ABSTRACT. L (FPS-NCSU 2002) was made by Dr.Sanghoon Lim from his graduate work. The

Transcription

1 ABSTRACT QIAN, HAO. The Study of Dilute Solution of Chitosan under the Effects of Salt and Acid Concentration. (Under the direction of Dr. Samuel Hudson, Dr. Wendy Krause.) Chitosan 00-ASSC-7676 was purchased from Vanson Inc. (Redmond WA). Chitosan L (FPS-NCSU 2002) was made by Dr.Sanghoon Lim from his graduate work. The intrinsic viscosities of two different chitosan samples, L and 00-ASSC-0767, were measured by testing the flow time of different chitosan solutions with Ubbelholde viscometer. The influence of salt and acid concentration on the intrinsic viscosity of each chitosan sample was analyzed by the comparison of six solutions with different solvents. Huggins and Kraemer equations were applied to calculate the values of Huggins k and Kraemer k. These parameters reflected the properties of solutions. To ensure the accuracy of the dilutions, serial dilution process was used in one batch. The important parameters, concentration, temperature, solvent, shear rate, salt concentration, ionic strength-relevant Salt Concentration (Cs) and temperaturerelevant d ln[η] / dt, that affect the intrinsic viscosity of chitosan solutions are discussed and it is concluded that intrinsic viscosity of chitosan solution is related to the flexibility of polymer chain and the conformation of chitosan polymer in solution. Temperature is relative to the radius of gyration and average molecular weight.

2 Copyright 2013 Hao Qian All Rights Reserved

3 The Study of Dilute Solution of Chitosan under the Effects of Salt and Acid Concentration by Hao Qian A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Textile Chemistry Raleigh, North Carolina 2014 APPROVED BY: Samuel Hudson Committee Co-chair Wendy Krause Committee Co-chair Xiangwu Zhang

4 DEDICATION To my dear parents, advisers, and girlfriend. ii

5 BIOGRAPHY Hao Qian was born in Shao Xing, Zhejiang Province, China. He has forged a deep bond with textiles. His father is a businessman in the clothing business. His hometown is the largest clothing trading city in China. He studied textile engineering in his undergraduate education in Shanghai, China. And he has learned about textile chemistry in NC State Univeristy under the direction of Dr. Samuel Hudson. iii

6 ACKNOWLEDGMENTS Thanks to Dr. Samuel Hudson for helping me overcome the difficulties on my experiment by providing excellent suggestions from theoretical and realistic aspects. I appreciate a lot for his kindness and patience. Thanks to Dr. Wendy Krause for being my committee co-chair. She is very kind and provided a lot of helpful advises on the experiment designing. I appreciate her kindness. Thank Dr. Xiangwu Zhang for taking the time to be my committee member. Last but not least, I want to thank my dear parents for their unconditional support, both financially and emotionally throughout my master degree. iv

7 TABLE OF CONTENTS LIST OF TABLES.vi LIST OF FIGURES..vii 1. INTRODUCTION LITERATURE REVIEW Characterization of chitosan Relationship between structure and property of chitosan Purification, acetylation and characterization of chitosan Applications of chitosan and its derivatatives Some other polyelectrolytes and their applications Viscosity Parameters influence viscosity Conclusion EXPERIMENTAL RESULTS DISCUSSION Salt effect Temperature effect Summary RECOMMENDATIONS FOR FUTURE RESEARCH.. 56 v

8 REFERENCE 53 vi

9 LIST OF TABLES Table 2.1 Relationship between DD/Molecular weight and properties Table 2.2 Degree of deacetylation of chitosan measured by EH-TPT method and by other methods: 1 H NMR, XRD and FT-IR method. 8 Table 3.1 Nomenclature Used in This Study. 18 Table 3.2 Original flow time for chitosan L Table 3.3 Original flow time for chitosan 00-ASSC Table 4.1 Relative, specific, reduced and inherent viscosities of L Table 4.2 Relative, specific, reduced and inherent viscosities of 00-ASSC Table 4.3 Mark-Houwink-Sakurada Constants for Chitosans with Varying of DA and Solvents of Different ph and Ionic Strength, µ. 44 Table 4.4 The estimated molecular weight referred to Rinaudo et al., , Rinaudo et al., , Rinaudo et al., Table 5.1 Huggins k and Kraemer k constants and intrinsic viscosity [η] refers to both equations for both L and 00-ASSC vii

10 Table 5.2 Intrinsic viscosities [η] of chitosans in 30 C and 40 C viii

11 LIST OF FIGURES Figure 2.1 Chemical structure of cellulose, chitin, chitosan (DD>75%) and trimethyl chitosan (chitosan s derivative)... 4 Figure 2.2 Deacetylation process of chitin 8 Figure 2.3 Chitosan and its applications. 10 Figure 2.4 Intrinsic viscosity evolution for different chemical structures of the polymer chain Figure 2.5 Different behavior of a polyelectrolyte in aqueous solution and a salt solution 15 Figure 2.6 Reduced viscosity ηred as a function of the concentration c for the polyelectrolyte sodium salt of poly(acrylic acid) (PAAcNa) in aqueous solution Figure 4.1 Kraemer and Huggins plots for L in HAc 0.1M/NaAc 0.01M solvent at 30 C (Datapoint with solute concentration 0.001g/mL is neglected.) 26 ix

12 Figure 4.2 Kraemer and Huggins plots for sample L in HAc 0.1M/NaAc 0.1M solvent at 30 C. 27 Figure 4.3 Kraemer and Huggins plots for sample L in HAc 0.1M/NaAc 0.3M solvent at 30 C 28 Figure 4.4 Kraemer and Huggins plots for sample L in HAc 0.1M/NaAc 0.3M solvent at 40 C Figure 4.5 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.01M solvent at 30 C 30 Figure 4.6 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.1M solvent at 30 C Figure 4.7 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.3M solvent at 30 C 32 Figure 4.8 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.3M solvent at 30 C using serial dilution x

13 Figure 4.9 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.3M solvent at 40 C.. 34 Figure 4.10 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.1M/NaAc 0.01M solvent at 30 C Figure 4.11 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.1M/NaAc 0.1M solvent at 30 C (Data points with solute concentration 0.005g/mL and 0.01g/mL are neglected.) 36 Figure 4.12 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.1M/NaAc 0.3M solvent at 30 C (Data points with solute concentration 0.002g/mL is neglected.).. 37 Figure 4.13 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.1M/NaAc 0.3M solvent at 40 C. 38 Figure 4.14 Kraemer plot for sample 00-ASSC-0767 in HAc 0.3M/NaAc 0.01M solvent at 30 C (No effective data for Huggins plot and data points with solute concentration 0.005g/mL and 0.01g/mL are neglected. The low salt xi

14 concentration contributes to this situation. See at low concentration in Figure 2.7.). 39 Figure 4.15 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.3M/NaAc 0.1M solvent at 30 C (Datapoints with solute concentration 0.004g/mL is neglected.) Figure 4.16 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.3M/NaAc 0.3M solvent at 30 C (Datapoints with solute concentration 0.001g/mL is neglected.) Figure 4.17 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.3M/NaAc 0.3M solvent at 40 C Figure 5.1 Huggins plot for solution of pectin-na in different aqueous NaCl solutions of constant composition at 27 C 47 Figure 5.2 Intrinsic Viscosity ([η]) as a function of Salt Concentration (Cs) refers to Huggins Equations for L and 00-ASSC-0767 in HAc solvent. 49 xii

15 Figure 5.3 Intrinsic viscosity ([η]) as a function of the temperature (T) for both L and 00-ASSC-0767 in solvent HAc 0.1M/NaAc 0.3M and HAc 0.3M/NaAc 0.3M... 5 xiii

16 1. INTRODUCTION Chitin, poly (β-(1 4)-N-acetyl-D-glucosamine), is an important natural polysaccharide with abundant source which exists in crabs and shrimp shells. It is biodegradable and widely distributed in nature, but it has low toxicity and relatively poor solubility. During industrial treatment, chitin is obtained by solubilizing proteins and dissolution of calcium carbonate. Usually the pigments are also removed to make the product colorless. In order to obtain a better solubility, alkaline is applied to generate NH2 functioned groups on the D-glucosamine repeat unit. The chitin derivative which is called chitosan is obtained by the partial deacetylation of chitin. Usually, the lowest degree of deacetylation of chitin must meet is 50% to ensure the solubilization of chitosan in aqueous acidic media. Due to its unique cationic character, chitosan has potential to form polyelectrolyte complexes with other polyanions such as polyacrylic acid, heparin, acidic glycosaminoglycans and carboxymethylcellulose. [1] [2] [3] [4] 1

17 2. LITERATURE REVIEW Polyelectrolytes are classified into natural, modified natural, and synthetic polymers which contain electrolyte groups as repeating units. According to the difference of their charge, there are three classifications of polyelectrolytes, polycations, polyanions and polyampholytes. Due to their highly charged structure and high molecular weights, polyelectrolytes exhibit both properties of salts and polymers, and are also called polymer-salts. Polyelectrolytes can be dissolved into solvents and their solutions are viscous and electrically conductive. Many biological molecules are polyelectrolytes, such as pectin and chitosan. The unique properties of chitosan attract many researchers all over the world. By deacetylation (most researchers often use more than 70% degree of deacetylation) of chitin, chitosan exhibits good solubility in aqueous acid solutions. Polyelectrolytes can be applied in various applications, e.g., stabilize colloidal suspensions, surface modification, conditioners, emulsifiers and drag reducers. Due to the good solubility in water, some of them can be applied in medical and biochemical fields. 2

18 2.1 Characterization of chitosan Chitosan which is also called poly-(d)glucosamine is a linear polysaccharide of β-( 1-4) linked 2 acetamido-2-deoxy- β-ɒ-glucopyranose and 2-amino-2-deoxy- β-ɒ-glycopyranose (Figure 2.2)[5]. It has one amino group and two hydroxyl groups in the repeating unit. Viewing from its carbohydrate backbone, chitosan is very similar to cellulose. Two different repeating units which are the amide N-acetyl-2-amino-2-deoxy-D-glucopyranose and 2-amino-2-deoxy- D-glucopyranose are connected by (1-4)-β-glycosidic bonds. The most attractive aspect is that it is easy to obtain chitosan by deacetylation of chitin, which is distributed worldwide as an element in the exoskeleton of shrimp, crabs, and cell walls of fungi [6]. Compared to chitin, chitosan s well performanced solubility in aqueous solutions and organic solvents is making it more and more popular in practical applications [5]. As far as the scientists know, chitosan is suitable to be used in the field of agriculture, industry, medicine, and winemaking. Meanwhile, its derivatatives are also widely used in nonviral gene delivery. (Figure 2.1)[7] 3

19 Figure 2.1 Chemical structure of cellulose, chitin, chitosan (DD>75%) and trimethyl chitosan (chitosan s derivative) [7] 4

20 2.2 Relationship between structure and property of chitosan The properties of chitosan are influenced by the diversity of the structure of chitosan, which is caused by several reasons. First, the difference proportions of N-acetyl- ɒ- glucosamine and ɒ-glucosamine residues in the structure of chitosan is decided by the degree of deacetylation (DD) and molecular weight, which influence the specific structure of chitosan. The structure parameters consideraby affect the properties a lot, such as solubility, viscosity and so on (Table 2.1). Cell adhesion is increased when the degree of deacetylation (DD) is increased, because of the presence of free amino groups. Also, the amino groups of chitosan are reactive and provide the versatility of chitosan. Chitosan is soluble in low ph and is insoluble in high ph [5]. The limit of chitosan is that it is not soluble in aqueous medium except below ph 5.6 [7]. Table 2.1 Relationship between DD/Molecular weight and properties 5

21 2.3 Purification, acetylation, and characterization of chitosan DA, the degree of acetylation, plays a significant role in reflecting the difference between chitin and chitosan which, otherwise, have the same chemical structure. (Figure. 2.2) It means the balance of β-(1-4) linked 2 acetamido-2-deoxy- β-ɒ-glucopyranose and 2-amino- 2-deoxy- β-ɒ-glycopyranose is decided by DA. Also, DA influences the conformations of chitosan in solution and its biological, physical, and physicochemical properties [8]. In order to obtain ideal chitosan, purification and deacetylation processes are necessary. First, the flakes of chitosan sample are dissolved and filtered through 0.45~1.2 µm membranes (Millipore). And aqueous ammonia is added to precipitate them. Then, the polymer is washed by deionized water and dried by lyophilization [9]. It was mentioned by Asako Hirai that 1 H NMR is an ideal method to obtain the DA of chitosan by analyzing the chemical structure of chitosan [10]. In M. Lavertu s paper, it proved that it is fast, precise and reproducible to use 1 H NMR method to determine the DA of chitosan sample by using the integrals of the peak of H1-D (proton H1of acetylated monomer) and H-Ac (protons of acetyl group) [11]. Acetic anhydride is chosen as reactive medium in a water or alcohol solution to adjust the DA, due to its fast reaction speed [12]. Besides, it is easy to control the process. XRD and FT-IR method are also used to determine the DA of chitosan. The characterization of chitosan can be obtained by size-exclusion chromatography (SEC), potentiometric measurement, capillary viscometry, thermogravimetric analysis (TGA) and mathematical modeling. By the scanning interferometric refractometer of size-exclusion chromatography, each DA s refractive index increment is measured. Potentiometric measurement is simple and low cost to determine the DA. [13] 6

22 By the effort of Yongqin Zhang, a novel potentiometric titration preceded by enzymatic pretreatment (EP-TPT) method is developed to determine DA of chitosan with a good agreement with data from 1 H NMR [14] (Shown in Table.2.2). It requires a certain time for a sample of chitosan solution to pass through a capillary tube. Capillary viscometry is used to measure the time which is called running time. If each sample is measured for specific times (at least 3 times), [η], the intrinsic viscosity can be calculated by extrapolation of the reduced viscosity to infinite dilution [15]. The capillary viscosity method is the simplest and fastest way to calculate the molecular weight by the help of [η] = K [η] M a (Mark-Houwink equation). TGA is used to evaluate the water content of chitosan by operating under helium while increasing the temperature [16]. Mathematical modeling is used to extrapolate more and obtain an accurate mathematic calculation and related curve analysis [17]. 7

23 Figure 2.2 Deacetylation process of chitin Table.2.2 Degree of deacetylation of chitosan measured by EH-TPT method and by other methods: 1 H NMR, XRD and FT-IR method 8

24 2.4 Applications of chitosan and its derivatatives Chitosan is widely used in the world for its good properties and easy accessibility. (Figure 2.3) First, due to its good resistance to abrasion and optical characteristics, chitosan is applied to form film in photography field. Second, the fungicidal and fungistatic properties of chitosan make it popular to produce creams and lotions in the cosmetics field. Third, chitosan can be developed as artificial skin for skin replacement due to its structural charactristics. Fourth, chitosan has potential as a wound dressing material for its degradation, which is beneficial for wound healing and regeneration of skin tissue. It is believed to be a novel breakthrough in the field of medical. What s more, chitosan is very useful in the field of water engineering, food and nutrition, ophthalmology, paper finishing, solid-state batteries, drugdelivery systems and biotechnology. In conclusion, chitosan has a wide range of use in various applications. Its excellent properties make it successful in many fields and promising in the future applications. [18] Although chitosan has many merits, it has a demerit in that chitosan is insoluble in aqueous medium above ph 5.6. The most popular method to eliminate this limit is trimethylation of chitosan to form a quarternary salt which is called trimethyl chitosan (TMC) and has increased solubility, especially in neutral ph values. [7] 9

25 Figure 2.3 Chitosan and its applications 10

26 2.5 Some other polyelectrolytes and their applications Poly(acrylic acid) (PAA) is an important polyelectrolyte with ionic side groups. Its anionic property makes it easy to absorb and retain water. Due to electro-static repulsion of the charged groups, its chain is isotropic when it swells. [19] Compared to chitosan, its polyelectrolyte behavior is similar to chitosan when it is dissolved in water. Its dissociation of the ionic groups also decides the coil expansion which is related to the viscosity of the solution. On the other hand, it can form polyelectrolyte complex or gel with chitosan called chitosanpoly acrylic acid (CS-PAA) complex as a counter poly ion to chitosan. Due to the fact that they are both biocompatible and biodegradable, it is viable to control the particle size by changing the weight ratio and solution concentration. [22] The chitosan-poly acrylic acid complex has been used in various applications for its changeable features. According to Visith s research, he found that it was possible to create specific chitosan-poly acrylic acid complex with controlled properties by adjusting the initial ph of the mixture of reacting chitosan and polyacrylic acid. [1] The applications of poly(acrylic acid) is various, e.g., ion exchange resins, adhesives and disposable diapers.[20] Xue s paper talked about the novel interpenetrating polymer network hydrogels based on konjac glucomannan (KGM) with poly(acrylic acid) which promoted the efficiency of drug delivery with its ph-dependent character. [23] Also, Jae-soon utilized poly(acrylic acid) and chitosan to develop a novel transmucosal drug delivery system by promoting the adhesive force and limited aqueous solubility of polymer complex which was formed by H-bonding between poly(acrylic acid) and chitosan. [25] In Carlos s article, his work illustrated the procedure of producing self-curing membranes of chitosan/paa 11

27 interpenetrating polymeric networks (IPNs) by radical polymerization and characterizations of the products. In his case, chitosan is used as a polymeric ionic template with ionizable acrylic acid. [24] 2.6 Viscosity Viscosity is an important property for determining molecular weight. It is used to calculate the molecular weight of polymer, e.g., polyelectrolytes. The viscosity of a solution is first defined as the ratio of the shear stress and the shear rate. To most researchers, however, the intrinsic viscosity [η] is the most relevant variable to describe the solution aspect of polymer viscous behavior. The solution is expected to be an ideally diluted solution where single unentangled polymer coils are found. What s more, most viscosimetric measurements are produced to determine intrinsic viscosity. The relation between the intrinsic viscosity [η] of a polymer and molar mass M can be showed in the Kuhn-Mark-Houwink-Sakurada-relationship, [η] = K [η] M a. 12

28 2.7 Parameters influence viscosity The viscosity is affected by many parameters, e.g., concentrations and molar mass, solvent, temperature, shear rate, chain flexibility, ph, chemical structure (Fig.2.4), salt effect and types of polyelectrolytes. Polyelectrolyte has different behaviors in aqueous solution and a salt solution. The addition of low molecular salts influences a polyelectrolyte solution significantly. (Fig.2.5) Osmotic pressure and coulomb forces are compensated by salts through shielding of the dissociated ionic groups. The result is that the reduced viscosity ηred becomes linearly increasing when the concentration of polyelectrolyte is gradually increased in the presence of salt. However, the reduced viscosity ηred is nonlinear without the addition of salt. [21] The coil expansion of polyelectrolytes such as chitosan is also determined by the degree of dissociation of the ionic groups. (Fig.2.6) [21]At very low polymer concentrations, the effect of repulsion between the ionic groups of the polymer chains results in expansion of the coil, which leads to the dramatic increase of the reduced viscosity ηred. At lower polymer concentrations, there exists a plateau of the reduced viscosity ηred. The reduced viscosity ηred also increases slightly with an increasing concentration. At high polymer concentrations, the entanglement of polymer chains makes the reduced viscosity ηred increase faster as the concentration rises. 13

29 Figure 2.4. Intrinsic viscosity evolution for different chemical structures of the polymer chain [21] 14

30 Figure 2.5.Different behavior of a polyelectrolyte in aqueous solution and a salt solution. [21] 15

31 Figure 2.6 Reduced viscosity ηred as a function of the concentration c for the polyelectrolyte sodium salt of poly(acrylic acid) (PAAcNa) in aqueous solution. [21] 16

32 2.8 Conclusion Chitosan has interesting properties as a poly electrolyte. Especially, viscosity properties are influenced by structure (DA, Mw), solvent (acid concentration, salt concentration.). These properties are important to understand preparation of chitosan products, gels, films, etc. 17

33 3. EXPERIMENTAL Chitosan is a polysaccharide consisting of of β-( 1-4) linked 2 acetamido-2-deoxy- β- ɒ-glucopyranose and 2-amino-2-deoxy- β-ɒ-glycopyranose as shown in Figure 2.2. The two different kinds of chitosan used in this research have different molecular weights by the deacetylation of different chitins. Chitosan 00-ASSC-7676 was purchased from Vanson Inc. (Redmond WA). Chitosan L (FPS-NCSU 2002) was made by Dr.sanghoon Lim during his graduate work. Chitosan samples were prepared individually for various concentrations (0.001, 0.002, 0.004, and 0.01g/mL) by dissolving a specific amount of polymer in a given acetic acid (HAc)/ sodium acetate (NaAc) solvent with different proportion. The samples were shaken for 30 minutes or centrifuged for 10 minutes at room temperature before they were stored in refrigerator for 24 hours. The flow time of each sample was measured by using an Ubbelohde capillary [26] (Cannon-Fenske, No.75). The properties of the chitosan samples were assessed by viscosity measurements. Various viscosity terms are listed in Table 3.1. Table 3.1 Nomenclature Used in This Study Kinematic viscosity of the solvent η0 mpa s or cst Kinematic viscosity of the solution η mpa s or cst Relative viscosity ηrel = η/η0 dimensionless Specific viscosity ηsp = ( η- η0)/η0 = ηrel -1~(t-to)/ to dimensionless Reduced viscosity ηred = ηsp/c = (ηrel -1)/C dl/g Inherent viscosity ηinh = (ln ηrel)/c dl/g Intrinsic viscosity [η] = (ηred/c)c 0 = (ηinh)c 0 dl/g Solution concentration C g/dl 18

34 To remove the influence of moisture, each chitosan sample was dried in a Fisher constant temperature oven at 80 C overnight. In order to explore the influence of salt and acid concentration on the viscosity of chitosan solutions, six different solvents were prepared with various proportions of acetic acid (HAc) and sodium acetate (NaAc), i.e. 0.1M HAc/0.01M NaAc, 0.1M HAc/0.1M NaAc, 0.1M HAc/0.3M NaAc, 0.3M HAc/0.01M NaAc, 0.3M HAc/0.1M NaAc and 0.3M HAc/0.3M NaAc. Dried samples were dissolved into the 6 different solvents at room temperature. Meanwhile, each solvent was used to prepare five different solutions with various chitosan concentrations, i.e g/mL, 0.002g/mL, 0.004g/mL, 0.005g/mL and 0.01g/mL. (shown in Table 3.2 and Table 3.3) To ensure the accuracy of the dilutions, a series of smaller dilutions is better than preparing individual solutions directly. So serial dilution process was used in one batch (Chitosan 00-ASSC-7676 dissolved in 0.3M HAc/0.3M NaAc solvent) as a matched group. Dilutions are made by mixing a known amount of sample with a known volume of diluent and each dilution is made using the same volume of material transferred to a series of tubes containing the same volume of diluent. [27] Thus, systematic errors are eliminated when the intrinsic viscosity versus solute concentration are plotted. All solutions were centrifuged (IEC HN-SII Centrifuge) for 10 minutes and stored at 5 C in the refrigerator. After the chitosan samples were fully dissolved in the solvents, the flow times of solutions were measured by viscometer at a constant temperature (controlled to within +/- 0.1 C). Solution is driven by gravity to flow through capillary. The flow time is defined as the running time of each solution between two points on the viscometer. Depending on the viscosity range of the chitosan solutions, an Ubbelohde capillary was used to make 19

35 measurement for its appropriate size such that the solution flow time was greater than 100sec. [28] The experimental temperature was maintained at 30 C or 40 C by placing chitosan solutions inside a constant temperature bath (Cannon CT-1000) for 1 hour before measuring the flow time. Flow time was measured for at least 3 times to make sure that the difference between each times was less than 1 sec. Flow time of solvent is t s. The average flow time of every solution was marked as t. Then the relative viscosity ηrel equals to t/ts, (flow time of each solution is shown in Table 3.2 and Table 3.3) 20

36 Table 3.2 Original flow time for chitosan L chitosan solute concentration solvent flow time flow time (g/ml) HAc/NaAc(M/L) solvent (S) (S) / / L / / / / / L / / / / / L / / / / / L / / / / / L / / / / / L / / /

37 Table 3.3 Original flow time for chitosan 00-ASSC-0767 chitosan solute flow concentration solvent flow time time (g/ml) HAc/NaAc(M/L) solvent (S) (S) / / ASSC / / / / / ASSC / / / / / ASSC / / / / / ASSC / / / / / ASSC / / / / / ASSC / / /

38 4. RESULTS 4.1 Intrinsic viscosity and polymer molecular weight The intrinsic viscosity, [η], is a characteristic property for a polymer molecule dissolved in a certain solvent [29]. In dilute chitosan solution, ionic strength plays a major role in determining the [η]. Chitosan solution with low ionic strength have an increased [η] by the formation of intra-molecular H bonds. On the other hand, [η] is constant with increased ionic strength by the screening of amino groups of the polymer which contribute to the appearance of H bonds [30]. The intrinsic viscosity is also influenced by molecular weight, polymer structure and conformation as well as by the solvent quality [31]. In brief, it reflects hydrogen bonding, dipole-dipole, hydrophobic interactions, and complexation ability of the polymer in the presence of different additives [32]. The intrinsic viscosity [η] can be obtained by using Huggins (1) and Kramer (2) equations by plotting ηsp/c and (ln ηrel)/c, respectively, against C, and subsequent extrapolation to zero concentration [29][31][33]. ηsp/c = [η] + k [η] 2 C (1) (ln ηrel)/c = [η] + k [η] 2 C (2) k and k are the Huggins and Kramer constants, respectively, and C is the concentration (g/dl) of the polymer solution. The experimental data and calculated results of L and 00-ASSC-0767 are shown in Table 4.1 and Table 4.2, respectively. The Kraemer and Huggins plots for both chitosan samples are shown in Figure 4.1~4.17. Notice that some data points are neglected due to deviation. 23

39 Table 4.1 Relative, specific, reduced and inherent viscosities of L Chitosan Solute concentration Solvent η(rel) η(sp) η(red) η(inh) (g/ml) HAc/NaAc (M/L) / / L / / / / / L / / / / / L / / / / / L / / / / / L / / / / / L / / /

40 Table 4.2 Relative, specific, reduced and inherent viscosities of 00-ASSC-0767 Chitosan Solute concentration Solvent η(rel) η(sp) η(red) η(inh) (g/ml) HAc/NaAc(M/L) / / ASSC / / / / / ASSC / / / / / ASSC / / / / / ASSC / / / / / ASSC / / / / / ASSC / / /

41 η sp /C(mL/g) & ln(η sp )/C(mL/g) 700 R² = η(red) η(inh) R² = C(g/mL) Figure 4.1 Kraemer and Huggins plots for L in HAc 0.1M/NaAc 0.01M solvent at 30 C (Datapoint with solute concentration 0.001g/mL is neglected.) 26

42 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = R² = η(red) η(inh) C(g/mL) Figure 4.2 Kraemer and Huggins plots for sample L in HAc 0.1M/NaAc 0.1M solvent at 30 C 27

43 η sp /C(mL/g) & ln(η sp )/C(mL/g) 450 R² = R² = η(red) η(inh) C(g/mL) Figure 4.3 Kraemer and Huggins plots for sample L in HAc 0.1M/NaAc 0.3M solvent at 30 C 28

44 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = R² = η(red) 50 η(inh) C(g/mL) Figure 4.4 Kraemer and Huggins plots for sample L in HAc 0.1M/NaAc 0.3M solvent at 40 C 29

45 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = η(red) η(inh) R² = C(g/mL) Figure 4.5 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.01M solvent at 30 C 30

46 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = R² = η(red) η(inh) C(g/mL) Figure 4. Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.1M solvent at 30 C 31

47 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = R² = η(red) η(inh) C(g/mL) Figure 4.7 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.3M solvent at 30 C 32

48 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = R² = η(red) 50 η(inh) C(g/mL) Figure 4.8 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.3M solvent at 30 C using serial dilution 33

49 η sp /C(mL/g) & ln(η sp )/C(mL/g) 350 R² = R² = η(red) 50 η(inh) C(g/mL) Figure 4.9 Kraemer and Huggins plots for sample L in HAc 0.3M/NaAc 0.3M solvent at 40 C 34

50 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = η(red) R² = η(inh) C(g/mL) Figure 4.10 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.1M/NaAc 0.01M solvent at 30 C 35

51 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = R² = η(red) 50 η(inh) C(g/mL) Figure 4.11 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.1M/NaAc 0.1M solvent at 30 C (Data points with solute concentration 0.005g/mL and 0.01g/mL are neglected.) 36

52 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = η(red) R² = η(inh) C(g/mL) Figure 4.12 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.1M/NaAc 0.3M solvent at 30 C (Data points with solute concentration 0.002g/mL is neglected.) 37

53 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = R² = η(red) 100 η(inh) C(g/mL) Figure 4.13 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.1M/NaAc 0.3M solvent at 40 C 38

54 ln(η sp )/C(mL/g) R² = C(g/mL) Figure 4.14 Kraemer plot for sample 00-ASSC-0767 in HAc 0.3M/NaAc 0.01M solvent at 30 C (No effective data for Huggins plot and data points with solute concentration 0.005g/mL and 0.01g/mL are neglected. The low salt concentration contributes to this situation. See at low concentration in Figure 2.7.) 39

55 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = η(red) R² = η(inh) C(g/mL) Figure 4.15 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.3M/NaAc 0.1M solvent at 30 C (Datapoints with solute concentration 0.004g/mL is neglected.) 40

56 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = η(red) η(inh) R² = C(g/mL) Figure 4.16 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.3M/NaAc 0.3M solvent at 30 C (Datapoints with solute concentration 0.001g/mL is neglected.) 41

57 η sp /C(mL/g) & ln(η sp )/C(mL/g) R² = R² = η(red) 100 η(inh) C(g/mL) Figure 4.17 Kraemer and Huggins plots for sample 00-ASSC-0767 in HAc 0.3M/NaAc 0.3M solvent at 40 C 42

58 Molecular weight of chitosan is important because [η] increases with it. This relationship is referred to as the Mark-Houwink-Sakurada equation (3) or [η]-m-relationship. [29] [η]= K M α (3) Where M is the viscosity average molecular weight (Mv) of the polymer in solution, and K and α are constants for a specific polymer-solvent pair at a particular temperature [31]. Many papers discuss the constants K and α in different solvents. Table 4.3 lists the values of Mark- Houwink-Sakurada equation constants, K and α, and molecular weight range from various papers for chitosan published in the literature [49]. The values of K range from 3.04*10-7 to 5.59*10-3. The values of α ranges from 0.58 to As a cationic polyelectrolyte, the positive charges (NH3 + ) on chitosan are influenced by DA, ph and ionic strength. Besides, the polymer conformation and the polymer-solvent interactions depend on the number of positive charges on chitosan for the reason that electrostatic repulsion contributes to the degree of expansion of chitosan [49]. The different values of α represent different conformations of the polymer chain. So α is relative to DA, ph and ionic strength. [50] In a good solvent, the value of α is 0.8 [51][52]. Due to the chain conformation of chitosan in solution is a random coil, the values of α of chitosan in solution is between 0.5~0.8. [50] We can use the values of K and α in Rinaudo s papers to estimate the molecular weight range of chitosan L and 00-ASSC-0767 (Results are shown in Table 4.4.). 43

59 Table 4.3 Mark-Houwink-Sakurada Constants for Chitosans with Varying of DA and Solvents of Different ph and Ionic Strength, µ 44

60 Table 4.4 The estimated molecular weight referred to Rinaudo et al., , Rinaudo et al., , Rinaudo et al., Chitosan K(mL/g) α Solvent (HAc/NaAc) MW 0.1M/0.01M M/0.1M ASSC M/0.3M M/0.01M M/0.1M L M/0.3M M/0.01M M/0.1M M/0.3M M/0.01M M/0.1M M/0.3M

61 5. DISCUSSION 5.1 Salt effect Usually, there exist high content of extra salts in many polyelectrolyte samples from the procedure of the synthesis. The intrinsic viscosity depends on salt concentration which also influences the chain expansion. So the influence of salt concentration in chitosan solution should be taken into account. It is necessary to remove salt or ensure the content of salt in chitosan solution. The effect of different salt concentrations on the specific viscosity (ηspec) of pectin-na is shown in Figure 5.1 for an aqueous solution. It is obvious that the addition of salt can reduce the ηspec of the solution at constant polymer concentration, because the ionic strength is changed by the effect of added salt. Especially, the ηspec decreases dramatically at low polymer concentration when the salt is added into the solution. It is a consequence of the fact that the electrostatic interaction between the charged polymer segments is still effective in extremely dilute systems, for which the viscosity difference between the solution and the solvent can no longer be measured accurately enough [53]. To get rid of the salt effect on the viscosity of chitosan samples, a method called isoionic dilution (serial dilution) was taken to avoid the error occurred in the measurement of the intrinsic viscosities by measuring the viscosity at various polymer concentrations in a series of electrolyte solutions in such a manner that the total ionic strength of the solution is kept constant [54]. The sample L in HAc 0.3M/NaAc 0.3M solvent at 30 C is used as serial dilution sample. The result of Kraemer and Huggins plots using serial dilution is shown in Figure

62 Figure 5.1 Huggins plot for solution of pectin-na in different aqueous NaCl solutions of constant composition at 27 C [53] 47

63 Table 5.1 Huggins k and Kraemer k constants and intrinsic viscosity [η] refers to both equations for both L and 00-ASSC chitosan solvent huggins kraemer [η](ml/g) [η](ml/g) (From (From HAc/NaAc(M/L) k k' Huggins Kraemer Equation) Equation) 0.1/ / L / / / / / ASSC / / /0.01 / / / /

64 [η] (ml/g) L ASSC Cs (M) Figure 5.2 Intrinsic Viscosity ([η]) as a function of Salt Concentration (Cs) refers to Huggins Equations for L and 00-ASSC-0767 in HAc solvent 49

65 With the experimental data and calculated results of L and 00-ASSC-0767 in Table 4.1 and Table 4.2, the Huggins k and Kraemer k constants and intrinsic viscosity [η] refers to both equations for both L and 00-ASSC-0767, and which are listed in Table 5.1. The plots of the salt concentration (Cs) vs intrinsic viscosity ([η]) for both L and 00-ASSC-0767, are shown in Figure 5.2. The intrinsic viscosity decreased and then increased with increased salt concentration. The explanation of this result is related to the ionic strength which is contributed to the expansion of the coils of chitosan. In high salt concentration of chitosan solution, the salt diffuses into the coils of polymer and more counter-ions which screen the protonated amine groups are generated [53]. The result of compactness contributes to the decrease of intrinsic viscosity [55]. However, in low salt concentration of chitosan solution, the salt is out of them and the third electro-viscous effect results in the repulsion that increase the chain expansion [56]. The result of repulsion contributes to the increase of intrinsic viscosity. We conclude that the influence of salt concentration is an important factor for both chitosan samples. The amplitude of the intrinsic viscosities changes of sample 00-ASSC-0767 is larger than L3-56-3, it shows that sample 00-ASSC-0767 is more sensitive than the other one by the addition of salt. 50

66 5.2 Temperature effect For the success of the experiment, the constant of the temperature (the error should be controlled under 0.1 C ) is a crucial requirement for the reason that it determines the intrinsic viscosity of chitosan solutions. Theoretically, the rheological properties of a Newtonian fluid or polymer liquid are consistent with the Arrhenius equation when the temperature is higher than the glass transition temperature (Tg) or melting point. [57] The Arrhenius equation (3) is shown as follows: [η] = Ae E/RT (3) Where [η] is the intrinsic viscosity, A is the characteristic constant for chitosan with a specific molecular weight, E is the activation energy, R is the gas constant, and T is the temperature ( K) [58]. If the Arrhenius equation is plotted at the natural logarithmic intrinsic viscosity (ln [η]) and the inverse of temperature (1/T), the slope of the Figure is E/R. Because stiffer polymers have larger E s, so the slope (d ln [η]/d (1/T)) which is related to E can be used as an index for stiffness of the chitosan [58][59]. The larger slope means the polymer is stiffer. [60] Table 5.2 Intrinsic viscosities [η] of chitosans in 30 C and 40 C Chistosan L ASSC Solvent [η]30 [η]40 HAc/NaAc(M/L) (ml/g) (ml/g) [η]30 / [η]40 -(d ln[η] / dt) 0.1M/0.3M M/0.3M M/0.3M M/0.3M

67 [η] (ml/g) / / / / T ( C) Figure 5.3 Intrinsic viscosity ([η]) as a function of the temperature (T) for both L and 00-ASSC-0767 in solvent HAc 0.1M/NaAc 0.3M and HAc 0.3M/NaAc 0.3M 52

68 To explore the relationship between the intrinsic viscosity and temperature, the intrinsic viscosity of both L and 00-ASSC-0767 were measured in 30 C and 40 C (data are shown in Table 5.2) There are three possibilities. If the value of d ln[η] / dt is positive, the intrinsic viscosity increases with the temperature. While if the value of d ln[η] / dt is negative, the intrinsic viscosity decreases with the temperature. The intrinsic viscosity is independent of the temperature if the value of d ln[η] / dt is 0. By comparing the intrinsic viscosities of the same chitosan samples in different temperature, it is obvious that the intrinsic viscosity decrease with increased temperature for the reason that the molecular flexibility of chitosan is higher with higher temperature, which cause the intrinsic viscosity to decrease [61][62]. The data show both chitosan sample solutions have negative values of d ln[η] / dt. However, for sample L in solvent 0.3M HAc /0.3M NaAc, the value of d ln[η] / dt is 0.04 which is almost close to 0. That means it is almost independent of temperature and temperature-relevant experiment is not work for it to explore the chain flexibility. Figure 5.3 demonstrates the result of intrinsic viscosity ([η]) as a function of the temperature (T) for both L and 00-ASSC-0767 in solvent HAc 0.1M/NaAc 0.3M and HAc 0.3M/NaAc 0.3M. The ratio of radius of gyration and average molecular weight decrease dramatically with an increased temperature, which contributes to higher compactness of the molecules and chain flexibility [61][62]. The more flexibility of chitosan chains, the less intrinsic viscosity. Pogodina et al. [63] mentioned the same result that the intrinsic viscosity of chitosan decreased with increasing temperature and the value of d ln[η] / dt was calculated to be 5.3*10 3. Rinaudo and Domard s group [64] transformed d ln[η] / dt to d ln[η] / d(1/t) and the value became 488, which means that chitosan is a stiff molecule in acid condition with 53

69 added salt. Chen and Lin s group [65] reported that the apparent viscosity of chitosan (1%) decreased with increasing temperature, and E=2.005*10 7 J/(kg*mol), which indicates that chitosan is quite stiff in 0.1 M acetic acid. Chen and Lin s group [65] also stated that the flexibility of chitosan increases with increasing solution ionic strength. Noguchi s group [66] found that the hydrogen-bonded hydration water influenced the intrinsic viscosity a lot. The intrinsic viscosity decreases with increased temperature which makes the reduction of hydrogen-bonded hydration. 54

70 5.3 Summary There are many parameters (C, T, solvent, shear rate, salt concentration) that affect the intrinsic viscosity of chitosan, among which salt concentration and temperature are crucial to understanding the flexibility of polymer chain and the conformation of chitosan polymer in solution. Ionic strength-relevant Cs and temperature-relevant d ln[η] / dt were calculated and discussed. Compared to literature, the characteristics of both chitosan samples are analyzed. Both of them have a sensitive response to variation of temperature. The salt concentration determines the ionic strength of the solution, which in turn influences the intrinsic viscosity of the chitosan solution. The small addition of salt makes a big difference on the compactness of the molecules. It indicates that the intrinsic viscosity can be controlled by adding proper amount of salt into the solution. The ionic side groups of polyelectrolytes will be influenced by the added salt, which contributes to the expansion of coils. Temperature is related to the radius of gyration and average molecular weight which can be described as compactness of the molecules. The value of d ln[η] / dt can be used as an index for the chain flexibility of chitosan. It helps us to decide if it is suitable to do temperaturerelevant experiment on specific chitosan sample. 55

71 6. RECOMMENDATIONS FOR FUTURE RESEARCH The intrinsic viscosities of chitosan solution with different acid salt and concentrations are relative to the chain conformation, which can be demonstrated by the some parameters. The intrinsic data can be used to calculate these parameters. The chain conformation is a huge field for the study of chitosan. The stiffness of polymer chain can be studied by worm-like chain model which is helpful to get a better understanding of chitosan. The polymer chains of chitosan can be determined by bond lengths and angles. The coil state can be described as unperturbed coil dimensions. Unperturbed coil dimensions can be discussed by employing the extrapolation methods of Kurata-Stockmayer-Fixman (KSF) and Berry equations. Hydrodynamic expansion factor and unperturbed state parameters can be obtained. 56

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