SULFATION OF KAPPA CARRAGEENAN WITH K 2 SO 4

Transcription

1 Journal of Engineering Science and Technology Special Issue on SMCHE 2014 & RSCE 2014 Conference, January (2015) School of Engineering, Taylor s University SULFATIN F KAPPA CARRAGEENAN WITH K 2 S 4 S. DISTANTINA 1, *, RCHMADI 2, M. FAHRURRZI 2, WIRATNI 2 1 Chemical Engineering Department, Sebelas Maret University Jalan Ir. Sutami 36 A Surakarta, 57126, Indonesia 2 Chemical Engineering Department, Gadjah Mada University Jalan Grafika No.2, Yogyakarta 55281, Indonesia *Corresponding Author: distantina@uns.ac.id Abstract Alkaline treatment in carrageenan recovery from seaweed is known as desulfation process and used to commercially enhance the gelation behavior by reducing sulfate content. In order to increase the number of hydrophilic groups, namely sulfate groups, the desulfated carrageenans from Kappaphycus alvarezii seaweed were sulfated using K 2 S 4 in sulfation process. Carragenan (2 gram) was dissolved in distilled water (30 ml) and then mixed with K 2 S 4 solution 0.1 N at 80 C for 30 min. After ethanol precipitation, the precipitated carrageenan was dried. This research studied the reaction mechanism of sulfation using the changes of infra red spectra (FTIR). It is found that sulfation was able increase sulfate groups in the desulfated carrageenan. The reaction between desulfated carrageenan and K 2 S 4 involved sulfation the hydroxyl group on carrageenan chain. From preliminary work, it is showed that the obtained of sulphated carrageenans can be used as a material for stable hydrogel. Keywords: Carrageenan, K 2 S 4, Sulfation, Infrared spectra. 1. Introduction Hydrogels are hydrophilic polymer network that can absorb and retain large quantities of water, saline or physiological solutions. Hydrogels have been widely used in agriculture, biomedical area, tissue engineering, biosensors, and sorbents for removal of heavy metals, and drug delivery. Recently, the hydrogels based on natural polymers, especially polysaccharides, have been investigated due to more biodegradable, less toxic, more biocompatible, renewable, and cheaper because the raw materials are locally abundant than synthetic polymer hydrogels. 96

2 Sulfation of KAPPA Carrageenan with K 2S 4 97 Kappa carrageeenan, sulfated polysaccharide, is an interesting alternative raw material for hydrogel synthesis. Kappa carrageenans are linear polysaccharides sulfatedgalactan extracted from red seaweed (Rhodopyta), such as Kappaphycus alvarezii (known as Eucheuma cottonii in industry) which are well cultivated in Indonesia. This natural polymers comprise of repeating units of (1,3)-Dgalactopyranose and (1,4)-3,6- anhidro-α-d-galactopyranose with sulfate groups in a certain amount and position [1]. The presence of hydroxyls and sulfate groups in carrageenan structure drive the carageenans tend to be hydrophilic. Kappa carrageenan has ability to form thermoreversible gel. Because of their gelling ability, carrageenans are widely used as agent for thickening and gelling in food and nonfood industries, and a potential as raw materials of hydrogels. Desulfation process using alkali in kappa carrageenan recovery from seaweed is used to produce gel forming structures, namely 3,6 anhydro galactose (3,6 AG) [1]. The reaction in desulfation process is written as Fig. 1. Desulfation involves sulfate release. In polymer, sulfate groups exhibit hydrophilic property and acid charged groups that can be ionized in certain ph environmental. As raw material of hydrogel, charged polymers are needed in order to prepare hydrogel having swelling properties that sensitive to the changes of ph and salt. These properties are very important in order to get hydrogel that can be applied in wide fields, such as in biomedical field and agriculture. Due to the sulfate groups are ionizable groups in a certain ph, therefore the sulfate addition or sulfation process into kappa or desulfated carrageenan chain is predicted may produce hydrogel having swelling property that can be applied in a certain ph. The swelling property that can respond to ph changes gives potential to be applied in biomedical application. The addition of sulfate groups or sulfation process in polymer chain can enhance the swelling ability [2,3]. Sulfation of carrageenan involved reaction between hydroxyl groups and sulfate group [4-6]. To our knowledge, there is no study of sulfation of kappa carrageenan with K 2 S 4. In this research, the sulfation of kappa or desulfated carrageenan extracted from Kappaphycus alvarezii was done using K 2 S 4 as the sulfate source. This research investigated the reaction mechanism of sulfation using the changes of infrared spectra (FTIR). The semi quantitative comparison of infrared absorbance of sulfate group and hydroxyl group may exhibit the changes of molecular structure before and after sulfation reaction. -3S H -3S H S3 - H- H H H H Mu-carrageenan H Kappa-carrageenan Fig. 1. Reaction in Desulfation.

3 98 S. Distantina et al. 2. Materials and Methods 2.1. Materials Seaweeds of Kappaphycus alvarezii were harvested from Makasar, South Sulawesi, Indonesia. The seaweeds were soaked in water for 2 hours, and then washed using tap water several times to eliminate all impurities such as salt and sand. After washing, the seaweeds were cut into about 1 cm length, and finally sun dried to constant weight. The clean seaweed sample was kept in a dry state until further processing was done. Technical grade of potassium hydroxide (purity 88%) was used as alkali treatment in desulfation process. Potassium sulfate (Merck) was used as reactant in sulfation process Desulfation The 30 gram of clean seaweed was soaked in distilled water for 15 minutes. After soaking, the water was separated from the seaweed by filtration. Firstly, 1500 ml of 0.5 M KH solution as the solvent was heated in a beaker as an extractor which emerged in a water bath equipped by a stirrer. If the temperature of solvent reached 80 C, the seaweeds then were added into solvent, and the time of extraction started to be counted. The speed of stirrer was set constant at 275 rpm. The constant ratio of seaweed weight to solvent volume (1/50; g/ml) was maintained by adding hot water. After 30 minutes desulfation, the filtrate was separated from residue and immediately poured into 3 volumes of cold (5 0 C) technical ethanol (90% w) resulting precipitated carrageenan. The precipitation was done for 30 minutes with stirring gently. The precipitated carrageenans were collected and then oven dried at C to a constant weight and named as desulfated carrageenans Sulfation Desulfated carrageenans were used as raw material in sulfation experiment. The 0.5 gram carrageenans were completely dissolved in 30 ml distilled water by heating. The resulting carrageenan solution were mixed with 100mL hot potassium sulfate aqueous in erlenmeyer glass for preparing sulfation reaction and the reaction time started to be counted. The temperature was kept constant at 86 C. After 30 minutes of sulfation reaction, the reaction was stopped by pouring the solution into 3 volumes of cold ethanol, so that the precipitated carrageenan could be collected. btained wet carrageenans were dried at C to a constant weight and called as sulphated carrageenas. In this work, potassium sulfate concentration varied, namely 0.01, 0.02, 0.05, and 0.1 M FTIR Molecular groups were identified using FTIR spectrometer (Shimadzu IR Prestige-21). Both desulfated and sulphated carrageenans were powdered. Infrared spectra were obtained by using KBr pellet method with 10 scans and 16 cm -1 resolution. Assignments of IR spectra of obtained carrageenans were based on spectroscopy data summarized by [7, 8]. Peak baselines and height were

4 Sulfation of KAPPA Carrageenan with K 2S 4 99 determined in transmittance (T) mode and peak height were converted to absorbance (A) [9]. Absorbance was calculated as Eq. (1). A= - log(t) (1) 2.5. Sulfate content determination The desulfated and sulphated carrageenans were analysed of their sulfate content. Percent sulfate content was determined using the method of sulfate hydrolysis followed by precipitation sulfate as barium sulfate [10]. 3. Results and Discussion Here, the desulfated carrageenan was used as raw material of sulfation process. The data of FTIR spectra changes were used to investigate the chemically changes during sulfation. Figure 2 shows the FTIR spectra of desulfated and sulfated carrageenan. The characteristic infrared peaks of samples are presented in Table 1. The study of desulfated carrageenan spectra by FTIR spectroscopy shows the presence of very strong absorption band in cm -1 region (due to the S= of sulfate esters), cm -1 (C- of 3,6-anhydro-D-galactose), and cm -1 (C--S 3 of D-galactose-4-sulfate). In this work, the infrared spectra of desulfated carrageenan from Kappaphycus alvarezii showed the main features of kappa carrageenan. The relative absorbance of some groups were evaluated through the absorbance of ratio groups peak to C-H peak, which remained almost constant during desulfation, as presented in Table 2. This absorbance ratio serves as semi quantitative index of chemical composition of carrageenan. From Table 2, it is showed that sulfate content in sulfated carrageenan was higher than that in desulfated carrageenan and hydroxyl content in sulfated carrageenan was lower than that in desulfated carrageenan. This indicates that desulfated carrageenan could bind sulfate. Figure 2 shows the presence of new peaks in sulfated carrageenan, namely 739 cm -1 and 619 cm -1. These new peaks are probably corresponding to sulfate groups. Table 2 also shows that gel forming structure resulted in desulfation process, namely 3,6 anhydro galactose, was still exist in sulfated carrageenan. The amount of 3,6 AG in sulfated carrageenan also was not different compared with that in desulfated carrageenan. This indicates that sulfation did not occur in the same sulfate position that released in desulfation process. The released sulfate in desulfation process formed stable 3,6 AG structure. Sulfation involved reaction between hydroxyl group and sulfate group [4, 6]. From Table 2, it is showed that sulfation resulted sulfated carrageenan having higher sulfate and lower hydroxyl amount. The relationship of reaction between hydroxyl group and sulfate group can be evaluated using mole ratio of hydroxyl and reacted sulfate. Table 3 shows the sulfate mole in desulfated carrageenan and in sulfated carrageenan. Sulfation with K 2 S and 0.02 M did not significantly increase the sulfate content. The significant amount of sulfate could be added with higher concentration K 2 S 4, namely 0.05 and 0.1 M. The low concentration of K 2 S 4

5 100 S. Distantina et al. caused reactivity of hydroxyl in carrageenan chain was low. Therefore, the ionic strength of K 2 S 4 affected the reaction of sulfation process. Fig. 2. FTIR Spectra of Desulfated and Sulfated Carrageenan. The difference between sulfate content in sulfated carrageenan and in desulfatedcrarageenan expressed the amount of reacted sulfate that substitute the hydroxyl group in carrageenan chain. In this research, the sulfation of mole monomer (carrageenan) with K 2 S M could add about mole sulfate at sulfated carrageenan chain. This indicates that one mole monomer was equivalent with one molesulfate. Therefore, it is predicted that sulfation occurred at one hydroxyl group.

6 Sulfation of KAPPA Carrageenan with K 2S Table 1. Characteristic Infrared Peaks Present in the Sample. Peak (cm -1 ) a Group Reference Desulfated carrageenan Peak (cm -1 ) b Sulfated carrageenan Ester sulfate [7] AG C- in C3 AG [7] AG C- in C6 AG [7] Galactose-4 -sulfate [7] C--S 3 in C6 AG [7] Sulfate [8] a : reference [7,8], b : this research. Table 2.Comparison of Groups Absorbance Ratio of Desulfated and Sulfated Carraggenan. Carrageenan 3,6AG 3,6AG sulfate H A2/A5 A3/A5 A4/A5 A6/A5 Desulfated Sulfated Note: A2= absorbance of C- in C6 3,6 AG A3= absorbance of C- in C3 3,6 AG A4= absorbance of ester sulfate A5= absorbance of C-H A6= absorbance of -H Table 3. Sulfate Content in Sulfated Carrageenan. mole S 4 K 2 S 4 (M) Desulfated carrageenan Sulfated carrageenan Reaction The sulfation activity on hydroxyl group is affected by hydroxyl position in carrageenan chain. Primary hydroxyl group tends to have higher activity compared with secondary hydroxyl group.sulfation carrageenan with K 2 S 4 was predicted involve reaction between sulfate and hydroxyls groups at C6 chain of (1,3)-β-D-galactose.The sulfation of desulfated carrageenan with K 2 S 4 can be predicted as depicted in Fig. 3. Sulfation at C6 chain of (1,3)-β-D-galactose can be read from absorption band in 739 cm -1 (Fig. 3). From the preliminary experimental, sulfated carrageenan exhibited as more stable hydrogel properties in water compare than that

7 102 S. Distantina et al. desulfated carrageenan. In the next paper, we will report the swelling properties of the obtained sulfated carrageenan. - 3 S H - 3 S S 3- K 2 S 4 H H H H Fig. 3. Sulfation of Desulfated Carrageenan. 4. Conclusions Sulfation was able to enhance the sulphate amount in kappa carrageenan chain without decreasing number of gel forming structure. Sulfation kappa carrageenan with K 2 S 4 was predicted involve reaction between sulfate and hydroxyls groups at C6 chain of (1,3)-β-D-galactose. Acknowledgment This work was supported by grant from Directorate General of Higher Education, Ministry of National Education, Indonesia, through research grant of Penelitian Unggulan Perguruan Tinggi , Sebelas Maret University and the scholarship of doctorate program (BPPS) at Gadjah Mada University. References 1. Campo, V.L.; Kawano, F.F.; Silva Junior, D.B.; and Carvalho, I. (2009). Carrageenans: biological properties, chemical modifications and structural analysis. Carbohydrate Polymers, 77(2), Lee, C.T.; Kung, P.H.; and Lee, Y.D. (2005). Preparation of poly(vinyl alcohol)-chondroitin sulfate hydrogel as matrices in tissue engineering. Carbohydrate Polymers, 61(3), Rhim, J.W.; Park, H.B.; Lee, C.S.; Jun, J.H.; Kim, D.S.; and Lee, Y.M. (2004). Crosslinked poly(vinyl alcohol) membranes containing sulfonic acid group: proton and methanol transport through membranes. Journal of Membrane Science, 238(1-2), Yuan, H.; Zhang, W.; Li, X.; Lu, X.; Li, N.; Gao, X.; and Song, J. (2005). Preparation and in vitro antioxidant activity of k-carrageenan oligosaccharides and their oversulfated, acetylated, and phosphorylated derivatives. Carbohydrate Research, 340(4), poku, G.; Qiu, X.; and Doctor, V. (2006). Effect of oversulfation on the chemical and biological properties of kappa carrageenan. Carbohydrate Polymers. 65(2),

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