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Small-angle neutron scattering study on irradiated kappa carrageenan
ARTICLE IN PRESS
Physica B 381 (2006) 103–108 www.elsevier.com/locate/physb
Small-angle neutron scattering study on irradiated kappa carrageenan Lucille Abada,b,c,, Satoshi Okabea, Satoshi Koizumic, Mitsuhiro Shibayamaa, a
Neutron Science Laboratory, Institute for Solid State Physics, University of Tokyo, 106-1 Shirakata, Tokai, Ibaraki 319-1106, Japan b Philippine Nuclear Research Institute, Commonwealth Ave., Diliman, Quezon City, Philippines c Advanced Science Research Center, Japan Atomic Energy Research Institute, 2-4 Shirane Shirakata, Tokai, Ibaraki 319-1195, Japan Received 22 November 2005; received in revised form 19 December 2005; accepted 19 December 2005
Abstract The structure of gamma-ray-irradiated k-carrageenan in aqueous solutions was investigated in terms of small-angle neutron scattering. The scattered intensity, I(q), of non-irradiated k-carrageenan solutions (5 wt%) was well described with an Ornstein–Zernike (OZ)-type function with the correlation length of 85 A˚, indicating that the k-carrageenan solution behaves just as a polymer solution in the semidilute regime. By increasing the irradiation dose (100 kGy), I(q) changed to a power-law function with the scattering exponent of 1.84. Further increase in dose results in a recovery of OZ-type function. This indicates that a progressive cleavage of k-carrageenan chains takes place randomly, leading to a self-similar structure at 100 kGy. This is followed by further segmentation of k-carrageenan chains. r 2006 Elsevier B.V. All rights reserved. PACS: 61.12.Ex; 87.15.v; 87.50.a Keywords: Small-angle neutron scattering; k-carrageenan; Gamma radiation
1. Introduction Radiation modification of natural polymers such as carrageenan, alginate, chitin, agar and cellulose have recently gained focus in various researches due to their widespread application in agriculture and in medicine. Radiation effects on these natural polymers result in drastic decrease in molecular weight. These low molecular weight polymers found valuable use as coatings in fruits and as plant growth promoter [1]. Irradiated carrageenans are among one of these promising polymers where they have been shown to exhibit optimum growth in rice plants at an irradiation dose of 100 kGy. Carrageenans are linear polymers of about 25,000 galactose derivatives with regular but imprecise structures, dependent on the source and extraction conditions. Different types are classified according to the number and position of sulfate groups. kCorresponding author. Philippine Nuclear Research Institute, Commonwealth Ave., Diliman, Quezon City, Philippines. Also to be corresponded to. E-mail addresses: [email protected] (L. Abad), [email protected] (M. Shibayama).
0921-4526/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.physb.2005.12.267
carrageenan is composed of alternating a(1,3)-D-galactose4-sulfated and b(1-4)-3,6-anhydro-D-galactose. Properties of k-carrageenan have been studied by several researchers [2–8]. It is known to form a strong thermoreversible gel in the presence of potassium ions where the conformation changes from a random coil to helix, leading to aggregation followed by gelation upon cooling. The structure of aggregating k-carrageenan from decreasing molar fractions from sonification have been investigated by dynamic light scattering (DLS) [9]. Previous work on gamma-irradiated k-carrageenan has also been done using the DLS. Results of this study correlated the relationship of a new fast mode peak observed at 100 kGy k-carrageenan from the decay time distribution function with its optimum biological activity [10]. Very few studies have been done on the small angle neutron scattering (SANS) of carrageenan [11–13]. The structure change of k-carrageenan gel with K+ in the gel to sol transition has been investigated by SANS. The SANS intensity decreased as it approached the gel-to-sol transition. Its scattering profile fitted with Guinier approximation formula [11]. This current study would investigate the structural changes of k-carrageenan with irradiation dose
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by SANS. The effect of K+ at varying temperatures on irradiated k-carrageenan will also be investigated. 2. Materials and methods 2.1. Materials Refined k-carrageenan (KK-100) was obtained from Shemberg Corporation, Philippines. Elemental analysis by XRF of this carrageenan reveals the presence of 12% S and 11% K as the major components. This was irradiated in air at ambient temperature using the Co-60 facility of the Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Gunma, Japan. Samples were irradiated at a dose of 10 kGy/h with an absorbed dose of 50, 100, 150 and 200 kGy. Non-irradiated and irradiated k-carrageenan were then purified by dissolving in distilled water and dialyzing it against distilled water for 24 h (Mol. Wt. cut-off ¼ 12,000–14,000). The dialyzed solutions were precipitated with isopropyl alcohol and freeze-dried. The purified non-irradiated k-carrageenan still contains 12% S and a reduced K content of 3%. 2.2. SANS of k-carrageenan Non-irradiated and irradiated k-carrageenan samples were dissolved in D2O and in 0.05 M KCl/D2O solutions at elevated temperatures at a fixed concentration of 5% by weight. The samples were then placed in drum cells with a sample thickness of 0.21 mm. SANS experiments were carried out with the SANS instrument, SANS-U, at the Institute for Solid State Physics, The University of Tokyo, located at the Japan Atomic Energy Research Institute, Tokai, Japan. A flux of cold neutrons with a wavelength of 7.0 A˚ was irradiated to the sample and the scattered intensity profile was collected with an area detector of 128 128 pixels. The sample to detector distances of 2.00 and 8.00 m were used which covered the accessible q range of 0.006–0.15 A˚1, where q is the scattering vector. The sample drum cells with quartz windows were placed in a copper chamber and the chamber was thermo-regulated with a circulating water bath. SANS experiments were initially done at 20 1C and the temperature was raised to 60 1C. The obtained intensities were corrected for the detector inhomogeneities, cell scattering, transmission, and incoherent scattering, and then were scaled to the absolute intensity with an intensity calibration standard sample (Lupolen).
to the k-carrageenan helix which reduces considerably its charge density, which then consequently promotes the formation of helix and induce aggregation/gelation [14]. The non-irradiated k-carrageenan in its purified state does not have a cold-set gelation (a hot solution is turned into gel upon cooling) even at a concentration of 1%. This is expected as the K content is reduced considerably after purification (11–3%). At higher concentration of 5%, gels were pasty-like, but were hard and brittle in the presence of 0.05 M KCl. Irradiated k-carrageenan gels on the other hand were soft and pasty-like (with or without 0.05 M KCl) as the radiation dose was increased. Works done on ultrasonically degraded k-carrageenan (5–100 kDa) also indicate no gelation upto 5% concentration [15]. Fig. 1 shows the k-carrageenan gels at varying irradiation doses. The picture indicates a clear homogeneous non-irradiated k-carrageenan gel. Turbidity increased with increasing irradiation, reaching its maximum between 50 and 100 kGy. Beyond this dose range, very clear gels were obtained. Radiation randomly cleaves off the glycosidic linkages of k-carrageenan resulting in a very heterogeneous system. Further cutting of the glycosidic linkages would result in a more homogeneous molecular size distribution as it reaches the minimum molecular size possibly attainable. DLS measurements indicate a flattening of decrease in molecular weight beyond a radiation dose of 100 kGy [16]. The sol–gel transitions of 5% k-carrageenan are shown in Fig. 2. Addition of K+ ions increased the gelation temperatures. Due to its high concentration, gelation temperature (47 1C–50 1C) did not vary so much up to a radiation dose of 150 kGy. A drastic decrease in gelation temperature of 29 1C was observed at 200 kGy. A similar trend was observed in k-carrageenan without KCl for the same concentration. Decrease in gelation temperature (33 1C) started at 150 kGy. At 200 kGy, no gelation was observed. Studies done previously using a semi-dilute concentration of 0.5% in 0.05 M KCl shows a decrease in gelation temperatures for irradiated k-carrageenan up to a
3. Results and discussion 3.1. Physical properties of irradiated k-carrageenan gels The gelation of k-carrageenan involves multiple aggregation of double helices. It produces a stronger gel especially in the presence of potassium ions. K+ ions bind
Fig. 1. k-carrageenan gels in 0.05 M KCl at 20 1C.
ARTICLE IN PRESS L. Abad et al. / Physica B 381 (2006) 103–108
55
5
No KCl
4
45
200kGy 150kGy 100kGy 50kGy 0kGy
ξ = 61 Å
10
3
10
40
2
ξ =195 Å
1
ξ = 85 Å
-1
10 I(q) /cm
Gelation Temperature (°C)
50
ξ =109 Å
10
With 0.05M KCl
105
35
10
30
0
10 25
0
50
100 150 Irradiation Dose (kGy)
200
10
-1
1
-2
0.1
10
Fig. 2. Gelation temperatures of 5% k-carrageenan.
10
dose of 50 kGy. Gelation is only observed at 100 kGy when the KCl is increased to 0.1 M. Beyond this dose no gelation takes place [16].
10
6 789 0.01
-3
2
6 7 8 9 0.01
3
4 5 6 7 89 0.1
2
3
4
-1
5
6 7 8 9 0.1
q /Å
3.2. SANS profile of k-carrageenan gels
Fig. 3. SANS intensity curves of k-carrageenan at different radiation doses.
Fig. 3 shows double logarithmic plots of the scattered intensity functions, I(q), observed for 5% k-carrageenan in 0.05 M KCl/D2O solutions irradiated at various doses. The figure shows that I(q) increased with increasing irradiation dose and reached a maximum at a dose of 100 kGy. Beyond this dose, a decrease in I(q) was observed. This increase and decrease in I(q) agrees with the visual observations of the gel where maximum turbidity was observed at 100 kGy (Fig. 1). This may be closely associated to the increasing heterogeneity of the polymer system with dose brought about by random cleavage of the polymer chains. Minimum intensity was found in nonirradiated k-carrageenan gels. Except for the cases of 100 and 150 kGy, the scattering functions approximate a close fitting to an Ornstein–Zernike (OZ)-type function as shown in the solid curves: IðqÞ ¼
Ið0Þ , 1 þ x2 q2
(1)
where I(q) is the scattering intensity and x is the correlation lengths indicating the extent of correlated concentration fluctuations. The solid lines in Fig. 3 are the fit of the experimental data with this equation. The obtained correlation lengths are shown in Table 1. No correlation length was evaluated from the carrageenan at 100 kGy since it had a power law behavior. Initially, x for 0 kGy is 85 A˚, which increased to 195 A˚ at 50 kGy. Further irradiation led to an annihilation of characteristic length scale and an appearance of fractal structure at 100 and 150 kGy as will be discussed later. Above these doses, x decreased to 109 A˚ (at 200 kGy). The value of x ¼ 195 A˚ at
Table 1 Correlation lengths of k-carrageenan at varying radiation doses Radiation dose (kGy)
x at 20 1C with 0.1 M KCl (A˚)
x at 60 1C with 0.1 M KCl (A˚)
x at 20 1C without KCl (A˚)
0 50 100 150 200
85 195 n.m. 61 109
64 190 n.m. 128 175
95 n.m. 71
n.m. – non-measurable.
50 kGy is somewhat larger than observed in other systems. However, it is understandable by the fact that it represents not only the mesh size but also the characteristic size of heterogeneity as well. While the fitting curves are essentially Lorentzian, i.e., an OZ function, certain deviations to this function were observed especially with irradiated k-carrageenan. The double logarithmic plot indicates also a power law behavior exclusively at a radiation dose of 100 kGy. Fig. 4 shows a very close power law fitting of k-carrageenan at 100 kGy. The scattering exponent was 1.84. Non-irradiated k-carrageenan and k-carrageenan irradiated at other doses do not exhibit any power law behavior. This phenomenological behavior can well be explained by looking at their collective fluctuations as illustrated in Fig. 5. The concentration at 0 kGy is high enough to form a polymer solution that fills the entire space with polymer
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chains overlapping each other (CbC*), where C* is the chain overlap concentration. Since k-carrageenan is a natural polymer, concentration fluctuations are strongly suppressed such that the free energy density remains at a minimum. This is attained by cooperative arrangement of cross-linking and/or helix formation between chains. Gamma-irradiation randomly cleaves the k-carrageenan chains, which leads to the formation of segments that are not able to pair with other segments to form cross-links and/or helices. As a result, concentration fluctuations increase. This is the case of k-carrageenan irradiated at 100 kGy. Interestingly enough, the scattering intensity function exhibits a power law behavior exclusively at this radiation dose. Unscreening of entangled and cross-linked polymer chains may take place exposing heterogeneous chains of polymers of varying cluster sizes. This may be why a power law is observed. Decreasing of the molecular weight takes place further as in the case of k-carrageenan irradiated at 200 kGy. The system then moves towards uniformity as a result of cleavage to low-molecular sizes achieved by high-radiation doses. The scattering function
2
100 kGy
-1
log [I(q) / cm ]
1
0 Slope = -1.84
3.3. The effect of temperature and K+ ions on the SANS profile Fig. 6 shows a comparison of the SANS profiles of k-carrageenan (in 0.05 M KCl) at 60 and 20 1C. As expected, I(q)s at 60 1C are lower than those at 20 1C. The largest deviation was seen at 200 kGy as shown in Fig. 6c. This shows that heterogeneities decrease as a result of a lowering of the number of cross-link points at higher temperatures. At 200 kGy, k-carrageenan is completely in its sol state at 60 1C. Thus, a large decrease in I(q) is observed. A SANS experiment on k-carrageenan done previously also showed a decrease in intensity with increasing temperature [11]. Even at 60 1C, a maximum intensity was still found at 100 kGy. Unlike the SANS profile of k-carrageenan at 20 1C, its profile at 60 1C does not perfectly fit an OZ-type function as shown in the dotted lines of Figs. 6a–c. Introduction of the squared-Lorentzian function (broken lines) was necessary to give a successful fitting curve that closely reproduced the observed I(q)s. The sum of a Lorentz (L) and squared-Lorentz (SL) functions (L–SL) can be expressed in this equation. IðqÞ ¼
-1
-2
-2.0
no longer exhibits a power law behavior at this dose. The loss of this self-similarity can be explained by the fact that high-irradiation doses of 200 kGy may destroy some of the galacto-pyranose rings of k-carrageenan leading to ring opening. This possible ring opening at high doses is also explained using dynamic light scattering results [10]. The value of x of this semi-dilute polymer solution is 109 A˚.
-1.6
-1.2
-0.8
-1
log [q / Å ] Fig. 4. Power law behavior of k-carrageenan (5% in 0.05 M KCl at 100 kGy).
ξ
(2)
where X is another correlation length describing the extent of spatial inhomogeneities [17]. Though the contribution of the SL component is quite minimal at all radiation doses as seen in the figures, its contribution is not negligible. Only the correlation lengths taken from the L function are shown in Table 1 for the main reason that its components are the major contributor to the fitting function. Table 1 shows that the correlation lengths, x, of these polymer solutions increased with irradiation dose. Decrease in x was observed beyond 100 kGy. A power law behavior was also
ξ
0 kGy
I L ð0Þ I SL ð0Þ þ , 2 2 1þx q ð1 þ X2 q2 Þ2
100 kGy
ξ
200 kGy
Fig. 5. Distribution of k-carrageenan polymer chains with radiation.
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0
20°C
-1
60°C
10 10
10 0 kGy
Without KCl With KCl I(q)/cm-1
ξ = 64 Å
-1
I(q)/ cm
107
-2
10
1
ξ = 95Å 0.1
-3
10
0 kGy
Lorentz Squared-Lorentz
-4
5
10
6 7
2
3
0.01 q /Å
4
5
(a)
6 7 0.1
-1
6 7 8 9 0.01
2
3
4
q / Å-1
6 7 8 9 0.1
10
(a)
Without KCl 2
With KCl
10
I(q)/cm-1
100 kGy 1
10
20°C
-1
I(q)/ cm
5
1 Slope = -1.82
0.1
0
10
60°C
-1
10
100 kGy 5
Slope = -1.87
6 7 8 9 0.01
(b)
2
3
4
5
q / Å-1
6 7 8 9 0.1
-2
10
6 7
2
3
0.01
4
5
0.1
-1
(b)
10
6 7
Without KCl
q /Å
I(q)/cm-1
With KCl
20°C
0
200 kGy
10
-1
gel ξ = 71Å
0.1
10
60°C ξ = 175 Å
-1
I(q)/ cm
1
-2
10
sol
200 kGy 5
-3
10
(c)
6 7 8 9 0.01
2 q / Å-1
3
4
5
6 7 8 9 0.1
Lorentz
-4
10
Fig. 7. The effect of K+ ions on the SANS intensity of 6-carrageenan (5%) at different radiation doses.
Squared-Lorentz
-5
10
6
7
2 0.01
(c)
3 -1
4
5
6 7 0.1
q /Å
Fig. 6. The effect of temperature on the SANS intensity of 6-carrageenan (5%) at different radiation doses.
observed exclusively at 100 kGy having a fractal dimension of 1.87. This trend is comparable to the one of kcarrageenan at 20 1C. Fig. 7 demonstrates the role of K+ ions in the ordering of the gels in k-carrageenan. We expected that I(q) for k-carrageenans irradiated at 0 and 200 kGy can be represented with a L function as shown by the dashed lines as was the case with KCl. The effects of the absence of
KCl are twofold. (1) In the absence of KCl, I(q) is smaller than those with KCl. (2) An upward trend with respect to the Lorentzian behavior was observed at low q region for k-carrageenans irradiated at 0 and 200 kGy. This suggests the presence of strong inhomogeneities of the k-carrageenan gels by aggregation. By nature, k-carrageenans are self-aggregating and form gels even in the absence of any ions. The presence of K+ ions facilitates the formation of well-ordered double helix domains. In effect, the absence of these K+ ions makes the gel more heterogeneous. At 200 kGy, k-carrageenan without the K+ ions is completely in the sol state. Thus, the fitting resembles back to an O–Ztype function. Its intensity though is much lower than k-carrageenan with KCl at 200 kGy. Polymer solutions have lesser number of cross-links than their gel
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counterparts. Correlation length at 0 kGy was determined to be 95 A˚, which decreased with irradiation dose of 200 kGy (71 A˚). I(q) at 100 kGy for k-carrageenan without KCl is very similar to the one of k-carrageenan with KCl except for a slight download deviation at the mid-q range. The characteristic power law behavior observed at this dose is also visible in k-carrageenan without KCl. Its fractal dimension is 1.82.
Dr. Fumio Yoshii for allowing the use of their gamma irradiation facility. This research was also made possible through the financial support of the Japan Atomic Energy Research Institute (JAERI) and the Forum for Nuclear Cooperation in Asia (FNCA). XRF analysis of k-carrageenan was done by the Physics Research Group of the Philippine Nuclear Research Institute.
4. Conclusion In summary, this work clearly demonstrates the following: k-carrageenan is subjected to degradation by gamma irradiation. The structure factor of non-irradiated kcarrageenan aqueous solutions is similar to those for polymer solutions in semi-dilute regime, i.e., an Ornstein– Zernike form. By gamma-ray irradiation, however, chain scission takes place randomly and destroys active sites capable of physical cross-linking. As a result, the molecular weight distribution becomes widely spread and chain clusters become self-similar and network formation with a minimal increase in concentration fluctuations becomes difficult. Hence a power-law behavior appears in I(q) which interestingly is observed at 100 kGy irradiated k-carrageenan. Further irradiation results in a progressive reduction in the molecular weight and a recovery of OZ behavior due to a loss of the self-similarity. Such kind of behavior is not observed in synthetic gels, such as polyacrylamide hydrogels. In synthetic polymer gels, I(q) cannot be described by an OZ function due to presence of inhomogeneities and a squared-Lorentz-type intensity function has to be added to describe them [18]. k-carrageenan seems to self-organize so as to keep the concentration fluctuations as low as possible by rearranging their cross-links. Degradation by irradiation unscreens the chain overlapping and makes explicit the architecture of the cluster structure. Acknowledgment The authors wish to thank the Takasaki Radiation Chemistry Research Establishment, JAERI, especially
References [1] T. Kume, N. Nagasawa, F. Yoshii, Radiat. Phys. Chem. 63 (2002) 625. [2] T. Hjerde, O. Smidsrød, B. Christensen, Carbohydr. Res. 288 (1996) 175. [3] M. Ciszkowska, J.G. Osteryoung, J. Am. Chem. Soc. 121 (1999) 1617 (Communication). [4] S. Cara, C. Tamerler, H. Bermek, O. Pekcan, Int. J. Biol. Macromol. 31 (2003) 177. [5] M. Ciszkowska, I. Kotlyar, Anal. Chem. 71 (1999) 5013. [6] I.S. Chronakis, J.L. Doublier, L. Picullel, Int. J. Biol. Macromol. 28 (2000) 1. [7] C. Viebke, J. Borgstro¨m, L. Picullel, Carbohydr. Polym. 27 (1995) 145. [8] M. Mangione, D. Giacomazza, D. Bulone, V. Martorana, P. San Biagio, Biophys. Chem. 104 (2003) 95. [9] V. Meunier, T. Nicolai, D. Durand, Int. J. Biol. Macromol. 28 (2001) 157. [10] L. Abad, I. Nasimova, L. Relleve, C. Aranilla, A.M. De la Rosa, M. Shibayama, Int. J. Biol. Macromol. 34 (2004) 81. [11] M. Sugiyama, C. Yuasa, K. Hara, N. Hiramatus, A. Nakamura, Y. Hayakawa, Y. Maeda, Physica B 999 (1998) 241. [12] G. Evmenko, E. Theunissen, K. Mortensen, H. Reynaers, Polymer 42 (2001) 29017. [13] N. Mischenko, B. Denef, K. Mortensen, H. Reynaers, Physica B 283 (1997) 234. [14] A. Michel, M. Mestdagh, M. Axelos, Int. J. Biol. Macromol. 21 (1997) 195. [15] L. Picullel, J. Borgstrom, I.S. Chronakis, P.O. Quist, C. Viebke, Int. J. Biol. Macromol. 21 (1997) 141. [16] L. Abad, I. Nasimova, C. Aranilla, M. Shibayama, Radiat. Phys. Chem. 73 (2005) 29. [17] M. Shibayama, Macromol. Chem. Phys. 199 (1998) 1. [18] M. Shibayama, K. Isono, S. Okabe, T. Karino, M. Nagao, Macromolecules 37 (2004) 2909.
Physica B 381 (2006) 103–108 www.elsevier.com/locate/physb
Small-angle neutron scattering study on irradiated kappa carrageenan Lucille Abada,b,c,, Satoshi Okabea, Satoshi Koizumic, Mitsuhiro Shibayamaa, a
Neutron Science Laboratory, Institute for Solid State Physics, University of Tokyo, 106-1 Shirakata, Tokai, Ibaraki 319-1106, Japan b Philippine Nuclear Research Institute, Commonwealth Ave., Diliman, Quezon City, Philippines c Advanced Science Research Center, Japan Atomic Energy Research Institute, 2-4 Shirane Shirakata, Tokai, Ibaraki 319-1195, Japan Received 22 November 2005; received in revised form 19 December 2005; accepted 19 December 2005
Abstract The structure of gamma-ray-irradiated k-carrageenan in aqueous solutions was investigated in terms of small-angle neutron scattering. The scattered intensity, I(q), of non-irradiated k-carrageenan solutions (5 wt%) was well described with an Ornstein–Zernike (OZ)-type function with the correlation length of 85 A˚, indicating that the k-carrageenan solution behaves just as a polymer solution in the semidilute regime. By increasing the irradiation dose (100 kGy), I(q) changed to a power-law function with the scattering exponent of 1.84. Further increase in dose results in a recovery of OZ-type function. This indicates that a progressive cleavage of k-carrageenan chains takes place randomly, leading to a self-similar structure at 100 kGy. This is followed by further segmentation of k-carrageenan chains. r 2006 Elsevier B.V. All rights reserved. PACS: 61.12.Ex; 87.15.v; 87.50.a Keywords: Small-angle neutron scattering; k-carrageenan; Gamma radiation
1. Introduction Radiation modification of natural polymers such as carrageenan, alginate, chitin, agar and cellulose have recently gained focus in various researches due to their widespread application in agriculture and in medicine. Radiation effects on these natural polymers result in drastic decrease in molecular weight. These low molecular weight polymers found valuable use as coatings in fruits and as plant growth promoter [1]. Irradiated carrageenans are among one of these promising polymers where they have been shown to exhibit optimum growth in rice plants at an irradiation dose of 100 kGy. Carrageenans are linear polymers of about 25,000 galactose derivatives with regular but imprecise structures, dependent on the source and extraction conditions. Different types are classified according to the number and position of sulfate groups. kCorresponding author. Philippine Nuclear Research Institute, Commonwealth Ave., Diliman, Quezon City, Philippines. Also to be corresponded to. E-mail addresses: [email protected] (L. Abad), [email protected] (M. Shibayama).
0921-4526/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.physb.2005.12.267
carrageenan is composed of alternating a(1,3)-D-galactose4-sulfated and b(1-4)-3,6-anhydro-D-galactose. Properties of k-carrageenan have been studied by several researchers [2–8]. It is known to form a strong thermoreversible gel in the presence of potassium ions where the conformation changes from a random coil to helix, leading to aggregation followed by gelation upon cooling. The structure of aggregating k-carrageenan from decreasing molar fractions from sonification have been investigated by dynamic light scattering (DLS) [9]. Previous work on gamma-irradiated k-carrageenan has also been done using the DLS. Results of this study correlated the relationship of a new fast mode peak observed at 100 kGy k-carrageenan from the decay time distribution function with its optimum biological activity [10]. Very few studies have been done on the small angle neutron scattering (SANS) of carrageenan [11–13]. The structure change of k-carrageenan gel with K+ in the gel to sol transition has been investigated by SANS. The SANS intensity decreased as it approached the gel-to-sol transition. Its scattering profile fitted with Guinier approximation formula [11]. This current study would investigate the structural changes of k-carrageenan with irradiation dose
ARTICLE IN PRESS 104
L. Abad et al. / Physica B 381 (2006) 103–108
by SANS. The effect of K+ at varying temperatures on irradiated k-carrageenan will also be investigated. 2. Materials and methods 2.1. Materials Refined k-carrageenan (KK-100) was obtained from Shemberg Corporation, Philippines. Elemental analysis by XRF of this carrageenan reveals the presence of 12% S and 11% K as the major components. This was irradiated in air at ambient temperature using the Co-60 facility of the Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Gunma, Japan. Samples were irradiated at a dose of 10 kGy/h with an absorbed dose of 50, 100, 150 and 200 kGy. Non-irradiated and irradiated k-carrageenan were then purified by dissolving in distilled water and dialyzing it against distilled water for 24 h (Mol. Wt. cut-off ¼ 12,000–14,000). The dialyzed solutions were precipitated with isopropyl alcohol and freeze-dried. The purified non-irradiated k-carrageenan still contains 12% S and a reduced K content of 3%. 2.2. SANS of k-carrageenan Non-irradiated and irradiated k-carrageenan samples were dissolved in D2O and in 0.05 M KCl/D2O solutions at elevated temperatures at a fixed concentration of 5% by weight. The samples were then placed in drum cells with a sample thickness of 0.21 mm. SANS experiments were carried out with the SANS instrument, SANS-U, at the Institute for Solid State Physics, The University of Tokyo, located at the Japan Atomic Energy Research Institute, Tokai, Japan. A flux of cold neutrons with a wavelength of 7.0 A˚ was irradiated to the sample and the scattered intensity profile was collected with an area detector of 128 128 pixels. The sample to detector distances of 2.00 and 8.00 m were used which covered the accessible q range of 0.006–0.15 A˚1, where q is the scattering vector. The sample drum cells with quartz windows were placed in a copper chamber and the chamber was thermo-regulated with a circulating water bath. SANS experiments were initially done at 20 1C and the temperature was raised to 60 1C. The obtained intensities were corrected for the detector inhomogeneities, cell scattering, transmission, and incoherent scattering, and then were scaled to the absolute intensity with an intensity calibration standard sample (Lupolen).
to the k-carrageenan helix which reduces considerably its charge density, which then consequently promotes the formation of helix and induce aggregation/gelation [14]. The non-irradiated k-carrageenan in its purified state does not have a cold-set gelation (a hot solution is turned into gel upon cooling) even at a concentration of 1%. This is expected as the K content is reduced considerably after purification (11–3%). At higher concentration of 5%, gels were pasty-like, but were hard and brittle in the presence of 0.05 M KCl. Irradiated k-carrageenan gels on the other hand were soft and pasty-like (with or without 0.05 M KCl) as the radiation dose was increased. Works done on ultrasonically degraded k-carrageenan (5–100 kDa) also indicate no gelation upto 5% concentration [15]. Fig. 1 shows the k-carrageenan gels at varying irradiation doses. The picture indicates a clear homogeneous non-irradiated k-carrageenan gel. Turbidity increased with increasing irradiation, reaching its maximum between 50 and 100 kGy. Beyond this dose range, very clear gels were obtained. Radiation randomly cleaves off the glycosidic linkages of k-carrageenan resulting in a very heterogeneous system. Further cutting of the glycosidic linkages would result in a more homogeneous molecular size distribution as it reaches the minimum molecular size possibly attainable. DLS measurements indicate a flattening of decrease in molecular weight beyond a radiation dose of 100 kGy [16]. The sol–gel transitions of 5% k-carrageenan are shown in Fig. 2. Addition of K+ ions increased the gelation temperatures. Due to its high concentration, gelation temperature (47 1C–50 1C) did not vary so much up to a radiation dose of 150 kGy. A drastic decrease in gelation temperature of 29 1C was observed at 200 kGy. A similar trend was observed in k-carrageenan without KCl for the same concentration. Decrease in gelation temperature (33 1C) started at 150 kGy. At 200 kGy, no gelation was observed. Studies done previously using a semi-dilute concentration of 0.5% in 0.05 M KCl shows a decrease in gelation temperatures for irradiated k-carrageenan up to a
3. Results and discussion 3.1. Physical properties of irradiated k-carrageenan gels The gelation of k-carrageenan involves multiple aggregation of double helices. It produces a stronger gel especially in the presence of potassium ions. K+ ions bind
Fig. 1. k-carrageenan gels in 0.05 M KCl at 20 1C.
ARTICLE IN PRESS L. Abad et al. / Physica B 381 (2006) 103–108
55
5
No KCl
4
45
200kGy 150kGy 100kGy 50kGy 0kGy
ξ = 61 Å
10
3
10
40
2
ξ =195 Å
1
ξ = 85 Å
-1
10 I(q) /cm
Gelation Temperature (°C)
50
ξ =109 Å
10
With 0.05M KCl
105
35
10
30
0
10 25
0
50
100 150 Irradiation Dose (kGy)
200
10
-1
1
-2
0.1
10
Fig. 2. Gelation temperatures of 5% k-carrageenan.
10
dose of 50 kGy. Gelation is only observed at 100 kGy when the KCl is increased to 0.1 M. Beyond this dose no gelation takes place [16].
10
6 789 0.01
-3
2
6 7 8 9 0.01
3
4 5 6 7 89 0.1
2
3
4
-1
5
6 7 8 9 0.1
q /Å
3.2. SANS profile of k-carrageenan gels
Fig. 3. SANS intensity curves of k-carrageenan at different radiation doses.
Fig. 3 shows double logarithmic plots of the scattered intensity functions, I(q), observed for 5% k-carrageenan in 0.05 M KCl/D2O solutions irradiated at various doses. The figure shows that I(q) increased with increasing irradiation dose and reached a maximum at a dose of 100 kGy. Beyond this dose, a decrease in I(q) was observed. This increase and decrease in I(q) agrees with the visual observations of the gel where maximum turbidity was observed at 100 kGy (Fig. 1). This may be closely associated to the increasing heterogeneity of the polymer system with dose brought about by random cleavage of the polymer chains. Minimum intensity was found in nonirradiated k-carrageenan gels. Except for the cases of 100 and 150 kGy, the scattering functions approximate a close fitting to an Ornstein–Zernike (OZ)-type function as shown in the solid curves: IðqÞ ¼
Ið0Þ , 1 þ x2 q2
(1)
where I(q) is the scattering intensity and x is the correlation lengths indicating the extent of correlated concentration fluctuations. The solid lines in Fig. 3 are the fit of the experimental data with this equation. The obtained correlation lengths are shown in Table 1. No correlation length was evaluated from the carrageenan at 100 kGy since it had a power law behavior. Initially, x for 0 kGy is 85 A˚, which increased to 195 A˚ at 50 kGy. Further irradiation led to an annihilation of characteristic length scale and an appearance of fractal structure at 100 and 150 kGy as will be discussed later. Above these doses, x decreased to 109 A˚ (at 200 kGy). The value of x ¼ 195 A˚ at
Table 1 Correlation lengths of k-carrageenan at varying radiation doses Radiation dose (kGy)
x at 20 1C with 0.1 M KCl (A˚)
x at 60 1C with 0.1 M KCl (A˚)
x at 20 1C without KCl (A˚)
0 50 100 150 200
85 195 n.m. 61 109
64 190 n.m. 128 175
95 n.m. 71
n.m. – non-measurable.
50 kGy is somewhat larger than observed in other systems. However, it is understandable by the fact that it represents not only the mesh size but also the characteristic size of heterogeneity as well. While the fitting curves are essentially Lorentzian, i.e., an OZ function, certain deviations to this function were observed especially with irradiated k-carrageenan. The double logarithmic plot indicates also a power law behavior exclusively at a radiation dose of 100 kGy. Fig. 4 shows a very close power law fitting of k-carrageenan at 100 kGy. The scattering exponent was 1.84. Non-irradiated k-carrageenan and k-carrageenan irradiated at other doses do not exhibit any power law behavior. This phenomenological behavior can well be explained by looking at their collective fluctuations as illustrated in Fig. 5. The concentration at 0 kGy is high enough to form a polymer solution that fills the entire space with polymer
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chains overlapping each other (CbC*), where C* is the chain overlap concentration. Since k-carrageenan is a natural polymer, concentration fluctuations are strongly suppressed such that the free energy density remains at a minimum. This is attained by cooperative arrangement of cross-linking and/or helix formation between chains. Gamma-irradiation randomly cleaves the k-carrageenan chains, which leads to the formation of segments that are not able to pair with other segments to form cross-links and/or helices. As a result, concentration fluctuations increase. This is the case of k-carrageenan irradiated at 100 kGy. Interestingly enough, the scattering intensity function exhibits a power law behavior exclusively at this radiation dose. Unscreening of entangled and cross-linked polymer chains may take place exposing heterogeneous chains of polymers of varying cluster sizes. This may be why a power law is observed. Decreasing of the molecular weight takes place further as in the case of k-carrageenan irradiated at 200 kGy. The system then moves towards uniformity as a result of cleavage to low-molecular sizes achieved by high-radiation doses. The scattering function
2
100 kGy
-1
log [I(q) / cm ]
1
0 Slope = -1.84
3.3. The effect of temperature and K+ ions on the SANS profile Fig. 6 shows a comparison of the SANS profiles of k-carrageenan (in 0.05 M KCl) at 60 and 20 1C. As expected, I(q)s at 60 1C are lower than those at 20 1C. The largest deviation was seen at 200 kGy as shown in Fig. 6c. This shows that heterogeneities decrease as a result of a lowering of the number of cross-link points at higher temperatures. At 200 kGy, k-carrageenan is completely in its sol state at 60 1C. Thus, a large decrease in I(q) is observed. A SANS experiment on k-carrageenan done previously also showed a decrease in intensity with increasing temperature [11]. Even at 60 1C, a maximum intensity was still found at 100 kGy. Unlike the SANS profile of k-carrageenan at 20 1C, its profile at 60 1C does not perfectly fit an OZ-type function as shown in the dotted lines of Figs. 6a–c. Introduction of the squared-Lorentzian function (broken lines) was necessary to give a successful fitting curve that closely reproduced the observed I(q)s. The sum of a Lorentz (L) and squared-Lorentz (SL) functions (L–SL) can be expressed in this equation. IðqÞ ¼
-1
-2
-2.0
no longer exhibits a power law behavior at this dose. The loss of this self-similarity can be explained by the fact that high-irradiation doses of 200 kGy may destroy some of the galacto-pyranose rings of k-carrageenan leading to ring opening. This possible ring opening at high doses is also explained using dynamic light scattering results [10]. The value of x of this semi-dilute polymer solution is 109 A˚.
-1.6
-1.2
-0.8
-1
log [q / Å ] Fig. 4. Power law behavior of k-carrageenan (5% in 0.05 M KCl at 100 kGy).
ξ
(2)
where X is another correlation length describing the extent of spatial inhomogeneities [17]. Though the contribution of the SL component is quite minimal at all radiation doses as seen in the figures, its contribution is not negligible. Only the correlation lengths taken from the L function are shown in Table 1 for the main reason that its components are the major contributor to the fitting function. Table 1 shows that the correlation lengths, x, of these polymer solutions increased with irradiation dose. Decrease in x was observed beyond 100 kGy. A power law behavior was also
ξ
0 kGy
I L ð0Þ I SL ð0Þ þ , 2 2 1þx q ð1 þ X2 q2 Þ2
100 kGy
ξ
200 kGy
Fig. 5. Distribution of k-carrageenan polymer chains with radiation.
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0
20°C
-1
60°C
10 10
10 0 kGy
Without KCl With KCl I(q)/cm-1
ξ = 64 Å
-1
I(q)/ cm
107
-2
10
1
ξ = 95Å 0.1
-3
10
0 kGy
Lorentz Squared-Lorentz
-4
5
10
6 7
2
3
0.01 q /Å
4
5
(a)
6 7 0.1
-1
6 7 8 9 0.01
2
3
4
q / Å-1
6 7 8 9 0.1
10
(a)
Without KCl 2
With KCl
10
I(q)/cm-1
100 kGy 1
10
20°C
-1
I(q)/ cm
5
1 Slope = -1.82
0.1
0
10
60°C
-1
10
100 kGy 5
Slope = -1.87
6 7 8 9 0.01
(b)
2
3
4
5
q / Å-1
6 7 8 9 0.1
-2
10
6 7
2
3
0.01
4
5
0.1
-1
(b)
10
6 7
Without KCl
q /Å
I(q)/cm-1
With KCl
20°C
0
200 kGy
10
-1
gel ξ = 71Å
0.1
10
60°C ξ = 175 Å
-1
I(q)/ cm
1
-2
10
sol
200 kGy 5
-3
10
(c)
6 7 8 9 0.01
2 q / Å-1
3
4
5
6 7 8 9 0.1
Lorentz
-4
10
Fig. 7. The effect of K+ ions on the SANS intensity of 6-carrageenan (5%) at different radiation doses.
Squared-Lorentz
-5
10
6
7
2 0.01
(c)
3 -1
4
5
6 7 0.1
q /Å
Fig. 6. The effect of temperature on the SANS intensity of 6-carrageenan (5%) at different radiation doses.
observed exclusively at 100 kGy having a fractal dimension of 1.87. This trend is comparable to the one of kcarrageenan at 20 1C. Fig. 7 demonstrates the role of K+ ions in the ordering of the gels in k-carrageenan. We expected that I(q) for k-carrageenans irradiated at 0 and 200 kGy can be represented with a L function as shown by the dashed lines as was the case with KCl. The effects of the absence of
KCl are twofold. (1) In the absence of KCl, I(q) is smaller than those with KCl. (2) An upward trend with respect to the Lorentzian behavior was observed at low q region for k-carrageenans irradiated at 0 and 200 kGy. This suggests the presence of strong inhomogeneities of the k-carrageenan gels by aggregation. By nature, k-carrageenans are self-aggregating and form gels even in the absence of any ions. The presence of K+ ions facilitates the formation of well-ordered double helix domains. In effect, the absence of these K+ ions makes the gel more heterogeneous. At 200 kGy, k-carrageenan without the K+ ions is completely in the sol state. Thus, the fitting resembles back to an O–Ztype function. Its intensity though is much lower than k-carrageenan with KCl at 200 kGy. Polymer solutions have lesser number of cross-links than their gel
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counterparts. Correlation length at 0 kGy was determined to be 95 A˚, which decreased with irradiation dose of 200 kGy (71 A˚). I(q) at 100 kGy for k-carrageenan without KCl is very similar to the one of k-carrageenan with KCl except for a slight download deviation at the mid-q range. The characteristic power law behavior observed at this dose is also visible in k-carrageenan without KCl. Its fractal dimension is 1.82.
Dr. Fumio Yoshii for allowing the use of their gamma irradiation facility. This research was also made possible through the financial support of the Japan Atomic Energy Research Institute (JAERI) and the Forum for Nuclear Cooperation in Asia (FNCA). XRF analysis of k-carrageenan was done by the Physics Research Group of the Philippine Nuclear Research Institute.
4. Conclusion In summary, this work clearly demonstrates the following: k-carrageenan is subjected to degradation by gamma irradiation. The structure factor of non-irradiated kcarrageenan aqueous solutions is similar to those for polymer solutions in semi-dilute regime, i.e., an Ornstein– Zernike form. By gamma-ray irradiation, however, chain scission takes place randomly and destroys active sites capable of physical cross-linking. As a result, the molecular weight distribution becomes widely spread and chain clusters become self-similar and network formation with a minimal increase in concentration fluctuations becomes difficult. Hence a power-law behavior appears in I(q) which interestingly is observed at 100 kGy irradiated k-carrageenan. Further irradiation results in a progressive reduction in the molecular weight and a recovery of OZ behavior due to a loss of the self-similarity. Such kind of behavior is not observed in synthetic gels, such as polyacrylamide hydrogels. In synthetic polymer gels, I(q) cannot be described by an OZ function due to presence of inhomogeneities and a squared-Lorentz-type intensity function has to be added to describe them [18]. k-carrageenan seems to self-organize so as to keep the concentration fluctuations as low as possible by rearranging their cross-links. Degradation by irradiation unscreens the chain overlapping and makes explicit the architecture of the cluster structure. Acknowledgment The authors wish to thank the Takasaki Radiation Chemistry Research Establishment, JAERI, especially
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