Developing Chitosan-Morphed Graphene Composite Based Functional Materials By

Home , Compression molding

Developing Chitosan-Morphed Graphene Composite Based Functional Materials by

Compression Molding and Laser Lithography

Amruta Narendra Raghatate

A Thesis submitted to the Department of Engineering Technology,

College of Technology

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCES

in Engineering Technology

Chair of Committee: Dr. Francisco Robles Hernandez

Co-Chair of Committee: Dr. Venkatesh Balan

Committee Member: Dr. Debora Rodrigues

Committee Member: Dr. Karim Alamgir

University of Houston

December 2020

ACKNOWLEDGMENTS

I would like to offer my sincere gratitude to my supervisor, Dr. Francisco Robles Hernandez for allowing me to work under his guidance for my master’s thesis. I am grateful that he kept me motivated throughout my research. He has been a source of inspiration and constantly encouraged me by sharing his constructive feedback, knowledge, and experiences. I appreciate his guidance and support throughout these two years both in academic and non-academic situations.

Special thanks to Dr. Venkatesh Balan for his explicit support and guidance which helped me understand this research. I appreciate all the time, help, and effort, which he has invested in me and my research.

I wish to thank the members of my thesis committee, Dr. Debora Rodrigues, from the civil and environmental engineering department, and Dr. Karim Alamgir from chemical and biomolecular engineering for their time, and guidance.

I gratefully acknowledge my graduate advisor Dawn Wolf-Taylor and others at the College of

Technology, the University of Houston for their support in my graduate study.

iii ABSTRACT

Chitosan could be chemically and enzymatically processed from Chitin that is widely present in crustaceans, mollusks, insects, and fungus. It has outstanding biodegradability, biocompatibility, nontoxicity, chemical reactivity, and finds applications in tissue engineering, artificial kidney, wound healing, burn treatment, biosensors, and electronics. The main goal of this study is to develop a fabrication process for producing Chitosan Morphed Graphene Composite (CMGC) using traditional sintering (compression molding) and study their properties. First, we produced

CMGC using established methods that combine mechanical milling and sintering at temperatures ranging from 120°C to 180°C. Second, the integrity of the chitosan matrix was studied by material characterization including scanning electron microscopy, Raman spectroscopy, Transmission

Electron Microscopy, and Fourier-Transform Infrared spectroscopy. Spectroscopy studies confirm that the processing conditions used in this study produced CMGC without degrading the chitosan molecule; although crystallinity is modified. Third, we studied different mechanical properties of

CMGC, that are then compared with conventional polymers. The compression modulus is comparable to that of polyethylene, the compression strength is similar to Nylon-6, while toughness and bulk density is superior to commercial polymers. Fourth, the CMGC is biodegradable and compostable when exposed to water or is mixed in moist soil. Also, it is non- toxic and can easily be used as compost to sustain plants without visible inhibitions. Fifth, CMGC can be processed by means of laser lithography to manufacture final products for a long range of applications which opens up an endless opportunity for fabricating biodegradable CMGC for different applications. Ideally, in future work, we plan to use the CMGC for applications such as computers, electronics, batteries, and defense weaponry such as tasers that will help to accomplish net zero emissions.

iv TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... iii ABSTRACT ...... iv LIST OF TABLES ...... vii LIST OF FIGURES ...... viii LIST OF ABBREVIATION ...... xi 1. CHAPTER I ...... 1 INTRODUCTION...... 1 2. CHAPTER II ...... 12 LITERATURE REVIEW ...... 12 2.1 HEAVY METAL ADSORPTION ...... 12 2.2 SYNTHETIC DYE REMOVAL ...... 15 2.3 MEMBRANE SEPARATION...... 18 2.4 SENSORS AND ELECTRODES ...... 20 2.5 BIOMEDICAL ENGINEERING ...... 25 2.6 BIOMATERIAL AND PACKAGING ...... 31 2.7 COMPRESSION MOLDING OF CHITOSAN BIOMATERIAL ...... 32 2.8 LASER PROCESSING FOR CHITOSAN BIOMATERIAL ...... 36 3. CHAPTER III ...... 38 EXPERIMENTAL METHODS AND TECHNIQUES ...... 38 3.1 MATERIALS ...... 38 3.2 MECHANICAL PROPERTIES ...... 40 3.3 MATERIAL CHARACTERIZATION TECHNIQUES ...... 43 3.4 PHYSICAL PROPERTIES TESTING ...... 44 3.5 COMPRESSION MOLDING ...... 48 3.6 LASER LITHOGRAPHY ...... 54 3.7 WATER DEGRADABILITY AND BIOTOXICITY EVALUATION ...... 55 4. CHAPTER IV ...... 57 RESULTS AND DISCUSSIONS ...... 57 4.1 MECHANICAL PROPERTIES ...... 57 4.2 MATERIAL CHARACTERIZATION TECHNIQUES ...... 64

v 4.3 PHYSICAL PROPERTIES TESTING ...... 72 4.4 COMPRESSION MOLDING ...... 77 4.5 LASER LITHOGRAPHY ...... 78 4.6 WATER DEGRADABILITY AND BIOTOXICITY EVALUATION ...... 80 4.7 DISCUSSION ...... 81 5. CHAPTER V ...... 86 CONCLUSIONS AND FUTURE WORK ...... 86 REFERENCES ...... 90

vi LIST OF TABLES

1.1 Physical properties of chitin and chitosan...... 4 2.1 Summary of chitosan and carbon composites for heavy metal adsorption application ...... 15 2.2 Summary of chitosan and carbon composites for dye adsorption application ...... 18 2.3 Summary of membrane composition, method and optimum carbon structure filler for pervaporation application ...... 20 2.4 Summary of chitosan-carbon composites for sensors and electrode applications ...... 24 2.5 Summary of chitosan-carbon composites for biomedical applications .....30 2.6 Summary of film composition, method and optimum GO filler for biomaterial and packing application ...... 32 3.1 Summarizes ASTM protocols used for composite compression testing .41 3.2 Dimensions of the circular cylindrical mold used for sintering ...... 47 3.3 Dimensions of Power screw mold ...... 50 3.4 Laser type and specifications of Trotec speedy 100 laser engraver and cutter ...... 54 3.5 The combinations of laser power and speed for laser cutting ...... 55 4.1 Summary of mechanical properties for chitosan and CMGC 5 wt% sintered at 180C ...... 59 4.2 Summary of mechanical properties for samples sintered at 120 and 180C for 3 h at 3.5 MPa pressure ...... 64 4.3 Summary of characteristic FT-IR spectral peaks of native chitosan ...... 68 4.4 Characteristics peaks of the Raman spectrum obtained from conventional and power screw mold ...... 72 4.5 Summary of sample specifications after sintering and resulting bulk density ...... 74 4.6 Theoretical density is calculated by the rule of mixtures and densification in percentage ...... 75 4.7 Mechanical properties of CMGC and conventional polymers ...... 82

vii LIST OF FIGURES

1.1 Different biological sources of chitin and its allomorph structural characteristics and conversion to chitosan. Here (a) Organisms containing chitin, (b) Chemical structure of chitin and chitosan biopolymer, (c) 13C NMR of different allomorph of chitin and (c) three polymorphic configures of α-chitin (antiparallel aligned polysaccharide chain), β-chitin (parallel aligned chains) and γ-chitin (a mixture of antiparallel and parallel aligned chains) ...... 3 1.2 Schematic diagram of different graphitic phases developed after the mechanical milling of carbon soot. Here carbon soot is milled using a high energy ball mill SPEX 8000M resulting in (a) development of Rh6 and Rh6- II phases and (b) sintered product after Spark plasma sintering...... 7 3.1 Image of sample sintering set up using a French press with a mold externally heated using electrical heater and data recorded by data acquisition software using a computer...... 39 3.2 Image of sintered bio composite using different ingredients. Here, (a) chitosan and (b) CMGC samples sintered using French press at 180C for 3 h...... 39 3.3 Schematic diagrams of sintered samples and their dimension before and after cutting. Here, (a) CMGC obtained directly from mold and (b) CMGC sample cut to appropriate dimension used for the compression test ...... 40 3.4 Image of the compression test setup. Here, (a) Interactive strength challenger machine and (b) custom-made compression sample platens ...42 3.5 Image of the sample that was subjected to a compression test with interactive strength challenger universal testing system...... 43 3.6 Schematic diagram of power screw mold ...... 50 3.7 Image of different parts of the power screw mold when disassembled and assembled. Here, (a) front view, (b) top view, and (c) assembled parts kept in box furnaces, heated at 180C for producing sintered chitosan biocomposite ...... 51 3.8 Schematic design of the cubical mold and fabricated stainless-steel molds. Here, (a), individual mold component design, (b) assembled mold components design ...... 52 3.9 Image of different parts of cubical mold when disassembled and assembled. Here, (a) front view, (b) top view, and (c) assembled parts kept in box furnaces, heated at 180C for producing sintered chitosan bio composite 53 3.10 Image of rectangular sheet mold with rounded corners...... 53

viii 3.11 Schematic design that was used as a template for making different shapes. Here, (a) UH logo, (b) Square plate with holes, and (c) Gear shape ...... 55 4.1 Images of the compression test samples, (a) chitosan and (b) CMGC 5 wt% and optical microstructures for the sintered (c) chitosan and (d) CMGC 5 wt% ...... 58 4.2 The plot for compression test curve for the sintered (a) chitosan and (b) CMGC 5 wt% at 180C ...... 59 4.3 Transmission electron microscopy of (a) pure chitosan, (b, c) low magnification CMGC 5 wt%, and (d) MG...... 61 4.4 The plots for (a) Young’s modulus, (b) 1st Onset, (c) 2nd onset, (d) compressive strength, and (e) Toughness for varying MG wt.% ...... 63 4.5 SEM images of (a) Raw chitosan and (b) milled chitosan...... 65 4.6 SEM images of sintered (a) Raw chitosan, (b) milled chitosan, and (c) CMGC ...... 66 4.7 SEM images of (a) chitosan, (b) CMGC 5wt% sintered at 120oC, (c) chitosan and (d) CMCG 5wt% sintered at 180oC ...... 67 4.8 FT-IR spectrum of (a) chitosan raw, (b) chitosan milled for 1.5 hours, (c) chitosan milled for 1.5 h followed by sintering at 180C, and (d) chitosan – milling along with 5% MG for 1.5 h followed by sintering at 180C ...... 69 4.9 Raman spectrum for chitosan with and without sintering in the presence and absence of morphed graphene. Here, (a) raw chitosan (unmilled), (b) milled chitosan, (c) sintered chitosan, and (d) CMGC 5wt% ...... 70 4.10 Raman spectrum for chitosan and CMGC samples sintered using power screw mold. Here, (a) chitosan 120C, (b) chitosan 180C, (c) CMCG – 5wt% at 120C, and (d) CMCG – 5wt% at 180C...... 71 4.11 Image of four-probe electrical conductivity testing of chitosan and chitosan- MG composite...... 72 4.12 Image of sintered samples (a) raw chitosan – no milled, (b) Milled chitosan, and (c) milled CMGC 5wt%...... 74 4.13 The plots for bulk density of chitosan and composites as a function of morphed graphene content and sintering temperature (a) sintering for 3 h, (b) sintering for 5 h ...... 77 4.14 Image of samples sintered at 180C for 3 h with a power screw mold (a) thickness 3 mm, and (b) thickness 10 mm and (c) top view and (d) front view of cubical mold samples with wall thickness 2 mm ...... 78

ix 4.15 Image of the laser cut samples of acrylic sheet, chitosan, and CMGC 5wt.% composite. Here the thickness of (a) UH logo design is 3 mm, (b) - (c) Gear shape is 2 mm, and (d) - (e) is 1.5 mm ...... 79 4.16 Image of the water degradability testing of (a) raw chitosan, (b) milled chitosan, and (c) CMGC 5 wt% samples in water ...... 80 4.17 Image of the experimental setup for the biotoxicity evaluation, (a) soil control, (b) chitosan raw, (c) chitosan milled, and (d) CMGC samples ....81 4.18 The Ashby plot for Young’s modulus E plotted against density. This graph is adopted from Ashby MF-Chapter 4: Material Property Charts ...... 83 4.19 The Ashby plot for Strength plotted against density. This graph is adopted from Ashby MF-Chapter 4: Material Property Charts ...... 83 4.20 The Ashby plot for Young’s modulus plotted against strength. This graph is adopted from Ashby MF-Chapter 4: Material Property Charts ...... 84 4.21 The Ashby plot for specific Young’s modulus plotted against specific strength. This graph is adopted from Ashby MF-Chapter 4: Material Property Charts ...... 85

x LIST OF ABBREVIATION

Abbreviation Meaning Ag Silver ASTM American Society for Testing and Materials (ATR)-FTIR Attenuated Total Reflectance FT-IR Au Gold BD Bulk Density BMC Bulk Molding Compound CMGC Chitosan Morphed Graphene Composite CNT Carbon Nano Tubes CO Carbon Monoxide

CO2 Carbon Dioxide COOH Carboxyl group Cr Chromium Cu Copper DA Degree of Acetylation DD D egree of De-acetylation DP Degree of Polymerization FTIR Fourier Transform-Infrared Spectroscopy GO Graphene Oxide HAP Hydroxyapatite hMSC human Mesenchymal Stem Cells MG Morphed Graphene MWNT Multi Walled Carbon Nanotube NMMO N-Methyl Morpholine-N-oxide NMR Nuclear Magnetic Resonance NOx Nitrogen Oxides OH Hydroxyl Pb Lead

xi PE Polyethylene Poly(3,4-Ethylene Di-Oxy-Thiophene) Poly(Styrene PEDOT-PSS Sulfonate) PEG Poly(Ethylene Glycol) PEO Poly(Ethylene Oxide) PET Poly(Ethylene Terephthalate) PHA Poly(Hydroxy Alkanoates) PLA Poly(Lactic Acid) PMMA Poly(Methyl Methacrylate) PP Poly(Propylene) PSS Poly(Styrene Sulfonic acid) PVA Poly(Vinyl Alcohol) PVDF Poly(Vinylidene Di-Fluoride) rGO Reduced Graphene Oxide SEM Scanning Electron Microscopy SLS Selective Laser Sintering SMC Sheet Molding Compound SWNT Single Walled Carbon Nanotubes TEM Transmission Electron Microscopy TPU Thermoplastic Polyurethanes UCS Ultimate Compressive Strength UH University of Houston VD Volumetric Density XRD X-Ray Diffraction

xii CHAPTER I

INTRODUCTION

Polymers are large macromolecules consisting of chains of repeating units made up of synthetic organic materials used as plastics and resins and they offer superior bulk physical and chemical properties at the macro and nanoscale (1). Widely used polymeric material such as epoxy, polyester, nylon, phenolic resins, epoxy, polyesters, polyethylene, polypropylene, glass-fibers, etc. are used for different applications in the packing industry, aerospace industry, automotive engineering, battery applications, and electronics manufacturing (2). Most of the synthetic polymers are produced using fossil fuel and are not biodegradable. Only 10% of the plastic in the developed countries are recycled due to logistics issues associated with collection and segregation from municipal waste. The majority of the plastics are dumped in the ocean producing soluble microplastics consumed by sea creatures and entering our food chain. When these plastics are dumped in the landfill, they take hundreds of years to decompose and occupy significant space when disposed of in landfills. Due to the growing concern of plastic pollution, extensive research is undertaken to produce biodegradable bioplastic using biopolymers such as poly-lactic acids produced using starch or different Polyhydroxyalkanoates (PHAs) produced using microorganisms that can be sustainably produced and has comparable mechanical properties as regular plastic (3). These bioplastics are designed to disintegrate in landfills benefiting our environment. Being able to compete with commercial plastics remains the greatest challenge for bioplastic research (4). One of the focuses of this research is to produce bio-composite using biopolymer that has superior mechanical strength, physical properties used for various application and at the same time has biodegradable characteristics that will benefit environment when disposed after their use.

Biopolymers are naturally produced by living organisms and broadly classified based on their backbones such as sugars (polysaccharides), aromatic molecules (lignin), amino acids (proteins), and nucleotides (nucleic acids). Most of the biopolymer stabilizes the exoskeleton of different organisms. The source of biopolymer includes plants (e.g., cellulose, lignin, starch, alginate, lipids, proteins, gums, carrageenan); animals (e.g., chitin, chitosan, hyaluronan, casein, whey, collagen, albumin, keratin, silk), and microorganisms such as bacteria, micro/macroalgae and fungi (e.g., a family of PHA, bacterial cellulose, hyaluronan, xanthan, curdlan, pullulan, alginate, agar, carrageenan, chitin, and chitosan). Among different biopolymers, chitin and chitosan are widely used to make different types of biomaterials and composites due to their unique properties to form polyoxysalts, biofilms, biocompatibility, biodegradability, non-toxicity, and molecular adsorption properties. Chitin is a crystalline sugar polymer made up of N-acetylglucosamine a derivative of glucose linked by β-(1-4) glycosidic bonds and its degree of acetylation (DA) is typically 0.90 with a molecular weight of 203.2 g/mol. Both inter- and intra-molecular network of hydrogen bonds that connect the sugar polymer strands giving the chitin-polymer matrix increased strength.

In nature, chitin is present in different allomorphs namely, α-chitin (antiparallel chains), β-chitin

(parallel chains), and γ-forms (a mixture of parallel and anti-parallel chains) (7, 8). The most abundant chitin allomorph is α-forms, the β-forms are present in squid pens and cuttlefish and γ- forms are present in the cocoon of the moth. Chitosan is a de-acetylated derivative of chitin which is a linear polysaccharide composed of randomly distributed β-linked D-glucosamine and N- acetyl-D-glucosamine (7). The DA in chitosan is considered to be below 50% and varies depending on the processing of chitin.

Chitin is widely present in cell walls of fungi, the exoskeletons of arthropods, such as crustaceans

(e.g., lobster, shrimp, crawfish, and crab), and insects (e.g., grasshoppers, beetle, and cricket).

Chitosan, a linear semi-crystalline polymer with molecular weight from about 10,000 to 1 million

Dalton and DA less than 0.35 (8). Molecular weight of chitosan has major effects on its physiochemical properties (9, 11). Chitosan is produced by de-acetylation reaction using the chemical or enzymatic treatment, as it is found in nature in limited quantities in certain fungi (7).

The acetyl group on chitosan provides different biocompatibility with a different medium (7,9).

Both chitin and chitosan form nanofibers with 50-500 nm diameter. The source of chitin affects the number of sugar chains in the biopolymer called the degree of polymerization (DP), its composition, and hence its physical and chemical properties. The structure of chitin and chitosan, its source, and their chemical properties are shown in Figure 1.1.

Figure 1.1. Different biological sources of chitin and its allomorph structural characteristics and

conversion to chitosan. Here, (a) Organisms containing chitin (10), (b) Chemical structure of

chitin and chitosan biopolymer (10), (c) 13C NMR of different allomorph of chitin (10) and (c)

three polymorphic configures of α-chitin (antiparallel aligned polysaccharide chain), β-chitin

(parallel aligned chains) and γ-chitin (a mixture of antiparallel and parallel aligned chains) (10).

The composition and properties of chitin and chitosan such as elemental formula, degradation temperature, source, degree of deacetylation (DD), and molecular weight are summarized in Table

1.1. The biopolymers used in the research were obtained for Alfa Aesar chemical company. The chemical and physical properties of chitin and chitosan are influenced by the source and method of extraction.

Table 1.1. Physical properties of chitin and chitosan

Chitin Chitosan

Formula (C8H13O5N)n (C6H11O4N)n Molar mass 209.1925 g/mol 10,000-190,000 Da (based on viscosity)

Degradation temperature 341- 406 °C 225 °C Source Crab shells Crab shell Degree of De-acetylation N/A 85%

Note: Chitin and chitosan used in the research were obtained for Alfa Aesar chemical company.

The DD for chitosan is 85%.

The class of material composite consists of two or more constituent materials with different chemical and physical properties. When these constituent materials are combined together, they produce a composite with superior properties than the individual constituent material (1). When a filler material or a stronger reinforcing phase is introduced in a polymer matrix, a polymer composite is formed. Composites can be stronger, less expensive, lighter, and chemically stable.

Therefore, they are preferred over the constituent materials. Widely used polymeric material such as epoxy, polyester such as nylon, polyethylene, polypropylene, glass-fibers, etc. is used for polymer composite applications in the aerospace industry, automotive engineering, battery applications, electronics manufacturing (2). Composite materials are traditionally made by combining petrochemical resin and reinforcement materials. Bio-composites on the other hand are 4 made using nature-made materials such as biopolymers having both resin and reinforcement properties. Biopolymer composites are gaining more importance due to their low environmental impact, renewable, and sustainable alternative. Different biopolymer composites are being developed in recent years to potentially replace conventional polymer material in engineering and biomedical applications (11). Some of the manufacturing techniques commonly used for bio- composite synthesis are extrusion, injection molding, compression molding, solution blending and casting, melt mixing, layer by layer assembly, and in-situ polymerization (13, 14). For practical applications, these biopolymers have relatively lower mechanical strength, thermal stability, permeability issues when compared to synthetic polymers. Hence, efforts are being made to explore the possible chemical modification of biopolymers followed by evaluating their ability to act as reinforcing filler materials to improve bio-composite, mechanical, physical, and structural properties (14).

High-speed ball milling is a solid-state synthesis process used to produce nanostructured material.

In this method, the particles of the material are initially deformed, fractured, and welded due to the high energy of the colliding balls. This eventually results in the formation of a nanostructure.

Mechanical milling ensures even dispersion of fillers in the polymer matrix. Different carbon structured materials such as graphene, graphene oxide, carbon nanoparticle, Carbon Nano Tubes

(CNT), graphene quantum dots, carbon soot, and fibers are very well known to have superior mechanical, electrical and thermal properties and when used as filler reinforcement with biopolymers can help enhance strength and properties of the resulting bio-composite (11–13). In this thesis, bio-composite was made using a morphed stake of graphene called Morphed Graphene

(MG) reinforced chitosan in different ratios, and their structural, chemical, mechanical, biodegradable properties were evaluated (15). In general, pristine carbon nanostructures are not

5 used in composites because their integrity and properties are compromised rather their functionalized derivatives are used. Morphed graphene on the other side, is ideal for composite production using milling because it sustains the milling without affecting its integrity and potential for outstanding reinforcement. Mechanical milling is an effective solid-state process that produces nanocomposites with consistent particle size and shape. MG has hexagonal carbon allotropes with

R-3m crystal symmetry was produced by mechanical milling of Fullerene soot as reported in the literature (15). Figure 1.2 shows a schematic diagram of different graphitic phases developed after milling of carbon soot. MG containing hexagonal carbon rings bridging graphene-like layers are evident from material characterization methods such as Transmission Electron Microscope (TEM) and Raman spectroscopy (15). Mechanical milling of Fullerene soot yields MG structures with

Rh6 (sp2) and the Rh6-II (sp3). These phases are more complex than graphene and they have higher strength, but lower elastic properties. Milling induces crystallinity in the soot particles and stronger

D and G bands in Raman spectra are developed. This method is a low cost and low energy route to synthesize carbon structures, hence the name MG and are produced mechanically at room temperature. Ideally, it is desirable to increase the toughness of the composite which is possible with MG carbon phases. After 0.5 and 2 h milling, the reported modulus of MG was reported as

18.5 GPa and 15.6 GPa respectively (16).

Sintering is a process that makes a powdered material coalesce into a solid or porous mass by heating at an elevated temperature at high pressure without melting the materials. This technique is widely used to make composite materials (17) and the principle of sintering involves diffusion of atoms.

Figure 1.2. Schematic diagram of different graphitic phases developed after the mechanical

milling of carbon soot. Here, carbon soot is milled using a high energy ball mill SPEX 8000M resulting in (a) development of Rh6 and Rh6-II phases (16) and (b) sintered product after Spark

plasma sintering (16).

Sintering is the important step to promote phase transformation to Rh6 across the boundaries of the particle, hence fusing them under application of temperature and pressure (2, 17). In one of the previous work, a chitosan bio-composite comprising of MG was produced by sintering at different temperatures (120, 150, 180, and 220oC), a varying amount of MG (0, 1, 3 and 5%wt) and heating time (3 and 5 h). Chitosan was subjected to milling for 1.5 h, MG for 3 h, and together chitosan and MG were milled together in the different ratio for an additional 1.5 h. This was followed by sintering at elevated temperature (up to 220oC) and pressure (3.5 MPa) using custom made French press using the principles of compression molding. These studies concluded that bio-composites with better physical and mechanical properties were achieved at 180oC in 3 h (18, 19). Overall, it took ~7 h to prepare the bio-composite from start to finish. Mechanical milling has several advantages including processing at low temperatures without the use of any solvent, helps to

7 achieve even particle size and homogenous dispersion of nano-fillers in a biopolymer matrix (20).

Since chitosan appears as a flaky structured biopolymer with irregular particle distribution, mechanical milling helps to produced chitosan nanoparticles with even particle size distribution and anchoring with MG (18).

In this study, we used similar processing conditions as reported earlier to produce the chitosan MG bio-composite (CMGC) to re-confirm their properties and produce different shapes of objects. The sintered CMGC samples were first tested for their mechanical properties such as Young’s modulus, compressive strength, and toughness by compression testing. Then their structure was analyzed for grain boundaries using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Chemical properties molecular vibrations analyzed using Raman spectroscopy, and Fourier-transform infrared spectroscopy (FT-IR). In addition, the dry bulk density and electrical conductivity were measured to evaluate probe resistivity.

Conventional mold sintering or compression molding is a very popular technology to produce commodities with powdered material. This technique follows the basic principles of powder metallurgy: powder pulverizing, die compaction, and sintering (21). Ball milling is an effective method of pulverizing material and for the production of nanostructured material as discussed earlier. Compaction refers to filling in the powdered material into the compression mold by application of pressure and it is generally carried out at room temperature. Sintering is performed at a higher temperature for material particles to fuse under the application of pressure and temperature. Owing to the excess surface energy available at solid-state sintering, the particles are bonded to the surface and take the desired shape of the die. The density of the output depends on the pressure applied. This processing generally applies to metals or ceramics (17).

Using compression molding techniques for the production of bioplastic commodities is quite an interesting concept and is the important goal of this research. We designed a new power screw mold for the first time that could produce multiple samples at the same time and increase manufacturability from the process. This mold design is just a proof of concept and a similar method can be used to make various shapes and sizes. In this design, a power screw would transfer torque to linear constant pressure to the mold at both ends to compress the sample. This would eliminate the mechanical movement due to French press operation instead it uses a thread mechanism similar to a bolt and a nut. The torque was measure with a digital torque wrench.

Multiple power screw molds can be tightened to the required pressure and can be directly put into the high temperature convection furnace for sintering. This new method of CMGC bio-composite manufacturing will help to significantly reduce the processing time and energy utilization.

Laser cutting is a thermal manufacturing technology that involves no contact machining of the material with the use of optical laser. Currently, laser cutting is gaining popularity as a machining process to manufacture composite materials (22). Laser optics and computer numerical control are used to direct a high-power laser beam on the material to facilitate the desired cut shape and size.

Generally, the CO₂ laser is used for laser cutting, boring, and engraving (23). Organic materials show high surface absorption for wavelength 10.6 m which is related to CO₂ lasers. Therefore, materials like glass, plastic, papers, fabrics, and other polymeric materials can be easily cut with this laser type with an output power of less than 500 W (23). Laser cutting is chosen over other manufacturing technologies due to its low-cost of operation, high surface finish, no contact cutting and hence no contamination with cutting media, and flexibility of the cut shapes and sizes. Three types of cutting processes have been reported on the above-mentioned materials: (i) fusion cutting,

(ii) vaporization cutting, and (iii) cutting via thermal degradation. Similar to metal cutting with

9 inert gases, thermoplastics have been cut by fusion laser cutting (24). The laser melts the area undertreatment and high-pressure gas are used to blow the molten material for the spot. The material is generally heated to its melting point. The power requirement in fusion cutting is much lesser than other processes. The cut surfaces are smooth visibly with some peaks due to molten material. Vaporization cutting is generally used for thermosets. Popularly Plexiglass is cut using vaporization laser cutting. This cutting process involved heating of the material surface with a beam of laser to its boiling point. As the material on the surface boils and generates vapors, it wears out the molten walls thus cutting even. Cutting via chemical degradation involves breaking the 3D lattice of the polymer with a high-power laser beam on the surface of the material unlike melting the linear chain of monomer in other processes. This process naturally consumes more energy than other cutting processes due to high energy requirements. However, the surface finish is better than other cuts and thin layer carbonaceous particles are found on the cut surface which can be cleaned easily (25–27). With laser facilitated cutting, composite materials can be cut with no force-induced manufacturing technique, and hence the structural integrity is confined, plus no contamination from the cutting media is achievable. Laser cutting is selected as a manufacturing process for sintered CMCG for the first time and is successfully demonstrated in the current work.

This research will open up a new avenue of biomanufacturing where the processed composite material can be cut into different shapes and sizes that can be used for different industrial and medical applications.

Finally, the water degradability of the CMGC was tested and its non-toxicity properties were confirmed by growing some plants after soaking them in water using the reported procedure (28,

29). Our studies demonstrate that chitosan and CMGC disintegrated quickly when in contact with water benefiting the environment. Just like sugars like glucose, sucrose, fructose chitosan also

10 decomposition during heat treatment. The rate of decomposition increases with prolonged heat treatment. The thermogravimetric method shows that most of the residual moisture in chitosan evaporates around 30–110 °C. The glass transition temperature of chitosan lies in the range of 180 to 240°C depending upon the molecular weight (30). Heating beyond glass transition temperature results in releasing toxic gases such as Carbon monoxide (CO), Carbon dioxide (CO2), Nitrogen oxides (NOx). Therefore, 180°C is selected as the maximum sintering temperature for the fabrication of CGMC. Through this study, we have demonstrated that the CMGC does not produce toxic byproducts on milling and sintering and could be used as a sustainable replacement for single-use commodity plastics products. The resistivity studies have proved that CMGC could also be used as disposable electronic components such as resistors, capacitors, circuit boards, and for making disposable batteries in the future.

The objective of this research is to develop a fabrication process for chitosan and CMGC using compression molding and laser lithography; to demonstrate the potential of CMGC composites and competitiveness when compared to commodity plastics. Here we demonstrate that a scalable technology is capable of producing a competitive product made using by-products is capable of substituting commodity plastics, with potential biodegradability and potential for applications from electronics, to electrochemistry, energy, defense, among others.

CHAPTER II

LITERATURE REVIEW

In this literature review, we discuss developments in chitosan and carbon structure nanocomposites based on their synthesis and applications. As described in the first chapter, biopolymers like chitosan are low-cost, abundantly available, and obtained from renewable resources. Its environment-friendly nature has led to growing research and development in areas like bone and tissue engineering, battery technology, electronics, drug delivery, food and agriculture, 3D printing, water, and effluent treatment systems, etc. Applications of chitosan as a benign biopolymer are limited by its mechanical strength, thermal stability, solubility, and difficulty in controlling pore size. With the developments in nanotechnology, nanocomposites with carbon structures as fillers are gaining importance in research. A wide variety of carbon structures such as activated carbon, char, Single-Walled Carbon Nano Tubes (SWNT), Multi-Walled Carbon

Nano Tubes (MWNT), graphitic carbon, graphene, graphene oxide (GO), functionalized graphene are mixed with chitosan for synthesizing nanocomposites. The availability of high surface area, electrical and thermal conductivity, low weight, mechanical strength makes the carbon structures an ideal filler or reinforcing composite material. A comprehensive review of the available synthesis techniques, optimum use of carbon material as filler, and challenges related to producing them are outlined here. In the end, we describe the infrastructure requirement and development of chitosan composite using compression molding and laser cutting.

2.1 Heavy metal adsorption

Ke et. al., was the first research group to synthesize chitosan and MWNTs composite film for heavy metal adsorption. They used milling to shorten MWNT and functionalized them by

12 covalently linking chitosan via nucleophilic substitution reaction (31). They found an increase in the crystallinity of chitosan after linking, however, did not provide further explanation. It is important to study the effect of milling and dispersion of MWNT to understand the efficiency of the composite for heavy metal removal application. Chen et. al., prepared eco-friendly chitosan and Graphene Oxide (GO) hydrogel adsorbent for removal of cationic and anionic dyes and heavy metal impurities (32). Based on their spectral investigation, an electrostatic interaction between the composite hydrogel and effluents is a potential mechanism for adsorption. They demonstrated the effectiveness of the composite hydrogel by constructing a water filtration column.

2.1.1 Adsorption of Lead (Pb2+) ions:

Chitosan composite materials were used for removing Pb2+ ions present in water for the first time.

Fan et. al., prepared magnetic chitosan incorporated with GO composite sheet for selective

2+ adsorption of Pb ions (33). To synthesize a magnetic chitosan composite sheet, they used Fe3O4 and linked with glutaraldehyde, and mixed with chitosan in an equal ratio. The composite material showed an increased surface area and the adsorption capacity depended on pH, contact time, and concentration of Pb2+ ions. The adsorption capacity was reported to be 76.9 mg/g, which was higher when compared to similar reference studies with different composite.

2.1.2 Adsorption of Gold (Au3+) and Lead (Pd2+) ions:

Liu et. al., followed the same synthesis technique as Fan et. al., for the adsorption of Au3+ and

Pd2+ ions from water (34). The adsorption capacity was pH-dependent [pH 3.0–5.0 for Au3+ and pH 3.0–4.0 for Pd2+]. Dispersing GO beyond 5wt.% was difficult to stir and they used sonication to overcome agglomeration. They used the same composition as they used for Pb2+ removal. They

13 failed to take into account the charge affinity of target metal and GO surface area required for effective adsorption. However, the pH dependency of adsorption selectivity is very well reported.

2.1.3 Adsorption of Chromium (Cr4+) ions:

Liu et. al., Magnetic modification of chitosan composite to study the adsorption of Cr4+ ions from the solution. Li et. al., prepared to GO functionalized magnetic cyclodextrin–chitosan composite for Cr4+ ion removal (35). The modification to the film was done by incorporating ionic liquid into the composition that improved the adsorption capacity. This film had limitations in terms of reusability and the efficiency goes down after the sixth cycle. Another study by Debnath et. al., worked on a batch adsorbed design using magnetic chitosan–GO nanocomposite for Cr4+ ion removal (36). They achieved an adsorption capacity of up to 92% after five cycles at an optimum pH of 3.0. The recent study in Cr4+ ion removal was reported by Zhang et. al. Unlike other groups, they chemically deposited Fe3O4 on the surface of GO, rather than chitosan (37). This helped to increase the chitosan surface area that provided a strong electrostatic attraction.

2.1.4 Adsorption of Copper (Cu2+) ions

Yu et. al., prepared chitosan - GO aerogel by lyophilization for Cu2+ removal. The regeneration of adsorbents was possible by filtration or low-speed centrifugation. The adsorption capacity reported was 101 mg/g and they pointed out even dispersion of GO in the composite is important for the interaction between GO and Cu2+ ions for effective adsorption (38). Table 2.1 summaries chitosan and carbon composites for heavy metal adsorption application.

Table 2.1 Summary of chitosan and carbon composites for heavy metal adsorption application.

Optimum carbon Year Material structure Method Application composition (wt.%) 2007 Nucleophilic Heavy metal Chitosan and CNT 42 (31) substitution absorption 2012 Heavy metal Chitosan and GO Hydrogel synthesis (32) absorption 2012 Chitosan, GO, Fe O Selective adsorption of 3 4 50 Crosslinking (33) and glutaraldehyde Pb2+ 2012 Chitosan, GO and Adsorption of Au(III) 5 Crosslinking (34) glutaraldehyde and Pd(II) and dye Chitosan, GO, 2013 Fe O , glutaraldehyde, 50 Crosslinking Cr4+ removal (35) 3 4 and cyclodextrin Chitosan, GO, 2013 Fe O , glutaraldehyde, 50 Crosslinking Cr4+ removal (36) 3 4 and Ionic liquid 2013 Aerogel by Chitosan and GO Cu2+ removal (38) lyophilization 2014 Chitosan, GO and Chemical Cr4+ removal (36) Fe3O4 modification

2018 Chitosan, GO and Chemical 50 Cr4+ removal (37) Fe3O4 modification

2.2 Synthetic dye removal

Several synthetic dyes are used in industries while producing textile, paper, and printing materials.

The effluents generated while producing these materials are carcinogenic and possess a threat to water bodies and hence human health. Techniques such as adsorption, membrane separation, ion exchange, and electrochemical conversions are used in water and effluent treatment systems (39).

Different adsorption matrix such as activated charcoal removal of such effluents is one of the efficient processes due to its low-cost and possible reuse. Chitosan is considered an excellent adsorption matrix to remove pollutants due to its active functional groups such as amine and

15 hydroxyl groups and is also considered as sustainable alternative other commonly used pollutant removal matrices (40). Limitations of chitosan application in the adsorption system include its low mechanical strength, difficulty to regenerate, and degradation in acid solutions. Several researchers have chemically modified the chitosan to produce nanocomposite material that could be efficiently recycled and suitable for water pollutant adsorption applications (41).

Dihydroxybenzene is one of the highly toxic environmental pollutant and endocrine disruptors.

The established electrochemical processes to remove this industrial water pollutant is very expensive (42). Amiri et. al., prepare carbon nanoparticle chitosan composite with solution casting

(42). Though the sensitivity and selectivity of dihydroxybenzene were found good, the adsorption was still weak. In another study, Cheng et. al., fabricated a 3D mesoporous composite using chitosan and graphite from waste sugarcane bagasse using a simple process with natural material to synthesize composite by stirring, filtering and vacuum drying (43). The composite was effective in removing 97.5% effluent in the first cycle. Fan et. al., used the prepared magnetic chitosan incorporated with GO sheets composite to remove methylene blue for water purification (44). To synthesize magnetic chitosan, it is treated with Fe3O4 and linked with glutaraldehyde. In another study, the chitosan-GO composite was prepared to absorb fuchsine (the deep red synthetic dye used as a biological stain and disinfectant) for up to five cycles without damaging the structure of the composite (35). The same composite has been demonstrated to be effective in both heavy metal and dye adsorption. Sheshmani et. al., the group used graphene instead of GO and investigated magnetic graphene/chitosan for removal of Acid Orange 7 (azo dye) (45). They pointed out the importance of initial pH of 3 and a longer contact time of 120 min for effective dye removal.

For fuchsin acid dye adsorption, Li et. al., used a wet-spinning process to prepare composite fibers from chitosan and GO (46). The wet spinning of chitosan is rather difficult, although fibers have

16 reported improved strength than films. They reported the mechanical strength of composite fiber with 4 wt.% GO loading was 165.8 MPa, whereas chitosan fiber was 96.7 MPa. A further modification to this composition was done by the addition of silica fiber. Du et. al., uniquely prepared chitosan/GO/silica fibers by wet spinning, followed by etching silica and creating chitosan/GO porous fibers. The maximum adsorption capacity reported was 294.12 mg/ with GO concentration up to 5 wt.% (47). Wang et. al., prepared porous chitosan - GO (up to 3 wt.%) aerogels. This structure reported the highest adsorption capacity of 686.9 mg/g for methyl orange and 573.5 mg/g for amido black 10B (amino acid staining azo dye) using. They utilized the lyophilization process to process the porous composite aerogel (48). Sabzevari et. al., prepared a

GO membrane crossed linked with chitosan (49). A well-dispersed composite is formed when chitosan interacts with GO and undergoes an amidation reaction with carboxyl groups. The ratio of GO to chitosan was 1:0.3 to maintain a minimum crosslinker percentage in the membrane.

More recently, Zhang et, al., fabricated a crosslinked chitosan and graphene nanoplates composite spheres for dye removal applications (50). The absorption efficiency of the composite was studied with methyl orange (color indicator) and acid red 1 (red azo dye). The crosslinking was achieved with glutaraldehyde and the reusability of the composite was demonstrated by uptake up to 90% for five cycles. Qi et. al., prepared chitosan, and GO sponge for filtering dye from water without the use of any cross-linkers (51). The dispersion of chitosan within GO was maintained between

9-26% without GO agglomeration. The electrostatic interaction and hydrogen bonding between chitosan and GO at lower chitosan concentration facilitated the gelation process for sponge structure formation. The reusability study of the composite sponge revealed the regeneration of up to 75%. Table 2.2 summarizes chitosan and carbon composites for different dye adsorption application.

Table 2.2 Summary of chitosan and carbon composites for dye adsorption application.

Optimum carbon Year Material structure Method Applications composition (wt.%) 2012 Chitosan Adsorption methylene 90 Solution casting (42) Carbon nanoparticle blue Chitosan and 2012 graphite from waste Vacuum drying Reactive Black 5 (43) sugarcane bagasse Chitosan, GO and 2012 Adsorption methylene Fe O 50 Crosslinking (44) 3 4 blue glutaraldehyde 2013 Chitosan, GO and Crosslinking Reactive Black 5 (52) Fe3O4 2014 Chitosan, GO and Industrial dye effluent 5 Wet spinning (47) Silica removal 2014 Adsorption of Fuchsin Chitosan and GO 4 Wet spinning (46) acid dye 2014 Chitosan, GO and Acid Orange 7 (45) Fe3O4 Adsorption of methyl 2015 Chitosan and GO 3 Lyophilization orange and amido black (48) 10B 2018 Chitosan and GO 0.3 N/A Dye removal (49) Chitosan, Graphene Syringe 2018 nanoplates and 5 dropping Dye removal (50) glutaraldehyde method 2018 Chitosan and GO 91 Freeze drying Dye removal (51)

2.3 Membrane separation

Nonporous polymeric membranes are popularly used in Pervaporation (processing method for the separation of mixtures of liquids by partial vaporization through a non-porous or porous membrane) to separate liquids due to their low cost and high efficiency associated with separation.

Chitosan is a hydrophilic polymer that provides good durability to organic solvents because of its 18 strong hydrogen bonding. Different carbon structured materials are used as a filler to complement the chitosan membrane with improved mechanical strength, hence producing a green bio-derived composite membrane. Most of the studies in the literature have reported a common solution casting synthesis technique for pervaporation membranes. The composite solutions are mechanically stirred and ultrasonicated for even distribution of the content, followed by the solution casting method. There are two main concerns for chitosan-carbon structure pervaporation membranes: (i)

Membrane selectivity and (ii) chitosan swelling. The even dispersion of carbon structure is very difficult because of agglomeration issues and its strong hydrophobicity. It is difficult to disperse raw MWNT because of their chemical inertness and tendency to agglomerate. Functionalizing

MWNT with polymer functional groups helps to inhibit the strong Van der Waal forces preventing agglomeration and leading to suitable hydrophilic modification. A highly selective pervaporation membrane has been used to separate benzene from benzene-cyclohexane mixtures. Peng et. al., first reported a poly(vinyl alcohol) (PVA) membrane incorporated with chitosan wrapped MWNT for benzene separation (53). The permeation flux and separation factor for the composite membrane was 3 and 5 times better when compared with the pristine PVA membrane. The improvements can be attributed to the modification of MWNT resulting in selective affinity toward benzene. Shen et. al., synthesized a chitosan and functionalized MWNT with silver ion (Ag+) that showed improve performance (54).

Multiple groups reported functionalizing MWNT to address the issue of MWNT bundling in the composite matrix. Qiu et. al., functionalized MWNT with a carboxyl group (-COOH) by treating it with mixed acid (55). The carboxylic MWNT was incorporated with chitosan for ethanol/water pervaporation. The improved performance of their membrane was based on the interaction of the carboxyl group (-COOH) from MWNT and hydroxyl (-OH) or amino groups (-NH2) on chitosan

19 resulting in even MWNT dispersion. Ong et. al., prepared a Poly(3-hydroxybutyrate) (PHB) functionalized MWNT – chitosan nanocomposite membrane (56) to separate 1,4-dioxane from water. Mechanical strength is the principal property of a membrane used for pervaporation due to pressure application in the process of separation. This group reported that PHB functional groups helped dispersion of MWNT in the chitosan matrix which improved the tensile strength by 41.8% and Young’s modulus by 24.1% compared to pure chitosan membrane. Table 2.3 summarizes the constituent membrane, method, and optimum carbon structure filler for pervaporation application.

Table 2.3 Summary of membrane composition, method, and optimum carbon structure filler for

pervaporation application.

Optimum carbon Tensile Elastic Year Material structure strength modulus Method Application composition (MPa) (GPa) (wt.%) Chitosan Separation of 2007 Solution MWNT 2 benzene/cyclohexane (53) casting PVA mixture Chitosan 2010 Solution Separation of Carboxylic 2 (55) casting ethanol/water mixture MWNT 2011 Chitosan Solution Separation of 1,4- 0.1 36.7 2.15 (56) PHB-MWNT casting dioxane/water mixture Separation of 2014 Chitosan Solution 1.5 benzene/cyclohexane (54) MWNT-Ag+ casting mixture

2.4 Sensors and Electrodes

Biocompatibility and chemical activity of chitosan, combined with mechanical strength, electrical conductivity, and high surface area of carbon structures are utilized for biosensors and electrode applications. The chitosan and carbon nanocomposites are used as electromechanical actuators for artificial muscles for robots, separation membranes, electrocatalysis, biofuel cells, biomimetic

20 flying, optical switches and microsensors, flexibly deployable reflectors, electrochemical biosensors for sensitive and fast determination of environmental pollutants, characterization of enzyme inhibitors, purification of biomass, and for pesticide analysis.

Initially, Du et. al., prepared an amperometric biosensor from chitosan/MWNT/glutaraldehyde to detect toxic enzymes like acetylthiocholine (57). They utilized the biocompatibility of chitosan and electrical conductivity of MWNT for enzyme-based electrochemical biosensors for sensitive and rapid determination of environmental pollutants. They cast a chitosan/MWNT/ glutaraldehyde film on a glassy carbon electrode and were able to achieve good hydrophilicity and adhesion. They used glutaraldehyde as crosslinkers, which may reduce available sensing sites in chitosan.

Following this study, Lau et. al., synthesized a porous, conductive, and biocompatible scaffold from chitosan and MWNT with the freeze gelation techniques (58) which provided an electron transfer path between pore walls and electrode. Although the maximum concentration of MWNT was 5 wt.%, they observed rougher and surface agglomeration with MWNT beyond 2.5 wt.%.

Addressing the dispersion problem Rassaei et. al., investigated chitosan and phenyl sulfonated surface functional groups - functionalized carbon nanoparticle composite film for sensing and electrocatalysis applications (59). They also used glassy carbon electrodes for layer-by-layer assembly. As the chitosan is cationic polyelectrolyte and functionalized carbon particles are negatively charged, they were able to produce an irreversible crosslinking and stable film deposits for applications in aqueous environments. Liu et. al., introduced a concept of using polymer modified CNTs instead of surface-functionalized CNTs with short chemical groups (60). They proved that polymer-modified CNTs provide better solubility in solvents, higher compatibility with polymers, and more reactive sites for further modifications. They used poly(styrene sulfonic acid)-modified CNTs to prepare the composite. The atom transfer radical polymerization reaction

21 was performed to modify CNT and further solution casting was used to prepare chitosan composite film. The strength for chitosan-modified CNT composite (30 MPa ) was three times better than the neat chitosan (11 MPa) and two times better than chitosan-CNT composite (12 MPa). A significant increase in the electrical conductivity from 2.1 x 10 -11 Siemens (S)/cm for neat chitosan to 1.2 x

10 -7 S/cm for the composite was observed. A composite film from chitosan and reduced GO was produced by Wang et. al., with vacuum filtration (61). The optimum concentration of 6 wt.% reduced GO was obtained to achieve electrical conductivity of 1.2 S/m. Mechanical strength improved to 6.3 GPa for Young’s modulus and tensile strength of 206 MPa and could find applications in biomimetics and tissue engineering which is the highest strength reported in the literature.

One of the aromatic compounds, 4-aminophenol is widely used in producing dyes, pharmaceuticals, and petroleum additives. The electrochemical behavior and voltammetric study by Yin et. al., suggest that the chitosan and graphene film resulted in a lowering of oxidation overpotential and a decrease in the peak-to-peak separation of redox peaks for 4-aminophenol (62).

In another study, Han et. al., prepared chitosan and graphene electrodes by in situ chemical reduction method and drop-casting procedure to detect the presence of ascorbic acid, dopamine, and uric acid simultaneously. The presence of chitosan triggered high electrocatalytic activity by providing binding sites and acted as dispersant and stabilizer for the biosensor electrode (63).

Li et. al., created an electromechanical actuator with a SWNT and chitosan in ionic liquid as an electrolyte layer for the first time (64). The chitosan electrolyte layer was prepared by mixing and stirring chitosan in acetic acid solution with ionic liquid 1-Ethyl-3-methylimidazolium tetrafluoroborate and cast in a plastic mold. The actuator was assembled by a hot pressing half- dried chitosan layer between two as grown SWNT electrode layers. The average mechanical

22 strength of the actuator was determined to be 50 MPa which is much better than the polymer actuator reported earlier. They reported a very high response of the actuator of 19 ms, high stress generating rate of 1080 MPa/s, and ultrahigh output power density 244 W/kg. The chitosan-SWNT actuator showed high performance compared to other available synthetic electroactive polymers

(e.g. PVDF). The mechanical and electrical properties of SWNT greatly complimented the chitosan biopolymer network for effective and faster ion transport mechanisms.

Lin et. al., developed a chitosan graphene-modified electrode for chiral sensing and recognition

(65). The biocompatible matrix is selective to a large amount of dopamine in ascorbic acid owing to π-π stacking interaction between the graphene surface and dopamine. Similarly, Rajabzadeh et. al., synthesis reduced GO-chitosan carbon glassy electrode for dimethyl disulfide electrochemical sensor (66). He et. al., prepared 1 wt.% GO incorporated chitosan nanocomposite by sonochemical

(ultrasound to chemical reactions and processes) method for electrochemical biosensors application (67). They followed a simple synergetic electrostatic interaction of chitosan and GO and hydrogen bonding in an aqueous medium for film formation. Most recently, Barra et. al., proposed a green method to prepare chitosan and reduced GO bio-composite (68). They were able to achieve 50 wt.% of reducing GO incorporation in the chitosan matrix using ultrasonication and solvent casting method. This is the highest reported GO concentration in literature with the ultrasonication and solvent casting method. The mechanical strength of 27 MPa and modulus of

2.5 MPa was achieved. The chitosan carbon structures for sensor and electrode applications is summarized in Table 2.4.

Table 2.4. Summary of chitosan-carbon composites for sensors and electrode applications.

Optimum Carbon Tensile Elastic Suggested Year Material structure strength modulus Method functions and composition (wt.%) (MPa) (GPa) improvements Chitosan, 2006 Solution Biosensors for MWNT and 1.2 (57) evaporation toxic enzymes Glutaraldehyde 2008 Chitosan and Freeze 2.5 (58) MWNT gelation Biofuel cells Electrocatalysis Chitosan and 2008 Solution Chiral selection Carbon 90 (59) evaporation Enatioselective nanoparticle drug sensors Chitosan, PSS- 2009 functionalized 1.5 30.4 2.8 Film casting Electrical (60) CNT conductivity Immobilizing 2009 Chitosan and 90 Film casting hemoglobin (69) Graphene protein 2010 Chitosan and Vacuum Biomimetic and 6 206 6.3 (61) GO filtration tissue engineering. 2010 Chitosan and 4-aminophenol 90 Film casting (62) Graphene detection Detection of 2010 Chitosan and Drop- ascorbic acid,

(63) Graphene casting dopamine, and uric acid Chitosan, Cast Electromechanical 2011 Ionic liquid molding Actuators 50 (64) and and heat Artificial muscles SWNT pressing for biomimetics 2014 Chitosan and chiral sensing and 90 Film casting (65) Graphene recognition. 2014 Chitosan and Sonochemic electrochemical 1 (67) GO al method biosensors dimethyl disulfide 2014 Chitosan and 90 Film casting electrochemical (66) Graphene sensor. 2019 Chitosan and 50 27 2.5 Film casting Biosensor (68) GO

2.5 Biomedical Engineering

Chitosan is biocompatible, hemostatic, osteoconductive, promotes mineralized bone matrix, and has a minimum inflammatory response on implantation. Chitosan is popularly used as an alternative to ceramic material in bone and tissue engineering substrates owing to its biodegradability, ability to form shapes, and mechanical strength. Besides these applications, chitosan is also used in the auto-sensing of cancer cells. Chitosan nanocomposites with carbon structures provide possibilities to expand chitosan applications in the biomedical field.

2.5.1 Solution casting and solvent evaporation method

Solution casting or solution evaporation is a popular method for studying the properties of chitosan and carbon composite film. Even dispersion of carbon has been the biggest concern so far. Carbon structures in their pristine or functionalized derivatives are surface enhanced to achieve better dispersion. In most cases, the ultrasonication process is used for mixing carbon and chitosan in the solution before casting. Wang et. al., prepared functionalized MWNT, and chitosan film using the same technique (70). With optimized time for ultrasonication, they confirmed that 0.8 wt.% is the maximum concentration of MWNT that can be used without agglomeration in the chitosan matrix.

The mechanical properties with this concentration were 93% and 99% improvement in the tensile modulus, and strength respectively compared to chitosan. Further improving the quality of carbon structure used, Fan et. al., used the N-doped graphene produced by the arc-discharge method to prepare graphene reinforced chitosan composite. The idea was to use graphene which is not chemically modified because it uses complex chemical reactions and leaves some residues. A pure graphene-chitosan composite is desirable for application in tissue engineering (71). With 2.3 wt.% graphene addition they reported elastic modulus for composite film increased 200% when

25 compared to neat chitosan film. However, their main focus was to study the biocompatibility of the film. Functionalizing MWNT with poly(3,4-ethylene di-oxythiophene) poly(styrene sulfonate)(PEDOT-PSS) to improve interfacial compatibility and dispersion for MWNT in the chitosan matrix was first demonstrated by Wu et. al., (72). Wu pointed out the importance of the duration and strength of ultrasonication to maintain a good length of CNT for inducing mechanical strength in the polymer matrix. The tensile strength, modulus, and hardness of the nanocomposites were improved by about 61%, 34%, and 31% respectively for 0.5% wt composite. The possible reason for polymer functionalizing MWNT was sulfonic acid groups of PSS provide ionic linkage with improved interfacial interaction with chitosan.

The next improvement to the solution casting method was performed by Lee et. al., where they synthesized chitosan/graphene nanocomposite film by cryomilling (usually liquid nitrogen or liquid argon) (73). Films with cryomilled and non-milled graphene were studied for improvements in mechanical properties. Results of the tensile test suggested that there was a 12% improvement in tensile strength and 33% in elastic modulus for cryomilled chitosan/graphene film. Milling is an effective solid-state process to achieve even particle size. Aryaei et. al., prepared a Chitosan-

MWNT film to study the bioactivity of the composite by observing non-toxicity on osteoblasts.

Their mechanical testing results are in agreement with the other reports that used solution casting techniques (74).

2.5.2 Solution precipitation and Freeze-drying method

Hydroxyapatite could be produced from eggshell is a mineral of the apatite group that is the main inorganic constituent of tooth enamel and bone are widely used in artificial bone and tissue engineering. It has excellent biocompatibility and bioactivity, but this ceramic is brittle and has

26 low fracture toughness. Li et. al., prepared Nano hydroxyapatite (HAP) on pristine and chitosan functionalized GO for biomedical application (75) by solution precipitation. The primary goal of the study was to prove the application of GO sheets in the biomedical area, to improve mechanical properties. They reported that chitosan functionalized GO used as a filler with HAP showed a high proliferation rate for L-929 and MG-63 cells due to the presence of chitosan. An improvement to their process was demonstrated by Mohandes et. al., by preparing hydroxyapatite (HAP) chitosan functionalized with graphene oxide (GO) (70: 29.9: 0.1% wt.) nanocomposite scaffolds by freeze- drying method (76). They tried to mimic the HAP and collagen (70:30) natural bone content. In the freeze-drying process, they hypothesized that the hydrophilicity of GO and NH2 and OH groups in chitosan could be protonated to polycationic materials in acid media, which leads to good interaction between chitosan polymer chains and GO sheets. Similarly, Dinescu et. al., wanted to emphasize GO application for cell proliferation for bone and tissue engineering. They prepared a chitosan-GO 3D scaffold using a similar method to study cytotoxicity, physicochemical properties, and cell support for metabolic activity (77) and reported similar results. The recent development was reported by Hermenean et. al. They prepared a similar scaffold with 3 wt.% GO highest reported concentration. The goal of their research was to study the regeneration of bone tissue and the repair capacity of chitosan-GO (78). The study was performed on a critical-sized mouse and it was found that the composite showed an increase of alkaline phosphatase activity and osteogenesis was promoted when compared to chitosan scaffold. They concluded the chitosan-GO scaffold is a promising biomaterial for reconstructing bone defects. From the reported studies, the chitosan and

GO composite has the potential to replace conventional bioceramics.

2.5.3 Electrodeposition and Electrospinning

Electrospinning is a method for producing fibers from polymers. In this process, the polymer solutions are jet spun in the presence of electric fields. This is a relatively new process that has received popularity for nanofiber synthesis. Electrospinning of chitosan remains a challenge for practical purposes due to low solubility, mechanical properties. Previously, a blend of chitosan with Poly-Ethylene Glycol (PEG), Poly-Vinyl Alcohol (PVA), Poly-Lactic Acid (PLA), and

Nylon was reported.

Hao et. al., produced a novel biocompatible conductive architecture from chitosan and carbon nanofibers for the first time (79). A film of chitosan doped with carbon nanofibers was prepared with electrodeposition. Before doping carbon nanofibers in chitosan, it was treated with nitric acid to form an oxygen-containing group on the surface. These groups further interacted with reactive amino and hydroxyl functional groups of chitosan to form a monodispersed chitosan-carbon nanofiber colloidal solution which supported deposition. They showed that the carbon nanofibers act as electron conductors and chitosan adheres to the electrode. It is a biocompatible electrode for impedance cell sensor application to study immobilization and cytosensing of K562 cells. Moving forward Lu et. al., used an electrospinning technique to prepare chitosan–PVA nanofibers with graphene for wound healing application for the first time (80). The concentration of PVA: chitosan was 70:30 and graphene was 4 wt.% of chitosan. Wound healing and antibacterial properties of this composite were tested on mice and rabbits. The presence of graphene had improved the performance of the composite, however possible mechanism and anti-diarrheal approach need more research. Ardeshirzadeh et. al., developed PEO/chitosan and GO nanofibrous scaffolds for controlled release of drugs (81). Due to the presence of chitosan, the scaffold was porous and showed higher drug loading and hydrogen bonding between GO and the doxorubicin drug

(antitumor antibiotic) used in the study is unstable in acidic medium and hence a faster drug release was achieved.

2.5.4 Other chemical modifications

Mazaheri et. al., synthesized GO and chitosan composite by layer by layer assembly for stem cell proliferation. With a 6 wt.% concentration of GO, it was possible to gain 80% and 45% improved strength and elastic modulus (82). They studied the antibacterial properties against Staphylococcus aureus and cytotoxicity with human mesenchymal stem cells (hMSCs). They found that the proliferation decreases with an increase in GO concentration, however, GO improved the bactericidal capacity of chitosan. The optimum GO concentration is 1.5 wt.% for superior biocompatibility. Justin et. al., applied a green reduction technique to reduce GO by a heating approach using chitosan. They were able to reduce GO at body temperature and prove stability in water, cell culturing, and pH sensitivity in the drug release system. The presence of chitosan provides biocompatibility and protection to drugs against heat applications and degradation (83).

For solid-phase extraction of protein Ding et. al., used functional guanidinium ionic liquid with magnetic chitosan and GO. The advantage of this system was that the functional magnetic chitosan-GO could be regenerated up to 94% after testing of adsorption conditions like the concentration of protein, pH, and temperature (84). The chemical modification makes the composite harmful for direct biocompatibility applications. The progress of chitosan-carbon composites for biomedical applications is noted in Table 2.5.

Table 2.5. Summary of chitosan-carbon composites for biomedical applications.

Optimum carbon Tensile Elastic Year Material structure strength modulus Method Application composition (MPa) (GPa) (wt.%) 2005 Chitosan and 0.8 74.9 2.08 Solution casting Bone Engineering (70) MWNT Chitosan and 2007 Impedance sensing Carbon 0.8 Electrodeposition (79) Cancer cytosensing nanofiber 2010 Chitosan and 2.3 ~6.5 Solution casting Bone Engineering (71) Graphene Chitosan and 2011 Solvent PEDOT-PSS 0.5 ~110 ~ 4 Bone Engineering (72) evaporation CNT 2012 Chitosan and Cryomilling and 1 65.17 1.48 Bone Engineering (73) Graphene Solution casting Chitosan and 2012 Graphene 4 Electrospinning Wound healing (80) PVA Cell proliferation 2012 Chitosan, GO Solution rate for L-929 and (75) And HAP precipitation MG-63 2014 Chitosan and 1 33% 3.5 Solution casting Bone Engineering (74) CNT Orthopedics 2014 Chitosan, GO Drug and gene 0.1 200% Freeze-drying (76) and HAP delivery Protein separation 2014 Chitosan and Bone and tissue Freeze-drying (77) GO engineering. 2014 Chitosan, GO Controlled release 0.7 Electrospinning (81) and PEO of drugs 2014 Chitosan and layer by layer Antibacterial 6 (82) GO assembly properties 2014 Chitosan and Chemical Drug release

(83) GO modification system. Solid-phase 2015 Chitosan, GO Chemical extraction of (84) and Ionic liquid modification protein 2017 Chitosan and Bone and tissue 3 Freeze-drying (78) GO engineering.

2.6 Biomaterial and packaging

The application of chitosan for biomaterial and food packing has been investigated due to chitosan’s excellent antibacterial, antimicrobial, and film-forming properties. Chitosan with carbon structures used as fillers provides the necessary mechanical strength to the film for all practical applications. However, it is important to investigate the effect of carbon addition to the biodegradable properties of chitosan for green material used for packaging.

Lim et. al., investigated the effect of GO with the small and large surface area on chitosan composite film for packing application (85). The GO has a large surface and binds the chitosan chain restricting its motion, thereby improving mechanical properties. They continued the study using reduced GO (rGO) to prepare the film for testing antibacterial properties using Pseudomonas aeruginosa bacteriacide. The results showed that rGO retards the growth of bacteria making the rGO large surface membrane the best choice for packing applications. The absence of oxide functional groups in rGO makes the wall surface very sharp, hence penetrating the bacterial cell wall easily to destroy their growth. This is a very important finding for antibacterial film application.

Han et. al., prepared chitosan, and GO composite films similar to previously reported studies using ultrasonication followed by stirring to study its mechanical strength (86). Chitosan and composite film were tested for stability in an aqueous medium for 30 days, where they found chitosan had swollen and eventually disintegrated, but composite membrane with GO was stable and maintained mechanical integrity even after 30 days. The tensile strength of the dry composite membrane was

1.7 times the chitosan and 3 times higher in the wet state. This could be well explained by the fact that water molecules interact with chitosan hydroxyl groups causing swelling and resulting in weakening of intermolecular h-bonding. Due to the incorporation of GO and potential interaction 31 with chitosan functional groups, the film does not weaken in a wet state. This irreversible crosslinking limits the applications of the film where self-healing of the composite is needed. This group further prepared a supramolecular hydrogel of chitosan and GO with self-healing property by reversible crosslinking (87). The smart material was responsive to temperature variation. With a high concentration of GO, it was possible to prepare the chitosan and GO composite hydrogel at room temperature whereas low concentration GO hydrogel was formed at a higher temperature up to 95 °C. Highly oxidized GO produced by Hummer’s method was dispersed in water by mild ultrasonication. They reported that noncovalent interactions between chitosan and GO are the driving force for the formation of the supramolecular structures. The conclusion was that hydrogel shows a thermo-reversible sol−gel transition when the composite with self-healing property on compression, cutting, or stretching. Table 2.6 summarizes the film composition, method, and optimum carbon structure filler for biomaterial and packing application.

Table 2.6 Summary of film composition, method, and optimum GO filler for biomaterial and

packing application.

Optimum carbon Year Composition structure Method Application composition (wt.%) 2010 Chitosan and Solution Membrane stability in a wet and 18 (86) GO casting dry state 2012 Chitosan and Solution 9 Antibacterial film (85) GO casting 2013 Chitosan and Self-healing property for smart 0.2 Hydrogel (87) GO material

2.7 Compression molding of chitosan biomaterial

Compression molding is popularly used for polymer composite for low cost and high-volume production. Molding processes can be done either in cold or hot conditions. For the cold process,

32 the pressure is applied, and curing occurs at room temperature whereas in hot molding both temperature and pressure applications are required, and heat is transferred from mold to the composite to initiate the curing process. The Bulk Molding Compound (BMC) and Sheet Molding

Compound (SMC) are two basic types of molding forms. BMC is 20–50 mm usually in the form of pellets, while on the other hand, SMC is up to 5 mm thickness sheets. Compression molding for biopolymers is a new upcoming process. Thermal degradation is a limitation for the hot molding of chitosan. Therefore, processing temperature and curing methods are important parameters to be studied for compression molding of chitosan.

Zhai et. al., prepared a starch and chitosan blend by compression mold. A semisolid gel-like mixture was prepared using starch and chitosan in acetic acid and glycerol by heating at 100 °C for 2 h (88). The hot gel was then cold-pressed to form a film of 0.5 mm thickness. The concentration of chitosan varied from 0-20% wt (increment of 5%) and showed better mechanical properties as chitosan concentration increased. The antibacterial property of the blend was studied against Escherichia coli. They reported that antibacterial properties were induced even with 5% of chitosan and the composite blend can be used for food packaging, compostable packages, and agriculture applications. Lopez et. al., obtained thermoplastic corn starch and chitosan/chitin film by melt-mixing and thermo-compression for industrial biodegradable packaging (89). They prepared a film with 1:20 and 1:10 using a hydraulic press at 140 °C for 6 min, and the pressure was increased every 2 min from 80, 140 to 180 kg/cm2. On testing the composite film for antimicrobial activity with Staphylococcus aureus and E. coli, a reduction in the growth of these microbes was found. They concluded that thermo-compression is an effective method to produce homogenous cross-sections without agglomerations of the material. Valencia-Sullca et. al., reported starch and chitosan bilayer film with essential oil produced by the thermo-compression

33 method for food coating and packing (90). The relative weight of starch: chitosan was 3:1. The starch film was prepared in a mold with hot plates in a hydraulic press at 160 °C and 1.2 x 107 Pa pressure. The bilayer was produced with the pressed starch film and chitosan film at 100 °C for 2 min. Although, they did not mention the procedure used to cast chitosan film and used plasticizer blend (glycerol: PEG) for the starch film. The obtained bilayer was consistent in cross-section and composition. The antimicrobial properties of the film showed effective protection for pork meat.

In a different study, they prepared a film by mixing starch and chitosan with a plasticizer blend

(glycerol: PEG). The maximum starch: chitosan ratio was 70:30 by weight. This time the thermo- compression parameters were 160 °C for 2 min at 5 MPa, followed by 12 MPa for additional 6 minutes. This film showed better permeability, rigidity, and was more resistant to break. Correlo et. al., investigated melt-based compression molding followed by particulate leaching for chitosan blended with synthetic aliphatic polyesters like polybutylene succinate, polybutylene succinate adipate, polycaprolactone, and polybutylene terephthalate adipate (91). The particulate used was common salt. The blend was prepared using a counter-rotating twin-screw extruder and the extruded strands were ground and injection molded. The concentration of chitosan in the blend was 25-75%. They were able to develop a 3D scaffold for hard tissue engineering application using chitosan which was proven non-toxic in cytotoxicity evaluation with L929 cells. Shih et. al., synthesized a 5 wt.% chitosan film with cellulose in N-methylmorpholine-N-oxide (NMMO). The film was prepared with a compression molding machine at 100 °C for 8 min at 70 kg/cm2 pressure.

The composite film showed slight antibacterial properties owing to the presence of chitosan.

Galvis-Sánchez et. al., prepared a chitosan film with a eutectic mixture of citric acid and choline chloride using thermo-compression molding (92). Here, they pointed to the chemical changes in the chitosan matrix using the eutectic mixture supported by FT-IR results and also stated that the thermal stability of chitosan film shifts on the lower side compared to pristine chitosan. The 34 chitosan, citric acid, and choline chloride were mixed to form paste-like consistency using an acetic acid solution. This paste was compressed using a hydraulic press at 125 °C. The film thus obtained reported better elasticity and high-water vapor permeability. Guerrero et. al., reported a thermo- compression molding process for synthesizing chitosan film to reduce production time and extensive use of organic solvents for processing (93). Here, chitosan was crosslinked with 10 and

20 wt.% citric acid, and 15 wt.% glycerol is used as a plasticizer to make a paste. This paste is hot- pressed between aluminum plates, at 125 °C, 2.5 MPa for 2 min to obtain the film. They mentioned that no film could be synthesized lower than the given temperature and pressure. The homogeneity of the film was confirmed by SEM and mechanical strength was determined by tensile testing. An increase of 80% for tensile strength was noted for a film containing 10% citric acid.

For the first time sintering of chitosan and its composite with carbon nanostructure has been reported by Brysch et al. They studied the mechanical properties of material by varying the sintering conditions (94). They reported that chitosan starts degrading at 220 C but can also degrade at lower temperature if the sintering time is longer. Based on Raman and XRD results they confirmed that 180 C and 3 h of sintering is the optimized time and temperature for chitosan sintering. Further, they synthesized composites with varying carbon nanostructure content and reported improvement in mechanical properties such as hardness (95). The improvement in mechanical properties was due to grain boundary reduction and even distribution of carbon nanostructure with milling and sintering. An increase in elastic property up to 50 % for sintering at 180 C with 5 wt% addition of fullerene soot (carbon nanostructure) was achieved with the thermomechanical process (96).

2.8 Laser processing for chitosan biomaterial

Laser processing such as cutting or engraving is a well-established process for polymers like polyamides, polycarbonate, polyester, polyethylene, polyimide, polypropylene, polyurethanes.

Some of the advantages of laser processing include precise operation, high-speed and local processing, minimum to no cutting waste, and no contamination by the cutting media. One major disadvantage of laser processing is localized heating which could thermally degrade the material.

Therefore, it is important to study the laser mechanism and material interaction with the laser irradiation, to be able to optimize operation parameters such as laser power, speed, etc.

Polysaccharides like chitosan and its composites were not cut before using lasers. However, Sun et. al., investigated selective laser sintering (SLS) as a processing method for natural polymers like chitosan (97). They prepared a composite membrane with chitosan and thermoplastic polyurethanes (TPU). The concept was to manually mix the polymer components, further when the composite powder is laser treated, the molten TPU wetted the chitosan on laser sintering. After curing, TPU combined with chitosan and a homogeneous membrane was obtained. They studied changes to chitosan and TPU structures by SEM, FT-IR, and XRD and reported no degradation or chemical changes to the polymer matrix using laser power up to 1.5 W and 200 m/s scanning speed. This composite membrane was used to study heavy metal adsorption like Pd2+and Cu2+ and showed good results of 19.6 mg/g and 30.4 mg/g respectively.

Conclusion

Considerable research has been conducted in the field of chitosan biomaterials over the past two decades. Undoubtedly, chitosan is a renewable, biodegradable, biocompatible polymer, proving its usability in a vast number of applications. The chitosan composites with carbon structures (GO,

Graphene, CNT, SWNT, and MWNT) showed significant improvements in mechanical strength, thus eliminating its major practical limitation. It is evident from the literature review that the electrostatic interaction between the functional groups of both the polymeric material has a significant effect on its performance as a functional composite material. Looking into the future, expanding laboratory level research to an industrial scale is the need of the time. The scope and prospect of synthesizing chitosan composite material on a larger economic scale are necessary.

The dispersion techniques such as ultrasonication, stirring, and centrifugation have been employed in most of the studies. Process limitations such as even dispersion of carbon structures, avoiding agglomeration is a primary problem to be addressed to increase the loading of carbon material.

Also, the effect of mechanical processes on structures of material hasn't been reported. Although some of the research group has optimized the sonication time, many studies failed to address the changes to carbon structure due to ultrasonication. The chemical modification of chitosan has compromised its bio-properties. The reactive sites of chitosan -NH2 and -OH groups are utilized during chemical processing such as crosslinking, hence limiting the efficiency of the composite.

For the successful development of biomaterials, the use of harsh and expensive chemical treatments needs to be investigated. Based on past studies, it can be noted that most synthesized processes have film and membrane formulation. Some of the studies fabricated 3D scaffolds, although the structural formation is limited to conducting studies rather than application. The applications of chitosan composite in other manufacturing processes like additive and subtractive manufacturing need to be explored. Though sintered chitosan composite preparation and their structural properties has been reported in the literature, no detailed study on producing these composited into different shapes has been reported which will be the major focus of the current study. Also, there is huge potential for using chitosan composite as computer inner components, disposable electronics, disposable batteries which will largely benefit the net zero emissions. 37

CHAPTER III

EXPERIMENTAL METHODS AND TECHNIQUES

This chapter discusses detailed protocols and procedures that are used to produce chitosan and

CMGC bio composite via solid-state synthesis technique, methods used to characterize the materials, measurement of physical and mechanical properties, manufacturing processes, and biotoxicity evaluation testing.

3.1. Materials

Commercial chitosan was purchased from Alfa Aesar research chemicals, Haverhill, MA with a medium molecular weight of 190–310 kDa and 85% de-acetylation. Commercial fullerene soot was obtained from SES Research Inc., Houston, TX. Soot is the byproduct of fullerene synthesis.

Milled MG was prepared by mechanical milling of soot for 3 h using a SPEX 8000M ball mill and used for making bio-composites by mixing with chitosan at different ratios.

3.1.1 Bio-composite preparation using sintering

Commercial chitosan was milled for 1.5 h in a SPEX 8000M high-energy ball mill using 50 ml stainless steel vial and 5 to 10 mm (diameter) stainless steel milling balls to obtain uniform nanoparticle size. Milled MG was mixed with chitosan nanoparticles with a varying weight percentage (0, 1, 3, and 5) and milled together for an additional 1.5 h to ensure even material dispersion. The milled composite mix was used for sintering at varying temperatures (120, 150, and 180C) in a stainless-steel circular cylindrical mold using a French press at a constant pressure of 3.5 MPa for 3 and 5 h are shown in Figure 3.1.

Figure 3.1. Image of sample sintering set up using a French press with a mold externally heated

using electrical heater and data recorded by data acquisition software using a computer.

The external heating arrangement was constructed around the mold and the temperature was measured at the core of the mold using a K-type thermocouple and monitored with a high speed, high-resolution data acquisition system from NIcDAQ-9174: National Instruments, Austin, TX.

The sintered bio-composite was carefully removed from the mold after cooling down. Based on the shape of the sintered mold a circular disk-shaped bio-composite with a dimension of 31 mm in diameter and ~3 mm in thickness was produced and weighed 3 g each (Figure 3.2).

Figure 3.2. Image of sintered bio-composite using different ingredients. Here, (a) chitosan and

(b) CMGC samples sintered using French press at 180C for 3 h.

3.1.2 Sample Preparation for compression test

A low-speed diamond cutter from Buehler’s sectioning equipment was used to cut samples for mechanical testing. Approximate 3 x 3 x 3 mm size was cut compression testing. Sample extracted for the compression test was polished for smooth and parallel surfaces using various grit sandpaper from 600 to 2400 grit size (Figure 3.3). To avoid carbide contamination, a final polishing was given by diamond grit paper of 12 m.

Figure 3.3. Schematic diagrams of sintered samples and their dimension before and after cutting.

Here, (a) CMGC obtained directly from mold and (b) CMGC sample cut to appropriate

dimension used for the compression test.

3.2. Mechanical Properties

3.2.1 Compression Strength

Compression strength is defined as the capacity of a material or structure to withstand loads tending to reduce the size, as opposed to withstanding loads that tend to elongate (98).

Compressive strength differs from tensile strength in a fundamental way. Tensile strength is defined as the resistance to pulling apart; while, compressive strength is the resistance to be pushed together. The tensile strength of a material is commonly studied to understand the mechanical properties of the material. The presented study is an attempt to test the resistance of chitosan and CMGC to compressive load on the addition of MG in varying weight percent. The

40 compressive stress test is typically conducted on materials having better resistance to compression rather than tension. In the compressive strength test both values, stress, and elongation are negative. However, for better visualization, here we will present them as positive values. The strength of the material greatly depends on its processing. To increase the strength of the material, various strengthening mechanisms were applied. With milling and sintering, even dispersion of MG in the chitosan matrix reduced grain size, and grain boundary refinement was achieved. With the decrease in grain size, the surface area to volume ratio increases which assists in shifting dislocations in the material along the boundary and ultimately increases its strength. This is known as Hall-Petch strengthening.

Compressing testing depends on various factors such as loading technique, size of the sample, and fixture design. Compression testing of plastic material has been well-established (98). The

ASTM protocols used for composite compression testing are listed in Table 3.1. The compression testing is carried out based on the type of loading and sample processing procedures such as bend compression, shear compression, end-loading compression testing, and a combination of either of these testing.

Table 3.1 Summarizes ASTM protocols used for composite compression testing

ASTM Description Type of loading and Protocol sample processing

ASTM Standard test method for compressive properties of Shear loaded, D3410/ polymer matrix composite materials with unsupported symmetrical sample D3410M gage section by shear loading

ASTM Standard test method for compressive properties of End-loaded, dogbone D695-15 rigid plastics shape sample

ASTM Standard test method for compressive properties of Bending load; D5467 unidirectional polymer matrix composite materials sample between a using a sandwich beam sandwich beam

For the present study, the ASTM D695-15 procedure was used (99). Figure 3.4 shows the compression test set-up using the Interactive strength challenger machine. The custom-made compression sample platens were prepared for the sample with dimensions 3 x 3 x 3 mm.

Figure 3.4. Image of the compression test setup. Here, (a) Interactive strength challenger

machine and (b) custom-made compression sample platens.

The maximum load that can be applied was 1250 lb. The testing speed of 0.5 mm/min was maintained. The testing machine has two loading heads, with one head movable perpendicular to the sample surface area. The sample was loaded between the fixtures designed with very flat surfaces. The sample surfaces were polished first with silicon carbide paper, 1200 (P-4000). It was further polished with aluminum oxide lapping film of 12 m to avoid any carbide contamination on the sample surface and sample under compression test (Figure 3.5). The test was performed in compliance with ASTM 695-15 standard protocol at room temperature (99).

Figure 3.5. Image of the sample that was subjected to a compression test with interactive

strength challenger universal testing system.

3.3. Material Characterization Techniques

3.3.1 Scanning Electron Microscopy (SEM)

The surface morphology, particle size, grain boundary, and location of failure in the compression test was studied using SEM. The Leo 1525 Gemini FEG with in-lens annular detector manufactured by Zeiss was used for the SEM study. The chitosan and CMGC samples were coated with an ultrathin gold layer with a low vacuum sputter coating for better conductivity. This way signal to noise ratio is improved for better topographic examination in the SEM. A carbon conductive tape was used to ground the sample to the SEM chamber stage using 2 x 10-5 푚푏푎푟 pressure in the vacuum chamber and an accelerating voltage of 3 kV was maintained.

3.3.2 Fourier Transform Infrared Spectroscopy (FTIR)

We used the Attenuated total reflectance (ATR)-FT-IR technique to analyze the chitosan and

CMGC samples. This technique is used to study the phase transition of chitosan during mechanical processes such as milling and sintering. ATR allows samples to be used as a solid or liquid form 43 without specific sample preparation for characterization. The Nicolet iS50 FT-IR from Thermo

Scientific was used to record the spectra between wavenumber 500 to 4000 cm-1. The sintered samples were ground to have uniform particle size before subjecting to characterization.

3.3.3 Raman Spectroscopy

Raman spectroscopy is a non-destructive testing technique that can be used to determine the chemical properties of a material. Raman is a complementary method when compared to FTIR and samples can be tested in water. Raman excites the chitosan molecules, which promotes characteristic vibrations that are identified as the bands for specific bonds. The result in a Raman spectrum is called ‘Chemical Fingerprint’ that corresponds to a specific molecular bond vibration.

Raman spectroscopy characterization was performed on XploRA, Horiba Scientific. Chitosan,

CMGC, and MG characterization were carried out using a 638 nm diode laser at 100 X magnification. Raman was performed to understand the effect of sintering on chitosan and CMGC.

Raman spectrum for samples sintered with conventional mold in the previous reported study and the power screw mold in the present study were compared.

3.4 Physical Properties Testing

3.4.1 Conductivity testing

The four-point probe technique was used to measure the electrical impedance of a semiconductor material either in bulk or in thin-film form. In the four-point probe method commonly known as

Kelvin sensing, a current is supplied with a pair of point connections of the probe which results in the generation of voltage drop. The impedance is measured according to Ohm’s law:

V = I x R …………………………………Equation 3.1

44 where V is the Voltage supplied and I is the measured current and R is the impedance or resistance.

This method can give high accuracy in low resistance measurement since the current and voltage measuring electrodes come in a separate pair which minimizes lead and contact resistance. For material with high resistivity, the input current must be kept low to reduce the output voltage. The recommended output voltage for semiconductors is less than 100 mV/mm. Jandel HM21 test unit was used to measure the resistivity of the composite. This test unit offers bulk resistivity measurement within the range of 10 milliohms. cm to 10 Kohm. cm. Following steps were carried out for successful and reliable results:

a. Uniform resistivity around the area of measurement was maintained, which was assured

by uniform mechanical dispersion of MG in the chitosan matrix.

b. Very fine polishing of the measurement surface was done to assure proper and defect-free

contact.

c. All four connection points were carefully placed on the surface area while resistivity

measurements were taken.

d. Samples were laid on the flat stage and the four-point connections of the test unit were

placed in contact with the smooth sample surface.

e. Samples were tested in the ascending order of weight percent i.e. from 1 wt.% through 5

wt.%.

f. All the samples, ranging from treatment temperature – 120, 150, 180, and 220oC were

tested in the given order.

g. A very low current of 10 µA was supplied to achieve accuracy in the output measurement.

3.4.2 Bulk Density Testing

Bulk density (BD) or volumetric density (VD) is defined as the mass occupied by the given sample

(Msample) divided by total volume (Vsample). The volume of the sample is a combination of particle volume, inter-particle space, and internal pores present in the sample. The compression and compaction characteristics of a material are directly affected or dependent on Bulk density. The measurements can be dry or wet type depending upon the moisture content in the sample. It is important to mention that both the chitosan and chitosan composites degrade relatively easily in water. For this reason, all the testing is carried out in dry form without any moisture.

The Bulk Factor (BF) measurement is the volume change that may occur during the fabrication of material/composite. In other words, BF is a measure of the volume change during processing.

ASTM D1895 - 17 (100) describes the method of measuring the bulk factor by determining the ratio of the volume of loose material to the volume of the same quantity of the material after molding or compaction. In this procedure, a funnel is inserted on the measuring cylinder and a certain amount of material is passed through a funnel to quickly fill the cylinder. The excess material is discarded without disturbing the cylinder and the weight of the material is calculated.

Based on the results BF is calculated by dividing the weight of the material by the known volume of the cylinder. The specific density of the material is defined as the density of the material relative to the density of reference material, in some cases water and it is also known as the specific gravity.

The reference material from specific density calculation is generally water Specific density (SD) is calculated in water based on ASTM D792- testing A and ratio of specific density and apparent density gives bulk factor (101). Another method to measure the bulk density of the material as a function of compressive stress is given by ASTM D6683-19 (102). In this procedure, a certain amount of material is filled in a density cup of known volume. The weight of the material filled in

46 the cup is carefully measured without disturbing the arrangement. The material in the cup is then compressed with the help of a plunger arrangement. The difference in height and weight is used to calculate volume occupied by compressed mass. More mass is added, and the same procedure is repeated until no change in height is measured. Using this method, the bulk density is calculated as a ratio of mass to volume and a range for bulk density for the given material is computed as a function of compressive stress.

In the present study, dry bulk density was calculated using ASTM D6683-19 procedure (102).

About 3g of powdered raw chitosan, milled chitosan, and CMGC 5 wt.% is sintered in a circular cylindrical mold. The powdered material was carefully filled in the mold and the mold was compressed using a custom-made French press at a constant pressure of 3.5 MPa. The mold was surrounded by an electrical heater to maintain 180C temperature. The sintering process was carried out for 3 h. After this time, the sintered samples were carefully pushed out of the mold, and the thickness of the sintered sample was measured. Dimensions of the circular cylindrical mold are summarized in Table 3.2 and the volume of sintered samples was calculated based on mold dimension and sample thickness for bulk density measurement. The dry bulk density was calculated based on the following formula:

푀푎푠푠 표푓 푡ℎ푒 푝표푤푑푒푟(푔푚) 푀 퐷푟푦 퐵푢푙푘 퐷푒푛푠푖푡푦 = = ...... 퐄퐪퐮퐚퐭퐢퐨퐧 ퟑ. ퟐ 푉표푙푢푚푒 표푐푐푢푝푖푒푑 푏푦 푡ℎ푒 푐표푚푝표푠푖푡푒(푚푚 ) 푉

Table 3.2. Dimensions of the circular cylindrical mold used for sintering

Dimensions (cm) Conventional Mold

Cylinder height 8

Wall thickness 0.65

Inner diameter 3.1

Outer Diameter 4.4

To calculate the actual density of the material and compared it with theoretical predictions the rule of mixtures can be used. In the rule of mixtures, we combined the volume fractions of each constituent as well as their respective densities. In Equation 3.3, the density (ρ) and the volume

(V) of each component, in this case, chitosan (m), and the respective reinforcement are the MG (f).

The theoretical density of chitosan as reported by the vendor Alfa Aesar is 0.25 g/mL. In the literature, the reported density of sintered chitosan is 1.48 g/mL and is used for theoretical calculations. For the MG, the density is 2 g/mL. The rule of the mixture is commonly used to determine the modulus and strength of composites. With the known volume fractions and densities of matrix and dispersed phase, the density of composite can be predicted (103).

휌 = 휌푉 + 휌푉...... 퐄퐪퐮퐚퐭퐢퐨퐧 ퟑ. ퟑ

휌, 휌, 휌 – Densities of the composite, matrix, and dispersed phase respectively;

Vm, Vf – Volume fraction of the matrix and dispersed phase respectively.

Further, the Bulk density of chitosan and CMGC as a function of MG concentration and sintering time and the temperature is plotted and compared for the same samples tested for the compression test carried out earlier.

3.5 Compression molding

3.5.1 Power screw mold

Conventional compression mold is used to produce samples using a pressing die commercially purchased. It was held in a vertical orientation in a custom-made French press that applied constant

48 pressure at 3.5 MPa. An external electrical heater was installed to maintain the required temperature for sintering. The milled chitosan and composite powder were compacted in the mold and then sintered. Since only one sample could be sintered at a time in the French press, it was difficult to expedite the sintering process. To produce more sintered material in a given time, the power screw mold was designed. Power screw mold works on a simple screw and nut thread mechanism. The torque due to the tightening of the thread of the screw is transferred as a linear pressure to the compression mold. The required torque of 5 MPa pressure was applied and measured using a digital wrench. The compression mold could be of different shapes and sizes.

Initially, a circular cylindrical mold with a power screw on both ends of the cylinder was designed.

This eliminated the use of the French press for applying constant pressure for the compaction of powder. To reach the sintering temperature, the power screw assembly could be directly put into a conventional heating furnace. Power screw design calculation was based on the following formula:

퐹 휂 = 푥 휋 푥 푇...... 퐄퐪퐮퐚퐭퐢퐨퐧 ퟑ. ퟒ 2퐿 where η is the efficiency of power screw

F is the force required to produced 5 MPa pressure on the unit area

L is the pitch length of the screw

T is the produced torque.

Assuming an efficiency of system η = 1, and for desired column length of 70 mm, the following dimensions were calculated, and a Power screw mold was designed. Table 3.3 shows the dimensions of the power screw mold calculated based on Equation 3.4 to produce the maximum linear pressure of 5 MPa.

Table 3.3. Dimensions of Power screw mold

Part Dimension (mm)

Cylinder height 90

Height of pressing disc 12

Wall Thickness 6.25

Screw thickness 6.30

Screw diameter 30

Screw height 20

Compression Nut length 37.2

Compression Nut diameter 20

Inner diameter 33

Outer diameter 42

Note: The power screw is made from stainless steel

The schematic design of the power screw arrangement to transmit a torque for sintering is shown in Figure 3.6. The sintering column in the middle has an arrangement for sintering multiple composite samples in the shape of a disc of diameter 31 mm.

Figure 3.6. Schematic diagram of power screw mold 50

The power screw mold was fabricated using stainless steel grade material (Figure 3.7). The cylinder column in the middle is screwed for one end. The pressing disc is placed next followed by a chitosan or composite powder. The quantity of powder is decided based on the desired thickness of the sample. The weight of powder required was calculated based on bulk density for previous samples. Another pressing disc is placed to enclose the powder and is screwed from the opposite end. Hence, the CMGC powder could be pressed between two power screws.

Figure 3.7. Image of different parts of the power screw mold when disassembled and assembled.

Here, (a) front view, (b) top view, and (c) assembled parts kept in box furnaces, heated at 180C

for producing sintered chitosan bio-composite.

The power screw assembly was directly put inside Thermofisher Scientific Lindberg/Blue M™

LGO box furnaces. Initially, the temperature inside the furnace was monitored with the K-type thermocouple, and data were analyzed via the LabVIEW Data acquisition system. It was observed that the actual temperature inside the furnace was 20C more than the set value of temperature.

The temperature was set according to the offset value for sample preparation. The samples were sintered for 3 h based on the optimum sintering time mentioned earlier. As the chitosan and MG particles fused during sintering, the samples get densified thus creating a void in the compression

51 mold. The mold was taken out of the furnace every 30 mins and tightened again to the required pressure and to reduce the void space.

3.5.2 Cubical mold design and making

A cubical mold of 1 x 1 x 1-inch dimension with a more complex design was machined to prove the feasibility of the power screw mold concept. Figure 3.8 (a & b) shows the schematic diagram of cubical mold fabricated with the same stainless-steel material. The mold consists of 3 parts which produced a four-walled cubical sintered product. The wall thickness of the mold was kept at 2 mm. The cubical mold was fabricated with stainless steel. The required weight of powdered material was calculated based on bulk density calculation. Approximately 4g of powdered material was used for sintering.

Figure 3.8. Schematic design of the cubical mold and fabricated stainless-steel molds. Here, (a),

individual mold component design, (b) assembled mold components design.

To pressurize this mold during sintering a 6-inch C-clamp in Figure 3.9 (c) was used to compress the cubical mold. The top and bottom face of the mold was held between the plate and base of the clamp. This system is capable of providing the required pressure of 3.5 MPa. The torque required for 3.5 MPa force was measured with a digital torque wrench. The whole assembly was kept in the box furnace at 180C for 3 h.

Figure 3.9. Image of different parts of cubical mold when disassembled and assembled. Here, (a)

front view, (b) top view, and (c) assembled parts kept in box furnaces, heated at 180C for

producing sintered chitosan bio-composite.

3.5.3 Rectangular sheet mold

A rectangular sheet mold with rounded corners in Figure 3.10 was fabricated and samples were sintered in a similar way to cubical mold. The dimensions of the mold were 10 x 5 cm. This mold was used to sintered sheets from chitosan and CMGC.

Figure 3.10. Image of rectangular sheet mold with rounded corners. 53

3.6 Laser Lithography

Acrylic is a thermoplastic, which is used as a transparent and shatterproof substitute for glass. It is widely used in laser cutting to create different patterns of various shapes and sizes. For demonstration purposes, the shapes were cut from a commercially available acrylic sheet, sintered chitosan sample, and sintered CMGC sample.

The physical dimensions of all the samples were kept consistent for clear comparison. A circular disc of diameter 31 mm and thickness 3 mm was used for laser cutting the UH logo, 2 mm thickness for gear shape, and 1.5 mm for the square with holes. The shapes were designed in AutoCAD software. A Trotec speedy 100 laser engraver and the cutter was used for laser cutting. Table 3.4 shows the type and specifications of the laser cutting machine. The combinations of laser power and speed used during the process are listed in Table 3.5. It was difficult to cut the material with different laser power and speed (combination I, II, and III) as did not cut the material through.

Therefore, combination IV was selected to cut all the shapes.

Table 3.4. Laser type and specifications of Trotec speedy 100 laser engraver and cutter

Laser Type CO₂ laser Work Area Between 24.0 x 12.0 & 40.0 x 24.0 inch Laser Power 50 W Laser speed 1 mm/s CO₂ Laser Wavelength 10.60 µm

The surface of the samples using for laser cutting was flat. All the samples were loaded together and cut with consistent parameters in a single setting. The AutoCAD images are shown in Figure

3.11 were fed to the laser cutting machine via computer software Universal Laser system interface.

Table 3.5. The combinations of laser power and speed for laser cutting Combinations Laser Power (%) Speed (%) I 40 20 II 50 15 III 60 10 IV 70 5

The samples were loaded onto the work area of the laser cutter. Then, the X and Y coordinates were adjusted according to the position of samples in the work area.

Figure 3.11. Schematic design that was used as a template for making different shapes. Here, (a)

UH logo, (b) Square plate with holes, and (c) Gear shape. Note: The proportionate dimension of

the square is 2.5 x 2.5 cm for each shape. The holes in (b) have a diameter of 2mm.

3.7 Water degradability and Biotoxicity evaluation

The chitosan and CMGC were tested to prove non-toxicity and water degradability. For the water degradability test, the raw un-milled chitosan, milled chitosan, and CMGC samples were immersed in normal tap water initially. The sintered samples disintegrate immediately after coming in contact with water (86,104). Later, the biotoxicity evaluation testing was set up by planting mustard seeds in the disintegrated samples. In this test, mustard seeds were planted in the crushed sintered unmilled chitosan, chitosan milled and CMGC 5wt.% samples all soaked in water. To compare the plant growth, a control sample with potted in soil. Plants were grown at room temperature (23-

25C) and humidity (40-45%). These testing conditions comply with ASTM D6400-test to certify 55 if a product can be composted and ASTM D6868 - test to determine if a biodegradable plastic is truly biodegradable (105, 106). A Dr. Meter S10 Soil Hygrometer Moisture Sensor was used for measuring moisture in the soil for the plant. The pointer scale in the moisture meter ranges from

1 to 10, where 1-3 indicated dry soil, 4-7 for medium moist soil, and 8-10 for high moisture. For our samples, medium moisture between 5-6 ranges was maintained to prevent drying. A drainage hole at the bottom of the pot was provided to ensure the drainage of excess water.

CHAPTER IV

RESULTS AND DISCUSSIONS

In this chapter, we present and summarize the results of the study and discuss the findings with reference to the goal of this research, which is to produce CMGC with compressing molding and laser cutting with superior physical and mechanical properties. The results outline the effect of

MG addition to chitosan on mechanical strength with respect to time, temperature, and pressure for sintering and milling. The produced CMGC ultrastructure and chemical properties were characterized using SEM, FT-IR, and Raman spectroscopy. Additionally, we evaluated physical properties such as bulk density, electrical conductivity, biotoxicity, biodegradability and propose possible applications of the CMGC.

4.1 Mechanical Properties

Compression Test

The compression test was performed based on ASTM 695-15 (99) standard to understand the effect of sintering time and temperature on strength of CMGC. The images of compression tested samples and microstructures are shown in Figure 4.1. The Young’s modulus and ultimate strength are the two important values obtained from a typical stress-strain curve. Young’s modulus for stiffer material has a steeper slope for the linear elastic curve. Yet, flexible material has a lower value of Young’s modulus as it changes shape considerably compared to stiffer material under the same applied load.

Figure 1.1. Images of the compression test samples, (a) chitosan and (b) CMGC 5 wt.% and

optical microstructures for the sintered (c) chitosan and (d) CMGC 5 wt.%. Note: All samples

are sintered at 180C and 3.5 MPa

Figure 4.2 shows an example of the experimental compressive test curve for chitosan and CMGC

5wt.% sintered at 180C. In GMGC, we observe more than one so-called ‘onset’ which is the irreversible deformation of composite materials. The presence of several onsets is not conventional; instead, we see a single maximum corresponding to the Ultimate Compressive

Strength (UCS). We identified three onsets, but it is not necessarily the case for all the investigated samples. Therefore, other samples may have more or less of those maximums in the compressive stress and strain curves. We decided to call the last one compressive strength because it is the

58 equivalent of the sample’s failure. The compressive test results show interesting phenomena due to potential collapse within the sintered composite. This curve is unusual, however, with further analysis, we present the reasons why they look so different from expectations. The reported results are: Young’s modulus, the elastic limit, the compressive strength which is typical of any stress- strain compression curve are presented in Table 4.1.

Figure 4.2. The plot for compression test curve for the sintered (a) chitosan and (b) CMGC 5

wt.% at 180C.

Table 4.1. Summary of mechanical properties for chitosan and CMGC 5 wt.% sintered at 180C.

Chitosan CMGC Percentage improvement % Young's Modulus (GPa) 10.14 8.82 -13 Elastic Limit (MPa) 34.10 39.94 17 First (1st) onset (MPa) 40.80 50.36 23 Second (2nd) onset (MPa) 18.67 32.55 75 Compressive strength, (MPa) 44.56 54.77 22 Toughness (KJ/m3) 1579.59 2522.45 60

The presence of onset is related to partial failure of the material. The fading of mechanical properties at onset is likely due to the crumbling of material. It is not a yield mechanism, but rather

59 the relatively rapid breakdown of the structure. In the case of pure chitosan, this phenomenon occurs because of the relatively larger grain boundaries pre-cracks, when the material collapses, it does not recover. On the other hand, after the first breakdown (onset) for composite, MG starts the recovery effect triggering a new strength building up. MG is responsible for carrying the load while the strength of chitosan fades. This self-healing is a reinforcement mechanism to carry the load again and to recover substantially. Since MG has higher mechanical properties than chitosan, the composite shows recovery on the second onset. Moving forward, a new breakdown occurs forming new cracks and activating this self-healing mechanism again to reach a new recovery strength. The combination of the multi-reinforcement mechanism has a complementary effect resulting in a clear strengthening improvement.

The MG reinforcement has a complementary effect to develop a self-healing mechanism. This phenomenon can be visualized in transmission electron microscopy (TEM) images (Figure 4.3).

The presence of MG is trans-granular, and it anchors the chitosan grains promoting a reinforcement mechanism. It is important to understand that MG doesn’t have the typical interlamellar spacing of graphene of 0.33 nm along the “c” direction; instead, the distancing resides in the ranges between 0.36 and 0.38 nm. In our results, the d-spacing clearly shows the existence of a non- graphene phase that is associated with MG instead (Figure 4.3 (d)). The improvement in mechanical properties is purely associated with the presence of MG as demonstrated in this work.

A major improvement (as seen in

Table ) is toughness which is from 1579.6 KJ/m3 for chitosan to 2522.5 KJ/m3 for CMGC 5 wt.%.

This is approximately a 60% increment in toughness suitable for some applications. For instance, the contribution of the fracture toughness may result in an exponential improvement when compared to endurance limit growth, which is responsible

Figure 4.3. Transmission electron microscopy of (a) pure chitosan, (b, c) low magnification

CMGC 5 wt.%, and (d) MG. Notes: The arrows in (b) show the location of the MG within the composite. The thick dotted line in yellow (c) represents the grain boundary, and the lighter in

grey is showing the MG transgranularly which anchors the chitosan grains and increase

strength during compression to prevent failure. Images courtesy of Dr. J. M. Herrera Ramirez

from CIMAV Chihuahua (reproduced with permission).

We performed a compression test and identified onsets for samples that are consistent with our previous work. The samples were sintered at 120, 150, 180, and 220°C at 3.5 MPa for 3h. Figure

4.4 shows the plots for (a) Young’s modulus, (b) 1st Onset, (c) 2nd onset, (d) compressive strength, and (e) Toughness for varying MG wt.% with respect to sintering temperature. The Young’s modulus decreases as the MG composition increases along with sintering temperature (Figure 4.4 61

(a)). The MG addition to the chitosan matrix increases the flexibility of the composite when compared to pure chitosan which seems stiffer with the highest value of Young’s modulus. The compressive strength of the composite improves with sintering temperature and increasing MG content. This is evident in Figure 4.4 (b), (c), and (d). The same is true for toughness (Figure 4.4

(e)). The samples sintered at 120C perform the least and the performance of samples sintered at

180C is the best which is evident from the higher value of 1st onset. The fact that 180C samples perform better than 220C, which could be because the glass transition temperature of chitosan is

225C and sintering close to this temperature could lead to polymer degradation. Table 4.2 summarizes mechanical properties of samples sintered at 120C and 180C for 3 h under a 3.5

MPa of pressure.

For the samples sintered at 120ºC, Young’s modulus chitosan is 12.5 GPa and CMCG 5 wt.% is

8.88 GPa which is 29% lower for composite. Similarly, for the samples sintered at 180ºC it is

10.14 GPa for chitosan and CMCG 5 wt.% is 8.82 GPa which is 13% lower for composite. In other words, pure chitosan is a stiffer material and the composite is more flexible. An additional parameter that improves the properties of the MG reinforced composites is the grain boundary reduction. The thermomechanical technique used in this work is effective to synthesize chitosan and their composites reinforced with different amounts of MG (1-5 wt.%) as they improve their mechanical properties. The overall improvement in toughness for composite sintered at 120 C is

97% and for 180C is 59% for toughness.

Figure 4.4. The plots for (a) Young’s modulus, (b) 1st Onset, (c) 2nd onset, (d) compressive

strength, and (e) Toughness for varying MG wt.%

Table 4.2. Summary of mechanical properties for samples sintered at 120 and 180C for 3 h at

3.5 MPa pressure.

120C MG Young's Elastic Limit 1st onset 2nd onset Compressive Toughness wt.% modulus (GPa) (MPa) (MPa) (MPa) Strength KJ/m3 (MPa)

0 12.5 23.33 30.41 9.44 18.11 751.02

1 10.83 17.44 19.00 12.89 34.38 1151.02

3 9.33 19.11 20.33 20 51.22 951.02

5 8.88 23.45 29.51 20.2 50.38 1479.59

Percentage improvement from 0 to 5 wt.% 97

180C 0 10.14 34.11 40.80 18.67 44.56 1579.59 1 9.56 24.78 35.22 21.22 50.11 2001.02

3 9.11 37.22 48.67 37.44 51.67 2332.65

5 8.82 39.94 50.37 32.55 54.77 2522.45

Percentage improvement from 0 to 5 wt.% 60

4.2 Material Characterization Techniques

4.2.1 Scanning Electron Microscopy

The general morphology of raw chitosan structure in SEM images shows irregular particle sizes

ranging from 10-50 µm. Figure 4.5 shows SEM images of Raw chitosan and milled chitosan. The

chitosan milled for 1.5 h shows relatively regular particle distribution 30–50 µm (107). From the

SEM images, it can be concluded that with high-energy ball milling it is possible to achieve

consistency in particle size and distribution.

Figure 4.5. SEM images of (a) Raw chitosan and (b) milled chitosan.

Note: Chitosan was milled for 1.5 h using a SPEX8000 Mill. The particle size of raw chitosan is

in the range of 10-50 µm. The SEM analysis was done using Leo 1525 Gemini FEG at 1000X

magnification.

Figure 4.6 shows SEM images of sintered (a) Raw chitosan, (b) milled chitosan, and (c) CMGC.

The sintering of these samples was done using a custom-made sintering unit that consists of a

French press with pressures reaching up to 4,000 psi which corresponds to 3.5 MPa of pressure for the geometry of our samples is approximate. Nevertheless, the French press can comfortably reach

5 MPa, but to be on the safe side we decided to use 3.5 MPa. The grain boundary in Figure 4.6

(a) clearly shows wider gaps for the raw chitosan when compared to milled chitosan as shown in

Figure 4.6 (b). The analysis performed in grain boundary measurement indicates that sintered raw chitosan is 12-19 µm, and milled chitosan is 8.7-7.5 µm. Hence, grain boundary reduction after milling is 38 -55 %. The further reduction of 80- 90 % in the grain boundary of CMGC (Figure

4.6 (c)) is observed to be 0.7-1.7 µm in the presence of 5 wt.% MG. It is well known that material diffusion and particle fusion during sintering results in grain boundary reduction that decreases porosity or allowing clear improvements in bulk density that can be an explanation for the strengthening improvements. 65

Figure 4.6. SEM images of sintered (a) Raw chitosan, (b) milled chitosan, and (c) CMGC

Note: The SEM analysis was done using Leo 1525 Gemini FEG at 10000X magnification. Samples were milled using SPEX8000 Mill, chitosan was milled for 1.5 h. For composite, milled chitosan was milled with 5% morphed graphene (milled for 3 h) for an additional 1.5 h. Sintering was done using a custom-made French press at a constant pressure of 3.5 MPa at 180°C

The morphology of Figure 4.7 (a) chitosan, (b) CMGC 5wt.% sintered at 120oC, (c) chitosan, and

(d) CMCG 5wt.% sintered at 180oC are shown. Grain boundary reduction from 120 to 180oC for chitosan is 30 – 40 % and for CMGC is 20-25 %. The consolidation between chitosan particles improved with an increase in temperature as observed from Figure 4.7 (a) and (c). The CMCG samples in Figure 4.7 (b) and (d) have MG coating on the surfaces of chitosan.

Figure 4.7. SEM images of chitosan and CMGC. Here, (a) chitosan, (b) CMGC 5wt.% sintered

at 120oC, (c) chitosan and (d) CMCG 5wt.% sintered at 180oC.

4.2.2 Fourier Transform Infrared Spectroscopy (FTIR)

Figure 4.8 shows FT-IR spectra for (a) chitosan raw (without milling), (b) chitosan (milled for

1.5 h), (c) chitosan milled for 1.5 h followed by sintering at 180oC and (d) chitosan milled along with 5% MG for 1.5 h followed by sintering at 180oC. The commercially available chitosan, 85% de-acetylated from Alfa Aesar is used for FTIR characterization. The vibration of the peak at

1035.1 cm-1 and 1065.01 cm-1 signifies C-O-C symmetric stretching which is related to glycosidic bonds in chitosan. The absorption peak at 1377.4 cm-1 represents C-H bond scissoring. symmetrical stretching peaks for C=O are seen at 1595.4 cm-1 and 1648.9 cm-1. Asymmetric stretching of the C-H bond is observed at 2877.4 cm-1 and 2925.1 cm-1. Asymmetric stretching of

N-H bond at 3291.5 cm-1 and hydroxyl vibrations O-H bond at 3364.7 cm-1 are evident. All the

FT-IR spectral peaks of native chitosan were similar to what was reported in the literature and are summarized in Table 4.3 (109).

Table 4.3. Summary of characteristic FT-IR spectral peaks of native chitosan.

FT-IR spectral peak Present study Reference study (109) C-O-C symmetric stretching 1035.1 and 1065.0 cm-1 1024 and 1065 cm-1 C-O-H stretching 1153.2 cm-1 1261 cm-1 C-H scissoring 1377.4 cm-1 1375 cm-1 C=O symmetrical stretching 1595.4 and 1648.9 cm-1 1574 and 1645 cm-1 C-H asymmetric stretching 2877.4 and 2925.1 cm-1 2875 and 2920 cm-1 N-H asymmetric stretching 3291.5 cm-1 3280 cm-1 O-H 3364.7 cm-1 3750 - 3000 cm-1

The C-H stretching band seen in FT-IR spectra is also known as the crystallinity band. The intensity of peak around 2877.4 cm-1 and 2925.1 cm-1 decreases with milling and further with sintering. This means that chitosan crystallinity decreases with milling and sintering. The peak at

3364.7 cm-1 corresponded to the stretching vibration of O-H groups reduces in intensity with milling. This effect may be due to the impact of intensive milling on the presence of water.

Sintering at 180C results in a further reduction in the intensity corresponding to the hydroxyl group that may imply further material’s dehydration, which may be responsible for the improved densification. Besides the changes in the hydroxyl group, we do not observe further changes in

FT-IR analysis. This is clear evidence, both milling and sintering did not alter or destroy other bonding or functional groups.

Figure 4.8. FT-IR spectrum of (a) chitosan raw, (b) chitosan milled for 1.5 h, (c) chitosan milled for 1.5 h followed by sintering at 180C, and (d) chitosan – milling along with 5% MG for 1.5 h

followed by sintering at 180C. Note: The chitosan raw used for analysis is not milled. The

milled chitosan was milled for 1.5 h using a SPEX8000M ball mill. Sintered chitosan and

chitosan -MG 5% composite sample was sintered at a constant pressure of 3.5 MPa at 180C

using a French press.

4.2.3 Raman Spectroscopy

Figure 4.9 shows the Raman spectrum of chitosan with and without sintering and in the presence and absence of MG. The samples are sintered using the conventional compression mold and a

French press. The wavenumber corresponding specific bonds stretching are given in bracket, N-H

-1 -1 -1 -1 bond (3301.5 cm ), C−Η bond (2887.0 cm ), CH2 bond (2722.6 cm ), C=O bond (1676.4 cm ).

The specific vibration corresponding to the C-H bond (1373.8 cm-1) resembles the scissoring of

69 the C-H bond. The rings stretching for C-H (1264.5 cm-1). For the C-O-C linkage asymmetric stretching (1095.3 cm-1) and symmetric stretching (909.1 cm-1). The Raman band vibrations for milled and sintered chitosan are consistent with reported results.8 The reduction in peak intensity

C=O bond, C-H bond vibrations, and stretching is correlated with the drop in crystallinity due to milling and sintering. For the CMGC 5 wt.% spectrum, the peaks at 1321.4 cm-1 and 1599.0 cm-1 for D and G carbon bands respectively. The D peak is referred to as a disordered carbon structure and presents a defect in the crystal and the G band represents the graphitic carbon structure. From the spectra, it is clear that the Raman intensity of MG is greater than that of chitosan. This confirms the fact that MG coats the chitosan particles during milling and is predominantly visible in the

Raman spectrum.

Figure 4.9. Raman spectrum for chitosan with and without sintering in the presence and absence

of morphed graphene. Here, (a) raw chitosan (unmilled), (b) milled chitosan, (c) sintered

chitosan, and (d) CMGC 5wt.%. Note: Milling was carried out for 1.5 h in SPEX8000M ball

mill. Sintered chitosan samples are milled and sintered at a constant pressure of 3.5 MPa at

180C using a French press dye.

Figure 4.10 represents the Raman spectrum of chitosan and CMGC samples sintered using the power screw mold. The wavenumber corresponding specific bonds stretching are given in bracket,

-1 -1 -1 N-H bond (3304.4 cm ), C−Η bond (2887.1 cm ), CH2 bond (2729.7 cm ), C=O bond (1493.1 cm-1). The specific vibration corresponding to the C-H bond (1373.5 cm-1) resembles the scissoring of the C-H bond. The rings stretching for C-H (1251.9 cm-1). For the C-O-C linkage asymmetric stretching (1090.6 cm-1) and symmetric stretching (976.8 cm-1). Here, the D and G carbon bands

CMGC 5 wt.% correspond to peaks at 1337.3 cm-1 and 1606.7 cm-1 respectively. We can note the predominance of the MG spectrum in the composite sample sintered in power screw samples too.

The peaks are consistent with the spectrum obtained from the conventional mold. Table 4.4 summarizes the Raman spectrum obtained from conventional and power screw mold.

Figure 4.10. Raman spectrum for chitosan and CMGC samples sintered using power screw

mold. Here, (a) chitosan 120oC, (b) chitosan 180oC, (c) CMCG – 5wt.% at 120oC, and (d)

CMCG – 5wt.% at 180oC. Note: The samples are sintered with power screw mold.

Table 4.4 Characteristics peaks of the Raman spectrum obtained from conventional and power

screw mold.

Functional Groups Conventional mold Power Screw mold N-H 3301.5 cm-1 3304.4 cm-1 C−Η 2887.0 cm-1 2887.1 cm-1 -1 -1 CH2 2722.6 cm 2729.7 cm C=O 1676.4 cm-1 1493.1 cm-1 C-H scissoring 1373.8 cm-1 1373.5 cm-1 C-H rings stretching 1264.5 cm-1 1251.9 cm-1 C-O-C asymmetric stretching 1095.3 cm-1 1090.6 cm-1 C-O-C symmetric stretching 909.1 cm-1 976.8 cm-1 D carbon bands 1321.4 cm-1 1337.3 cm-1 G carbon bands 1599.0 cm-1 1606.7 cm-1

4.3 Physical testing

4.3.1 Conductivity Testing

It was observed that the measured resistivity does not fall within the available bulk resistivity measurement of 10 milliohms. cm to 10 Kohm. cm from the Jandel HM21 Test Unit. Figure 4.11 shows the result as a contact limit for all the samples with varying MG wt.% which is common when insulators are tested for bulk resistivity.

Figure 4.11. Image of four-probe electrical conductivity testing of chitosan and chitosan-MG

composite

Hence, it can be concluded that the composite containing 1-5 wt.% of MG are insulators. Various studies report improvement in electrical conductivity on the addition of carbon structures as low as 1 wt.%. The mechanism involved with carbon fillers like GO, it is possible to provide an electron transfer path, π-π stacking interaction between the graphene surface, or synergetic electrostatic interaction of chitosan. However, these synthesis processes involve chemical modification of bonds or even crosslinking. With the solid-state processing of chitosan and MG in our study, MG does not alter the bonding structure, nor does it react chemically. Besides, it is believed that percolation is not exactly the best event though the appearance of the composite with 1 wt.% MG or less looks very homogeneous. Since Chitosan is an insulators, increase the MG concentration will lead to better percolation for electrical conductivity; however, 5 wt.% is already a massive amount that may have two detrimental effects: i) excessive amounts of stress concentrators and carbon agglomeration and ii) high cost. Certainly, the current optimized 5 wt.% of MG is not enough to establish conductance in the composite. Hence CMGC can be used only as insulation material.

4.3.2 Bulk Density Testing

Figure 4.12 shows the sintered raw chitosan, milled chitosan, and CMGC 5wt.%. The sample thickness obtained after sintering is summarized in Table 4.5. Bulk density was calculated based on the formula:

푀푎푠푠 표푓 푡ℎ푒 푝표푤푑푒푟(푔푚) 푀 퐷푟푦 퐵푢푙푘 퐷푒푛푠푖푡푦 = = ...... 퐄퐪퐮퐚퐭퐢퐨퐧 ퟒ. ퟏ 푉표푙푢푚푒 표푐푐푢푝푖푒푑 푏푦 푡ℎ푒 푐표푚푝표푠푖푡푒(푚푚 ) 푉

Figure 4.12. Image of sintered samples (a) raw chitosan – no milled, (b) Milled chitosan, and

180C and weigh 3 g each.

Table 4.5. Summary of sample specifications after sintering and resulting bulk density

Raw Chitosan Milled Milled Chitosan with No milling Chitosan 5% MG

Weight (g) 3 3 3

Thickness of sample (cm) 0.31 0.29 0.25

Diameter (cm) 3.1 3.1 3.1

Volume (cm3) 2.33 2.1 1.88

Bulk density in (g/cm3) 1.28 1.37 1.59 Note: Milling was done using Spex Sampleprep 8000M Mill. Chitosan and Morphed Graphene

were milled for 1.5 and 3 h respectively. About 5 wt.% MG was milled with chitosan for an

additional 1.5 h.

To calculate the actual density of the material and compared it with theoretical predictions the rule of mixtures can be used. In the rule of mixtures, we combine the volume fractions of each constituent as well as their respective densities. In Equation 4.2, the density (ρ) and the volume

(V) of each component, in this case, chitosan (m), and the respective reinforcement are the MG (f).

The theoretical density of chitosan as reported by the vendor Alfa Aesar is 0.25 g/mL. In the literature, the reported density of sintered chitosan is 1.48 g/mL (110) and is used for theoretical

74 calculations. For the morphed graphene, the density is 2 g/mL. Using the rule of mixture, the theoretical values obtained are stated in Table 4.6.

휌 = 휌푉 + 휌푉...... 퐄퐪퐮퐚퐭퐢퐨퐧 ퟒ. ퟐ

휌, 휌, 휌 – Densities of the composite, matrix, and dispersed phase respectively;

Vm, Vf – Volume fraction of the matrix and dispersed phase respectively.

Table 4.6. Theoretical density is calculated by the rule of mixtures and densification in percentage.

Density (g/cm3) from Density (g/cm3) Material Densification (%) Equation 4.2 After sintering Chitosan raw 1.48 1.28 86.5 Chitosan milled 1.48 1.37 92. 6 CMGC-1wt.% 1.49 1.50 101.4 CMGC-3wt.% 1.50 1.48 99.0 CMGC-5wt.% 1.51 1.59 105.6

Table 4.6 summarizes the theoretical density as calculated by the rule of mixtures and bulk density calculation after sintering. Ultimately the densification is calculated in percentage. It is evident that sintering is an effective process responsible for densification. This is a clear indication of a reduction in porosity. For samples sintered at 180C the improvement is 86, 92, 101, 98, and 105% for chitosan raw, milled, 1, 3, and 5 wt.% content MG respectively. The densification is calculated based on reference density value 1.48 g/cm3 reported by Lima, et al (110). The improvement in densification is then relative to the reference. It can be concluded that with milling and sintering, the morphed graphene particle anchors the chitosan matrix, filling the void spaces in the polymer.

This effect is further responsible for the improvement in mechanical properties as MG strengthened the chitosan matrix on even dispersion.

Figure 4.13 shows the Bulk density of chitosan and composites as a function of MG content and sintering temperature. Composite samples were prepared with varying MG content 0, 1, 3, and 5 wt.%. MG was milled for 3 h separately, and together they were milled for an additional 1.5 h based on the required composition. The chitosan and composite milled powder were sintered at

120, 150, 180, and 220C for 3 to 5 h. The powdered material was carefully filled in the mold and the mold was compressed using a custom-made French press at a constant pressure of 3.5 MPa.

The bulk density was calculated according to Equation 4.1. It can be concluded that the bulk density increases with an increase in morphed graphene addition and sintering temperature. With an increase in MG wt.%, the matrix becomes denser as morphed graphene is dispersed evenly in the chitosan matrix filling void spaces, reducing grain boundary. With an increase in sintering temperature, the particle fusion improves.

Here, we need to point out that the conditions used for sintering were selected based on thermal analysis conducted on our chitosan (18). The logic behind the sintering conditions is the determination of the degradation temperature that is approximately 225C. The rule of thumb for material sintering is approximately 0.8Tmelting. In our case, we are not melting, but the chitosan degrades rapidly above 225C; therefore, we used 0.8Tdegradation temperature for sintering that is

180C and based on several experimental tests we demonstrated that this is the optimum sintering temperature for this material.

The Bulk density for chitosan sintered at 180C for 3 h is 1.37 g/cm3 and for 5 h is 1.45 g/cm3.

Similarly, for CMGC 5 wt.% it is 1.59 and 1.66 g/cm3 for 3 and 5 h respectively. An increase in sintering time shows an increase in Bulk density. Sintering for various times was also practiced and we concluded that the best conditions for 180C is of 3 h. Further time may result in excessive chitosan degradation due to dehydration and ultimately chitosan degradation. 76

Figure 4.13. The plots for bulk density of chitosan and composites as a function of morphed

graphene content and sintering temperature (a) sintering for 3 h, (b) sintering for 5 h. Note: the

composition of samples was chitosan pure, chitosan: MG – 99:1, 97:3, and 95:5 by weight.

The bulk density range for commercially available plastic is 0.4-0.7 g/cm3, biomass is 0.42-0.82 g/cm3. For electronic circuit boards, PMMA is used which has a reported density of 1.18 g/cm3

(111). Bulk density of raw chitosan milled chitosan and composite produced with sintering at

180C is 1.28, 1.37, and 1.56 g/cm3 respectively. The synthetic polymer shows a lower density than sintered chitosan and composite samples. Thus, sintering is an effective method for the synthesis of highly dense components. Sintering time and temperature should be carefully investigated before it is optimized for this or other materials. Our optimum conditions allowed us to maintain chitosan integrity while improving densification above the theoretical for pure chitosan.

4.4 Compression molding

Figure 4.14 shows samples sintered at 180C for 3 h with a power screw and cube molds. Here we demonstrate that our composites are ready to produce final products of variable thickness while 77 maintaining the consistency of the samples. The sample in Figure 4.14(a) has a thickness of 3 mm and a weight of 3.5 g and Figure 4.14(b) has a thickness of 10 mm and weighs 12 g. For the cubical mold samples, the weight is 4g. The thickness and weight of samples agree with the bulk density reported earlier. With compression molding principles and optimized sintering time and treatment, it is possible to produced different shapes and sizes with a proper mold design.

Figure 4.14. Image of samples sintered at 180oC for 3 h with a power screw mold (a) thickness 3 mm, and (b) thickness 10 mm and (c) top view and (d) front view of cubical mold samples with

wall thickness 2 mm.

4.5 Laser lithography

As seen from Figures 4.15 the cut surface of chitosan and CMGC looks very similar and clean when compared to the acrylic sheet. During the laser cutting process, the CMGC samples sintered

78 for 1 and 2 h resulted in carbon fumes, this could be related to the loose packing of MG. The samples sintered for 3h did not show signs of any fumes resulting from the cutting. This is a clear indication that MG was confined very well to the chitosan matrix. Also, in the previously reported work, it was demonstrated that MG graphene is a fire retardant (112). This shows the effectiveness of fusion during the sintering process. The sintering condition 180C for 3 h is ideal for producing chitosan bio-composite. The laser power and processing speed are two important parameters for effective laser cutting. The combination of higher laser power and slower speed results in effective depth cuts. However, the power of the laser should be optimized to avoid the burning of samples with the laser. We utilized 70% power of laser power and 5% the scanning speed of a 10W CO2 laser (10.6 nm wavelength) for laser cutting of our samples. Laser cutting for chitosan-based bio- composite is demonstrated for the first time and is a successful manufacturing process to make commodity products of different shapes and electronic circuit boards using CMGC.

Figure 4.15. Image of the laser cut samples of acrylic sheet, chitosan, and CMGC 5wt.% composite. Here the thickness of (a) UH logo design is 3 mm, (b) - (c) Gear shape is 2 mm, and

(d) - (e) is 1.5 mm. Note: The proportionate dimension of the square is 2.5 x 2.5 cm for each

shape. The holes in (d-e) have a diameter of 2 mm.

4.6 Water degradability and Biotoxicity evaluation

Figure 4.16 presents the water degradability of chitosan and CMGC samples in water. The chitosan containing samples swells and disintegrates quickly as soon as it comes in contact with water. Considering the challenges related to land pollution by plastic waste, it is important to develop a biomaterial that nurtures soil and promotes the growth of vegetation when discarded as a waste material on land. After the thermomechanical process, a drop in crystallinity is observed based on FT-IR and Raman analysis. A decrease in crystallinity is directly is related to an increase in the degradation rate of chitosan (113). Chitosan degrades via oxidation, chemical, or enzymatic hydrolysis producing non-toxic oligosaccharides (30). In our water degradation study, no fumes were observed when chitosan comes in contact with water.

Figure 4.16. Image of the water degradability testing of (a) raw chitosan, (b) milled chitosan, and

Figure 4.17 shows the experimental setup (a) soil control, (b) chitosan raw, (c) chitosan milled, and (d) CMGC samples for the biotoxicity evaluation. The level of samples in the pot was initially the same. However, after watering, (b) chitosan raw, (c) chitosan milled and (d) chitosan composite samples swelled up. The milled samples (c) and (d) show more swelling than (b) owing to the reduced crystallinity and potential hydroxyl reduction or dehydration as observed in FT-IR.

Water holding capacity increase with decreases in crystallinity (114). From Figure 4.18 it is

80 evident that chitosan and the respective composite samples support plant growth without any inhibition. It is safe to conclude that no toxicity seems to be induced by the thermomechanical processing of the chitosan samples. At least it is not truly apparent for the plant growth. A further advantage is a fact that chitosan and the composites have a superior water holding capacity than soil. Soil tends to dry within a few h if kept indoor. Chitosan samples showed good water retention over a period of 12 h.

Figure 4.17. Image of the experimental setup for the biotoxicity evaluation, (a) soil control, (b)

chitosan raw, (c) chitosan milled, and (d) CMGC samples.

4.7 Discussion We presented results related to the physical and mechanical properties of chitosan and CMGC material. Table 4.7 below summarizes the mechanical properties of our material and conventional polymers. The compressive strength of CMGC is better than the commercially available plastics 81 like PE and PP (115–118). The toughness of CMCG is greatly influenced by the self-healing mechanism evident from the compression test.

Table 4.7. Mechanical properties of CMGC and conventional polymers (115–118). CMGC PE PP Nylon-6 PMMA PLA

Compressive Modulus (GPa) 8.82 0.7 1.5 2.3 3 3.6 Compressive Strength (MPa) 54.77 20 40 55 95 17.9 Toughness (KJ/m3) 2522.45 N/A N/A N/A N/A N/A Density (g/cm3) 1.59 0.99 0.91 1.17 1.19 1.15

Note: CMCG- Chitosan Morphed Graphene Composite, PE- Polyethylene (High density), PP- Polypropylene, PMMA- Poly(methyl methacrylate), and PLA- Polylactic acid

To get a better understanding, we present Ashby plots for a clearer comparison of various material classes. An Ashby plot is a scatter plot that displays two or more properties of many materials or classes of materials (119). Design of any component does not necessarily depend on one property of a material, rather it requires a combination of properties such as density, modulus strength, conductivity, etc. Ashby plot helps get a clear idea during the material selection process and are plotted on a logarithmic scale to display more information. Figure 14.18 shows a typical Modulus- density Ashby chart. The CMGC material is located in the polymer class region. As seen from the graph, the CMCG could be used for an application where polymers such as PP, PE, PET, Epoxies, etc. are used.

Figure 4.18. The Ashby plot for Young’s modulus E plotted against density. This graph is

adopted from Ashby MF-Chapter 4: Material Property Charts (120).

Figure 4.19. The Ashby plot for Strength plotted against density. This graph is adopted from

Ashby MF-Chapter 4: Material Property Charts (120).

Figure 4.19 shows a plot of Strength - density of the material. CMGC is located again in the polymer class region. The density of CMGC is greater than other polymers and elastomers in that 83 group with approximately equivalent strength. Below is the plot for Modulus- strength in Figure

4.20. CMGC could perform better than many polymers and elastomers where high strength is required.

Figure 4.20. The Ashby plot for Young’s modulus plotted against strength. This graph is

adopted from Ashby MF-Chapter 4: Material Property Charts (120).

Many applications where moving parts are involved may require high stiffness and strength with minimum weight. The specific properties of material play an important role in determining the mechanical efficiency of the material which can perform a similar function with the least mass.

Here, CFRP is an emerging material in the composite class of material. Although their brittleness limits their structural use. CMGC could be a smart alternative owing to its less stiffness and comparable specific strength as seen in Figure 4.21.

Figure 4.21. The Ashby plot for specific Young’s modulus plotted against specific strength. This graph is adopted from Ashby MF-Chapter 4: Material Property Charts (120).

It is important to note that improvement in the strength of the material should not be compromised by an increase in brittleness. As concluded by locating CMGC position on various Ashby plots, it stands as a promising composite material with improved modulus and strength than commercial polymers. Chitosan and CMGC composites are fabricated with a simple solid-state synthesis technique. The process does not involve the use of any harsh and toxic chemicals and has been proven for nontoxicity and biodegradability. We have successfully prepared different shapes and sizes from the composite using compression molding. The chitosan products are fabricated with laser cutting for the first time and have the potential to be scaled up for industrial-level manufacturing.

CHAPTER V

CONCLUSIONS AND FUTURE WORK

Synthesis of bio-composite with superior mechanical properties is desirable, but yet remains challenging due to chemical and physical limitations of biopolymers. The potential to account for manufacturability and to compete with commercially available polymers for various commodity applications remains far achievable. Abundantly available chitin and chitosan biopolymers are underutilized and sustainable substrates for replacing synthetic polymers with comparable mechanical properties that are today a major environmental concern. CMGC on the other hand is the idea material for net zero emissions as it can be produced purely from byproducts that qualifies it as a negative emission.

This work re-confirm the improvement in mechanical properties of chitosan by the addition of varying percentages of morphed graphene followed by conventional sintering consisting of thermomechanical processes. As concluded from the compression test results, the contribution of

MG reinforcement is by no means negligible. On the contrary it is a major contributor to improve the mechanical properties of CMGC. The compression modulus and strength of CMGC are superior to conventional polymers like PE and PP and comparable to Nylon and PMMA that itself gives our composites marketable visibility. Here we demonstrated that 5 wt.% MG additions improve toughness over 60%. CMGC stands out with the highest bulk density when compared to other polymers. In perspective, the mechanical properties of CMGC are far superior to PLA which is a widely used biodegradable polymer. CMGC has compressive strength of 54.77 MPa which is

200 % more than that of PLA 17.90 MPa (115–118). In addition, CMGC is easily compostable when it is in contact with water, unlike PLA. CMGC can support plant growth without apparent

86 inhibition. We can produce unique water degradable architectures with compression molding and laser lithography (e.g. cutting) without the use of any toxic solvents or chemicals.

We saw improvement in the mechanical properties as the temperature is raised from 120oC to

180oC grain due to boundary reduction and even dispersion of MG in CMGC. However, we saw a decrease in mechanical strength when the temperature was increased from 180C to 220C. This could be due to the disintegration of chitosan as its glass transition temperature is 225 C as mentioned earlier.

The material characterizations Raman and FT-IR show the integrity of chitosan after the thermomechanical processing is intact. The addition of MG does not affect bonding in chitosan, rather it anchors the grains allowing higher densification by reducing grain boundary, which improvs matrix densification. Both the FT-IR and Raman spectroscopies demonstrate that chitosan do not seem to have major effects on structural decomposition such as the amino, and acetyl groups that are not affected by the presence of carbon or the milling process. On the other hand, it seems that the hydroxyl group is unavoidably affected by the thermomechanical process. Yet, there is no indication of detrimental effects of any kind. In addition, crystallinity seems to decrease as the chitosan FT-IR peaks lower their intensity and become broader. SEM images provide some evidence about a reduction in the grain boundary.

The bulk density of CMCG has improved with thermomechanical processing. Optimizing sintering time and the temperature to 180 ºC has led to the densification of samples without damage to chitosan natural structure. The theoretical bulk density calculated by rule of the mixture and the determined bulk density shows near full densification of the sintered CMCG. The density of the pure chitosan is also considerably high. Fr instance, chitosan shows 87% and the CMCG with 5 wt.% MG additions shows 105% densification with respect to the reference chitosan sample as 87 reported in the literature. The milling time, sintering time, and temperature influence the densification of the sintered samples that seems to increase with temperature, having a 180 ˚C limit. The properties of the chitosan composites are comparable to commercially available

Plexiglas (PMMA), Teflon, sand, and glass reinforced composite materials. Electrical conductivity testing showed that CMGC is an insulator material. The 5wt.% MG composite is not enough to induce conductivity in the polymer matrix since there is no chemical bonding network between

MG and chitosan.

Biopolymers tend to lose crystallinity when subjected to physical or chemical changes. Chitosan showed loss of crystallinity due to milling and sintering. However, this is directly related to the released of the hydroxyl groups (likely water) stored within the structure chitosan is well known for its holding capacity of water. Due to this water capacitance potential the chitosan and CMGC samples absorb water to a point of disintegration immediately after coming in contact. This effect is key for the plant growth potential as observed in the herbs we produce. Therefore, the CMGC does not seem to have negative effects or toxic byproducts potential to the environment and all the processing we used does not seem to affect its biodegradability and biocompostability. In addition, the fact that both, chitosan and MG are byproducts, our research is a solid contributor to negative emission; hence, contributing directly to net zero emissions.

Compression molding is an effective technique for mass manufacture of chitosan bio-composite.

With innovative mold designs, it was possible to bring down manufacturing time as compared to the conventional pressing mold. Multiple samples can be sintered at the same time that can be assisted by laser lithography techniques, products of various shapes and sizes can be manufactured.

Laser cutting of chitosan material is successfully demonstrated for the first time and can play a pivotal role in scaling up bio-composite manufacturing.

Future Work

Although the presented methodologies and protocols for compression molding and laser cutting of chitosan is an effective and simple technique for manufacturing of biomaterials, there are some improvements that we would like to concentrate on in the future. The future work concerns a simulation study of compression molds which would allow us to study pressure gradient while compressing samples of higher thickness. Milling and sintering can be investigated for time and temperature conditions to produce chitosan composite with varying porosity for various applications like biodegradable battery as electrodes, membrane separation, environmental engineering, and gas filtration systems (121). A more detailed analysis of biodegradability can be done to prove the degradation mechanism of chitosan composite in natural and landfill environments. Water sensitivity of chitosan bio-composite can limit its application to a moisture- free setting. This can be eliminated by coating the material with a hydrophobic coating that supports biodegradability. For laser cutting applications, future researchers can explore more complex designs, such as square bulk samples cut into thin sheets, and etching of sheet samples to simulate biodegradable mother boards, printed circuit boards for electronics application.

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