3.1. MATERIALS
The commercial products were used as received if not otherwise stated.
3.1.1. Solvents and Reagents
Amylene stabilized chloroform, acetic anhydride, and butyric anhydride were supplied by Aldrich Chemical Co., Germany. Sulphuric acid was purchased from J.T. Baker, The Netherlands. Hydrochloric acid, ethanol, sodium hydroxide, and potassium hydroxide were obtained by Carlo Erba Chemical Co., Italy. Octadecyldimethyl(3- trimethoxysilylpropyl) ammonium chloride (ODSP) was supplied by ABCR GmbH &
Co., Germany.
3.1.2. Process Additives
Tetrakismethylene(3,5-di-t-butyl-4-hydroxyhydrocinnamate) methane (Anox 20TM) and tris(2,4-di-t-butylphenyl) phosphite (Alkanox 240 TM) were kindly supplied by Great Lakes Chemical Corporation, USA. Stabaxol® P200 was kindly supplied by Lanxess S.r.l., Italy.
3.1.3. Fillers
Organic fillers. Sugar cane bagasse (SCB) was purchased by Cosan S/A Indùstriale e Comèrcio, Brazil. Cellulose (CELL) and cellulose acetate (CA) were obtained by Aldrich Chemical Co., Germany. Inorganic fillers. Sodium-montmorillonite (Na-OMMT) and montmorillonite modified with dimethylbenzyl tallow ammonium (BTA), and dimethyl ditallow ammonium (DTA) were kindly supplied by Laviosa Mineraria SpA, Italy.
3.1.4. Polymers
BIOCYCLE® poly(3-hydroxybutyrate) (PHB), microbially produced from Burkholderia
3.2. PHB STABILIZATION
3.2.1. PHB Stabilization with Anox 20TM and Alkanox 240TM
Before melt compounding, PHB, Anox 20TM, and Alkanox 240TM were mixed physically and dried at 80°C in a laboratory oven with air circulation. Melt processing was performed in a torque rheometer W 50 EHT (with roller blade) connected to a Plastograph Can-Bus Brabender at 170°C and 30 rpm by 7 minutes. Composition and identification codes of the mixtures are presented in Table 3.1. Pristine PHB was also processed in the same conditions as blank. All mixtures were prepared in replicate.
Table 3.1. Compositions and identification codes of PHB stabilized with Anox 20TM and Alkanox 240TM
Sample Anox 20TM wt-% Alkanox 240TM wt-%
PHB — —
50A 0.50 —
33A17P 0.33 0.17
25A25P 0.25 0.25
17A33P 0.17 0.33
50P — 0.50
50A50P 0.50 0.50
100P — 1.00
Films of 200-500 µm were obtained by compression moulding in a laboratory press at 170°C with a pressure of 100 Bar by 5 minutes. Before films preparation, the mixtures were dried at 80°C in a laboratory oven with air circulation.
3.2.2. PHB Stabilization with Stabaxol® P200
Before melt compounding, PHB and Stabaxol® P200 were dried at 80°C in a laboratory oven with air circulation. Melt processing was performed in a torque rheometer W 50 EHT (with roller blade) connected to a Plastograph Can-Bus Brabender at 175°C and 30 rpm by 7 minutes. Composition and identification codes of the mixtures are presented in Table 3.2. Pristine PHB was also processed in the same conditions as blank. All mixtures were prepared in replicate.
Table 3.2. Compositions and identification codes of PHB stabilized with Stabaxol®
P200
Sample PHB wt-% Stabaxol® P200 wt-%
PHB 100 —
SL1 99 1
SL3 97 3
SL5 95 5
Films of 200-500 µm were obtained by compression moulding in a laboratory press at 170°C with a pressure of 100 Bar by 5 minutes. Before films preparation, the mixtures were dried at 80°C in a laboratory oven with air circulation.
3.3. PHB – NATURAL FIBRES COMPOSITES
3.3.1. Modification of Natural Fibres
Prior to any experiment, SCB was divided in three different fractions with a mechanical sieve: BNT (average diameter between 50 and 800 µm), BNTf (average diameter < 50 µm) and BNTg (average diameter > 800 µm).
3.3.1.1. Alkalization of Sugar Cane Bagasse
Ca. 50 g of BNT were immersed in a 18% NaOH solution (material:liquor ratio 1:20 w/w) at 140°C for 30 minutes. The fibres were then washed with distilled water until the pH of the filtrates remained unchanged. The filtrated fibre (BD) was dried under vacuum until constant weight. The procedure was repeated several times, in order to obtain the desired amount of fibre.
3.3.1.2. Esterification of Natural Fibres
Natural fibres esterification was performed adapting the procedure suggested by Frisoni et al. [202]. The reactions were performed at the constant temperature of 30°C for 7 hours. In a 1-l glass reaction vessel 45 g of natural fibres (BNT, BD and CELL) were totally immersed in 300 ml of acetic or butyric anhydride in the presence of 1 ml sulphuric acid as reaction catalyst. Esterified fibres were washed to neutrality with water and dried under vacuum until constant weight. Identification codes of esterified natural fibres are presented in Table 3.3.
Table 3.3. Identification codes of esterified natural fibres
Fibre Acetylation Butyrilation
BNT BNTa BNTb
BD BDa BDb
CELL CELLa —
3.3.2. Preparation of PHB – Natural Fibres Composites
Before melt compounding, PHB and natural fibres were mixed physically and dried at 80°C in a laboratory oven with air circulation. Melt processing was performed in a torque rheometer W 50 EHT (with roller blade) connected to a Plastograph Can-Bus Brabender at 170°C and 30 rpm by 7 minutes. Pristine PHB was also processed in the same conditions as blank. Composites composition and identification codes are presented in Table 3.4.
Table 3.4. Compositions and identification codes of PHB – natural fibres composites
Fibre content
Fibre 10 wt-% 20 wt-%
BNT 10BNT 20BNT
BNTa 10BNTa 20BNTa
BNTb 10BNTb 20BNTb
BD 10BD 20BD
BDa 10BDa 20BDa
CELL 10CELL 20CELL
CELLa 10CELLa 20CELLa
CA 10CA 20CA
BNTf 10BNTf 20BNTf
BNTg 10BNTg 20BNTg
Composite films of 300-500 µm were obtained by compression moulding in a laboratory press at 170°C with a pressure of 100 Bar by 5 minutes. Before films preparation, the composites were dried at 80°C in a laboratory oven with air circulation.
3.4. PHB – ORGANOPHILIC MONTMORILLONITE COMPOSITES
3.4.1. Preparation of PHB – Organophilic Montmorillonite Composites by Solution-Casting
Before solutions preparation, organophilic montmorillonites (OMMT) were dried or not under vacuum for two days. Three OMMTs were used: DTAl (38 wt-% of dimethyl dihydrogenated tallow quaternary ammonium), DTAh (46 wt-% of dimethyl dihydrogenated tallow quaternary ammonium), and BTA (32 wt-% of dimethyl benzyl hydrogenated tallow quaternary ammonium). The amylene stabilized chloroform solutions containing PHB (95 wt-%) and selected OMMT (5 wt-%), after 2 or 6 days ageing, were stirred vigorously at room temperature for 5 minutes. The solutions were spread onto a Petri glass plate (8.5 cm diameter), and the solvent was evaporated for 1 week in saturated atmosphere. The films were then dried under vacuum for 2 days.
Pristine PHB film was also prepared in the same conditions as blank. Composites identification codes are presented in Table 3.5.
Table 3.5. Identification codes of PHB – organophilic montmorillonite composites prepared by solution casting
Composite OMMT type Ageing Drying
B-DTAl DTAl no no
B-DTAh DTAh no no
B-BTA BTA no no
Ba-DTAl DTAl yes no
Ba-DTAh DTAh yes no
Ba-BTA BTA yes no
B-DTAld DTAls no yes
B-DTAhd DTAhs no yes
B-BTAd BTA no yes
Ba-DTAld DTAl yes yes
Ba-DTAhd DTAh yes yes
Ba-BTAd BTA yes yes
3.4.2. Preparation of Silane Modified Organophilic Montmorillonite
Silane modified organophilic montmorillonite (OMMTSi) was prepared adapting the procedure suggested by Zhao et al. [203]. Na-OMMT (50 g) was mixed with anhydrous methanol (750 mL) and stirred under a nitrogen flow to form a uniformly dispersed suspension. Then octadecyldimethyl(3-trimethoxysilylpropyl) ammonium chloride (ODSP) equivalent to 1.2 CEC of Na-OMMT was added into the dispersion. The admixture was stirred at 70 °C for 72 h under a nitrogen flow, filtered, washed, and water suspended to lyophilize obtaining OMMTSi as a light powder.
3.4.3. Preparation of PHB – Organophilic Montmorillonite Composites by Melt Processing
Before melt compounding, PHB and OMMTSi were physically mixed and dried under vacuum at 80°C. Melt processing was performed in a torque rheometer W 50 EHT (with roller blade) connected to a Plastograph Can-Bus Brabender at 170°C and 30 rpm by 7 minutes. Pristine PHB was also processed in the same conditions as blank. Composites composition and identification codes are presented in Table 3.6.
Table 3.6. Compositions and identification codes of PHB – organophlic montmorillonite composites prepared by melt processing
Composite PHB wt-% OMMTSi wt-%
PHB 100 0
1-OMSi 99 1
2-OMSi 98 2
3-OMSi 97 3
5-OMSi 95 5
10-OMSi 90 10
Composites films of 200-500 µm were obtained by compression moulding in a laboratory press at 170°C with a pressure of 100 Bar by 5 minutes. Before films preparation,
3.5. CHARACTERIZATION
3.5.1. Transmission Fourier Transform Infrared Spectroscopy (FT-IR)
Transmission infrared spectra of PHB – Anox 20TM – Alknanox 240TM and PHB – Stabaxol® P200 mixtures (Perkin Elmer FT-IR Spectrometer mod. Spectrum One) and Modificated Natural Fibres (Jasco FT-IR Spectrometer mod. FT/IR-410) were recorded in the mid-IR region (4000-400 cm-1) at 4 cm-1 resolution using 32 scans. Samples were pressed in a KBr pellet or casted on a KBr crystal plate.
3.5.2. Photoacoustic Fourier Transform Infrared Spectroscopy (PAS-FT-IR)
Photoacoustic infrared spectra were recorded using a Bruker IFS 66 Spectrometer equipped with an MTEC (Model 200) photoacoustic cell under helium purge in the mid- IR region (4000-400 cm-1) at 8 cm-1 resolution using 128. Scan velocity was fixed in 2.2 KHz. Carbon black spectrum was collected as reference.
3.5.3. Nuclear Magnetic Resonance Spectroscopy (NMR)
13C NMR spectra were obtained with a Varian Gemini VRX 200 operating at 50.3 MHz.
3.5.4. Polarized Optical Microscopy (POM)
POM observations were performed with a Reichert-Jung Polyvar, equipped with an hot- stage METTLER FP 52. After complete melting, samples were cooled down to crystallization temperature. Pictures were taken after complete crystallization. Light polarization, heating and cooling rates, and images magnifications were decided according to sample nature.
3.5.5. Scanning Electron Microscopy (SEM)
Adhesion and morphology of two phases composites were observed with a SEM JEOL 5600 LV operating at 12kV. Prior to observation composites were fractured in liquid nitrogen and then vacuum metallized.
3.5.6. Wide Angle X-ray Scattering (WAXS)
Wide angle X-ray diffraction patterns were obtained using a Kristalloflex810 diffractometer (SIEMENS) using a Cu Kα (1.5406Å) with a rotation velocity of 0,016°/min and a measuring interval of 1 s. The patterns were recorded at room temperature.
3.5.7. Gel Permeation Cromatography (GPC)
Jasco PU-1580 liquid chromatograph connected to Jasco 830-RI and Perkin-Elmer LC-75 spectrophotometric (λ = 260 nm) detectors and equipped with two PLgel 5 µ mixed-C columns was used to obtain molecular weights and polydispersities. Amylene stabilized chloroform was used as eluent at 1 ml/min flow rate. Due to the their high degree of crystallinity, some samples were melted prior dissolution. Monodisperse polystyrene standards were used for calibration.
3.5.8. Thermogravimetric Analysis (TGA)
A TA Thermogravimetric Analyzer TGA Q500 was employed for TGA analysis.
Evaluations were performed on 10-20 mg samples at 10°C/min under a 60 ml/min gas flow according to the following protocols:
PHB – Anox 20TM – Alknanox 240TM and PHB – Stabaxol® P200 mixtures:
- From 30°C to 500°C under nitrogen flow.
Modificated Natural Fibres and PHB – Natural Fibres composites:
- From 30°C to 700°C under nitrogen flow.
PHB – Organophilic Montmorillonite composites prepared by melt processing:
A Mettler Thermogravimetric Analyzer TA 400 equipped with a Mettler TG50 furnace, a Mettler M3 microbalance and a TA72 GraphWare software was employed for TGA analysis of PHB – Organophilic Montmorillonite composites prepared by solution casting. Evaluations were performed on about 20 mg samples from 25°C to 900°C at 10°C/min under a 60 ml/min air flow.
3.5.9. Differential Scanning Calorimetry (DSC)
A Mettler TA 4000 System instrument consisting of a DSC-30 differential scanning calorimeter and a TA72 GraphWare software was employed for thermodynamic characterization. The samples of around 10 mg were placed in 40 µl aluminum pan. An empty pan was used as reference. Measurements were carried out under a 80 ml/min nitrogen flow rate according to the following protocols:
PHB – Anox 20TM – Alknanox 240TM and PHB – Stabaxol® P200 mixtures:
- First heating from -30°C to 210°C at 10°C/min.
- First cooling from 210°C to -30°C at 100°C/min followed by a 3 min isotherm at - 30°C.
- Second heating from -30°C to 210°C at 10°C/min.
- Second cooling from 210°C to -30°C at 10°C/min with 3 min isotherm at -30°C.
- Third heating from -30°C to 350°C at 10°C/min.
Modificated Natural Fibres:
- First heating from 25°C to 200°C at 10°C/min followed by a 3 min isotherm at 200°C.
- First cooling from 200°C to -30°C at 100°C/min.
- Second heating from -30°C to 200°C at 10°C/min.
PHB – Natural Fibres composites:
- First heating from 25°C to 200°C at 10°C/min followed by a 3 min isotherm at 200°C.
- First cooling from 200°C to -30°C at 100°C/min followed by a 3 min isotherm at - 30°C.
- Second heating from -30°C to 200°C at 10°C/min followed by a 3 min isotherm at 200°C.
- Second cooling from 200°C to -30°C at 10°C/min.
PHB – Organophilic Montmorillonite composites prepared by solution casting:
- First heating from 0°C to 200°C at 10°C/min.
- First cooling from 200°C to 0°C at 10°C/min.
- Second heating from 0°C to 200°C at 10 °C/min.
- Second cooling from 200°C to -30°C at 100°C/min followed by a 3 min isotherm at -30°C.
- Third heating from -30°C to 200°C at 10°C/min.
PHB – Organophilic Montmorillonite composites prepared by melt processing:
- First heating from -30°C to 210°C at 10°C/min.
- First cooling from 210°C to -30°C at 100°C/min followed by a 3 min isotherm at - 30°C.
- Second heating from -30°C to 210°C at 10°C/min.
- Second cooling from 210°C to -30°C at 10°C/min with 3 min isotherm at -30°C.
- Third heating from -30°C to 210°C at 10 °C/min.
The phase transitions temperatures were taken as corresponding to the maximum temperature in the relevant enthalpic peaks. The glass transitions temperatures were taken at the inflection point of polymer devitrification. Temperature and energy calibrations were carried out using standard samples of tin, indium and zinc.
3.5.10. Dynamic Mechanical Thermal Analysis (DMTA)
Dynamic mechanical properties were evaluated using a Rheometrics DMTA V with a single cantilever bending geometry. Rectangular strip specimens (22x5x0.4 mm) were investigated from -50°C to 100°C (PHB – Natural Fibres composites) or from -50°C to 150°C (PHB – Organophilic Montmorillonite composites prepared by melt processing) at a heating rate of 4 °C/min, under nitrogen atmosphere. At a frequency of 1Hz, linear viscoelasticity was observed for strain at 0.05%.
3.5.11. Mechanical Tests
Tensile strength, elongation at break, and Young modulus were measured in tensile deformation with a universal machine for mechanical test Instron model 5564 (Instron
preconditioned for at least one week at 25°C and 50% RH inside desiccators containing saturated solutions of magnesium nitrate. Samples thickness was measured with a digital micrometer. Testing protocols were based on ASTM Standard D1708-93 and D638M-93.
Pneumatic grips of the testing machine were set at an initial separation of 21.6 mm.
Crosshead speed was set at 1 mm/min. At least 12 specimens for each sample were tested.
Statistical evaluations (mean, standard deviation, Student t test, and ANOVA) were performed on the best 10 specimens from normality plot.
3.5.12. Oxygen Permeability
Oxygen permeability tester PermeO2 was assembled in in the Istituto Nazionale di Fisica della Materia (INFM) of the University of Pisa. The instrument was connecetd to an oxygen transducer Rapidox 2000 (Cambridge Sensotec). According to the ASTM Standard F 1307-02, during the measurements oxygen was carried by a 15 ml/min nitrogen flow rate. Films diameter was 8 cm with a thickness of around 200 µm. Oxygen permeability values were calculated as the average of the last 50 measurements normalized to a film thickness of 25 µm.
3.5.13. Determination of the Degree of Substitution of Esterfied Natural Fibres
The degree of substitution (DS) of esterified natural fibres was determined according to the procedure proposed by Samios et al. [204]. Approximately 1 g of the dry fiber was weighed accurately in a weighing bottle. The sample was then transferred to a Erlenmeyer flask, and the bottle was reweighed to determine the exact sample weight.
Ethanol (40 ml of 75% v/v) was then added to each sample, and reagent blanks were set up and carried through the rest of the procedure. The flasks, loosely stoppered, were heated for 30 min at 60°C. Then, a 0.5 N potassium hydroxide solution was added to each of the flasks, which were then heated again at 60°C for 15 min. The flasks were then stoppered tightly and allowed to stand at room temperature (below 30°C) for 72 h. The excess alkali was then titrated with hydrochloric acid (0.5 N) using phenolphtalein as indicator. An excess of acid was added (1 ml), and the alkali was allowed to diffuse from the fibres overnight. The disappearence of the pink colour indicated the complete neutralization of the alkali. The small excess of acid was then back titrated with
potassium hydroxide (0.5 N) to the phenolphtalein end point. After the solution acquired a faint pink colour, the flask was stoppered and shaken vigorously. Because the colour might fade because of acid diffusing from the fibres, the addition of alkali and the shaking were continued until the faint pink end-point persisted. The weight percent gain (WPG) of the fibres was then calculated as follows:
WPG (ACETYL) = [(A – B)NB– (C – D)NA] ×4.3 / W
and:
WPG (BUTYRIL) = [(A – B)NB– (C – D)NA] ×7.1 / W
where A is the amount in millilitres of potassium hydroxide added to the sample, B is the amount in millilitres of potassium hydroxide added to the blank, Nb is the normality of the potassium hydroxide solution, C is the mount in millilitres of hydrocloric acid added to the sample, D is the amount in millilitres of hydrocloric acid added to the blank, Na is the normality of the hydrocloric acid solution, W is the weight of the sample in grams and 4.3 and 7.1 are the factors needed to calculate the two WPGs. The average number of ester groups per anhydro-D-glucose unit of cellulose (degree of substitution, DS) of pure cellulose and commercial cellulose acetate was calculated as follows:
DS = (3.86 ×WPG) / 102.4 - WPG)
