Journal of Engineering Science and Technology
Special Issue on SOMCHE 2014 & RSCE 2014 Conference, January (2015) 96 - 103
© School of Engineering, Taylor’s University
SULFATION OF KAPPA CARRAGEENAN WITH K2SO4
1,
2
2
S. DISTANTINA *, ROCHMADI , M. FAHRURROZI , 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: [email protected]
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 K2SO4 in sulfation process. Carragenan (2 gram)
was dissolved in distilled water (30 mL) and then mixed with K2SO4 solution
0.1 N at 80OC 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 K2SO4 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, K2SO4, 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.
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Sulfation of KAPPA Carrageenan with K2SO4
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
K2SO4. In this research, the sulfation of kappa or desulfated carrageenan extracted
from Kappaphycus alvarezii was done using K2SO4 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.
-O3SO
-O3SO
OH
O
O
OH
O
OSO3-
OH-
O
O
O
HO
OH
O
O
O
OH
OH
OH
Mu-carrageenan
Kappa-carrageenan
Fig. 1. Reaction in Desulfation.
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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.
2.2. 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 KOH 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 80OC, 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 0C)
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 50-60OC to a constant weight and named as
desulfated carrageenans.
2.3. 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
86OC. 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. Obtained wet carrageenans were dried at 50-60OC 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.
2.4. 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
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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 1210-1260 cm-1 region (due to the
S=O of sulfate esters), 925-935 cm-1 (C-O of 3,6-anhydro-D-galactose), and
840-850 cm-1 (C-O-SO3 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 K2SO4 0.01 and 0.02 M did not significantly increase
the sulfate content. The significant amount of sulfate could be added with higher
concentration K2SO4, namely 0.05 and 0.1 M. The low concentration of K 2SO4
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S. Distantina et al.
caused reactivity of hydroxyl in carrageenan chain was low. Therefore, the ionic
strength of K2SO4 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 0.0013
mole monomer (carrageenan) with K2SO4 0.1 M could add about 0.002 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.
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Table 1. Characteristic Infrared Peaks Present in the Sample.
Peak (cm-1)b
Peak (cm-1)a
Reference
1220-1260
Ester sulfate
[7]
Desulfated
carrageenan
1242.16
1070
3.6 AG C-O in C3 AG
[7]
1064.71
928-933
3.6 AG C-O in C6 AG
[7]
933.35
840-850
Galactose-4 -sulfate
[7]
840.96
815-820
C-O-SO3 in C6 AG
Sulfate
[7]
[8]
618
a
Group
Sulfated
carrageenan
1118.76
926.84
845.82
739
671.23
619.16
b
: reference [7,8] , : this research.
Table 2.Comparison of Groups Absorbance
Ratio of Desulfated and Sulfated Carraggenan.
Carrageenan
3,6AG
3,6AG
sulfate
OH
A2/A5
A3/A5
A4/A5
A6/A5
Desulfated
0.912
1.024
0.938
1.172
Sulfated
1.018
1.807
0.846
Note: A2= absorbance of C-O in C6 3,6 AG
A3= absorbance of C-O in C3 3,6 AG
A4= absorbance of ester sulfate
A5= absorbance of C-H
A6= absorbance of O-H
Table 3. Sulfate Content in Sulfated Carrageenan.
K2SO4 (M)
0.01
0.02
0.05
0.10
Desulfated
carrageenan
0.00060
0.00060
0.00060
0.00060
mole SO4
Sulfated
carrageenan
0.00062
0.00066
0.00252
0.00262
Reaction
0.000002
0.000005
0.001921
0.002022
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 K2SO4 was
predicted involve reaction between sulfate and hydroxyls groups at C6 chain of
(1,3)-β-D-galactose.The sulfation of desulfated carrageenan with K2SO4 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
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S. Distantina et al.
desulfated carrageenan. In the next paper, we will report the swelling properties
of the obtained sulfated carrageenan.
- O SO
3
OH
- O SO
3
O
K 2 SO 4
O
O
O
O
OSO 3
O
O
O
OH
O
O
OH
OH
OH
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 K2SO4 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 2013-2014, Sebelas Maret University and the
scholarship of doctorate program (BPPS) at Gadjah Mada University.
References
1.
2.
3.
4.
5.
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), 167-180.
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), 348-354.
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), 143-151.
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), 685-692.
Opoku, G.; Qiu, X.; and Doctor, V. (2006). Effect of oversulfation on the
chemical and biological properties of kappa carrageenan. Carbohydrate
Polymers. 65(2), 134-138.
Journal of Engineering Science and Technology
Special Issue 1 1/2015
Sulfation of KAPPA Carrageenan with K2SO4
103
6.
Araujo, C.A.; Noseda, M.D.; Cipriani, T.R.; and Goncalves, A.G. (2013).
Selective sulfation of carrageenan and the influence of sulfateregiochemistry
on anticoagulant properties. Carbohydrate Polymers. 91(2), 483-491.
7. Pereira, L.; Amado, A.M.; Critley, A.T.; Van de Velde, F.; and RibeiroClaro, P.J.A. (2009). Identification of selected polysaccharides
(phycocolloids) by vibrational spectroscopy (FTIR-ATR and FT-Raman).
Food Hydrocolloids, 23(7), 1-7.
8. Amamou, W.; Guerfel, T.; and Mhiri, T. (2012). Crystal structure and
physicochemical properties of 2-diisopropylammonium ethylammoniumsulfate
dehydrate. Crystal Structure Theory and Applications, 1, 40-45.
9. Smith, B.C. (2011). Fundamental of Fourier Transform Infrared
Spectroscopy. CRC press, Boca Raton.
10. Jeffery, G.H.; Bassett, J.; Mendham, J.; and Denney, R.C. (1989). Vogel’s
Textbook of Quantitative Chemical Analysis. Bath press, Avon, Great Britain.
Journal of Engineering Science and Technology
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