Chapter 5: Preparation and characterization of environmentally safe and
5.2 Materials and methods
5.2.3 Characterization
5.2.3.1. Physico-chemical characteristics
Dispersion of Gr-NPs and morphology of the PHB/Gr-NPs nanocomposites and pristine PHB were studied using imaging techniques - atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM) and field-emission transmission electron microscopy (FETEM). PHB micro-grain structure was analysed by using AFM (Oxford, Cypher model, United Kingdom) operated under non-contact mode. 1 mL of PHB dissolved in chloroform was cast on a coverslip prior to the AFM analysis. For FESEM
analysis, either PHB or PHB/Gr-NPs nanocomposite was cast directly on double-sided carbon tape and mounted on a stainless steel stub. These stubs were gold-sputtered to form a thin layer of gold on the sample. Gold-coated samples were finally analysed using FESEM (Zeiss, Sigma model, Germany) operated at 2 kV. For FETEM analysis, the samples were dropped directly onto a copper grid and dried overnight. These dried samples were used for observation under FETEM (JEM 2100F, JEOL, Japan) operated at 200 kV.
Fourier transform infrared (FTIR) (Shimadzu, IR-affinity 1 model) analysis under attenuated total reflection (ATR) mode was carried out to identify the functional groups present in the pristine PHB and PHB/Gr-NPs nanocomposites. An average of 60 scans with a resolution of 4 cm-1 was performed for all the samples over the wavelength range of 400 to 4000 cm-1.
Contact angle analysis was carried out using Drop shape analyser – DSA 25, Kruss model, Germany. The droplet size of 4 µL with a dropping rate of 0.16 mL/min was maintained for all the analysis. Image for the contact angle analysis was captured after 30 s from the dropping of the liquid. X-ray diffraction (XRD) analysis of pristine PHB and PHB/Gr-NPs nanocomposites was carried out under an ambient atmosphere using powder XRD (Rigaku, SmartLab, USA) instrument operated at 40 kV and 40 mA with Cu-K
radiation wavelength of 0.15406 nm. The samples were scanned in the 2- theta degrees ranging from 5 to 60o along with a scanning rate and step size of 0.1o s-1and 0.5 s, respectively.
5.2.3.2. Oxygen permeability and water vapour permeability
Oxygen permeability (OP) analysis was carried out using Ox-Tran Modular System, India operated at 25 oC and relative humidity of 50%. Prior to this analysis, the pristine PHB and PHB/Gr-NPs nanocomposites films were equilibrated for a total period of 2 days at a
relative humidity value of 50% (Chandra Mohan et al., 2018). Polymeric films were mounted between two stainless steel mould having a circular opening in the centre so as to have an active surface area of 5 cm2. On one side of the stainless steel mould, oxygen was passed while on the other side, nitrogen gas was passed. Nitrogen gas connected to the coulometric sensor and analysis was carried out after attaining steady-state to calculate oxygen transmission rate (OTR). Finally, oxygen permeability (OP) values were estimated by using equation (5.1) (Hema Prabha and Ranganathan, 2018):
Oxygen permeability (OP) =OTR×L ΔP
(5.1)
Where OP is the oxygen permeability (cm3·mm/m2·d2·atm), OTR is the oxygen transmission rate (cm3/m2·d), ΔP is the difference in partial pressure of oxygen on both sides of the film (atm) and L is the film thickness (mm).
Water vapour permeability (WVP) analysis of the samples was carried out as per the ASTM E96/E96 M gravimetric method, which is generally referred as “cup method”.
For this analysis, the respective films were placed over a steel mould having a circular opening of 2.9 cm2. Free space in the cup underneath the polymeric film was filled entirely with calcium chloride having a relative humidity value of 0% (Jha et al., 2019). This permeation cup was thereafter placed in a desiccator having a surrounding temperature of 25 oC and relative humidity of 75% maintained through sodium chloride solution. The increase in weight of the permeability cup was measured for 2 days with a regular time interval of 12 h. The increase in weight of the permeation cup was measured as a function of time divided by the transfer area to get the water vapour transmission rate (WVTR).
Finally, water vapour permeability (WVP) values were estimated by using equation (5.2) (Hema Prabha and Ranganathan, 2018):
Water vapour permeability (WVP) =WVTR×L ΔP
(5.2)
Where WVP is the water vapour permeability (g·mm/m2·d2·atm), WVTR is the water vapour transmission rate (g/m2·d), ΔP is the difference in partial pressure of oxygen on both sides of the film (atm) and L is the film thickness (mm).
5.2.3.3. Shelf life (Ɵ) simulation test
Shelf life (Ɵ) simulation of the pristine PHB and PHB/Gr-NPs nanocomposites was determined for the packaging of potato chips and milk products and by interrelating the oxygen permeability values with the rate of oxidation as specified in the following models (eqs. 5.3 and 5.4):
2 2
2(max)
O O
O
k p
(5.3)
2 2
O O
0.21× OP ×A p = d
k + OP ×A d
(5.4)
O2 (max) is the maximum concentration of oxygen that can react with the food items and can finally lead to its spoilage. Oxidation rate constant (kO2) values of the potato chips and milk products were obtained based on the literature (Andersson H, 2006; QUAST et al., 1972). MatlabTM 2015a software was used for this shelf simulation study. In equation (5.4) the term pO2, OP, A and d are the partial pressure of oxygen, oxygen permeability value, surface area and thickness of the film, respectively. For this simulation study, the thickness of the PHB/Gr-NPs composite films was fixed as 100 µm and the film surface area of 1L capacity was taken as 0.1 m2.
5.2.3.4. Thermal characteristics
The thermo-gravimetric analysis (TGA) was performed under a nitrogen atmosphere with a flow rate of 40 mL/min on a TG 209 F1, Libra Analyser, Germany with a heating rate of 10 oC/min and a temperature range of 30-500oC. The sample was placed in a 900 µL ceramic crucible for analysis. Differential thermogravimetric analysis (DTG) was carried out to find the maximum thermal degradation temperature (Tmax) of the samples.
Differential scanning calorimetry (DSC) measurement was done on Mettler Toledo - 1 series, Switzerland in the temperature range of 25-200 oC with a heating rate of 5 oC/min.
For this analysis, all the samples were first heated from 25 to 200 oC and maintained at the same temperature for 5 minutes to eliminate the processing and thermal history associated with the samples. Melting temperature (Tm) of the samples was later obtained from the DSC thermographs.
5.2.3.5. Transmittance and tensile characteristics
Transparency of the pristine PHB and PHB/Gr-NPs nanocomposites films was analysed using a UV-visible spectrophotometer (Perkin Elmer, Lambda 35 model, USA). For this analysis, the samples were scanned in the wavelength ranging from 200 - 600 nm with a scan rate and bandwidth of 50 nm/min and 2 nm, respectively. BaSO4 coated plate was used as a reference for this analysis. Tensile test of the pristine PHB and PHB/Gr-NPs nanocomposites films was measured using 5 kN Electromechanical Universal Testing Machine (Z005TN model, Zwick Roell, USA). Samples for this analysis were prepared according to ASTM D882-12 standard. The dimensions of the sample specimens are as follows: length 80 mm, gauge length 50 mm, width 5 mm and thickness 0.2 mm. Sample thickness was measured using digital micrometre (Mitutoyo, Japan), and the sample elongation rate for all the analysis was maintained at 5 mm/min. Tensile stress ( ) and
elongation () were calculated from the stress-strain curve by using the following equations (5.5 and 5.6) (Chandra Mohan et al., 2018):
Fmax
= A
(5.5)
=ΔL×100
L (5.6)
Where is tensile stress in MPa, Fmax is the maximum force (MPa) needed to pull the sample apart, A is the initial cross-sectional area of the sample in m2, is the elongation in percentage, ΔL is the extension of the sample at the time of rupture in mm and L is the original length of the sample, which is 50 mm for the present study. Change in surface morphology of the polymeric samples during the course of elongation was analysed only for PHB/Gr-NPs nanocomposite with 0.7 wt% Gr-NPs loading using FESEM analysis.