4.1 FUNCTIONAL CHARACTERIZATION OF CHITOSAN MEMBRANE

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1 RESULTS AND DISCUSSIONS CHAPTER IV 4.1 FUNCTIONAL CHARACTERIZATION OF CHITOSAN MEMBRANE Among the novel families of biological macromolecules, whose relevance is becoming increasingly evident are chitin and its main derivative, chitosan. Potential and usual applications of chitin, chitosan and their derivatives are estimated to be more than 200 (Kumar, 2000). Chitosan is derived from chitin, by a deacetylation reaction using an alkali. Chitosan is therefore a copolymer of glucosamine and N-acetyl glucosamine. It is composed of β-(1, 4)-2 amino-2-deoxy-d-glucopyranose (glucosamine units) and β-(1, 4)-2-acetamido-2-deoxy-Dglucopyranose (acetyl glucosamine units). The term chitosan refers to chitin that has been deacetylated to greater than 60%. Chitosan has many properties that have generated interest in its use such as biodegradability, biocompatibility and its nontoxic nature (Varma, 2004). The deacetylated product, chitosan, has an amine functional group, which is strongly reactive with metal ions. This has initiated research into the use of chitosan in metal uptake. The deacetylation degree will control the content of glucosamine and therefore the fraction of free amine groups available for metal binding. These reactive amine groups interact with the metal ions in different ways, such as by chelation or electrostatic attraction depending on parameters such as ph, and total composition of the solution. These groups are more reactive than the acetamide groups present on chitin. A complete Physical, Chemical and Physiochemical characterization of chitosan and its complexes are not possible without using spectroscopic techniques like FTIR, XRD, UV- Vis, SEM etc., (Kumirska et al., 2010) Fourier Transform Infrared (FTIR) Spectroscopy Infrared (IR) spectroscopy is one of the most important and widely used analytical techniques available to scientists working on chitin and chitosan. It is based on the vibrations of the atoms of a molecule. The infrared spectrum is commonly obtained by passing infrared electromagnetic radiation through a sample that possesses a permanent or

2 induced dipole moment and determining what fraction of the incident radiation is absorbed at a particular energy (Stuart 2004). The energy of each peak in an absorption spectrum corresponds to the frequency of the vibration of a molecular part, thus allowing qualitative identification of certain bond types in the sample. An IR spectrometer usually records the energy of the electromagnetic radiation that is transmitted through a sample as a function of the wavenumber or frequency. Fourier-transform infrared (FTIR) spectroscopy has dramatically improved the quality of infrared spectra and minimized the time required to obtain data (Smith 1996, Stuart 2004). The infrared spectrum is commonly plotted in one of three formats such as transmittance, reflectance, or absorbance. If a fraction of light is transmitted through the samples, then the transmittance of the sample at frequency ω (T ω ) is defined as I ω t (4.1.1) T = I 0 ω where I t is the intensity of transmitted light and I 0 is the intensity of the incident light. In the present work, absorbance spectra were used to quantify the characteristic peaks of Chitosan in the middle infrared region (4000 cm -1 to 400 cm -1 ) with a resolution of 2 cm -1 for 8 to 128 scans at room temperature using a Bruker IFS - 66V spectrometer. Since all the samples under study were transparent and freestanding films, the samples were placed directly on the sample holder for the analysis. The analyses were carried out in the three regions ( , and cm -1 ) of the pure chitosan (CHP) and chitosan acetate (CHA) spectra (Fig 4.1.1). Pure chitosan means, chitosan membranes / films free from solvent or dopant molecules and chitosan acetate means, chitosan membranes / films in the presence of acetic acid (solvent). There are no significant peaks observed in the range of cm -1. The first region ( cm -1 ) has no significant peaks except small peaks at 1153 cm -1 (C-O-C vibrations), 900 cm -1 and 647 cm -1 (NH wag primary and secondary amines) found in pure chitosan, which is gradually broadened and obscured when it is dissolved in acetic acid. The peak positions and the corresponding band assignments of pure chitosan film / membrane are shown in table

3 The spectra of pure chitosan (CHP) and chitosan acetate (CHA) films in the region between 1200 and 1800 cm -1 show (Fig ) significant peaks for analysis. This is the region where the carbonyl, C O /NHR, amine, NH 2 and ammonium, NH + 3 band, OH and CH deformation are situated. The band at 1380 cm -1 is due to the CO stretch of the primary alcoholic group (CH 2 -OH) and at 1262 cm -1 represents free primary amino group (NH 2 ). The band at 1153 cm -1 and 1082 cm -1 are due to the anti-symmetric stretching of C-O-C bridge and skeletal vibrations involving C-O-C stretching. The band at 1650 cm -1 represents acetylated amino group, which is due to the C=O stretching vibrations of (Amide I) O-C-NHR. The band at 1590 cm -1 is assigned to NH 2 bending vibrations (amide II) (Osman 2003). CHA Transmittance (Arbitrary Units) CHP Wave Number (cm -1 ) Fig FTIR spectra of Chitosan pure (CHP) and Chitosan acetate (CHA) The NH 2 bending vibrations (1590 cm -1 ) for CHP disappeared and new absorption band characteristic of NH + 3 bending vibrations appeared at 1630 cm -1 and 1568 cm -1. These results suggest that the NH 2 groups in the chitosan chains were protonated by the H + supplied by acetic acid. In this work, the carbonyl band for the pure chitosan (CHP) spectrum is observed at 1650 cm -1, the amine (NH 2 ) band at 1590 cm -1 and OH and CH deformation band at 1420 cm

4 Table Infrared band assignment for pure Chitosan membrane Peak Position (cm -1 ) Assignment 900 NH wag primary amine 1153 C-O-C vibrations 1262 CH wag (ring) 1323 OH and CH deformation ring 1380 CH symmetrical deformation bend 1420 OH and CH deformation ring 1590 NH 2 deformation 1650 Amide I (C=O) 2878 C-H asymmetrical stretching 2913 C-H symmetrical stretching 3317 N-H 2 asymmetrical stretching 3369 N-H 2 symmetrical stretching 3441 O-H Stretching 64

5 Due to some interaction between the acetic acid and the nitrogen donors of the chitosan polymer, the carbonyl band has shifted to 1630 cm -1, the amine band to 1568 cm -1 and OH and CH deformation moved to 1410 cm -1 in the chitosan acetate film (CHA). This is due to the formation of salt (chitosan acetate) produced due to a reaction between the acetic acid and chitosan as have been reported (Kaneko, 1997), where the H + of acetic acid has formed a dative bond with nitrogen of the chitosan functional group. This is in good agreement with the literature survey (Yahya et al., 2002]. In the third region of the spectra (Fig cm -1 ), the broad band around 3200 cm -1, confirms the conversion of NH 2 groups into NH + 3 groups by the protonation. It is seen from various reports (Miya et al., 1980., Mima et al., 1983 Brugnerotto et al., 2001) that the infrared C-H stretching bands shift to lower wave number and became sharper as crystallinity increases. However, the peak position of aliphatic C-H stretching band in this study (2878 cm -1 ) remains same, but its sharpness is reduced when it is dissolved in acetic acid. This property indicates that the acetic acid alter the crystallinity of the pure chitosan film X- Ray Diffraction (XRD) Spectroscopy X-ray spectroscopy is a powerful and flexible tool and an excellent complement to many structural analysis techniques such as UV-Vis, IR, NMR or Raman. It is unarguably the most versatile and widely used means of characterizing materials of all forms (Guo 2009). There are two general types of structural information that can be studied by X-ray spectroscopy: electronic structure (focused on valence and core electrons, which control the chemical and physical properties, among others) and geometric structure (which gives information about the locations of all or a set of atoms in a molecule at an atomic resolution). Clark and Smith (1937) were the first to make crystal studies of chitin and chitosan using X-ray diffraction (XRD). Main parameter that determines properties of chitosan, apart from molecular mass and deacetylation degree is polymer crystallinity. The chitosan is a semi-crystalline material shows polymorphism depending on its physical state. Recently, true crystal structure and configuration of chitosan is intensively 65

6 investigated. The structures for different forms including an anhydrous form, a hydrated form and various salts were obtained by X-ray diffraction analyses (Ogawa et al., 1992, 1993, 2004; Yawo-Kuo Twu et al., 2005; Trang Si Trung et al., 2006). The small angle X-ray diffraction study carried out on chitosan powder obtained from crab shells, show regular packing of the molecules in parallel bundles. The data indicate that the interactions of chitosan macromolecules along the b-axis give rise to a fibrous structure (Ogawa et al. 2004). The XRD study on chitosan and its complexes will reveal many important parameters that characterize the polymer and show significant information about the physicochemical properties of these materials. 400 (110) 10.3 o, Intensity (cps) (020) Chitosan Two theta ( degrees) Fig XRD spectrum of pure Chitosan (CHP) 66

7 X-ray diffractograms of pure chitosan film measured in the range of 2θ=10-40 (Fig ) showed two characteristic reflections at 10.3 and 20.2 that are typical fingerprints of semi crystalline Chitosan indexed as (020) and (110) known as hydrated crystalline structure and an amorphous structure of chitosan, respectively (Ogawa et al. 1984, Wang et al., 2005). In 1990, Focher et al., used XRD to study chitin and postulated the following equation for determining the crystallinity index (CI): where I 110 (arbitrary units) is the maximum intensity of the (110) peak at around 2θ = 20, and I am (arbitrary units) is the amorphous diffraction at around 2θ = This expression had in fact been employed three years earlier by Struszczyk (1987) to determine the CI of chitosan. Currently this equation is routinely applied during investigations of chitin, chitosan and their derivatives (Kumirska et al., 2010). The degree of crystallinity for pure chitosan film in the investigated range through X-ray diffractometry using the equation 4.1.2, which was employed to determine the crystallinity index (CI) for chitin and chitosan (Zang et al., 2005, Serkan Keleolu 2007, Kumirska et al., 2010) was found to be CI 110 = 56.25%. On the basis of X-ray powder diffractograms of chitin and chitosan with different degrees of N-acetylation, Zhang et al., (2005) noted two maximum peaks of the following intensities: one at the (020) reflection and the other at the (110) reflection and postulated a crystallinity index (CI) expressed by equations and : Further chitin and chitosan studies indicated that crystallinity could also be assigned from an X-ray diffractogram by dividing the area of the crystalline peaks by the total area under the curve (background area) (Kumirska et al., 2010). The calculated value of CI 020 is 8.75% for pure chitosan membrane. In these calculations, the crystallinity percentage supplied information based on relative crystallinity. Studies of Yawo-Kuo Twu (2005) refer to crystallinity of the chitosan formed as scaffolds. The scaffolds were formed by 67

8 electrolysis of chitosan using different solvents (acetic and formic acid). Crystallinity of the chitosan scaffolds is lower than crystallinity of initial polymers and it drops with an increase of the acid concentration ,15.7 0, Intensity (cps) Chitosanacetate Two theta ( degrees) Fig XRD spectrum of Chitosan acetate (CHA) Fig shows the X-ray diffractogram of chitosan acetate film (i.e.) chitosan scaffold, which exhibit two sharp peaks at 11.5 and 15.7, and a suppressed amorphous hump (indicated in dotted line) exhibit many minor peaks around as reported earlier (Puteh et al., 2005, Modrzejewska et al., 2006). It is clearly observed that the peak positions of pure chitosan membrane were shifted to new position ( , and distributed over a range) when chitosan converted to chitosan acetate (Fig and 4.1.3). When chitosan films were formed with acetic acid as solvent, the peak intensity ratio of pure chitosan membrane is reduced (comparing intensity ratio of Fig and 4.1.3). 68

9 Table Crystal Index of pure chitosan and chitosan acetate films Crystal Index Film Type CI 110 CI 020 CHP 56.25% 8.75% CHA 16.12% 32.24% The above result is an indication of reduction of crystallinity of chitosan. The decrease in the crystallinity of chitosan acetate films are due to the formation of hydrogen bonding between acetic acid that leads to their good compatibility. The changes in the crystal index as shown in table informed the reduction of CI 110 and raise in CI 020 explains reordering of glucosamine molecules in the chitosan acetate which is different from the initial polymer. However, the total crystal index (CI) of chitosan scaffold calculated as per the equations and are lower than the pure chitosan due to the fact that when chitosan dissolved in acetic acid the interaction between the acetic acid and the nitrogen donors of the chitosan polymer (NH 2 groups in the chitosan chains were protonated by the H + supplied by acetic acid results in the formation of NH + 3 ) disrupt the crystallinity of the pure chitosan Ultra Violet and Visible (UV-Vis) Spectroscopy The steps in the structural analysis of chitosan and its complexes by UV-Vis spectroscopy are very similar to those in the FTIR methodology (Section 4.1.1). The main difference is connected with the aim of these analyses. IR spectroscopy is used mostly for determining the molecular structure of chitosan and its complexes, whereas UV-Vis spectroscopy is more often applied to the study of covalent and non-covalent interactions. Since certain functional groups present in organic molecules absorb light at characteristic wavelengths in the UV-Vis region, this technique is applied qualitatively to identify the presence of these groups in samples, supporting structural information obtained from other spectroscopic methods, especially IR. As mentioned, one of the most important applications of UV-Vis techniques is the characterization of interactions between chitosan and its solvents or dopants. 69

10 Chitosan has two far-uv chromophoric groups namely, N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), since chitosan cannot be deacetylated completely. The UV-Vis spectra obtained for pure chitosan (CHP) and chitosan acetate (CHA) films showed (Fig transmittance spectra and inset show the absorbance spectra) two characteristic absorptions at circa 210 and 260 nm revealing the presence of pure chitosan (Tyagi et al., 1996). The spectrum of the chitosan acetate (CHA) exhibited a new absorption centered at circa 320 nm indicates the conversion of glucosamine (GlcN) into glucosamine acetate unit. The λ max of CHP (260 nm) shifts to longer wavelength demonstrated the chemical interaction between chitosan and acetic acid even at room temperature. A similar effect was reported by Sharma et al., (2003) who had studied the interaction of chitosan with ammonium sulfate and t-butanol. 320 nm CHA Transmittance (a.u) 260 nm absorbance (a.u) 320 CHP Wave length (nm) Wave length (nm) Fig UV-Vis Spectra of Chitosan Pure (CHP) and Chitosan acetate (CHA) 70

11 The shifts to longer wavelength in UV spectra are known to be associated with increase in hydrogen bonding. Both inter- and intra-molecular hydrogen bonding are possible in these cases. It is already observed that the IR bands of chitosan acetate are shifted to lower frequency (Section 4.1.1). This again fits in well with increase in intra- or inter-molecular hydrogen bonding which decreased the frequency of amine and hydroxyl bonds. The XRD result also showed the reordering of glucosamine (GlcN) unit under the action of solvent. Therefore, the FTIR, XRD and UV-Vis spectroscopic methods characterize the interaction between chitosan and ions of solvents or dopants leads to various applications to be discussed in the later part of the thesis Optical Absorption Analysis of Heavy Metal Ions The problems of the ecosystem are increasing with developing technology. Heavy metal pollution is one of the main problems. Toxic metal compounds coming to the earth's surface not only reach the earth's waters (seas, lakes, ponds and reservoirs), but can also contaminate underground water in trace amounts by leaking from the soil after rain and snow. Therefore, the earth's waters may contain various toxic metals. Drinking water is obtained from springs which may be contaminated by various toxic metals. One of the most important problems is the accumulation of toxic metals in food structures. As a result of accumulation, the concentrations of metals can be more than those in water and air. The contaminated food can cause poisoning in humans and animals. Although some heavy metals are necessary for the growth of plants, after certain concentrations heavy metals become poisonous for both plants and heavy metal microorganisms. Another important risk concerning contamination is the accumulation of these substances in the soil in the long term. Heavy metals are held in soil as a result of adsorption, chemical reaction and ion exchange of soil. In recent years, chitosan and agar have been commonly used to remove heavy metals and organic compounds from water and waste water (Muzzarelli, 1977; Knorr, 1991; Taguchi et al., 1999; Yuh Shan Ho, 2004) in bulk and film forms. 71

12 λ max =514 nm [Fe]/µg Wave length (nm) Fig Linear variation of Fe 2+ ions absorption by the chitosan films λ max =455nm Wave length (nm) [Cu] /µg Fig Linear variation of Cu 2+ ions absorption by the chitosan films 72

13 λ max =414 nm Wave length (nm) [Co]/µg Fig Linear variation of Co 2+ ions absorption by the chitosan films The purpose of this preliminary study is to investigate the heavy metal absorbing capacity of pure chitosan in film form with specific thickness and to study its variation of absorption with concentration. The optical absorption analysis of the chitosan films of typical thickness (50 µm) coated with Iron (II), Copper (II) and Co (II) solutions were carried out using UV-Vis spectrophotometer in the wavelength range of nm. The absorption spectra for different concentrations of metal ions (Fe 2+, Cu 2+ and Co2 + ) were taken and the maximum absorptions were measured from its characteristic peaks. The insets show the absorption spectrum of Fe 2+, Cu 2+ and Co2 + metal ion adsorbed in chitosan films at maximum metal ion concentration. The figures show the linear variation of metal ions absorption for various concentrations by the pure chitosan films. The absorption values get saturated at higher concentration and it limits the analysis. The preliminary experiments on higher thickness improve the absorption capacity. However, interest is taken to study the absorption property in thin film form. Therefore the study is limited and reported only for the optimized thickness, which gives the preliminary support for fabrication of heavy metal sensor for effluent and water treatment. 73

14 Selective Adsorption Property of Chitosan Membrane Rapid industrialization, large scale urbanization involves the use of chemicals containing toxic elements and heavy metal ions that resulted in the increased contamination of our environment. Metallic pollution specifically caused potential danger to the mankind as the heavy metal ions are dumped into the surroundings in the form of industrial effluents. A lot of research has been carried out to alleviate or at least minimize the effect of the heavy metal ions from industrial waste waters in environmental pollution. Several ways and means including filtration, chemical precipitation, ion exchange, adsorption, electro deposition and the use of membrane systems have been developed (Taguchi et al., 1999; Yuh Shan Ho, 2004). Each of the above methods has its own advantages and disadvantages. Any method chosen for that matter need to be environment friendly as the method itself should not be counterproductive to the surroundings. Adsorption method is found to be relatively convenient and economical in addition to our prime objective of not to affect the environment. In recent years, studies on chitin and chitin derivatives, which adsorb metal ions, have increased substantially and that attract greater interest in terms of their efficiency, wider availability and environmental safety. The effectiveness of chitosan to remove lead and cadmium in drinking water has been demonstrated by Knorr, (1991). Chitosan from treated crab shells have also been used effectively to treat effluents from electroplating industry and for the removal of hexavalent chromium (Muzzarelli, 1977). Therefore, studies on the interaction of ions with chitin and chitosan are of importance in ecology not only in connection with water pollution but also with ionic equilibria in uncontaminated natural waters. A systematic analysis of literatures reveals that studies on heavy metal adsorption by chitin and chitosan are plenty. However the cost involved in this process is heavy, since it requires materials in bulk form. At the same, time for certain specific applications, chitin and chitosan in bulk form is unsuitable (Meyers et al., 2000). Therefore, preparing this material in thin film form is valuable and improves its physical and chemical character. 74

15 Wavelength (nm) Fig peaks of the effluent sample Wavelength (nm) Fig peaks of the chromium solution 75

16 Time in minutes Wavelength (nm) 0 Fig Optical absorbance spectra of pure and chromium adsorbed chitosan 1.0 membranes 0.9 CHP+(Cr-VI) 329 nm CHP 30 min min Wavelength (nm) Fig a Optical spectra of pure and 30 min. immersed chitosan membranes 76

17 CHP+(Cr-VI) min. 90 min min Wavelength (nm) Fig b Optical spectra of 60, 90 and 120 min. immersed chitosan membranes Time (min) Fig Variation of absorbance maximum with time 77

18 It is very difficult to dissolve chitin to make it as thin films. Therefore chitosan is selected as a suitable candidate, which can be easily dissolved in organic solvents and can be made as free standing film, which can act as membrane for metal adsorption. The chitosan membranes thus prepared were subjected to selective adsorption property of chromium ions present in the leather effluents. Leather industrial effluent mainly contains chromium III (Cr-III), chromium VI (Cr- VI), proteins and some minerals. Out of these various components Cr-VI is the carcinogenic species that cause cancer in living beings. In order to study the selective adsorption property, the chitosan membranes were immersed in the effluent samples for different time intervals (0, 30, 60, 90 and120 minutes) collected in a leather industry (NMZ Tanners - Ambur). The optical absorption properties of the effluent (Fig.4.1.8) samples show peaks at 326, 415 and 578nm. The peak at 326nm is due to chromium VI species that was well documented in several literatures (Muzzarelli, 1977; Knorr, 1991; Taguchi et al., 1999; Yuh Shan Ho, 2004). The peaks 415 and 578nm are due to chromium III species which was verified with the help of standard chromium solution (Fig 4.1.9). The complete absorption spectra of chitosan membranes show the absorption peak around 330nm as shown in Fig This indicates that the chitosan films selectively adsorbed the dangerous species of the chromium (Cr-VI) in the effluent. Fig a and b show the selective part of the graph drawn to show the correct peak positions. Since the instrument used is having an accuracy of ± 2nm, the error in the measurement of the peak positions are within the limits. The absorption starts immediately after the immersion of the chitosan films in the effluents. The absorption gets saturated around two hours. The maximum absorption found within 30 minutes as shown in Fig The SEM image shows porous nature of the chitosan membrane (Fig ) which is the pre-requisite for membrane filtration (Serkan Keleolu, 2007). Figure shows Cr-VI adsorbed chitosan membrane surface. The absorption mechanism of chitosan can be explained as follows. Chitosan have different functional groups, such as hydroxyls and amines (anions) to which the chromium metal ions (cations) can bind either by chemisorptions or by physi-sorption process. Since the chitosan is fabricated in thin film form provide enormous surface area 78

19 per unit volume which enhances the capacity for attachment of the chromium metal ion on the surface of the film. If a stack of chitosan films arranged in an effluent unit may facilitate the removal of this carcinogenic Cr-VI species completely from the effluent. Further studies on various parameters of heavy metal absorption in chitosan films both qualitatively and quantitatively will throw more light on this material. Fig Scanning Electron Micrograph of the porous surface of pure chitosan membrane Fig Scanning Electron Micrograph of the Cr-VI adsorbed surface of chitosan membrane 79

20 4.1.6 Permeability and Porosity of Chitosan Membrane Permeability is the most important physical property of porous medium, while porosity is the most important geometric property. Permeability describes the conductivity of the porous medium with respect to fluid flow, whereas porosity is a measure of fluid storage capacity of a porous material. The permeability coefficient of any porous medium can be directly obtained from Darcy s law, which states that where Q Flow rate, K permeability coefficient, n fluid viscosity, P Pressure difference, L Flow length or Thickness of the test sample and A Area of the sample. The porosity can be calculated from the formula % 100 / where V t is the total volume of chitosan (cm 3 ), V a the actual volume of chitosan (cm 3 ) and W t is the mass of chitosan (g) and ρ is the density of chitosan (1.342 g/cm 3 ). A fully automated Gas Permeability Tester (GPT LYSSY L ) was used to analyze the permeability and porosity of pure chitosan (CHP) samples of thickness 40 µm with the CO 2 flow rate of 100 bars on an area of cm 2. The permeability and porosity was calculated using equations 4.4 and 4.5 and corresponding values are cm 3 / (m s Pa) or 205 ml / m 2 day and Similar results were reported for chitosan by Wen-Chuan Hsieh et al., (2007) who studied the microporous structure for the development of cell culture. It should be noted that the porosity does not give any information concerning pore sizes, their distribution, and their degree of connectivity. However, the porosity value indicates that 88% of the membrane area is porous as seen from the SEM image (Fig ). From the SEM image it was found that the pore size ranges from µm suitable for water and effluent filtration. The maximum capacity of single membrane to withstand the gas pressure was 800 bars. Therefore stack of chitosan membrane may be arranged for the removal of Cr-VI in an effluent unit. 80