ULTRASONIC DEGRADATION OF XANTHAN AND LOCUST BEAN GUMS IN AQUEOUS SOLUTIONS: RHEOLOGICAL AND KINETIC STUDIES RUOSHI LI

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1 ULTRASONIC DEGRADATION OF XANTHAN AND LOCUST BEAN GUMS IN AQUEOUS SOLUTIONS: RHEOLOGICAL AND KINETIC STUDIES by RUOSHI LI Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Donald L. Feke Department of Chemical Engineering CASE WESTERN RESERVE UNIVERSITY January, 2014

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Ruoshi Li Candidate for the Doctor of Philosophy degree*. (signed) Dr. Donald L. Feke (chair of the committee) Dr. John Blackwell Dr. Uziel Landau Dr. Chung-Chiun Liu (date) 08/14/2013 *We also certify that written approval has been obtained for any proprietary material contained therein.

3 Ultrasonic Degradation of Xanthan and Locust Bean Gums in Aqueous Solutions: Rheological and Kinetic Studies Abstract by RUOSHI LI This work consists of experimental studies and analytical modeling of the ultrasonic degradation of polysaccharides in both the dissolved and suspended phases. In the experimental part of this work, natural xanthan gums, modified xanthan gums, locust bean gums, and waste coffee grounds have been sonicated in aqueous solutions for specific times and ultrasonic field and chemical conditions. Three models, including the Huggins and the Solomon-Ciuta equations, have been applied and compared for estimation of intrinsic viscosity of gum solutions, and the experimental results confirm the existence of a disorderorder conformational transition during the degradation process. Besides performing the sonication in deionized distilled water, two categories of salts with different behavior have been utilized, and their influences on molecular conformation, molecular interaction, and degradation efficiency have been carefully screened and studied by comparing a series of parameters, including the Huggins constant, the power-law exponent, the salt tolerance parameter, and the relative chain stiffness parameter. The results of the experiments confirm that the influence of salt species and concentration is more significant in the ultrasonic degradation of polyelectrolyte-type polysaccharides than that of non- I

4 polyelectrolyte type polysaccharides. The function of pyruvate groups located on xanthan gum side chains has also been studied by comparing the degradation results of a series of blends of natural xanthan gum with pyruvate-free xanthan gum. These results furthered the basic understanding of how pyruvate groups affect the stability of xanthan gum molecular backbones, while demonstrating the influence of the salt on molecular conformation and subsequently the resulting differences in degradation efficiency. Two kinetics models have been applied and compared to understand the degradation rate and the factors that impact degradation efficiency. Negative reaction orders with respect to concentration confirm that the ultrasonic degradation occurs mainly via a mechanical mechanism, in which the degradation rate increases with decreasing the polymer concentration. Reaction rate constants, the advantages and disadvantages caused by different factors, including salt species, salt concentration, polymer concentration, degradation temperature, functional groups, have been clearly quantified, and this enables the analysis and prediction of degradation efficiency. II

5 Acknowledgment I would like to express my sincere gratitude to my advisor and mentor, Dr. Donald L. Feke, for his enthusiasm, guidance, understanding, and patience throughout my thesis work. Meeting with him solidified my intent to pursue an advanced degree in Chemical Engineering. He gave me the opportunity to work on many exciting projects, and allowed me the autonomy to perform my work to the best of my abilities. His expertise, supervision, and teaching were an important asset to the success of this work. I was fortunate to have Dr. Uziel Landau, Dr. John Blackwell, Dr. Chung-Chun Liu, Dr. J. Adin Mann, and Dr. Daniel J. Lacks as my thesis and proposition committee members. Their valuable suggestions and insightful comments made the dissertation more complete. I d like to acknowledge Dr. Heidi B. Martin and Dr. Syed Qutubuddin for the help they provided in my graduate life. Dr. Dale Ray s help with performing the 600 MHz NMR is also appreciated. I also would like to graciously thank Nestlé R&D in Marysville, OH for their financial support, which enables me to successfully complete my graduate studies. Finally, I would like to dedicate my work to my fiancée, Dangdan (Crystal) Zhuang, and my family for their patience, support, encouragement, and unshakable faith in my abilities. They are the ones who inspire me to finish my graduate studies in 3 years. III

6 Contents Chapter 1 Introduction Polysaccharide Properties and Applications The Construction of Polysaccharides The Structures of Polysaccharides The Properties of Polysaccharides The Applications of Polysaccharides Basics of Ultrasound Processing Ultrasound Intensity and Pressure Amplitude The Applications of Ultrasound Processing The Mechanisms and Impact Factors of Ultrasonic Degradation Generation of Ultrasound in Liquid Cavitation Free Radical Reactions Frictional Force The Impact Factors Motivation for This Work Chapter 2 Rheological Study of the Degradation of Xanthan Gum Introduction Background and Theory Xanthan Gum Properties Viscosity Measurement and Determination of Intrinsic Viscosity Evaluation of Degradation Kinetics Models Evaluating the Influences of Temperature and Salt Species on the Ultrasonic Degradation of Xanthan Gum Materials and Methods Experimental Results and Discussion Conclusions Evaluating the Influences of the Salting-in and Salting-out Behavior Salts on the Ultrasonic Degradation of Xanthan Gum Materials and Methods Experimental Results IV

7 2.4.3 Discussion Conclusions Chapter 3 Rheological Study of the Degradation of Modified Xanthan Gum Introduction Background and Theory Pyruvate-free Xanthan Gum Properties Salt Tolerance and Relative Chain Stiffness Evaluating the Influences of the Pyruvate Group on the Ultrasonic Degradation of Xanthan Gum Materials and Methods Experimental Results Discussion Conclusions Chapter 4 Rheological Study of the Degradation of Locust Bean Gum Introduction Background and Theory Locust Bean Gum Properties The Power-Law Equation and the Coil Overlap Parameter Evaluating the Influences of Salt Species on Ultrasonic Degradation of Locust Bean Gum Materials and Methods Experimental Results and Discussion Conclusions Evaluating the Influences of the Salting-in and Salting-out Behavior Salts on the Ultrasonic Degradation of Locust Bean Gum Materials and Methods Experimental Results Discussion Conclusion Chapter 5 Ultrasonic Degradation and Extraction of Waste Coffee Grounds Introduction Background and Theory Ultrasonic Degradation and Extraction of Waste Coffee Grounds Materials and Methods V

8 5.3.2 Experimental Results and Discussion Conclusion Chapter 6 Summary and Future Work Summary Future Work References VI

9 List of Figures Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 2.1 Figure 2.2 Figure 2.3 Interconversion between the different forms of a free sugar mutarotation by using glucose as an example.2 The characterization of orientation of covalent bonds with two dihedral angles (ϕ, ψ) and one torsion angle (ω) in a locust bean gum repeating unit.4 Artist s conception of the space occupied by the gyration of a linear polysaccharide and a branched polysaccharide with the same molecular weight...6 Schematic diagram of sonochemical apparatus...11 Idealized representation of micro bubble cavitation of solutions under an ultrasonic field.13 Proposed free radical reactions of water during the ultrasonic processing 15 Molecular structure of repeating unit for xanthan gum...24 Conformational ordering of xanthan gum in solution.25 A schematic of Ubbelohde capillary viscometer.27 Figure H NMR spectrum of xanthan gum in D2O (polymer concentration 5.0 g/l, T = 25 o C).37 Figure H NMR spectrum of xanthan gum in D2O (polymer concentration 5.0 g/l, T = 45 o C).37 Figure H NMR spectrum of xanthan gum in D2O (polymer concentration 5.0 g/l, T = 55 o C).38 Figure H NMR spectrum of xanthan gum in D2O (polymer concentration 5.0 g/l, T = 65 o C).38 Figure H NMR spectrum of xanthan gum in D2O (polymer concentration 5.0 g/l, T = 75 o C).39 Figure H NMR spectrum of xanthan gum in D2O (polymer concentration 5.0 g/l, T = 85 o C).39 VII

10 Figure H NMR spectrum of xanthan gum in D2O (polymer concentration 5.0 g/l, T = 90 o C).40 Figure 2.11 Plots of intrinsic viscosity vs. sonication time. Ultrasonic degradation of xanthan gum solutions with polymer concentration of 3.0 g/l at 25 o C or 35 o C for 2, 5, 10, 30, 60, and 120 min 41 Figure 2.12 Plots of dynamic viscosity vs. sonication time. Ultrasonic degradation of xanthan gum solutions (3.0 g/l) with 0.5 M of salts (NaCl, NaI, NaSCN, NaNO3, NaOH, NaClO4, NaHCO3) at 35 o C for 2, 5, 10, 30, 60, and 120 min...44 Figure 2.13 Interactions among anions, xanthan gum (using pyruvate group part as an example), and hydration water 45 Figure 2.14 Plots of reduced viscosity vs. xanthan gum concentration...48 Figure 2.15 Plots of relative viscosity vs. xanthan gum concentration...50 Figure 2.16 Plots of reduced viscosity vs. gum concentrations for xanthan gum with different concentration of NaCl addition.51 Figure 2.17 Plots of reduced viscosity vs. gum concentrations for xanthan gum with different concentration of Na2SO4 addition.52 Figure 2.18 Plots of intrinsic viscosity vs. sonication time for xanthan gum with different concentration of NaCl (Equation 2.13) Figure 2.19 Plots of intrinsic viscosity vs. sonication time for xanthan gum with different concentration of Na2SO4 (Equation 2.13) Figure 2.20 Plots of intrinsic viscosity vs. sonication time for xanthan gum with different concentration of NaCl (Equation 2.18) Figure 2.21 Plots of intrinsic viscosity vs. sonication time for xanthan gum with different concentration of Na2SO4 (Equation 2.18) Figure 2.22 Plots of initial degradation rate constant ratio vs. NaCl concentration 63 Figure 2.23 Plots of Huggins parameter b vs. sonication time. 1.0 g/l of xanthan gum solution was sonicated with NaCl, and all NaCl concentrations were adjusted to 0.1 M after sonication 65 VIII

11 Figure 2.24 Plots of Huggins parameter b vs. sonication time. 1.0 g/l of xanthan gum solution was sonicated with Na2SO4, and all Na2SO4 concentrations were adjusted to 0.1 M after sonication 66 Figure 2.25 Plots of parameter φ1vs sonication time. 1.0 g/l of xanthan gum solution was sonicated with NaCl, and all NaCl concentrations were adjusted to 0.1 M after sonication...69 Figure 2.26 Plots of parameter φ1vs sonication time. 1.0 g/l of xanthan gum solution was sonicated with Na2SO4, and all Na2SO4 concentrations were adjusted to 0.1 M after sonication...70 Figure 2.27 Plot of parameter φ2 vs. sonication time. 1.0 g/l of xanthan gum solution was sonicated with NaCl, and all NaCl concentrations were adjusted to 0.1 M after sonication.70 Figure 2.28 Plot of parameter φ2 vs. sonication time. 1.0 g/l of xanthan gum solution was sonicated with Na2SO4, and all Na2SO4 concentrations were adjusted to 0.1 M after sonication 71 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure H NMR spectra of natural xanthan gums and pyruvate-free xanthan gums in D2O (polymer concentration 5.0 g/l, T = 90 o C)...81 Plots of intrinsic viscosity vs. inverse square root of ionic strength for natural xanthan gum 100% with NaCl and Na2SO4 addition 81 Plots of intrinsic viscosity vs. inverse square root of ionic strength for natural xanthan gum 80% pyruvate-free xanthan gum 20% (v/v) with NaCl and Na2SO4 addition...80 Plots of intrinsic viscosity vs. inverse square root of ionic strength for natural xanthan gum 20% pyruvate-free xanthan gum 80% (v/v) with NaCl and Na2SO4 addition...82 Plots of intrinsic viscosity vs. inverse square root of ionic strength for pyruvate-free xanthan gum 100% with NaCl and Na2SO4 addition.. 83 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of NaCl.84 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of Na2SO4.85 IX

12 Figure 3.8 Figure 3.9 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of NaCl (sonication time 0.5 min).87 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of Na2SO4 (sonication time 0.5 min).88 Figure 3.10 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of NaCl (sonication time 2 min) Figure 3.11 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of Na2SO4 (sonication time 2 min) Figure 3.12 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of NaCl (sonication time 5 min) Figure 3.13 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of Na2SO4 (sonication time 5 min) Figure 3.14 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of NaCl (sonication time 10 min)..92 Figure 3.15 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of Na2SO4 (sonication time 10 min) Figure 3.16 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of NaCl (sonication time 30 min)..94 Figure 3.17 Plots of intrinsic viscosity vs. pyruvate-free xanthan gum percentage with the addition of Na2SO4 (sonication time 30 min) Figure 3.18 Plots of reaction rate constant k vs. pyruvate-free xanthan gum with the addition of NaCl 98 Figure 3.19 Plots of reaction rate constant k vs. pyruvate-free xanthan gum with the addition of Na2SO4 99 Figure 4.1 Figure 4.2 Figure 4.3 Molecular structure of repeating unit for locust gum 105 Schematic of dilute regime, critical overlap concentration, and concentrated regime of polymer coils in solutions 107 Plots of dynamic viscosity vs. sonication time. Ultrasonic degradation of locust bean gum solutions (3.0 g/l) with 0.5 M of salts (NaCl, NaI, NaSCN, NaNO3, NaOH, NaClO4, NaHCO3) at 35 o C for 2, 5, 10, 30, 60, and 120 min 110 X

13 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Plots of reduced viscosity vs. locust bean gum concentration Plots of relative viscosity vs. locust bean gum concentration.114 Plots of relative viscosity vs. gum concentrations for locust bean gum with different concentration of added NaCl Plots of relative viscosity vs. gum concentrations for locust bean gum with different concentration of added Na2SO Plots of intrinsic viscosity vs. inverse square root of ionic strength for locust bean gum with NaCl and Na2SO4 addition 117 Plots of intrinsic viscosity vs. sonication time for locust bean gum with different concentration of NaCl (Equation 2.17) 121 Figure 4.10 Plots of intrinsic viscosity vs. sonication time for locust bean gum with different concentration of Na2SO4 (Equation 2.17). 122 Figure 4.11 Figure 4.12 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Figure 5.6 Plot of coil overlap parameter β vs. sonication time. 1.0 g/l of locust bean gum solution was sonicated with NaCl 125 Plot of coil overlap parameter β vs. sonication time. 1.0 g/l of locust bean gum solution was sonicated with Na2SO4 125 Plots of sugar mixture concentration vs. immersion time 134 Plots of sugar mixture concentration vs. sonication time (the waste coffee grounds were sonicated with the amplifier percentage adjusted at 30%, 45%, 60%, and 75% ). 135 Plots of sugar mixture concentration vs. energy input Plots of sugar mixture concentration vs. sonication time (the waste coffee grounds with different particle sizes were sonicated).137 Plots of sugar mixture concentration vs. sonication time (the waste coffee grounds with concentration of 2.5 g/ L and 5.0 g/l were sonicated) Plots of degradation percentage vs. sonication time (the waste coffee grounds with concentration of 2.5 g/ L and 5.0 g/l were sonicated) XI

14 List of Tables Table 2.1 Table 2.2 Table 2.3 Table 2.4 Table 2.5 Table 2.6 Table 2.7 Table 2.8 Table 2.9 Ubbelohde viscometer s size, viscometer constant, and range of measurement The effects of temperature on ultrasonic degradation of xanthan gum solutions Comparison of intrinsic viscosities and correlation values obtained from two models Critical overlap concentrations of xanthan gum solutions with different salt addition Intrinsic Viscosity [η] (dl/g, 25 o C, Huggins Equation) of Xanthan Gum Solution with NaCl addition..55 Intrinsic Viscosity [η] (dl/g, 25 o C, Huggins Equation) of Xanthan Gum Solution with Na2SO4 addition...56 Intrinsic Viscosity [η] (dl/g, 25 o C, Equation 2.7) of Xanthan Gum Solution with NaCl addition...56 Intrinsic Viscosity [η] (dl/g, 25 o C, Equation 2.7) of Xanthan Gum Solution with Na2SO4 addition...57 Reaction rate constant k obtained from Equation Table 2.10 Reaction rate constant and reaction order of ultrasonic degradation of xanthan gum under different Mark-Houwink constant..59 Table 2.11 Reaction rate constant k and reaction order n obtained from Equation Table 3.1 Table 3.2 Table 3.3 Salt tolerance and relative chain stiffness parameters of xanthan gum blends 83 Intrinsic viscosity [η] (dl/g, 25 o C, Huggins equation) of xanthan gum blend solution with the addition of salts. 86 Intrinsic viscosity [η] (dl/g, 25 o C, Huggins equation) of xanthan gum blend solution with the addition of salts. Sonication time was 0.5 min. 89 XII

15 Table 3.4 Table 3.5 Table 3.6 Table 3.7 Table 3.8 Intrinsic viscosity [η] (dl/g, 25 o C, Huggins equation) of xanthan gum blend solution with the addition of salts. Sonication time was 2 min.90 Intrinsic viscosity [η] (dl/g, 25 o C, Huggins equation) of xanthan gum blend solution with the addition of salts. Sonication time was 5 min. 92 Intrinsic viscosity [η] (dl/g, 25 o C, Huggins equation) of xanthan gum blend solution with the addition of salts. Sonication time was 10 min.93 Intrinsic viscosity [η] (dl/g, 25 o C, Huggins equation) of xanthan gum blend solution with the addition of salts. Sonication time was 30 min.95 Reaction rate constant k and reaction order n for 100% natural xanthan gum solutions obtained from Equation Table 3.9 Reaction rate constant k and reaction order n for 80% natural xanthan 20% pyruvate-free xanthan blend solutions obtained from Equation Table 3.10 Reaction rate constant k and reaction order n for 20% natural xanthan 80% pyruvate-free xanthan blend solutions obtained from Equation Table 3.11 Reaction rate constant k and reaction order n for 100% pyruvate-free xanthan gum solutions obtained from Equation Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Intrinsic viscosity of locust bean gum solutions with different salt addition, but with no sonication treatment..116 Salt tolerance and relative chain stiffness parameters of native xanthan gum and locust bean gum.118 Intrinsic viscosity [η] (dl/g, 25 o C, Equation 2.7) of locust bean gum solution with NaCl addition.119 Intrinsic viscosity [η] (dl/g, 25 o C, Equation 2.7) of locust bean gum solution with Na2SO4 addition.119 Reaction rate constant k and reaction order n obtained from Equation XIII

16 Table 4.6 Table 4.7 Coil overlap parameter β of locust bean gum solution with the addition of NaCl Coil overlap parameter β of locust bean gum solution with the addition of Na2SO XIV

17 Nomenclature English symbols a B b C Cp Cv The constant of power-law equation [Dimensionless] The relative stiffness parameter [Dimensionless] The Huggins parameter [Dimensionless] The polymer concentration [M] The specific heat of gas at constant pressure [J/(mol K)] The specific heat of gas at constant volume [J/(mol K)] C* The critical overlap concentration [M] c The speed of sound [m/s] cvis The viscometer constant [mm 2 /s 2 ] E The volumetric energy density [J/m 3 ] f fs I The frictional force [N] The frequency of the sound wave [Hz] The ionic strength [M] Is The intensity of the sound field [W/m 2 ] K k k' The Mark-Houwink constant [dl/g] The reaction rate constant [Depends on the reaction order] The reaction rate constant [Depends on the reaction order] k" The reaction rate constant [Depends on the reaction order] L l M0 The length of polymer molecular chain [nm] The distance between two monomer units [nm] The initial molecular weight [g/mol] XV

18 Mn Mt Mv M n P0 PA Pa PB Pin Pmax Pn PV R0 R 2 The number-average molecular weight [g/mol] The molecular weight at sonication time t [g/mol] The viscosity average molecular weight [g/mol] The limiting molecular weight [g/mol] The reaction order [Dimensionless] The external pressure [Pa] The maximum acoustic pressure [Pa] The pressure in the medium [Pa] The Blake threshold pressure [Pa] The initial pressure of noncondensible gas inside the cavity [Pa] The maximum pressure of violent shock waves [Pa] The degree of polymerization [Dimensionless] The vapor pressure of the medium [Pa] The equilibrium radius of the microscopic bubbles [Å] The correlation coefficient [Dimensionless] q The shear rate [s -1 ] r S T Tmax t te v v0 The radius of sphere occupied by a polymer chain [Å] The salt tolerance parameter [M 0.5 dl/g] The temperature of the solution [K] The maximum temperature of violent shock waves [K] The sonication time [min] The efflux time [s] The fluid velocity [m/s] The maximum particle velocity [m/s] XVI

19 Greek symbols α β γ η ηrel ηs ηsp [η] [η]0 [η]t [η] [η] π ρ σ ν φ1 φ2 ϕ ψ ω The Mark-Houwink constant [Dimensionless] The coil overlap parameter [Dimensionless] The ratio of the specific heat at constant pressure to the specific heat at constant volume of the gas inside the bubbles [Dimensionless] The dynamic viscosity of the solution [cp] The relative viscosity of the solution [Dimensionless] The viscosity of the solvent [cp] The specific viscosity of the solution [Dimensionless] The intrinsic viscosity of the solution [dl/g] The initial intrinsic viscosity of the solution [dl/g] The intrinsic viscosity of the solution at sonication time t [dl/g] The intrinsic viscosity of the solution at infinite ionic strength [dl/g] The limiting intrinsic viscosity of the solution [dl/g] The ratio of a circle's circumference to its diameter [Dimensionless] The fluid density [g/l] The surface tension of the solution [N/m] The kinematic viscosity [mm 2 /s] The ratio of the reduction in the intrinsic viscosity at sonication time t to the total potential reduction [Dimensionless] The degree close to limiting degradation [Dimensionless] The dihedral angle of chemical bonds [Radian] The dihedral angle of chemical bonds [Radian] The torsion angle of chemical bonds [Radian] XVII

20 Chapter 1 Introduction 1.1 Polysaccharide Properties and Applications Polysaccharides, which are polymerized saccharides, are a large family of long chain carbohydrate molecules, which are formed by the condensation of monosaccharide residues through hemi-acetal or hemi-ketal linkages. They also exist as short oligosaccharide sequences or polymeric repeat units and can be linked with other biological units to form more complicated biopolymers, such as lipopolysaccharides, glycoproteins, glycolipids, peptidoglycans, proteoglycans, etc. [Ramesh 2003]. Polysaccharides are widespread in nature, and play important roles in all life stages of organisms. In addition, polysaccharides are broadly used in the food industry as thickeners and stabilizers, because of their low cost and nontoxic properties. Polysaccharides are also used for nonfood applications, due to their special properties. For example, as natural polymers and the most abundant renewable resource on the earth, polysaccharides also play an increasingly important role of replacing oil resources in industrial use. As a result, they have been widely studied in recent years, wherein the focus has been on different aspects such as the macromolecular variety, function, structure, and chemical modification, because of their existing and potential commercial importance The Construction of Polysaccharides Polysaccharides are composed of a series of building units with a heterocyclic structure, which has one oxygen atom and several carbon atoms on the ring. The building units, glycosyl units, naturally would stay in open-chain forms and close-chain (ring) 1

21 forms. The open chain forms of glycosyl units are quite flexible, being capable of rotating about each of the single C C bonds. For the ring forms, based on the point of attack and the number of atoms on the ring, the glycosyl units can be classified into four groups: seven-membered rings, six-membered rings, five-membered rings, and four-membered rings. The most common repeating building units of natural polysaccharides is a sixmembered ring, while five-membered rings can be found in nucleic acids and some microbial polysaccharides. On the other hand, the seven- and four-membered ring structures are very rare, and play minor but important roles in biological systems [Lapasion 1995a]. Figure 1.1 Interconversion between the different forms of a free sugar mutarotation by using glucose as an example. The closing of the linear glycosyl unit generates a ring with a new chiral center at C-1, called the anomeric carbon, and results in two possibilities, which are designated 2

22 with either an α or β prefix [Pérez 1996]. The cyclization is a reversible reaction, and the energy generated from Brownian collision at ordinary temperatures is adequate to induce the spontaneous conversion of isomers, for example, from α to β and vice versa. This equilibrium between the various forms, known as mutarotation of sugars, is shown in Figure 1.1. Besides the mutarotation, the sugar monomers also are asymmetric. They exist in nonsuperposable mirror images, or enantiomers, known as the L- or D-sugars [Lapasion 1995a]. The linear or branched chains of polysaccharides are composed of several hundred to several thousand glycosyl units. If the polysaccharides are made up of a single type of glycosyl unit, they are called homopolysaccharides. Examples of homoglycans are cellulose, starch amylose, and starch amylopectin. On the other hand, if the polysaccharides are composed of two or more glycosyl unit types, they are named as heteropolysaccharides. To date, the number of glycosyl unit types found in any polysaccharides is no more than six. Also, even in a hexaheteropolysaccharide, the organization of glycosyl is not random but follows a certain order [Lapasion 1995a] The Structures of Polysaccharides Chemical structures of polysaccharides are of prime importance in determining their physico-chemical and biological properties. Therefore, a good understanding of the primary or covalent structure of polysaccharides, as well as secondary, tertiary and quaternary structure, has essential meaning for explaining and predicting the properties of polysaccharides, which have similar structures. The primary structure represents the covalent sequence of glycosyl units along the molecular chain. The shape of a particular glycosyl unit has been considered as fixed in 3

23 the solid state, in order to stay in a stable form. As a result, the geometry of polysaccharide molecular chain is determined by the covalent bond, which links two single glycosyl units together. The covalent linkage between two glycosyl units is not completely flexible, and is very dependent on the connection positions located on the two glycosyl units. The relative orientations of two linked glycosyl units could be evaluated by two dihedral angles (, ψ) and one torsion angle (ω), depending on the connection type. The 1,4-linked covalent bond can be characterized by two dihedral angles, while the 1,6-linked covalent bond need an additional torsion angle to describe the freedom of the glycosyl units. The relative orientations of different covalent bonds are shown in Figure 1.2, where the angles are limited in a narrow range by intramolecular hydrogen bonding and the bulk of glycosyl units. Figure 1.2 The characterization of orientation of covalent bonds with two dihedral angles (ϕ, ψ) and one torsion angle (ω) in a locust bean gum repeating unit. The secondary structure of polysaccharides, known as conformation, is highly dependent on the primary structure. The secondary structure can be represented as ribbons or helices, which describes the interactions between molecular chains (e.g., hydrogen bonding, dipole-dipole forces, Coulomb forces, and Van der Waals forces). 4

24 Compared with polysaccharides with branch-on-branch structures, the linear polysaccharides or simple branched polysaccharides have a tendency to form helical structures in solutions. As a result, the interactions between molecular chains have significant influence on solution properties of polysaccharides. The tertiary and quaternary structures are used to characterize higher level of organization of polysaccharide molecular chains, which is associated with the specific ordered structures or compacted structures [Lapasion 1995a] The Properties of Polysaccharides It has been broadly accepted that solutions with dissolved linear polysaccharides have a higher viscosity than those with dissolved branched polysaccharides of the same number of units. A visual conception of the space occupied by the gyration of a polysaccharide with linear structure or a branched structure is shown in Figure 1.3, where a larger sphere encompasses an extended linear rod-like polysaccharide molecular chain as it gyrates in solution. The viscosity of the solution is attributed to the collisions between this extended polysaccharide molecule and the adjacent molecules. On the contrary, a gyrating highly branched (bush-shaped) polysaccharide with the same molecular weight will sweep out a much smaller sphere and result in less frequent collisions, which lead to a lower viscosity of the corresponding solution [Whistler 1993]. Based on the above theory, if the viscosity of the solution is considered as a function of the molecular chain s length, the degree of molecular chain breakage could be estimated by measuring the viscosity of the solution. For example, considering a linear polysaccharide molecule with a chain length L, the region it sweeps out can be expressed as a function of L 3, hence the viscosity of the solution could be represented as η = f (L 3 ). 5

25 If the molecular chain is broken from the middle point, the size of the sphere will reduce to 1/8 of the original size, which could theoretically lower the viscosity of the solution by 87.5%. Linear Polysaccharide Branched Polysaccharide Figure 1.3 Artist s conception of the space occupied by the gyration of a linear polysaccharide and a branched polysaccharide with the same molecular weight [adapted from Whistler (1993)]. As mentioned in the previous section, many polysaccharides can form helical or ribbon-like secondary structures in solutions, where the ordered form makes the molecular chains more compacted and stiffer, and the disordered form results in more flexible and expanded molecular chains. Therefore, the viscosity of solutions will be highly affected by the secondary structure of dissolved polysaccharides, where the polysaccharide in a disordered structure may present a higher solution viscosity than that of polysaccharides in an ordered structure The Applications of Polysaccharides In nature, polysaccharides hold a wide range of different functions. From the viewpoint of their physiological functions, they can be classified as energy storage materials, structural materials, and protective substances. Starch, glycogen, and some 6

26 plant seed polysaccharides (locust bean gum and guar gum) are well known examples for energy storage, and can be metabolized rapidly. On the other hand, polysaccharides such as cellulose and chitin play important roles in enhancement of the structural integrity and mechanical strength of plant tissues by generating a hydrated cross-linked threedimensional network. In addition, matrix polysaccharides, such as hyaluronate, chondroitin sulfate, and other related glycosaminoglycans provide size, shape, and rigidity to the cell-wall matrix [Lapasion 1995a]. Protective polysaccharides, including the antigenic and immunogenic exocellular microbial polysaccharides, can prevent plants from bacterial infections by sealing off the injured parts. Nowadays, polysaccharides are broadly used in the food industry as thickeners and stabilizers, because they are widely available, usually of low cost, and nontoxic. Examples of commercial polysaccharides for food applications are xanthan gum, locust bean gum, guar gum, gum arabic, etc. Moreover, their use in nonfood applications depends on the unique special properties they provide, often at costs below those of synthetic polymers. For example, cellulose has been broadly used in making paper, textiles, films, and novel biomaterials [Chang 2011; Shi 2011]; xanthan gum can be used for making cosmetics and improving oil recovery [Kabir 1980]; carrageenan has been used for making room deodorants; and, starch could be used as emulsifying agent or a green fuel resource. 1.2 Basics of Ultrasound Processing Ultrasound Intensity and Pressure Amplitude Ultrasound is a sound wave that can be transmitted through any elastic substance, during which the sound field is applied in a given medium (gas or liquid), and generates 7

27 molecule collisions in the medium by changing the surrounding pressure. In onedimensional space, the interactions can be imagined as continuous collision between any two adjacent molecules, acting as a set of billiard balls hitting one another in a line. The molecules will return to their initial resting positions after being displaced by the advancing wave. Within the medium, fluid-particles which are displaced along the direction of sound propagation, results in longitudinal waves, while motion of fluidparticles perpendicular to the sound propagation direction is known as a transverse wave. The pressure in the medium caused by the sound wave at time t and at fixed point can be expressed as Equation 1.1, where Pa is the pressure in the medium, PA is the maximum acoustic pressure, and fs is the frequency of the sound wave. 2 (1.1) The intensity of the sound field is the product of the volumetric energy density and the speed of sound in the fluid, which is defined as energy flux with units of W/cm 2. The expression of intensity is given in Equation 1.2, where c is the speed of sound in the fluid, ρ is the fluid density, v is the fluid velocity, and E is the volumetric energy density. 1 2 (1.2) For a plane progressive wave, the particle velocity in the fluid is related to the acoustic pressure. As a result, the maximum particle velocity v0 is related to the maximum acoustic pressure, and the relationship can be expressed as Equation 1.3. (1.3) 8

28 Therefore, the intensity of the sound wave (Is) can be calculated from the maximum acoustic pressure of the sound field as shown in Equation (1.4) Normally, the intensity of the sound wave is highly related to the ultrasound processing efficiency, whereas high intensity is expected to lead to better ultrasonic processing results, but this also consumes more energy per unit time and unit volume. As a result, one of the essential objectives of ultrasound studies is to balance the energy input and ultrasonic processing results The Applications of Ultrasound Processing High intensity ultrasound generates various physico-chemical effects, including radiation pressure, streaming, cavitation, and interface instabilities [Mulet 2002]. In the last few decades, a great deal of practical work has been done concerning possible applications of ultrasound technique to clinical and industrial areas. One of the remarkable renaissances for clinical analysis and study is 3-D ultrasonic diagnostic imaging technique, which is based on detection of reflected sound waves [Fenster 1996; O Brien Jr. 2007]. On the other hand, ultrasound is also broadly being used in industrial areas for the welding of thermoplastics, the cleaning of instruments, the cutting and etching of materials, and dispersion of particles in a suspension [Rashli 2013; Challis 2005]. For research in sonochemistry, ultrasound has a relatively recent history. There has been a considerable rise in the applications of high-intensity ultrasound for organic and organometallic modification or synthesis, which may improve the rate and yield 9

29 significantly, and also change the reaction mechanisms [Bradley 2005; Price 1996a; Suslick 1999]. Among all ultrasound applications, the most well-known application of ultrasound in sonochemistry is the degradation of long-chain polymers suspended in organic or inorganic solutions [Basedow 1977], which provides a convenient and highly efficient option for industries to lower the particle size and modify the particle shape. 1.3 The Mechanisms and Impact Factors of Ultrasonic Degradation Ultrasonic degradation techniques have been widely used in laboratory studies to generate fine particles. Degradation could be applied to natural or synthetic polymers, and in aqueous or organic solutions. Compared with other degradation methods, such as enzyme involved fermentation, acid or base induced hydrolysis, and thermal degradation, ultrasonic degradation has been considered to be the best way to control molecular weight and produce fragments of definite molecular size. In addition, the degraded polymers have no major change in their chemical structures under ultrasonic processing, whereas the other methods might alter the chemical composition and thereby the solution behaviors. It is well-known that degradation of long-chain polymers occurs when the solutions are irradiated under ultrasound field. A number of mechanisms for ultrasonic degradation have been suggested. The major effects of ultrasound that lead to breakage of polymer chains are cavitation and free radical reactions [Doulach 1978; Riesz 1992], although others have shown that frictional forces between the solvent and polymer molecules can cause the breakage of polymer chains [Mostafa 1958]. 10

30 1.3.1 Generation of Ultrasound in Liquid For most ultrasonic degradation processes, ultrasonic vibrations are transferred from a solid source into the liquid phase. A schematic of an ultrasonic degradation apparatus is shown in Figure 1.4, where the tip of the horn is placed just below the surface of the sample solutions in the cooling cell, and then the sample particles are degraded by the ultrasound vibration. Figure 1.4 Schematic diagram of sonochemical apparatus: (a) ultrasound generator/amplifier; (b) piezoelectric transducer; (c) horn/micro-tip; (d) sample solutions; (e) cooling cell/sample container; (f) cooling bath; (g) connections to thermocouple; (h) equipment holder. During sonication, the electrical energy is generated and transported to the transducer, which is converted to mechanical energy introduced to the sample solutions. The transducer could be made of piezoelectric or magnetostrictive materials, such as quartz, barium titanate, and zirconate titanate, and could be used for generation of high 11

31 intensity ultrasound. Using horns could avoid the direct contact of the liquid that is being irradiated with the transducer [Basedow 1977]. The ultrasonic apparatus has to be placed in water bath with thermal controller during the sonication in order to keep the temperature constant. Moreover, the sample solutions are also put in a reaction vessel with a special shape, known as a cooling cell, which could improve the temperature stability considerably Cavitation Many recent studies indicate that the degradation of polymers arises as a result of the physical (mechanical) and chemical effects caused by ultrasound are usually attributed to cavitation, though there is no direct evidence that there exists any interaction between the ultrasound waves and the polymer chains. Cavitation is the rapid formation and collapse of microscopic bubbles in the liquid. Technically, cavitation occurs at an ultrasound frequency ranging from 18 khz to 1 MHz. Each cavitation event occurs over several acoustic cycles, and each acoustic cycle is composed of two actions: an expansion cycle, during which liquid molecules are being pulled apart; and a compression cycle, during which the liquid molecules are being compressed. To initiate growth of a cavitation bubble, the expansion cycle should generate enough energy to overcome the forces which hold the liquid molecules together, and an acoustic pressure above the socalled Blake threshold pressure has to be applied [Kuijpers 2002]. The maximum acoustic pressure (PA) depends on the intensity provided by ultrasonic device (I), the density of the liquid (ρ), and the velocity of sound c. The expression is derived from Equation 1.4, and given in Equation

32 2 (1.5) The expression of the Blake threshold pressure (PB) is shown in Equation 1.6, where P0 is the external pressure, PV is the vapor pressure of the medium, σ is the surface tension of the solution, and R0 is the equilibrium radius of the microscopic bubbles (1.6) Figure 1.5 Idealized representation of micro bubble cavitation of solutions under an ultrasonic field [adapted from Suslick (1990) and Riesz (1992)]. Cavitation is a three-step process consisting of nucleation, bubble growth, and bubble collapse. However, cavitation could not normally occur in pure liquids. As 13

33 mentioned above, the liquid pressure has to be sufficiently negative to overcome the forces of natural cohesion [Noltingk 1950]. As a result, many experimental results have confirmed that the presence of micro-particles, dissolved gases, or other cavitation nuclei are necessary for the onset of cavitation in order to lower the solution s strength. The whole process of cavitation is shown in Figure 1.5. During the cavitation process, bubbles may exist for several acoustic cycles, and their size will expand to two to three times of their original size, after that the bubbles will implode. Those micro bubble implosion are utilized to break the molecular chains during the ultrasonic processing Free Radical Reactions Another broadly accepted mechanism for ultrasonic degradation is free radical reaction. Normally, cavitation is treated as an adiabatic process, because the density changes in the vibrating liquid are so rapid that the heat cannot be transferred from the compressed region to the surroundings and vice versa. Thus, the collapse is nearly adiabatic, and results in a localized hot spot [Riesz 1992]. As a result, the violent shock waves caused by cavitation may have a high temperature of about 5000 K, and a high pressure of around 1000 bar, which are sufficiently high to easily break chemical bonds between monomer units [Flint 1991]. The expressions of maximum pressure (Pmax) and temperature (Tmax) are given in Equations 1.7 and 1.8, where γ =Cp/Cv is the ratio of the specific heat at constant pressure to the specific heat at constant volume of the gas inside the bubbles, Pin is the initial pressure of noncondensible gas inside the cavity when the radius reaches maximum, and T is the temperature of the liquid. 14

34 1 (1.7) 1 (1.8) In liquids, using water as an example, the high temperature and high pressure will lead to the formation of hydroxyl radicals ( OH) and hydrogen radicals ( H). The decomposition of water is considered to be initiated by the ejection of an electron from the water molecule due to the action of ionizing radiation, which is accomplished by the emission of visible and ultraviolet light during ultrasonic processing, and then followed by a series of secondary processes [Basedow 1977]. The proposed free radical reactions caused by ultrasonic processing are shown in Figure 1.6. These equations were proven through isotope exchange experiments. Not only for solvents, but also polymers in solutions were confirmed to form free radical fragments by electron spin resonance analysis, mass spectrometric analysis [Kawasaki 2007], and degradation kinetics modeling studies. Thus, these free radicals will subsequently react with the polymer fragments to help the degradation in a chemical manner [Riesz 1992]. Figure 1.6 Proposed free radical reactions of water during the ultrasonic processing. 15

35 In addition, free radical reactions due to ultrasonic processing also occur in organic solvents, especially if trace of water is present. However, the ultrasonic processing is more complicated in this case. Besides the depolymerization of polymers and decompositions of medium, different macro-radicals formed through the chain scission of polymers can recombine into copolymers [Price 1996b]. Consequently, copolymerization can proceed along with depolymerization during the degradation process Frictional Force In order to explain the action of ultrasonic degradation, many mechanisms based on the consideration that the ultrasound directly acted on the polymer chains have been proposed. But none of them can completely explain all of the effects observed during the ultrasonic processing. As a result, another mechanical mechanism based on frictional force between solvent and polymer molecules was proposed. In this theory, the ultrasound was considered to generate different vibrations on solvent molecules and polymer chains. Therefore, the difference of vibration may result in the generation of frictional forces between macromolecules and solvent molecules, and hence may break the chemical bonds. The first expression of this frictional force (f) was suggested by Schmid, in which the polymer chains are considered to be rigidly fixed at one end. The expression is given in Equation 1.9, where Pn is the degree of polymerization, ηs is the viscosity of the solvent, r is the radius of sphere occupied by a polymer chain, and v0 is the maximum velocity of the solvent molecules. 16

36 6 (1.9) On the other hand, another expression of the frictional force was reported, in which the polymer chains were considered to be rigid, and consequently the force acts on a molecular chain with both ends fixed [Jellinek 1951]. The expression is given in Equation 1.10, where ρ is the density of the solvent, l is the distance between two monomer units, and c is the velocity of sound. 4 (1.10) Although this kind of theory can well explain some experimental results, it also has some disadvantages. For example, the polymer chains should not move freely in order to guarantee that the molecular chains are rigid enough during the vibration. As a result, a sufficient polymer concentration should be used to ensure the long chain molecules sufficiently entangled with each other, and no degradation can be expected to occur by friction in extremely dilute solutions [Mostafa 1958] The Impact Factors Factors influencing ultrasonic degradation have been studied and confirmed in many previous studies, in which the ultrasound frequency, ultrasound intensity, environmental temperature, solvent species, polymer concentration, and dissolved gases are considered to be essential for understanding the ultrasonic degradation results. - Effect of Ultrasound Frequency It is well known that free radicals are produced during the ultrasonic processing, and the yields of H and OH radicals are highly depend on the ultrasound frequency and intensity. Chemical effects due to radicals are prominent in the frequency range of

37 600 khz [Mark 1998]. The ultrasonic degradation of natural polymers in aqueous solutions, such as starch, chitosan, methyl cellulose, have been reported to be more efficient when the sonication was processed at high frequency of khz instead of low frequency of 20 khz [Czechowska-Biskup 2005; Koda 2011]. For example, it was found that the degradation processed at 500 khz sonication is much faster than that in 20 khz sonication. This result was considered mainly caused by chemical effects, which are reinforced at higher sonication efficiency. Furthermore, hydroxyl radicals were confirmed to play important roles in water-soluble polymer degradation, which are highly related to the sonication efficiency. However, increasing the frequency of ultrasound may also increase the cavitation thresholds, which suggests it is essential to select different sonication frequencies when using different solvents, in order to achieve better results. - Effect of Ultrasound Intensity The ultrasound intensity is one of the most important factors which could remarkably determine the degradation efficiency. It has been reported that the higher ultrasound intensity could result in a lower limiting molecular weight for the same sonication time [Price 1993a]. Since the maximum radius of the microscopic bubbles was considered to be proportional to the square root of the intensity above the cavitation threshold, increasing the intensity leads to larger bubbles and generates higher shear forces on collapse. As a results, a lower limiting molecular weight could be reached at higher ultrasound intensity. Moreover, the number of microscopic bubbles per unit volume would also increase by increasing the ultrasound intensity. However, above a certain value of microscopic bubble number, a higher bubble density will inhibit the ultrasound transport, and subsequently lower the degradation efficiency. Furthermore, a 18