cellulose phosphate from oil palm...
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CELLULOSE PHOSPHATE FROM OIL PALM BIOMASS AS POTENTIAL
BIOMATERIAL
BY
MOHAMAD HAAFIZ BIN MOHAMAD KASSIM
Thesis submitted in fulfillment of the requirements for the degree of Master of
Science
APRIL 2011
ACKNOWLEDGEMENT
Alhamdulillah, I am grateful and thankful to The Great Almighty, Allah
SWT, for his guidance and blessing until I complete my master project, and thank to
my beloved family members for their continuous support and encouragement. I
would like to dedicate my appreciation and thank you to my supervisor Professor
Wan Rosli Wan Daud for his guidance, persistence encouragement and associated
aid throughout this study.
I would also like to express my sincere gratitude to my co-supervisor Dr Md
Azmaan Seeni Mohd in encouragement me in completing this master study. Thank
also to all staff at the Universiti Sains Malaysia in general and in particular the
School of Industrial Technology has been involved in and provides assistance
directly or indirectly. Thank to Universiti Sains Malaysia for provided financial
support in the grant scheme 1001/PTEKIND/8140151, 1001/PTEKIND/833050
Kementerian Pendidikan Tinggi (KPT) and USM RLKA sponsor ship. Also to my
beloved wife Marliana Binti Mohd Mahadar, who give me the strength to keep up
with study encourage and helping me that leading towards the success of this project.
A special dedication also goes to all craniofacial staff that assists me during
worked there. In addition thank to my entire friend at USM. who had shared the
precious moment together during my study period. I really appreciated all the
contribution and this master project had given me a lot of knowledge and experience
that I believe I could not get elsewhere.
Thank you so much.
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TABLE OF CONTENTS
ACKNOWLEDGEMENT 11
TABLE OF CONTENTS 111
LIST OF FIGURES Vll
LIST OF TABLES Xll
LIST OF ABBREVIATES Xlll
ABSTRACT xiv
ABSTRAK XVI
CHAPTER ONE: INTRODUCTION 1
1.1 General Introduction 1
1.2 Objectives 5
CHAPTER TWO: LITERATURE REVEIW 6
2.1 Oil Palm Tree 6
2.1.1 Oil palm Industry in Malaysia 8
2.1.2 Oil palm Lignocellulocis By-Product 11
2.1.3 Oil Palm Empty fruit Bunch (OPEFB) 13
2.1.4 Application of OPEFB B_iomass 14
2.2 Biodegradable Polymer 16
2.3 Natural Polymers 19
2.3.1 Polysaccharides 21
2.3.1.1 Cellulose 22
2.3 .1.1.1 Cellulose chemistry 23
2.3 .1.2 Micro Crystalline cellulose (MCC) 25
2.3.1.3 Cellulose derivative 27
2.3 .1.4 Cellulose and its derivatives in medical applications 29
2.3 .2 Cellulose Phosphate as Biomaterial 31
2.3.2.1 Nature of Cellulose phosphate 32
2.3 .2.2 Production of Cellulose phosphate 33
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2.4 Biocompatibility
2.4.1 In Vitro Study
2.4.1.1 Cell Viability
2.4.1.2 MTS Assay
2.4.1.3 Trypan Blue Exclusion Method
2.5 Bioactivity
2.5.1 Simulated Body Fluid (SBF)
CHAPTER THREE: MATERIAL AND METHODS
3 .1 Material
3.1.1 Oil Palm Empty Fruit Bunch (OPEFB)
3.2 Preparation of Dissolving Pulp
3.2.1 Pre-Hydrolysis process
3.2.2 Soda Pulping 26%
3.2.3 Preparation of oxygen-ozone-peroxide (OZP) pulp
3.2.3.1 Oxygen Bleaching (0)
3.2.3.2 Ozone Bleaching (Z)
3.2.3.3 Peroxide Bleaching (P)
3.3 Production of Microcrystalline Cellulose (OPEFB-MCC)
3.4 Preparation of Cellulose Phosph~te (OPEFB-CP)
3.4.1 Phosphorylation Process
3.5 Cellulose Phosphate Characterization
3.5.1 Fourier Transform Infrared Spectroscopy (FTIR)
3.5.2 Phosphorus Contents
3.5.2.1 Digestion process
3.5 .2.1.1 Preparation of Standard Phosphate Solution
3.5 .2.1.2 Calibration Curve
3.5.3 Scanning Electron Microscopic (SEM)
3.5.4 Water Absorption
3.5.5 X-Ray Diffraction Analysis (X-RD)
3.5.6 Thermo Gravimetric Analysis (TGA)
3.5.7 Differential Scanning Calorimetry (DSC)
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3.6 Biocompatibility
3.6.1 Cell culture
3 .6.1.1 Growth media preparation
3.6.1.2 Thawing frozen cell
3.6.1.3 Cell passage (Sub-Culture)
3 .6.2 Extraction process
3.6.3 Cell Counting
3.6.4 Cell Seeding and treatment
3 .6.4.1 MTS analysis
3 .6.5 Trypan Blue Exclusion method
3 .6.6 Cell proliferation
3.7 Bioactivity of Cellulose Phosphate (OPEFB-CP)
3. 7.1 Preparation of Simulated Body fluid (SBF)
3.7.1.1 Preparation of solution A
3. 7 .1.2 Preparation of solution B
3.7.1.3 Mixing of solution A and solution B
3.7.2 Bioactivity assay evaluation
3.7.2.1 FTIR analysis
3. 7.2.1 SEM-EDX analysis
3.7.2.2 X-Ray Diffraction analysis (XRD)
CHAPTER FOUR: RESULTS AND DISCUSSION
4.1 Characterization of Cellulose Phosphate
4.1.1 Scanning Electron Microscopic (SEM)
4.1.2 Energy Dispersion analysis (EDX)
4.1.3 Degree of Substitutions (DS)
4.1 4 Fourier Transform Infra Red (FTIR)
4.1.5 X-Ray Diffraction
4.1.6 Differential Scanning Calorimetry (DSC)
4.1. 7 Thermal Gravimetric analysis
4.1.8 Water absorption
4.2 Biocompatibility of Cellulose Phosphate
4.2.1 In vitro Cytotoxicity MTS assay
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4.2.2 Trypan Blue Exclusion assay
4.2.2.1 Cell morphology
4.2.3 Cell Proliferation rate
4.3 Bio-activity Evaluation
4.3.1 Fourier Transform Infrared (FTIR) analysis
4.3.2 Scanning Electron Microscopy (SEM)
4.3.3 Energy Dispersion analysis (EDX)
4.3.4 X-ray Dispersions analysis (X-RD)
CHAPTER 5: CONCLUSION AND RECOMMENDATION
5.1 Conclusion
5.2 Recommendations
REFERENCES
APPENDIX
PUBLICATION
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Figure 2.1
Figure 2.2
Figure 2.3
Figure 2.4
Figure 2.5
Figure 2.6
Figure 2.7
Figure 2.8
Figure 2.9
LIST OF FIGURES
Oil palm tree
Oil palm fresh fruit bunch and the structure of the palm
fruit
Annual production of palm oil between Malaysia and other
producers
EFB dumping waste at oil palm mills
Oil palm empty fruit bunches
Structure of polysaccharides
Structure of wood cell showed middle lamella (ML),
primer wall (P), second wall layer (S1, S2, &S3) and lumen
The cellulose molecule- (C6 H10 Os) n configuration.
Cellulose chain (chair comformation ). Dash line
connecting between cellulose ring showed the intra and
inter molecular bond.
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7
10
12
13
21
22
24
24
Figure 2.10 Schematic of crystalline and amorphous region in cellulose 25
chain
Figure 2.11 Schematics formati~m of microcrystalline cellulose 26
Figure 2.12 General structure (a) cellulose and (b) cellulose derivative. 27
Figure 2.13 Examples of polysaccharides ester produced by the 28
reaction of active hydroxyl groups
:Figure 2.14 Structures of MTS tetrazolium salt and its formazan 39
product
Figure 3.1 Oil palm empty fruit bunch fibres 43
Figure 3.2 Oxygen bleaching machine 45
Figure 3.3 Ozone bleaching machine with modified rotating vessels 46
Figure 3.4 Systematic arrangement of apparatus used for 48
phosphorylation process
Figure 3.5 Calibration curve of standard phosphate 51
Figure 3.6 Mouse Skin Fibroblast cell L929 56
Figure 3. 7 Illustration of extraction process 58
vii
Figure 3.8
Figure 3.9
Figure 3.10
Layout of hemocytometer counting chamber
Illustration of hemocytometer counting chamber
1x104 cells were placed inside each well of 96 well/plates
(A-H)
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Figure 3.11 Serial of extraction dilution; undiluted, 100 %, 50 %, 25 %, 61
12.5 %, 6.25% and 3.125 %. (A-G)
Figure 3.12 lx105 ofL929 Cell seeding inside 6 well plates 62
Figure 4.1 Micrograph SEM; A) OPEFB-MCC, B) OPEFB-CP) 69
Figure 4.2 FTIR spectra of (A) OPEFB-MCC and (B) OPEFB-CP. 72
Figure 4.3 XRD patterns of(a) OPEFB-MCC and (b) OPERFB-CP. 73
Figure 4.4 DSC curves for a) OPEFB-MCC, and OPEFB-CP. 74
Figure 4.5 TGA and DTG curves of a) OPEFB-MCC and OPEFB-CP 77
under N2 atmosphere at 20 °C/min.
Figure 4.6
Figure 4.7
Figure 4.8
Figure 4.9
Water absorption ofOPEFB-MCC and OPEFB-CP.
Cytotoxicity assay of extracts (indirect method) OPEFB
CP after 72 hours cultured with Mouse Skin Fibroblast
Cells (L929).
Percentage of cell viability and percentage of death cell of
L929 at different dilution concentration(%) of OPEFB-CP
extraction dilution as evaluated by Trypan Blue. exclusion
method.
Cell concentration (lx104 cell/ml) of L929 at different
dilution concentration (%) of OPEFB-CP extraction as
evaluated by Trypan Blue Exclusion method
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Figure 4.10 Cell morphology of L929 after 72hours in culture medium 87
at different OPEFB-CP concentration(%)
Figure 4.11 Percentage of Cell viability on Cell proliferation usmg 88
L929 up to 8 days with IC50 concentration ofOPEFB-CP.
Figure 4.12 Cell concentration of L929 (lx104cell/ml) on Cell 89
proliferation up to 8 days with ICso concentration of
OPEFB-CP as compares to control (Cell in DMEM).
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Figure 4.13 L929 Cell morphology of L929 cell line after 8 days 90
treated with OPEFB-CP IC50 concentration.
Figure 4.14 FTIR analysis of OPEFB-CP after soaked inside SBF for 93
30 days. (a) 0 day; (b) 1 days; (c) 3 days; (d) 6 days; (e) 15
days; (f) 30 days.
Figure 4.15 FTIR analysis OPEFB-CP-Ca after soaked inside 94
Simulated Body Fluid for 30 days. (a) 0 day; (b) 1 days; (c)
3 days; (d) 6 days; (e) 15 days; (f) 30 days.
Figure 4.16 OPEFB-CP after immersed in SBF, (A) Oday; (B) 1 day; 96
(C) 3 days; (D) 6 Days; (E) 15 Days; (F) 24 Days; and (G)
30 Days
Figure 4.17 SEM photographs of OPEFB-CP surfaces coated with 97
Ca(OH)2 before and after soaking in the SBF for various
periods (A) 0 day; (B) 1 days; (C) 3 days; (D) 6 days;
(E)15 days; (F) 24 days and (G) 30 days. (A-G
magnification 1 OOOx), (H magnification at 1 OOOOx).
Figure 4.18 Changing trend of Ca to P ratio on the surface of OPEFB- 98
CP-Ca samples after immersed in SBF, as detected by
SEM-EDX.
Figure 4.19 XRD patterns of the OPEFB-CP-Ca before immersed in 100
SBF and after immersed in SBF for A 0 day; B 3 days; C,
15 days; and D 30 days.
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LIST OF TABLE
Table 2.1 Exports of palm oil annually: 2004 - 2008 10
Table 2.2 Annual dry weight of trunk and fronds 11
Table 2.3 Chemical composition of OPEFB 15
Table 2.4 Molecular weight and degree of polymerization for cellulose 23
Table 2.5 Ion concentrations of the simulated body fluid and human 42 blood plasma
Table 4.1 EDX Analysis ofOPEFB-MCC and OPEFB-CP. 70
Table 4.2 Degree of substitution of OPEFB-CP as evaluation by 71
energy dispersion analysis and ascorbic acid method
Table 4.3 Thermal properties of OPEFB-MCC and OPEFB-CP 76
Table 4.4 Changing trend of Ca to P ratio on the surface of OPEFB- 99
CP-Ca after 30 days in SBF.
X
CP
DP
DS
EFB
Et3P04
H3P04
IC3o
ICso
L929
MCC
OD
-OH
OPEFB
OPEFB-MCC
OPEFB-CP
p
P20s
-P04
SBF
LIST OF ABBREVIATES
Cellulose phosphate
Dissolving pulp
Degree of substitutions
Empty Fruit Bunch
Tri-ethyl phosphate
Phosphoric acid
70% cell growth
50% cell inhibited
Mouse Skin fibroblast cell line
Microcrystalline cellulose
Optical density
Hydroxyl group
Oil Palm Empty Fruit Bunch
Microcrystalline cellulose from Oil Palm Empty Fruit Bunch
Cellulose phosphate from Oil Palm Empty Fruit Bunch
Phosphorus contains
Phosphorus Pentoxide
Phosphate group
Simulated Body Fluid
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CELLULOSE PHOSPHATE FROM OIL PALM BIOMASS AS POTENTIAL
BIOMATERIAL
ABSTRACT
OPEFB-CP with a DS of 2.4 was synthesized from oil palm biomass via the
phosphorylation of microcrystalline cellulose (OPEFB-MCC) using the H3POJ
P20sl Et3POJ hexanol method. Characterization of OPEFB-CP was performed using
scanning electron microscopy (SEM), energy dispersive analysis (EDX), Fourier
Transform Infrared (FTIR), X-ray Diffraction (XRD), Differential Scanning
Calorimetry (DSC), Thermogravimetry (TG) and water absorption. The
biocompatibility in terms of cytotoxicity of OPEFB-CP was performed on mouse
skin fibroblast cells (L929) by MTS Assay analysis and Trypan Blue Exclusion by
indirect extraction method. The proliferation rate of L929 cell on OPEFB-CP IC50
concentration was accessed up to 8 days. The bioactivity of OPEFB-CP was studied
by immersion of OPEFB-CP in Simulated Body Fluid (SBF) for 30 days in order to
observe its applicability to form apatite nuclei.
Results showed that phosphorylation has changed the OPEFB-MCC surface
morphology, where OPEFB-CP has a more sponge-like surface character and
compact structure. EDX confirmed the presence of phosphorous with a 16.4%
increase in atomic weight of P upon phosphorylation. The presence of new FTIR
peaks at 2380 cm-1, 1380 cm-1 and a shoulder at 920-1000 cm-1 are indications of a
successful grafting of the phosphate groups on the cellulose backbone. XRD data
revealed that most of the diffraction bands of OPEFB-MCC were depressed or absent
demonstrating the loss of crystallinity of the material after phosphorylation. OPEFB
MCC have a higher thermal stability as compared to OPEFB-CP, nevertheless
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OPEFB-MCC presents a total loss of 90% while the OPEFB-CP showed a loss of
only 62%; the smaller rate of mass loss of the latter is attributed to some kind of
thermal protection of the phosphate on the product. OPEFB-CP has also higher water
absorption capacity.
OPEFB-CP showed non cytotoxicity reaction on L929 cells depending on the
dilution concentration of extract used with an IC50 value of OPEFB-CP of 45% after
72 hours of cell exposure. No change in cell morphology can be observed when the
concentration used is below the IC50 concentration, however with concentrations
higher than IC5o, the cells became swollen and lost their membrane integrity. After
immersion in SBF for 30 days, apatite formation was formed on OPEFB-CP surfaces
which was treated with Ca (OH)2. However there is no sign of the apatite layer on
untreated OPEFB-CP surface. The sharp peak at 874 em -I is indicative of the
deposition of apatite layer on the surface of OPEFB-CP-Ca. SEM-EDX analysis
confirmed the formation of apatite with Ca to P molar ratio of 1.85; a characteristic
peak of apatite at 28= 26.1 oa further strengthened the evidence of apatite formation
on OPEFB-CP-Ca. Results demonstrate that cellulose phosphate from oil palm
lignocellulosic residual has the potentials to be applied in biomedical applications.
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SELULOSA FOSFAT DARIPADA BIOJISIM KELAPA SA WIT SEBAGAI
BIOBAHAN BERPOTENSI
ABSTRAK
OPEFB-CP dengan DS 2.4 telah disintesis daripada biojisim kelapa sawit
melalui fosforilasi selulosa mikrohablur (OPEFB-MCC) dengan mengguna kaedah
H3P04 I P20s I Et3P04 I heksanol. Pencirian bagi OPEFB-CP telah dilakukan
menggunakan mikroskop pengimbas elektron (SEM), tenaga dispersif analisis
(EDX), spek:trofotometer transfomasi inframerah fourier (FTIR), X-ray difraksi
(XRD), kalorimetri pengimbas perbezaan (DSC), Termogravimetri (TG) dan
penyerapan air. Penilaian toksik bagi sample OPEFB-CP dijalankan dengan
menggunakan sel fibroblas kulit tikus (L929), dan analisis esei MTS dan teknik
perwarnaan trypan biro, berasaskan kaedah ekstraksi tidak langsung digunakan
sebagai kaedah analysis. Kadar proliferasi L929 terhadap ekstrak OPEFB-CP dengan
menggunakan kepekatan IC50 selama 8 hari juga dilakukan. Sifat bioaktif OPEFB-CP
dikaji dengan merendam OPEFB-CP di dalam SBF selama 30 hari dalam usaha
meneliti kebolehupayaan OPEFB membentuk pembentukan tulang.
Keputusan kajian menunjukkan fosforilasi telah mengubah morfologi
permukaan OPEFB-MCC, di mana OPEFB-CP memiliki sifat permukaan berongga
seperti span dan struktur yang lebih padat. Analisis EDX mengesahkan kehadiran
fosforus dengan peningkatan 16.4% dalam berat atom P selepas fosforilasi.
Kehadiran puncak FTIR barn di 2380 cm_I, 1380 cm_I dan bahu di 920-1000 cm_I
adalah penunjuk kejayaan pencantuman kumpulan fosfat pada tulang belakang
selulosa. Analisis XRD menunjukkan fosforilasi mengubah struktur hablur OPEFB
MCC di mana sebahagian besarnya hilang dan mengalami kemelesetan. OPEFB
MCC mempunyai kestabilan terma yang lebih baik berbanding dengan OPEFB-CP,
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namun OPEFB-MCC menunjukan 90% jumlah kehilanga berat akhir sedangkan
OPEFB-CP menunjukkan kehilangan hanya 62 %; tahap kehilangan yang lebih kecil
oleh OPEFB-CP dikaitkan dengan perlindungan terma daripada fosfat yang terdapat
pada produk. OPEFB-CP menunjukkan sifat tahap penyerapan air yang lebih tinggi
daripada OPEFB-MCC.
Sifat toksik OPEFB-CP ke atas sel L929 adalah berga..'ltung pada pencairan
kepekatan ekstrak yang digunakan dengan nilai IC50 OPEFB-CP adalah pada 45%
· selepas 72 jam di dedahkan kepada sel. Tiada sebarang perubahan yang dapat di
perhatikan ke atas morfologi sel apabila kepekatan digunakan di bawah kepekatan
ICso- Namun dengan kepekatan yang lebih tinggi daripada ICso, sel-sel menjadi
bengkak dan kehilangan integriti membran. Setelah direndam dalam SBF selama 30
hari, terdapat pembentukan apatit pada permukaan OPEFB-CP-Ca yang di rawat
dengan Ca (OH)2. Namun tidak ada tanda lapisan apatit pada permukaan OPEFB-CP
yang tidak dirawat. Puncak di 874 cm-1 menunjuk pengendapan lapisan apatit pada
permukaan OPEFB-CP-Ca. SEM-EDJ;C analisis membuktikan pembentukkan apatit
dengan nisbah molar Ca kepada P ialah 1.85; puncak apatit · pada 28= 26.10°
menguatkan lagi bukti pembentukkan apatit pada OPEFB-CP-Ca. Keputusan
menunjukkan bahawa selulosa fosfat daripada kelapa sawit sisa lignoselulosa
mempunyai potensi dalam aplikasi bioperubatan.
XV
CHAPTER!
INTRODUCTION
1.1 General
The growth of oil palm industry in Malaysia has been one phenomenal, and
the country is now one of the largest producer and exporter of palm oil in the world.
Malaysia is blessed with huge quantities of oil palm biomass, with more than 4.69
million hectares land under oil palm cultivation, and producing 17.73 million tonnes
of palm oil annually (MPOB 2009). Due to this situation, enormous amounts of
lignocelluloses residues from trunks, fronds and the empty fruit bunches (EFB) were
generated from palm oil industry, with the latest estimate of more than 15 million
tons (Abdul Khalil et al.,2007 ). Oil constitutes only about 10 % of the palm
production while the rest is biomass which is available throughout the year as EFB
and pruned fronds (Rozman et al., 1998; Amin et al., 2007).
Attempts have been made in turning these wastes into value added product,
converting this lignocellulosic residue to paper-making pulp. is an example.
Nevertheless it is a relatively "low end" one, where the price per tonne is rather low
(WanRosli et al., 1998, 2007). Hence, other utilization alternatives are sought, one of
which is conversion into cellulose derivatives, such as carboxymethylcellulose
(CMC), cellulose acetate and cellulose phosphate (CP), and the latter is of significant
importance due to its potentials to be used as biomaterials.
The term biomaterial refers to any materials, either natural or man-made that
are intended to interface with biological systems or biomedical device for evaluating,
treating, augmenting, or replacing any tissue, organ, or function of the body
(Agrawal, 1998; Matthew, 2002; Nair & Laurencin, 2005). Biomaterial elements
1
encompass in medicine, biology, chemistry, tissue engineering and material science.
Biocompatibility and bioactivity are the essential properties to qualify any material
as a biomaterial (Entcheva et al., 2004).
Biocompatibility is the ability of a material to perform with an appropriate
host response in a specific application. From clinical perceptive these materials were
not producing a toxic or injurious reaction and not causing ,immunological rejection
(Matthew 2002; Vepari and Kaplan 2007; Correlo eta!., 2008). Bioactivity on the
other hand refers to the capability of material to mineralize in physiological
environment. In orthopaedic application bioactivity was referred to the ability of
material to induce the formation of an apatite layer (calcium phosphate layer) in
simulated plasma solutions (Granja et al., 2001 b, 2005).
Several classes of materials such as ceramics, metal, glasses, natural and
synthetic polymeric materials have been investigated as biomaterials for medical use
especially in tissue engineering and orthopedic applications (Burg, et al., 2000, Nair
& Laurencin 2005, Barbosa et al., ~005; Correlo et al., 2008). Some of these
materials have drawbacks and limitation such as metallic implants used as bone
repairing which will cause stress shielding and bone resorption due to the elasticity
mismatch with the surrounding bone. Meanwhile ceramic materials are not
favourable for bone repairing material because of its poor resistance against fatigue
failure and low fracture toughness. Although modulus of elasticity of less rigid
titanium is five times higher than human bone, it still has limitation for their
extensive application in bone reconstruction (Li et al., 1997; Nair & Laurencin
2005).
2
Due to socioeconomic situation of the modern world and environmental
concerns, interests in using natural biodegradable polymer are on the rise. These are
due to their biodegradability, low toxicity and low disposal costs that make them
excellent candidates in biomedical application (Li et al., 1997; Barbosa eta!., 2005;
Vepari and Kaplan 2007). Therefore the desire to develop and create a new
biomaterial for medical application using natural biodegradable polymer such as
cellulose is always rational approach (Nair & Laurencin 2005; Barbosa et al., 2005;
Alriols et al., 2008).
Cellulose is the world's most abundant natural occurring polymer which is
produced by plants, as well as by microorganism. It is a linear homopolymer of
glucose (C6Hw0s)n that is insoluble in water and degradable in nature by microbe
and fungal enzymes (Granja et al., 2001 b; Muller et al., 2006). Cellulose is one of
the natural polymer groups with the longest and widest medical applications
experience. Non-toxicity (monomer residues are not hazardous to health), water
solubility or high swelling ability by simple chemical modification, stability to
temperature and pH variations made cellulose as being the wid~st use in medical
applications (Fricain et al., 2001; Granja et al., 2001 b,2005; Barbosa et al., 2005;
Nair &Laurencin, 2007).
In biomedical applications cellulose derivatives have been extensively
investigated as dressings in treating surgical incisions, burns, wounds, hemodialysis
membranes, coating materials for drugs, drug-releasing scaffolds, and various
dermatological disorders (Entcheva et al., 2004; Nair & Laurencin 2005; Granja et
al., 2005). Oxidized cellulose was used as a wound dressing and has been proposed
for bone regeneration. Regenerated cellulose hydrogels (cellulose regenerated by the
viscose process, CRV) have been investigated as implantable materials in
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orthopaedic surgery, as sealing materials for the femoral component m hip
prostheses, in place of the acrylic cement (Granja et al., 2005).
Nevertheless, a full bioactive character cannot be attributed to normally
occurring cellulose because of its lack of osteoinduction. Phosphorylation was
therefore envisaged as the mean to enhance cellulose bioactivity. Once implanted,
phosphorylated cellulose could promote the formation of calcium phosphate (which
has a closer resemblance to bone functionality), hence ensuring a satisfactory
bonding at the interface between hard tissue and biomaterial. The derivatives of
pentavalent phosphorus such as H3P04, P20 5, organic phosphate and POCh, were
most frequently used to obtain phosphorus containing cellulose derivatives (Suflet et
al., 2006). Phosphorylated material based on cellulose regenerated by viscous
process (CRY) were found to be non-cytotoxic in cultured human osteoblast as well
as fibroblasts, having the ability to induce the formation of an appetite layer in
simulated physiological condition (Granja et al., 2005; Jayakumar et al.,2009).
This present study concerns the investigation of cellulose from oil palm
biomass as potential raw material for the synthesis of CP for used as biomaterial. As
a potential biomaterial, tissue compatibility has to be studied. For this purpose, CP
synthesized will be exposed to mouse skin fibroblast (L929) and the cytotoxicity and
proliferation accessed. Bioactivity of the material will be carried out by immersing
the material in a simulated body fluid (SBF), where the ion concentration of the
solution is approximately equal to human blood plasma, for 30 days.
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1.2 Objectives
• To produce and characterize cellulose phosphate from oil palm empty fruit
bunch.
• To investigate the biocompatibility of cellulose phosphate on connective
mouse skin fibroblast (L929).
• To assess the bioactivity of cellulose phosphate m physiological
environment.
5
2.1 Oil palm
CHAPTER2
LITERATURE REVIEW
The oil palm tree, (Elaeis guineensis), is one of the most important
agriculture plants in Malaysia. It's originated from West Africa where it was growing
and later developed into an agricultural crop (Law et a!., 2007). The oil palm is an
erect monoecious plant that produced separate male and female inflorescences (Teoh,
2002). The oil palm trees are shown in Figure 2.1.
Figure 2.1: Oil palm tree
6
Climatic conditions which are hot and humid throughout the year
make oil palm cultivation suitable in Malaysia. This view has been supported
by Fitzherber et al. (2008) where climatic condition was important factor in
growing oil palm, because oil palm need high-rainfall, low-lying areas, a
zone naturally occupied by moist tropical forest, the most biologically diverse
terrestrial ecosystem on Earth. Figure 2.2 showed the oil palm fresh fruit
bunch and the structure of the palm fruit.
Botanically, oil palm is classified thusly:
Kingdom :Plantae
Sub-Kingdom : Tracheopionta
Division : Angiospermae
Class : Monocotyledones
Subclass : Arecidae
Order : Arecales
Family : Arecaceae
Genus : Elaeis
Figure 2.2: Oil palm fresh fruit bunch and the structure of the palm fruit
7
This plant was first introduced to Malaysia in early 1870's, as an ornamental
plant, and in 1917 first commercial oil palm estate in Malaysia was set up at
Tennamaran Estate, Selangor. The cultivation of oil palm rapidly increased
beginning in the sixties under the government's agricultural diversification program
which to reduce the country's economic where dependence on rubber and tin. Later
in the 1960s, the government introduced land settlement schemes for planting oil
palm as a means to eradicate poverty for the landless farmers and smallholders. The
oil palm plantations in Malaysia are based on the estate management system and
small holders' scheme (Mohamad et al., 1985; MPOC, 2010).
In terms of hectare, the total area under oil palm cultivation is over 2.65
million hectares, which producing over 8 million tonnes of oil annually. The oil
consists of only 1 0 % of the total biomass produced in the plantation. The remainder
consists of huge amount of lignocellulosic materials such as oil palm fronds (OPF),
oil palm trunks (OPT) and oil palm empty fruit bunch (OPEFB). The residues are
Figure up as follows:
• 7.0 million Tonnes of oil palm trunks.
• 26.2 million Tonnes of oil palm fronds.
• 23 % of Empty Fruit Bunch (EFB) per tonne of Fresh Fruit Bunch
(FFB) processed in oil palm mill (ASEAN, 2010).
2.1.1 Oil palm Industry in Malaysia
Malaysia oil palm industry started in 1917 and grew slowly until late 1950s
due to switch over from rubber to oil palm during agricultural diversification policy.
From the onward, the industry started to grow rapidly and currently very little room
remains for any significant increase in oil palm plantations in peninsular Malaysia.
8
All future growth of oil palm is expected to be in Sabah and Sarawak (Abdul Khalil
et al., 2001).
Despite this enormous production, the oil is only a minor fraction of the total
biomass produced in the plantation. The remainder consists of a huge amount of
lignocellulosic material in form of OPF, OPT, and EFB. The year 1985 was
considered as the start of a major replanting era in the oil palm industry, and from
1985 to 1995, there was a steady increase in oil palm replanting. By 1997, the
replanting era reach maximum, yield over 27 million tonnes of biomass. As such, the
oil palm industry must be prepared to take advantage of the situation and utilize the
available biomass in the best possible manner (Rozman et al., 2004; Yusof, 2007).
The status of oil palm biomass in Malaysia during 2006 as stated by Anis et
al. (2007) showed that the total area of oil palm trees planted was 4.17milion
hectares. Oil palm mills generally generate large amount of biomass waste. The
amount of biomass produced by an oil palm tree included lignocellulosic material an
average of 231.5 kg dry weight/year. In the year 2008, OPEFB and OPT are the
major contributor of oil palm biomass, whereby about 15.8 and 8.2 million tonnes,
respectively, have been produced annually (Sumathi et al., 2008).
To date, 4.49 million hectares of land in Malaysia is under oil palm
cultivation; producing 17.73 million tonnes of palm oil and 2.13 tonnes of palm
kernel oil. Malaysia is one of the largest palm oil producers and exporters in the
world, accounting for 11 %of the world's oils & fats production and 27% of export
trade of oils & fats (Abdul Khalil et al., 2008; MPOC 2010). Figure 2.3 Comparison
of Annual Production of Palm Oil between Malaysia and other producers.
9
Now palm oil industry has become an important source of income to the
country. In Malaysia more than 17 million tons per year of palm oil crude produced
and has been exported to over 150 countries, where China was a major importer,
Table 2.1 summarised the annually oil palm export from Malaysia.
mw b~ . <D ....
16 0) ..., :i ..,.., M
M ~ M ..... 14 <-i
~ ~ .... E
~
;; IL
12
6 10 ---o• • 8 c: c: •Others OS::: -o -u ... 6 clndonesla i§
t~ 4 •Malaysian ;; :I 2 c c c
0
2009 2008 2007 2006 2005
Year
Figure 2.3: Annual production of palm oil between Malaysia and other producers (Source: MPOB, 2009).
Table 2.1: Exports of palm oil annually: 2004 - 2008
Year Crude palm oil Processed palm oil Palm oil
2004 1,324,479 11,257,313 12,581,792
2005 1,611,621 11,833,890 13,445,511
2006 2,376,542 12,046,626 14,423,168
2007 1,935,927 11,810,895 13,746,823
2008 2,336,577 13,075,935 15,412,512
Source: MPOB, 2009
10
2.1.2 Oil palm lignocellulosic By-products
Besides crude palm and kernel oils, palm oil mill also generates massive
amounts of lignicellulosic residues such as OPT, OPF during replanting and pruning,
EFB and the mesocarp fibers during milling. These by-products, if not utilised and
managed properly, will impose a disposal problem to the oil palm mill. In 1980,
depending on the type of residue, oil palm residues is utilised as mulch, boiler fuel,
or as fertiliser in the fields in order to reduced the environment impact paving the
way toward a zero-waste policy (Abdul Khalil et al., 2008).
The economic life span of the palm is about 25 years. However, the main
economic criteria for felling (with the purpose for replanting) are the height of palm
which reaches 13m and above and annually yield of bunches falling below 10 to 12
tonne per hectare. This felling activity under the project of replanting produces a
large quantity of lignocellulosic by product, in the form of stem, as well as frond
(Sumathi et al., 2008). The estimate of trunks and crown fronds at the time of
replanting and from annual pruning froJV a hectare of oil palm are given in Table 2.2.
Table 2.2: Annual dry weight of trunk and fronds
Trunk at felling (OPT)
Frond at felling (OPF)
Annual pruning of fronds (OPF)
Source: Sumathi et al., 2008
Dry weight/area (tonne/hectare)
11
75.5-84.0
14.5- 16.0
10.4- 11.0
The dry weight of OPT and OPF per hectare area are varied due to the
different number of palm planted per hectare. Sumathi et al. (2008) has pointed out
that based on the mature hectarages, assuming that 3 percent of the palm oil in
Malaysia is replanted annually. The annual estimated quantity of oil palm trunk and
frond available from replanting are more than 6 million and 1.2 million tonne
respectively. In addition, the dry weight of frond obtained during annual pruning and
harvesting is estimated to be about 29.3 million tonnes.
Figure 2.4: EFB dumping waste at oil palm mills
Besides trunks, a large amount of fronds, also generated during replanting.
Indeed, the annual production of felled and pruned fronds exceeds from trunk.
Currently, a certain amount of pruned fronds is used as interior mulching to conserve
soil moisture in the plantations. However, this utilization has been found to attract
rats, snakes, insect pests and thus limited its application. As a result a large quantity
of lignocelluloses resource is still available for exploitation.
1.2
OPEFB are partly dried in the sun and later used as fuel, if not incinerated or
applied to the fields. An economic use of OPEFB is to return them to the plantation
as a mulch to enhance moisture retention and organic matter in the soil. On the other
hand, the ash recovered from the incinerated OPEFB can also be sold or used as
fertilizer in the palm plantations. The incineration of OPEFB emits particulates into
the surrounding atmosphere and indiscriminate dumping of OPEFB causes additional
methane emission into the atmosphere. The OPEFB, have traditionally been burnt
and their ash recycled into the plantation as fertilizer. However, due to the pollution
problem, incineration of EFB has been discouraged. Instead EFB is returned to the
field to act as mulch (Nasrin et al., 2008).
2.1.3 Oil Palm Empty Fruit Bunch (OPEFB)
OPEFB is one of such solid waste generated in oil palm planted throughout
Malaysia over 4 million hectares land, which remains unutilized commercially.
Hence, it has been verified in engineering properties and construction products made
from OPEFB satisfy the requirement of consumers' trend using products made from
eco-friendly and natural waste.
Figure 2.5: Oil palm empty fruit bunches
13
As a source of both edible and industrial oils, oil palm is one of the most
important tree species in Southeast Asian countries like Malaysia. However, woody
fibrous residues, which remain after oil is extracted, have not been effectively
utilized so far. These so-called EFB (Figure 2.5) could serve as an alternative source
for cellulose pulp, or dissolving pulp, (DP) which could be used as a raw material of
cellulose derivatives and regenerated cellulose (WanRosli eta/., 1998, 2003, 2004).
2.1.4 Application of OPEFB Biomass.
Recently, utilization of biomass resources has been the subject of various
studies. Among the oil palm fiber residues, OPEFB offer the best prospect for
commercialize since its ready available at the palm oil mill, which can minimize
transportation and procurement cost. In Malaysia, OPEFB is one of the biomass
materials, which is a by-product from the palm oil industry. The production of
OPEFB was around 5.3 million per year (Rozman et al., 2004; Myrtha eta/., 2008).
OPEFB has been investigated .as a raw material for building materials and are
now used for other value-added products like pulp, medium density board, wood
composite product and fiberboard. In general, utilization of biomass in
lignocellulosic composite has been attributed to several advantages such as having
low density, greater deformability, less abrasiveness to equipment, biodegradable
and low cost (Rozman et al., 2004).
Through the initial review is believed to be a profitable method in producing
innovative products, from OPEFB. Now varieties products produced from OPEFB,
such food packaging, pulp, fertilizer and medium density fiberboard and for energy
purpose such as ethanol, and biodiesel bio-methane (Yokoyama and Matsumuya,
2008). OPEFB contains high cellulose, lignin and hemicelluloses. The chemical
14
composition (Table 2.3) showed that OPEFB have a good potential usage in various
industries where cellulose as raw material in producing product.
Table 2.3: Chemical composition ofOPEFB
Composition %DryEFB
Holocelulose 82.4±1.4
Cellulose 62.9 ± 2.0
Hemicellulose 28.0
Lignin (non dissolved in acid) 18.8 ± 0.3
Free Ash Lignin (non dissolved in 17.8 ±
acid) 0.02
Dissolved Material 1% NaOH 14.5 ± 2.7
Material Dissolved in Hot Water 7.5 ± 0.8
Extractive 3.7 ± 0.3
ASH 1.3 ± 0.2
Source: Law et al., 2007
As example product that successfully produced from OPEFB cellulose-based
1s Palmcell ® which is carboxylmethylcellulose (CMC). This material has the
potential to be used in paper manufacturing process and as additives to improve
paper strength. As a result the strength of paper produced increase either in the dry
and wet conditions. The products not only efficient to increase the strength of the
paper, but the cost for production Palmcell ® are relatively low compared to other
15
additives. This is because the use of OPEFB as a mam ingredient m the
manufacturing process.
Law et al. (2007) has made a study of some fiber derived from oil palm tree,
and concluded the dissolved pulp derived from EFB are suitable as raw materials for
produce various grades of paper. Continuing research and study has increased the
diversity of applications of OPEFB as an alternative source. For example filling
mattresses and cushions, footwear and automotive hardware already used OPEFB as
raw material. OPEFB products applied in the manufacturing process has now been
successfully commercialized and exported. Looking to the efforts done, OPEFB
could be important source of biomass alternative in many industries.
2.2 Biodegradable Polymer
Over the last few decades, the development of biocompatible and
biodegradable materials also known as biomaterials used for biomedical applications
have been made (Nair and Laurencin, 2005). These materials were intended to
replace part of a living system or to function closely with living tissue. The
requirements for use in medical applications, the biodegradable polymer must be
biocompatible and biodegradable (Correlo et at., 2008; Liuyun, et al., 2009;
Salmoria et al., 2009).
The term biocompatibility was referred to the ability of biomaterial to
provoke an acceptable cellular and biological response from the host environment,
and in a clinical context this entails not producing a toxic or injurious reaction and
not causing immunological rejection (Matthew, 2002; Vepari et al., 2007; Correlo et
al., 2008).
16
On the other hand, the term biodegradable polymers refer to material that can
degrade in vitro and in vivo, either into products that are normal metabolites of the
body or into products that can be completely eliminated from the body with or
without further metabolic transformations (Dumitriu, 2002; Nair and Laurencin,
2005).
Several classes of inorganic materials, such as metals, ceramics and glasses
also polymeric material either natural or synthetic have been approved for medical
used (Nair and Laurencin, 2005; Correlo et a/., 2008). Some of synthetic material
those physical properties most similar to natural tissue was successful used for tissue
replacement (Bhat, 2005).
Metal because of their excellent electrical and thermal conductivity and also
good mechanical properties, made it suitable being biomaterial, three main alloys,
which is titanium-aluminium, stainless steel and cobalt-chromium alloys were used
for the high load bearing application in skeletal system. Conducting metal like
platinum and platinum-iridium alloys was used for electrical stimulation heart, also
for muscles and nervous tissue. In orthodontics application nitinol. an alloy of nickel
and titanium were already used (Peng et al., 2006; Bedi eta/., 2009).
Ceramic defined as the art and science of making and using solid articles that
have essential inorganic non-metallic material components (Bhat, 2005; Habraken, et
al., 2007). Alumina which used in orthopaedic joint replacement is extremely stable
and inert ceramic material. The chemical inertness and high abrasive resistance
provide improvements over the widely used metal. In improving the surface
properties of alumina and metal alloys bio-glass was employed. The degradable
ceramics, which are almost invariably used on calcium phosphates, find application
in hard tissue regeneration. In cardiovascular application, inert carbons are used to
17
improve blood compatibility and also find in dental applications (Kokubo et a/.,
1992; Habraken eta/., 2007).
Particles and fibers reinforcement has been used to improve properties of
biomaterials, for example barium sulphate particles in Poly(methylmethacrylate)
(PMMA) cement improves stiffness and fatigue life considerably. In improving the
strength for rubber which used in catheters very fine particles of silica (Si02) was
usually reinforced. In modern dental composite materials were blend of glass or
ceramic particles dispersed in polymeric organic resin matrix with interfacing saline
coupling agents. The composite such as graphite fibers in epoxy resin can be strong
as steel but much lighter. However, this material is complaint when the loaded
transversely to the fibers (Marques et al., 2009).
A number of synthetic polymers find application as biomaterial. They include
polyolefins, polyamide, polyesters, polyurethanes, polyacylates, polysulfone,
polyethers, and silicon rubbers. Some of these materials are also used as sutures,
tissue adhesives, shunts, catheters and space fillers. Biodegradable polymers, such as
natural, synthetic polyesters, and polyamides were employed a.s bone plates which
provide temporary scaffolding or support respectively, while natural tissue
regeneration take place (Chandra and Rustgi, 1998; Nair and Laurencin, 2007).
Among them, polymeric materials are widely used in clinical applications
because of their unique physical and chemical properties. According Bhat, (2002)
polymers have physical properties that almost close to soft tissue and this class of
material were used to replace the function of soft tissue including skin, tendon,
cartilage, vessel walls, lens, breast and bladder. On the other hand because of its
ability to be structurally and functionally modified, also their biodegradation easy to
control and because they are relatively inexpensive to manufacture made polymeric
18
material as candidates for various medical applications (Barbosa eta/., 2005; Correlo
et a/.,2008; Salmoria et al., 2009; Liuyun et al., 2009).
2.3 Natural Polymer
The used of structural material from natural polymer or biopolymer is not a
new phenomena. Materials derived from animals or plants world already used as
biomaterials. Due to similarity of this material with material to human bodies gives
natural polymer huge advantage (Barbosa et al., 2005; Jayakumar et al., 2009). The
natural polymer materials do not offer the problems of toxicity that often faced by
synthetic material. Some of natural material may carry specific protein binding sites
and other biochemical signals that may assist in tissue healing or integration. One of
the examples of a natural material was collagen, which existed mostly in fibril form,
has a characteristic triple-helix structure and the most prevalent protein in the animal
world (Jayakumar et al., 2009).
Other natural materials under consideration include coral, chitin from insects
and crustaceans, keratin from hair and various natural biodegradable polymers such
as polysaccharides which is currently being investigated as matrices for controlled
drug delivery or as scaffolds for tissue engineering (Nair and Laurencin, 2005;
Barud, 2007).
Due to their excellent biocompatibility, natural biodegradable polymers seem
to be the choice for biomedical applications. This is because they closely mimic
native cellular environments, have unique mechanical properties, and are
biodegradable by an enzymatic or hydrolytic mechanism (Granja et al., 2005;
Barbosa et al., 2005; Nair and Laurencin, 2005; Wan et al., 2006).
19
According to Chu, (2002), biodegradable polymer do not elicit permanent
chronic foreign body reaction due to the fact that they would be gradually absorbed
by human body, and also they didn't permanently retain trace of residual in the
implantation sites. Furthermore some of them have been found to be able to
regenerate tissues, through the interaction of their biodegradation with immunologic
cells like macrophages (Li et al., 1997; Bhat, 2002).
Advantages of biodegradable polymers are once implanted they no need for a
second surgical procedures as well as eliminate the long-term biocompatibility
concern. The biodegradation may offer other advantages in many short-term medical
applications; in orthopaedic applications mechanically incompatible implants such as
metallic implants can sometimes lead to stress shielding, whereas biodegradable
implants may slowly transform the load as it degrades (Li et al., 1997; Nair and
Laurencin, 2005).
Natural biodegradable polymers remain attractive because they are
economical, readily available, capable of chemical modifications, and potentially
degradable and compatible due to their origin. In addition degradation of natural
biodegradable polymers into physiological metabolites makes them excellent
candidates for a wide range of applications, such as drug delivery (Satturwar et al.,
2003).
However, natural biodegradable polymers not been fully exploited in the
biomedical field, this is due to the inherent disadvantages such as risk of viral
infection, antigenicity, unstable material supply, and batch-to-batch variation in
properties. Another problem faced by these materials, is their tendency to denature or
decompose at temperatures below their melting points. This severely limits their
20
fabrication into implants of different sizes and shapes (Stillwell et al., 1997; Nair and
Laurencin, 2005).
2.3.1 Polysaccharides
Polysaccharides are high molecular weight polymers, which is having one or
more monosaccharide repeating units (Figure. 2.6). Polysaccharides, have excellent
properties such as non-toxicity (monomer residues are not hazardous to health),water
solubility or high swelling ability by simple chemical modification, stability ·to pH
variations, and a broad variety of chemical structures which make these group of
polymer the longest and widest experience in medical applications (Nair and
Laurencin, 2005). All of these advantages able to overcome some of its
disadvantages such as low mechanical properties, temperature and chemical stability,
and enzymatic degradation (Barbosa et al., 2005). ·
- - n
Starch Q!llulose
Chitin
Figure 2.6: Structure of polysaccharides. Source: Chandra and Rustgi, 1998.
21
Furthermore with wide availability, cost effectiveness, easily modified with
the presence of reactive functional groups along the polymer chain, biodegradability,
biocompatibility, and water solubility, combined with the ability to form hydrogels,
make them excellent candidates for tissue engineering and drug delivery applications
(Nair and Laurencin, 2005,2007; Bhat, 2002; Muller et al., 2006).
2.3.1.1 Cellulose
Cellulose is the main component of plant cell walls and one of the most
abundant, renewable and biodegradable organic compounds on earth. In wood
contents about 40-50 % cellulose, 98 % of cellulose was in cotton fibers, 70 % in
bast fiber like hem and located predominantly in the secondary wall (Figure. 2.7).
Cellulose can be produced from various other plant fibers, such as com cobs or
bagasse (sugar cane stalks), rice hulls, bamboo, and fibers such as EFB, kenaf, flax,
pineapple leaf etc (Kamide, 2005). The used of cellulose in industry can be
considered spacious, pulp and paper was the largest industries that used cellulose as
raw materials in production process.
Secondary Wall Layers
S2
Sl
Primary wall
Figure 2. 7: Structure of wood cell showed middle lamella (ML ), primer wall (P), second wall layer (S., S2, &S3) and lumen. Source: David, 2003.
22
2.3.1.3.1 Cellulose chemistry
Cellulose is a white fibrous material composed of the elements Carbon (C),
Hydrogen (H) and Oxygen (02). The chemical formula for cellulose is (C6H120s) n,
where n is the degree of polymerization (DP) or the number of repeating glucose
monomer per cellulose chain with the average between 700 to 10000. DP of cellulose
is different depending on the source and the process that cellulose gone through
(Entcheva et a!., 2004). Cellulose applications are depending on DP value own by
cellulose. Table 2.4 showed the different characterization of cellulose from source,
molecular weight, and degree of polymerization.
Table 2.4: Molecular weight and degree of polymerization for cellulose from different source
Source Molecular weight DP
Pure Cellulose 600 000- 1 500 000 3 500- 10 000
Cotton 80 000 - 500 000 500-3 000
Wood Pulp 80 000 - 340 000 500-2 100
Filaments rayon 57 000 - 73 000 350-450
Source: Nissan et al., 1965
Cellulose is a linear polymer with repeating units consisting of d-glucose in
4C1 conformation (Figure. 2.8). It forms the structural framework of plants and was
isolated in the form of micro fibrils. Hydrogen bonds either inter and intra (Figure.
2.9), made cellulose molecule interact with one and other (Entcheva et al., 2004;
Barbosa et al., 2005; Granja et al., 2005; Barud et al., 2007).
23
The intra-molecule hydrogen bond, limit the movement of glucopyranose unit ·
which is around acetyl bond (glycoside). However (1 ~4)-J3 bond need 180° rotation
for single glucose unit for fulfil the J3 configuration which attached to hemiacetyl
bond. Therefore cellulose molecule gives a stiff and rigid linear chain like rod
(Nissan et a/., 1965).
Cellulose
(13-1,4 linkages)
Figure 2.8: The cellulose molecule- (C6 Hw Os) n configuration.
H
0
0 ••••••••·H~ • • •
t. ········•~'"''~ • • • H
. .0
H H
H
Figure 2.9: Cellulose chain (chair comformation ). Dash line connecting between cellulose ring showed the intra and inter molecular bond.
24