaction, we did grafting reaction on BC surfaces with
optimized condition (size of the BC surfaces is 7 cm × 5 cm × ≈
30 0.20 mm) along with control experiment. The tensile strength of
bacterial cellulose films depend on the thickness of surfaces and
processing methods. So, we have chosen same thickness of BC
materials for all reaction, in order to understand the changes of
mechanical property during polymer grafting reaction. Fu et al.
35 reported that the δ and ɛ values of wet bacterial cellulose film is
1.96 MPa and 23.00 % (for 2.0 mm thickness) and dry bacterial
cellulose film is 10.32 MPa and 9.00 % (for 0.55 mm thickness)
respectively.38 We obtained the δ and ɛ values (average values of
two specimen of samples) of our prepared pristine bacterial
40 cellulose respectively are 5.61 MPa and 6.08 % (for 0.22 mm
thickness) (ESI table S3 and figure S17). After polymer
functionalization (QBC surface) by “graft to” technique, the
tensile strength (δ) increased almost two times (10.27 MPa)
compared to neat BC. The elongation at break (ɛ) values also
45 increased to 21.69 %. The improvements in mechanical
properties may be attributed to the crosslinking of the adjacent
glucose units in cellulose by quaternization as well as surfaces
functionalization with increment of amorphous nature through
“graft to” reaction.
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Figure 9. AFM height images of different surfaces before and after
functionalization: (A) BC, (B) QBC, (C) QBCC, (D) QBCP and (E) QBCCP
surfaces
5
2.2.8. Surface wettability study by contact angle meter
Wettability properties of pristine surfaces and
polymer grafted surfaces were analysed by water contact angle
meter with sessile drop (volume is 10 µL) method (showed in
figure 10). Both the pristine BC and BCC surfaces showed
10 hydrophilic characteristics with water contact angle values of less
than 40 degree because of the presence of a number of hydroxyl
groups on surfaces.31 After sillylation the contact angle increased
to above 60 degree which further increased to a range of 90
degree to 120 degree after functionalization with different
15 polymers (figure 10). This may be attributed to the reaction on
exposed hydroxyl groups and cross linking of cellulose and vinyl
pyridine-based polymers leading to blocking of some of the pores.
This increase is significant considering the detrimental effect of
water on pristine BC-based materials.
50 2.2.10. Study of antibacterial properties
The antibacterial activity was assayed with gram +ve bacteria
Staphylococcus aureus (MTCC 0096) and gram -ve bacteria
Escherichia coli (MTCC 0045) by observing the zone of inhibition in
disc diffusion method. For investigation petri dishes (90 mm in
55 diameter) containing Luria-Bertani agar medium and Nutrient
agar medium were inoculated and spread with S. aureus and E.
coli respectively. Subsequently, approximately 1.5 cm (diameter)
circular pieces of BC-polymer conjugates 5 were placed on the
surface of the bacteria inoculated culture plate and incubated at
60 37±1°C for 48h. Antibacterial effect was evaluated by observing
the formation of inhibition zone around the polymer film after
desired period of incubation. The entire assay was repeated for
three times to obtained conclusive result.
20
Figure 10. Images of water droplets on various bacterial cellulose surfaces
before and after immobilization of polymers
2.2.9. Mechanical properties of BC and polymer grafted BC
surfaces
No distinct zone of inhibition was observed around the BC-
65 polymer conjugate films in presence of both bacteria, indicating
the absence of antibacterial characteristic of the conjugate. This
may be attributed to high degree of biocompatibility of the BC
films.
25
The mechanical properties of BC materials were analysed
by INSTRON 3369/J7257 instrument (showed in ESI figure S17).To
compare the values of tensile strength (δ) and elongation at break
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9
In a nitrogen-degassed 100 ml round bottom flask, 100 mg
(0.952 mmol) of poly-4-vinyl pyridine and 25 mL of anhydrous
methanol was charged. It was stirred for five minutes under N2
atmosphere. Then 27.6 mg (0.095 mmol, 10 mmol%) of (3-
45 iodopropyl) trimethoxy silane was added and refluxed for 3 hours.
After that 585 mg (0.856 mmol, 90 mmol%) of compound PBz (3)
((2,5-dibromo-4-(bromomethyl) benzyl) triphenyl phosphonium
bromide) was added, maintaining at reflux condition for 21 hours
in N2 atmosphere. After completion of reaction, solvent was
50 evaporated by rotary evaporator to obtain thick oil mass. The
crude was dissolved in methanol, reprecipitated with acetone and
washed with diethyl ether to obtain green solid (460 mg, 64.6%)
coded as QP4VP-PBz, depicted in reaction scheme 1.20
3. Experimental
3.1 Materials
2,5-Dibromo-p-xylene was purchased from Alfa Aesar
5
company. Poly(4-vinylpyridine) (P4VP-60,000 MW), (3-iodopropyl)
trimethoxy silane, N-bromosuccinimide, triphenylphosphine, dry
toluene, dry methanol and deuterated solvents (CDCl3 and DMSO-
d6) were purchased from Sigma-Aldrich. NaHCO3, NaOH,
anhydrous Na2SO4, diethyl ether and ethyl acetate solvent were
10
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59
60
10 purchased from Merck. Chloroform (CHCl3) and petroleum ether
(60-80) were purchased from Loba Chemie. The bacterial cellulose
was prepared and purified using the method described
elsewhere.7 Briefly BC pellicles were obtained by fermentation
method, washed many times with distilled water followed by
15 treatment with 2% sodium hydroxide at 80°C for 1 h followed by
rinsing with distilled water.
3.5 In Situ preparation of cotton-bacterial cellulose (BCC)
55 material:
Cotton-BC composite was prepared by
a modified
literature procedure.39 In brief, a layer of cotton was placed in the
BC culture medium as shown in figure 11. The medium was
maintained at the static condition for 6 days. We observed
60 bacterial cellulose gel formed with cotton in the upper layer of
the medium. The cotton-BC materials were washed with plenty
amount of water to remove the BC medium residues. It was
purified using the same procedure as described above for BC
material using aqueous sodium hydroxide and hot water (coded
65 as BCC).
3.2 Synthesis of (2,5-dibromo-4-(bromomethyl) benzyl) triphenyl
phosphonium bromide (PBz) 3:
In a 50 ml nitrogen-degassed flask, 500 mg (1.18 mmol) of
20 1,4-dibromo-2,5-bis(bromomethyl)benzene (compound 2), 321.77
mg (1.22 mmol) of triphenylphosphine and 20 mL of ethyl acetate
were charged and stirred at room temperature for 36 hours. A
precipitation was observed. It was filtered and washed with ethyl
acetate, re-dissolved in chloroform and reprecipitated with
25 diethyl ether. After filtration, a white solid was obtained which
was dried in vacuum to yield 680 mg, 83% of white solid
compound 3 (scheme 1).
3.3 Model reaction: synthesis of poly-4-vinyl pyridinium polymer
(QP4VP-Bz) 4:
30
In a 50 ml dry and degassed round bottom flask, 100 mg
(0.95 mmol) of poly-4-vinyl pyridine, 400 mg (1.14 mmol) of
compound
1 (1,4-dibromo-2-(bromomethyl)-5-methylbenzene)
Figure 11. Schematic representation of preparation of BC and BCC films.
3.6 Grafting of polymers on BC and BCC materials:
and 10 mL of anhydrous methanol was refluxed for 24 h in N2
atmosphere. The solvent was then removed in rotary evaporator
3.6.1 “Graft to” method: immobilization of poly-4-vinyl pyridine
70 (P4VP) on BC and BCC surfaces:
35 and precipitated with chloroform: acetone mixture. Finally, we
got white solid (320 mg, 75.3%) coded as QP4VP-Bz polymer,
which was analysed by FT-IR, NMR, TGA and DSC (shown in
reaction scheme 1).20
i) Preparation of BCTMS and BCCTMS surfaces:
Reaction procedure was same for both BC and BCC
surfaces using 3-iodopropyltrimethoxy silane. First, in a clean and
dry 100 mL flat bottom flask equipped with condenser, several
3.4 Synthesis of phosphonium salt-based poly-4-vinylpyridinium
40 polymer (QP4VP-PBz) 5:
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pieces of BC or BCC surfaces with sizes 1 cm × 1 cm square shape 30 completion of the reaction the surfaces were removed to wash
were taken (weight 30 mg, 0.185 mmol). It was charged with 15
ml of anhydrous methanol and 0.537 mg (1.85 mmol) of 3-
iodopropyltrimethoxysilane and heated at 70°C under stirring
condition for 24 hours. After completion of the reaction, the
with 20 mL of methanol and dried in oven at 50 °C for 6 h (coded
as QBCP (phosphonium salt-based poly-4-vinylpyridinium polymer
grafted bacterial cellulose) and QBCCP (phosphonium salt-based
poly-4-vinylpyridinium polymer grafted cotton-bacterial cellulose)
3
4
5
6
5
7
8
9
surfaces were taken and washed with methanol under 5 minutes 35 surfaces which were depicted in scheme 2.
of sonication. The surfaces were dried at 50°C for 12 hours under
10
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59
60
4. Conclusions
vacuum to obtain sillylated BC or BCC materials coded as BCTMS
Cationic and zwitterionic polymers with pyridinium or
(3-iodopropyl trimethoxy silane grafted bacterial cellulose) and
phosphonium groups were immobilized on bacterial cellulose (BC)
surfaces by “graft to” or “direct graft” technique. Cotton-bacterial
40 cellulose surface was also used for grafting for the comparison
and to expand the area of applications. While ATR-IR and solid-
state NMR indicated the success of functionalization of BC
surfaces, the spectroscopic studies using a fluorescent dye
indicated the high degree of quaternization in both cases. TGA
45 data indicated moderate decrease in thermal stability after
functionalization while DSC data indicated a change in the pattern
of phase transition in the 65-90°C ranges. The XRD data indicated
the significant decrease in crystallinity after functionalization,
possibly due to random arrangements of polymers, while SAXS
50 data indicated the separation and cross-linking of well-defined
fibers after polymerization. This was corroborated from FESEM
and AFM microscopic studies where pattern of microscopic fibers
were clearly understood. Finally, the usefulness of
functionalization was clearly realized from the appreciable
55 amount of improvements in both mechanical properties and
hydrophobicity, possibly due to cross linking by a number of
10 BCCTMS (3-iodopropyl trimethoxy silane grafted cotton-bacterial
cellulose) shown in scheme 2.
ii) Preparation of QBC and QBCC surfaces:
In a 100 mL flat bottom flask, 50 mg (0.1402 mmol) of
sillylated surfaces (BC-TMS or BCC-TMS) was taken and added
15 14.72 mg (0.1402 mmol) of poly-4-vinyl pyridine and 10 mL of
anhydrous methanol. The reaction medium was degassed with N2
gas and refluxed for 24 hours. It was cooled down and both the
surfaces were taken out to wash with methanol under 5 mins of
sonication. Finally, after drying at 50°C for 12 hours under vacuum
20 we got pale yellow surfaces named as QBC (poly-4-
vinylpyridinium polymer grafted bacterial cellulose) and QBCC
(poly-4-vinylpyridinium
polymer
grafted
cotton-bacterial
cellulose) surfaces as depicted in scheme 2.
3.6.2 “Direct graft” method: preparation of QBCP and QBCCP
25 surfaces:
100 mg of pristine BC or BCC surfaces were taken into the
100 mL flat bottom flask and added 40 mg of phosphonium salt
based quaternized poly-4-vinyl pyridine polymer (QP4VP-PBz) and
20 mL of dry methanol. It was refluxed for 24 hours. After
functional
groups
in
polymers.
5. Acknowledgement
60 Financial support from DST project (ECR/2015/000219) and CLRI MLP1805 is gratefully acknowledged. We are thankful to CATERS
Department of CLRI for assisting with various characterizations and discussions. CSIR-CLRI communication No. is A/2019/PLY/DST-
CLRI/1318.
†Electronic
Supplementary
Information
(ESI)
available:
[spectra,
experimental
detail]
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6. Notes and references
14
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Abbreviations
P4VP
: Poly-4-vinyl pyridine (scheme 1)
QP4VP-Bz : poly-4-vinyl pyridinium polymer (scheme 1)
35 QP4VP-PBz: phosphonium salt-based poly-4-vinylpyridinium polymer (scheme 1)
BC
: bacterial cellulose
BCC
: cotton-bacterial cellulose
BCTMS
: 3-iodopropyl trimethoxy silane grafted bacterial cellulose (scheme 2)
BCCTMS : 3-iodopropyl trimethoxy silane grafted cotton-bacterial cellulose (scheme 2)
40 QBC
: poly-4-vinylpyridinium polymer grafted bacterial cellulose (scheme 2)
: poly-4-vinylpyridinium polymer grafted cotton-bacterial cellulose
QBCC
QBCP
: phosphonium salt-based poly-4-vinylpyridinium polymer grafted bacterial cellulose (scheme 2)
QBCCP : phosphonium salt-based poly-4-vinylpyridinium polymer grafted cotton-bacterial cellulose(scheme 2)
14 | Journal Name, [year], [vol], 00–00
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DOI: 10.1039/C9NJ02199J
Graphical Abstract
Immobilization Of Quaternized Polymers on Bacterial Cellulose by
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Different Grafting Techniques†
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P. Ramar,a,b Sourita Jana,a,c Sandipan Chatterjee,d S. N. Jaisankar,*a and Debasis Samanta*a,b
aPolymer Science & Technology Department, CSIR-Central Leather Research Institute (CSIR-CLRI), Adyar, Chennai-600020,
India. Fax: 91-44-24911589, Tel: 91 -44 24422059 E-mail: debasis@clri.res.in (DS), snjsankar@clri.res.in (SNJ)
bAcademy of Scientific and Innovative Research (AcSIR), India
cUniversity of Madras, Chennai-600005, India
dRCED, CLRI, Kolkata, India
Different polymers were immobilized on bacterial cellulose surfaces using grafting techniques to
improve mechanical properties and surface hydrophobicity.