ULTRASOUND MODIFICATION OF CELLULOSE AND
CURDLAN
WONG SHEN SIUNG
NATIONAL UNIVERSITY OF SINGAPORE
2011
ULTRASOUND MODIFICATION OF CELLULOSE AND
CURDLAN
WONG SHEN SIUNG
(B. Tech. (Hons.), USM)
A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF CHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2011 ACKNOWLEDGEMENTS
I would like to express my gratitude to my supervisor, Professor Stefan
Kasapis, and Associate Professor Huang Dejian for their valuable guidance, encouragement and support.
The help and cooperation of all fellow friends and laboratory colleagues have been truly appreciated. Especially, I would like to thank Ms Mabelyn Tan, Ms Sun
Yuting and Ms June Ngiam for their contributions in various experiments. The generous assistance of the following laboratory staff members is also much appreciated: Ms Lee Chooi Lan, Ms Lew Huey Lee and Mr Abdul Rahaman from the
FST lab; Mdm Francisca Lim Guek Choo from the chromatography lab; Ms Toh Soh
Lian and Ms Sanny Tan Lay San from the applied chemistry lab; Mdm Leng Lee Eng and Ms Zing Tan Tsze Yin from the elemental analysis lab; and Mdm Han Yanhui from the NMR lab. Not forgetting a note of gratitude to Mr Michael Riches for help in the grammatical department.
The support and encouragement from my parents and family members are priceless. A special thanks to Mr Gus Teoh for being a supportive and helpful friend.
Finally, I would like to thank NUS for the award of research scholarship to make my
PhD study a dream come true in NUS.
Wong Shen Siung
January 2011
i TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS i
TABLE OF CONTENTS ii
SUMMARY vi
LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS xi
CHAPTER 1 INTRODUCTION 1
1.1 Background and Rationale 2
1.2 Objectives and Scope of the Thesis 5
1.3 References 7
CHAPTER 2 LITERATURE REVIEW 11
2.1 Ultrasonication 12
2.1.1 Introduction and definition 12
2.1.2 Principles of ultrasound depolymerisation 13
2.1.3 Application of ultrasonication 17
2.2 Ultrasonication of Polymer 19
2.3 Ultrasonication of Cellulose 22
2.3.1 Fundamental structure and physicochemical properties of 22
ii cellulose
2.3.2 Ultrasonication of cellulose ether 26
2.3.3 Ultrasonication of cellulose ester 28
2.3.4 Ultrasonication of native cellulose 29
2.4 Ultrasonication of Beta Glucan 31
2.4.1 Fundamental structure and physicochemical properties of β- 31
glucan from microbial origin, curdlan
2.4.2 Ultrasonication of liner and branch microbial β-D-glucan 34
2.4.3 Fundamental structure and physicochemical properties of 38
chitin and chitosan
2.4.4 Ultrasonication of chitin and chitosan 40
2.5 Sulfation of Beta Glucan 46
2.6 References 48
CHAPTER 3 ULTRASONICATION OF BACTERIAL AND PLANT 65
CELLULOSE
3.1 Introduction 66
3.2 Experimental 68
3.2.1 Materials 68
3.2.2 Size exclusion chromatography (SEC) 70
3.2.3 X-ray powder diffraction studies (XRPD) 71
3.2.4 Thermogravimetric analysis (TGA) 72
3.2.5 Fourier transformed infrared (FTIR) spectroscopic analysis 72
3.2.6 Transmission electron microscopy (TEM) 73
3.3 Results and Discussion 74
iii 3.3.1 Effect of ultrasound sonication on the weight average 74
molecular weight and polydispersity index
3.3.2 X-ray diffraction profile and crystallinity index (CrI) 79
3.3.3 Thermal degradation of cellulosic materials 82
3.3.4 Fourier transform infrared analysis (FTIR) 86
3.3.5 Transmission electron microscopy (TEM) 89
3.4 Conclusion 91
3.5 References 91
CHAPTER 4 MOLECULAR WEIGHT AND CRYSTALLINITY 96
ALTERATION OF CELLULOSE VIA PROLONGED ULTRASOUND
FRAGMENTATION
4.1 Introduction 97
4.2 Experimental 100
4.2.1 Materials 100
4.2.2 Size exclusion chromatography (SEC) 101
4.2.3 Calculation of rate coefficient 101
4.2.4 X-ray powder diffraction studies (XRPD) 102
4.2.5 Fourier transformed infrared (FTIR) spectroscopic analysis 103
4.2.6 Statistical analysis 103
4.3 Results and Discussion 103
4.3.1 Molecular weight profile of ultrasonicated cellulose 103
4.3.2 Kinetics of cellulose degradation via ultrasonication 107
4.3.3 X-ray diffraction profile and crystallinity index (CrI) 110
4.3.4 Fourier Transform Infrared Analysis (FTIR) 113
iv 4.4 Conclusion 116
4.5 References 116
CHAPTER 5 NOVEL SULFATION OF CURDLAN ASSISTED BY 120
ULTRASONICATION
5.1 Introduction 121
5.2 Experimental 123
5.2.1 Materials 123
5.2.2 Ultrasonication of curdlan 123
5.2.2.1 Aqueous system 123
5.2.2.2 Organic solvent system 123
5.2.3 Elemental analysis of curdlan 124
5.2.4 Fourier transformed infrared (FTIR) spectroscopic analysis 125
5.2.5 Nuclear magnetic resonance (NMR) spectroscopy 125
5.2.6 Size exclusion chromatography (SEC) 125
5.2.7 Statistical Analysis 126
5.3 Results and Discussion 126
5.3.1 Sulfur content and degree of sulfation (DS) 126
5.3.2 Fourier transform infrared (FTIR) spectroscopy 129
5.3.3 NMR spectroscopy 130
5.3.4 Size exclusion chromatography (SEC) 136
5.4 Conclusion 137
5.5 References 138
CHAPTER 6 SUMMARY AND CONCLUSIONS 143
APPENDIX: LIST OF PUBLICATIONS AND PRESENTATIONS 148
v ULTRASOUND MODIFICATION OF CELLULOSE AND CURDLAN
SUMMARY
Current study aims to elucidate the effectiveness of ultrasound in the
modification of polysaccharides, namely cellulose and curdlan. It is anticipated that
better understanding will be gained on the effect of ultrasound beyond the
conventional concept of polymeric chain fragmentation. The first part of the thesis
outlined the physicochemical characterisation of the crystalline cellulose produced via ultrasonication of native bacterial cellulose (BC) and plant cellulose (PC) in a cuprammonium hydroxide solution (CUAM). Ultrasound induced depolymerisation, causing a reduction in molecular weight (MW) as shown by size exclusion chromatography study, yet the molecular structure of sonicated BC and PC were unaltered; they remained as (14)--D-glucan. It was interesting to found that ultrasonication promotes the increment of the crystallinity index (CrI) of sonicated
BC in a relatively short period of 30 minutes. Highly crystalline cellulose is desired since there is always a demand for precision engineered cellulosic materials to incorporate in bio-nanocomposites. The subsequent chapter described the same ultrasonication process of BC and PC with prolonged ultrasonication time (up to 90 minutes), with particular focus on elucidating the MW and CrI change of the products.
Limiting molecular weight (Mlim) of BC and PC was achieved after 60 minutes of
ultrasonication. Ultrasonication significantly (p < 0.05) increased the CrI of both BC
and PC. The increment stops at samples (BC and PC) sonicated for 60 minutes. This
corresponds with the Mlim values of both samples, confirming the correlation of CrI to the MW of these materials. The final chapter of the thesis describes an alternative, non-conventional method to prepare curdlan sulfate assisted by ultrasonication, based
vi on green chemistry considerations. Curdlan was suspended in 50% (v/v) sulfuric acid at 40°C with or without sonication. Ultrasonication has significantly (p < 0.05) increased the degree of sulfation (DS) value of sonicated samples by almost fourfold; at the same time, it achieved MW reduction by one order of magnitude. This is due to the combined effect of ultrasonication and sulfuric acid treatment, which successfully produced curdlan sulfate with low MW (~49 kDa) that is of application interest. As final concluding remarks, the current study has demonstrated the ability of ultrasound in modification of cellulose and curdlan. In addition, present projects have also confirmed the ability of utilising ultrasound beyond conventional depolymerisation experiments. It is believed that these results will provide a new insight into polysaccharides modifications and eventually expand the methodologies of polysaccharides modifications.
vii LIST OF TABLES
Tables Title Page
2.1 Examples of ultrasound application in various industries. Adapted 18 from Mason and Lorimer (2002).
2.2 Various (13)--D-glucans of microbial origin, having (13)-- 32 D-glucopyranose as the polymer backbone with different branch points. Adapted from Lee (2002) and Soltanian et al. (2009), with modifications.
3.1 Crystallinity index (CrI) of bacterial cellulose (BC) and plant 82 cellulose (PC) ultrasonicated for different times.
3.2 Peak onset temperature, Te (°C) and peak maximum temperature, 86 Tmax (°C) of bacterial cellulose (BC) and plant cellulose (PC) ultrasonicated at different times.
4.1 Crystallinity index (CrI) of bacterial cellulose (BC) and plant 112 cellulose (PC) ultrasonicated for different times.
5.1 Sulfur content, degree of sulfation (DS) and yield of curdlan 128 sulfates.
5.2 Characteristic chemical shifts assignments (ppm), and integration 133 intensity of 1HNMR signals for curdlan samples.
5.3 Chemical shifts (ppm) assignments of 13C NMR signals for curdlan 134 samples.
5.4 Molecular weight for curdlan samples. 137
viii LIST OF FIGURES
Figures Caption Page
2.1 Mechanism for ultrasound-induced polymer chain scission: (a) 16 gradual bubble formation as a result from pressure variations induced by the acoustic field; (b) rapid bubble collapse generates solvodynamic shear. Adapted from Caruso et al. (2009), with modifications.
2.2 Molecular representation of cellulose chain structure, a (14)-- 23 D-glucan. n indicates the number of repeating disaccharide units (cellobiose) in a given cellulose chain.
2.3 Classifications and examples of a few important cellulose solvents 24 have been used to date. Adapted from Heinze and Liebert (2001), with modifications.
2.4 Molecular representation of curdlan chain structure, a (13)--D- 33 glucan. n indicates the number of repeating disaccharide units (laminaribiose) in a given curdlan chain.
2.5 Molecular representation of chitin and chitosan chain structure, a 40 (14)--N-acetyl-D-glucosaminoglycan. x and y indicates the number of repeating monosaccharide units in a given chain. If x > y, the chain represents chitosan. If y > x, the chain represent chitin.
3.1 SEC elution patterns of ultrasonicated (a) BC and (b) PC in 75 DMAc/0.5% LiCl. BC-X / PC-X denotes sample sonicated for X amount of time.
3.2 Weight-average molecular weight variations of plant and bacterial 77 cellulose as a function of ultrasonication time.
3.3 Polydispersity-index variations of plant and bacterial cellulose as a 78 function of time of ultrasonication.
3.4 X-Ray diffraction pattern of (a) bacterial cellulose and (b) plant 80 cellulose sonicated at different times, and compared to those of MCC.
3.5 Thermogravimetric (TG) and derivative TG (DTG) curves of 83 ultrasonicated samples of bacterial cellulose (a, b).
ix
3.6 Thermogravimetric (TG) and derivative TG (DTG) curves of 84 ultrasonicated samples plant cellulose (a, b).
3.7 FTIR spectra of (a) bacterial cellulose and (b) plant cellulose 87 ultrasonicated at different times, and compared to those of MCC.
3.8 TEM micrographs of ultrasonicated BC and PC, and MCC 90 samples: a) BC-0; b) PC-0; c) BC-30; d) PC-30, e) MCC. Horizontal scale bar is 1 m.
4.1 Change of weight average molecular weight ( Mw ) of the celluloses 105 in cuprammonium hydroxide (CUAM) solution over the course of ultrasonication. Values are means ± standard deviation (n=3). Data points within a sample with the same alphabet are not significantly different (p > 0.05).
4.2 Variation of 1/ Mw as a function of ultrasonication time (min). 108
2 4.3 Variation of 1/ Mw as a function of ultrasonication time (min). 108
4.4 X-Ray diffraction patterns of (a) bacterial cellulose and (b) plant 111 cellulose sonicated at different time periods, as compared to MCC.
4.5 FTIR spectra of bacterial cellulose ultrasonicated at different times, 114 and compared to that of MCC.
4.6 FTIR spectra of plant cellulose ultrasonicated at different times, 115 and compared to that of MCC.
5.1 FTIR spectra of curdlan samples: Curdlan, 0_360 (acid treatment 130 only) and 360_360 (acid + ultrasonication).
5.2 300.13 MHz 1H NMR spectra of curdlan samples: Curdlan, 0_360 132 (acid treatment only) and 360_360 (acid + ultrasonication).
5.3 Sulfation of curdlan assisted by ultrasonication. Substitution 133 happened preferably at position OH-6. n indicates the number of repeating disaccharide units (laminaribiose) in a given curdlan chain.
5.4 75.48 MHz 13C NMR spectra of curdlan samples: Curdlan, 0_360 135 (acid treatment only) and 360_360 (acid + ultrasonication).
x
LIST OF ABBREVIATIONS
Abbreviation Caption
BC Bacterial cellulose
CrI Crystallinity index
CUAM Cuprammonium hydroxide
Da Dalton
DMAc N,N-dimethyl acetamide
DMSO Dimethyl sulfoxide
DP Degree of polymerization
DS Degree of sulfation
DTG Derivative thermogravimetric
FTIR Fourier transform infrared h Hour
IPA Isopropyl alcohol
LODP Level-off degree of polymerisation
M Molar
MCC Microcrystalline cellulose
Mlim Limiting molecular weight
Mw Weight average molecular weight
Mn Number average molecular weight
MW Molecular weight min Minute
N Normal
NMR Nuclear magnetic resonance
xi PC Plant cellulose
PI Polydispersity index s Second
SEC Size exclusion chromatography
TGA Thermogravimetric analysis
TG Thermogravimetric
TEM Transmission electron microscopy
xii
CHAPTER 1
INTRODUCTION
1 1.1 BACKGROUND AND RATIONALE
Polysaccharide modification has been receiving a continuous interest since mid nineteen century with the aim to tackle drawbacks related to utilizations of native unmodified polysaccharides. To date, various approaches have been taken to alter the molecular structure of native polysaccharides in order to obtain novel functionalised macromolecules with better physicochemical properties. Usually, derivatised polysaccharides (i.e. carboxymethyl guar gum, hydroxypropyl cellulose, etc.) are more readily to dissolve in water compared to their native counterparts. On the other hand, depolymerised polysaccharides with lower viscosity will be more favourable in food applications when high solid content is needed. In general, polysaccharides modification can be classified into three main modes: chemical, physical and enzymatic modifications.
Chemical modifications of polysaccharides are probably the oldest way to modify a biopolymer. Reports on acid hydrolysis or etherification of cellulose could be traced back to the late nineteen century. Lately, more sophisticated chemical derivatisation of polysaccharides has been done to specifically substitute certain hydroxyl group on the monosaccharide unit of the polysaccharides backbone with a desired functional group.
For example, regioselective substitution of the hydroxyl group of glucose in cellulose at position 2, 3 or 6 will produce derivatised cellulose with specialised functional properties.
Nevertheless, chemical modification of polysaccharides usually involves toxic chemicals, as either the precursors or by-products of the reaction which have been perceived as a less environmentally friendly process. On the other hand, enzymatic modification could possibly achieve similar or even better specificity in the final
2 modified product, in a more ecologically friendly way. However, utilization of enzyme usually increases the cost of the whole process substantially. Moreover, repeating using of enzyme will involve more tedious work to immobilize the enzyme.
Meanwhile, physical modification of polysaccharides is a relatively new method compared to the former two modes. Nowadays, advances in mechanical and electronic engineering in the manufacturing of advanced instruments to deliver various physical aids have made this option more amenable for further research. Physical aids that have been employed include gamma irradiation, electron beam irradiation, microwave irradiation, extrusion, and ultrasound irradiation (ultrasonication). There is a noticeable limitation for physical modification: the process usually can not be precisely controlled in order to obtain certain functional macromolecules with specific substitution and / or molecular structure. However, studies have shown that ultrasound irradiation seems to be a more promising method in controlling the molecular structure of the modified moiety compared to its peers. Various findings (Chen et al., 1997; Marx-Figini, 1997; Liu et al.,
2006) confirmed that ultrasonication is able to reduce the polydispersity index of the sonicated polysaccharides to form a product with more homogenous molecular weight distributions, which could not be done by other means of physical modifications.
Ultrasound is defined as sound having a frequency higher than the human hearing range, i.e. > 20 kHz (Mason and Lorimer, 2002). Ultrasound has found its application in various areas, including ultrasound imaging in the medical field, cleaning and drilling of teeth in dentistry, homogenization, cell disruption in biology, and molecular synthesis, degradation in chemistry. Literatures have shown that application of ultrasound in chemical and polymer processes could be traced back to the early 1930s (Flosdorf and
3 Chambers, 1933; Szent-Györgyi, 1933). Ultrasound delivers a form of energy for the modification of chemical reactivity which is different from that of conventionally used forces, i.e. temperature and pressure. Though ultrasound has been employed in polymer modification since decades ago, its used tends to focus on polymer degradation, usually synthetic polymer and to a lesser extent, natural polymer, such as polysaccharides.
To date, most studies on ultrasound degradation of polysaccharides have dealt with water soluble or dilute acid / base soluble; either natural or modified polysaccharides. Majority of the reported works dealt with chitosan, which is soluble in dilute acid (Chen et al., 1997; Tsaih and Chen, 2003; Trzcinski and Staszewska, 2004;
Tsaih et al., 2004; Baxter et al., 2005; Czechowska-Biskup et al., 2005; Liu et al., 2006;
Kasaai et al., 2008; Li et al., 2008; Wu et al., 2008; Yue et al., 2008), and dextran (Szu et al., 1986; Lorimer et al., 1995; Portenlanger and Heusinger, 1997; Cote and Willet, 1999).
Other reported polysaccharides including water soluble beta glucan, schizophyllan
(Tabata et al., 1981; Kulicke et al., 1993), agarose, carrageenan (Lii et al., 1999), guar gum (Tayal and Khan, 2000), xanthan (Kulicke et al., 1993), and cellulose derivatives
(e.g. hydroxyethyl cellulose (Ni et al., 2001) hydroxypropyl cellulose (Malhotra, 1982) and carboxymethyl cellulose (Gautier and Lecourtier, 1991; Gronroos et al., 2008)).
It is clear that various studies have dealt with ultrasonication of cellulose derivatives. However, to the best of my knowledge, scientific publications on ultrasonication of native, dissolved cellulose is still scarce and lacking. Even though
Striegel (2007) has reported the ultrasonication of native cellulose in N,N-dimethyl acetamide (DMAc) / 0.5% LiCl, the author only examined plant cellulose and did not further characterise the samples in solid state to evaluate the physicochemical changes on
4 cellulose after ultrasonication. Therefore, it is desirable to investigate the ultrasound degradation of dissolved plant cellulose (PC) and bacterial cellulose (BC) and to further characterise the ultrasonicated fragments.
As mentioned earlier in this section, a majority of the publications related to ultrasonication of polysaccharides are limited to the study of polymer degradations.
Nevertheless, the recent work by Cízová and his team (2008) on esterification of carboxymethyl starch with octenylsuccinic anhydride suggested that ultrasound is able to promote the esterification process. Meanwhile, though considerable amount of works have reported the synthesis of curdlan sulfate, these studies tend to utilize harmful and / or toxic precursors such as pyridine-SO3 complex and pyridine-chlorosulfonic acid, thus presenting a clear need in seeking a new environmental friendly method to synthesize curdlan sulfate. Therefore, it is reasonable to employ ultrasound as a tool to assist the synthesis of curdlan sulfate, utilizing sulfuric acid as a non-toxic precursor.
1.2 OBJECTIVES AND SCOPE OF THE THESIS
The aim of this project was to elucidate the effectiveness of ultrasound in modification of polysaccharides, namely cellulose and curdlan. It is hypothesised that ultrasonication able to reduce the molecular weight of native cellulose and curdlan; in addition, to promote sulfation of curdlan. The specific objectives were i. To perform physicochemical characterization on the crystalline cellulose produced
via ultrasonication of native bacterial and plant cellulose;
5 ii. To evaluate the effect of prolonged ultrasonication on the molecular weight and
crystallinity of ultrasonicated bacterial and plant cellulose samples and to compute
the degradation kinetics; iii. To develop an alternative method in preparing curdlan sulfate assisted by
ultrasonication
Ultrasonication has been perceived as a promising alternative to the chemical modification in the context of green chemistry. The present work provides a viable option to depolymerise crystalline celluloses (PC and BC) via ultrasonication. In addition, the current project has demonstrated that ultrasonication is capable of assisting esterification of curdlan which is beyond conventional application of ultrasound on polymer degradation. What is of particular interest is the combination of ultrasonication and sulfuric acid in synthesis of curdlan sulfate, which appears to be the first reported in the literature. This has opened up a new avenue for polysaccharides modifications and could shed new light on the effort of seeking alternatives to conventional chemical synthesis.
In this thesis, two polysaccharides, namely cellulose (of plant and bacterial origin) and curdlan were the subjects of study. Chapter 3 reports the ultrasonication process of the plant cellulose (PC) and bacterial cellulose (BC) followed by physicochemical characterization of the ultrasonicated PC and BC. Chapter 4 describes the same ultrasonication process of PC and BC with prolonged ultrasonication time (up to 90 min), with particular focus on elucidating the crystallinity and molecular weight change of the products. In addition, from the molecular profiles obtained, kinetics of the ultrasound degradation will be computed. Finally, Chapter 5 reports the sulfation of curdlan assists by ultrasonication, followed by a brief structural elucidation studies.
6
1.3 REFERENCES
Baxter, S., Zivanovic, S. and Weiss, J. (2005). Molecular weight and degree of
acetylation of high-intensity ultrasonicated chitosan. Food Hydrocolloids, 19(5),
821-830.
Chen, R. H., Chang, J. R. and Shyur, J. S. (1997). Effects of ultrasonic conditions and
storage in acidic solutions on changes in molecular weight and polydispersity of
treated chitosan. Carbohydrate Research, 299(4), 287-294.
Cízová, A., Sroková, I., Sasinková, V., Malovíková, A. and Ebringerová, A. (2008).
Carboxymethyl Starch Octenylsuccinate: Microwave- and Ultrasound-assisted
Synthesis and Properties. Starch - Stärke, 60(8), 389-397.
Cote, G. L. and Willet, J. L. (1999). Thermomechanical depolymerization of dextran.
Carbohydrate Polymers, 39(2), 119-126.
Czechowska-Biskup, R., Rokita, B., Lotfy, S., Ulanski, P. and Rosiak, J. M. (2005).
Degradation of chitosan and starch by 360-kHz ultrasound. Carbohydrate
Polymers, 60(2), 175-184.
Flosdorf, E. W. and Chambers, L. A. (1933). The Chemical Action of Audible Sound
Journal of the American Chemical Society, 55(7), 3051-3052.
Gautier, S. and Lecourtier, J. (1991). Structural characterization by 13C nuclear magnetic
resonance of hydrolysed carboxymethylcellulose. Polymer Bulletin, 26(4), 457-
464.
7 Gronroos, A., Pirkonen, P. and Kyllonen, H. (2008). Ultrasonic degradation of aqueous
carboxymethylcellulose: Effect of viscosity, molecular mass, and concentration.
Ultrasonics Sonochemistry, 15(4), 644-648.
Kasaai, M. R., Arul, J. and Charlet, G. (2008). Fragmentation of chitosan by ultrasonic
irradiation. Ultrasonics Sonochemistry, 15(6), 1001-1008.
Kulicke, W.-M., Otto, M. and Baar, A. (1993). Improved NMR characterization of high-
molecular-weight polymers and polyelectrolytes through the use of preliminary
ultrasonic degradation. Die Makromolekulare Chemie, 194(3), 751-765.
Li, J., Cai, J. and Fan, L. (2008). Effect of sonolysis on kinetics and physicochemical
properties of treated chitosan. Journal of Applied Polymer Science, 109(4), 2417-
2425.
Lii, C.-y., Chen, C.-H., Yeh, A.-I. and Lai, V. M. F. (1999). Preliminary study on the
degradation kinetics of agarose and carrageenans by ultrasound. Food
Hydrocolloids, 13(6), 477-481.
Liu, H., Bao, J., Du, Y., Zhou, X. and Kennedy, J. F. (2006). Effect of ultrasonic
treatment on the biochemphysical properties of chitosan. Carbohydrate Polymers,
64(4), 553-559.
Lorimer, J. P., Mason, T. J., Cuthbert, T. C. and Brookfield, E. A. (1995). Effect of
ultrasound on the degradation of aqueous native dextran. Ultrasonics
Sonochemistry, 2(1), S55 - S57.
Malhotra, S. L. (1982). Ultrasonic Degradation of Hydroxypropyl Cellulose Solutions in
Water, Ethanol, and Tetrahydrofuran. Journal of Macromolecular Science-
Chemistry, A17(4), 601-636.
8 Marx-Figini, M. (1997). Studies on the ultrasonic degradation of cellulose
macromolecular properties. Angewandte Makromolekulare Chemie, 250(1), 85-92.
Mason, T. J. and Lorimer, J. P. (2002). Applied Sonochemistry: The uses of power
ultrasound in chemistry and processing Wiley-VCH Weinheim, Germany
Ni, X., Hu, Y., Liu, B. and Xu, X. (2001). Mechanical degradation and mechanochemical
copolymerization of hydroxyethyl cellulose. European Polymer Journal, 37(1),
201-206.
Portenlanger, G. and Heusinger, H. (1997). The influence of frequency on the mechanical
and radical effects for the ultrasonic degradation of dextranes. Ultrasonics
Sonochemistry, 4(2), 127-130.
Striegel, A. M. (2007). Influence of Anomeric Configuration on Mechanochemical
Degradation of Polysaccharides: Cellulose versus Amylose. Biomacromolecules,
8(12), 3944-3949.
Szent-Györgyi, A. (1933). Chemical and biological effects of ultra-sonic radiation.
Nature, 131(3304), 278.
Szu, S. C., Zon, G., Schneerson, R. and Robbins, J. B. (1986). Ultrasonic irradiation of
bacterial polysaccharides. Characterization of the depolymerized products and
some applications of the process. Carbohydrate Research, 152, 7-20.
Tabata, K., Ito, W., Kojima, T., Kawabata, S. and Misaki, A. (1981). Ultrasonic
degradation of schizophyllan, an antitumor polysaccharide produced by
Schizophyllum commune fries. Carbohydrate Research, 89(1), 121-135.
9 Tayal, A. and Khan, S. A. (2000). Degradation of a Water-Soluble Polymer: Molecular
Weight Changes and Chain Scission Characteristics. Macromolecules, 33(26),
9488-9493.
Trzcinski, S. and Staszewska, D. U. (2004). Kinetics of ultrasonic degradation and
polymerisation degree distribution of sonochemically degraded chitosans.
Carbohydrate Polymers, 56(4), 489-498.
Tsaih, M. L. and Chen, R. H. (2003). Effect of degree of deacetylation of chitosan on the
kinetics of ultrasonic degradation of chitosan. Journal of Applied Polymer Science,
90(13), 3526-3531.
Tsaih, M. L., Tseng, L. Z. and Chen, R. H. (2004). Effects of removing small fragments
with ultrafiltration treatment and ultrasonic conditions on the degradation kinetics
of chitosan. Polymer Degradation and Stability, 86(1), 25-32.
Wu, T., Zivanovic, S., Hayes, D. G. and Weiss, J. (2008). Efficient Reduction of
Chitosan Molecular Weight by High-Intensity Ultrasound: Underlying
Mechanism and Effect of Process Parameters. Journal of Agricultural and Food
Chemistry, 56(13), 5112-5119.
Yue, W., Yao, P., Wei, Y. and Mo, H. (2008). Synergetic effect of ozone and ultrasonic
radiation on degradation of chitosan. Polymer Degradation and Stability, 93(10),
1814-1821.
10
CHAPTER 2
LITERATURE REVIEW
11
2.1 ULTRASONICATION
2.1.1 Introduction and Definition of Ultrasound
Frequencies of sound are recorded in units of Hertz (1 Hertz = 1 cycle per second). The hearing range of a human being is from about 20 Hz to 20 kHz; the upper limit reduces with age. By reference to this, ultrasound can be defined as sound waves having a frequency beyond the human hearing range, i.e. > 20 kHz.
Application of ultrasound could be divided into two main areas based on the frequency range. First, low amplitude (higher frequency) sound, also known as “high frequency ultrasound”, usually operates in the range of 2 to 10 MHz and is normally employed in medical imaging. Secondly, high energy (low frequency) sound, also known as “power ultrasound”, lies between the range of 20 to 100 kHz and is widely used for cleaning, plastic welding and sonochemistry (Mason and Lorimer, 2002).
Although the field of ultrasound was established in the late 1800s – when
Curie discovered the effect of piezoelectric energy in 1880, and Galton subsequently invented the ultrasonic whistle in 1893 – the first commercial application of ultrasound did not appear until the late 1910s (Lorimer and Mason, 1987).
Piezoelectric material was first used to construct ultrasound transducers (devices that convert electrical energy into a mechanical signal); today, the majority of modern ultrasonic devices rely on transducers (Mason and Lorimer, 2002). Therefore, the discovery of the effect of piezoelectric energy marked the inauguration of the ultrasound era. Subsequently, the investigations of Richards and Loomis (1927) demonstrated the effect of ultrasound on chemical compounds upon being exposed to acoustic waves.
12
Research on ultrasonication have never stopped since then. The availability of more publications on ultrasound and acoustic waves enables subsequent researchers to gain further understanding on the utilisation and control of ultrasound. Moreover, the advancement in electronic circuitry and transducer design resulted in the rapid expansion of the application of power ultrasound to chemical reactions and processes, a subject that has become known as “sonochemistry”. Recently, Mason and Lorimer
(2002) have covered broadly and in-depth on various aspects related to the field of ultrasonication. Meanwhile, Caruso and colleagues (2009) have summarised and reinstated the relatively new concept of the “mechano-chemical nature of ultrasonication”, particularly explaining this effect on macromolecules degradation.
The general principles and discussions of mechano-chemical activation of ultrasound will be covered in the following sections.
2.1.2 Principles of Ultrasound Depolymerisation
Being a sound wave, ultrasound can be transmitted through any medium or substances with elastic properties such as solids, liquids or gases. However, since the acoustic energy cannot be absorbed by molecules, it must be transformed to a chemically usable form via a complex phenomenon known as cavitation (Kardos and
Luche, 2001). It has been generally accepted that, when liquid is irradiated with ultrasound, very small bubbles (with diameter of a few hundred µm) will be generated in the liquid. These bubbles will oscillate according to the acoustic pressure: they grow during the expansion phase (rarefaction) and shrink slightly during the compression phase of sound waves. Eventually these bubbles will undergo a violent collapse that generates pressure of hundreds of atmosphere, and a temperature of a few thousand Kelvin. This transient process is call cavitation and happens within a
13
few hundreds of µsec (Figure 2.1a). The “Hot-spot Theory” was constructed based on
this transient cavitation and the temperature pressure fluctuation (Mason and Lorimer,
2002).
Some earlier literature assumed that the effect of ultrasound depolymerisation is a result of cavitation-induced transient temperature and pressure fluctuation.
Nevertheless, recent investigations lead to the conclusion that ultrasound imposes a combination of mechanical and chemical effects, which give rise to the term
“mechano-chemical”. To further explain this, it is important to understand the concept of limiting molecular weight (Mlim). Limiting molecular weight (Mlim), or level-off
degree of polymerisation (LODP), could be defined as the Mw which beyond it no
further degradation will happen. According to Caruso et al. (2009), the presence of
limiting molecular weight (Mlim) during the course of ultrasound degradation of
polymer is strong evidence of the mechano-chemical degradation. This is because, if
cleavage is a result of temperature and / or pressure fluctuation, this fluctuation will
still be present as long as ultrasound is prolonged, and the degradation will not be
stopped. Therefore, limiting molecular weight cannot be achieved. As a result,
mechano-chemical degradation is in favour with the Hot-spot Theory. A clear
example with detailed discussion is available in section 4.3.1.
The term “mechano-chemical” and accompanying explanations have been
available in the literature from as early as 1977 (Basedow and Ebert, 1977).
Nevertheless, even the findings were acceptable at that time, many debates were
ongoing. Only until recently Striegel (2003) had re-visit the term “mechano-
chemical” by providing an in-depth discussion on the mechano-chemical effect of
ultrasound depolymerisation by using long chain branching polystyrene as an
example. Subsequently, most of the recent findings on ultrasonication agreed on the
14
mechano-chemical effect of degradation (Czechowska-Biskup et al., 2005; Striegel,
2007).
Although there is still some debate about the precise mechanism of mechanical activation, it is generally accepted that the chain scission of polymers is the result of solvodynamic shear created by cavitation, which involves a three-step process: the nucleation, growth, and collapse of micro bubbles in solution. In solution, polymer chain or segments present in the high-gradient shear filed near the collapsing bubble move at a higher velocity than those segments further away from the collapsing cavity. This velocity gradient causes the polymer chain to become elongated, and tension develops along the polymer backbone, which finally leads to chain scission, a result of mechanical effect (Figure 2.1b). At the same time, when the polymer chain is stretched, thermal fluctuation occurs as a result of transient cavitation. When this thermal fluctuation overcomes the required energy barrier that covalently links two atoms together, bond scission happens; the strain energy is converted into chemical energy, resulting in the chemical effect of ultrasound degradation (Caruso et al., 2009).
Degradation of polymer during the course of ultrasonication is believed to be a non-random process that mainly occurs near the mid-point of the polymer backbone
(within the middle 15% of the chain). Therefore, it should be safely confirmed that the chain scission process is not a thermal process, since thermal induced degradation should happen randomly (Caruso et al., 2009). However, whether the degradation happens at mid-point or randomly occurs along the polymer backbone is still debatable, and controversial results are sometimes reported (Tayal and Khan, 2000;
Wu et al., 2008). It has been proven that high intensity ultrasonication will induce the homolytic degradation of polymer that formed macro-radical (Czechowska-Biskup et
15
al., 2005), which could contribute to the random degradation of polymer. Since Wu et al. (2008) conducted their work using a high-intensity ultrasonicator, it could be postulated that this contributes to the random degradation that they reported .
(a) Time
Increase Sound wave Decrease
Pressure wave
Bubble size
Figure 2.1 Mechanism for ultrasound-induced polymer chain scission: (a) gradual bubble formation as a result from pressure variations induced by the acoustic field; (b) rapid bubble collapse generates solvodynamic shear. Adapted from Caruso et al. (2009), with modifications.
All in all, most of the recent research findings agree that ultrasound
depolymerisation is a non-random process, and chain scission will only occur at a
specific location along the polymer backbone. This location, however, might not be
16
near the mid-point of the polymer backbone. Striegel (2007) had proposed a “revised
path theory” for ultrasound degradation according to the experimental results
obtained. According to this theory, in order for ultrasound degradation of a polymer to happen, a minimum continuous “path length” within the polymer backbone is needed.
Moreover, this path must have sufficient chain flexibility and conformational
freedom. With this theory, it can be deduced that, if ultrasound degradation is random,
it should not be restricted by the minimum path length that is present on the polymer
backbone.
2.1.3 Application of Ultrasonication
To date, ultrasound has been widely use in the industry, as summarised in
Table 2.1. Lately, there is an increased in the utilisation of ultrasound in chemistry – a
subject which is known as “sonochemistry”. Actually, the application of ultrasound in chemistry, particularly in polymer degradation, has been reported in the early 1930s
(which will be discussed in the subsequent section). Nevertheless, it was only until
1960s when sonochemistry has become more popular, due to the more commonly
available ultrasound cleaning bath. As a result, ultrasound expanded its application in chemistry to other area besides polymer chemistry. Sonochemistry provokes the interest of chemist owing to the fact that ultrasound provides a form of energy via cavitation of bubbles, for the modification of chemical reactivity, which is different from the normally used, i.e. heat, light and pressure (Mason and Lorimer, 2002).
Ultrasound has been perceived as a promising alternative to conventional
chemical methods in chemical synthesis, either to create an alternative synthetic route
or useful to aid in synthesis process. For example, Gronroos et al. (2004a) had
reported the acceleration of bile acid esterification via ultrasound, with a 19-fold
17
increase in reaction rate. As an unconventional method in chemical synthesis, it could
be useful in substituting conventional chemical reagent or catalyst, which results in
toxic waste reduction. This is in connection with the emerging concept of “Green
Chemistry” to achieve the objectives of designing non-polluting products and processes (Kardos and Luche, 2001).
Table 2.1 Examples of ultrasound application in various industries. Adapted from Mason and Lorimer (2002).
Field Application Biology, Cell disruption and homogenisation: Power ultrasound is used to Biochemistry rupture cell walls in order to release contents for further study Engineering Ultrasound has been used to assist drilling, grinding and cutting. It is particularly useful Dentistry Cleaning and drilling of teeth, also for curing glass ionomer fillings Geography, Pulse / echo techniques are used in the location of mineral and Geology oil deposit and in depth gauges for seas and oceans. Echo ranging at sea has been used for many years (SONAR) Medicine Ultrasonic imaging (2 – 10 MHz) is used, particularly in obstetrics, for observing the foetus and for guiding subcutaneous surgical implements. In physiotherapy lower frequencies (20 – 50 kHz) are used in the treatment of muscle strains, dissolution of blood clots and cancer treatment General industrial Pigments and solids can be easily dispersed in paint, inks and resins. Engineering articles are often cleaned and degreased by immersion in ultrasonic baths. Two less widely used applications are in acoustic filtration and metal casting.
Other application of ultrasound in chemistry including (Mason and Lorimer,
2002):
1. Reactions involving metal or solid surfaces
2. Reactions involving powders or other particulate matter
3. Generating fine emulsion via homogenisation
4. Homogeneous and heterogeneous reactions in chemical synthesis
18
2.2 ULTRASONICATION OF POLYMER
Reports on ultrasonication of polymer can be traced back to the early 1930s.
Szent-Györgyi (1933) had mentioned in his short report about the irreversible reduction of viscosity in the ultrasound irradiated starch, gum arabic and gelatine, indicating depolymerisation occurred. At the same time, Flosdorf and Chambers
(1933) also found that when agar and starch solution being irradiated with ultrasound, dextrin and reducing sugars were produced. First two reports available in the literature are related to natural / bio-polymer; however, subsequent studies on ultrasonication in the 1950s and 1960s were concentrated more on synthetic polymer, majority using polystyrene (Weissler, 1950; Smith and Temple, 1968), polyethylene (Bernhardt,
1954), and polymethyl methacrylate (Melville and Murray, 1950) as the main precursor. The obvious advantage of utilising synthetic polymer in ultrasonication studies would be the more precise control on molecular structure of the initial sample.
This is due to the feasibility of obtaining synthetic polymer with more define molecular structure, such as more uniform molecular distribution with more precise molecular weight.
Earlier studies on ultrasonication of polymer were concerned on understanding how ultrasound could reduce molecular weight and viscosity of polymer solutions.
Mostly, researchers tried to confirm the depolymerisation was caused by cavitations, and trying to understand the mechanism underlying the cavitations and / or the cavitation induced depolymerisation (Melville and Murray, 1950; Weissler, 1950;
Borgstedt, 1964). Subsequently, more studies are related to understanding the factors that affecting the ultrasonication, i.e. ultrasound frequency (Mostafa, 1958), type of
19
solvent (Basedow et al., 1978), temperature (Malhotra, 1982), concentration of polymer (Jellinek and White, 1951), initial molecular weight (Nguyen et al., 1997), intensity of ultrasound and external pressure (Price and Smith, 1993). At the same time, researchers started to elucidate the effect of ultrasonication on the physicochemical properties of sonicated polymers, namely molecular weight (MW), polydispersity index (PI) / molecular weight distribution (MWD) (Nguyen and
Kausch, 1998), viscosity, etc (Glynn et al., 1972). Meanwhile, Basedow and Ebert
(1977) have prepared an extensive report on general aspect of ultrasonic degradation of polymers in solution, from the approach of elementary physical chemistry. They discussed the effects of high intensity ultrasonication in liquids and reviewed a few important parameters that affect ultrasonication process, such as intensity and frequency of ultrasound. They also reviewed the kinetic analysis of ultrasound degradation reaction and the degradation models that have been proposed previously.
Most importantly, they discussed the concept of mechanochemical nature of ultrasound depolymerisation.
To date, numerous reports related to ultrasonication of natural and synthetic polymers have been well documented. These include, but not limited to, water soluble or aqueous preparation of polysaccharides / hydrocolloids (Milas et al., 1986; Szu et al., 1986; Lorimer et al., 1995; Portenlanger and Heusinger, 1997; Cote and Willet,
1999; Lii et al., 1999; Tayal and Khan, 2000; Miyazaki et al., 2001; Vodeničarová et al., 2006; Zhou and Ma, 2006), starches (Czechowska-Biskup et al., 2005; Cízová et al., 2008; Luo et al., 2008; Zuo et al., 2009), peptides (Sakakura and Takayama,
2009), proteins (Wang et al., 2009), deoxyribonucleic acid (DNA) (Fukudome et al.,
1986a; Fukudome et al., 1986b); as well as various synthetic polymers including polystyrene (Price and Smith, 1991; Price et al., 1994; Nguyen et al., 1997), polyvinyl
20
acohol (Gronroos et al., 2001), poly(vinyl acetate) (Madras and Chattopadhyay,
2001), polypropylene (Desai et al., 2008), poly(ethylene oxide) (PEO) (Vijayalakshmi
and Madras, 2005), poly(D,L-lactide) (PLA) and poly(D,L-lactide-coglycolide)
(PLGA) (Reich, 1998). Since the present project only focus on cellulose and curdlan
(microbial -glucan), other natural and synthetic polymers shall not be covered in the
subsequent sections.
After the extensive researches have been done throughout the years, it is
generally agreed that ultrasound will induce the following physicochemical changes
of the resulting sonicated fragments:
1. Depolymerisation, causing MW reduction
2. Reduction of polydispersity index (PI), or the narrowing of molecular
weight distribution (MWD)
3. Obtaining limiting molecular weight (Mlim), or level-off degree of
polymerisation (LODP)
4. Viscosity reduction, as a result of MW reduction
As mentioned early, factors that affecting the ultrasonication include initial molecular weight of the polymer, concentration of polymer, type of solvent, external pressure, reaction temperature, intensity and frequency of ultrasound. More explanations on relevant factors, using cellulose and its derivatives and several β- glucans will be covered in the following sections.
21
2.3 ULTRASONICATION OF CELLULOSE
2.3.1 Fundamental Structure and Physicochemical Properties of Cellulose
Cellulose is the most abundant biopolymer on earth, recognised as the major component of plant biomass. Cellulose is obtained via photosynthesis in plants, present as a natural bio-composite together with hemicelluloses and lignin in plant cell walls. A rather pure form of cellulose could also be obtained from cotton linter
(seed hair of the cotton plant). Apart from plants, certain bacteria, algae and fungi are also able to synthesise cellulose. The bacterial cellulose (BC) synthesised by
Gluconacetobacter xylinus (acetic acid bacteria) is identical to that made by plants, in respect to molecular structure, both being 14-β-D-glucan (Heinze, 2005). BC has an advantage over plant cellulose for being a faster renewable cellulose resource; in addition to being present in a nearly pure form without other associated biogenic compounds, such as lignin, pectin and hemicelluloses, which are usually present in plant cell walls.
Figure 2.2 shows the molecular structure of cellulose as a carbohydrate polymer generated from repeating β-D-glucopyranose molecules that are covalently linked through acetal functions between the equatorial OH group of C-1 and the C-4 carbon atom (14-β-D-glucan), which is, in principle, the manner in which cellulose is biogenetically formed. As a result, cellulose is an extensive, linear-chain homopolymer of β-D-glucopyranose with a large number of hydroxyl groups (three per anhydroglucose (AGU) unit, at C-2, C-3 and C-6) present in the
4 thermodynamically preferred C1 chair conformation (Pérez and Samain, 2010).
Hydroxyl groups are positioned in the ring plane (equatorial) while the hydrogen
atoms are in the vertical position (axial). To accommodate the preferred bond angles
22
of the acetal oxygen bridges, every second AGU ring is rotated 180 in the plane. In
this manner, two adjacent structural units define the disaccharide cellobiose (Klemm
et al., 2005). The 14-β diequatorial configuration results in a rigid and linear
structure for cellulose. The presence of abundance hydroxyl groups facilitate the
forming of intra- and intermolecular hydrogen bonds, resulting in the formation of
linear aggregates of cellulose polymer. This contributes to the strength shown by
cellulose-containing structures in plants and also to the virtual insolubility of cellulose
in water and common organic solvents (i.e. hexane, methanol and dimethyl
sulfoxide). Though, it swells in many polar protic and aprotic liquids.
CH OH CH OH 2 O OH 2 O OH HO HO O HO OH HO O HO O O O OH CH OH OH CH OH 2 n 2
Figure 2.2 Molecular representation of cellulose chain structure, a (14)--D- glucan. n indicates the number of repeating disaccharide units (cellobiose) in a given cellulose chain.
Nevertheless, to date, many research works have led to the discoveries of
various cellulose solvents. Figure 2.3 shows the classifications and examples of a few
important cellulose solvents that have been used to date. The use of specific cellulose
solvents, which disrupts the extensive hydrogen bonds and thus dissolves the cellulose
polymer chain, enables the analytical study of cellulose in dissolve condition, as well
as to perform a homogeneous reaction. In this context, a solution of LiCl in N,N-
dimethyl acetamide (DMAc) (usually DMAc / 8% LiCl) is one of the most important
solvent systems for cellulose in organic syntheses as well as for analytical purposes
(Dupont, 2003). Lately, more cellulose solvents have been proposed, such as dimethyl
23
sulfoxide / ammonium fluorides (Köhler and Heinze, 2007), LiOH / urea, NaOH/ urea
(Cai and Zhang, 2005); and the research is still ongoing.
Figure 2.3 Classifications and examples of a few important cellulose solvents have been used to date. Adapted from Heinze and Liebert (2001), with modifications.
Cellulose dissolved in cuprammonium hydroxide solution (CUAM) has been used in the textile industries since early 1850 as a coating reagent on cotton cloth
(Kamide and Nishiyama, 2001). Therefore, it is relatively easier to recover or regenerate cellulose from CUAM or other aqueous medium, such as 10% NaOH, especially when a large quantity of cellulose is involved. However, the use of CUAM as cellulose reaction medium is relatively limited. This might due to the complex nature of the solvent with a few compounds (i.e. copper and ammonium ion) present
24
together, which have the tendency to become inhomogeneous during a homogeneous
reaction of cellulose (Heinze, 2005).
As a linear polysaccharide with enormous free hydroxyl groups present,
cellulose chain formed various intra- and inter-molecular hydrogen bonding, and
consequently gives rise to various ordered crystalline arrangement. In solid state,
highly ordered crystalline areas are interspersed between less-ordered amorphous
zones. These amorphous zones are regions in which the hydroxyl groups are more
readily available for reaction than in the more highly ordered crystalline areas, which
are less reactive (Pérez and Samain, 2010). Four principal allomorphs have been
identified for cellulose: I, II, III, and IV. Each of these forms can be identified by its
characteristic X-ray diffraction pattern. In the native state cellulose is in the form
known as Cellulose I, which has a monoclinic unit cell containing two cellulose chain
in a parallel orientation in line with the b-axis (Klemm et al., 2002). Native cellulose from plant and bacterial origin are having Cellulose I conformation, thus making
Cellulose I the most abundant allomorph present.
Cellulose I can be transformed into the thermodynamically more stable
Cellulose II by subjecting to two distinctive treatments: mercerisation, which involve intra-crystalline swelling of cellulose in concentrated aqueous NaOH (17-20% w/v) follow by washing and re-crystallisation; or dissolution in any cellulose-dissolving solvent (e.g. CUAM) and subsequently regeneration / precipitation (Pérez and
Samain, 2010). This transformation is irreversible, indicating Cellulose II is a more stable allomorph. Cellulose II has a monoclinic crystal structure with two anti-parallel
cellulose chains in the unit cell, and the c-axis (fibre axis) is lengthened while a-axis
shortened.
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2.3.2 Ultrasonication of Cellulose Ether
According to Caruso et al. (2009), ultrasound degradation of polymer is
machano-chemical in nature. Therefore, for a polymer to be effectively degraded by
ultrasound, the polymer is required to be fully dissolved in a solution. This presents an
obvious hurdle to the ultrasound degradation of native cellulose, since native cellulose
with high crystallinity and extensive inter and intra-molecular hydrogen bonding
could not be readily dissolved into various commonly used solvent. As a result, early
investigations on ultrasound degradation of cellulose were conducted on modified
cellulose. Chemical modification that could be employed on cellulose includes
etherification and esterification. Upon chemical etherification such as
carboxymethylation, dissolution could be achieved especially in water to facilitate the
investigations.
The cellulose ethers that have been investigated include hydroxyethyl cellulose (Sato and Nalepa, 1977; Ni et al., 2001), hydroxypropyl cellulose (Malhotra,
1982; Goodwin et al., 2011), hydroxypropylmethyl cellulose (Sato and Nalepa, 1977;
Goodwin et al., 2011), hydroxyethylsulfoethyl cellulose, sulfoethyl cellulose,
carboxymethylsulfoethyl cellulose (Schittenhelm and Kulicke, 2000) and
carboxymethyl cellulose (Gautier and Lecourtier, 1991; Gronroos et al., 2004b;
Gronroos et al., 2008). Among these modifications, carboxymethylation was chosen
as a favourable modification method lately since the resulting product is water soluble
and can be easily obtained commercially. Nevertheless, it is generally known that after derivatisation, cellulose backbone will experience noticeable depolymerisation.
Depolymerised fragments with lower molecular weight will exhibit different degradation patterns compared to those with higher molecular weight segments
(Mason and Lorimer, 2002; Caruso et al., 2009). Therefore, using cellulose ether as a
26
precursor to examine the effect of ultrasound on cellulose could be less appropriate,
since cellulose ethers usually have lower molecular weight than cellulose and thus,
could not accurately represent the latter.
Molecular weight profiles of ultrasonicated cellulose ethers were the subject of
interest in the earlier literature; since ultrasonicated polymers will have a different
degradation pattern compared to other means, e.g. acid hydrolysis. Molecular weight
profiles of hydroxypropyl cellulose (HPC) ultrasonicated in different types of
solvents, namely water, ethanol and THF, have been reported by Malhotra (1982). By
studying molecular weight–ultrasound irradiation time relationship, the author found
that degradation of HPC is faster in water, followed by ethanol and THF. On the other
hand, Brookfield viscosity and intrinsic viscosity ([η]) were used as indicators to
gauge the extent of ultrasonic degradation of hydroxyethyl cellulose (HEC) and hydroxypropylmethyl cellulose (HPMC) in water (Sato and Nalepa, 1977). More recently, Schittenhelm and Kulicke (2000) have reported an interesting finding on ultrasound degradation of cellulose ethers by employing a more advanced size- exclusion chromatography coupled with multi-angle laser light scattering and a refractive index (SEC/MALLS/DRI) detector. They confirmed that ultrasonication capable of producing hydroxyethylsulfoethyl cellulose with a more homologous series of molar masses, i.e. lower polydispersity index. In addition, they revealed that conformation of cellulose derivatives in solution plays an essential role in the degradation process by comparing the linear cellulose derivatives with the strongly coiled hydroxyethyl starch, with the latter having a significant slower degradation rate compare to the former.
Meanwhile, Gronroos and colleagues (2004b; 2008) have studied the factors that influenced the ultrasound degradation of carboxymethyl cellulose (CMC).
27
Concentration, molecular weight and dynamic viscosity of the CMC were compared
before and after ultrasonication. They found that CMC with higher molecular weight
and higher concentration degrade faster than their counterparts. Understanding factors
that affect ultrasonication of a particular polymer is important in order to monitor the
progress of degradation and obtain desired end products. Nonetheless, it is arguable
that the outcome of these studies was predictable, since factors that affect polymer
degradations via ultrasonication are similar in general; thus, these studies did not shed much new light on the future research.
2.3.3 Ultrasonication of Cellulose Ester
Compared to the numerous reports on cellulose ether, publications on ultrasonication of cellulose ester are few and far between. However, the earliest available publication on ultrasonication of cellulose actually utilized cellulose nitrate as the precursor, a type of cellulose ester that could be readily dissolved in organic solvent such as ethyl acetate. In the 1950s, Thomas and Alexander (1955; 1957) were the first group of researchers to report the ultrasonication of cellulose nitrate in ethyl acetate. Similar to some subsequent reports on cellulose ethers, they studied the factors that affected the ultrasound degradation of cellulose esters. From the degree of polymerization (DP) – ultrasonication time relationship, they found that after a certain period of time, prolonging ultrasonication would not further induce depolymerisation, with the curve of DP – ultrasonication time reaching an asymptote. This was an important observation and subsequently other researchers described the phenomenon as the ultrasonicated samples reaching a ‘level-off degree of polymerization’ (LODP) or, to use the more contemporary term ‘limiting molecular weight’ (Mlim).
28
After almost 40 years, Marx-Figini (1997) conducted similar experiments by employing a more advanced ultrasound generator to degrade cellulose nitrate. In addition, the author utilized a better instrument (SEC) to characterize the molecular weight profile of the ultrasonicated samples. The results were much expected and similar to those reported by Thomas and Alexander (1955; 1957), except that samples reached LODP earlier (about 60 min) than in the previous study (more than 120 min).
Nonetheless, based on the narrowed molecular weight profiles and the reduction of polydispersity index, Marx-Figini (1997) postulated that ultrasonication induces a non-random chain scission process to the cellulose backbone, which is similar to that of synthetic polymers. Furthermore, based on the obtained polydispersity index, the author concluded that the chain scission could happen at the centre or very close to the centre of the cellulose backbone. This useful information has become fundamental data for further investigation by other subsequent researches.
2.3.4 Ultrasonication of Native Cellulose
Due to the fact that dissolution is a prerequisite for ultrasound depolymerisation as mentioned in section 2.3.2, published literature on ultrasound depolymerisation of native unmodified cellulose is very much scarce and lacking.
Only until recently, now that the methodologies of native cellulose dissolution have been well developed, scientific publication on native cellulose ultrasonication has been encountered. To date, only Striegel (2007) has reported the ultrasonication of native cellulose in N,N-dimethyl acetamide (DMAc) / 0.5% LiCl. Cellulose could be fully dissolved in DMAc / 0.5% LiCl, producing a colourless solution with negligible degradation (section 2.3.1), thus providing an excellent system for ultrasonication experiments. However, the major drawback of utilising this solvent is the difficulty in
29
recovering the sonicated products for further solid state (powdered form) characterisations, such as solid state CP/MAS 13C NMR spectroscopy and thermogravimetric analysis (TGA).
In the report, Striegel (2007) had proposed a modified “path theory” that is suitable to apply to cellulose degradation via ultrasonication, based on a size exclusion chromatography (SEC) study coupled with a multi-angle light scattering
(MALS) detector. As mentioned in section 2.1.2, this fairly novel idea explained that in order for ultrasound degradation to happen, a minimum continuous “path length” within the polymer backbone is needed. Moreover, this path of polymer must have sufficient chain flexibility and conformation freedom. The latter concept is consistent with the work reported by Schittenhelm and Kulicke (2000) on cellulose ethers and starch ethers. Nevertheless, the author did not further characterise the sample due to the technical difficulties in recovering the ultrasonicated cellulose from DMAc / 0.5%
LiCl as mentioned earlier in this section. Thus, several important details on ultrasonicated unmodified cellulose could have been missing from this publication. In addition, the degradation was only pronounced after two hours of ultrasonication and did not reach the limiting molecular weight within the observed time (13 hours), suggesting the solvent used is less suitable for ultrasonication experiments, although samples attained full dissolution in the solvent.
Although there are fewer publications on native cellulose ultrasonication, a few researchers have reported the effectiveness of ultrasound in isolating cellulose from industrial waste such as sugar cane bagasse (Sun et al., 2004; Liu et al., 2006a) and wheat straw (Sun and Tomkinson, 2004). Usually, these ultrasonication experiments were performed in gently heated water (55 – 60 C) or dilute KOH solution (0.5 M). However, less or no depolymerisation was encountered, confirmed
30
by the fact that dissolution is needed. Moreover, most of these studies did not aim to depolymerise the cellulose; they only intended to disintegrate the cellulose microfibrils at the supramolecular and / or microscopic level in order to facilitate the isolation of cellulose microfibrils from the plant tissue matrix.
2.4 ULTRASONICATION OF BETA GLUCANS
2.4.1 Fundamental Structure and Physicochemical Properties of Beta Glucan
from Microbial Origin, Curdlan
Beta glucan is a class of polysaccharides that consist of repeating -D- glucopyranose unit connected by -glycosidic linkages. Large varieties of -glucan presents in the nature, including that of plant origin such as cereal cell wall beta glucan, and of microbial origin; each characterised with different type of -glycosidic linkages. Some of these polysaccharides contain branching side chain while some others are essentially linear. The sources of -glucans that have been reported in the literature are summarised in Table 2.2. Due to the presence of large variety of - glucan, this section will only focus on -glucan of microbial origin, particularly - glucan from bacteria – curdlan; which is also the subject of interest in this thesis.
Curdlan is a bacterial synthesised exo-polysaccharides that contains almost solely -D-glucopyranose joint by (13)--glycosidic linkages (Figure 2.4). This linear, neutral homoglycan of -D-glucopyranose is synthesised by non-pathogenic and non-toxicogenic strain of Agrobacterium biovar 1 (formerly known as
Alcaligenes faecalis var. myxogenes strain 10C3). It was first discovered by Harada and his colleagues (1966) when they tried to identify microorganism capable to utilise
31
Table 2.2 Various (13)--D-glucans of microbial origin, having (13)--D- glucopyranose as the polymer backbone with different branch points. Adapted from Lee (2002) and Soltanian et al. (2009), with modifications.
Source (Origin) Glucan type Mw (Da) Reference Bacteria Curdlan Linear (13)--D-glucan 5.3 × 104 Nakata et al. (Agrobacterium without side chain - 2.0 × 106 (1998) sp.) Fungi Grifolan One (16)-β-D-glucopyranosyl 4.5 × 105 Okazaki et (Grifola side chain attached to almost al. (1995) frondosa) every third backbone unit Lentinan Two (16)--D- 5.6 × 105 Zhang et al. (Lentinus glucopyranosyl side chain (2011) eeodes) attached to almost every third backbone unit Schizophyllan Single (16)-β-D- 4.3 × 106 Tabata et al. (Schizophyllum glucopyranosyl side chain (1981) commune) attached to almost every third backbone unit Scleroglucan One (16)-β-D-glucopyranosyl 1.6 × 106 Fariña et al. (Sclerotium side chain attached to almost (2001) glucanum) every third backbone unit SSG (Sclerotinia Highly branch (13)--D- 2.0 × 105 Okazaki et sclerotiorum) glucan - 2.0 × 106 al. (1995) Pachyman (Poria Small amount of (16)-β-D- 8.9 × 104; (Zhang et cocos) glucopyranosyl side chain 2.1 × 105 al. (1997) Krestin (Coriolus Protein bound; (14)--D- 9.4 × 104 Masanori et versicolor) glucans with (13)-β-D- al. (1988) glucopyranosyl and (16)-β-D- glucopyranosyl side chain Cinerean Single (16)-β-D- 1.2 × 1010 Stahmann et (Botrytis glucopyranosyl side chain al. (1995) cinerea) attached to almost every third backbone unit
Yeast Linear (13)--D-glucan with 1.5 × 104 Williams et (Saccharomyces very few side chain al. (1992) cerevisiae) Euglenoids Paramylon Linear (13)--D-glucan 2.7 × 105 Tamura et without side chain al. (2009) Brown algea Laminarin Very few (16)-β-D- 5.4 × 103 (Koizumi et (Laminaria glucopyranosyl side chain al., 1993) digitata)
32
petrochemical materials, and they isolated Alcaligenes faecalis var. myxogenes strain
10C3 from soil. Curdlan was given its name because of its ability to “curdle” when
heated (Lee, 2002). Curdlan has an average degree of polymerisation (DP) of
approximately 450; with an average MW (in 0.3 N NaOH) of 5.3 × 104 to 2.0 × 106
Da (Nakata et al., 1998).
CH OH CH OH CH OH CH OH 2 O 2 O 2 O 2 O HO HO HO HO HO O O O OH OH OH OH OH n
Figure 2.4 Molecular representation of curdlan chain structure, a (13)--D-glucan. n indicates the number of repeating disaccharide units (laminaribiose) in a given curdlan chain.
Curdlan is insoluble in water, alcohol and most organic solvents, but dissolve
in dilute NaOH (i.e. 0.25 N NaOH), formic acid, dimethyl sulfoxide (DMSO) and
aprotic reagent such as N-methylmorpholino-N-oxide, 1,3-dimethyl-2-
imidazolidinone (DMI) and N,N-dimethylacetamide (DMAc) with lithium chloride
(LiCl) (Nakata et al., 1998; Tamura et al., 2009). Although it does not dissolve in
water, aqueous suspension of curdlan will slowly swell and subsequently forms a gel
when heated up (Nishinari and Zhang, 2004). Curdlan forms two distinctive gels
when heated up in aqueous suspension, and thus gained a special attention in food
related research. The high-set, thermo-irreversible gel is usually formed by heating
the suspension to 80 C, or to 100 C to attain complete irreversibility. The low-
set, thermo-reversible gel is created by heating an aqueous suspension to
approximately 60 C follow by cooling to below 40 C (Konno and Harada, 1991).
33
Curdlan molecules, as with most other (13)--D-glucans shown in Table
2.2, have been reported to exist as a triple helix, single helix or single chain (random coil) depending mainly on degree of hydration, heating temperature and solvent conditions (Zhang et al., 2002). In DMSO, curdlan is fully dissolved, exhibit as random coil structure. Although curdlan is dissolve in dilute NaOH, it will only exhibit random coil of single chain in solution of > 0.25 N NaOH. At concentration of
NaOH below 0.25 N, curdlan exist as a single helical conformation (Nakata et al.,
1998; Futatsuyama et al., 1999). The conformation difference of curdlan and some
microbial β-glucans (i.e. scleroglucan) in different solution have been confirmed by various MW determination experiments; the MW of curdlan in NaOH < 0.25 N is almost three fold higher than curdlan in DMSO, confirming the association of triple helix of curdlan in dilute NaOH (Yanaki and Norisuye, 1983).
2.4.2 Ultrasonication of Linear and Branch Microbial Beta Glucan
As stated in section 2.4.1, most β-glucans are not capable to dissolve in water.
Nevertheless, if compared to cellulose ((14)--D-glucan), majority of the β-glucans are more readily to dissolve in simple aqueous system, such as dilute NaOH (i.e. 0.25
N NaOH). As a result, ultrasound depolymerisation of β-glucans is supposed to be accomplished much easier compared to cellulose. However, due to the possible gel forming (especially for curdlan) under various situation as mentioned earlier, it impose several difficulties on ultrasonication and most importantly to recover the sonicated fragment for further characterisation. Therefore, to the best of my knowledge, literature information on ultrasound degradation of microbial β-glucans is scarce and lacking. Earlier studies on ultrasonication of microbial β-glucans were not emphasised on the effect of ultrasound to the depolymerisation and the subsequent
34
physicochemical properties. Most work are concentrated on understanding the molecular conformation of these β-glucans in different type of solvent and ultrasonication is just a tool to reduce molecular weight of the β-glucans to obtain a series of samples with different molar mass.
It was only until early 1980s when the first article on ultrasonication of microbial β-glucans, schizophyllan being published by Tabata and co-workers (1981).
Since schizophyllan is water soluble, it is much easier to perform ultrasonication experiments. By measuring the intrinsic viscosity, they confirmed the molecular weight reduction of the sonicated fragment. In addition, the sonicated samples exhibit narrow molecular weight distribution, which is in agreement with the knowledge on ultrasonication. Tabata and co-workers (1981) also performed total acid hydrolysis and gas liquid chromatography (GLC) on the sonicated samples and concluded that no alteration of glycosidic linkages happen during ultrasonication. Depolymerised samples remain as (13)--D-glucan with single side branch of (16)--D- glucopyranosyl residues. After 2 years, Yanaki et al. (1983b) also conducted a research on ultrasonication of schizophyllan. They employed a more advance GPC system coupled with a low-angle laser light scattering (LALLS) detector to obtain more information on molecular weight. They have established a relationship between intrinsic viscosity, [] and weight average molecular weight. Throughout the course of ultrasonication, the polydispersity index has gradually deceased, which is in agreement with literature information on other ultrasonicated polymer.
It appears that schizophyllan is the polysaccharide of interest in the early
1980s, owing to its ability to dissolve in water compared to other -glucan, as well as its potential biological activity. In another publication by Yanaki et al. (1983a), they studied the antitumor activity of a series of ultrasonicated schizophyllan.
35
Ultrasonication of schizophyllan in water was carried out for a relatively long period of time (500 hours) in ambient temperature. With such an extensive period of ultrasonication, the depolymerisation was not enormous; only about 5-9 folds of molecular weight reductions were observed. This suggested that the triple helical structure of schizophyllan in water had a higher resistant on ultrasound degradation.
They also found that antitumor potency of schizophyllan is independent of molecular weight for sample with molecular weight > 6 × 104, and become less potent when the
molecular weight falls below 6 × 104.
Subsequent work on ultrasonication of schizophyllan by Kulicke et al. (1993)
demonstrated the ability of ultrasound in the reduction of molecular weight yet at the
same time did not produce fragments of monosaccharide. In addition, they also found
that the triple helical structure of schizophyllan remain intact after ultrasonication.
With the molecular weight reduction and thus decrease in viscosity, it facilitate the
dissolution of ultrasonicated fragment in suitable NMR solvent and subsequent
structural elucidation via NMR has become more feasible. Meanwhile, it was found
that ultrasonication having the ability to reduce molecular weight yet at the same time,
did not disrupt the tertiary triple helical structure. It was suggested that the rigid triple
helical structure of certain β-glucans is important for its physiological activity.
Apart from schizophyllan, ultrasonications of a few other less common
microbial β-glucans have also been reported. Stahmann et al. (1995) have reported the
ultrasonication of cinerean, a (13) (16)--D-glucan from fungus Botrytis cinerea.
Their study was concentrated on molecular weight (MW) and conformational
characterisation. It was found that native cinerean has MW of about 1.2 × 1010 Da as
determined by low-angle laser light scattering. To obtain fragment with the size range
comparable with dextran standards in order for GPC characterisation, a sample with
36
250 kDa was prepared from native cinerean. This 250 kDa cinerean was subsequently
subjected to ultrasonication, and an approximately exponential decrease of MW with
the sonication duration down to 50 kDa was observed. Most interestingly, they
discovered that prolonged ultrasonication (up to 38 h) capable to produce cinerean
with a more rigid, rod like, triple helical ordered structure compared to the native
counterparts, which seems to be important for its physiological activity.
Attaining aqueous solubility before ultrasonication is always preferable for all
polysaccharides. Therefore, similar to cellulose as discussed in section 2.3.2,
derivatisation could be performed on β-glucans prior to ultrasonication. Machová et al. (1995) have performed carboxymethylation on a type of water insoluble (13)--
D-glucan from cell wall of yeast Saccharomyces cerevisiae in order to achieve solubility. Two carboxymethylated β-glucans were obtained with DS of 0.56 and
0.91, both attained water solubility. Subsequently, they investigate the degradation of carboxymethylated glucan by comparing ultrasound and enzymatic depolymerisation.
They concluded that ultrasonication is a more effective way for the production of lower molecular weight fragment, though both process produced fragments with similar molecular weight. Ultrasonication having the advantage of avoids the needs to separate treated samples from the enzyme.
Due to the ability of depolymerisation and thus molecular weight reduction, ultrasonication could be employed as a useful method in fractioning polymer into different molecular weight for the study of molecular weight dependence parameters.
In addition, ultrasonication able to narrow the molecular weight distribution of the sonicated polymer, which is the added advantage to facilitate characterisation. Nakata et al. (1998) had ultrasonicated curdlan samples and subsequently fractionated the sonicated fragments into samples with a series of molecular weight from low to high.
37
Since curdlan is insoluble in water, it was dissolved in dimethyl sulfoxide (DMSO) /
5% lithium chloride (LiCl) prior to ultrasonication at ambient temperature for 24
hours. Following that, the sonicated samples were recovered and subjected to static light scattering measurement in dilute NaOH solutions (0.3 M) to elucidate the conformation of curdlan in dilute NaOH solutions. Nevertheless, Nakata et al. (1998) did not emphasize on characterisation of the sonicated fragments nor established the kinetics of degradations. Recently, Ma et al. (2008) have also utilised ultrasonication as a tool to depolymerise a newly discovered water soluble β-glucan from Auricularia auricula-judae, a type of edible fungus. Interestingly, it has been detected that this β- glucan composed of a main chain of (14)-linked D-glucopyranosyl with D- glucopyranosyl side groups at O6. Usually, (14)-β-glucan with side branch is barely found in the fungal cell wall. Ultrasonication was conducted for 16 h at room temperature. MW reduction is not extensive, about nine-fold after relatively long hours of sonication. Nevertheless, ultrasonication has effectively produced a series of molecular weight for this branched β-glucan and the structural elucidation has been realised.
2.4.3 Fundamental Structure and Physicochemical Properties of Chitin and
Chitosan
Chitin, the second most abundant biopolymer in the biosphere after cellulose, is a unique aminopolysaccharides that widely synthesised by various living organism.
Chitin occurs in nature as ordered crystalline microfibrils forming structural components in the exoskeleton of crustaceans or in the cell walls of fungi. Chitin is a polymer of linear (14)- linked 2-acetamido-2-deoxy-D-glucopyranose which resembles that of cellulose (Figure 2.2), except the C-2 hydroxyl group of cellulose is
38
replaced by an acetamide group in chitin (Figure 2.5). Due to the liner diequatorial
arrangement of the repeating 2-acetamido-2-deoxy-D-glucopyranose (N-acetyl-D-
glucosamine), chitin also forms rigid semi-crystalline polymorph similar to cellulose
in solid state: α, β, and γ form. A detailed review on different chitin allomorphs has
been reported by Rinaudo (2006).
CH OH NH2 CH OH NH2 2 O 2 O HO HO O HO OH HO O HO O O O NH CH2OH NH CH2OH OC OC
CH3 CH3 x y
Figure 2.6 Molecular representation of chitin and chitosan chain structure, a (14)- -N-acetyl-D-glucosaminoglycan. x and y indicates the number of repeating monosaccharide units in a given chain. If x > y, the chain represents chitosan. If y > x, the chain represent chitin.
The “ideal structure” of chitin comprise of entirely N-acetyl-D-glucosamine.
Nevertheless, chitin present in nature as partially deacetylated form, with a degree of
deacetylation (DD) of about 0.05 to 0.15 (5 to 15%); or in other words, degree of
acetylation (DA) of about 0.85 to 0.95. When the DD of chitin reaches about 50%
(depending on the origin of chitin), it becomes soluble in aqueous acidic solution and
is called chitosan (Rinaudo, 2006). It has also been proposed to define chitosan as the
moiety of 1% w/v that dissolved in 0.1 M acetic acid, while chitin does not dissolved
in it (Vårum and Smidsrød, 2005). Therefore, DD is an important indicator of the
chitin / chitosan polymer and in fact, it affects the physicochemical properties of
chitin and chitosan. Chitosan attain solubility in dilute acid due to the protonation of
the amine group (–NH2) present at C-2 of the D-glucosamine unit, whereby the polysaccharides in transform into a cationic polyelectrolyte. This confers the
39
uniqueness of chitosan since it is the only pseudo-natural cationic polysaccharides
present.
As shown in Figure 2.5, chitosan is a copolymer of 2-acetamido-2-deoxy-D-
glucopyranose and 2-amino-2-deoxy-D-glucopyranose. It usually obtained via
deacetylation of chitin using concentrated NaOH. Commercial available chitosan
normally possess DD ranging from 70 to 95%. Chitosan are characterised by a DD
value which in turn determine its solubility. Nevertheless, the solubility of chitosan
also affects by the distribution of acetyl groups along the polymer backbone. In
addition, the solubility of chitosan of a given DD and MW, will have different solubility in aqueous acidic solution of various pH. In general, chitosan is soluble in
dilute acidic solution of pH lower than 6.
2.4.4 Ultrasonication of Chitin and Chitosan
Chitin and chitosan possess (14)--glycosidic linkages similar to those of
cellulose. Therefore, it is postulated that ultrasonication of chitin and chitosan will
have some similarity to ultrasonication of cellulose, which is the intention of
preparing a brief review in this section. Since chitosan has a higher practical impact
and application interest compared to chitin, almost all publications on ultrasonication
are concentrated on chitosan but not chitin. In addition, chitosan also has the
advantage of easier dissolution in a simple solvent system (dilute acidic medium) due
to the presence of free amine groups (–NH2) in the molecular structure, as mentioned
earlier in section 2.4.3. Therefore, it is expected that chitosan is more feasibly
degraded via ultrasonication compared to chitin.
Nevertheless, Huang and co-workers (2010) had chemically derivatised chitin
to obtain carboxymethyl chitin (CM chitin) in order to facilitate in dissolution before
40
further subjecting to depolymerisation via ultrasonication. They argued that
carboxymethylation is a better alternative to modify chitin as opposed to deacetylation
of chitin (in which chitosan will be obtained). Although both CM chitin and chitosan
are readily dissolved in an aqueous system, CM chitin with a higher degree of
substitution (DS > 0.60) and more even distribution of carboxymethyl groups will
have the additional advantage of being able to dissolve in water over a wide range of
pH (Chen et al., 2002). The deacetylated derivative of chitin, chitosan can only
dissolve in dilute acid but not in water. In addition to achieving solubility, the
molecular structure of carboxymethylated chitin also resembles hyaluronic acid,
which is another added advantage for carboxymethylation. Hyaluronic acid is a type of glycosaminoglycan that has been widely used in the pharmaceutical and cosmetic field, yet it is costly and thus limited in its application.
It has been reported that carboxymethyl chitin with a MW higher than 2.48 ×
105 has the potential to be used as a HA substitute (Chen et al., 2002). To obtain CM
chitin with such molecular weight, conventional acid hydrolysis was employed to
degrade CM chitin. However, it was found that the progress of acid hydrolysis is
difficult to control and all hydrolysed fragments possessed a very low molecular
weight. By dissolving CM chitin into water and subsequently subjecting it to
ultrasonication, the fragment obtained had a higher molecular weight (2.77 × 106) and
therefore could be utilised as a hyaluronic acid substituent.
Similar to ultrasound depolymerisation of cellulose, most researchers
employed ultrasonication to aid in depolymerisation of chitosan in the hope of
achieving a lower MW fragment. Lower MW chitosan or chito-oligosaccharides are
reported to have more desirable biological activity such as antimicrobial activity (No
et al., 2002; Zheng and Zhu, 2003; Vishu Kumar et al., 2004) and antitumour
41
property (Qin et al., 2002) compared to the high MW counterparts. In addition, chitosan with MW of less than 10 kDa will be water soluble (Cravotto et al., 2005).
Ultrasonication has been perceived as a promising alternative tool to substitute for the
conventional acid hydrolysis method.
Modifications of polysaccharides are done in the hope of achieving
functionalised materials with desirable physicochemical properties and thus better
application. Therefore, it is always of interest to investigate the parameters that
govern a modification process, as well as the physicochemical properties of the modified products. Moreover, knowing the preferred combination of experimental
settings will eventually lead to an optimised product yield and/or shorter reaction
time. As a result, numerous works have been done to understand the effects of various
experimental conditions on the progress of ultrasound degradation of chitosan, and
subsequently the characterisation of the sonicated fragments (Chen et al., 1997; Tsaih
and Chen, 2003; Tsaih et al., 2004; Kasaai et al., 2008; Li et al., 2008).
Chen and colleagues (1997) studied the effect of ultrasonication conditions
and parameters including those of chitosan concentration, reaction temperature, type
of solvent, sonication time and storage in acidic solution) on the changes in the
molecular weight (MW) and polydispersity (molecular weight distribution) of treated
chitosan. Tsaih et al. (2004) also conducted similar research, but during the process of
ultrasonication, small degraded fragments were continuously removed by
ultrafiltration. Both groups of researchers discovered that chitosan was degraded
faster in dilute solutions than in concentrated solutions and faster in lower temperature
solutions than in higher temperature solutions. Chen et al. (1997) also reported that
polydispersity of the modified chitosan decreased as a function of ultrasonication time
for all ultrasonication conditions. In the meantime, Tsaih et al. (2004) concluded that
42
removing the smaller degraded molecules during ultrasonication resulted in higher
reaction rate constant / coefficient and thus a more efficient degradation.
It is known that the degree of deacetylation (DD) of chitosan will greatly affect its physicochemical properties; hence, a few studies have concentrated on the effect of ultrasonication on DD of sonicated chitosan, as well as to obtain a relationship between DD and the ultrasound degradation rate coefficient.
Interestingly, the results obtained by various researchers are somewhat controversial.
It has been confirmed that DD of chitosan will not be affected by a relatively short duration of ultrasonication (up to 30 minutes) and power level of the sonicator (Tang
et al., 2003; Baxter et al., 2005). Nevertheless, Liu and co-workers (2006b) argued
that work done by previous researchers is not conclusive since they did not employ
chitosan with different DD as their precursor. To provide a more accurate
understanding on this, Liu et al. (2006b) investigated the ultrasonication of four
chitosan samples with a wide range of initial DD (61.9% – 91.6%). They discovered
that the process of ultrasonication changed the DD of the sonicated fragment when the
initial chitosan possessed a lower DD (< 90%); but DD of the sonicated fragment will
remain constant if the initial chitosan has a higher DD (> 90%). The latter confirmed
the findings by Tang et al. (2003) since they employed chitosan with an initial DD of
96%. On the contrary, Baxter et al. (2005) used chitosan with an initial DD of 78%,
yet they found that DD has not been significantly altered after ultrasonication, which
is opposed to the findings by Liu et al. (2006b). This could possibly be due to the
prolonged ultrasonication (up to 100 hours) conducted by Liu et al. (2006b) that led to
the change in DD. It is possible that if Tang et al. (2003) and Baxter et al. (2005)
prolonged their ultrasonication duration, they could have obtained similar results.
43
Meanwhile, kinetics of ultrasound degradation is also a subject of interest.
Often, researchers only incorporate kinetic study as a minor part of their experimental work. Nevertheless, Trzcinski and Staszewska (2004), Wu et al. (2008) and Popa-Nita
et al. (2009) provided extensive reports on kinetics of ultrasonication of chitosan. In
the research work by Wu and co-workers (2008), they explained that it is necessary
to determine the limiting molecular weight (Mlim) of chitosan prior to comparing
different kinetic models that are suitable to explain the ultrasonication chitosan. They
have tested two different kinetic models, namely a random scission polymer
degradation model and a midpoint chain scission degradation model, by using
molecular weight results obtained via a gel permeation chromatography (GPC) study.
They found that the MW results fit better into the random scission model and
therefore results of subsequent experimental work (i.e. solution properties that
affected the degradation process) were derived according to this model. Meanwhile,
Popa-Nita et al. (2009) found that the pH and ionic strength of the chitosan solution
has no influence on ultrasound reaction kinetics; on the contrary, the reactor geometry
and acoustic parameters (sonicator probe diameter, ultrasound output power and
frequency) played a significant role in influencing the mechanism of
depolymerisation. Subsequently, they constructed a “master curve” by incorporating
the reactor geometry and acoustic parameters into a plot of 1/DPn (number-average
degree of polymerisation) as a function of ultrasonication time. The master curve
allows a comprehensive quantification of various main parameters influencing the
ultrasonication depolymerisation kinetics, which has not been done previously by
other researchers.
Trzcinski and Staszewska (2004) also conducted a chitosan depolymerisation
study by combining ultrasonication and acetic acid hydrolysis. They discovered that
44
the rate of ultrasound degradation is two-fold higher in the more concentrated acetic acid solution (1.0 M) compared to the dilute solutions (0.1 M), and concluded that ultrasonication accelerates the acid hydrolysis of chitosan. Their work could have inspired subsequent researchers in utilising several other possible factors during ultrasonication to achieve synergistic effects in the degradation of chitosan. Yue et al.
(2008) studied the effect of combining both ultrasonication and ozone treatment on chitosan depolymerisation. They compared the combinatory effect with solely acid hydrolysis (HCl) or ozone treatment and concluded that ozone has a promising synergistic effect in enhancing the ultrasound depolymerisation of chitosan.
Moreover, with the combined treatment, the degradation could happen in ambient temperature as opposed to solely relying on acid hydrolysis, which requires a higher reaction temperature (~70 °C).
On the other hand, Taghizadeh and Abdollahi (2011) studied the effect of combining ultrasonication with a catalyst (TiO2), and with / without the presence of
UV irradiation, on the molecular weight reduction of sonicated chitosan. They investigated the chitosan depolymerisation based on the following experimental combinations: (1) ultrasonication; (2) ultrasonication + TiO2 (sonocatalytic); (3) UV irradiation + TiO2 (photocatalytic); (4) ultrasonication + UV irradiation + TiO2 (sono- photocatalytic). It was confirmed that the combined effect in experiment (4) gives the highest degradation rate constant and thus is most efficient in depolymerisation of chitosan. They explained that this could be attributed to the increase of catalytic activity due to ultrasound de-aggregating the catalyst particles, as well as the enhancement of mass transfer between the liquid phase and the catalytic surface with the aid of ultrasonication. Nevertheless, they did not clearly describe the contribution of UV irradiation to the results obtained.
45
2.5 SULFATION OF MICROBIAL BETA GLUCAN
Polysaccharides derivatisation is an interesting subject to pursue, owing to the presence of free hydroxyl groups on the bio-polymer backbone, providing viable positions for various functional groups to be substituted. Mainly, the types of functional groups will in turn determine the physicochemical and even physiological properties of the polysaccharides derivatives. Usually, the functionalised polysaccharides could attain the desired water solubility, which is particularly useful for polysaccharides that originally do not dissolve in water. Therefore, it could be understandable that it is relatively of interest to study the derivatisation of β-glucans, since several native β-glucans of microbial origin (i.e. curdlan, pachyman) are not water soluble. Particularly, a lot of reports concentrated on sulfation of β-glucans.
– This is due to the sulfate (–SO3 ) derivatives of polysaccharides possessing several
important physiological properties such as antitumor, antivirus and anticoagulant
properties. Moreover, β-glucans, particularly (13)--D-glucan, and their derivatives
have been identified as a biological response modifier that is capable of enhancing or
restoring normal immune defences (McIntosh et al., 2005).
Sulfation of polysaccharides has been well discussed in the literature since the
last century. After the discovery of more microbial β-glucans in the past few decades,
such as curdlan and lentinan, reports on synthesising curdlan and other β-glucan
sulfate have gradually increased, due to the application interest of these sulfate
derivatives in medical and pharmaceutical areas. To date, three general methods for
sulfation of β-glucans have been reported. These include the use of SO3-pyridine
46
complex (Gao et al., 1997; Koumoto et al., 2004; Sun et al., 2009), piperidine N- sulfonic acid (Yoshida et al., 1990; Miyano et al., 1992; Uryu et al., 1996) and chlorosulfonic acid in pyridine (Cirelli et al., 1989; Nie et al., 2006). It has been found that by using these conventional chemical synthesis agents, the degree of substitution obtained is usually relatively high; ranging from 0.8 by using piperidine
N-sulfonic acid (Yoshida et al., 1990) to 1.79 by using SO3-pyridine complex (Gao et
al., 1997). Yoshida et al. (1995) have also compared the above-mentioned three
methods in their subsequent work. They found that by employing a suitable amount of
sulfation agent per molecule of glucose, a desirable DS of as high as 2.9 could be
obtained by using chlorosulfonic acid in pyridine. Nevertheless, it seems that SO3- pyridine complex is a better option among the three choices, since the sulfation process can occur below 100°C and thus less depolymerisation; at the same time, with a relatively high DS of > 2.0.
Apart from the well established sulfation methods, Williams et al. (1992) and
Wang et al. (2005) have reported the use of a non-conventional sulfation agent in synthesising (13)--glucan sulfate. Both groups of researchers have employed sulfuric acid as the sole sulfating agent. The group of Williams (1992) used a concentrated sulfuric acid in a DMSO / urea mixture while Wang and co-workers
(2005) employed sulfuric acid in n-propanol. However, the noticeable drawback of using these methods is the relatively low DS obtained: 0.34 and 0.36, respectively. It has been reported that, curdlan sulfate with DS = 0.35 is hardly soluble in water
(Yoshida et al., 1990). Therefore, it presents a clear disadvantage for this type of sulfation work, especially if water solubility is desired for subsequent physiological characterisation. Moreover, the reaction between sulfuric acid and (13)--D-glucan can only occur at a subzero temperature (–6 °C) in n-propanol (Wang et al., 2005),
47
thus imposing some practical difficulties in experimental work. As a result, there is a
need to seek for catalysts or other forms of aids to increase the DS of these reactions.
Lately, more works have been conducted in the hope of finding better
alternative routes in derivatising polysaccharides. It was recently discovered that by
employing a conventional chemical derivatisation method combined with microwave
or ultrasound irradiation, the process of modification is able to progress faster. Feng et
al. (2010) have reported a rapid and efficient microwave-assisted sulfation of lentinan. They employed chlorosulfonic acid in pyridine as the conventional sulfation agent for a 6-hour reaction time at 45°C. Meanwhile, the same reaction mixture was subjected to microwave heating for only about 4.5 minutes. The DS obtained by these two methods is 1.45 and 1.57, respectively. This clearly indicated that comparable DS could be obtained under microwave irradiation, with a significant shorter time. In addition, another key advantage of using a microwave device is the easy availability of this inexpensive household electrical appliance.
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64
CHAPTER 3
ULTRASONICATION OF BACTERIAL AND PLANT
CELLULOSE
65 3.1 INTRODUCTION
Cellulose is the principle cell wall component of higher plants and hence the most abundant biopolymer on earth. It is a high molecular weight, linear, water insoluble homoglycan of repeating -D-Glucopyranosyl units joined by (14)--D glycosidic linkages (Klemm et al., 2005). For many centuries, plant-derived cellulose has been utilised extensively by the paper and textile industries leading to a significant demand on wood biomass. Recently, the pharmaceutical, cosmetic and food industries have considered utilisation of plant cellulose but its high molecular weight and water insoluble character has somehow restricted direct incorporation in various applications. To address this, a depolymerised form of cellulose known as microcrystalline cellulose (MCC) and various water or nonpolar-solvent soluble derivatives of the material have been developed as structuring or suspending aids (El-
Sakhawy and Hassan, 2007).
Owing to its high crystallinity, unique mechanical properties and renewable
nature, the utilization of crystalline cellulose and its derivatives as a reinforcing filler
in polymer composites has gained wide attention. Commonly, MCC is produced by acid hydrolysis of -cellulose (Klemm et al., 2005). The process cleaves the polymer backbone preferably at the less crystalline regions, leaving fragments with lower
molecular weight and high crystallinity. Obtaining crystalline cellulose via acid
hydrolysis has been extensively studied and it is a well established industrial process
(Roman and Winter, 2004; Wang et al., 2007). Nevertheless, energy saving environmentally friendly methods, and yet capable of retaining desired functionality, in particular thermostability and crystallinity in the end product, are sought after alternatives to chemical modification.
66 Ultrasound irradiation (or sonication) affords a promising alternative to conventional hydrolysis methods. Mason and Lorimer (2002) define ultrasound as sound having a frequency higher than the human hearing range, i.e. > 20 kHz. The ability of ultrasound in degrading polymeric sequences has been well documented, particularly in synthetic materials dissolved in various solvents. In the case of biopolymers, studies have dealt with chitosan (Chen et al., 1997; Baxter et al., 2005;
Liu et al., 2006), cellulose derivatives (e.g., carboxymethylcellulose (Gronroos et al.,
2004), cellulose nitrate (Marx-Figini, 1997) hydroxyethylsulfoethyl cellulose, sulfoethyl cellulose, carboxymethylsulfoethyl cellulose (Schittenhelm and Kulicke,
2000)) and other water soluble polysaccharides such as guar gum (Tayal and Khan,
2000), dextran (Lorimer et al., 1995; Portenlanger and Heusinger, 1997; Cote and
Willet, 1999), agarose and carrageenans (Lii et al., 1999).
To the best of our knowledge, scientific works on the effect of ultrasound treatment on the structural properties of water-insoluble plant cellulose (PC) and bacterial cellulose (BC) are rather scarce. Marx-Figini (1997) has conducted a study on ultrasound degradation of cellulose nitrate in ethyl acetate. As for the sulfate groups present in carrageenans (Lii et al., 1999), the presence of nitrate groups in the cellulose backbone imparts charge density and steric hindrance, which affect the degradation profile. Moreover, the ester linkage joining the nitrate group with the cellulose backbone could susceptible to hydrolysis while being irradiated with ultrasound, a similar phenomenon to the deacetylation of chitin upon ultrasonication
(Liu et al., 2006).
In the present study, the degradation patterns of underivatized PC and BC are examined in an effort to control the molecular weight distribution of these materials
67 whose structural properties will then be characterised using an armoury of
complementary physicochemical techniques.
3.2 EXPERIMENTAL
3.2.1 Materials
Plant cellulose (PC) (Cellulose fibrous medium, from cotton lint), powdered
microcrystalline cellulose (MCC) were purchased from Sigma-Aldrich, Germany.
N,N-dimethylacetamide (DMAc) (99%, Sigma-Aldrich, USA), lithium chloride
(LiCl) (anhydrous, ACS reagent, 99.0%, Fluka, USA), sodium alumino-silicate
molecular sieve (0.4 nm effective pore size, Riedel-de Haën, Germany), copper (II)
sulfate pentahydrate (CuSO4.5H2O) (GR for analysis, MERCK, Germany) were used
in this study. Unless otherwise specified, all other chemicals were of reagent grade
and were used without further purifications.
Bacterial cellulose was obtained from fermentation process as follows:
Gluconacetobacter xylinus (ATCC® 700178, Manassas, USA) stored previously in a
frozen MicrobankTM (Pro-Lab Diagnostics, Richmond Hill, Canada) was used. Yeast
glucose carbonate (YGC) medium consisting of 5% (w/v) D-glucose, 0.5% (w/v)
yeast extract and 1.25% (w/v) calcium carbonate was used for the inoculum
preparation and BC production. All media and apparatus were autoclaved at 121°C
and 1.02 atm for 15 min. A single bead was drawn from a frozen MicrobankTM, promptly cultured on a YGC agar plate (with 1.5% (w/v) bacteriological agar) and incubated at 26°C for 72 h.
68 Separate colonies were aseptically transferred to 500 ml of a seed fermentation medium followed by static incubation at 26°C for 72 h. Thirty six ml of this medium were inoculated aseptically into an aluminium tray (260 mm width × 345 mm length × 55 mm height) containing 1164 ml YGC medium. The tray was covered with aluminium foil and incubated statically at 26°C for 12 days. Bacterial cellulose pellicle formed at the liquid-air interface of the fermentation medium was removed, rinsed thoroughly with deionised water and boiled in deionised water for 15 min.
Following this, the pellicle was immersed four times in a solution of 0.1 N NaOH held each time at 80°C for 2 h, rinsed, boiled (15 min), soaked overnight and washed repeatedly with deionised water until washings became neutral to methyl red, and lyophilised.
Purified bacterial cellulose and plant cellulose were then ultrasonicated. That required the following preparation of cuprammonium hydroxide solution (CUAM)
(method of Kamide and Nishiyama (2001), with some modifications): Sixty grams of
CuSO4.5H2O were dissolved into 1 l of hot water (90°C) followed by addition of 23
ml of 25% (w/v) ammonia solution (50°C) to form the basic copper sulfate
precipitate. The precipitate was washed with hot deionised water and the supernatant was discarded. Two hundred ml of 20% (w/v) NaOH were added to form the blue
copper hydroxide precipitate, which was washed twice with cold deionised water.
The precipitate was vacuum filtered (Whatman No. 1) and the retentate was further washed with cold deionised water until the filtrate was free from sulfate ions (tested with 1 M CaCl2). Copper hydroxide was oven dried overnight at 50°C, transferred
into a screw cap bottle in the presence of 1400 ml of 20% (w/v) ammonia solution
and the mixture was shaken vigorously. The precipitate of excessive copper
69 hydroxide was removed by filtering under vacuum through Whatman glass microfiber
GF/B and the dark blue filtrate was the CUAM solution used.
Homogeneous solutions of 0.125% (w/v) PC and BC were made in CUAM and left at ambient temperature for 2 h. Ultrasonication (Elmasonic S 60H, ELMA,
Singen, Germany) of both materials was carried out at a frequency of 37 kHz and effective ultrasonic power of 150 W for the time periods of 5, 10, 15 and 30 min at ambient temperature. After the desired ultrasonication time, PC and BC were recovered from solution by addition of 1 M H2SO4, which led to the formation of a white precipitate. That was filtered under vacuum, washed with deionised water until the filtrate became neutral to methyl red, dialysed and lyophilised to produce neutral cellulosic materials. Control sample of the ultrasonication process was prepared in the similar manner except without subjected to ultrasound irradiation. For the ease of explanation in the text, samples were denoted by abbreviation according to the type of sample (BC or PC) and duration of sonication (0, 5, 10, 15 and 30 min). For example,
BC-0 denotes control sample for bacterial cellulose.
3.2.2 Size Exclusion Chromatography (SEC)
Pre-dissolution activation steps were necessary to facilitate the dissolution of underivatised cellulose samples. In the present study, polar medium exchange activation was used. The method of Dupont (2003) with some modifications was used to prepare PC, BC and MCC for size exclusion chromatography: Deionised-water preparations of cellulose (0.6% w/v) were held at approximately 40 °C for 2 h, and subsequently subjected to consecutive exchanges/activation steps of methanol (two –
1 h each) and dry N,N-dimethyl acetamide (DMAc) (two – 1 h and overnight) with constant stirring. Following each steps, activation liquids were discarded by filtering
70 under vacuum, and DMAc / 8% LiCl was added to form dissolved cellulose solutions
of 1.2% (w/v). Dilution with dry DMAc gave a final concentration of 0.24% (w/v)
with respect to the cellulosic sample and 1.6% (w/v) with respect to LiCl.
The SEC system with manual injector included a Waters 515 HPLC Pump and
a Waters 410 refractive index (RI) detector (Waters Corporation, Milford, USA).
Prior to injection, samples and mobile phase (DMAc / 0.5% LiCl) were filtered
through a PTFE membrane filter with a pore size of 0.2 m. The injection volume
was 250 l, separation was performed on a series of three Phenogel mixed bed
columns (300 × 7.8 mm; particle size 10 m) at 65°C, and at the flow rate of 0.8
ml/min. Each sample run lasted for 50 min. Nine pullulan standards of narrow
polydispersity, i.e., 788k, 404k, 212k, 112k, 47.3k, 22.8k, 11.8k, 5.9k and 0.67k Da
(Polymer Laboratories, Church Stretton, UK) were used to calibrate the columns. The
linear coefficient of determination (r2) between the weight average molecular weight
( Mw ) of the standards and elution time was 0.999. Data acquisition and processing
were done by using Waters Empower data software Version 5.00.
3.2.3 X-Ray Powder Diffraction Studies (XRPD)
X-Ray powder diffraction patterns were obtained using a SIEMENS D5005
X-Ray Diffractometer (Bruker AXS, Karlsruhe, Germany) equipped with Cu-Kα
(1.54 Å) radiation. An accelerating voltage and current of 40 kV and 40 mA, respectively, in combination with a scan rate of 0.8°C/min were employed. The diffractograms were recorded in a 2θ range between 5° and 45°, and subsequently analysed using the Bruker Advanced X-Ray Solutions software, DIFFRACplus
Evaluation (Eva), version 10.0 revision 1.
71 Diffraction profiles of the samples were fitted with a Gaussian function to resolve into separate diffraction peaks and subsequent integration was performed on each peak. PC and BC spectra were resolved into three peaks corresponding to 101,
10Ī and 002 crystal planes but the MCC spectrum exhibited an additional 040 crystal
plane. The crystallinity index (CrI) of samples was determined following the method
reported by Wang et al. (2007) with slight modifications:
ACrystal CrI = 100 (3.1) ATotal
where, ACrystal is the sum of the areas under the crystalline diffraction peaks and ATotal represents the total area under the diffraction curve between 2θ of 5° and 45°.
3.2.4 Thermogravimetric Analysis (TGA)
This was carried out to determine the thermal stability of our materials by employing a thermogravimetric analyzer, SDT 2960 (TA Instruments, New Castle,
DE). Samples were heated from ambient temperature to 600°C in a nitrogen current of 100 ml/min at a heating rate of 10°C/min. Thermogravimetric (TG) curves and derivative TG (DTG) curves were analysed with the aid of the TA Instruments
Universal Analysis Software (Version 3.9A) in an effort to determine the peak onset temperature and peak maximum temperature.
3.2.5 Fourier Transformed Infrared (FTIR) Spectroscopic Analysis
Samples were dried in oven at 50 °C overnight before being analyzed by
Fourier Transformed Infrared (FTIR) spectroscopy. FTIR spectra were obtained with a Spectrum One FTIR Spectrophotometer (Perkin-Elmer, Norwalk, USA). Prior to analysis, the solid samples were incorporated in a potassium bromide (KBr) pellet.
72 Since KBr is hygroscopic, it needs to be stored in a desiccator and oven dried at 50
°C, overnight before use. KBr pellets were prepared by mixing samples with KBr in a
weight ratio 1: 100. The mixture was finely ground with the aid of a pestle and
mortar, and then placed in a KBr pellet die, which subsequently subjected to
approximately 640 MN/m2 pressures for approximately 1 min in a specially
constructed manual hydraulic press unit (Specac Ltd, London, UK). A transparent
disk is produced, which is removed from the die with tweezers and placed in a special holder, and ready for analysis. A single background scan was performed, before 100 scans on sample were taken with a resolution of 4.00 cm–1 in order to produce the
spectra. Spectra were recorded from the wavenumbers range of 4000 cm–1 to 400
cm–1. For data acquisition, software Spectrum v5.0.1 (Perkin-Elmer Instrument LLC,
USA) was used.
3.2.6 Transmission Electron Microscopy (TEM)
Disintegrated cellulose crystals, which were produced by the combined
CUAM / ultrasonication treatment, were suspended in deionized water. This was
followed by deposition of a few droplets of the suspension onto a Formvar coated
copper grid with a mesh size of 200. Copper grids with cellulose crystals were loaded
into a sample chamber following air drying. Electron micrographs of tested materials
were recorded by a Philip CM10 transmission electron microscope (Philip,
Eindhoven, Netherlands) operated at 100 kV. Micrographs were taken using Kodak
Electron Microscopy film 4489 (Eastman Kodak Company, New York, USA) and
converted into a digital form with a conventional digital photo scanner.
73 3.3 RESULTS AND DISCUSSION
3.3.1 Effect of Ultrasound Sonication on the Weight Average Molecular
Weight and Polydispersity Index
Figure 3.1 reproduces the SEC elution patterns of ultrasonicated bacterial and
plant cellulose in DMAc/0.5% LiCl. The treatment has affected the retention time,
with the ultrasonicated samples having longer retention times compared to that of the control (BC-0 and PC-0). Slowly eluting materials possess small molecular weight and Figure 3.2 summarizes the effect of ultrasonication time on the degradation patterns (weight average molecular weight) of BC and PC.
The decrease in Mw is attributable to scission of the (14)--D glycosidic
linkages. Mason and Lorimer (2002) dealt in detail with the hydrodynamic force and
shear stress generated from the cavitation of bubbles, which are the outcome of the
ultrasound treatment of polymeric solutions. The treatment amounts to alternating
compression and rarefaction processes that make bubbles to rapidly form and collapse within the solution. Tested materials have been subjected to a relatively short period of treatment (maximum of 0.5 h). Therefore, further reduction in molecular weight is anticipated upon prolonged periods of ultrasonication, which is the subject of interest
in the subsequent chapter. Sonication of chitosan (Liu et al., 2006) and cellulose
nitrate (Marx-Figini, 1997) solutions has been reported for 100 h and 1.5 h, respectively, and within this time period of experimentation, the decrease in molecular weight approaches asymptotically a “level-off degree of polymerization”.
74
Figure 3.1 SEC elution patterns of ultrasonicated (a) BC and (b) PC in DMAc/0.5% LiCl. BC-X / PC-X denotes sample sonicated for X amount of time.
75 Another issue to consider is the use of cuprammonium hydroxide as the solvent of underivatised cellulose. CUAM can dissolve the polymer without having to perform pre-dissolution activation steps. Moreover, sonicated materials can be recovered readily from the aqueous solution for further characterizations, as compared to organic solvents (e.g. DMAc / LiCl) which has been used by Striegel
(2007). However, ammonia in CUAM is volatile and according to Price and co- workers (Price et al., 1994), ultrasound degradation is less efficient in such a highly volatile solvent. This observation may bear an effect on the ultrasonication profile of plant and bacterial cellulose. Nevertheless, degradation has been observed in a relatively short period of time (within 0.5 h) in the current study (Figure 3.2), unlike the report by Striegel (2007) where degradation became pronounced in 2 h using
DMAc/LiCl. This further confirms that CUAM is a suitable solvent for the ultrasound degradation of cellulose.
The weight average molecular weight of bacterial cellulose shows a drastic fall at the beginning of ultrasonication (≈ 64% reduction in the first 10 min), which is followed by a gradual decrement at longer times (Figure 3.2). In comparison, Mw reduction of PC followed a steady declining trend throughout the experiment period of observation, and the reduction was relatively limited (≈ 21% within 10 min of ultrasonication). It appears that as for chitosan (Liu et al., 2006), high molecular weight bacterial cellulose is amenable to ultrasonicated depolymerisation. The higher proportion of such high molecular weight fractions at the beginning of ultrasonication warrantees high degradation rates at the first stage of experimentation. In addition,
Gronroos et al. (2004) postulated that below a certain limit of molecular weight, the short polymer chains might follow flexibly the ultrasonic vibration thus escaping covalent-bond cleavage.
76
Figure 3.2 Weight-average molecular weight variations of plant and bacterial cellulose as a function of ultrasonication time.
The second part of this section examines the polydispersity index (PI), which is an indication of the segmental size distribution of a particular polymer defined as the ratio of weight to number ( Mn ) average molecular weight. Figure 3.3 depicts the
effect of increasing ultrasonication time on the PI of plant and bacterial cellulose. The
reduction in the polydispersity index of PC is consistent with the idea that prolonged
sonication yields chain segments that can not be further degraded, an outcome which
tends to create homogeneous systems with a relatively narrow molecular weight
distribution (Gronroos et al., 2004). This is in agreement with the work on chitosan
(Chen et al., 1997), where degradation stops and low values of the polydispersity
77 index are obtained once a limiting molecular weight for this system is reached at 1.8
× 105 Da.
Figure 3.3 Polydispersity-index variations of plant and bacterial cellulose as a function of time of ultrasonication.
In contrast to the results of the preceding paragraph, the PI of bacterial
cellulose exhibited a noticeable increase during the course of experimentation (Figure
3.3). The rapid reduction in Mn as compared to that of Mw is rather unexpected for
ultrasonication treatments. This is commonly encountered in enzymatic (Klemanleyer
et al., 1994; Tayal and Khan, 2000), and acid hydrolysis (Shibazaki et al., 1995) of
polysaccharides, which is attributed to a random scission process. Plant and bacterial
cellulose are chemically identical but the supramolecular structure, i.e., the
morphological characteristics of the cellulose microfibrils are quite distinct (Klemm
et al., 2005). In addition, the susceptibility of polysaccharides to ultrasonic
78 degradation is subject to their conformational state in solution (Lii et al., 1999). Thus,
“single” helices of carrageenan in solution offer greater resistance to degradation than their random-coil counterparts (Lii et al., 1999). Bacterial cellulose in this work was properly dissolved in preparation for ultrasonication and, therefore, the conformational characteristics of the material in CUAM may prove to be of critical importance.
The molecular dimensions of plant/bacterial cellulose were compared to those of a sample of microcrystalline cellulose. MCC was found to possess a weight average molecular weight of ≈ 99.30 kDa with a polydispersity index of ≈ 3.60. This is distinct from the Mw of PC and should be attributed to natural or processing variations of the material. Furthermore, MCC samples obtained from different sources of cellulose are expected to exhibit dissimilar physicochemical properties
(such as molecular weight) even though similar acid-hydrolysis conditions are employed. Interestingly, bacterial cellulose ultrasonicated between 10 and 15 min possesses similar Mw to that of MCC. The broad agreement in the molecular dimensions of BC and MCC is encouraging as a first step for considering industrial applications for the molecular fractions of bacterial cellulose.
3.3.2 X-Ray Diffraction Profile and Crystallinity Index (CrI)
Figure 3.4a illustrates the X-Ray diffraction patterns of bacterial cellulose sonicated at different time periods (0, 5, 10, 15, and 30 min) at ambient temperature.
Three major diffraction peaks at 2θ equal to 12.0°, 20.0° and 21.8° were resolved from the deconvolution process and these become sharper with increasing time of sonication. X-Ray studies of plant cellulose reveal similar diffraction patterns for each time period of observation with some noticeable differences in peak intensity
79
Figure 3.4 X-Ray diffraction pattern of (a) bacterial cellulose and (b) plant cellulose sonicated at different times, and compared to those of MCC.
80
(Figure 3.4b). According to the literature, this X-Ray diffraction profile is compatible with what is known as the “Cellulose II crystal” (Shibazaki et al., 1995; Oh et al.,
2005). Conversely, MCC shows four diffraction peaks, which are ascribed to the crystallographic planes of 101, 10Ī, 002 and 040 at 2θ equal to 14.8°, 16.4°, 22.5° and
34.4°, respectively. This is known in the literature as the conformation of the
“Cellulose I crystal” (Cao and Tan, 2004).
The mutant strain of Gluconacetobacter xylinus, ATCC® 23769, has been
reported to produce the so called “native band cellulose”, which exists as the
Cellulose II allomorph (Shibazaki et al., 1995) but, normally, natural occurring cellulose (BC and PC) exists in the thermodynamically less stable Cellulose I allomorph (Yamamoto et al., 2006). Besides ultrasonication, cellulosic materials of the present work have been subjected to dissolution and subsequent regeneration, a course of action that should transform Cellulose I to the more stable Cellulose II structure. Thus the mercerization process, which involves dissolution / swelling of cellulose in aqueous NaOH followed by regeneration / neutralization with HCl, is known to transform Cellulose I into Cellulose II (Shibazaki et al., 1997). There is scant literature addressing the effect of CUAM but Miyamoto and co-workers
(Miyamoto et al., 1996) have shown that plant cellulose recovered from the solvent
projects an X-ray diffraction profile with two major peaks at 2θ equal to 20.0° and
21.9°, which is associated with the Cellulose II allomorph.
For the crystalline component of cellulosic materials, the first observation is
that the crystallinity index of microcrystalline cellulose is the highest (87.6%; Table
3.1). Furthermore, the CrI of non-sonicated BC (55.6%) is well below that of PC
(72.2%), with the latter being comparable to data obtained by Miyamoto et al. (1996).
The crystalline fraction of plant cellulose remains unaffected by the duration of
81 ultrasonication, an outcome which is attributed to the rapid formation of the stable
Cellulose II allomorph within three minutes of mercerization (Shibazaki et al., 1997).
It appears, however, that ultrasonication is capable of increasing considerably the level of crystallinity of bacterial cellulose to 75.4% within thirty minutes of treatment.
It is known that increasing exposure of bacterial cellulose to aqueous NaOH raises the crystalline content of the recovered material (Shibazaki et al., 1997). Exposure of BC to copious amounts of OH- from CUAM used in the present study should contribute
towards the increment of CrI. Nevertheless, all materials were exposed roughly to the
same dissolution time in CUAM hence it is argued that the variable time of
ultrasonication also plays a significant role in promoting crystallinity in bacterial
cellulose by reducing its molecular weight distribution.
Table 3.1 Crystallinity index (CrI) of bacterial cellulose (BC) and plant cellulose (PC) ultrasonicated for different times.
Ultrasonication Ultrasonication Sample CrI (%) Sample CrI (%) Time (min) Time (min)
MCC – 87.6 BC-0 0 55.6 PC-0 0 72.2 BC-5 5 63.6 PC-5 5 72.3 BC-10 10 71.0 PC-10 10 71.6 BC-15 15 73.2 PC-15 15 71.2 BC-30 30 75.4 PC-30 30 71.4
3.3.3 Thermal Degradation of Cellulosic Materials
To evaluate the thermostability of ultrasonicated samples, thermogravimetric
analysis (TGA) was performed on bacterial and plant cellulose, and results were
compared to those of the commercial microcrystalline cellulose. TG and derivative
82
Figure 3.5 Thermogravimetric (TG) and derivative TG (DTG) curves of ultrasonicated samples of bacterial cellulose (a, b).
83
Figure 3.6 Thermogravimetric (TG) and derivative TG (DTG) curves of ultrasonicated samples plant cellulose (a, b).
84
TG (DTG) curves of BC and PC are depicted in Figure 3.5(a-b) and 3.6(a-b), respectively. Experimental profiles in Figures 5a and 6a exhibit sharp steps of weight loss indicative of degradation processes in the macromolecules. DTG curves of the experimental data show two peaks throughout the pyrolytic scan (Figures 3.5b and
3.6b).
The low temperature event centres at about 50°C and it is a relatively small peak that corresponds to the evaporation of water molecules adsorbed to the polymeric sequences. The dominant peak between 300 and 350°C is likely to be caused by the weight loss arose from concurrent cellulose degradation and formation of charred residues. These include depolymerization, further dehydration, degradation of the glucopyranosyl units and subsequent oxidation leaving behind charred residues
(Roman and Winter, 2004). The degradation patterns in terms of shape and temperature band appear to be quite similar for all materials. Within this cluster of traces, MCC and BC-0 exhibit the highest and lowest thermal stability, respectively.
Data have been tabulated to afford unambiguous comparisons of the peak onset temperatures (Te) and peak maximum temperatures (Tmax) of materials at different
stages of ultrasonication. Wang et al. (2007) suggested that the more ordered the cellulose the higher the energetic requirements for polymeric degradation. Table 3.2 indicates such a trend for the extreme cases of BC-0 and MCC (lowest and highest
CrI) but, in general, the Te and Tmax values of all materials are comparable.
Normally, chemical alteration of the polymer affects the degradation profile which spreads over a wide temperature range causing noticeable broadening of the
DTG peak. Roman and Winter (2004) reported that the presence of sulfate groups in cellulose accelerates the onset and broadens the temperature fingerprint of degradation. Furthermore, the presence of an oxidized moiety in cellulose will yield
85 multimodal steps of thermal decomposition, an outcome which is confirmed by the present of additional peaks in DTG figure as confirmed by Varma and Chavan in their work (Varma and Chavan, 1995). In the absence of such “anomalies” and since the thermal degradation is largely insensitive to the changing component of crystallinity, it can be argued that chemical oxidation has not taken place during dissolution, regeneration or ultrasonication of the cellulosic materials investigated presently.
Table 3.2 Peak onset temperature, Te (°C) and peak maximum temperature, Tmax (°C) of bacterial cellulose (BC) and plant cellulose (PC) ultrasonicated at different times.
Ultrasonication Peak onset Peak maximum Sample 1 1 Time (min) temperature, Te (°C) temperature, Tmax (°C)
MCC – 312.0 0.3 337.1 0.2 BC-0 0 301.1 0.2 324.7 0.3 BC-5 5 314.9 0.4 332.2 0.2 BC-10 10 311.1 0.1 333.8 0.4 BC-15 15 308.9 0.2 333.7 0.4 BC-30 30 313.1 0.3 333.4 0.5 PC-0 0 302.1 0.4 329.8 0.4 PC-5 5 307.6 0.5 330.9 0.3 PC-10 10 308.1 0.3 328.9 0.4 PC-15 15 305.6 0.1 326.3 0.2 PC-30 30 311.3 0.5 334.0 0.1 1Means of readings standard deviation (n=3).
3.3.4 Fourier Transform Infrared Analysis (FTIR)
Figure 3.7a and 3.7b depict the FTIR spectra of bacterial and plant cellulose at distinct durations of ultrasonication, which are then compared with those of microcrystalline cellulose. FTIR spectroscopy is a useful tool in elucidating the
86
Figure 3.7 FTIR spectra of (a) bacterial cellulose and (b) plant cellulose ultrasonicated at different times, and compared to those of MCC.
87 functional groups of organic macromolecules, as well as understanding the bonding interactions of these functional groups. Thus it is utilised to provide key information on the nature of molecular forces that dominate the transformation of the cellulose crystal during the present treatment. The detailed designation of each wavenumber band to a specific functional group has been reported previously by others (Carrillo et al., 2004; Oh et al., 2005).
Visual examination of the FTIR spectra of BC and PC with or without ultrasonication shows a broad adsorption peak that bottoms out at about 3447 cm–1.
This is attributed to the stretching of hydroxyl groups involved in intramolecular hydrogen bonding of the Cellulose II crystal. MCC, on the other hand, shows the –
OH stretching peak at about 3350 cm–1 hence indicating the conformation of the
Cellulose I crystal. Both observations confirm the argument of the X-Ray diffraction
study (Section 3.2) that acid hydrolysis of cellulose retains in MCC the Cellulose I
allomorph whereas dissolution and regeneration convert the material into the
thermodynamically stable Cellulose II crystal.
The lack of an absorption band in the range of 1740 to 1745 cm–1 for all
materials argues for the absence of carbonyl groups (C=O), which are usually present
in oxidized cellulose either as part of the carboxyl (COO–) or aldehyde group (HC=O)
(Kim et al., 2000; Son et al., 2004). A peak at 1740 cm–1 can be detected in oxidized
cellulose with a degree of oxidation as low as 0.12% (Kim et al., 2000). Furthermore,
the presence of a pattern of peaks between 894 cm–1 and 897 cm–1 for BC, PC and
MCC reveals the stretching of C–O–C bonds at β-glycosidic linkages (Oh et al.,
2005). Results discussed in this paragraph are in agreement with the observations from the thermogravimetric study (Section 3.3) that material preparation in CUAM and subsequent ultrasonication did not cause oxidative reactions on the chain
88 segments of cellulose. It should be noted that Cao and Tan (2004) using the enzyme cellulase to depolymerise plant cellulose reported FTIR spectra in accordance with the original untreated sample.
3.3.5 Transmission Electron Microscopy (TEM)
There is scant information in the literature on the microscopic morphology of ultrasonicated cellulosic materials especially those that have been dissolved and subsequently regenerated. In this case, the resulting particle characteristics are expected to be influenced by both ultrasonication and regeneration. Figure 3.8 depicts the TEM micrographs of selected ultrasonicated BC and PC samples, and microcrystalline cellulose. These appear to be discrete and isolated particles, and
MCC possesses the most regular and ordered structures with defined edges, an outcome which should be attributed to the high level of crystallinity (87.6% in Table
3.1) of this material. Clearly the current process of ultrasound sonication is capable of yielding small-size particles within 30 min of treatment in accordance with the variation in molecular weight of plant and bacterial cellulose in Figure 2. During the regeneration process, addition of sulfuric acid neutralises the hydroxyl ion and forms copper sulfate in CUAM thus making dissolution of cellulose molecules unfavourable. It seems that the short period (about 60 s) of the regeneration process in the present investigation results in a spontaneous aggregation of the microfibrils of bacterial and plant cellulose, which possess relatively irregular particle shapes.
Furthermore, the abundant positive charge of the solution contributed by the excessive levels of sulfuric acid added during regeneration should further enhance the stability of the small-size (about 1 m) particles via electrostatic repulsion.
89
Figure 3.8 TEM micrographs of ultrasonicated BC and PC, and MCC samples: a) BC-0; b) PC-0; c) BC-30; d) PC-30, e) MCC. Horizontal scale bar is 1 m.
90
3.4 CONCLUSIONS
The present work has achieved an efficient depolymerisation process of cellulose without having to prior derivatize the biopolymer. Findings concluded that ultrasonication of cellulose in CUAM will not induce undesirable chemical modifications (e.g., oxidative reactions) and the shorter polymeric segments of the sonicated product remain (14)--D-glucan. It is of interest to observe that fractionated plant and bacterial celluloses are of the type II crystal whereas microcrystalline cellulose possesses the crystal I conformation. Furthermore, it is argued that long ultrasonication times yield a high crystallinity index for the ultrasonicated BC, an outcome that merits further investigation. Highly crystalline cellulose is desired since there is always a demand for precision engineered cellulosic materials to incorporate in bio-nanocomposites.
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Wang, N., Ding, E. and Cheng, R. (2007). Thermal degradation behaviors of spherical
cellulose nanocrystals with sulfate groups. Polymer, 48(12), 3486-3493.
Yamamoto, H., Horii, F. and Hirai, A. (2006). Structural studies of bacterial cellulose
through the solid-phase nitration and acetylation by CP/MAS 13C NMR
spectroscopy. Cellulose, 13(3), 327-342.
95
CHAPTER 4
MOLECULAR WEIGHT AND CRYSTALLINITY
ALTERATION OF CELLULOSE VIA PROLONGED
ULTRASOUND FRAGMENTATION
96 4.1 INTRODUCTION
Depolymerisation of polysaccharides is a subject of interest since decades ago.
The main purpose to perform these experiments is to obtain fragments of depolymerised polysaccharides with low molecular weight that could possess different physico-chemical properties hence functional properties. For instance, native guar gum (with high molecular weight) will induce high viscosity when dissolved in water, thus preventing the incorporation of large amounts of this material into food products. Hydrolysed guar gum, however, will produce low viscosity when dissolved making it possible to utilise as a source of soluble dietary fibre in beverages.
For cellulose, due to its extensive hydrogen bonding between polymer backbone and high crystallinity, it is useful to be employed as a natural fibre reinforced composite.
Nevertheless, most natural celluloses have limited tensile strength with the presence of an amorphous region. Therefore, essential improvements are required to produce high crystallinity and high purity single cellulose fibres (“whiskers”) that are suitable for fibre related applications (Kohler and Nebel, 2006). Microcrystalline cellulose (MCC), an acid depolymerised (usually by hydrochloric acid) form of cellulose, possess high crystallinity with a desired crystal aspect ratio (length, L to diameter, d ratio), which is suitable in fibre reinforce composites. Alternative attempts have been performed to produce crystalline cellulose, including enzymatic hydrolysis combined with high pressure homogenisation (Pääkkö et al., 2007), sulfuric acid hydrolysis (Elazzouzi-Hafraoui et al.,
2008) and other means of which ultrasonication is a promising technology.
Ultrasound degradation of water soluble polysaccharides has been an interesting alternative to depolymerise materials as compared to conventional acid hydrolysis.
However, research on ultrasound depolymerisation of water insoluble cellulose presents
97 obstacles due to the insolubility of cellulose in many conventional solvents. It has been
confirmed in Chapter 3 the ability of ultrasound to depolymerise bacterial and plant
cellulose within 30 min sonication time in cuprammonium hydroxide (CUAM) solution to produce low MW fragments. This provides an interesting alternative to other
conventional depolymerisation methods (such as acid hydrolysis). Nevertheless, the
sonicated fragments did not attain limiting molecular weight (Mlim) within 30 min of
sonication time. Therefore, it would be of interest to investigate the effect of prolonged
ultrasonication on the molecular weight of bacterial cellulose (BC) and plant cellulose
(PC). Based on the results obtained in Chapter 3, it is thus, hypothesised that the Mlim of
BC and PC will be achieved with prolonged ultrasonication of more than 30 min.
Studies on kinetics of polymer degradation have generally been done to complement the fundamental information obtained from the characterisation of molecular weight distribution. Kinetics of ultrasound degradation of various polysaccharides has been reported in the literature, namely for chitosan (Tsaih et al., 2004; Baxter et al., 2005;
Wu et al., 2008), dextran (Lorimer et al., 1995), agarose and carrageenan (Lii et al.,
1999). Meanwhile, Schittenhelm and Kulicke (2000) studied the kinetics of degradation for a few water soluble cellulose derivatives (hydroxyethylsulfoethyl cellulose, sulfoethyl cellulose, carboxymethylsulfoethyl cellulose). Most of the reported kinetics were based on first order reaction, with the assumption that the scission of glycosidic linkages of polysaccharides during ultrasonication is similar to an acid hydrolysis process (Lii et al.,
1999). Based on first order reaction kinetics, ultrasound depolymerisation of polysaccharides could be represented by the following equation:
1 1 = + k1t (4.1) M t M0
98 where M0 and Mt are the weight average molecular weight (Da) of polysaccharides
- before ultrasonication (0 min) and after ultrasonication for t min, respectively, and k1 (Da
1 min–1) is the rate coefficient (Lii et al., 1999; Tsaih et al., 2004; Wu et al., 2008). On contrary, though most reported ultrasound degradation kinetics were following equation
2 4.1, Wu et al. (2008) had also reported that 1/ Mw could be used. They found that chitosan with high initial Mw fit into equation 4.2 better, as below:
1 1 = + k t (4.2) 2 2 2 M t M0 where M0 and Mt are the weight average molecular weight (Da) of polysaccharides
- before ultrasonication (0 min) and after ultrasonication for t min, respectively, and k2 (Da
2 min–1) is the rate coefficient.
In contrast to reports on other polysaccharides, kinetic studies related to unmodified cellulose are relatively scarce and lacking. Although Striegel (2007) investigated the ultrasound degradation of plant cellulose in DMAc/0.5% LiCl, the process is time consuming (>780 min) and the experiments did not attain a limiting molecular weight. Therefore, it would be useful to study the kinetics of ultrasound degradation of both native plant and bacterial cellulose in a more potent solvent.
Meanwhile, the increment of crystallinity index (CrI) of the sonicated PC and BC in a relatively short period of 30 min sonication time has been reported in section 3.3.2. It would definitely be worthwhile to investigate the effect of prolonged ultrasonication on the CrI of sonicated PC and BC. In short, the current investigation aims to unveil the effect of prolonged ultrasonication (more then 30 min) on the Mw and CrI of BC and PC, and to compare the trend of Mw reduction and CrI increment. In addition, the kinetics of degradation will be computed based on first order reaction kinetics.
99
4.2 EXPERIMENTAL
4.2.1 Materials
Plant cellulose (PC) (Cellulose fibrous long, from cotton lint) and powdered microcrystalline cellulose (MCC) were purchased from Sigma-Aldrich, Germany.
Bacterial cellulose was a kind gift from Wong Coco(s) Pte. Ltd. Unless otherwise specified, all other chemicals were of reagent grade and were used without further purifications. Bacterial cellulose (BC) was received as wet pellicles after the fermentation. Individual pellicles were washed with deionised (DI) water before subsequently being boiled in deionised water for 20 min. The pellicles were heated in
0.5N NaOH held at 80 °C for 2 hours. After the desired period of time, fresh NaOH was used to replace the previous solution and the process was repeated until no yellowish colour was observed from the NaOH solutions with the BC pellicles being transformed into white colour. Eventually, the pellicles were boiled in DI water for another 15 minutes before thorough rinsing with DI water until neutral pH was attained, and the purified BC pellicles were subsequently lyophilized.
Cuprammonium hydroxide (CUAM) solution was used to dissolve the celluloses prior to ultrasonication. Details of CUAM preparation has been reported previously in section 3.2.1. Homogeneous solutions of 0.1% (w/v) BC and PC were prepared in CUAM by stirring of the celluloses in CUAM at ambient temperature for
2 h. Ultrasonication (Elmasonic S 60H, ELMA, Germany) of both materials were performed at a frequency of 37 kHz and effective ultrasonic power of 150 W for the time period of 5, 10, 15, 30, 60 and 90 min at ambient temperature. After the desired period of time, BC and PC were recovered from CUAM solution by the addition of 1
100 M H2SO4, which led to the formation of white precipitate. The precipitate was filtered under vacuum, washed with DI water until the filtrate became neutral to methyl red, extensively dialysed and finally lyophilized to produce neutral, powdered, sonicated cellulosic materials. Control sample of the ultrasonication process was prepared in the same manner except without subjected to ultrasound irradiation. Triplicate sample preparation was done, and a total of 42 samples (including control samples, for both bacterial and plant cellulose) have prepared.
4.2.2 Size Exclusion Chromatography (SEC)
The SEC system with manual injector included a Waters 515 HPLC Pump and a Waters 2414 refractive index (RI) detector (Waters Corporation, Milford, USA).
Prior to injection, samples and mobile phase (DMAc / 0.5% LiCl) were filtered through a PTFE membrane filter. The injection volume was 250 l, separation was performed on a series of three Phenogel mixed bed columns (300 × 7.8 mm; particle size 10 m) at 65°C, and at the flow rate of 0.8 ml/min. Each sample run lasted for 50 min. Nine pullulan standards of narrow polydispersity, i.e., 788k, 404k, 212k, 112k,
47.3k, 22.8k, 11.8k, 5.9k and 0.67k Da (Polymer Laboratories, Church Stretton, UK) were used to calibrate the columns. The linear coefficient of determination (r2) between the weight average molecular weight ( Mw ) of the standards and elution time was 0.999. Data acquisition and processing were done by using Waters Empower data software Version 5.00.
4.2.3 Calculation of Rate Coefficient
Equation 4.1 was used to determine the rate coefficient (k1) of ultrasound depolymerisation of cellulose in CUAM:
101 1 1 = + k1t (4.1) Mt M0
By plotting 1/ Mw (of each sample) versus time of ultrasonication (t), the slope of the regression line will give the rate coefficient (k1).
For comparison purpose, Equation 4.2 was also employed to determine the rate coefficient (k2).
1 1 = + k t (4.2) 2 2 2 M t M0
2 By plotting 1/ Mw (of each sample) versus time of ultrasonication (t), the slope of the regression line will give the rate coefficient (k2).
4.2.4 X-Ray Powder Diffraction Studies (XRPD)
X-Ray powder diffraction (XRPD) patterns were obtained using a SIEMENS
D5005 X-Ray Diffractometer (Bruker AXS, Karlsruhe, Germany) equipped with Cu-
Kα radiation. Samples were pressed into pellet and held in a 20 mm diameter sample holder. An accelerating voltage and current of 40 kV and 40 mA, respectively, and scan rate of 0.8° min–1 were employed. The diffractograms were recorded in a 2θ range between 5° and 45°, and subsequently analysed using the Bruker Advanced X-
Ray Solutions software, DIFFRACplus Evaluation (Eva), version 10.0 revision 1.
Diffraction profiles of the samples were fitted with a Gaussian function to resolve into separate diffraction peaks before integration was subsequently performed on each peak. All samples were resolved into 3 peaks corresponded to the 101, 10Ī and 002 crystal plane, except for MCC that had an additional 040 crystal plane.
Crystallinity index (CrI) of the samples were determined by following the method reported by Wang et al. (2007) as follows (equation 4.3), with slightly modifications:
102 ACrystal CrI = 100 (4.3) ATotal where ACrystal is the sum of the areas under the crystalline diffraction peaks and ATotal represents the total area under the diffraction curve between 2θ = 5° and 45°.
4.2.5 Fourier Transformed Infrared (FTIR) Spectroscopy Analysis
FTIR spectra of curdlan samples were obtained with a Spectrum One FTIR
Spectrophotometer (Perkin-Elmer, Norwalk, USA) using a KBr pellet. Each sample was scanned once with a resolution of 4.00 cm–1, within the range of 4000 cm–1 to 400 cm–1. Samples within same sonication duration, i.e. triplicate samples of PC-60, having spectra that is superimpose. Therefore, only single spectrum from each sonication time was presented. Detailed sample preparation steps have been reported in Chapter 3 section 3.2.5.
4.2.6 Statistical Analysis
Where necessary, the test data were statistically analyzed by one-way
ANOVA (for comparing more than two means) using SPSS (Statistical Package for
Social Science) for Windows Version 17.0.0 (SPSS Inc., Chicago, Illinois). Duncan test was also carried out to perform comparison of means at the 5 % significance level.
4.3 RESULTS AND DISCUSSION
4.3.1 Molecular Weight Profile of Ultrasonicated Cellulose
103 Ultrasound is capable to induce cavitation of bubbles in solution. This in turn generates transient elongational flow that is sufficient to cleave (14)--D glycosidic linkages of the cellulose backbone, resulting in depolymerisation and subsequent reduction of molecular weight. Figure 4.1 illustrates the change of weight average molecular weight ( Mw ) of the celluloses in cuprammonium hydroxide (CUAM) solution over the course of ultrasonication. The figure clearly reveals that for both plant cellulose (PC) and bacterial cellulose (BC), a continuous reduction in Mw can be observed when ultrasonication progresses. It also confirms that ultrasound had significantly (p < 0.05) reduced the Mw of both BC and PC, as compared to the control sample. BC has a higher initial Mw , showing a significant reduction in molecular weight at the beginning of ultrasonication compared to PC. This is in agreement with previous findings that polymer chain scission caused by ultrasonic degradation occurs more rapidly for polymers with higher molecular weight (Caruso et al., 2009). Similar results with detailed discussions have been reported previously in section 3.3.1, confirming that depolymerisation of celluloses (PC and BC) has happened.
Nevertheless, in contrast to the previous results, current observations also reveal that for both samples, Mw reduction proceeds and then levels off asymptotically approaching a final value at the end of the ultrasonication period. This is when the sample is said to reach a level-off degree of polymerization (LODP) or the limiting molecular weight (Mlim). For both BC and PC, the Mlim reported is ~47 and ~46 kDa, respectively, after 60 min of ultrasonication. It can be found in the literature that for ultrasonication of other cellulose derivatives, such as cellulose nitrate (Marx-Figini, 1997), hydroxyethylsulfoethyl cellulose, sulfoethyl cellulose and
104
Figure 4.1 Change of weight average molecular weight ( Mw ) of the celluloses in cuprammonium hydroxide (CUAM) solution over the course of ultrasonication. Values are means ± standard deviation (n=3). Data points within a sample with the same alphabet are not significantly different (p > 0.05).
carboxymethylsulfoethyl cellulose (Schittenhelm and Kulicke, 2000), level-off DP could be observed within the ultrasonication time ranging from 60 min to 240 min.
However, for native non-derivatized cellulose ultrasonicated in DMAc / 0.5% LiCl studied by Striegel (2007), the sample did not reach the limiting molecular weight, even after 780 min of ultrasonication. It is claimed that it is not possible to reach Mlim within the experimental time frame. This suggests that CUAM is a more appropriate solvent in ultrasound depolymerisation of cellulose, since current set up has successfully led to the LODP of both PC and BC, which are materials of native non- derivatized cellulose.
105 Meanwhile, Mlim obtained for both PC and BC is almost analogous, though with a different initial Mw . This is because both cellulose samples possessed identical chemical structure, that is, (14)-β-D-glucan. Current results are consistent with the findings reported by Schittenhelm and Kulicke (2000) that carboxymethylsulfoethyl cellulose has a higher initial molecular weight (473 kDa) than sulfoethyl cellulose
(286 kDa) and that after ultrasonication, both of them achieve similar Mlim of 42 and
36 kDa respectively.
It has been generally agreed that beyond Mlim further ultrasonication will not induce subsequent degradation (Striegel, 2007; Caruso et al., 2009). According to
Gronroos and co-workers (2004), when a polymer has a molecular weight that is similar to or lower than the Mlim, the polymer chain will follow the ultrasonic wave vibration upon irradiation being unable to degrade further. Caruso and co-workers
(2009) further explained that below Mlim, ultrasonic wave could not introduce the minimum force that is required for bond cleavage onto the polymer backbone which is too short. Consequently, no depolymerisation will occur below Mlim. According to the revised path theory proposed by Striegel (2007), in order for ultrasound degradation of a polymer to happen, a minimum continuous ‘path length’ within the polymer backbone is needed. Moreover, this path must have sufficient chain flexibility and conformational freedom. This obviously explains why below Mlim, a polymer chain will no longer have a minimum continuous ‘path length’ to further degrade.
According to Caruso and co-workers (2009), the presence of Mlim in ultrasonicated products indicates that ultrasonication is a molecular weight- dependence process. Moreover, this also provides strong evidence that ultrasound
106 bond cleavage is mechano-chemical in nature and not a result of ultrasound induced temperature and / or pressure fluctuation. If degradation is caused by temperature and
/ or pressure fluctuation, Mlim will not be observed within the process of ultrasonication and degradation will continue unabated due to continuous changes of temperatures and / or pressures.
4.3.2 Kinetics of Cellulose Degradation via Ultrasonication
By plotting 1/ Mw versus time of ultrasonication (Figure 4.2), the slope of the regression line will give the rate coefficient (k1) of this mechanism. Data points of 90 min ultrasonication were omitted since there was no degradation from 60 min to 90 min of ultrasonication, as discussed in the previous section. As shown in Figure 4.2,
-7 -7 -1 -1 rate coefficient (k1) of PC and BC was 1.94 × 10 and 2.46 × 10 Da min , respectively. This implies that degradation of BC is faster than for PC under the same reaction condition. The result is consistent with the literature that polymers with higher molecular weight will degrade faster (Lii et al., 1999; Wu et al., 2008). Caruso et al. (2009) have also pointed out that the rate coefficient increases with increasing molecular weight of the polymer.
Nevertheless, as shown in Figure 4.2, it seems that coefficient of BC (R2 =
0.9154) is slightly low. In order to investigate other possible degradation kinetics that could better represent the degradation of BC, Figure 4.3 was constructed by plotting
2 1/ Mw versus time of ultrasonication. It was found that the liner relationship of
2 2 1/ Mw versus t provide a better liner correlation (R = 0.9892) between these two parameters, as revealed in Figure 4.3. This could due to the high initial molecular weight of the sample, similar to the work reported by Wu et al. (2008) on high Mw chitosan.
107
Figure 4.2 Variation of 1/ Mw as a function of ultrasonication time (min).
2 Figure 4.3 Variation of 1/ Mw as a function of ultrasonication time (min).
108
Bacterial cellulose being chemically identical to plant cellulose is known to have a more ordered ultrafine fibre structure compared to native plant cellulose
(Bielecki et al., 2002). Before the BC solution was subjected to ultrasonication, it was fully dissolved in CUAM. However, the conformation of BC in CUAM is not known to date nor is available in the literature, due to the high volatility of CUAM that precludes detailed elucidation of its conformation in this solution. Bearing this in mind, it could be postulated that the cellulose backbone of BC still forms micro- aggregates of smaller crystalline domains in CUAM that have high resistance to the ultrasound degradation. According to Caruso et al. (2009), the chain scission rate relates to polymer chain dimensions (conformation) in solution. As a result, although the initial Mw of BC is significantly higher then that of PC, its rate of degradation is not much higher than the latter.
Several equations have been used in the literature to describe the ultrasonic degradation of polysaccharides and to determine the rate coefficient of the reaction.
Basically, most equations are generated based on the assumption that ultrasound depolymerisation is a first order reaction. Earlier publications (Price et al., 1994) suggested the incorporation of limiting molecular weight (Mlim) into the degradation kinetics as shown below:
1 1 1 1 M lim ln = ln – k t (4.4) M lim M t M lim M i cm0 where Mlim is the limiting molecular weight, Mi is the initial value and Mt the molecular weight at the degradation time, t, and m0 the molecular weight of the repeating unit (monosaccharide) in solution with concentration c. Lately, Mlim has been omitted and a simplified equation has been proposed to account the kinetics of
109 ultrasound degradation. According to Striegel (2007), removing Mlim in computing the degradation kinetics will eliminate the possible error caused by uncertainty in the determination of Mlim. In addition, to be able to utilise equation 4.4, the Mlim reading has to be smaller than Mw reading at any reaction time. As a result, it is better to use equation 4.1 and 4.2 and it was chosen to represent the degradation kinetics in the present work.
4.3.3 X-ray Diffraction Profile and Crystallinity Index (CrI)
Figure 4.4a depicts the X-ray diffraction patterns of bacterial cellulose sonicated at different time periods (0, 5, 10, 15, 30, 60 and 90 min). For plant cellulose, similar diffraction profiles were shown for each time period of sonication
(Figure 4.4b). The diffraction profile of MCC was used as a comparison. After the deconvolution process, three major diffractions peaks at 2θ = 12.0°, 20.1° and 21.6° were obtained for both BC and PC samples. The results were in a good agreement with the findings from previous chapter, indicating all sonicated samples existed in the Cellulose II crystal conformations. Current results also unveil that prolonged ultrasonication did not alter the crystal conformation of both bacterial and plant cellulose. Native cellulose exhibits Cellulose I conformation, but dissolution and regeneration of cellulose from CUAM resulted in the transformation of Cellulose I to
Cellulose II crystal allomorph. Detailed explanation of this process has been discussed previously in Chapter 3.
As illustrated in Figure 4.4, the difference in diffraction profiles for each sample over the course of ultrasonication was less visible. However, subsequent integration revealed that the crystallinity index (CrI) for BC and PC had slightly
110
Figure 4.4 X-Ray diffraction patterns of (a) bacterial cellulose and (b) plant cellulose sonicated at different time periods, as compared to MCC.
111
Table 4.1 Crystallinity index (CrI) of bacterial cellulose (BC) and plant cellulose (PC) ultrasonicated for different times.
Ultra- Ultra- Sample sonication CrI (%)1 Sample sonication CrI (%)1 Time (min) Time (min)
MCC – 90.7 ± 0.6 BC-0 0 64.6 ± 0.4a PC-0 0 65.7 ± 0.5a BC-5 5 66.0 ± 0.2b PC-5 5 66.3 ± 0.5b BC-10 10 68.2 ± 0.6c PC-10 10 68.3 ± 0.3c BC-15 15 69.7 ± 0.4d PC-15 15 68.6 ± 0.3c BC-30 30 70.5 ± 0.2e PC-30 30 69.5 ± 0.1d BC-60 60 71.4 ± 0.2f PC-60 60 70.4 ± 0.4e BC-90 90 71.3 ± 0.1f PC-90 90 70.5 ± 0.1e 1Means ± standard deviation (n = 3). Means with the same superscripts in the same column are not significantly different (p > 0.05)
increased as a function of ultrasonication time, as summarised in Table 4.1. Statistical analysis reveals that ultrasonication significantly (p < 0.05) increase the CrI of both
PC and BC. The increment stops at samples sonicated for 60 min; i.e. no significant (p
> 0.05) increment was observed between BC-60 and BC-90 or PC-60 and PC-90. This is in correlation with the Mlim values of both samples, i.e., the sonicated fragments attain their Mlim after 60 min of ultrasonication.
By comparing the profiles of molecular weight reduction (Figure 4.1) and the
CrI increment pattern, it can be said that when the molecular weight is reduced during ultrasonication from 0 to 60 min, the CrI increases significantly (p < 0.05). After the samples reached their Mlim at 60 min and 90 min of ultrasonication, no further depolymerisation occurred and similarly no significant changes of CrI observed (p >
0.05). This outcome leads to the conclusion that CrI changes were affected by the
Mw profiles. A recent finding by Öztürk et al. (2009) also reveals a similar trend:
112 Mw reduction of cellulose (lyocell fibres) treated with NaOH and simultaneously CrI increment. The authors explained that NaOH treatment resulted in mild depolymerisation and thus Mw reduction; predominantly the amorphous region was removed during depolymerisation, and thus CrI increment in the treated samples.
Ultrasonication of celluloses resulted in depolymerisation and thus molecular weight reduction. During the regeneration process after ultrasonication, fragments with short molecular weight will rearrange differently than the longer counterparts in the crystallite producing distinct CrI values. During the dissolution of cellulose into
NaOH, the crystallite size will decrease as a function of dissolution time until a minimum point is reached due to the gradual disappearance of the Cellulose I allomorph (Wang and Deng, 2009). Subsequently, the crystallite size will slowly increase indicating the formation of the Cellulose II allomorph. Similar findings were reported by Shibazaki et al. (1997), explaining the development of crystallinity in regenerated bacterial celluloses that were subjected to a prolonged NaOH treatment.
These results suggest that ultrasonication-induced depolymerisation exposes BC to
OH- from CUAM, which results in incremental CrI contents of cellulose in the present study.
4.3.4 Fourier Transform Infrared Analysis (FTIR)
FTIR spectroscopy is a fast and convenient way to check the functional group of a macromolecule, hence the important structure of the molecule. Figure 4.4 and
Figure 4.5 illustrates the FTIR spectrum of ultrasonicated bacterial cellulose and plant cellulose samples obtained in present work, respectively. Similar results were obtained in Chapter 3; detailed explanation of major peaks observed and relevant functional groups have been discussed in section 3.3.4. As illustrated in Figure 4.4, all
113 spectrum almost super imposable. In addition, the presence of peaks around 890 cm–1 for all samples indicated that sonicated samples still remain as (14)--D-glucan (Oh et al., 2005). This is the most significant information obtained from current FTIR results as it implies that prolonged ultrasonication of celluloses in CUAM (> 60 min) did not alter the molecular structure, particularly polymeric sequence of the sonicated cellulose.
Figure 4.5 FTIR spectra of bacterial cellulose ultrasonicated at different times, and compared to that of MCC.
Meanwhile, it has been reported by Striegel (2007) that prolong ultrasonication (up to 780 min) will not induced other chemical reaction that resulted in undesirable modification of the cellulose backbone. However, the previous work by
114 Striegel (2007) employed DMAc / LiCl as the solvent for cellulose whereas current work utilised CUAM, a solvent with high hydroxyl ion (OH–) content. Even though it is known that cellulose is generally stable in CUAM, it was uncertain that prolonged ultrasonication may trigger any side reactions, such as -elimination and “peeling reaction” during pulping of cellulose in alkaline condition (Klemm et al., 2006).
Anyway, results from FTIR have confirmed that no other undesirable reactions happen, besides, depolymerisation. The chemical purity of prolonged sonicated samples has also been confirmed by thermogravimetric analysis (TGA). Sonicated samples and native samples are having similar degradation profile, peak onset temperature, Te (°C) and peak maximum temperature, Tmax (°C) (data not shown).
Figure 4.6 FTIR spectra of plant cellulose ultrasonicated at different times, and compared to that of MCC.
115
4.4 CONCLUSION
Current investigation demonstrated the effect of prolonged ultrasonication on the molecular weight profile and crystallinity index of cellulose. Ultrasonication significantly (p < 0.05) reduced the weight average molecular weight ( Mw ) of bacterial and plant cellulose. Limiting molecular weight (Mlim) of BC and PC was
~47 and ~46 kDa, respectively, after 60 min of ultrasonication. This confirmed the hypothesis that prolonged ultrasonication will lead to Mlim of both samples. Both BC and PC were not further degraded after 60 min of ultrasonication, confirming that the samples had reached their Mlim. Study of the kinetics of ultrasound degradation reveals that BC degraded slightly faster than for PC. A continuous increment of CrI was observed for both BC and PC. This increment stops at samples sonicated beyond
60 min, confirming the correlation of CrI to the molecular weight of these materials.
Results of FTIR confirmed the polymer backbone remained unaltered as (14)--D- glucan, as well as the chemical purity of the sonicated products.
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Preparation and anticoagulation activity of sodium cellulose sulfate.
International Journal of Biological Macromolecules, 41(4), 376-382.
Wu, T., Zivanovic, S., Hayes, D. G. and Weiss, J. (2008). Efficient Reduction of
Chitosan Molecular Weight by High-Intensity Ultrasound: Underlying
Mechanism and Effect of Process Parameters. Journal of Agricultural and
Food Chemistry, 56(13), 5112-5119.
119
CHAPTER 5
NOVEL SULFATION OF CURDLAN ASSISTED BY
ULTRASONICATION
120
5.1 INTRODUCTION
Polysaccharide modification has been an interesting subject to pursue with a view to achieving novel functionalized macromolecules. Searching for desirable non- conventional modification methods is always of contemporary interest, especially in connection with the emerging concept of green chemistry. Ultrasonication has been described as a useful aid in chemical processes to achieve the aim of waste and polluting-product reduction (Kardos and Luche, 2001), thus offering a promising alternative to currently established protocols.
Ultrasound is defined as a sound wave having a frequency higher than the human hearing range, i.e. > 20 kHz (Mason and Lorimer, 2002). Polymer depolymerisation via ultrasonication has been extensively studied throughout the years. For polysaccharides, studies have focused on chitosan (Kasaai et al., 2008), cellulose (Striegel, 2007) and its derivatives (e.g., cellulose nitrate (Marx-Figini,
1997), hydroxypropyl cellulose (Goodwin et al., 2011) and carboxymethylcellulose
(Gronroos et al., 2008)), starch (Luo et al., 2008), and water soluble polysaccharides including guar gum (Tayal and Khan, 2000), dextran (Cote and Willet, 1999), agarose and carrageenans (Lii et al., 1999). Nevertheless, these studies were limited to depolymerization experiments. A recent finding by Cízová and his team (2008) showed that by employing ultrasonication, the reaction time required for octenylsuccinoylation of carboxymethyl starch was significantly shortened. That seminal study provided fundamental insights on the ability of ultrasound to assist derivatization of polysaccharides.
Curdlan is a bacteria synthesized linear (13)-β-D-glucan obtained from
Alcaligenes faecalis var. myxogenes strain 10C3 (McIntosh et al., 2005). Since its
121 discovery by Harada in 1966 (Harada et al., 1966), it has received interest in food and non food related research due to its unique physicochemical properties. Curdlan possesses the ability to form ‘high-set’ thermo-irreversible gels and ‘low-set’ thermo- reversible gels at two distinctive temperatures, which is a unique type of behaviour compared to other heat-set gels (Nishinari and Zhang, 2004). In addition, (13)-β-D- glucan belongs to a group of compounds known as biological response modifiers that enhance or restore normal immune defences (McIntosh et al., 2005). Therefore, most non-food related studies were concentrated on immuno-modulation and anti-tumour activity of this bacterial exo-polysaccharide (Gao et al., 1997).
Since the early 1990s, studies have shown that sulfated derivatives of curdlan possess antiviral properties on top of antitumour ability (Yoshida et al., 1990; Yutaro et al., 1990). Thus, considerable amount of work has dealt with the synthesis of curdlan sulfate and the corresponding biological activity (Gao et al., 1997). To the best of my knowledge, curdlan and other β-glucan-sulfation processes involved the utilization of harmful chemicals such as pyridine-SO3 complex (Gao et al., 1997),
pyridine-chlorosulfonic acid (Yoshida et al., 1995) and / or piperidine-N-sulfonic acid
(Yoshida et al., 1990). Moreover, most of these reactions will generate hazardous by-
products such as pyridine. Clearly, it is of interest to seek alternative methods based on green chemistry considerations of producing curdlan sulfate, which is the subject of this chapter.
A few researchers have reported the hydrolysis of cellulose with 30% (v/v) sulfuric acid (Wang et al., 2007) or even more concentrated sulfuric acid (65%)
(Roman and Winter, 2004). This process had introduced the sulfate groups onto the
cellulose backbone, besides breaking down the polymeric chain. Therefore, it could
122 be hypothesised that treating curdlan with concentrated sulfuric acid could also achieve the sulfation process, which will be investigated in this chapter.
5.2 EXPERIMENTAL
5.2.1 Materials
Commercial curdlan samples were kindly donated by Takeda-Kirin Foods
Corporation, Tokyo, Japan. DMSO-d6 (99.96%) and spectroscopic grade KBr were purchased from Merck, Whitehouse Station, NJ, USA. Unless otherwise specified, all other chemicals were of reagent grade and were used without further purification.
5.2.2 Ultrasonication of Curdlan
5.2.2.1 Aqueous system
Curdlan was thoroughly suspended in 50% (v/v) sulfuric acid to form a 1 %
(w/v) suspension before being subjected to ultrasonication for 360 min at 40 °C, using an ultrasound cleaning bath (Elmasonic S 60H, ELMA, Germany) operate at frequency of 37 kHz and effective ultrasonic power of 150 W. Control samples were prepared in a similar way by suspending curdlan for 360 min at 40 °C, without subjecting to ultrasonication. The samples were subsequently neutralised, extensively dialysed and eventually lyophilized. The sonicated sample was denoted as 360_360, while the non-sonicated sample was denoted as 0_360. Samples were prepared in triplicate and the results reported are an average.
5.2.2.2 Organic solvent system
123
Mix solvent system was also employed in ultrasonication of curdlan. Isopropyl alcohol (2-propanol) and concentrated sulfuric acid were pre-freeze at -30 °C for 3 hours before used. Fifty ml of sulfuric acid was slowly pour into a container consist of
150 ml of isopropyl alcohol (IPA). The resulting 2 layers of solvent were slowly swirled to form a homogenous mixture and left at 15 °C for a few hours before used.
Curdlan was thoroughly suspended in the IPA: sulfuric acid mixture to form a 1 %
(w/v) suspension before being subjected to ultrasonication for 360 min at 15 °C.
Control samples were prepared in a similar way by suspending curdlan for 360 min at
15 °C, without subjecting to ultrasonication. The samples were subsequently neutralised, extensively dialysed and eventually lyophilized. The sonicated sample was denoted as 360_360-IPA, while the non-sonicated sample was denoted as 0_360-
IPA. Samples were prepared in triplicate and the results reported are an average.
5.2.3 Elemental Analysis of Curdlan
Lyophilized curdlan samples were analysed for sulfur content (%S) by elemental analysis using Elementar Vario Micro (Elementar, Analysensysteme,
Germany). Elemental analysis has been widely used lately for determination of %S of sulfated polysaccharides (Bae et al., 2009; Chen et al., 2010), due to the fast and rapid method as well as easier sample preparation when compared to chemical method.
Prior reduction of the sulfate groups were achieved in the instrument before subsequent combustion. Degree of sulfation (DS) was calculated according to the following equation (Bae et al., 2009):
%S 0.162 ( ) DS = 32 (5.1) 80 100 ( ) %S 32
124 where, %S is the sulfur content (%); 162 is the MW of monosaccharide unit, 80 is the
– MW of –SO3 , and 32 is the mass of sulfur atom. Results from aqueous and organic
solvent system will be compared; however, only samples with higher DS content were
subjected to the subsequent analysis.
5.2.4 Fourier Transformed Infrared (FTIR) Spectroscopy
FTIR spectra of curdlan samples were obtained with a Spectrum One FTIR
Spectrophotometer (Perkin-Elmer, Norwalk, USA) using a KBr pellet. Samples were
scanned with a resolution of 4.00 cm–1, within the range of 4000 cm–1 to 400 cm–1.
Detailed steps have been reported in Chapter 3 section 3.2.5.
5.2.5 Nuclear Magnetic Resonance (NMR) Spectroscopy
1H and 13C NMR spectra of curdlan samples were obtained with a Bruker
AV300 NMR Spectrometer (Bruker Biospin, Germany) operating at 300.13 MHz and
75.48 MHz, respectively, by employing DMSO-d6 as sample solvent. NMR experiments were performed at ambient temperature (~300 K). DMSO-d6 signal (δ =
2.50 ppm and δ = 39.5 ppm) were used as internal reference for 1H and 13C, respectively. For data acquisition, software 1D WIN-NMR was used.
5.2.6 Size exclusion chromatography (SEC)
Samples (0.03 g) were suspended in dry N,N-dimethyl acetamide (DMAc) for
1 hour at 40 °C, filtered and subsequently dissolved in 2.5 ml of DMAc / 8% LiCl for
24 hours at same temperature. Prior to injection, samples and mobile phase
(DMAc/0.5% LiCl) were filtered through a PTFE membrane filter. The SEC system with manual injector included a Waters 515 HPLC Pump and a Waters 2414
125 refractive index (RI) detector (Waters Corporation, Milford, USA). Separation was performed on a series of three Phenogel mixed bed columns (300 7.8 mm; particle size 10 m) at 65 °C, with flow rate of 0.8 ml/min. Nine pullulan standards of narrow polydispersity, i.e., 825 (American Polymer Standards Corporation, USA) 404, 212,
112, 47.3, 22.8, 11.8, 5.9 and 0.67 kDa (Polymer Laboratories, Church Stretton, UK) were used to calibrate the columns. For data acquisition, Waters Empower data software Version 5.00 was used.
5.2.7 Statistical Analysis
Where necessary, the test data were statistically analyzed by t test (for comparing two means) using SPSS (Statistical Package for Social Science) for
Windows Version 17.0.0 (SPSS Inc., Chicago, Illinois), at the 5 % significance level.
5.3 RESULTS AND DISCUSSION
5.3.1 Sulfur Content and Degree of Sulfation (DS)
Prior experiments have been conducted using different sulfuric acid concentrations (30, 40, 50, 60 and 70% v/v). It was found that at higher sulfuric acid concentration, the sample yield is significantly low, due to the extensive hydrolysis of curdlan. Meanwhile, at lower sulfuric acid concentrations, sulfation did not happen
(result not shown). Table 5.1 reveals the sulfur content and DS of curdlan sulfate prepared in the current study. The extent of sulfation for all samples is relatively low compared with previous work, as deduced from the elemental analysis results. The highest DS value obtained for curdlan sulfate in the present study was 0.115 0.005.
126
This means for every 1,000 units of β-D-glucopyranosyl, there are about 115 sulfate groups present as a substituent. Previous studies have shown that curdlan sulfate prepared by the conventional chemical method showed a minimum %S of 5.6%
(Yoshida et al., 1990) and a maximum of 20% (Yoshida et al., 1995), which corresponding to DS of 0.35 and 3.0, respectively.
It seems that conventional chemical synthesis is in favour when a high DS of sulfated curdlan is required. However, conventional chemical syntheses (i.e. utilising pyridine-SO3 complex) could be quite tedious; some requiring many hours of reflux
and / or multi-step preparations, in addition to the generation of harmful waste
(pyridine). A recent report published by Bae and his team (2009) on the sulfation of
citrus pectin using chlorosulfonic acid, yields %S values comparable to ours (i.e.
2.68). This could imply that sulfation with conventional chemicals does not warrant
the high DS value; other reaction parameters also played an influential role in
controlling the DS value, which could possibly be optimised. Meanwhile, Yoshida et
al. (1995) had commented that an excessive sulfating agent is required in order to
obtain a higher DS for curdlan sulfate. Moreover, Wang et al. (2005) conducted their
sulfation process using sulfuric acid and n-propanol in the ratio of 1:1, and the DS
obtained was not satisfactory. Current work used a much lower sulfuric acid ratio
(organic system: sulfuric acid: IPA = 1:3; aqueous system: sulfuric acid: H2O = 1:1),
therefore this could contribute to the lower DS in present work.
Nevertheless, the process of ultrasonication has significantly (p < 0.05)
increased the DS value of samples sonicated under aqueous system (360_360) by
almost fourfold as revealed in Table 5.1. Meanwhile, for samples sonicated in isopropyl alcohol (360_360-IPA), the DS obtained is also higher than the non- sonicated counterpart. Moreover, it might be possible to obtain a higher degree of
127 sulfation by employing systematic optimisation processes such as response surface methodology. Therefore, it seems promising to utilise sulfuric acid in aqueous reaction systems to prepare curdlan sulfate.
Table 5.1 Sulfur content, degree of sulfation (DS) and yield of curdlan sulfates.
Sulfur content Degree of sulfation Yield (%) Sample (%)1 (DS)1
Curdlan < 0.50 0a 80 12; 89 13 0_360 0.58 0.02 0.030 0.001b 26 3 360_360 2.15 0.09 0.115 0.005c 18 2 0_360-IPA 0.71 0.12 0.037 0.006d 43 2 360_360-IPA 1.25 0.08 0.065 0.004e 29 1 1 Means of readings standard deviation (n=3). Means with the same superscripts in the same column are not significantly different (p > 0.05). However, comparison of means are not made between aqueous and IPA system. 2 Curdlan (control) recovered from aqueous system. 3 Curdlan (control) recovered from organic solvent system.
It has been confirmed that curdlan will remain in a triple helical structure in a
dilute aqueous solution of NaOH (< 0.25 N), while fully dissociating into a random
coil only in DMSO or DMSO/LiCl (Nakata et al., 1998; Futatsuyama et al., 1999).
Thus, curdlan could still retain a similar triple helical structure in an IPA system. This
could result in less efficiency of the sulfation process, since a triple helical structure
will result in less exposure of the available hydroxyl groups for substitution.
Therefore, using DMSO as a solvent could possibly improve the DS obtained.
Nevertheless, using an organic solvent, especially a harmful reagent, is not in
accordance to the current objective of “green chemistry”. On the other hand, sulfation of curdlan in present studies seems to proceed homogeneously under an aqueous sulfuric acid system, since it was fully dissolved and no residue was retained after filter with 0.2 µm pore size PTFE membrane. Nonetheless, it was unclear whether the
128 curdlan polymer preserves its triple helical structure while dissolved in the acidic condition, thus with relatively lower DS obtained.
Moreover, utilising a high-intensity ultrasonicator could possibly improve the
DS obtained in present work. According to the report obtained by Cravotto et al.
(2005) on carboxymethylation of chitosan, they employed a cup-horn sonicator operated under high intensity and successfully achieved carboxymethylation in one hour compared with conventional methods that required six hours. In addition, Wang and colleagues (2009) have confirmed that high-intensity ultrasound managed to double the DS of p-acetamidobenzoylate chitosan. These findings suggested that a high-intensity ultrasonicator is a better alternative in ultrasound-assisted polysaccharides derivatisation, compared with a conventional ultrasonicator bath used in present studies.
5.3.2 Fourier Transform Infrared (FTIR) Spectroscopy
As mentioned in section 5.2.3, subsequent study will be continued with curdlan sulfate having higher DS, which are the samples obtained from aqueous reaction system (0_360, 360_360). Figure 5.1 shows the FTIR spectra of various curdlan samples. All samples showed an absorption peak at around 890 cm-1, which is
the characteristic of β-glucan (Sandula et al., 1999; Ma et al., 2008). This confirmed
that ultrasonication of curdlan under concentrated sulfuric acid (50% v/v) did not alter
the polysaccharide backbone. Presence of peak around 1644 cm-1 indicates the existence of absorbed water in all samples, as been reported in previous study (Jin et
al., 2006).
Both acid treated samples (0_360 and 360_360) exhibit an absorption peak at
1249 cm-1, which is ascribed to the asymmetrical S=O stretching vibration (Vikhoreva
129 et al., 2005; Bae et al., 2009). The presence of a peak at 1060 and 1061 cm-1 for
360_360 and 0_360, respectively, represent the symmetrical S=O stretching vibration
(Vikhoreva et al., 2005). These spectra confirmed that acid and ultrasonication treatments have incorporated sulfate groups into the curdlan backbone. Results from elemental analysis (Table 5.1) suggested that the sulfate-group presence is relatively low compared to other chemical moieties, thus peaks attributed to sulfate groups will be less pronounced and slightly overlapping with other chemical fingerprints.
Figure 5.1 FTIR spectra of curdlan samples: Curdlan, 0_360 (acid treatment only) and 360_360 (acid + ultrasonication).
5.3.3 NMR Spectroscopy
It is generally agreed that 1H NMR spectra of curdlan sulfate are rather
difficult to analyse in detail due to the heavily overlapping chemical shifts in the
130 region between 3.0 to 5.5 ppm (Yoshida et al., 1995). Polysaccharides are relatively high in MW, causing the chemical shifts overlapped and become broader. Figure 5.2 reproduces the 1H NMR spectra of curdlan samples. The residual water signal can be
seen at around 3.35 ppm for all samples. The proton signal that appears at 4.50 ppm
for curdlan (see also Table 5.2) is ascribed to the anomeric H1 chemical shift (Ensley
et al., 1994; Mo et al., 2009). Similar signals were recorded for the spectra of sulfated
curdlan (0_360 and 360_360), an outcome that confirms that the modified species
remain in (13)-β conformation, which also in accordance to the results of FTIR in
previous section.
Sulfation of curdlan in current study is expected to happen randomly on any
available hydroxyl groups on the -D-glucopyranosyl unit; since no protecting groups
were substituted to a particular hydroxyl group (to prevent sulfate group substitution)
prior to sulfation. However, hydroxyl group at C-6 (OH-6) is expected to be the
preferred position to be substituted compared to hydroxyl group at C-2 and C-4
(Figure 5.3). This is because, being a primary hydroxyl group, OH-6 has a relatively
lower steric hindrance and more accessible by the sulfating agent (Yoshida et al.,
1990). As a result, integration of the chemical shift of OH-6 could provide useful
information for the occurrence of sulfation.
Integration of the proton signal of the hydroxyl group which is attached to C-6
(OH-6) of the -D-glucopyranosyl unit shows a decrease in the intensity after
sulfation, as shown in Table 5.2 for 360_360 in relation to the intensity of H-1, which
is set to 1.0. This further confirms that sulfation has occurred, since esterification of
the sulfate group with OH-6 reduces the total amount of protons present in this group.
No obvious change was observed for the integration intensity of 0_360 owing to the
lower DS value compared to 360_360. As for curdlan, the intensity of H-1 and OH-6
131
Figure 5.2 300.13 MHz 1H NMR spectra of curdlan samples: Curdlan, 0_360 (acid treatment only) and 360_360 (acid + ultrasonication).
132 is suppose to be same, since there is no substituent present at OH-6 and there is only a single proton each present at C-1 and OH-6 respectively.
Table 5.2 Characteristic chemical shifts assignments (ppm), and integration intensity of 1HNMR signals for curdlan samples.
Chemical shift (ppm)a (Integration intensity)b Sample H-1 OH-6
Curdlan 4.50; (1.00) 5.20; (1.01) 0_360 4.51; (1.00) 5.41; (0.99) 360_360 4.51; (1.00) 5.36; (0.86) a Only characteristic protons chemical shifts are shown here b Intensity of H-1 was set at 1.00.
OH OH O CH CH 2 O 2 O HO S OH HO HO O O O OH OH
n 40 oC, 6 h Curdlan
- O OSO
OH O CH CH 2 O 2 O HO HO O O OH OH n
Curdlan sulfate
Figure 5.3 Sulfation of curdlan assisted by ultrasonication. Substitution happened preferably at position OH-6. n indicates the number of repeating disaccharide units (laminaribiose) in a given curdlan chain.
Figure 5.4 illustrates the 13C NMR spectra of curdlan samples. Clearly, the
spectra unveil the presence of six chemical shifts for each sample, which are
attributed to carbon atoms from positions one to six of the -D-glucopyranosyl unit of
133 the curdlan backbone. This confirms that ultrasonication in sulfuric acid neither altered the chemistry of the glucan backbone nor destroyed the -D-glucopyranosyl unit. Chemical shifts of untreated curdlan are in good agreement with results in the literature (Delattre et al., 2009; Tamura et al., 2009). Chemical shifts of the sulfated samples (0_360 and 360_360) did not significantly different from that of the original curdlan sample shown in Table 5.3, which is in accordance with the modest degree of sulfation for both samples (Table 5.1). Previous work by Williams and co-workers utilising chemical synthesis reported no obvious change in the 13C NMR chemical
shifts for both original and sulfated (13)-β-D-glucan even with a %S as high as 5.69
(Williams et al., 1992).
Table 5.3 Chemical shifts (ppm) assignments of 13C NMR signals for curdlan samples.
Chemical shift (ppm) Sample C-1 C-2 C-3 C-4 C-5 C-6
103.06 72.90 86.20 68.43 76.40 60.88 Curdlan 103.60a 73.85a 86.73a 68.82a 76.64a 61.35a 0_360 103.04 72.84 86.20 68.42 76.40 60.87 360_360 103.10 73.10 86.20 68.50 76.50 61.20 aReported by Delattre et al. (2009)
Meanwhile, a downfield shifting of signal (i.e. to higher ppm values) for the
carbon with the hydroxyl group that covalently links to sulfate group is anticipated in
an ideal situation, as argued for sulfated curdlan (Gao et al., 1997) and (13)-β-D-
glucan (Sun et al., 2009) samples. This is due to the electron withdrawing ability of
– the sulfated group (–SO3 ) producing a de-shielding effect on the adjacent carbon.
According to Gao and colleagues (1997), however, an obvious shift of the signal is
134
Figure 5.4 75.48 MHz 13C NMR spectra of curdlan samples: Curdlan, 0_360 (acid treatment only) and 360_360 (acid + ultrasonication).
135 only visible when the particular hydroxyl group is fully substituted by a sulfate group, hence rationalising the non-obvious shifting in this work, i.e. the relatively low degree of sulfation obtained presently. Moreover, in the work reported by Yoshida et al.
(1995) on curdlan sulfation, they commented that incomplete downfield shifting of C-
6 signals (δ = 69.5) will happen on partially sulfated curdlan; which means there will be a signal remain in the original position (δ = 63.0) that resemble the “non- substituted C-6”. As a final comment, curdlan sulfate obtained in present study did not undergo other unexpected reaction though current reaction was performed under a relatively harsh condition of 50% v/v sulfuric acid. This could be deduced by confirming the absence of significant shift beyond 110 ppm or before 25 ppm (data not shown), as well as being verified from the FTIR spectra.
5.3.4 Size Exclusion Chromatography (SEC)
Table 5.4 shows the molecular weight of curdlan and its sulfated derivatives obtained in present study. Molecular weight reduction of the sulfated derivatives are expected, due to depolymerisation of the (13)-β-D-glucan, caused by ultrasonication and / or acid hydrolysis. Sample 360_360 (curdlan sonicated in 50% v/v sulfuric acid) has the smallest MW (49.0 1.9 kDa) among the samples, due to the combine effect of both treatment. Sulfated polysaccharide, particularly (13)-β-
D-glucan, with lower molecular weight could be more favourable in clinical application. According to Alban and Franz (2001), linear (13)-β-D-glucan sulfate with the sulfation position occurred at C-2 and C-4 and MW between 18 and 50 kDa, found to be most suitable as heparin alternative in vivo.
Ultrasonication has been employed to depolymerise β-glucan since long time ago, as being outlined in Chapter 2 section 2.4.2. Nevertheless, sonication of β-glucan
136 in water usually takes longer time, up to 16 hours (Ma et al., 2008) or 24 hours
(Nakata et al., 1998), with only about nine-fold and ten-fold of MW reduction, respectively. Present work manages to achieve MW reduction of curdlan by one order of magnitude within 6 hours of sonication time in sulfuric acid, as well as obtained sulfated derivative of curdlan. Therefore, the sulfation method employed in present study is of application interest, since it can achieve sulfation and depolymerisation at the same time.
Table 5.4 Molecular weight for curdlan samples.
Sample Molecular weight (kDa)1
Curdlan 806.7 10.7a 0_360 75.6 13.8b 360_360 49.0 1.9b 1 Means of readings standard deviation (n=3). Means with the same superscripts in the same column are not significantly different (p > 0.05).
5.4 CONCLUSION
An alternative, non-conventional method has been developed to prepare curdlan sulfate assisted by ultrasonication. Ultrasonication has significantly (P < 0.05) increased the DS value of samples sonicated under aqueous system (360_360) by almost four fold. Structural elucidation via FTIR and NMR confirmed the presence of sulfate group in the modified curdlan, and the polysaccharide backbone was unaltered.
In conclusion, current work contributes to a better understanding on sonochemical process of polysaccharides modification, particularly, beyond conventional depolymerisation.
137
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142
CHAPTER 6
SUMMARY AND CONCLUSIONS
143 In order to elucidate the effectiveness of ultrasound in the modification of polysaccharides, namely cellulose and curdlan, ultrasonication of both samples has been carried out using different approaches. Firstly, ultrasound depolymerization of plant and bacterial cellulose has been done in cuprammonium hydroxide (CUAM) solution and its physicochemical properties were subsequently characterized. On the other hand, sulfation of curdlan was assisted by ultrasonication to develop a novel environmental friendly method in preparing curdlan sulfate. It is hypothesised that ultrasonication able to reduce the molecular weight of cellulose and curdlan; in addition, to aid in sulfation of curdlan.
In Chapter 3 and 4, crystalline plant cellulose (PC) and bacterial cellulose (BC) have been recovered from cuprammonium hydroxide (CUAM) solution after ultrasonication. It was found that ultrasonicated PC and BC possessed lower molecular weight (MW), but did not change chemically as shown in thermogravimetric analysis
(TGA) profile. FTIR spectra further affirmed that different ultrasonication durations did not impose any obvious change on the absorbance band for both PC and BC. Thus, it was confirmed that the ultrasonicated sample did not change chemically, which remained as
(14)-β-D-glucan. In Chapter 4, prolonged ultrasonication (up to 90 min) resulted in
Mlim of both PC and BC, suggesting CUAM is a better solvent compared to DMAc /
0.5% LiCl for ultrasonication experiments. Moreover, it is relatively easy to recover sonicated cellulose from CUAM. A continuous, significant (p < 0.05) increment of crystallinity index (CrI) was observed for both ultrasonicated BC and PC. The increment stops at samples sonicated beyond 60 min. This interesting result offered a new potential application for the sonicated crystalline cellulose in bio-nanocomposite. Kinetics of the cellulose degradation unveiled that BC degrades slightly faster than PC.
144 The current study has successfully offered a new avenue in the depolymerization of cellulose. In addition, it is the first time to report the characterization of ultrasonicated cellulose in solid state. The present work has also suggested that ultrasonication capable of producing crystalline cellulose that is comparable to microcrystalline cellulose (MCC).
MW reduction in PC and BC as reported in both Chapter 3 and 4 has re-affirmed the first hypothesis of present thesis.
Even though the current study has depolymerised native, non-derivatised plant and bacterial cellulose via ultrasonication in cuprammonium hydroxide (CUAM) solution, there are still some technical complications when dealing with CUAM. As mentioned earlier in section 3.3.1, CUAM is a volatile solvent and it is less favourable to perform ultrasonication in a volatile solvent. Moreover, as being mentioned in the literature review, cellulose, being highly crystalline and having extensive inter and intra molecular hydrogen bonding, could not be dissolved in conventional solvent. This implies a major drawback when trying to perform ultrasonication studies on native and dissolved cellulose. Nonetheless, there is still a limited selection of cellulose solvent other than CUAM that could be used to carry out ultrasonication experiments. Cai and
Zhang (2006) have reported the dissolution of cellulose in 7% NaOH / 12% urea system.
Meanwhile, 20% NaOH is another promising solvent that can be used. The latter has been described as one of the most simple and environmental friendly solvents for cellulose. In addition, it could be a better alternative if the ultrasonicated cellulose is to be used in food application. Therefore, it could be of interest to perform ultrasonication of celluloses in 20 % NaOH and / or 7% NaOH / 12% urea system as the future research direction. It will be worthwhile to explore the possibility of ultrasonication to further
145 increase the crystallinity index (CrI) of celluloses in the aforementioned aqueous solvent system.
Chapter 5 examined the efficacy of using ultrasound in assisting the synthesis of curdlan sulfate by employing sulfuric acid. The process of ultrasonication significantly increased the degree of sulfation (DS) value of samples by almost fourfold. This is in consistent with the hypothesis of the ability of ultrasound in assisting curdlan sulfation.
Nuclear magnetic resonance (NMR) and FTIIR studies confirmed that sulfation had occurred. This had offered a very promising alternative to the conventional chemical synthesis of curdlan sulfate from the context of green chemistry. Moreover, current ultrasonication process also produced curdlan sulfate with lower Mw concurrently.
Nevertheless, the DS of curdlan sulfate obtained is still noticeably less than that obtained through conventional chemical synthesis. This could due to the inadequacy of delivering ultrasound wave into the reaction mixture during ultrasonication. Therefore, choosing a horn type, high intensity ultrasound probe as the ultrasound generator could be a better alternative compared to a conventional ultrasound cleaning bath, though the latter is usually cheaper. This is because probe type device will deliver the ultrasound wave directly into the reaction mixture as opposed to the ultrasound cleaning bath. In addition, it is possible to employ response surface methodology (RSM) to systematically optimize the reaction conditions in order to obtain higher DS values. Meanwhile, yield of curdlan sulfates in present study was less desirable. This is due to the extensive hydrolysis of curdlan that take place in the sulfuric acid. Therefore, minimising hydrolysis in order to increase yield could be the subsequent challenge. It may be possible to achieve higher
146 yield by holding the reaction mixture at sub-zero temperature, as outlined by Wang et al.
(2005) in sulfation of 1,3-β-glucan.
As final concluding remarks, the current project has demonstrated the ability of ultrasound in modification of polysaccharides. In addition, the current study has also explored the feasibility of utilising ultrasound beyond conventional depolymerisation. It is believed that these results will provide a new insight into polysaccharides modifications and eventually expand the methodologies of polysaccharides modifications.
REFERENCES:
Cai, J. and Zhang, L. (2006). Unique Gelation Behavior of Cellulose in NaOH/Urea
Aqueous Solution. Biomacromolecules, 7(1), 183-189.
Wang, Y.-J., Yao, S.-J., Guan, Y.-X., Wu, T.-X. and Kennedy, J. F. (2005). A novel
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and its structural elucidation. Carbohydrate Polymers, 59(1), 93-99.
147 APPENDIX:
LIST OF PUBLICATIONS AND PRESENTATIONS
1. Refereed Journal Publications
Wong, S.-S., Kasapis, S., & Huang, D. (2011). Molecular weight and crystallinity
alteration of cellulose via prolonged ultrasound fragmentation. Food
Hydrocolloids, in press.
Wong, S.-S., Ngiam, Z. R. J., Kasapis, S. and Huang, D. (2010). Novel sulfation of
curdlan assisted by ultrasonication. International Journal of Biological
Macromolecules, 46(3), 385-388.
Wong, S.-S., Kasapis, S. and Tan, Y. M. (2009). Bacterial and plant cellulose
modification using ultrasound irradiation. Carbohydrate Polymers, 77(2), 280-
287.
2. Conference Proceedings
Wong, S.-S., Sun, Y., Ngiam, Z. J., Kasapis, S., & Huang, D. (2009). Polysaccharide
ultrasonication – beyond depolymerization. In: P.A. Williams & G.O. Phillips
(Eds.), Gums and stabilizers for the food industry 15. Proceedings of the 15th
Gums and Stabilizers for the Food Industry Conferences held in Wrexham,
UK on 22 – 25 June 2009. Cambridge, England: The Royal Society of
Chemistry, 77-83.
148 3. Oral Presentations
Wong, S.-S., Kasapis, S., & Huang, D. (2010). Molecular weight alteration of
crystalline cellulose via prolonged ultrasound fragmentation – A kinetic study.
Presented in the 10th International Hydrocolloids Conference held in Shanghai,
China on 20 – 24 June 2010.
Wong, S.-S., Sun, Y., Ngiam, Z. J., Kasapis, S., & Huang, D. (2009). Polysaccharide
ultrasonication – beyond depolymerization. Presented in the 15th Gums and
Stabilizers for the Food Industry Conferences held in Wrexham, United
Kingdom on 22 – 25 June 2009.
Wong, S.-S., Kasapis, S. and Tan, Y. M. (2008). Crystalline cellulose obtained via
ultrasound fragmentation. Presented in the 9th International Hydrocolloids
Conference held in Singapore on 15 – 19 June 2008.
Wong, S.-S., Kasapis, S. and Tan, Y. M. (2007). Size exclusion chromatography of
ultrasound fragmented cellulose. Presented in the 5th Singapore International
Chemistry Conference (SICC) held in Singapore on 16 – 19 December 2007.
149