Effect of chemical crosslinking on properties of chitosan-montmorillonite composites

Accepted Manuscript Effect of chemical crosslinking on properties of chitosan-montmorillonite composites Magdalena Gierszewska, Ewelina Jakubowska, Ewa Olewnik-Kruszkowska

PII:

S0142-9418(19)30041-8

DOI:

https://doi.org/10.1016/j.polymertesting.2019.04.019

Reference:

POTE 5872

To appear in:

Polymer Testing

Received Date: 8 January 2019 Revised Date:

26 March 2019

Accepted Date: 20 April 2019

Please cite this article as: M. Gierszewska, E. Jakubowska, E. Olewnik-Kruszkowska, Effect of chemical crosslinking on properties of chitosan-montmorillonite composites, Polymer Testing (2019), doi: https:// doi.org/10.1016/j.polymertesting.2019.04.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Effect of chemical crosslinking on … Magdalena Gierszewska et al.

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EFFECT OF CHEMICAL CROSSLINKING ON PROPERTIES OF CHITOSAN-MONTMORILLONITE COMPOSITES Magdalena Gierszewska1,*, Ewelina Jakubowska1, Ewa Olewnik-Kruszkowska1 Nicolaus Copernicus University in Toruń, Faculty of Chemistry, Chair of Physical Chemistry and

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1

Physicochemistry of Polymers, Gagarin 7 street, 87-100 Toruń, Poland; [email protected] (M.G.); [email protected] (E.J.); [email protected] (E.O.-K.)

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*Correspondence: [email protected]

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Abstract In order to determine the effect of crosslinking agent on the chitosan-based composites, new materials consisting of chitosan (Ch) as polymer matrix, montmorillonite (MMT) as nanofiller and glutaraldehyde (GA) as crosslinking agent were obtained. The structure and surface morphology of obtained materials

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were determined using Infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM) coupled with energy-dispersive X-Ray spectroscopy (EDX), transmission electron microscopy (TEM) and X-ray diffraction (XRD). Thermal properties were characterized based on

thermogravimetric results. Surface hydrophilicity was also studied. Obtained results indicate that the

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addition of glutaraldehyde beneficially affects properties of chitosan and its composites. AFM and SEM results indicate that the incorporation of glutaraldehyde into the pure polymer as well as in Ch-MMT

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systems reduces changes in the surface morphology. Moreover, covalent crosslinking with GA improves thermal resistance of investigated materials. Simultaneous modification of chitosan with MMT and GA leads to significant decrease in total surface energy (SFE).

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Keywords: chitosan; nanocomposite; crosslinking agent; montmorillonite; glutaraldehyde

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1. Introduction Chitosan (Ch) - the cationic copolymer of (1-4)-2-amino-2-deoxy β-D-glucopyranose and (1-4)-2acetamido-2-deoxy-β-d-glucopyranose - belongs to a group of biopolymers which are replacing synthetic polymers in various applications. It is industrially produced in varying quality grades from chitin which is

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the second most abundant polysaccharide in nature [1]. In aim to improve mechanical and thermal properties of chitosan as well as to change the hydrophobic-hydrophilic properties, nanofillers are

introduced into the polymer matrix. Currently, the most popular nanofillers are carbon nanotubes and

modified layered silicate such as, for example, montmorillonite (MMT) [2–4]. Montmorillonite belongs

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to a group of clay minerals which swell in water and possess high cation-exchange capacities. The

theoretical formula for montmorillonite is (OH)4Si8Al4O20·nH2O [5]. Composites based on chitosan and

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unmodified and modified montmorillonite were the object of prior studies [6,7]. Katti et al. [8] have obtained novel chitosan/montmorillonite/hydroxyapatite nanocomposite for bone tissue engineering. Barrier properties of chitosan-nanoclay materials were investigated by Giannakas et al. [7], while Lavorgna et al. [1] established estimated structural properties and antibacterial potential of chitosan filled with MMT-supported Ag nanoparticles. In the case of investigated materials it has been determined that

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the incorporation of nanofiller into polymer matrix decreases water permeability while increasing antimicrobial properties. Furthermore it should be noted that a number of researches have devoted their efforts to obtain chitosan-clay films with an addition of synthetic polymers such as poly(vinyl alcohol)

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[9,10] and hydroxyl-aluminum oligomeric cations [11] or biopolymers, with pectin being one such example [12]. However, we have to bear in mind that the optimal content of nanoclay should not exceed

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5%. It has been established that with a nanofiller load exceeding 5%, the obtained materials become brittle - their overall mechanical properties decrease significantly. Crosslinking is one of the method applied in order to improve mechanical properties of polymers [13]. This type of stabilization can be divided into physical and chemical modifications. In physically crosslinked materials crosslinkers that establish ionic interactions between the polymer chains, are used [14]. In the case of chitosan-based materials the most popular ionic crosslinking agents are: calcium chloride and pentasodium tripolyphosphate [15,16]. Chemical crosslinking is however as far more popular method. In the case of chemical modifications, permanent networks with a covalent bonding between the polymer chains and crosslinking agents are formed [14,17]. In recent years epichlorohidrine, glutaraldehyde (GA), genipin,

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dextran sulfate, oxidized cylclodextrins, as well as ethylene glycoldiglyceryl and ether, ethylene glycol diglycidyl ether (EGDE), have been studied for the purpose of using them as crosslinking agents [14,17,18]. In spite of extensively documented study devoted to chitosan-based composites as well as to crosslinking

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of chitosan, there has been no focus on research into the influence of both the nanofiller and the crosslinking agent on the properties of chitosan-based materials. There is no indication of any paper

reporting whether a synergistic effect occurs resulting in a significant increase in desired properties of

crosslinked composites, or if the addition of both components impedes possible applications of chitosan-

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based materials.

For this reason the aim of this study was to investigate the influence the introduction of a nanofiller in the

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form of montmorillonite and a glutaraldehyde as crosslinker into the chitosan matrix has got on surface, structural and thermal properties of obtained films. Structural changes in the obtained crosslinked composites were determined by means of Fourier transform infrared (FTIR) spectroscopy. Thermogravimetry (TG) technique was used in aim to study thermal stability and calculate the oxygen index of the samples. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were

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used in order to observe changes in morphology after addition of montmorillonite and glutaraldehyde. Energy Dispersive X-Ray analysis (EDX) was employed in the identifying of the elemental composition of materials. Surface hydrophilicity was also studied by contact angle measurements.

2.1.1. Materials

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2.1. Materials and methods

Commercially available chitosan from crab shells was purchased from BioLog Heppe GmbH (Garmany)

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and glutaraldehyde (25 wt% solution in water) of analytical grade was purchased from Sigma-Aldrich (Germany). The natural clay mineral—montmorillonite (MMT) used in this study was provided by the Riedel-de Haen. In the process of polymer films formation, acetic acid (POCh Poland) was used as a solvent.

2.1.2. Chitosan characterization The degree of deacetylation (DDA) of chitosan determined by potentiometric titration was 72.25±0.77 % and the viscosity average molecular weight (Mv) of chitosan solutions was 148±26 kDa. The details of determinations of chitosan parameters were described elsewhere [19].

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2.1.3. Preparation of chitosan based materials All chitosan films were prepared by casting and solvent evaporation technique. To obtain non-modified one-component chitosan film (Ch) 1% (w/v) chitosan solution in 2% (w/v) acetic acid was prepared, filtered, degassed and cast on a clean glass plate, then evaporated to dryness at 37 °C. Crosslinked

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chitosan/glutaraldehyde (ChGA) films were prepared by adding dropwise the 0.25 wt% aqueous glutaraldehyde to chitosan solution under gentle stirring (0.5 wt% of GA in casting mixture). Ch/GA mixture was left for 1h to reaction take place, then cast and dried as Ch films.

Chitosan nanocomposite films were prepared by intercalation of chitosan from solution. Montmorillonite

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was dispersed in 2% (w/v) acetic acid to obtain a 0.25 wt% clay solution (at room temperature), left for 24h under continuous mechanical stirring and then sonicated for 1h in a bath-type ultrasound sonicator

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(Elma D-78224 Singen/Htw., Germany). Chitosan/montmorillonite (ChMMT) and

chitosan/montmorillonite/glutaraldehyde (ChMMTGA) films were formed as Ch and ChGA, except that the known amount of MMT dispersion was added to chitosan solution, homogenized using magnetic stirring plate for 24h before casting or crosslinking and casting, respectively. Montmorillonite content in nanocomposite films was equal to 1, 3 and 5 wt% and was denoted in sample names as MMT1, MMT3

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and MMT5.

2.1.4. Fourier transform infrared spectroscopy (FTIR) Fourier transform infrared (FTIR) spectra of chitosan, chitosan/glutaraldehyde and nanocomposite

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(ChMMT and ChMMTGA) films in ATR (Attenuated Total Reflectance) mode with diamond crystal as well as of montmorillonite powder in a form of a KBr disc were recorded on Bruker Vertex 70 spectrometer in range 400-4000 cm-1. All spectra were recorded at the resolution of 4 cm-1 and 16 scan

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passes and analyzed using OPUS 7.5 software. 2.1.5. Thermogravimetric analysis (TG) TA Instruments, SPT 2960 simultaneous DSC-TGA was used for studying thermal behavior of investigated materials. TGA traces were monitored from room temperature to 600 °C at 10 °C·min-1 under air and nitrogen flow of 40cm3·min-1. To estimate the tendency of a material to sustain a flame, limiting oxygen index (LOI) was also calculated in accordance with Fenimore and Martin equation [20]: LOI = 17.5 + 0.4 CR500

(1)

where CR500 represents char yield at 500 °C under nitrogen atmosphere.

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2.1.6. Scanning electron microscopy (SEM) imaging and energy-dispersive X-Ray (EDX) analysis The morphology of each thoroughly dried film surface was observed by SEM using a LEO1430 VP scanning electron microscope (Leo Electron Microscopy Ltd., England). The cross-section elemental composition of thoroughly dried MMT modified films were examined using

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Energy Dispersive X-Ray (EDX) Spectrometer Quantax 200 with XFlash 4010 Detector (Brucker), installed in an environmental scanning electron microscope. The specimens for the EDX images of the cross-sections of the films were prepared by fracturing the films in liquid nitrogen. EDX data were

2.1.7. Atomic force microscopy (AFM) measurements

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collected and analyzed using Bruker Quantax software (QUANTAX ESPRIT v1.8.2).

AFM was used to visualize the topological morphology and to gather information on the surface

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roughness. The photograph of the nanocomposite film surface topography was made by means of a microscope with a scanning SPM probe of the NanoScope MultiMode type (Veeco Metrology, Inc., Santa Barbara, USA) operating in the tapping mode, in air, at room temperature. The roughness parameter such as the root mean square (Rq) was calculated for scanned area (5µm×5µm) using Nanoscope v6.11software.

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2.1.9. Transmission electron microscopy (TEM) imaging

Transmission electron microscopy (TEM) has been chosen to evaluate both: the dispersion of nanoclay in the chitosan matrix and characterization the type of nanocomposites. Ultrathin films for TEM observation

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were prepared via cutting at room temperature from the nanocomposite sheet embedded in epoxy resin with a Leica EM UC7 ultramicrotome with a diamond knife. Thin specimens were collected and placed on 200-mesh copper grids. The TEM observations were performed using TEM type Tecnai F20 X-Twin

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(FEI Europe) under an accelerated voltage of 100 kV. 2.1.10. X-ray diffraction

The structure of pure montmorillonite, chitosan as well as chitosan-based composites were determined by an X’Pert Philips Analytica, diffractometer type X-Pert PRO System, using Cu Ka filtered radiation (λ = 1.5418 Å) at 40 kV and 40 mA. The scattering angles ranged from 1° to 12°. The divergence slit size was 0.125, and the scan step time was 25 s. 2.1.11. Contact angle measurements and surface free energy (SFE) calculation

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To determine changes in hydrophilicity of chitosan films, contact angles (CA) of two test liquids of different polarity: diiodomethane (D) and glycerol (G) were measured at a constant temperature (24 °C). A drop of 10 µl was placed on the film surface and CA measurements were performed by taking a photograph of the drop of liquid. At least 10 photographs were taken of each film. Then CA values were

accuracy of ±2°. The surface tension

and its polar

and dispersive

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determined using microscope image processing software (ImageJ, NIH-freeware version) with an components were calculated

according to geometric mean by Owens–Wendt–Rabel–Kaelble [21]. The polar and dispersive

3. Results and discussion 3.1. Fourier transform infrared spectroscopy (FTIR)

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components of surface tension of the testing liquids were taken from literature [22].

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FTIR spectra of montmorillonite powder and films comprising chitosan, chitosan/glutaraldehyde and its nanocomposites have been presented in Figure 1.

Location of Figure 1

Certain differences between non-modified chitosan (Figure 1(A)) and nanocomposite or/and crosslinked films (Figures 1(C-E)) have been observed. According to our previous findings [23] some differences in

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the range between 3500-3150 and 1800-1550 cm-1 can be seen after crosslinking of chitosan with GA (Figure 1(C)). After adding of glutaraldehyde the band at about 3274 cm-1, resulting from O-H and N-H vibrations of chitosan functional groups engaged in hydrogen bonds [24], becomes more intense and

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wider. This indicates that some hydrogen bonds within chitosan structure are destroyed but new ones between Ch and GA are formed. The addition of GA also results in a change in position of characteristic Ch bands [25]: the band at 1640 cm−1 (C=O stretching in amide group, amide I vibration) shifts to 1633

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cm−1 and the band at 1547 cm−1 (N–H bending in amide group, amide II vibration) shifts to 1541 cm-1. In ChGA spectrum we cannot observe the band characteristic of the Schiff bases (C=N stretching band, depending on the compound, absorb in the range 1620–1660 cm−1 [26]) but it is justified to assume that the band at 1633 cm−1 relates most probably to the amide I band of chitosan and the C=N stretching band of the Schiff base. Additional confirmation that in the experimental conditions GA completely reacted with chitosan is the lack of a band at ca. 1715 cm−1, related to the free aldehyde group [24], and change of the chitosan film color from transparent to yellow.

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The spectrum of the ChMMT5 film revealed bands characteristic of both its components: chitosan and montmorillonite (Figure 1(D). After the addition of montmorillonite into chitosan matrix, a visible decrease in intensity of bands, resulting from vibrations of MMT and chitosan functional groups engaged in hydrogen bonds [24] in 3600-3000 cm-1 region, can be observed. These findings seems to indicate that

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some of hydrogen bonds in chitosan structure are destroyed but a new one between chitosan functional groups and Al-OH / Si-OH of montmorillonite is formed. Moreover, changes in the position and an

increase in the intensity of bands in the 1250-950 cm-1 region occur. This phenomenon can be related to overlapping of chitosan 1055 cm-1 (antisymmetric stretching of the C–O–C bridge) and 1024 cm-1

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(skeletal vibrations involving the C–O stretching [23]) bands with very strong 1054 cm-1 MMT band (SiO stretching [27]). New bands at 521 and 452 cm-1 in FTIR spectrum can be attributed to Si-O bending in

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montmorillonite structure.

In FTIR-ATR spectrum of the ChMMT5GA film (Figure 1(E)) bands characteristic of both ChMMT5 and ChGA films can be observed. New hydrogen bonds formed between Ch, GA and MMT result in a change in the position of 3274 cm-1 chitosan band to 3193 cm-1; as a result of the reaction between Ch and GA, the band at 1640 cm-1 shifts to 1633 cm-1 and the band at 1547 cm−1 shifts to 1542 cm-1. There is also

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a lack of band corresponding to vibration in free aldehyde group. Incorporation of MMT into ChGA matrix caused, as in the case of the ChMMT5 films (Figure 1 (D)), an increase in intensity of bands at 1250-950 cm-1. New bands corresponding to Si-O bending at 519 and 456 cm-1 can also be noticed.

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The FTIR analysis of ChGA and ChMMT5GA films confirmed that in the relevant reaction conditions amine groups of chitosan react with aldehyde groups of glutaraldehyde. Changes in the position and the intensity of FTIR bands observed in ChMMT5 and ChMMT5GA spectra justify the assumption that

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montmorillonite was successfully intercalated in biopolymer matrix. This has also been confirmed by XRD analysis presented in Figure 10. Based on above findings we have proposed a schematic structure of chitosan/montmorillonite nanocomposite crosslinked with glutaraldehyde – as presented in Figure 2. Location of Figure 2 Most of the methods in order to determine the chitosan crosslinking density are based on the assessment of decreasing number of chitosan functional groups being involved in the reaction of crosslink formation. According to what had already been established by Navarro and Monsan [28] and confirmed by Monteiro

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and Airoldi [29], in a normal occurrence of glutaraldehyde reaction with a primary amine group in acidic medium an imine bond is immediately formed, and less than 1h is apparently enough to complete the crosslinking process. Based on the well-known mechanism of chitosan crosslinking with glutaraldehyde it can be assumed that when the molar ratio of the amino groups in chitosan and glutaraldehyde is 2:1 the

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crosslinking degree is defined as 100%. Knowing the DDA value of chitosan and polymer to glutaraldehyde mass ratio we found that in the case of crosslinked Ch films -NH2 the glutaraldehyde

molar ratio to be 83:1. Therefore the crosslinking density of chitosan in ChGA and ChMMTGA is 2.4%. 3.2. Thermogravimetric analysis

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The results obtained by researchers indicate that the introduction of montmorillonite into polymer matrix influences thermal properties obtained materials. In the case of clay nanofillers however a number of

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factors such as structure of nanocomposite material, the degree of MMT dispersion or chemical constitution of surfactant molecule affect thermal stability [30]. In our work the effect of the presence of crosslinking agent and nanoclay on thermal stability of chitosan-based films was analysed by means of the thermogravimetric method. The degradation temperatures at 5 %, 10 % and 50 % mass loss (T5%, T10%

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and T50%) in air were chosen and presented in Table 1.

Table 1. TG data for chitosan (Ch) and its composites Temperature/˚C at mass loss a)

Sample

T10%

T50%

66.9

124.4

332.0

32.81

67.4

124.9

332.2

31.36

ChMMT1

58.5

110.1

328.1

32.09

ChMMT3

64.4

117.7

335.0

31.88

ChMMT5

61.6

112.6

332.2

31.92

ChMMT1GA

63.1

114.7

331.8

31.86

ChMMT3GA

66.1

121.4

335.5

31.90

ChMMT5GA

70.4

129.1

340.9

32.08

Ch

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ChGA

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T5%

LOI b)

a) b)

in air atmosphere in N2 atmosphere

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Moreover, TG curves of chitosan, chitosan with 5 wt% of nanofiller as well as the same samples containing an additional component – a crosslinking agent have been presented in Figure 3. TG results show that the incorporation of glutaraldehyde into chitosan matrix (Ch) does not noticeably change values of analyzed parameters while the addition of only the unmodified nanofiller causes a shift toward lower

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values when compared to neat chitosan. The reduction in the thermal stability in air (T5% and T10% values) of the films consisting of Ch and MMT is strongly connected with quantity of the nanofiller. It is well known that MMT may lead to accumulation of thermal energy in a nanocomposite material and to a degradation of the polymer [30]. However, in the case of studied materials the decrease in thermal

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stability values can be strongly related to the presence of free water and interlayer water. It has been

determined by Greene-Kelly [31] that the region of the mass loss of this kind of water occurs between 50

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and 300 °C. Moreover the films were obtained from a water solution, for this reason on the thermal curve of pure chitosan the T5% values below 100 °C. It has been indicated that in a range between 50 and 130 °C moisture vaporization can be observed [32].

Location of Figure 3

The obtained results of T50% in the case of chitosan/montmorillonite systems indicate that an addition of

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nanofiller up to 3 % can improve thermal stability of the chitosan matrix in air. On comparing the changes occurring in the thermograms a shift in thermogravimetric curves, towards higher temperatures has been observed in the case of samples containing nanofiller and crosslinking agent

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in comparison with pure chitosan as well as materials consist of chitosan and montmorillonite. Such behavior suggests that glutaraldehyde plays an important role in thermal stability of obtained materials. In the case of sample containing nanofiller and crosslinking agent evidently the discussed parameters: 5 %

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loss temperature (T5%), 10 % loss temperature (T10%) as well as the 50 % loss temperature (T50%) of the composites summarized in Table 1 are higher than those of neat chitosan. The most significant changes were observed in the case of sample containing 5 % of nanofiller. This means that crosslinking strongly influences the structure of chitosan based composites and volume of comprised water. This phenomenon indicates that glutaraldehyde has a stabilization effect on chitosan/MMT composites. Based on the results obtained by means of thermogravimetric method in nitrogen the flammable properties of the investigated materials were determined by calculating the value of oxygen index (LOI) according to Eq. 1 formulated by Fenimore and Martin [20].

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The values of LOI listed in Table 1 indicate that all studied materials are non-flammable or flame resistant (LOI > 21). The flame resistance does not change significantly after addition of a nanofiller or a crosslinking agent. At this point it should be mentioned that chitosan is used as a material which can improve thermal resistance [33]. It is well known that chitosan with an addition of nanoclays can be

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applied as a flame retardant. In the work of Hassan et al. [34] we can find information about synergistic effect of chitosan and modified clay on the flammability of low-density polyethylene. Furthermore,

chitosan can play role of modifier of montmorillonite and in this way can improve flame retardancy of polypropylene-based composites [35].

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Flame retardancy is related to its structure, especially to the presence of nitrogen. Nitrogen, aluminium as well as silica in the sample influence formation of char on the surface of the investigated material during

3.3. Atomic force microscopy

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heating which has already been observed and discussed [36].

Atomic force microscopy belongs to the most useful techniques providing further information on the surface morphology. For this reason the surface roughness of the obtained materials was observed using AFM method. In Figure 4 the morphology of chitosan and chitosan with an addition of unmodified

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montmorillonite are shown. The roughness parameters such as Ra and Rq are presented in Table 2. Location of Figure 4

Table 2. The roughness parameters (Ra and Rq) of chitosan (Ch) and its composites with and without

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addition of crosslinking agent

Ra [nm]

Rq [nm]

Ch

0.532 ± 0.012

0.677 ± 0.024

ChGA

0.480 ± 0.062

0.613 ± 0.053

ChMMT1

0.611 ± 0.057

0.759 ± 0.049

ChMMT3

0.541 ± 0.015

0.849 ± 0.008

ChMMT5

0.808 ± 0.041

1.03 ± 0.048

ChMMT1GA

0.476 ± 0.018

0.603 ± 0.026

ChMMT3GA

0.539 ± 0.022

0.707 ± 0.029

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ChMMT5GA

0.570 ± 0.043

0.714 ± 0.061

The images depict differences between the surface of pure chitosan (Ch) and chitosan-based composites (ChMMT1, ChMMT3, ChMMT5). The results indicate that introduction of nanofiller significantly

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influences the surface properties of discussed samples. Moreover, it needs to be mentioned that the values of Ra and Rq parameters increased according with the increase quantity of montmorillonite in the polymer matrix. The observed changes may be connected with the formation of MMT aggregates in the polymer matrix resuluting from hindered dispersion of nanofiler in Ch solution. In aim to establish the influence of

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crosslinking agent on the surface morphology of chitosan and chitosan/MMT composites in Figure 5,

examples of photographic images of chitosan as well as chitosan/MMT composites with an addition of

corresponding samples are showed.

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glutaraldehyde are presented. Moreover, in Table 2 the values of the roughness parameters for

Location of Figure 5

Based on the obtained results it was revealed that an addition of crosslinking agent decreases values of Ra and Rq parameters in the case of all samples containing glutaraldehyde. This phenomenon can be

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connected with rearrangement of macro-chains during formation of crosslinked materials and could result in lower values of surface roughness. The decrease in the roughness of surface is exceptionally significant in the case of the sample consisting of chitosan, 5 wt% of MMT and the crosslinking agent. In

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comparison with the ChMMT5GA sample chitosan filled with 5 wt% of MMT (ChMMT5) is characterized by a more corrugated surface and the Rq parameter is about 0.316 nm higher.

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3.4. SEM imaging and EDX analysis

Scanning electron microscopy (SEM) is one of the most useful techniques for studying texture, configuration, structure and chemical characteristics of clay samples as well as nanoscale materials. For this reason in aim to estimate the dispersion in the polymer matrix, the SEM method has been chosen. In Figure 6 the surfaces of chitosan and samples containing different amounts of nanofiller, are presented. Location of Figure 6 Based on obtained results it can be seen that the surface of pure chitosan is smooth, uniform and flat without cracks, irregularities, which was also observed by the other researchers [6]. However after introduction of montmorillonite into chitosan SEM microphotographs showed brighter areas indicating

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the presence of aluminosilicate structure elements with higher atomic numbers (e.g. Fe, Al, Si, Mg) which correspond to the nanofiller particles. In aim to determine the chemical composition of each sample and the distribution of the introduced clay, the EDX analysis was performed in cross-section. The study of the materials focused on establishing the distribution of elements such as aluminum (Al), silicon (Si), calcium

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(Ca), sodium (Na) and magnesium (Mg). The presence of these elements hints to the presence of clay. When using the EDX technique, the presence of aluminum derived from the equipment used during scanning electron microscopy should be taken into account. Figure 7 shows EDX analysis of the unmodified chitosan, chitosan filled with 5% of MMT and the same samples with an addition of

materials contain a nanofiller.

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Location of Figure 7

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glutaraldehyde. The analysis indicated the presence of Si and Na elements confirming that studied

The amount and size of observed domains in Figure 6 is directly linked to the increase in the amount of the nanofiller in the polymer matrix. The size of the particles ranges between 10 and 50 µm, however in the case of the ChMMT5 sample a few points of aggregated MMT with the size of about 70 µm can be seen. Obtained results indicate that the compatibility between chitosan and pure montmorillonite is not

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sufficient resulting in an increase in the size of MMT aggregate [37]. Figure 8 shows SEM microphotographs of the surface materials with an addition of glutaraldehyde as crosslinker. Location of Figure 8

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The surfaces of samples consisting of chitosan and glutaraldehyde appear relatively flat and smooth as does the pure polymer. However, in the case of polymer films containing nanofiller and a crosslinking agent it could be observed that the surfaces of the investigated materials were covered by oval-shaped

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particles of the 15–70 µm order. The number of aggregates on the surface increase significantly, especially in the case of the composites with a higher MMT quantity. Moreover, the size of some microdomains is greater in relation to composites without the crosslinker. Based on the obtained results it should be mentioned that the introduction of GA into a chitosan/MMT system increases the melt viscosity of the composites and in this way affects the dispersion of nanofiller in the polymer matrix. This phenomenon suggests that an increase in viscosity can influence the morphology modification by increasing the aggregation of nanofiller in chitosan [38].

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Furthermore, the observed changes in the SEM microphotographs do not correspond with the AFM results. It is well known that Rq parameter is the root mean square average of height deviations taken from the data plane. For this reason, certain discrepancies between SEM and AFM results can suggest that on the surface of crosslinked materials the aggregates of MMT are more numerous. The distances between

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the highest and the lowest data points in the image however are significantly lower in the case of samples containing glutaraldehyde. 3.5. Transmission electron microscopy (TEM)

The clay dispersion within the chitosan matrix was characterized by TEM imaging which is the most

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popular technique used in order to establish the internal morphology of composites. Figure 9 illustrates the TEM micrographs of chitosan/montmorillonite (ChMMT) and

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chitosan/montmorillonite/glutaraldehyde (ChMMTGA) films containing 3 and 5 wt% of nanofiller. Location of Figure 9

The dark lines/platelets represent clay tactoids while the gray base corresponds to the chitosan matrix. Obtained results indicate that MMT dispersed in the chitosan matrix forms intercalated (well-ordered multilayer structure composed of polymer chains intercalated into the silicate layers) and exfoliated

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structure (silicate layers fully homogenously dispersed in the polymer matrix), what is consistent with the observations made by Wang et al. [39] and Dias et al. [40]. It is not a novelty that chitosan exhibits good miscibility with MMT and thus can be easily intercalate into the interlayers of nanofiller (Figures 9 A, B).

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In the case of ChMMT5GA system besides intercalated and exfoliated structures some aggregates of MMT can be observed (Figure 9 C, D). Crosslinking of chitosan with glutaraldehyde result in increase viscosity of casting solution as well as in decrease of available hydrophilic amino groups of chitosan.

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Moreover, covalent crosslinks formation causes lower chitosan chain mobility. For this reason, dispersion of nanofiller in crosslinked matrix as well as intercalation of chitosan chains can be more difficult in comparison to pure chitosan matrix. Observations made on the basis of images obtained by means of TEM technique are in accordance with results presented by XRD curves (Figure 10). Obtained results clearly indicate that the introduction of pure montmorillonite into chitosan allows formation of intercalated composites [40]. The characteristic diffraction peak for pure MMT at about 2θ = 6.23° Location of Figure 10

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corresponds to the spacing between the individual layers of analyzed montmorillonite d = 14.18 Å. However, after incorporation of MMT into the chitosan matrix the shift of the described peak towards the lower angles (5.17°, d = 17.08 Å in the case of ChMMT5GA sample) was observed. This indicates that an expansion of the MMT layers has taken place and it proves that intercalated composites were obtained.

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Moreover, apart from the movement of signal to lower angles, the broadening of the peak can be seen. It can be attributed to the presence of simultaneously exfoliated and intercalated structures [39]. In the case of samples with an addition of the crosslinking agent the same tendencies are visible. 3.6. Contact angle measurements and surface free energy calculation

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To estimate an effect of nanofiller addition on hydrophilicity of chitosan film surface, diiodomethane (D) and glycerol (G) contact angles were measured at room temperature and used in order to calculate surface

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free energy (SFE) according to Owens–Wendt–Rabel–Kaelble method [21]. The experimental results would also aid the understanding of the thermodynamic behavior of nanocomposite films in contact with a droplet of liquid. The obtained contact angle is the average value from 10 tests, with error normally within ± 2°. Both values of contact angle of D and G as well as the total surface energy, the disperse and the polar components of films SFE are listed in Table 3.

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Table 3. Values of contact angles (°), surface free energy ( components

Contact angle [°]

) and dispersive (

)

Surface-energy components [mN·m-1]

G

D

76.1 ± 0.2

54.1 ± 0.4

33.5±0.2

32.0±0.3

1.5±0.3

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Ch

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Sample

) and its polar (

ChGA

89.1 ± 1.4

58.5 ± 0.2

29.5±0.8

29.4±1.0

0.1±0.7

ChMMT1

77.7 ± 1.2

57.5 ± 1.4

31.6±1.1

29.9±1.2

1.7±1.4

ChMMT3

81.6 ± 0.6

61.3 ± 0.9

29.1±0.6

27.9±0.9

1.1±0.7

ChMMT5

82.1 ± 0.8

63.5 ± 2.0

28.0±1.5

26.7±1.6

1.2±1.3

ChMMT1GA

79.5 ± 0.7

58.2 ± 1.6

30.7±1.1

29.5±1.3

1.2±0.9

ChMMT3GA

83.6 ± 0.8

62.5 ± 0.8

28.1±0.8

27.2±0.8

0.9±0.7

ChMMT5GA

84.1 ± 0.4

67.1 ± 0.3

25.9±0.3

24.5±0.3

1.4±0.4

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The accuracy of the contact angle measurements can be influenced by film surface structure [41]. As indicated by AFM analysis (Table 2), Ra values are lower than 0.1 µm, therefore the roughness of the analyzed surface does not cause significant thermodynamic hysteresis. The smaller the contact angle, the better the surface wettability and vice versa. The comparison between

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contact angle values of D and G liquids indicates that the surfaces of non-modified, MMT modified and/or covalently crosslinked chitosan films are rather hydrophobic. The lowest contact angle of glycerol on chitosan film surface suggests that it is most hydrophilic among others. With an increasing amount of MMT, contact angles of glycerol increase and as a result the hydrophobicity of chitosan films also

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increases. Similar phenomenon was observed earlier by others in the case of MMT modified chitosan and polyvinyl alcohol films [10], methyl cellulose/montmorillonite [42] and cellulose/montmorillonite

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nanocomposite films [43]. Taking into consideration chemical structure of chitosan, that exhibits a large number of hydrophilic functional groups (-OH, -NH2), it can be concluded that these groups are hidden below the film’s surface. Moreover, a reduction in surface’s hydrophilic properties, observed after MMT addition, is due to the presence of the silicate layers of the nanoparticles.

It can be also seen that the GA crosslinking effect of decreasing surface wettability by glycerol is

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analogous in the case of both pure- and MMT-modified chitosan films. It is a result of decreased number of chitosan –NH2 functional groups that reacted with glutaraldehyde in crosslinking reaction process. Changes in chitosan surface structure after modification can be also determined based on SFE values. The

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γS for Ch is low and corresponds with data presented in literature [44]. After the addition of either MMT or glutaraldehyde or both of these substances, the films’ total SFE decreases. The reduction in surface component’s value while the polar

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energy is mostly related to the decrease in the dispersive

component changed little with the variation of the MMT content. The observed effect of MMT nanofiller on surface free energy corresponds with the data presented by Gao et. al [45] for chitosan polyacrylonitrile films. 4. Conclusions

In this study, novel chitosan/ montmorillonite nanocomposites non-modified as well as modified with glutaraldehyde as crosslinking agent were obtained. Based on the FTIR analysis it was confirmed that chitosan and glutaraldehyde form chemical crosslinks and montmorillonite, if added, is entrapped within this polymeric network. Thermogravimetric analysis indicated that crosslinked nanocomposites exhibit

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improved thermal stability than corresponding ChMMT films. Moreover, formation of chemical network reduced surface roughness and beneficially affected the dispersion of nanofiller in the polymer matrix. Analysis of surface free energy and its components indicated that addition of glutaraldehyde improved hydrophobic character of studied materials in relation to materials without crosslinking agent.

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Comparison of TG, AFM, SEM and SFE results obtained in the case of all of the investigated films proved that addition of glutaraldehyde has got a beneficial effect on selected physicochemical properties of new materials and can be an efficient way of manufacturing chitosan-based nanocomposites with improved properties.

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5. Funding

This work was supported by Nicolaus Copernicus University in Toruń, Faculty of Chemistry, statutory

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funds. 6. Data Availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study and due to time limitations.

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Figure captions Fig. 1. FTIR spectra of chitosan (A), montmorillonite (B) and ChGA (C), ChMMT5 (D), ChMMT5GA (E) films Fig.2. Schematic structure of chitosan/montmorillonite nanocomposite crosslinked with glutaraldehyde

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Fig. 3. TG curves of non-crosslinked and glutaraldehyde crosslinked chitosan and chitosan/montmorillonite films.

Fig. 4. AFM images of the surface of chitosan (Ch) and its composite with montmorillonite Fig. 5. AFM images of the surface of crosslinked chitosan (ChGA) and its composite with

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montmorillonite

Fig. 6. SEM microphotographs of the surface of chitosan and chitosan/MMT composites

and chitosan/montmorillonite films

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Fig. 7. EDX cross-sectional microanalysis of non-crosslinked and glutaraldehyde crosslinked chitosan

Fig. 8. SEM microphotographs of the surface of crosslinked chitosan (ChGA) film and crosslinked chitosan composites

Fig. 9. TEM images of ChMMT3 (A), ChMMT3GA (B) and ChMMT5GA (C, D) films

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Fig. 10. XRD patterns of chitosan, montmorillonite, ChMMT and ChMMTGA nanocomposites

Table captions

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Table 1. TG data for chitosan (Ch) and its composites Table 2. The roughness parameters (Ra and Rq) of chitosan (Ch) and its composites with and without

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addition of crosslinking agent

Table 3. Values of contact angles (°), surface free energy (

) and its polar (

) and dispersive (

)

components

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ACCEPTED MANUSCRIPT Highlights • Glutaraldehyde (GA) was used to reinforce chitosan/montmorillonite (Ch/MMT) films. • TEM analysis confirmed intercalated and exfoliated structure of Ch/MMT and Ch/MMT/GA nanocomposites. • GA crosslinking resulted in improved thermal stability and reduced surface roughness of

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Ch/MMT films. • Aggregation of nanofiller particles in Ch matrix took place for 5 wt% MMT content. • GA crosslinking results in decreasing surface wettability of pure chitosan and

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chitosan/montmorillonite films by glycerol.