Assessment of kinetic release of thymol from LDPE nanocomposites obtained by supercritical impregnation: Effect of depressurization rate and nanoclay content

Accepted Manuscript Macromolecular Nanotechnology Assessment of kinetic release of thymol from LDPE nanocomposites obtained by supercritical impregnation: Effect of depressurization rate and nanoclay content Adrián Rojas, Alejandra Torres, Francisca Martínez, Leonardo Salazar, Carolina Villegas, María José Galotto, Abel Guarda, Julio Romero PII: DOI: Reference:

S0014-3057(17)30412-3 http://dx.doi.org/10.1016/j.eurpolymj.2017.05.049 EPJ 7910

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

7 March 2017 26 May 2017 26 May 2017

Please cite this article as: Rojas, A., Torres, A., Martínez, F., Salazar, L., Villegas, C., José Galotto, M., Guarda, A., Romero, J., Assessment of kinetic release of thymol from LDPE nanocomposites obtained by supercritical impregnation: Effect of depressurization rate and nanoclay content, European Polymer Journal (2017), doi: http:// dx.doi.org/10.1016/j.eurpolymj.2017.05.049

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Assessment of kinetic release of thymol from LDPE nanocomposites obtained by supercritical impregnation: effect of depressurization rate and nanoclay content Adrián Rojasa,b, Alejandra Torresb, Francisca Martínezb, Leonardo Salazarb, Carolina Villegasb, María José Galottob , Abel Guardab, Julio Romeroa* a) Laboratory of Membrane Separation Processes (LabProSeM), Department of Chemical Engineering, University of Santiago de Chile. b) Food Packaging Laboratory, Department of Food Science and Technology, Center for the Development of Nanoscience and Nanotechnology (CEDENNA), University of Santiago de Chile. *Corresponding Author: Chemical Engineering Department, University of Santiago de Chile, Santiago, Chile, Phone: +56(2) 27181827, Email: [email protected]

ABSTRACT LDPE nanocomposites prepared with different concentrations of an organo-modified montmorillonite (OM-MMT) (2.5 and 5.0% (w/w)) were impregnated with thymol using supercritical carbon dioxide, with the aim of obtaining an antimicrobial packaging. Impregnation assays were carried out at 12 MPa, 1 h, 313 K and two depressurization rates, 10.0 and 1.0 MPa min-1, with impregnation yields in the range of 0.36 to 1.19% (w/w). The highest incorporation percentages of thymol were obtained employing the lowest depressurization rate. Moreover, thymol incorporation was favored with the presence of nanoclays. XRD results indicated the improvement of nanocomposites intercalation impregnated with thymol. Simultaneously, a phenomenological mass transfer model has been used to describe the release of thymol from the nanocomposite in ethanol 95% (v/v) solution. From the model, the effective diffusion coefficient of thymol through the nanocomposite seem to be independent of the depressurization rate employed. These values

were ranged from 2.7 to 3.5 x 10 -13 m2 s-1 and are significantly lower than the values obtained for free nanoclay LDPE films, which shows values ranged from 1.0 to 1.3 x 10 -12 m2 s-1. This result could be explained by the formation of a polymer-nanoclay intercalated structure that slows down the thymol transfer.

Keywords: Supercritical impregnation process; LDPE nanocomposite; thymol mass transfer.

1. Introduction In the last decades several factors, among which are the need to avoid losses and food waste and to increase the consumption trend of minimally processed food, have motivated the development of new packaging technologies. One of them is active packaging, an innovative concept that involves an active protection for food through the establishment of beneficial interactions between food, packaging and the environment in order to extend the shelf life, increase the safety and/or enhance sensory properties, while the quality of the products is preserved [1]. Among the various active packaging types, those that possess the ability to release antimicrobial substances have gained great attention. For the development of an antimicrobial packaging, great emphasis has been given to the use of natural compounds because the strategies currently used to reduce food spoilage by microorganisms show severe disadvantages as the consumer rejection grows towards the use of synthetic additives and the mode of application of these is useless [2]. A widely used type of natural compounds are essential oils because these compounds generally have low toxicity and are recognized as safe for human consumption [3]. Thymol is one of the most abundant component of the essential oils of thyme and oregano and has received considerable attention for the development of antimicrobial materials for food packaging [4, 5]. The effectiveness of an antimicrobial packaging is primarily determined through the release rate of the antimicrobial compound from the polymer material. Thus, the active compound should reach a minimal inhibitory concentration as fast as possible. It must be considered that a very slow release could mean that microbial growth is not inhibited, while a very rapid release could involve that the inhibition is not sustained.

An alternative to modify the release rate of an active compound is offered by nanotechnology, a term that encompasses a range of technologies that understand and control matter and processes at nanoscale (1-100 nm). It is a fairly new technology, and one of its first commercial applications within the food sector is in packaging, with the development of nanocomposites [6]. The nanocomposite materials consist of at least two individual constituents. One is usually the matrix and the other corresponds to nanoparticles used as reinforcing phase. The matrix surrounds and holds in place the nanoparticles, as these provide the composite of specific chemical or physical properties [7]. In the 1990s, research into the use of nanocomposites for food packaging started with the use of montmorillonite clay type as nanofiller in various polymers, offering extraordinary benefits to improve fundamental characteristics in food packaging such as thermal, mechanical and barrier properties [8, 9]. A feature of the polymer matrix modification using nanoclays is the generation of a tortuous path that could produce an increase on the path length for diffusion of solute molecules modifying the mass transfer processes through the polymer (permeability and migration). Thus, one of the most important aspects to be taken into account to obtain the best results in the polymer properties is how the nanoclay is dispersed in the matrix [10]. In an intercalated structure, one or more extended polymer chains are intercalated between the clay layers, resulting in a well-ordered multilayer morphology, while in an exfoliated nanocomposite, the silicate layers are completely separated from one another and are well dispersed. The exfoliated structure has been shown to exhibit the most significant improvement over the physical properties of the material [11].

One aspect to be taken into account for the development of active polymers is the method of incorporation of the active compound in the plastic matrix. Extrusion process it is the most common method used in the industry. Nevertheless, its application is limited for nonthermosensitive active compounds [12]. The second alternative is the incorporation by means of coating, having as main disadvantage the requirement of great amounts of active compounds and the excessive volatilization of active compounds with high vapor pressure [13]. A new alternative for active compound incorporation has arisen, the supercritical fluid impregnation. A supercritical fluid is a substance at a temperature and pressure conditions above their respective critical values [14]. In a supercritical state, a fluid shows physical properties intermediate between its gas and liquid phase. The similarity of the density of a solvent on its supercritical state with the density on its liquid state provides a high solvent power, while a viscosity and diffusivity similar to those from its gaseous phase with a surface tension near to zero provides excellent transport properties to the supercritical fluid. The supercritical impregnation process is basically the reverse process to supercritical extraction, where a substance is dissolved in the supercritical fluid and the high diffusivity and low surface tension of the fluid allow the polymer to swell and deposit or promote absorption of a compound within a polymeric matrix [15]. Among impregnation applications, especially those linked to the food and pharmaceutical industries, the most used fluid is carbon dioxide (CO2), because it is relatively inert towards reactive compounds, non-flammable, tasteless, odorless, has a relatively low cost and has relatively low values of critical pressure and critical temperature (P>Pc= 7.38 MPa and T>Tc= 304.15 K) [16]. In this sense, the critical point is not extremely high, particularly in terms

of temperature. Therefore, organic compounds dissolved in supercritical CO 2 (scCO2) are not susceptible to thermal degradation. Another advantage of scCO2 is its high diffusivity combined with its easily adjustable solvent power. Moreover, CO 2 is gaseous at ambient conditions of temperature and pressure, which facilitates the recovery of the analyte and provides solvent-free analytes [17]. The supercritical impregnation process has been employed as an alternative method for the development of controlled release materials, with applications in patches [18] and medicated contact lenses [19-21] and in the last years for the development of active polymers for food packaging employing natural compounds [22-28]. In this framework, this work is focused on the characterization of the thermal, morphological and structural properties of LDPE nanocomposites, containing different concentrations of an organo-modified montmorillonite (OM-MMT), impregnated with thymol by means of scCO2. Moreover, on the experimental and theoretical characterization of mass transfer during the release of thymol from LDPE nanocomposites to a fatty food simulant. Thus, mass transfer properties of thymol within the nanocomposites were analyzed and explained in terms of the results obtained from the thermal and structural characterization of the nanocomposites. To our knowledge, this is the first study on supercritical impregnation of thymol in a nanocomposite material and its kinetics release assessment to be applied in food packaging.

2. Experimental section 2.1 Materials LDPE (MFI: 0.75 g/10 min at 190 ◦C/2.16 kg, 923 kg m−3) was obtained from Borealis (Vienna, Austria). The commercial OM-MMT Cloisite® 20A (C20A) (90meq/100g) was provided by Southern Clay Products (Texas, United States), thymol (≥ 99.5%) was purchased from Sigma-Aldrich (Darmstadt, Germany), Toluene (≥ 99.9% HPLC grade) from Merck (Darmstadt, Germany), methanol (≥ 99.9% HPLC grade) from J.T. Baker (Center Valley, Unites States) and carbon dioxide from Linde (Santiago, Chile).

2.2 Nanocomposite preparation LDPE films without and with the OM-MMT C20A at different concentrations (2.5 and 5.0% (w/w)) were obtained by melt extrusion in a pilot scale extruder Scientific LabTech LTE20 model (Samutprakarn, Thailand) installed in the Food Packaging Laboratory (LABEN) at University of Santiago de Chile. The temperature profile used was 170 – 185 °C with a screw speed of 20 rpm and a chill roll speed equal to 1.7 rpm.

2.3 Supercritical fluid impregnation LDPE films and LDPE nanocomposites with different concentrations of OM-MMT C20A (2.5 and 5.0% (w/w)) were impregnated with thymol in order to study the effect of nanoclay content on thymol incorporation by means of a supercritical impregnation process. Supercritical fluid impregnation tests were implemented using the apparatus schematically described in Fig.1. These impregnation assays were carried out in a 100 mL high-pressure view cell, where previous tests were developed in order to determine the initial amount of thymol to be loaded in the cell to reach complete miscibility of thymol in

dense carbon dioxide at the impregnation conditions: 12 MPa and 313 K. From these experiments, it was possible to observe the total dissolution of 1 g of thymol in the 100 mL view cell filled with scCO2. This result is in agreement with the solubility reported in literature by Milavanovic and coworkers [29], who quantified the solubility of thymol in dense CO2 at 12 MPa and 313 K obtaining a value equal to 1.447 g in 100 mL. Supercritical impregnation tests were carried out following the procedure applied in previous studies by Rojas and coworkers [22] and Torres and coworkers [27]. Thus, 8 films with a surface of 25 cm2 were placed into the high-pressure cell and separated by metal supports, in order to avoid direct contact between the films and ensuring homogeneous impregnation of both sides of the polymer. Once sealed, the high-pressure cell was repeatedly purged with gaseous CO2 in order to remove the contained air. Subsequently, liquid CO2 was loaded in the system and once the cell was filled, the system was isothermally pressurized by means of a syringe pump Teledyne Isco 500D (Nebraska, United States), which operated at a constant pressure regime during the impregnation runs. The temperature of the high-pressure cell was controlled using a thermostatic electric resistance Electrothermal model MC 810 (Staffordshire, United Kingdom) around the cell. Supercritical impregnation runs were done at 12 MPa, and two different depressurization rates (1.0 and 10.0 MPa min-1) maintaining a constant temperature of 40 °C for 1 hour. After this time, the cell was depressurized by means of a micrometering needle valve Autoclave Engineers® model 10VRMM2812 (Pennsylvania, United States). Finally, impregnated films were submitted to the characterization assays.

2.4 Determination of thymol amount impregnated in LDPE and LDPE nanocomposites The average concentration of thymol in LDPE films and LDPE nanocomposites after the supercritical impregnation was determined according the method described by Galotto with some modifications [29, 30]. 0.2 g of impregnated LDPE films or nanocomposites were dissolved into a centrifuge tube with 20 ml of toluene. These bottles were placed in a thermostatic bath Heidolph model Hei-VAP (Schwabach, Germany) at 90 °C until there was complete dissolution of the polymer. Subsequently, after the temperature of the solution reached a value near 20 °C, 30 mL of ethanol was added into the bottle to produce the precipitation of the polymer. Then, the solution was centrifuged using a Hettich centrifuge model 32R (Tuttlingen, Germany) at 4500 rpm and 20 °C for 10 min in order to separate the phases. Finally, the supernatant was recovered by means of a syringe with a 45 µm internal filter and its thymol concentration was analyzed by high performance liquid chromatography, following the method explained in section 2.6.1. Before thymol determination assays, impregnated films were superficially washed and cleaned using a paper towel in order to avoid mistaken quantification of initial concentration of thymol in the nanocomposite. This procedure removed superficial residues caused by thymol re-solidification when the cell is depressurized.

2.5 Characterization of the nanocomposites 2.5.1 X-ray diffraction (XRD) XRD analysis was performed to analyze the interlayer spacing in nanoclay particles and to evaluate the capacity of the supercritical processing method to improve clay intercalation. These assays were carried out in a Siemens Diffractometer model D5000 (Texas, United States) (30mA and 40Kv) using CuKa (ʎ=1.54Å) radiation at RT. All scans were

performed in a 2θrange 2-12° at 0.02°seg-1. Inter-laminar distances were calculated using Bragg´s law: (1) Where ʎ is the wavelength of the radiation and θ the measured diffraction angle.

2.5.2 Thermal properties Thermal properties of LDPE films and LDPE nanocomposites were determined by means of a Differential Scanning Calorimeter using a Mettler Toledo DSC model 822e (Schwarzenbach, Switzerland). Film samples of 10 mg were placed in hermetically sealed DSC capsules and heated from 25 to 200 °C at a rate of 10 °C min-1. All experiments were done under purge of dry nitrogen. Commercial samples of indium (99.999% purity) with a melting point of Tm =156.68 °C and a melting enthalpy of ΔH m = 34.8 J g-1 were used as a calibration standard. The crystallinity (%) of the materials was estimated from the melting enthalpy. Thermogravimetric analyses (TGA) were carried out using a TGA/DSC GC20 Mettler Toledo thermal analyzer (Schwarzenbach, Switzerland) in order to study the influence of nanoclays and the supercritical impregnation process of thymol on the thermal stability of the materials. Samples of around 7 mg were heated from 25 to 600 °C under a nitrogen atmosphere.

2.5.3 Attenuated total reflectance - Fourier transform infrared (ATR–FTIR) spectroscopy FTIR spectra of the different materials were done by means of a Bruker Alpha spectrometer (Massachusetts, United States) equipped with an attenuated total reflectance diamond crystal accessory. The spectra were obtained with a resolution of 4.0 cm−1 in a wave-

number range from 4000 to 400 cm−1 with 30 scans. The spectra analysis was done using OPUS® Software Version 7.

2.6 Kinetic release of thymol from nanocomposites 2.6.1 Release test procedure The last step of the material characterization implied the experimental and theoretical description of the release kinetics of thymol, which was studied by means of experimental specific migration analyses, with the aim to study the transport and thermodynamic properties of the active compound. Migration experiments were carried out in accordance with the European Committee for Standardization [31] according to the methodology detailed in previous work [22, 27, 30]. Impregnated nanocomposite samples with a surface area of 50 cm2 were totally immersed into glass tubes filled with 130 ml of ethanol 95% v/v solution (fatty food simulant). Films were placed in the tubes using metal supports in order to avoid direct contact between samples and with the tube’s wall. These tubes were placed in an oven at 40 °C during at least 4 days. Food simulant samples of 1 mL were collected from the migration tubes as a function of time without replacement since its replacement would generate a thymol quantification error higher than its value without refill [32]. Thymol quantification was performed through an HPLC method carried out in a Hitachi LaChrom Elite machine (Tokyo, Japan) equipped with a diode array detector Hitachi L-2455 and autosampler Hitachi L-2200. The chromatographic column used was a Chromolith FastGradient RP-18 end capped 50–2 mm from Merck (Darmstadt, Germany). The mobile phase was acetonitrile: distilled water (30:70, v/v) mixture with a flow rate of 1.0 mL/min and an injection volume of 5 µL. Thymol detection was performed at a wavelength of 283 nm. The

calibration curve was made by fitting peak area and thymol concentration of standard solutions from 5 to 500 mg kg–1 (ppm), with three samples for each thymol concentration. Thymol was identified by comparison of its retention time and UV spectra with those of an injected pure standard using the same HPLC conditions. Release experiments were carried out until the equilibrium condition was observed, when concentration of thymol becomes practically constant in time in at least two continuous consecutive measurements. The release rate of thymol from LDPE and LDPE nanocomposites was analyzed in terms of Cloisite 20A content and the depressurization rate after the supercritical impregnation process. Thymol release kinetics was expressed as a function of the thymol amount released to the food simulant per surface area of the material sample.

2.6.2 Determination of partition and diffusion coefficients of thymol in impregnated LDPE nanocomposites Fig. 2 shows an outline of the system analyzed in this work. The Figure 2.a shows thymol transfer through an ideal LDPE film represented as a homogenous material, in which the mass transfer is driven by the concentration gradient of thymol with profiles that are discontinuous at the interface where the thermodynamic equilibrium between the polymer and liquid simulant phase is established. Meanwhile, Fig. 2.b shows thymol transfer through LDPE nanocomposites, which are represented as a non-continuous system because of the nanoclay inclusion in the polymer matrix. In this work, this system will be described by means of an effective Fick’s law approach where the polymer-nanoclay ensemble is considered a continuous medium in terms of its mathematical description. Therefore, the mass transfer process for both analyzed systems can be explained by means of a

resistances-in-series approach based on the one-dimensional simplification of Fick's Law [3, 29, 33, 34]. Thus, instantaneous mass transfer of the released compound can be estimated by molecular diffusion through the materials. This mechanism can be described through equation 2:

JI 

Deff L2



M M  Cthymol ( x  0, t )  Cthymol ( x  L / 2, t )



(2)

Where JI is the mass transfer flux (kg m-2 s-1) of the released thymol through the material (LDPE films or LDPE nanocomposites), Deff is the effective diffusion coefficient of thymol M

in the materials (m2 s-1), Cthymol (kg m-3) is the concentration of thymol in the material bulk, and L is the film thickness (m). In this case, the configuration of the release experiments allows the symmetrical description of the mass transfer with equation 2 from both sides of the film. The dimensionless distribution coefficient of thymol between the material and the food simulant is represented by the ratio of concentrations in the interface: K M / FS 

M Cthymol ( x  L / 2, t ) FS Cthymol ( x  L / 2, t )

(3)

This value can be directly estimated from the results of the release experiments at the FS x  L / 2, t  is the equilibrium concentration of thymol equilibrium condition, where Cthymol M x  L / 2, t  is its value at the interface in at the interface in the food simulant and Cthymol

the material. This last value can be estimated as a function of time through mass balance. Finally, the next step in the release process is the transfer through the boundary layer in the food simulant phase at the proximities of the interface. The transfer mechanism considered

in the model was natural convection because the food simulant solutions were not stirred during the release assays. Thus, this step can be described by equation. 4: FS FS J II  k  Cthymol ( x  L / 2, t )  Cthymol ( x  , t )

(4)

FS

Where Cthymol is the thymol concentration in the food simulant bulk (kg m-3) and k (m s-1) is the thymol mass transfer coefficient that quantifies natural convection in the food liquid phase. A few assumptions had to be made in order to use this model: 1.- The initial thymol concentration in the materials is known and is homogeneously distributed, C thymol x  o, t   C thymol,0 ; 0  x  L/2. M

M

2. - The food simulant is initially thymol free: Cthymol x,0  0 . FS

3.- There is no chemical interaction between food simulant and polymer (no swelling). 4.- There is no mass transfer limitation in the food, so thymol is always homogeneously distributed in the food simulant. In this work, we suppose that thymol concentration released from the materials to the food simulant is homogeneously distributed. This assumption was considered valid taking into account the value of the diffusion coefficient of thymol in the food simulant, which is equal to 5.67×10−10 m2 s−1, estimated with a correlation applied in a previous work [30]. This value is significantly higher than the value for thymol in both materials, which could easily be two orders of magnitude lower. Thus, the mass transfer rate is mainly controlled by the diffusion through the polymer, and the transferred compound can be assumed as homogeneously distributed in the food simulant solution.

2.6.3 Mass transfer model numerical solution These mathematical model equations describing molecular diffusion of thymol through the materials, as well as the material/simulant distribution coefficient and transfer through the boundary layer in the food simulant phase at the proximities of the material/food interface were applied in a previous study developed by the same working group [3, 22, 29, 33] . Mass transfer equations were solved under steady-state condition for an instantaneous time. Regula Falsi method was applied in order to reduce the number of interfacial concentration values of thymol iterations. Taking into account that the initial thymol concentration in the material and the simulant bulk are known, an iterative calculation can be done to estimate the mass transfer flux through the interface at steady-state condition (JI = JII) for a specific time step. Iterative variables in this calculation are the interfacial concentrations of thymol in both phases. When this numerical solution was identified, the bulk concentrations of thymol in the polymer and in the food simulant were recalculated by mass balance, starting a new iterative calculation for the next time step. These calculations were implemented by means of a program built in Matlab 7.1. Simulations were developed, using experimental values of the KM/FS defined in Eq. (3). Different De values of thymol in the materials were considered involving the lowest values of RMSE (root of the mean-square error) between experimental data and values predicted by the mathematical model. The root of the mean-square error (RMSE (%)) was calculated using Eq. (5). This measures the fit between experimental and estimated data [30] :

RMSE 



1 1 N      M FS ,t exp erimental,i  M FS ,t  predicted,i M M ,0  N  i 1



2

(5)

Where N is the number of experimental points for each release curve; i is the observations number; MM,0 is the initial amount of thymol in the material (µg) and MFS,t is the thymol amount in the food simulant at time t (µg).

3. Results and discussion 3.1 Incorporation of thymol in nanocomposites Supercritical impregnation tests were carried out according to the experimental procedure described in section 2.3. In this case, the experimental variables were the depressurization rate (10.0 and 1.0 MPa min-1) after impregnation and the concentration of the OM-MMT C20A in the nanocomposites with values of 0, 2.5 and 5.0% (w/w). Meanwhile, the impregnation time, pressure and temperature were maintained constant and equal to 1 hour, 12 MPa and 313 K, respectively. Table 1 shows the thymol incorporation percentage in LDPE films and nanocomposites as a function of different nanoclay concentrations and different depressurization rates. From these results, it can be seen that there is a slight influence of the depressurization rate on the thymol incorporation percentage. Previous studies [22, 23] report a higher active compound incorporation percentage in polymers when depressurization rate increases. Both Rojas and coworkers [22] and Goñi and coworkers [23] have reported higher incorporation percentages of 2-nonanone and eugenol in linear low-density polyethylene films, respectively, when the depressurization rate increases. These authors explained the positive effect of the slower depressurization rate because of the higher stability of the precipitated thymol into the polymer phase when the solvent power of CO2 as well as its concentration decrease slowly [36, 37].

In this study, table 1 does not show a significant effect of the depressurization rate on the thymol incorporation under the studied conditions. However, it can be seen a higher influence of the concentration of the OM-MMT on the thymol incorporation. Thus, when the concentration of OM-MMT is equal to 5% (w/w) the thymol incorporation percentage increases close to three times respect to the nanoclay-free LDPE. This effect could be related to the interaction between thymol and the silicate layers of the nanoclay because of its hydrophobic characters [38]. Indeed, thymol could show sorption in other hydrophobic nanoclays such as Cloisite® 30B [10] and in some less hydrophobic nanoclays like D43B [35] where the thymol presence could even generate swelling of the nanoclay stacks.

3.2 Nanocomposites characterization Fig. 3 shows the x-ray diffraction patterns of OM-MMT C20A and nanocomposites with different concentrations of C20A treated by supercritical impregnation process with different depressurization rates. X-ray diffraction analysis allows examining the interlayer spacing in the structure of clay particles, which is related to the grade of intercalation of the polymer chains between the organoclay stacks [36]. From the results showed in Fig. 3, intercalation of the polymer chains between nanoclays was verified for all nanocomposites through an increase of the interlaminar distance respect to its value in the pure organoclay. Furthermore, the interlaminar distance was slightly increased for all nanocomposites impregnated with thymol using scCO2. Thus, thymol seems to be a plasticizer agent of the LDPE increasing the molecular mobility of its polymer chains [10, 37, 38]. A maximum interlaminar distance of 3.49 nm was obtained for the sample with the highest concentration of thymol, which was equal to 1.19% (w/w).

On the other hand, this contribution includes XRD analysis of nanocomposite samples exposed to pure supercritical carbon dioxide (Fig. 3). From these results, it can be verified that the presence of scCO2 itself did not change the interlaminar distance in the nanoclay structure. In this way, the interlaminar distance of the nanoclay stacks contained in the nanocomposite was exclusively increased by the presence of thymol. Nevertheless, recent studies on supercritical soaking process with CO2 show a homogenous polymer-clay dispersion and even an improvement of this interaction. Soaking with dense CO2 is a process that considers higher temperature and pressure (80ºC and 17.2 MPa) and faster depressurization rates (300 MPa min-1) [39, 40] Another step of the materials characterization considered differential scanning calorimetry (DSC) in order to determine its thermal properties before and after the supercritical impregnation process, and thermogravimetric analyses (TGA) in order to study the influence of the supercritical impregnation process over the thermal stability of the materials. With the purpose of identifying the effect of scCO2, separate from which could cause thymol incorporation, over thermal properties of LDPE and LDPE nanocomposites, previous tests were done only submitting the samples to scCO2 without thymol. Table 2 shows the effect of the CO2 application at 12 MPa and 313 K and the use of different depressurization rates on thermal properties of LDPE and LDPE nanocomposites with different contents of nanoclay. From these results, it can be seen that nanoclay incorporation does not affect the melting temperature (T m) of LDPE. However, it increases slightly the melting enthalpy of the polymer nanocomposite. This effect could be attributed to the property as nucleating agent that has been reported to clays, whose effect of nucleation could promote a higher crystallinity in a polymeric matrix [41, 42]. Nonetheless,

there were no significant differences in the increase of melting enthalpy when the nanoclay content was increased from 2.5 to 5.0% (w/w). Probably due to less dispersion as the clay content increases to 5.0% (w/w), which involves the formation of clay agglomerates, these structures have a smaller amount of potential nucleation sites compared to one that could present a well-dispersed or exfoliated structure [43]. From the results showed on Table 2, it can also be seen that the application of high-pressure CO2 does not affect the melting temperature of the samples. Nevertheless, it decreases slightly its melting enthalpy with no differences for the two depressurization rates employed. These results show that the high-pressure CO2 treatment, through its swelling and plasticizing effect, could produce deformations in the crystalline and amorphous regions of the polymer. Other authors have reported this effect even for lower CO 2 pressures. For example, Torres and coworkers [27] for LLDPE films pressurized with CO2 at pressures between 7 and 12 MPa at 40 °C and Goñi and coworkers for LLDPE films pressurized with CO2 at pressures between 12 and 15 MPa at 45 °C [23]. The melting enthalpy decrease is more evident for the nanocomposites, this effect is increased for the nanocomposite with the highest nanoclay content (5.0% (w/w)). This effect could be related to the formation of a nanocomposite of a less compatibilized structure given at high levels of clay agglomerations, this structure could be more susceptible to deformations by effect of high-pressure CO2. Morawiec and coworkers [44], reported a higher decohesion of LDPE from nanoclay particles for a non-compatibilized system (high content of nanoclay aggregates) than the obtained for a compatibilized system (high nanoclay dispersion) during a deformation to elongation. Finally, Table 3 shows that the addition of thymol by means of scCO2 does not change the melting temperature neither generates an additional change over the melting enthalpy of the

LDPE samples without nanoclays, although it causes a decrease on the fusion enthalpy of the nanocomposite with 2.5% (w/w) of nanoclay, probably because a higher percentage of incorporation could cause an additional plasticizing effect than the produced for CO 2 (see Table 2). However, the thymol addition to the nanocomposites with 5.0% (w/w) of clay produces an inverse effect. Its incorporation improves crystallite formation in the nanocomposites counterbalancing the negative effect of supercritical CO 2. Thus, probably this effect could be related to the better dispersion of nanoclays evidenced due to thymol incorporation, which could improve the amount of potential nucleation sites for the formation of crystals in the nanocomposite. For example, Persico and coworkers [45] reported the increment of LDPE crystallinity due to the presence of a modified montmorillonite and carvacrol. To investigate how the nanoclay content and the supercritical impregnation process affect the thermal stability of LDPE nanocomposites, thermogravimetric analyses were done following the procedure showed in section 2.5.2. As thermograms on Fig. 4.a and values obtained from the first derivate on Table 2 show, the addition of nanoclay decreases the thermal stability of the polymer. In fact, the decrease of thermal stability is more pronounced for the nanocomposite with 2.5% (w/w) of C20A (Tdeg = 470 °C) than for the nanocomposite with 5.0% (w/w) of C20A (Tdeg = 473 °C). These results show that a higher level of dispersion of the nanoclay decreases the thermal stability of the polymer. Marazzato and coworkers [46] reported the decrease of thermal stability of LDPE nanocomposites when they possess Cloisite 15A with a high exfoliation degree. These authors explained that this behavior is given because a better nanoclay dispersion improved the dispersion of the protonated silicate residue remaining after the thermal degradation of nanoclay, which in turn catalyzes the degradation of the polymer. Thus, a higher dispersion

of the residue in the exfoliated nanocomposite leads to an increase of catalytic sites for the degradation of the polymer. On the other hand, from Fig. 4.a and Table 2 it can be seen that the treatment of LDPE with high-pressure CO2 does not affect the thermal stability of the polymer. Nevertheless, it affects the thermal stability of nanocomposites. All nanocomposites submitted to highpressure CO2 show a decrease on thermal stability without differences for the depressurization rate employed and the nanoclay content. Thus, this behavior would be related to the fact that the nanocomposites treated with scCO2 show a more amorphous structure (less crystalline) than the nanocomposites untreated. This structure could allow a better dispersion of the protonated silicate residue after the degradation of nanoclay. Thereby, the nanocomposite with the lowest thermal stability (Tdeg = 463 °C) was the nanocomposite with the lowest crystallinity (19.44%). Finally, a thermal protective effect of thymol on nanocomposites, possibly related to their antioxidant and radical scavenger character or to the decreasing action of clays due to the interaction with thymol [38], can be seen in Fig.4.b and results are shown in Table 3. Fourier transform infrared (FTIR) spectroscopy was performed on the samples with the aim to verify the incorporation of the nanoclay and thymol into the LDPE matrix and to obtain information about the nature of the molecular interactions between thymol and nanoclays. The spectrum of the nanocomposites (see Fig.5) shows characteristic absorption bands corresponding to aluminosilicate minerals: ~1047 cm-1 (Si-O stretching) and ~ 520 cm-1 (Si–O–Al bending) [47], confirming the incorporation of nanoclays into the LDPE matrix. On the other hand, the active compound incorporation into the LDPE matrix was verified through the appearance of the characteristic band between 800 and 815 cm-1 assigned to ring vibrations of thymol (see Fig.5) [48, 49], the intensity of the band was directly related

with thymol incorporation. Thus, the nanocomposite with the highest thymol content (1.19% (w/w)) showed the highest band intensity. An interesting result is that in the LDPE matrix, the band associated to the vibration of the aromatic ring from thymol consists of a multitude of smaller bands, which would indicate that thymol could be interacting with the polymer through weaks interactions. While in the nanocomposites, this band appears as a lower amount of smaller bands as a single one when a depressurization rate of 1.0 MPa min-1 is used after the impregnation of the nanocomposite with 5.0% (w/w) of C20A. Revealing the great affinity between thymol and modified nanoclays, which would be related to the greater thymol incorporation in nanocomposites than in LDPE without nanoclays (see section 3.1).

3.3. Thymol release kinetics Thymol release kinetics have been characterized by means of specific experimental assays in order to describe the mass transfer from impregnated LDPE films and LDPE nanocomposites with different nanoclay concentrations (2.5 and 5.0% (w/w) C20A) obtained at different depressurization rates (1.0 and 10.0 MPa min -1). These experiments were described in sections 2.6.1 and 2.6.2 and conducted in order to determine the partition coefficient of thymol at the interphase between the material and food simulant (KM/FS) as well as the effective diffusion coefficient (Deff) of thymol in the different materials. Release tests were done using a 95% (v/v) ethanol solution as food simulant. Material/food simulant partition coefficient, KM/FS, was experimentally estimated under equilibrium after each release run. Table 4 shows the initial concentrations of thymol in the materials, the values for the material/food simulant partition coefficients, KM/FS, and for the effective diffusion coefficients of thymol in the different materials. The diffusion coefficients were estimated

through the correlation of the simulated transfer rate with the experimental data. In the last column of Table 4, the value of root mean square error (RMSE) of the model solution related to experimental data is reported for each release test. Fig. 6 shows the thymol release kinetics from impregnated LDPE films and nanocomposites with different nanoclay concentrations (2.5 and 5.0% (w/w) C20A) obtained at different depressurization rates after the impregnation process (1.0 and 10.0 MPa min-1) into ethanol 95% (v/v), expressed as a function of the thymol amount released into the food simulant per surface area of the material sample. Results represent experimental and theoretical estimations, which were obtained through the transfer model with correlated diffusion coefficients of thymol in the materials. From the results, it can be seen that thymol release from impregnated LDPE films reaches equilibrium after 3 h and the depressurization rate does not affect the release rate of thymol from LDPE films. These results are in accordance with results obtained from DSC assays for these materials, from which can be confirmed that the establishment of the different depressurization rates generates similar decreases in LDPE crystallinity (see Table 2). Moreover, thymol incorporated, using both depressurization rates, does not generate an additional decrease of LDPE crystallinity to the obtained through the application of high-pressure CO2 (see Table 3). Therefore, changes in the deposition and control zones of the solute diffusion in the LDPE matrix are similar. Thus, Deff of thymol through LDPE films was equal to 1.0 x 10-12 and 1.5 x 10-12 m2 s-1 for 1.0 and 10.0 MPa min-1, respectively (see Table 4). The Deff values were lower than those obtained for the thymol diffusion through cellulose acetate films (8 x 10-12 m2 s-1) [10] and polylactic acid films (1-100 x 10-11 m2 s-1) [50] , which could be explained due to a higher crystallinity degree of impregnated LDPE films. In another work, the coefficient diffusion of thymol in polypropylene, a polymer with high crystallinity, was

estimated in a range between 1-2 x10-14 m2 s-1 [51], a value 65 times lower than that found in our study for thymol through LDPE. In a special case, the Deff of thymol impregnated in LDPE films obtained in this study was slower than thymol incorporated in LLDPE by means of supercritical impregnation [27]. A polymer with higher crystallinity, but that suffered a higher loss of crystallinity (33.9 to 28.1%) than LDPE (21.7 to 19.8%) due to the supercritical treatment. This indicates that in LLDPE the amorphous zones were more exposed to possible plasticizing effect, a situation that would explain the higher diffusion of thymol in LLDPE than in LDPE. From Fig. 6, it can be seen that thymol release equilibrium for both nanocomposites (2.5 and 5.0% (w/w) of C20A) was reached after a time between 9-11 h, a higher time than the obtained for thymol release from LDPE films. These results indicate that the impregnated nanocomposites present a structure that makes difficult the diffusion of thymol in the polymeric matrix. Thus, the Deff of thymol for both nanocomposites was independent of the depressurization rate employed and estimated in a range between 2.7 and 3.5 x 10-13 m2 s-1 (see Table 4). These values are in accordance with XRD results from which was confirmed that the impregnated nanocomposites showed a similar degree of intercalation and that the depressurization rate does not affect this intercalation degree (see Fig.3). Thus, nanocomposites presented a similar intercalated structure that makes a tortuous path that could make difficult the thymol diffusion through the polymer structure. On the other hand, from the release curves showed in Fig. 6, it can be seen that there is almost no difference in the amount of thymol migrated among the impregnated LDPE samples obtained at different depressurization rates, this is explained because the samples show similar thymol concentrations (Table 1). Meanwhile, it can be seen that the total amount of thymol released from the nanocomposites was directly related with the initial

thymol concentration in the polymer. Thus, the nanocomposites obtained at a depressurization rate of 1.0 MPa min-1 showed the highest amount of released thymol.

Conclusions LDPE films and LDPE nanocomposites with different concentration of C20A (2.5 and 5.0% (w/w)) were impregnated with thymol as active compound by means of supercritical impregnation using carbon dioxide as impregnation medium. Thymol incorporation was favored by the nanoclays presence and the lowest depressurization rate used. Thus, the highest incorporation percentage of thymol (1.19% (w/w)) was obtained on the nanocomposite with the highest nanoclay content (5.0% (w/w)) using 1.0 MPa min-1 depressurization rate. XRD results showed that supercritical impregnation is an effective method to improve intercalation of LDPE into C20A through the promotion of the plasticizing effect of thymol and the improvement of the interaction between thymol and nanoclays. The nucleating effect of nanoclays was verified through an increase in the crystallinity degree of nanocomposites, while the supercritical impregnation process caused a decrease in the crystallinity of the different materials, mainly linked to the effect of the pressurized CO2. The crystallinity decrease was more evident for the nanocomposites, and this effect is enhanced for the nanocomposite with the highest nanoclay content (5.0% (w/w)). The nanocomposites showed lower thermal stability than LDPE films. The treatment of LDPE samples with pressurized CO2 does not generate changes on their thermal stability, although it causes a thermal stability decrease on nanocomposites. Experimental and theoretical characterizations of thymol transfer from the LDPE films and nanocomposites with different nanoclay concentration (2.5 and 5.0% (w/w) of C20A) into a 95% (v/v) ethanol solution was analyzed. Meanwhile, a phenomenological mass transfer

model based on a resistances-in-series approach was proposed and solved in order to correlate the diffusion coefficient of thymol in the different materials during the release experiment. The diffusion coefficients obtained for thymol in LDPE was independent of the depressurization rate employed with values between 1.0 and 1.5 x 10-12 m2 s-1. In the nanocomposites, the diffusion coefficient of thymol was slower than the obtained in LDPE and, again, independent of the depressurization rate, indicating that the intercalated structure present in the impregnated nanocomposites could make difficult the diffusion of thymol through the polymeric matrix. Thus, the diffusion coefficient of thymol in the nanocomposites takes values between 2.7 and 3.5 x 10-13 m2 s-1. Finally, this work shows that nanoclay presence improved thymol incorporation in LDPE by the supercritical impregnation process, decreasing its diffusion coefficient during release test allowing a sustained release over time of the active compound.

Acknowledgements This work has been developed in the framework of the project FONDECYT 1150592, the Basal Financing Program for Scientific and Technological Centers of Excellence (grant number FB0807) and Program of Insertion of Advanced Human Capital (grant number 79150059), with the support of the University of Santiago de Chile. The CONICYT scholarship (number 21150969) for the Ph.D. student Adrián Rojas Sepúlveda is gratefully acknowledged.

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Table 1. Incorporation percentages of thymol in LDPE films and LDPE nanocomposites

with

different

nanoclay

content

submitted

at

different

depressurization rates after the impregnation process with thymol at 12 MPa and 40 °C.

Depressurization rate (MPa min-1)

1

10

Cloisite 20A content (% (w/w))

Film thickness (µm)

Thymol impregnation percentage (% (w/w))

0

104.6 ± 1.2

0.36 ± 0.04

2.5

104.7 ± 1.8

0.65 ± 0.07

5

89.1 ± 1.9

1.19 ± 0.14

0

104.7 ± 1.8

0.34 ± 0.08

2.5

92.9 ± 1.8

0.42 ± 0.08

5

89.4 ± 1.9

0.95 ± 0.11

Table 2. Effect of supercritical CO2 at 12 MPa and 40 °C on thermal properties of LDPE films and LDPE nanocomposites with different nanoclay content at different depressurization rates after the high-pressure carbon dioxide exposure.

Cloisite 20A content (% (w/w))

0

2.5

5

Depressurization rate (MPa min-1)

Tm (°C)

ΔHm (Jg-1)

Crystallinity (%)

Tdeg (°C)

-

109

63.82

21.74

480

1

109

60.55

20.62

482

10

109

59.76

20.35

480

-

109

67.30

22.92

470

1

109

60.47

20.60

465

10

109

61.64

20.99

466

-

109

67.26

22.91

473

1

109

57.07

19.44

463

10

109

56.23

19.15

465

Table 3. Effect of supercritical impregnation with thymol at 12 MPa and 40 °C on thermal properties of LDPE films and LDPE nanocomposites with different nanoclay content at different depressurization rates after the impregnation process.

Cloisite 20A content (% (w/w))

Depressurization rate (MPa min-1)

Tm (°C)

ΔHm (Jg-1)

Crystallinity (%)

Tdeg (°C)

1

109

60.50

20.60

483

10

109

58.16

19.81

481

1

109

57.47

19.57

479

10

109

59.41

20.24

471

1

109

60.30

20.54

479

10

109

61.73

21.02

468

0

2.5

5.0

Table 4. Partition and diffusion coefficients and RMSE values of thymol from LDPE films and nanocomposites with different nanoclay content submitted at different depressurization rates after the impregnation process with thymol at 12 MPa and 40 °C.

Food Simulant

Depressurization rate (MPa min-1)

1 EtOH 95% (v/v) 10

Cloisite 20A content (% (w/w))

-1

(mg kg )

KM/FS ± SD

Deff (m2 s-1)

RMSE (%)

0

3250

87 ± 9

1.0 x 10 -12

7.72

2.5

5900

65 ± 3

3.2 x 10-13

3.29

5.0

10700

11 ± 1

3.5 x 10-13

5.46

0

3380

100 ± 11

1.5 x 10 -12

1.51

2.5

3600

70 ± 6

3.0 x 10-13

8.59

5.0

9800

51 ± 5

2.7 x 10-13

7.23

FIGURES CAPTIONS Figure 1. Outline of the experimental setup for the supercritical impregnation process. Figure 2. Scheme of the thymol release process: concentration profiles and equilibrium conditions at the material/ food simulant interphase for a) LDPE film and b) LDPE nanocomposite. Figure 3. X-ray diffraction (XRD) patterns of (a) Cloisite 20A (C20A), nanocomposites with different C20A concentration: (b) LDPE/C20A 2.5% (w/w) and (c) LDPE/C20A 5.0% (w/w), nanocomposites submitted to high-pressure carbon dioxide: (d) LDPE/C20A 2.5% (w/w)_10.0 MPa min-1 and g) LDPE/C20A 5.0% (w/w)_10.0 MPa min-1, and nanocomposites submitted to supercritical impregnation with thymol at different depressurization rates: (e) LDPE/C20A 2.5% (w/w)/thymol_1.0 MPa min -1, (f) LDPE/C20A 2.5% (w/w)/thymol_10.0 MPa min-1, (h) LDPE/C20A 5.0% (w/w)/thymol_ 1.0 MPa min-1 and (i) LDPE/C20A 5.0% (w/w)/ thymol_10.0 MPa min-1. Figure 4. TGA and derivate curves (inset) of LDPE and LDPE nanocomposites films: a) effect of nanoclay content and high-pressure CO2 on thermal degradation, b) effect of thymol incorporation on thermal degradation. Figure 5. Fourier transform infrared spectra of LDPE film and LDPE nanocomposites. Figure 6. Effect of nanoclay content and depressurization rate over thymol release from LDPE and LDPE nanocomposites to 95% (v/v) ethanol at 40 °C: mathematical model, experimental results:

: LDPE/C20A 5.0% (w/w)/thymol_1.0 MPa min-1, -1

LDPE/C20A 5.0% (w/w)/thymol_10.0 MPa min , /thymol_1.0 MPa min-1 ,

: LDPE/C20A 2.5% (w/w)

: LDPE/C20A 2.5% (w/w)/thymol_10.0 MPa min-1,

LDPE/ thymol _1.0 MPa min-1 and

:

: LDPE/thymol_l0.0 MPa min-1.

:

FIGURES

Figure 1. Outline of the experimental setup for the supercritical impregnation process.

Figure 2. Scheme of the thymol release process: concentration profiles and equilibrium conditions at the material/ food simulant interphase for a) LDPE film and b) LDPE nanocomposite.

i) h) g)

2 = 2.58 (d=3.43 nm) 2 = 2.53 (d=3.49 nm) 2 = 2.60 (d=3.41 nm)

In te n s ity (a .u )

f)

e) d) c)

2 = 2.60 (d=3.41 nm) 2 = 2.59 (d=3.41 nm)

2 = 2.63 (d=3.35 nm) 2 = 2.60 (d=3.41 nm)

b)

2 = 2.63 (d=3.35 nm) a)

2

2 = 3.33 (d=2.65 nm) 4

6

2T heta (degree)

Figure 3. X-ray diffraction (XRD) patterns of (a) Cloisite 20A (C20A), nanocomposites with different C20A concentration: (b) LDPE/C20A 2.5% (w/w) and (c) LDPE/C20A 5.0% (w/w), nanocomposites submitted to high-pressure carbon dioxide: (d) LDPE/C20A 2.5% (w/w)_10.0 MPa min-1 and g) LDPE/C20A 5.0% (w/w)_10.0 MPa min-1, and nanocomposites submitted to supercritical impregnation with thymol at different depressurization rates: (e) LDPE/C20A 2.5% (w/w)/thymol_1.0 MPa min -1, (f) LDPE/C20A 2.5% (w/w)/thymol_10.0 MPa min-1, (h) LDPE/C20A 5.0% (w/w)/thymol_ 1.0 MPa min-1 and (i) LDPE/C20A 5.0% (w/w)/ thymol_10.0 MPa min-1.

Figure 4. TGA and derivate curves (inset) of LDPE and LDPE nanocomposites films: a) effect of nanoclay content and high-pressure CO2 on thermal degradation, b) effect of thymol incorporation on thermal degradation.

Figure 5. Fourier transform infrared spectra of LDPE film and LDPE nanocomposites.

Figure 6. Effect of nanoclay content and depressurization rate over thymol release from LDPE and LDPE nanocomposites to 95% (v/v) ethanol at 40 °C: model, experimental results:

: LDPE/C20A 5.0% (w/w)/thymol_1.0 MPa min-1,

LDPE/C20A 5.0% (w/w)/thymol_10.0 MPa min-1, /thymol_1.0 MPa min-1 ,

mathematical

: LDPE/C20A 2.5% (w/w)

: LDPE/C20A 2.5% (w/w)/thymol_10.0 MPa min-1,

LDPE/ thymol _1.0 MPa min-1 and

:

: LDPE/thymol_l0.0 MPa min-1.

:

Graphical Abstract

Highlights:    

LDPE nanocomposites were impregnated with thymol by supercritical CO 2. Impregnated materials were characterized through DRX, DSC, TGA, FTIR-ATR, SEM and release experiments. Nanoclay concentration and depressurization rates affects the amount of thymol impregnated. Polymer-nanoclay intercalated structure slows down the thymol transfer decreasing the thymol diffusion coefficient.