Microbial Screening of Thermally stable
N-Vinyl Carbazole–co-Methoxyethyl methacrylate Copolymers: Synthesis and Characterization
R. P. Meena1,2, K. Abdul Wasi2,
K. Anver Basha2*
1PG Department of Chemistry, L. N. Govt. College,
Ponneri, Tamilnadu, India.
2PG & Research Department of Chemistry, C. Abdul
Hakeem College, Melvisharam, 632 509, Tamilnadu, India.
*Corresponding
Author E-mail: kanverbasha@gmail.com
ABSTRACT:
The scope of the present study is focused
to explore the antimicrobial properties of thermally stable poly
(NVC-co-MOEMA). Copolymers of N-Vinyl Carbazole (NVC) with Methoxy
ethylmethacrylate (MOEMA) at different feed compositions were prepared by free
radical solution polymerization at (60 ± 5)°C using benzoyl peroxide (BPO) as
an initiator. The characterization of the copolymers was carried out by FTIR
and 1H-NMR spectroscopy. The high thermal stability and higher Tg
values of the copolymers were predicted by Thermo gravimetric analysis (TGA)
and Differential scanning calorimetry (DSC). Antimicrobial activities of the
copolymers were also investigated against the selected pathogenic bacteria’s
(Staphylococcus aureus, Escherichi coli, Bacillus subtilis, Salmonella
Paratyphi) and fungi’s (Candida albicans, Aspergillus niger). Microbial
screening of both the Homopolymer and Copolymers were carried out and their
activities studied. The antimicrobial activity of the Copolymers was higher
than that of the Homopolymer. This shows that copolymerization with methoxy
ethylmethacrylate moiety plays a very important role in the antimicrobial
activity.
KEYWORDS: NVC-co-MOEMA; Copolymer; Thermo gravimetric analysis;
Antimicrobial activity; Microbial screening.
INTRODUCTION:
In the 1990s, antimicrobial polymer systems
were surveyed by Worley and Sun and Afinogenov and Panarin. 1, 2
Varieties of antimicrobial polymers have been considered in several
comprehensive reviews during the past decade. Synthesis of antimicrobial polymers
such as polyionenes (polymers with positively charged nitrogen atoms located in
the backbone of a macro chain) was carried out. Copolymerization techniques
have a number of advantages in controlling the degree of functional groups in
the product, controlling its structure, and introducing the required properties
into the polymer system.
Pleurdean and co-workers reported the synthesis of
acrylic esters that are useful as biocides, by reacting pentachlorophenol,
p-chloro-m-cresol and o-phenyl phenol with acryloyl chloride3. Oh
and co-workers synthesized 2,4,4’-trichloro-2’-acryloyloxydiphenyl ether and
its homo- and copolymers with methyl methacrylate (MMA), styrene and
2-hydroxyethyl methacrylate and tested bactericidal properties against Staphylococcus
aureus and Pseudomonas aeruginosa microorganisms.4,5 Thus, growth
inhibition of these bacteria by the copolymers N-vinyl carbazole (NVC) with
ethyl methacrylates (EMA) has been studied. In the present investigation,
N-Vinyl carbazole (NVC) was copolymerized with Methoxy ethylmethacrylate
(MOEMA) which may be useful in pharmaceuticals, pesticides, paints, coatings,
inks, adhesives and cosmetics.6 This article discusses the synthesis
and characterization of the copolymers using different feed ratios. FTIR and 1H-NMR
spectroscopy were used to characterize the copolymers. The copolymers showed
high thermal stability and higher Tg. It was predicted by the
Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)
values. Homo and copolymers have been characterized in their antimicrobial
activity against microorganisms. Their activies were tested with bacteria’s
such as Staphylococcus aureus, Escherichi coli, Bacillus subtilis and
Salmonella Paratyphi and fungi’s such as Candida albicans, and Aspergillus
niger.
MATERIAL AND METHODS:
All chemicals used were of analytical
reagent grade. Solvents and monomers were purified by the conventional methods.7
Synthesis of poly (N-Vinyl
Carbazole-co-Methoxy Ethylmethacrylate):
The copolymers were synthesized from the appropriate
amounts of different monomer using BPO as initiator. The reactants were
dissolved in 25mL of chlorobenzene to obtain homogeneous solutions in a
standard polymer tube. Dry nitrogen gas was flushed in to the reaction mixture.
The reaction vessel was then immersed in a thermostatic water bath maintained
at 60±5oC. After desired period, the tubes were removed from the
water bath and cooling was done under running tap water. The solutions were
then poured into ice-cold methanol to precipitate the copolymers. The
copolymers were purified by repeated precipitation of chloroform from solution
in chloroform and dried in vacuum oven at 45oC for 24 hrs. The
schematic representation of the synthesis of copolymers is shown below.
Table 1: Different feed ratios of the monomers.
|
S. NO.
|
Monomer
1:Monomer 2 Ratio
|
N-Vinyl
Carbazole (NVC)
|
Methoxy
Ethylmethacrylate (MOEMA)
|
YIELD
|
|
1`
|
0.20:0.80
|
0.2577
|
0.7743
|
80%
|
|
2
|
0.50:0.50
|
0.3865
|
0.2904
|
83%
|
|
3
|
0.80:0.20
|
0.3865
|
0.0725
|
79%
|
Fig.-1: The schematic representation of the
synthesis of poly (NVC-co-MOEMA) is shown below,
RESULTS AND DISCUSSION:
FT-IR SPECTRUM:
The FT-IR Spectrum of Poly (NVC–co-MOEMA) is
shown in Fig.2. The band at 3056 cm-1 can be assigned to the
aromatic C–H asymmetric stretching. The bands at 1450 and 1482 cm-1
may be attributed to the ring vibration of NVC moiety. The C–H in-plane
deformation of aromatic ring has been observed at 1500 cm−1
-1400 cm−1. The band at 3056 cm−1 can be
assigned to the −CH3 stretch vibration. CH2 stretch
vibration appears at 2973 cm−1.8 A very strong band
at 1727cm−1 belongs to the carbonyl group and
−C−O−C− bands at 1244 and 1223 cm−1.9
The aromatic ring and the oxygen bond appears around 1725 cm-1,
while aromatic C-C appears around 1596cm-1. The peak positions of
methoxy can be summarized as follows, asymmetric C-H stretching of the
terminal OCH3 group in the side chain of the methoxy were observed
at 2968 cm-1.10 The asymmetric stretching of the OCH2 is
observed at 2973cm-1 respectively.11 The C-O frequency
can be attributed to the band at 1329 cm-1. The -CH bending mode of
vinyl group appears at 861cm-1, while the rocking mode at 620cm-1.
Fig.-2: FT-IR SPECTRUM OF POLY
(NVC-co-MOEMA)
Fig.-3:1H NMR SPECTRUM OF POLY
(NVC-co-MOEMA)
THERMOGRAVIMETRIC ANALYSIS OF POLY (NVC–co-MOEMA):
The thermal decomposition of the copolymer was
characterized by TG curves. The TGA curves Fig.4 clearly indicate that
all the copolymers undergo single step decomposition process. The measured
results were shown in the Table 2. Different copolymer ratio exhibit
excellent thermal stability with decomposition temperature ranges from 0 to 600
OC.13 The copolymers exhibited no significant weight loss
upon being heated to 600 OC. The thermal stability of the copolymer
increases as the N- vinyl carbazole moiety is increased.14-16
Table 2: TGA analysis data of poly (NVC–co-MOEMA)
|
S.NO.
|
POLY (NVC–co-
MOEMA)
COMPOSITION
|
IDT
|
Decomposition
temperature range(OC)
|
TEMPERATURE(OC)
of WEIGHT LOSS (%) of the copolymers
|
|
10
|
30
|
50
|
70
|
90
|
|
1
|
20:80
|
250
|
0-600
|
297
|
336
|
389
|
415
|
480
|
|
2
|
50:50
|
256
|
0-600
|
299
|
341
|
390
|
420
|
487
|
|
3
|
80:20
|
257
|
0-600
|
302
|
349
|
395
|
423
|
495
|
Fig. 4: TGA Spectrum of poly (NVC–co-MOEMA)
DSC OF POLY (NVC–co-MOEMA):
The glass transition temperature of the polymer was
studied by DSC in the temperature range 0°C to 300°C under nitrogen atmosphere.17
The glass transition temperature (Tg) of poly (NVC–co-MOEMA) was
found to be 84.92°C as in Fig.5.
Fig. 5: DSC Spectrum of poly (NVC–co-MOEMA)
MICROBIAL SCREENING:
The synthesized homo- and copolymers were
tested against different microorganisms which are commonly employed for
biodegradability tests.18 Bacterial strains (Staphylococcus aureus,
Escherichia coli, Bacillus subtilis, and Salmonella Paratyphi) fungi’s (Candida
albicans, and Aspergillus niger) were grown in Nutrient broth (N-broth) and
Subourand’s dextrose broth medium.19-22
ANTIBACTERIAL ACTIVITY:
Screening of acrylic copolymers for
antibacterial activity:
The inoculums for the experiment were prepared in
fresh Nutrient broth from preserved slant culture. McFarland standards were
used to standardize the inoculums by adjusting the turbidity of the culture. By
the addition of sterile saline or broth, we can adjust the turbidity of the
culture.23, 24 Cotton wool swabs on wooden applicator or plastics
were prepared and sterilized by autoclaving or dry heat (only for wooden swabs)
by packing the swabs in culture tubes, papers or tins etc. By dipping in
alcohol and burning off the alcohol, we have sterilized the forceps. The
standardized inoculums was inoculated in the plates prepared earlier
(aseptically) by dipping a sterile in the inoculums. The excess of inoculums
were removed by passing, pressing and rotating the swab firmly against the side
of the culture tube above the level of the liquid. Finally, streaking the swabs
all over the surface of the medium 3 times. The plates were then rotated
through an angle of 60ş after each application. At last, pass the swab round
the edge of the agar surface. The drying of inoculums was done at room
temperature by the lid closed.
In each Petri dish sample disc such as NVC: MOEMA
(20:80), NVC: MOEMA (50:50), NVC: MOEMA (80:20) and HOMO (100µg) discs (discs
are soaked overnight in sample solution) and STD Ciprofloxacin 10µg, were
placed with the help of sterile forceps. Diffusion was done by placing the
Petri dishes in the refrigerator at 4ş C or at room temperature for 1 hour and
Incubated at 37 ş C for 24 hours. The zone of inhibition produced by different
samples was observed. Measure it using a scale and record the average of two
diameters of each zone of inhibition.
Table 3: Antimicrobial activity of Poly (NVC- co-MOEMA)
against bacterial pathogens
|
S. NO.
|
ORGANISMS
|
Zone of
Inhibition(mm)
|
|
STD
CIPROFLOXACIN
(10μg/disc)
|
SAMPLES
(100μg/disc)
|
|
HOMO
|
NVC:MOEMA
(20:80)
|
NVC:MOEMA
(50:50)
|
NVC:MOEMA
(80:20)
|
|
1
|
Staphylococcus
aureus
|
30
|
02
|
09
|
09
|
09
|
|
2
|
Escherichia
coli
|
25
|
05
|
09
|
08
|
08
|
|
3
|
Bacillus subtiles
|
30
|
01
|
13
|
05
|
06
|
|
4
|
Salmonella paratyphi
|
15
|
04
|
16
|
20
|
19
|
Fig. 6: Antibacterial activity of poly (NVC-co-MOEMA)
ANTI FUNGAL ACTIVITY:
Screening of acrylic copolymers for antifungal
activity:
The inoculum for the experiment was prepared in fresh
sabouraud’s broth from preserved slant culture. By adjusting the turbidity of
the culture, the inoculum was standardized to that of McFarland standards. The
turbidity of the culture may be adjusted by the addition of sterile saline or
broth. 23, 24 Cotton wool swab on wooden applicator or plastics were
prepared and sterilized by autoclaving or dry heat (only for wooden swabs) by
packing the swabs in culture tubes, papers or tins etc. The forceps was sterilized
by dipping in alcohol and burning off the alcohol. Inoculation was done in the
plates prepared earlier (aseptically) by dipping a sterile in the inoculums and
the excess of inoculums was removed by passing and pressing and rotating the
swab firmly against the side of the culture tube above the level of the liquid.
Finally, the swab was streaked all over the surface of the medium 3 times. The
plates were then rotated through an angle of 60 şafter each application. Then
pass the swab round the edge of the agar surface. The inoculums were left to
dry at room temperature by closing with a lid.
In each Petri dish, STD Clotrimazole (10µg) was placed
in center of the plate and sample disc such as NVC: MOEMA (20:80), NVC: MOEMA
(50:50), NVC: MOEMA (80:20) and HOMO (100µg) discs (discs are soaked overnight
in sample solution) with the help of sterile forceps. Then Petri dishes were
placed in the refrigerator at 4ş C or at room temperature for 1 hour for
diffusion. Incubated at 28 ş C for 48 hours. Observed the zone of inhibition
produced by different Antibiotics. Measured it using a scale and recorded.
Table 6.8: Antimicrobial activity of Poly (NVC-
CO-MOEMA) against fungal pathogens
|
S.NO.
|
ORGANISMS
|
Zone of
Inhibition(mm)
|
|
STD CLOTRIMAZOLE
(10μg/disc)
|
SAMPLES(100μg/disc)
|
|
HOMO
|
NVC:MOEMA
(20:80)
|
NVC:MOEMA
(50:50)
|
NVC:MOEMA
(80:20)
|
|
1
|
Candida
albicans
|
32
|
09
|
13
|
12
|
18
|
|
2
|
Aspergillus
niger
|
27
|
08
|
19
|
09
|
08
|
Fig.7: Antifungal activity of poly (NVC-co-MOEMA)
CONCLUSION:
The microbial screening of the Homopolymer and
copolymers show that the copolymer was most effective in inhibiting
microorganism growth. As the percentage of NVC in the copolymers increases, the
effectiveness of the copolymers to inhibit the growth of the microorganisms
mostly decreases except in few cases. As expected, poly (NVC-co-MOEMA) is
effective in inhibiting the growth of microorganisms. Although the nitrogen
present in the polymers appears to be the most important component to impart antimicrobial
properties, it is to be remembered that the conformation of the polymers
acquired under experimental conditions is a factor for their antigrowth
activity. This might account for the reversal in the trend of activity of the
copolymers in a few cases. Summarizing, the antimicrobial activity of the
polymers has been attributed to the presence of nitrogen-containing aromatic
heterocyclic ring in the polymers and their conformational changes took
place during the copolymerization.
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7.18-7.54ppm are due to aromatic protons of NVC. The
signals at 
1.55-2.16ppm are due to the - methylene protons and
methine protons of NVC unit. While the signals at