I J P A C Global Research Publications

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1 I J P A C Global Research Publications International Journal of Pure & Applied Chemistry Vol. 6 No. 2 April-June 2011 pp Study on Dye-Binding Interactions of Chitosan Obtained from the Fish Scale of Tilapia (Tilapia nilotica) Nanthawan Uawonggul, Supalak Kongsri and Saksit Chanthai * Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science Khon Kaen University, Khon Kaen 40002, Thailand ABSTRACT: In this study, a white powder of chitosan was produced from its chitin, which was extracted from the fish scale of Tilapia (Tilapia nilotica). Proximate analysis (moisture and ash) and average molecular weight (~69 kda) of the chitosan were determined. Structural data of the chitosan determined by FT-IR were compared with those of chitin concerning the degree of deacetylation. For dye binding study, the chitosan soluble in 2% (v/v) lactic acid was mixed with each of dye solutions (Congo red, Methylene blue and Sunset yellow FCF). The visible absorption resulting in the interactions of the dye-chitosan complex was monitored at their maximum wavelengths; 483, 488 and 662 nm for Congo red, Methylene blue and Sunset yellow FCF, respectively. The following parameters attributed to their interactions including concentration of dye and chitosan, ph, ionic strength, incubation time and temperature were investigated in detail. These results are due insight of the molecular interactions of these dye-chitosan complexes, and can further be implied for the removal efficiency of toxic waste dyes. Keywords: Dye adsorption, Interactions, Chitin, Chitosan, FT-IR INTRODUCTION Many industries such as textile, paper, plastics and dyestuffs, consume substantial volume of water, and also use chemicals during manufacturing and dyes to color their products. As a result, they generate a considerable amount of polluted wastewater, especially color. Color is the first contaminant to be recognized in wastewater. It is known that wastewaters containing dyes are very difficult to treat, since the dyes are recalcitrant molecules particularly azo dyes, resistant to aerobic digestion, and are stable to oxidizing agents. Another difficulty is treatment of wastewaters containing low concentrations of dye molecules [1]. A large variety of non-conventional adsorbent materials has been proposed and studied for their ability to remove dyes. However, high adsorption capacities of adsorbents are still under development to reduce the adsorbent dose and minimize disposal problems. To improve the efficiency of the adsorption processes, it is essential to develop the * To whom correspondence be made: sakcha2@kku.ac.th more effective and cheaper adsorbents with higher adsorption capabilities [1-3]. In recent years, from the viewpoint of environmental safety, much attention has been paid to the adsorption of dyes on various kinds of biomass, such as cellulose, chitin and chitosan. Among these natural polymers, chitin is most abundant natural aminopolysaccharide, poly[β- (1 4)-2-acetamido-2-deoxy-D-glucopyranose], which is a natural polymer. The main derivative of chitin is chitosan, obtained by alkaline deacetylation of chitin giving amino group as shown in Figure 1. Chitosan is usually found as polymer chains containing a unique monomer (Nacetyl-D-glucosamine or D-glucosamine). The presence of the amido/amino groups in the polymer chain allows the interaction of the biopolymer with dye, therefore it can be used as sequestering agent in polluted effluents [3-5]. Chitosan has been investigated by several researchers as a biosorbent for the capture of dissolved dyes from aqueous solutions. This natural polymer possesses several intrinsic characteristics that make it an effective biosorbent for the removal of color. Advantages of biosorbent are low cost compared to commercial

2 140 Nanthawan Uawonggul, Supalak Kongsri & Saksit Chanthai activated carbon, silica and zeolite and its outstanding chelation behavior, one of the major applications of this aminopolymer is based on its ability to tightly bind pollutants. Other useful features of chitosan include its abundance, non-toxicity, hydrophilicity, biocompatibility, biodegradability, and anti-bacterial property [1, 5]. is responsible or has a stronger influence on the adsorption phenomena observed [13]. In the present research an alternative adsorbent biomaterial was tested, the fish scales from Tilapia (Tilapia nilotica). This fish species is commercially exploited and consumed by the population of Thailand. The amount of fish scale generated as waste from this consumption allows an abundant source of biomaterial. The use of cheap waste materials in the treatment process of wastewaters containing dyes has raised considerable interest recently. Much attention is paid to the elaboration of technologies with the use of natural and effective sorbents [7]. In this work, the treatment of fish scales from Tilapia (Tilapia nilotica) as chitosan polymer was done from the waste fish scale from food industry to obtain adsorbent materials and used to study the interactions of this biopolymer with dyes. EXPERIMENTAL Figure 1: General Structures of Cellulose, Chitin and Chitosan (Majeti, 2009) The removal of reactive dyes by various kinds of chitosan has revealed notable variability in the dye binding capacities of various chitin and chitosan from crustacean shell wastes [1, 4-12]. However, attempt of isolation of chitin and chitosan from the fish scales waste is introduced. Fish scales are composed of both organic and inorganic matter. Specific studies with fish scales from cod, porgy, and flounder have established that the proteins, the organic fraction present in fish scales, seem to be the major factors governing the adsorption ability, because of the nitrogencontaining ligand present [2]. Of the proteins present in the organic fraction, it seems that keratin with its chitosan, may also be responsible for the adsorptive properties of fish scales. However, other studies reported that hydroxyapatite (HAP), Ca 10 (PO 4 ) 6 (OH) 2, was found and extracted from bones, sea shells and fish scales, and similar materials can also remove metallic ions from wastewater. Thus, it is still not totally clear which fraction of the fish scales of the organic fraction, mainly composed of proteins, or the inorganic fraction, mainly composed of HAP Fish Scale and Dyes The fish scales of Tilapia (Tilapia nilotica) were collected from the waste of fish available in local markets in Khon Kaen. This material was washed clean with tap water and then dried at 60 o C before use. The anionic dyes studied include Congo red and methylene blue (both AR grade) and Sunset yellow FCF (NaCl 53.32%, Ponceau 4R 5% and Sunset yellow FCF 41.68%) as food additive dye, a practical grade used in the experiment as shown in Figure 2. (a) Congo red (b) Sunset yellow FCF

3 Study on Dy-Binding Interactions of Chitosan Obtained from the Fish Scale of Tilapia Figure 2: Deproteinization The chitosan is prepared following the method as described elsewhere [4, 13-21]. The fish scales (40 g) of Tilapia were dissolved in 700 ml of 5% (w/v) NaOH solution. The aqueous solution with stirring 5 h at 70 C was left to stay further overnight for deproteinization, and then the sample was washed with distilled water until neutral ph. Demineralization Demineralization was carried out in dilute HCl solution. All of samples were treated with 0.1 M HCl solution with a solution-to-solid ratio of 40 ml g -1 at ambient temperature for 2 h and then the sample was washed with distilled water until neutral. It was finally washed with ethanol to remove any impurities, and then the demineralized samples were dried at 60 o C overnight. Deacetylation (c) Methylene blue Chemical Structures of (a) Congo Red, (b) Sunset Yellow FCF and (c) Methylene Blue Chitin was treated with 50% (w/v) NaOH (15 ml g -1 ) by stirring at 100 C for 1 h. After that the obtained chitosan was subjected to wash to neutral ph and dried at 60 C overnight. Characterization of Chitin and Chitosan Determination of Moisture and ash Contents Moisture content of the chitosan was determined by gravimetric method [20] as follows: weigh and record weight of dish, place 1.0g of chitosan sample in duplicates in aluminum dish, record weight of dish with sample, take into oven temperature at 105 o C and dry the sample for 2 h. Moisture content is then calculated from difference in the mass loss. The ash content was determined by heating a sample of raw material (1 g) at 600 C for 3 h and weighing the remaining product after cooling in dessicator. Ash content is also calculated from the difference in the mass loss [20]. Fourier Transform Infrared Spectroscopy The FTIR spectra were measured as KBr pellet in the transmission mode in the range cm -1 using Fourier Transform spectrophotometer [19]. The degree of deacetylation (DD) was also determined by FTIR. The method is based on the relationship between the absorbance at 1655 cm -1, attributed to amide groups, and the corresponding value of the hydroxyl band at 3450 cm -1 by applying the following equation [20]: DD = 100 [(A 1655 / A 3450 ) x 100 / 1.33] Determination of the Intrinsic Viscosity of Chitosan For the determination of viscosity-average molecular weight (Dalton), the chitosan was dissolved in a mixture of 2% (v/v) acetic acid with 0.1 M NaCl, then the automated solution viscometer (Viscometer CT 1450) was used to measure the intrinsic viscosity ( η). The Mark- Houwink equation relating to intrinsic viscosity with empirical viscometric constants (K and a) for chitosan was used to calculate the molecular weight using this equation [20]: [η] = KM a [η] ; intrinsic viscosity (dl g -1 ) = dl g -1 K = cm 3 g -1 a = 0.93 Study on Dye-binding Interactions of Chitosan Preparation of Dye and Chitosan Solutions Each powdered dye (0.15 g) of Congo red, methylene blue and Sunset yellow FCF was dissolved in 100 ml de-ionized water. Five ml of the dye solution was added into volumetric flask 100 ml and finally, adjust the final volume with suitable buffer solution. Ammonium buffer solution was used for Congo red and methylene blue, while acetate buffer solution was used for Sunset yellow FCF. The dye concentration was estimated by using UV Visible spectrophotometer (Jenway 6400, U.K.). Their absorb ance was measured at 483, 488 and 662 nm for Congo red, Sunset yellow FCF and methylene blue, respectively. Chitosan (0.25 g) was dissolved in 50 ml deionized water and 2 ml of 87.5% lactic acid. After that, the solution was stirred for 1 h and adjusted to final volume 100 ml with de-ionized water. The stock concentration of chitosan solution used in this experiment was 5000 mg L -1.

4 142 Nanthawan Uawonggul, Supalak Kongsri & Saksit Chanthai Effect of Chitosan and Dye Concentrations The effect of chitosan concentration was carried out by mixing various chitosan concentrations ( mg L -1 ) with 3 ml of each dye solution. The dye-binding solution of chitosan was shaken and incubated at 25 C for 1 h in water bath prior to absorption measurement by UV-Visible spectrophotometer. The effect of dye concentration was done by mixing mg L -1 of chitosan with 3 ml of various concentrations (3-60 mg L -1 ) of dye solutions for Congo red and Sunset yellow FCF. For methylene blue solution, its concentration of mgl -1 was used. The solution was shaken and incubated at 25 C for 1 h in water bath. Effect of ph and Ionic Strength The effect of ph was conducted by mixing mg L -1 of chitosan with 3 ml of the dye solution (75 mg L -1 ). The ph value ranging between 2 and 12 was kept constant throughout the sorption process by addition of HCl or NaOH. The solution was shaken and incubated at 25 C for 1 h in water bath. The effect of ionic strength was done by mixing mg L -1 of chitosan with 3 ml of the dye solutions used. Various concentrations of sodium chloride ( M) were studied. The solution was shaken and incubated at 25 C for 1 h in water bath following the absorption measurement as mentioned above. Effect of Temperature and Incubation Time The effect of temperature was studied by mixing mg L -1 of chitosan with 3 ml of the dye solutions. The solution was shaken and incubated using water bath at various temperatures (5-65 C) for 1 h in water bath. The effect of incubation or contact time was also carried out by mixing mg L -1 of chitosan with 3 ml of the dye solutions. The solution was shaken and incubated at optimum temperature with various contact times (1-12 h) prior to analysis. RESULTS AND DISCUSSION Physical Property and Chemical Structure of Chitin and Chitosan The white and fine solids of chitin and chitosan were proximately analyzed for moisture and ash contents. The moisture contents of chitin and chitosan are 2.12 and 2.98%, respectively. It is low enough comparing with that of the commercial chitosan products (10%) [20]. Ash contents of chitin and chitosan are found to be 6.56 and 8.76%, respectively. This is an indicator of not the effectiveness of the demineralization step for removal of calcium carbonate. Elimination of the demineralization resulted in products both chitin and chitosan gave rather high ash contents. The ash content in chitosan is an important parameter. Some residual ash of chitosan may affect their solubility, consequently contributing to lower viscosity, or can affect other more important characteristics of the final product and the larger mineral content also causes an increase in the gas emissions. A high quality grade of chitosan should be less than 1% of ash content [18]. Molecular weight (MW) of chitosan varied depending on sources and also methods of preparation. The MW of native chitin is usually larger than one million Daltons (Da) while the commercial chitosan products fall between 10 to 120 kda [21]. Several factors during chitosan production, including high temperature, concentration of alkali, reaction time, previous treatment of the chitin, particle size, chitin concentration, dissolved oxygen concentration and shear stress may influence the MW of chitosan can cause degradation of chitosan [18, 21]. In this work, the average molecular weight of this chitosan (6.88 x10 4 Da) was also roughly estimated by calculation using the Mark-Houwink equation. FTIR Spectra of chitin and chitosan from the fish scale of Tilapia ( Tilapia nilotica) are shown in Figure 3, giving typical chitin and chitosan peaks. The fingerprint bands of the spectra are related to two distinct regions from 3500 to 2900 cm -1 and from 1700 to 1000 cm -1. These regions are normally used to obtain the deacetylation degree of chitin and chitosan biopolymers. Thus, the obtained % deacetylation was about 80-90% for the chitosan. In addition, the deacetylation data are very important to understand correctly the differences in adsorbing abilities of these materials. Although most of the amino groups within chitosan are acetylated, free amino groups are also present to some extent because of deacetylation during deproteinization process in the alkaline medium. Therefore, chitosan samples have different degrees of deacetylation depending on their sources of origin and mode of isolation [19].

5 Study on Dy-Binding Interactions of Chitosan Obtained from the Fish Scale of Tilapia These studies provide information regarding the functional groups and the bonds present the band at ~3450 cm -1 is due to the elongation of the N H and O H bonds, These functional groups of chitosan such as amino and hydroxyl groups are very important for adsorption. Therefore, it can be assigned to several functional groups present in the sample as RNH 2 primary amines, R 2 HN secondary amines, and alcohols. The band at ~2854 cm -1 is due the C H (CH 3, CH 2 ) bond elongation, this band is a characteristic of the materials with saturated carbons or sp 3 hybrid orbital. The band at ~1660 cm -1 is a characteristic of the C=O bond of an amide, since the chitosan was prepared from chitin by partial deacetylation. The band at ~1640 cm -1 is due to the flexion of the amine and amide N H bond, plus the band observed at ~1460 cm -1 is caused by the bond flexion of methylene groups. Also the band around 1500 cm -1 is due to the N H bond flexion. The band at ~1427 cm -1 is due to the C O H bond flexion; this band generally appears very close to the CH 2 bands. The presence of primary, secondary and tertiary alcohols can be made out by means of the bands observed in the region from 1260 to 1000 cm -1. Finally, at ~1030 cm -1 band is related to the presence of C O elongation of a primary alcohol [8, 19-20, 22]. Dye-binding Interactions of Chitosan The interactions of chitosan with dyestuffs may simultaneously be characterized by van der Waals forces, hydrophobic bonding, hydrogen bonding, or electrostatic interactions, depending on the nature Figure 3: FTIR Spectra of Chitin (top) and Chitosan (bottom) Obtained from the Fish Scale of substrate (ionic charge) and ph of the medium. The color removal from the water used in textile industry is a major environmental problem because of difficulty of treatment by conventional chemical coagulation/flocculation and biological methods. Sulphonate groups are the functional groups of acid dyes used in our study, their reactivity are based on the ready nucleophilic displacement by the amine groups in chitosan after protonated [17]. The effect of the chitosan concentration on the adsorption of Congo red, Sunset yellow FCF and methylene blue onto chitosan was investigated. Figure 4 shows the effect of dye-adsorption onto chitosan. In this results, when more than 100 mg L -1 of chitosan was added. Each of Congo red, Sunset yellow FCF and methylene blue was quantitatively adsorbed. The concentration of chitosan more than 1000 mg L -1 used could make the reaction solution precipitating. Thus, mg L -1 of chitosan was employed for the proposed procedure. The effect of ph on the adsorption of Congo red, Sunset yellow FCF and methylene blue onto chitosan was investigated as shown in Figure 5. The absorbance of Congo red, Sunset yellow FCF and methylene blue each was measured in the ph range of 2 to 12 of their buffer solutions. The absorbance of Congo red and Sunset yellow FCF upon its complexes was increase with an

6 144 Nanthawan Uawonggul, Supalak Kongsri & Saksit Chanthai Simultaneously, the reactive dye molecule is dissociated as follows: Figure 4: increasing in the ph, because free amine groups in chitosan are much more reactive and effective for chelating pollutants than the acetyl groups in chitin, especially the hydroxyl groups in C-3 position may contribute to adsorption. However, at the neutral ph, about 50% of total amine groups remain protonated and theoretically available for the adsorption of dyes. The existence of free amine groups may cause direct complexation of dyes coexisting with anionic species, depending on charge of dye [17]. It was noted that the absorbance of methylene blue-chitosan complex along this ph range was found no difference. Figure 5: Effect of Chitosan Concentration on Absorbance of the Dye-chitosan Solution Effect of ph on Absorbance of the Dye-chitosan Solution The amino groups of chitosan are protonated under acidic conditions according to the following reaction: R NH 2 + H + R NH 3 + (1) H 2 O D SO 3 Na D SO 3 +Na + (2) As a result, the sorption process proceeds through electrostatic interaction between the two counter ions (R NH 3 + and D SO 3 ): R NH D SO 3 R NH 3 + O 3 S D (3) Increasing ph of the solution, electrostatic interactions decrease due to the deprotonation of amino groups [8] The effect of dye concentrations on the absorbance of Congo red, Sunset yellow FCF and methylene blue onto chitosan was studied as shown in Figure 6. In this result, with a fixed adsorbent dose, the amount of adsorbed dye increased with an increasing concentration of the solution. This is due to the increase in the driving force of the concentration gradient with higher dye concentrations. At low concentration the adsorption of dyes by chitosan is very intense and reaches equilibrium very quickly. This indicates the possibility of the formation of monolayer coverage of the molecules at the outer interface of the chitosan. The ratio of number of dye moles to the available adsorption sites is low and subsequently the fractional adsorption becomes independent of the initial concentration. At higher concentrations the number of available adsorption sites becomes lower and subsequently the removal of dyes depends on the initial concentration. At high concentrations, it is not likely that dyes are only adsorbed in a monolayer at the outer interface of chitosan. As a matter of fact, the diffusion of exchanging molecules within chitosan particles may govern the adsorption rate at higher concentrations [1]. Figure 7 shows the effect of ionic strength on the absorbance of Congo red, Sunset yellow FCF and methylene blue onto chitosan. Sodium chloride affected the adsorption via two mechanisms, either by screening the Coulombic potential between the adsorbing molecule and charged adsorbents, or by adsorbing preferentially on the active sites of the adsorbent. In the presence of salt, cation and anion from the salt probably interact with binding sites of the chitosan via electrostatic interaction [1, 23]. The results show that the adsorption of Congo red and Sunset yellow FCF onto chitosan was not affected by the presence of sodium chloride,

7 Study on Dy-Binding Interactions of Chitosan Obtained from the Fish Scale of Tilapia indicating the role of the dye structure. So, it is concluded that the adsorption of Congo red and Sunset yellow FCF also involves hydrophobic interactions between the sulfonate groups in the dye and the hydrophobic functions in this adsorbent. Figure 7: Effect of Ionic Strength on Absorbance of the Dye-chitosan Solution Figure 6: (a) Methylene blue (b) Congo red and Sunset yellow FCF Effect of Concentration of Each Dye on Absorbance of the Chitosan Solution with Methylene Blue (a), and Congo Red and Sunset Yellow FCF (b) On the other hand, the retention of methylene blue solution increases with an increasing amount of NaCl. These results indicate that the salt seems to have a beneficial effect on complexation and the binding sites of chitosan. Moreover, observing that it can accelerate the adsorption. Also, the increase in NaCl concentration can reduce the dose of chitosan [1]. The effect of temperature for the adsorption of Congo red, Sunset yellow FCF and methylene blue onto the chitosan was studied (Figure 8). The adsorption capacity of chitosan for removing dyes from aqueous solution is unstable with increasing temperature. The adsorption capacity of chitosan had a little increase when the temperature was less than 35 C. However, the absorbance of dye adsorption had a large decrease if the temperature exceeded 45 C. This was probably due to the configuration of chitosan that was broken and the amine groups would probably deviate from the chitosan while the temperature was increased. This might also be the interaction force between dye and the adsorbent particles that had been destroyed at higher temperatures [22]. Thus the temperature was an important factor in removing the dyes for chitosan. Further research would be carried out to investigate this event. Chitosan could achieve the maximum dye adsorption at the temperature range from 5 to 35 C. At higher temperatures (45 C), the absorbance of dye adsorption also increased. Since the chitosan particles and dye droplets would probably be more active at higher temperatures compared with at lower temperatures, thus the flocs could be formed easily. But when the temperature exceeded 45 C, the construction of chitosan might be destroyed. So, the removed dyes by chitosan from aqueous solutions would decrease if the energy concerns exceeded this level. In addition, the effect of incubation time on the absorbance of Congo red, Sunset yellow FCF and methylene blue onto the chitosan was also investigated as shown in Figure 9. From this result, it is indicated that the adsorption capacity increases with time and the sorption process is initially very fast and then slowly reached

8 146 Nanthawan Uawonggul, Supalak Kongsri & Saksit Chanthai Figure 8: Effect of Temperature on Absorbance of the Dye-chitosan Solution and Sunset yellow FCF 15 mg L -1. The appropriate temperature at 25 C and incubation time about 60 min were required for these interactions. The use of chitosan for dye sequestration can be constituted as a way of waste colorisation and pollution remediation. But the utilization of fish scale waste for a singular use as chitosan source may not be completely cost-effective. This study has clearly demonstrated that this fish chitosan can be used as an effective adsorption medium for dyes, resulting in the molecular interactions of the dye binding complex. Therefore, these results can be implied for the removal efficiency of toxic waste dyes. Acknowledgments The Center for Innovation in Chemistry (PERCH-CIC), Commission of Higher Education, Ministry of Education and the Hitachi Scholarship Foundation, Tokyo, Japan is gratefully acknowledged for financial support. Figure 9: equilibrium. The adsorption behavior of anionic dyes was directly related to the dimensions of the dye organic chains, the amount and position of the sulfonate groups [1, 9]. CONCLUSION Effect of Incubation Time on Absorbance of the Dye-chitosan Solution The natural polymer chitosan produced from the fish scale of Tilapia (Tilapia nilotica) has shown a good efficiency to react with dye from aqueous solution. This raw adsorbent has high sorption capacity for dye by its amino groups. Interactions between the chitosan and dye were found to be strongly dependent on ph of the media. The optimal ph for dye removal from solution was at ph 10 for Congo red and Sunset yellow FCF, But that for methylene blue was no influence of its removal from the solution. Dye concentrations for their sorption capacities of the chitosan used were as follows: methylene blue 0.15 mg L -1, Congo red 15 mg L -1 References [1] Gregorio C, Application of Chitosan, a Natural Aminopolysaccharide, for Dye Removal from Aqueous Solutions by Adsorption Processes using Batch Studies: A Review of Recent Literature, Prog. in Polymer Sci., 33, 2008, [2] Yang T C and Zall R R, Absorption of Metals by Natural Polymers Generated from Seafood Processing Wastes, Ind. Eng. Chem. Prod. Res. Dev., 23, 1984, 168. [3] Yoshinari B, Hiroshi N, Rie N and Yohichi M, Preconcentration of Chitosan Derivatives Containing Methylthiocarbamoyl and Phenylthiocarbamoyl Groups and their Selective Adsorption of Copper(II) over iron(iii), 18, 2002, [4] Majeti N V R K, A Review of Chitin and Chitosan Applications. Reactive & Functional Polymers, 46, 2000, [5] Ming-Shen C, Pang-Yen H and Hsing-Ya L, Adsorption of Anionic Dyes in Acid Solutions using Chemically Crosslinked Chitosan Beads. Dyes and Pigments, 60, 2004, [6] Crini G, Martel B and Torri G, Adsorption of C.I. Basic Blue 9 on Chitosan-based Materials. Int. J. Environ. Pollut., 33, 2008, 1 4. [7] Filipkowska U, Efficiency of Reactive Dyes Adsorption onto chitin, chitosan and chitosan beads. Polish Chitin Soc.. Monograph XI, 2006, [8] George Z and Nikolaos K, Reactive and Basic Dyes Removal by Sorption onto Chitosan Derivatives. J. Colloid and Interface Sci., 331, 2009, [9] Giles C H, Hassan A S A and Subramanian R V R, Adsorption at Organic Surfaces: IV Adsorption of Sulphonated azo Dyes by Chitin from Aqueous Solution, J. Soc. Dyes Colour, 74, 1958, [10] Knorr D, Dye Binding Properties of Chitin and Chitosan, J. Food Sci., 48, 1983,

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