Montmorillonite reinforced polymer nanocomposite

Applied Clay Science 108 (2015) 40–44

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Applied Clay Science journal homepage: www.elsevier.com/locate/clay

Research paper

Montmorillonite reinforced polymer nanocomposite antibacterial film Lemiye A. Savas, Mehmet Hancer ⁎ Department of Materials Science and Engineering, Engineering Faculty, Erciyes University, Talas 38039, Kayseri, Turkey

a r t i c l e

i n f o

Article history: Received 24 October 2014 Received in revised form 17 February 2015 Accepted 18 February 2015 Available online xxxx Keywords: Antibacterial Montmorillonite Silver Polyethylene Nanocomposite

a b s t r a c t In this work, montmorillonite (Mt) was first ion-exchanged with silver ions (Ag + Mt) then organically modified with cetyltrimethylammonium bromide, CTAB (Ag + OMt). Active carrier Mt-layers were then compounded into low density polyethylene (LDPE) polymer using a high shear force co-rotating twin screw extruder. The order of Ag ion and CTAB adsorption on to Mt was revised in order not to form AgBr nano particles of which existence was reported in previous studies. Antibacterial activity of the Ag + OMt–LDPE nanocomposite films was then investigated against powerful gram negative Escherichia coli (E. coli) bacteria. It seems there is strong synergetic effect between silver ions and OMt nanoparticles as evidenced by the bacterial inhibition properties of thin blow molded Ag + OMt–LDPE nanocomposite film samples using liquid surface bacterial testing at a much lower active component concentration than similar studies appeared in the literature. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Combining materials with different properties to form new composite materials is widely used in many fields. Nanostructured materials; especially with nano-sized reinforcements are known to be more effective due to their high surface area/volume ratios (Girase et al., 2011; Xu et al., 2011; Echegoyen and Nerín, 2013). Food packaging, for example has been one of the most successful application of this field. Natural Mt nano layers, dispersed into the plastics and films, form a barrier against oxygen, and carbon dioxide and to prevent moisture into the food resulting in extended storage. By 2015, nanotechnology based food packaging products are expected to reach 19% of the total nanotechnology products produced in the world. The applications of nanocomposites in food packaging can be in the form of film and coating. Active carrier nanocomposite doped materials, with antibacterial and antioxidant properties have been used for some time now in food industry, plastic and cardboard packaging (Dallas et al., 2011; Silvestre et al., 2011). Most recently, the ceramic materials with antibacterial and antioxidant properties are also sold by the companies originated from US, China and Europa (Iris Ceramica, Ceramics, Bo-Hua, Fiandre etc.). These products are 3–5 times more expensive than conventional products and therefore are significant. Furthermore, in some developing countries legislations were introduced for the mandatory usage of antibacterial plastic, ceramic and metal surfaces in the public buildings

⁎ Corresponding author. Tel.: +90 5076870747. E-mail address: [email protected] (M. Hancer).

http://dx.doi.org/10.1016/j.clay.2015.02.021 0169-1317/© 2015 Elsevier B.V. All rights reserved.

(hospitals, schools, new born units etc.) due to their superior hygienic properties and health benefits. 2. Experimental 2.1. Materials Low density polyethylene (LDPE, MFI: 0.75–1.0 g/min at 190 °C/ 2.16 kg and density of 0.919–0.923 g/cm3 at 23 °C) pellets (G08-21 T) were purchased from Petkim of Turkey. Natural sodium montmorillonite (Na + Mt), with specific surface area about 250 m2/g, silver nitrate (AgNO3, reagent grade) and cetyltrimethylammonium bromide (CTAB N % 98) were all purchased from Sigma-Aldrich. 2.2. Preparation of organophilic montmorillonite (OMt) Pure Mt was first grounded using a Fritsch pulverisette 7 planetary nano ball mill in a 100 ml sintered alumina bowl and balls at 800 rpm for 4 h using isopropanol alcohol (IPA). The milled slurry was then heated at 80 °C to evaporate IPA. A sample from the remaining dry powder was dispersed using sonication and the size distribution of milled Mt was determined using dynamic light scattering method (Malvern Nano ZS 90). The average Mt particle diameter (d50) was measured to be 310 nm with 0.246 polydispersity index. The milled dry Mt powder was modified using CTAB according to the reported methods (Pettarin et al., 2008; He et al., 2010). For this purpose, 10 g of Mt (138.86 meq/ 100 g cation exchange capacity) was dispersed into 800 ml of distilled

L.A. Savas, M. Hancer / Applied Clay Science 108 (2015) 40–44

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water with continuous stirring for 1.5 h. 5.166 g CTAB was dissolved in 200 ml of distilled water at 80 °C. The dissolved CTAB solution was then poured into Mt-water slurry under vigorous stirring for 1.5 h to yield a white precipitate. The precipitate was collected on a fine filter paper (Whatman grade 2), and washed three times with distilled water. The product was then dried at 80 °C for overnight and then dry grounded shortly using the Fritsch pulverisette 7 planetary nano ball mill in order to achieve fine dispersion. 2.3. Preparation of Ag ion-exchanged organophilic montmorillonite (Ag + OMt) The pure hydrophilic Mt was ion-exchanged in an AgNO3 solution of 10−2 M at 25 °C for 1.5 h with continuous stirring (Tokarský et al., 2010; Costa et al., 2011). At the end of the reaction, the product was centrifuged at 4000 rpm and the sediment was washed three times with distilled water. The product was then dried at 80 °C overnight and dry grounded using Fritsch pulverisette 7 planetary ball mills for the third time. The silver modified Mt (Ag + Mt) was then modified with CTAB as described above in OMt preparation.

2.4. Preparation of Ag + OMt–LDPE nanocomposite as used clay polymer nanocomposite (CPN) A series of CPN was prepared in a co-rotating twin screw extruder (Gulnar Machines, Turkey: screw diameter 16 mm, L/D = 40). The Ag + OMt powder and LDPE granules were dried at 80 °C for 3 hr in a vacuum oven prior to compounding. First, a masterbatch of Ag + OMt and LDPE at 1:3 ratios (by mass) were compounded using the twin screw extruder. In the second step, the masterbatch was diluted with pure LDPE in order to yield 1.25, 2.25 and 5% (by mass) CPN. Third time, these products were then blown into films of 25–35 μm thickness using single screw extruder (Gulnar Machines, Istanbul Turkey with a screw diameter of 16 mm, screw L/D ratio of 24). The masterbatch compounding and film blowing process conditions are presented in Table 1. The OMt–LDPE nanocomposites were also prepared using the same method described above.

Fig. 1. XRD reflections of a) Mt, b) OMt and c) Ag + OMt.

3.3. Determination of the antimicrobial activity Antimicrobial activity of Mt, OMt, Ag + OMt powders and CPN thin films were determined using the E 2149 test of the American Society for Testing and Materials (ASTM). The average initial microbial culture (ATCC 11230) concentration was 106 CFU/ml. The Mt, OMt and Ag + OMt powders of 0.5 g were added to the separate flasks, each containing the test culture of 1 ml and the buffer solution (0.3 mM phosphate buffer, pH = 7.2) of 19 ml. The same procedure was conducted for CPN films of 5 × 5 cm (4 pieces in one flask). The flasks were then shaken for 5 h at 350 rpm (37 °C) in an orbital shaker. An inoculum of cell suspension in a flask without a test film was used as a control. After a series of dilutions of the bacterial solutions using the recovery diluent, 0.1 ml of the solution taking from the dilution of 1.10−4 was plated in nutrient agar. The inoculated plates were then incubated at 37 °C for 18 h and surviving cells were counted. The antimicrobial activity was expressed as % reduction of the organisms after contact with the test specimen compared to the number of bacterial cells surviving after contact with the control.

4. Results and discussions 4.1. X-ray diffraction

3. Characterization 3.1. X-ray diffraction (XRD) X-ray diffraction patterns of Mt, OMt, Ag + OMt powders and the CPN thin films were recorded at room temperature, on a Bruker D8 Advance diffractometer with Cu λ = 1.54 Å. The scan rate was 1.2°/min, over a diffraction angle 2θ ranging between 2° and 12°. The interlayer spaces were calculated using the Bragg's equation.

The XRD results of the pure Mt, OMt and Ag + OMt powders are shown in Fig. 1. The XRD patterns of Mt, OMt and Ag + OMt were revealed reflections at 2θ = 6.9°, 2θ = 4.6° and 2θ = 3.6° respectively. Using these values, the clay interlayer distance (d001) was calculated by the Bragg's equation as 1.26 nm, 1.88 nm and 2.43 nm respectively. These basal spacing values prove the successful intercalation of Mt layers. The results are also significant of which demonstrate Ag + adsorbed effectively into the interlayer of Mt along with CTAB.

3.2. Scanning electron microscopy (SEM) SEM micrographs of CPN thin films were taken using a Zeiss EVO® LS 10, at 3.89 × 10− 5 Torr, 5 kV in inert atmosphere with 3500 × magnifications. Table 1 CPN processing condition used in this study. Equipment type

Processing condition

Twin screw extruder (For the first masterbatch) Twin screw extruder (For the second masterbatch) Single screw extruder (For composite film production)

Screw speed: 200 rpm Barrel temp.: 40, 160, 160, 181, 164, 160 °C Screw speed: 400 rpm Barrel temp.: 40, 160, 160, 181, 164, 160 °C Screw speed: 250 rpm Barrel temp.: 40, 160, 160, 160, 165 °C

Fig. 2. XRD reflections of a) pure LDPE b) 1.25% CPN, c) 2.25% CPN and d) 5% CPN films.

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Fig. 3. SEM micrographs of surface of a) pure LDPE, b) 1.25%, c) 2.25% and d) 5% CPN.

The thin film forms of pure LDPE and CPN with varying concentrations (1.25, 2.25 and 5% Ag + OMt) were also measured with XRD. It is seen from Fig. 2 that all CPN show a clear reflection around 2θ angle of 4–6°, which can only be associated with the interlayer distance (d001) of the clay layers incorporated into the polymer matrix since pure LDPE did not present this characteristic pattern.

4.2. Scanning electron microscopy (SEM) SEM analysis was used to visualize the surface morphology of CPN thin films as shown in Fig. 3. SEM micrographs indicate that with increasing Mt amount, particle agglomeration becomes more pronounced as evidenced by the surface texture of the films. It should be noted that

Fig. 4. EDX spectrums of a) 1.25%, b) 2.25% and c) 5% CPN.


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Fig. 5. Antibacterial effect of E. coli on powder form of a) Mt, b) OMt and c) Ag + OMt.

this behavior is not unusual since higher concentration of nanoparticles dispersion into polymer matrix becomes difficult even under high shear force mixing conditions (Ataeefard and Moradian, 2011). 4.3. EDX analysis Energy dispersive X-ray spectroscopy (EDX) was used to analyze the elemental constitution of CPN thin films. Fig. 4 displays the spectrum of CPN films obtained by elemental microprobe analysis of EDX. The results show that carbon, silicon, aluminum and silver were the principal elements present in of CPN films. It is important to note that no Br was detected on these CPN film surfaces. It was reported that Br anion which exist in the CTAB molecule can react with silver cation to form AgBr nanoparticles. It is critical to note that unlike the present work, the CTAB and silver ions were reported to be added at the same time so that Br ion released from the CTAB molecule can react and form AgBr nanoparticles. In the present work, AgBr nanoparticle formation was not preferred due to the extreme sensitivity of AgBr to light (discoloration) and possible health effects (halogens) when CPN films used for

food packaging. As explained in the experimental procedure above, in the present study the Ag ions were first adsorbed on clay surfaces, the excess Ag ions were removed and then CTAB molecules were adsorbed with no chance to form AgBr (Liu et al., 2007; Moosavi et al., 2012) nanoparticles. EDX analysis, therefore, provides direct evidence that silver is embedded in the CPN films. EDX analysis also confirmed the existence of elemental silver at concentration varying from 0.28 to 0.59% measured on CPN thin film surfaces. 4.4. Antibacterial activity of CPN films The antibacterial efficacy of Mt, OMt and Ag + OMt against E. coli was tested in powder as well as CPN thin film forms. The powder form of Mt and OMt in the absence of silver (control), as expected, did not show any antibacterial activity (Fig. 5). Ag + OMt on the other hand, were found to exhibit good antibacterial activity as presented in Fig. 5. Fig. 6 also depicts the antibacterial activity of CPN films with varying Ag + OMt powder filler ratios. It is apparent from this figure that CPN

Fig. 6. The photographs above shows nutrient agar plates on E. coli containing different concentrations of CPN film: a) pure LDPE b) 1.25% CPN c) 2.25% CPN and d) 5% CPN according to ASTM E-2149 standard.

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Table 2 The effect of Ag + OMt concentration in CPN films for the reduction of bacteria. Ag + OMt% in LDPE

Number of colonies (in petri dish)

Percent reduction of bacteria

1.25 2.25 5 Control: 281

197 149 84

29.89 46.97 70.10

films containing silver exhibits potent antibacterial activity against E. coli with increasing Ag + OMt concentration. While the composite film with 1.25 (30% reduction) and 2.25% (47% reduction) of silver content exhibited slight bactericidal activity, CPN film with 5% of Ag + OMt content was showed a strong bacterial effect (70% reduction) against E. coli as presented in Table 2. 5. Conclusions Hydrophilic Mt with a size less than 310 nm was first ion-exchanged with silver ion followed by organic modification with quaternary amine CTAB at Mt surface and interfaces. The silver ion adsorbed and organically modified Mt (Ag + OMt) was then compounded with LDPE at 1.25, 2.25 and 5% (by mass) Ag + OMt concentrations using a high shear force twin screw extruder followed by blow molding using single screw extruder in order to achieve thin CPN films of 25–35 μm in thickness. XRD results were revealed the successful incorporation of both Ag+ and CTAB ions as evidenced by the increase of interlayer distance (d001) of the Mt layers from 12 Å to 24 Å. A minute amount of elemental silver at concentration varying from 0.28 to 0.59% was found on CPN thin film surfaces using the SEM/EDX analysis with no Br ion present. The antibacterial CPN film surface testing in liquid E. coli containing cultures demonstrated an effective bacterial reduction as much as 70%. These results are significant and indicate that much higher bacterial reduction is possible on surface contact testings of which may represent a more realistic bacterial adhesion schemes on surfaces.

Acknowledgment Financial support for this work was provide by Erciyes University, Scientific Research Division (BAP) project number FBA-11-3772 and is highly acknowledged.

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