- Email: [email protected]
Radiation crosslinking of methylcellulose and hydroxyethylcellulose in concentrated aqueous solutions
Nuclear Instruments and Methods in Physics Research B 211 (2003) 533–544 www.elsevier.com/locate/nimb
Radiation crosslinking of methylcellulose and hydroxyethylcellulose in concentrated aqueous solutions Radoslaw A. Wach b
a,*
, Hiroshi Mitomo a, Naotsugu Nagasawa b, Fumio Yoshii
b
a Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu 376-8515, Japan Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki 370-1292, Japan
Received 24 February 2003; received in revised form 19 May 2003
Abstract The effects of ionizing radiation on aqueous solutions of cellulose ethers, methylcellulose (MC) and hydroxyethylcellulose (HEC) were investigated. The well-established knowledge states that cellulose and its derivatives belong to degrading type of polymers. However, in our study intermolecular crosslinking initiated by gamma rays or electron beam leaded to the formation of insoluble gel. This is an opposite effect of irradiation to the degradation. Paste-like form of the initial specimen, i.e. concentration 20–30%, when water plasticizes the bulk of polymer; and a high dose rate were favorable for hydrogel formation. Gel fraction up to 60% and 70% was obtained from solutions of HEC and MC, respectively. Produced hydrogels swell markedly in aqueous media by imbibing and holding the solvent. Radiation parameters of irradiation, such as yields of degradation and crosslinking and the gelation dose, were evaluated by sol–gel analysis on the basis of Charlesby–Rosiak equation. Despite of the crosslinked structure, obtained hydrogels can be included into the group of biodegradable materials. They undergo decomposition by the action of cellulase enzyme or microorganisms from compost. 2003 Elsevier B.V. All rights reserved. PACS: 61.25.H; 61.82.P; 81.05; 82.70.G Keywords: Hydrogels; Methylcellulose; Hydroxyethylcellulose; Crosslinking; Radiation yield; Biodegradation
1. Introduction Irradiation of polymeric materials generates some effects depending on the kind of polymer, parameters of irradiation, the state of the material under processing, etc. The two main reactions, which determine the final properties of the poly* Corresponding author. Present address: Nuclear Engineering Research Laboratory, The University of Tokyo, 2-22 Shirakata-Shirane, Tokai-mura, 319-1188 Ibaraki-ken, Japan. Tel.: +81-29-287-8984; fax: +81-29-287-8488. E-mail address: [email protected] (R.A. Wach).
mer, include (a) scission of main chain, leading to the diminishing of the molecular weight of macromolecules and (b) crosslinking, the opposite process ending with macroscopic, insoluble material [1]. The yields of scission and crosslinking, or more precisely the mutual ratio of these two parameters, determine the outcome. To achieve the desired aim, irradiation of polymeric material can be conducted under special conditions. Particularly, the state of material under processing is of a great significance. Thus, an elevated temperature, the polymer is in molten state, is often use [2]. Dose rate can be the variable; irradiation with EB
0168-583X/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-583X(03)01513-1
534
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
gives higher local concentration of radicals. Furthermore, irradiation of water-soluble polymers in aqueous solution, due to the high yield of radicals, is often use for formation of hydrogels [3]. Gamma or EB irradiation has been regarded as a very useful method in order to get macroscopic threedimensional able to swell lattice. Polymeric network, which exhibits the ability to absorb and retain a significant amount of water within its structure, but which does not dissolve is defined as hydrogel. One can regard hydrogel-like material possessing both liquid-like and solid-like properties. The former one results from the fact that the main constituent is water. Thus, penetration of solvent or low molecular weight solutes throughout the bulk of gel is possible. The later one is due to the difference in osmotic pressure (Donnan equilibrium), elastic strength of the network maintains the shape of gel but allows for increasing its dimensions [4]. Hydrophilicity of the polymer to be used to the formation of hydrogel is of great importance for the final properties of the product. To change hydrogel water uptake an addition of hydrophobic segments can be applicable as accompanying copolymer or grafted chain [5,6], or as in interpenetrating polymer network by incorporation of another polymer, which creates independent lattice in the matrix [7,8]. Crosslinking density, the average molecular mass between the two junction points, affects water absorption characteristic of the hydrogel. In the synthesis of gels by chemical method, i.e. by bi/ multifunctional monomers, crosslinking density is controlled by the concentration of this crosslinker, reaction time, temperature and others. While, for radiation method it is determined by the absorbed dose, which means by the irradiation time. Moreover, crosslinking by the chemical method can be performed only in liquid state, which is in distinct contrast with the later one. Since the ionizing radiation is highly penetrating, it is possible to initiate chemical reactions in liquid or in solid state. In cellulose, the most abundant renewable raw material, the presence of active hydroxyl groups has been utilized in a variety of chemical modifications to produce derivatives. Among them cellulose ethers are commercially important products due to
their superior properties with respect to the native material. Chemically crosslinked gels of hydroxyethylcellulose (HEC) with carboxymethylcellulose (CMC) have been synthesized by divinylsulphone crosslinker. The chemical composition and manufacturing procedures have the influence on equilibrium water content [9]. Swelling ability of products enhanced with an increase of CMC content in the blend. It has been proposed that the crosslinker reacted with the unsubstituted groups on the cellulose ethers [10]. Polysaccharides and their derivatives exposed to ionizing radiation, gamma rays or electron beam (EB) undergo degradation [11]. It is apparent that polymers of natural origin, irradiated in solid state suffer scission of acetal linkages in main chain. Some mechanisms of the processes have been proposed [12–14]. Even after the first successful attempts of Leavitt to crosslink cellulose ethers by ionizing radiation only a little interest of researchers in this subject had been reported [15,16]. Possibility of radiation-induced crosslinking of methylcellulose (MC) was mentioned by the occasion of hydrogel fabrication from synthetic polymers [17]. The authors achieved a swellable gel by EB irradiation of MC aqueous solution (10% w/w). The degree of swelling equilibrium of the resulted hydrogel was about 20 g of water per gram of polymer. The key factor for successful intermolecular bonding of MC and HEC, according to the results, was a high dose rate of electron radiation [16]. The aim of this study was to provide more systematic data concerning crosslinking of cellulose derivatives, MC and HEC in order to fill the gap in knowledge on radiation processing of cellulosics. In addition some results on hydrogels degradation by the action of enzyme and naturally occurring microorganisms are reported.
2. Experimental 2.1. Materials Ethers o cellulose: HEC obtained from Sumitomo Seika Co. Ltd. Japan and MC from Shin Etsu Co. Ltd. Japan were used in our experiments.
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544 Table 1 Characteristic of cellulose ethers Sample
Degree of substitution (DS)
Intrinsic viscosity (dL/g)
Weight-average molecular weight
MC5 MC6 HEC7 HEC8 HEC9
1.8 1.8 2.0 2.0 2.0
1.96 7.58 3.72 5.39 9.35
1.20 · 105 14.0 · 105 2.02 · 105 3.09 · 105 5.82 · 105
Scheme 1. Chemical structure of 2 repeating units of cellulose, MC and HEC connected by glycosidic linkage at 1 and 4 positions with b configuration.
Characteristic of these polymers is summarized in Table 1 and the structure is presented in Scheme 1. Average molecular weights were determined by measuring intrinsic viscosity, see below. All chemicals used were analytical grade. 2.2. Molecular weight of polymers Weight-average molecular weights of the polymers were determined from intrinsic viscosity on the basis of the Mark–Houwink equation ½g ¼ K DPa for HEC or ½g ¼ K Mwa for MC, where K and a are constants, DP is the weightaverage degree of polymerization and Mw is the weight-average molecular weight. The viscosity of polymer solution was measured by an Ubbelohde viscometer in water at 25 C. The intrinsic viscosity was found by plotting the obtained reduced viscosity gsp =c and lnðg=gsp Þ=c against concentration (in g dl1 ) and extrapolation to zero concentration.
535
For this polymers and measurement conditions constant K equals to 3.16 · 103 , 1.1 · 102 and a to 0.55, 0.87 for MC [18] and HEC [19], respectively. 2.3. Sample preparation and irradiation Polymer was mixed with an appropriate amount of deionized water and the material was kept for 7 days at room temperature to ensure complete dissolution and homogeneity of the specimen. Higher concentrated solutions had a tixotropic paste-like properties, while those of lower concentrations were extremely viscous however, exhibited slow gravity flow. Irradiation was conducted with gamma rays generated from 60 Co source at a dose rate of 10 or 1 kGy/h at inert temperature. For irradiation by high-energy electrons, the 2 MeV accelerator was used at the following irradiation parameters: current 1 mA, voltage 1 MeV and the dose per pass 10 kGy. Irradiation was carried out in polyethylene bag. Alternatively, for air-free irradiation, to avoid the penetration of additional oxygen during irradiation, specimens were sealed in poly(vinylidene chloride) bag after removal of the air by a vacuum pump. 2.4. Gel content and swelling properties of hydrogel Amount of gel content and swelling properties of resulted hydrogels were determined gravimetrically. Gel content in the dried hydrogels was estimated by measuring insoluble part after extraction in deionized water for 7 days at room temperature. Gel fraction was calculated as follows: Gel fraction ½% ¼ ðGd =Gi Þ 100;
ð1Þ
where Gi is the initial weight of polymer, Gd is the weight of the dried insoluble part after extraction. Hydrogels were swollen in deionized water, NaCl solution or buffers of certain pH value. An equilibrium water uptake was reached, when the mass of two consecutive weighing did not differ more than 0.5%. The surface of hydrogel was blotted by filtration paper prior to weighing. If Gs is the weight of hydrogel in swollen state, swelling is presented as follows:
536
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
Swelling ½grams of water=1 g of dried gel ¼ ðGs Gd Þ=Gd :
ð2Þ
2.5. Biodegradation Enzymatic degradation was carried out in an acetic acid–NaOH buffer, at pH 5.0, by cellulase enzyme, from penicillium funiculosum. About 10 mg of dried film of gel, with a thickness about 0.3 mm, was immersed in 2.5 ml of the enzyme solution for given time. The concentration of the cellulase enzyme was 0.1 mg ml1 . Tests were performed at the most appropriate temperature for the enzyme activity, 37 C, with shaking. After incubation, the samples were washed and kept in an excess of distilled water to wash away the degraded polymer then, dried at 35 C under vacuum. The result of enzymatic degradation is expressed as percentage of the initial sample weight: Degradation ð%Þ ¼ Ge =Gd 100%;
ð3Þ
where Ge and Gd are the weights of films after and before enzymatic degradation, respectively. Microbial degradability of MC and HEC in soil under controlled conditions was evaluated by measuring CO2 production. Especially designed apparatus – Microbial Oxidative Degradation Analyzer (MODA) [20], comprised of 4 independent lines of columns, was used. 10 grams of the sample was mixed with rinsed sea sand – 450 g and compost – 130 g and then placed in a heated reactor. Inside the column monitored temperature was 35 C and the flow in of the carbon dioxidefree but moisturized air was 30 ml min1 . After flowing through the sample inside the reactor, the air carrying formed due to polymer disintegration CO2 was passing series of columns filled in turn with silica gel, calcium chloride, soda lime and calcium chloride. Ammonia, which could be formed from the decomposing sample, was trapped in sulfuric acid solution, and water vapor was absorbed into first two columns (silica gel and calcium chloride). The CO2 was collected quantitatively by soda lime and water produced during the reaction was caught in the last CaCl2 column.
Thus, the mass of produced carbon dioxide was calculated as a difference in the weight of two last columns (containing soda lime and calcium chloride) at the beginning and during the test. Pure compost mixed with sea sand was used as a blank and cellulose powder as a reference sample.
3. Results and discussion 3.1. Radiation crosslinking Examined polymers exposed on c-rays in solid state and in aqueous solutions of low concentrations undergo degradation, in the atmosphere of air as well as in the absence of oxygen. The results of degradation are closely similar to those reported previously for another cellulose derivatives CMC and HPC thus, there is no reason to present them in the present paper [21,22]. It just has to be mentioned that the main reason of the low yield of the intermolecular recombination in diluted solutions of concentration less than 10% is caused by insufficient number of chains in close vicinity one to each other. In the solid state, extremely limited mobility of chain segments bearing radicals prevents their mutual recombination. In both cases scission is the predominant reaction leading to the diminishing of degree of polymerization of cellulose ethers. Irradiation of MC and HEC at moderate or high concentrations ends with the formation of insoluble gel by intermolecular crosslinking. The relation of gel fraction to irradiation dose of hydrogel produced from 20% HEC aqueous solution initiated by EB and gamma rays is presented in Fig. 1. For all samples processes by EB the gel fraction increases with absorbed dose and then stabilize at the maximum value. Gelation dose, at which first insoluble material appears, and the gel fraction depend on the initial molecular weight of HEC. Gel fraction reaches about 58%, 53% and 49% for HPC9, HPC8 and HPC7, respectively. Since crosslinking and scission takes place simultaneously, macromolecules of the longest chains are more prone to bond each other before they undergo substantial degradation. Even if scission undergoes with the same yield for every molecular
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544 60 HEC 9 HEC 8 HEC 7 HEC 9 HEC 8
Gel Fraction [%]
50 40 30 20 10 0
0
50
100
150
200
Dose [kGy] Fig. 1. Influence of irradiation dose on the resulted gel fraction of HEC hydrogels. Hydrogels formed by c-rays (solid points) and EB (open points) irradiation of HEC 20% aqueous solution in air-free atmosphere.
weight fraction, to reduce the size of the biggest molecules, it is necessary more scission events to occur than for the degradation of short chains. For HEC9 and HEC8 irradiated by gamma rays without access of oxygen at dose rate of 10 kGy/h the maximum gel fraction is significantly lower than that for EB irradiation. Moreover, the gel part of HEC8 reaches the maximum and then diminishes – apparently, for higher doses scission prevails over intermolecular crosslinking. In Fig. 2, there is presented data concerning MC. The distinction between the two molecular
weight samples irradiated by gamma rays is the most pronounced in the gelation dose and the value of gel fraction. The gel starts to arise at a dose lower than 10 kGy for MC6, while for MC5 gelation begins at the dose as high as 70 kGy. Irradiation by EB results in higher gel fraction than that by gamma rays. For MC6 it reaches about 60% in comparison to less than 40% obtained by gamma irradiation. During all irradiation process, up to 200 kGy, crosslinking dominates over cleavage of mainchain bonds, and no deterioration of the gel is observed. Influence of dose rate, irradiation at 1 kGy/h, and air atmosphere on the gel fraction is depicted in Fig. 3. Diminishing of the gel fraction takes place for HEC9 from 30% (10 kGy/h) to 20% (1 kGy/h). No apparent change in gelation of MC6 is observed as a function of the dose rate. MC resembles the behavior of another cellulose ether, CMC examined previously, for which also no evident diversification was found [23]. Probably, the lifetime of radicals plays an important role in the share of crosslinking – scission reactions. Radicals formed on MC macromolecules seem to be more stable than those on HEC chains and in consequence, there is a higher probability for their recombination with the other fitting macroradical, before their transformation with the rupture of mainchain. Radicals on MC and HEC was
60
70
Gel Fraction [%]
Gel Fraction [%]
60 50 40 30 20 MC6 MC5
10 0
0
20
40
60
80 100 120 140 160 180 200
537
1kGy/h MC 6 HEC 9 Air MC 6 MC 5
50 40 30 20 10 0
0
50
100
150
200
Dose [kGy]
Dose [kGy] Fig. 2. Influence of irradiation dose on the resulted gel fraction of MC hydrogels. Irradiation in air-free atmosphere by c-rays, MC 25% aqueous solution (solid points); and EB irradiation, MC 20% aqueous solution (open points).
Fig. 3. Influence of irradiation conditions (c-rays) on the resulted gel fraction of hydrogels. Irradiation of 20% aqueous solutions of MC6 and HEC9; dose rate – 1 kGy/h; air-free atmosphere. Irradiation of 25% MC aqueous solutions; dose rate – 10 kGy/h; air atmosphere.
538
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
investigated by ESR technique, described elsewhere [24]. Ionizing radiation in means of electromagnetic and charged-particle radiation interacts with matter by distinctly different physical processes, although the final overall effects – generation of a mixture of excited and ionized species – are qualitatively similar [25]. Beam of ionizing radiation, independently on its origin, causes the cascade of primary and later secondary electrons. Such electrons have the same character for all kinds of ionizing radiation. However, they may differ in their kinetic energy. The next steps of the attenuation are in turn: (a) formation of positive ions, excited molecules and thermal electrons, (b) formation of free radicals and (c) chemical effects. The number of created radicals is two orders of magnitude higher for EB than for gamma irradiation at dose rate 10 kGy/h. Thus, if the polymer concentration is high enough, much higher density of macroradicals exist in the same time, and intermolecular recombination is favored over chain scission. According to this, the dose rate has a significant influence on the gel fraction, and in general, on overall gelation process. Another important factor, which affects the hydrogel formation, is presence of oxygen in the solution during irradiation. In the above investigations the poly(vinylidene chloride) bags were used which, is assumed to protect against penetration of air into the bulk during irradiation. Similarly to irradiation without air the gel fraction of MC increases throughout the applied dose range, but is greatly reduced. It corresponds to the results obtained for CMC [21]. It is due to the reaction of oxygen with macroradicals, which transform to peroxy-macroradicals. Subsequently, formed unstable compounds decompose and affect the main chain of polymer causing rupture of glycosidic linkages [26]. The shear of scission reaction greatly increases. 3.2. Radiation yield of crosslinking and scission The main chemical effects on polymers subjected to ionizing irradiation are crosslinking and chain scission. Both processes occur simultaneously and their yields determine the final results
of irradiation [27]. If crosslinking predominates over scission, an insoluble macroscopic gel is formed. In such case, the sol–gel analysis allows to estimate parameters of the irradiation process [1]. The yield value (G) is a number of chemical events or molecules changed, e.g. crosslink bonds or scission acts, per 100 eV of absorbed energy [28]. Deviations of the initial molecular weight distribution of irradiated polymers from the random one can affect calculated yields values. Thus, in the present study, to avoid an inaccuracy resulting from unknown molecular weight distribution of used polymers the Charlesby–Rosiak equation (4) was used [29]. This equation allows for estimation of radiation parameters of linear polymers of any initial weight distribution as well as is applicable to systems when an initial material is monomer or branched polymer. pffiffi p0 p0 Dv þ Dg sþ s¼ þ 2 ; ð4Þ q0 q0 D v þ D GðxÞ ¼
4:8 105 ; M w0 Dg
GðsÞ=GðxÞ ¼ 2p0 =q0 :
ð5Þ ð6Þ
In the above equations s is the sol fraction, p0 is the degradation density, q0 is the crosslinking density, D is the absorbed dose, Dg is gelation dose and Dv is the virtual dose – a dose required to change the distribution of molecular weight of the certain polymer in such a way that the relation between weightaverage and number-average molecular weight would be equal to 2 (for detailed explanation see [30,31]). The data for examined polymers irradiated under selected conditions is listed in Table 2. According to calculated results, gelation dose (Dg ), a dose when the first insoluble gel appears, diminishes for higher molecular weight fractions of the same cellulose ether. Dg is significantly lower – gelation starts earlier – also for material irradiated by EB. Degradation to crosslinking density, p0 =q0 corresponds directly to the obtained gel fraction. If the parameter decreases, the crosslinking takes greater share; one can expect higher part of gel after irradiation. It is evident for the example of HEC9, for which Dg and p0 =q0 enhance greatly for gamma processing in contrast to EB irradiation.
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
539
Table 2 Radiation parameters of irradiated polymers, evaluated on the basis of sol–gel analysis No.
Sample/concentration
Irradiation and conditions a
Dgb [kGy]
p0 =q0 b
GðxÞb [1/100 eV]
GðsÞb [1/100 eV]
1 2 3 4 5 6 7 8 9 10 11
MC5/20% MC5/25% MC5/25% MC6/20% MC6/25% MC6/25% MC6/20% HEC7/20% HEC8/20% HEC9/20% HEC9/20%
EB c, vacuum c, air EB c, vacuum c, air c, vacuum (1 kGy/h) EB EB EB c, vacuum
41.3 76 148 4.5 7.1 24.2 6.6 21 9.6 3.56 9.1
0.98 1.02 1.85 0.80 0.96 1.38 1.02 1.00 1.08 1.07 1.54
0.01 0.05 0.03 0.08 0.05 0.014 0.05 0.11 0.16 0.23 0.09
0.19 0.11 0.1 0.12 0.09 0.04 0.11 0.22 0.35 0.5 0.28
The influence of oxygen during irradiation on the obtained results is apparent after comparison of p0 =q0 for MC5 and MC6 (compare rows 2 and 3; 5 and 6). The share of degradation increases considerably. Gelation dose doubles for samples irradiated in air. The comparison of gelation parameters indicates that gelation occurs more effectively by EB processing. No significant diverse between gamma irradiation of MC with the dose rates 1 and 10 kGy/h was observed, row 5 and 7. The gelation process of these two samples proceeds similarly with only a little advantage of maximum gel fraction of the irradiation at 10 kGy/h. 3.3. Swelling of hydrogels The main feature of hydrogel is its ability to absorb and hold in its structure an amount of solvent. Swelling, usually presented as weight of solvent absorbed per 1 g of dried gel, depends on the hydrophilicity of the polymer, density of intermolecular links i.e. molecular weight of chain part between crosslinks and others. The important factor is also a composition of a solvent if it is not distilled water. Presence of low molecular weight solutes, e.g. inorganic salt, as well as pH of the solvent can affect greatly the swelling of hydrogel. Fig. 4 shows swelling of HEC hydrogel made from 20% aqueous solution by EB as a function of
Swelling [g water / g dry gel]
p0 =q0 – degradation to crosslinking density, Dg – gelation dose, GðxÞ, GðsÞ – radiation yield of crosslinking and radiation yield of scission, respectively. a If not indicated, the dose rate of gamma radiation was 10 kGy/h. b Values calculated by Charlesby–Rosiak (4), and Eqs. (5), (6), indicated in text.
175 HEC 9 HEC 8 HEC 7
150 125 100 75 50 25 0
0
50
100
150
200
Dose [kGy] Fig. 4. Swelling in distilled water of hydrogels formed by EB irradiation of 20% HEC aqueous solutions.
absorbed dose. Obtained shape of swelling curves is common for gels formed by ionizing radiation. Swelling is the highest just after the dose oversteps gelation point. To form a gel, statistically one crosslink per chain is necessary to form an insoluble macroscopic gel [1]. Then, the network is weak and susceptible to break, but because of a relatively low number of intermolecular bonds, more solvent can penetrate inside the crosslinked polymer matrix. With continuous increasing of the density of crosslinks, due to further irradiation the absorption ability of the gel decreases, voids accessible for
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544 500
Swelling [g water / g dry gel]
water shrink. The structure of hydrogel becomes more firmly connected and rigid. For HEC swelling drops sharply with the dose and finally remains at the level of 20–40, for samples irradiated at 200 kGy. Obviously, higher initial molecular weight fraction is characterized by lower swelling after crosslinking. Changes in crosslinks density for crosslinking type polymers, are reflected by the extent to which the material is swollen by soaking in a compatible solvent – this is determined by comparison the weight of the solvent swelled material with its dry weight. During irradiation the density of crosslinks increases consequently leading to a decrease in the solvent absorption ability of hydrogel. When the scission prevails, opposite process occurs. The percentage of crosslinked polymer that is insoluble in the appropriate solvent may decrease. The process is presented schematically in Fig. 5. An increase of soluble fraction – sol – reflects degradation, whereas a reduced soluble fraction indicates crosslinking. Swelling of hydrogels formed in air-free atmosphere by c-rays at a dose rate of 1 kGy/h from 20% MC and HEC aqueous solutions is presented in Fig. 6. The amount of solvent imbibed by MC6 hydrogel decreases gradually with dose increase. It is in accordance with the fact that the gel fraction does not decrease with the dose. Thus, it can be concluded that throughout all irradiation process the reaction of intermolecular crosslinking overweighs over scission and the density of crosslinks increase or, at least does not reduce. The process stops at the B step from Fig. 5. Swelling of HEC9 reveals different characteristic in response to irradiation with a low dose rate, 1 kGy/h from that observed for MC6. It is apparent that HEC9 degrade slightly with continuous irradiation
HEC 9 MC 6
400 300 200 100 0
10
20
30
40
50
60
70
Dose [kGy] Fig. 6. Swelling of hydrogels formed by c-rays irradiation at low dose rate. Conditions: MC6 and HEC9 20% aqueous solution; 1 kGy/h; air-free atmosphere.
(compare Fig. 3). Breaking of chains inside the gel is directly reflected in swelling. Evidently, swelling starts to increase at higher doses, over 30 kGy. The C step from Fig. 5 is reached. Equilibrium swelling of hydrogels in NaCl aqueous solutions of various ionic strengths and of several pH values is presented in Fig. 7. Gels were prepared from 20% aqueous solutions in air-free
pH of the solution 40
Swelling [g water / g dry gel]
540
0
2
4
6
8
10
12
35 30 25 20 15 10 5 0.0
0.2
0.4
0.6
0.8
1.0
Ionic strength [M]
Fig. 5. Schematic representation of intermolecular crosslinking of polymer chains and their degradation with continuous irradiation.
Fig. 7. Equilibrium swelling of hydrogels in aqueous solutions of various ionic strength and pH. Gels were prepared from 20% aqueous solutions of HEC9 (solid points) and MC6 (open points) by EB irradiation in air-free atmosphere at 50 and 20 kGy, respectively.
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
3.4. Biodegradation of hydrogels Biodegradable polymers can be converted, to carbon dioxide and water as end products, with releasing of energy. Degradation is a desirable feature of hydrogels utilized in medicine, agriculture and other fields. However, most hydrogels with high water absorption capacity prepared from synthetic polymers are tough to decompose naturally. It is well known that cellulose and its derivatives disintegrate in soil and under controlled
100
80
Degradation [%]
atmosphere by an EB at doses 50 kGy (HEC9) or 20 kGy (MC6). HEC9 hydrogel reveals continuous deswelling with the increase of solutions pH from 38 at pH 1.7 to about 30 at pH 7, and then the gel volume increases slightly at basic pH. The solution uptake is independent on the presence of NaCl at various ionic strengths. Hydrogel of MC6 presents similar character of swelling as a function of solution pH, but much less pronounced. On the contrary, the uptake decreases with an increase in ionic strength. This diverse swelling manner, despite that both polymers are nonionic, results from different hydrophilic/hydrophobic character of their functional groups. MC is more hydrophobic in nature than HEC because of its pendant methylene groups. MC swelling is less responsible to the pH, but displays dependence on the ionic strength. This could be due to the fact that salt competes with the polymer for water molecules, which cause easier hydrophobic aggregation of chains [18]. As a hydrophobic and nonionic gel, MC is hardly affected by changes of pH value of the solution. HEC provides numerous sites for hydrogen bonding, thereby facilitating the formation of highly organized structure consisting of a layer of water molecules around the polymer backbone and the ethylene glycol side chains. Obviously these hydrogen bonding occur in neutral pH, resulting in contraction of the gel. In acidic and basic media the presence of hydrogen cations or hydroxyl anions is evidently conducive to higher swelling. It might be due to disruption of hydrogen bonds structure and, for low pH values, formation of –OHþ 2 ions, which induces osmotic swelling pressure and imbibes the solution.
541
HEC 20 kGy HEC 50 kGy HEC 100 kGy MC 20 kGy MC 50 kGy
60
40
20
0
0
20
40
60
80
Time [h] Fig. 8. Biodegradation of hydrogels by the cellulase enzyme C0901 from P. funiculosum in an acetic acid – NaOH buffer (pH 5.0). Hydrogels were prepared by EB irradiation of 20% MC6 and HEC8 aqueous solutions in air-free atmosphere. Degradation of the hydrogels did not exceed 5% for blank tests (without the enzyme).
conditions by using enzymes or by means of acidic hydrolysis by cleavage of glycosidic linkages [32,33]. Therefore, especially suitable for those purposes are cellulose ethers. Results of degradation tests carried out by cellulase enzyme, in acetic acid–NaOH buffer of pH 5.0, are presented in Fig. 8. Hydrogels were prepared by EB irradiation of MC or HEC in 20% aqueous solution and only gel part, after extraction of sol, was subjected to the enzymatic hydrolysis. Blank test in buffer solution without the enzyme diminished the mass of each gel by less than 5%. Taking into consideration the applied dose of irradiation, a tendency of decomposition of the both polymers is identical. Hydrogel of lower crosslinking density degrades faster than that crosslinked more tightly at higher dose. It is because of greater number of intermolecular bonds in the latter. MC6 and HEC8 formed at 20 kGy posses relatively high gel fraction, 60% and 30%, respectively. However, they degrade completely at less than 24 of incubation. Those irradiated to 50 kGy become fully soluble before 72 h of enzymolysis. For HEC sample irradiated at 100 kGy the degradation is 15% (85% of remained weight) after 72 h of incubation. Similar rate of enzymatic degradation was achieved for MC and HEC samples instead of that
542
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
MC6 had the initial molecular weight more that four times higher that HEC8. Obviously, size and the number of substituent group as well as its kind played the crucial role in the degradation mechanism. The higher DS hinders biodegradation [34]. Access of the enzyme in to the proximity of glycosidic bond was not hampered by a spatial hindrance of such small group as –CH3 , but it was balanced by a relatively high DS, 1.8. Thus, the degradation rate of these two hydrogels (MC and HEC) is comparable. Microbial degradability was evaluated by assessment of carbon dioxide produced from the decomposition of the cellulosic material. Samples of MC6 and HEC8, also cellulose as a reference material, were investigated and the results are presented in Table 3. Cellulose undergoes the fastest degradation, which reaches 80% after one month (after subtraction of the carbon dioxide produced in blank test, the sample comprised of only compost). Cellulose ethers decompose much slower. Radiation processing of MC polymer improves its susceptibility for bacterial digestion comparing to initial MC powder. MC6 irradiated in 20% at 50 kGy yields faster decomposition in spite of a number of introduced crosslinks. The gel fraction of the tested hydrogels was about 65%. It was reported that treatment by ionizing radiation of cellulose before its enzymatic hydrolysis induces faster degradation [35]. At the dose as low as 10 kGy, HEC8 irradiated in 20% aqueous solution does not form a rigid gel. After placing the gel into excess of water, the system had a form of viscous slurry without any specific shape. Apparently, the gelation process has started, but scission has not
occurred. This restrained susceptibility to biodegradation. It was reported that enzymatic hydrolysis of cellulose of various origin yields glucose, which is a measure of degradation, in smaller amount after radiation treatment at low doses than without irradiation or after irradiation at high dose [36]. Comparing the data collected by the two methods it can be noticed that the degradation rate of individual material by enzymatic test and in composted soil varies. It may be due to the difference in the entity to be determined. In the case of enzymolysis, a decreasing weight of the insoluble fraction was measured. Thus, one scission event of the end linked chain or two places of degradation in the chain between the two closest joint points resulted in significant mass reduction of the specimen. On the other hand, for CO2 evolution a total disintegration of molecules is essential. In the former case, cellulase enzyme was employed but degradation in composted soil engaged number of natural microorganisms, which activities toward certain cellulose ether could differ. It was proposed that the cleavage of main chain bonds is the fastest between two adjacent unsubstituted anhydroglucose units [37]. The lower probability of an enzyme attack and slower degradation rate was postulated to occur next to a single, isolated unsubstituted ring. Possibly, degradation also takes place at linkages involving single, unsubstituted unit at one end of the chain. Thus, the higher DS reduces the probability for the presence of unsubstituted glucose unit, and consequently obstructs degradation. Lower rate of microbial degradation was detected for MC6, in
Table 3 Microbial oxidative degradation of cellulose derivatives Sample
Irradiation
Time [days]
CO2 [g]
Degradation [%]
Control (compost) Control (compost) Cellulose MC6 MC6 HEC8 HEC8
– – Unirradiated Unirradiated 20%, EB, 50 kGy Unirradiated 20%, EB, 10 kGy
30 20 30 30 30 20 20
0.90–0.93 0.59–0.61 13.4 2.0 2.4 6.5 5.32
– – 80 5.9 8.1 33.5 26.8
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
which every average anhydroglucose repeating unit was substituted directly in the number of 1.8 replacements. Such high DS hampers biodegradation. In the case of HEC, which is characterized by MS 2.0, its average DS might vary in the range 0.8–1.2 [38]. Hence, in HEC the access of microorganisms in the vicinity of glycosidic bond and the glucose ring is unrestrained in such an extent like for MC. 4. Conclusion Formation of hydrogels from polysaccharides of two popular kinds, MC and HEC was achieved by radiation-induced crosslinking. Hydrogels were chemically pure, since neither crosslinking agents nor monomers were used. It is a significant advantage of the product for applications in the biomedical field. The most effective gelation occurred by irradiation of highly concentrated aqueous solutions of the polymers. Swelling of gels in distilled water was the highest for low irradiation doses and the amount of imbibed solvent decreased with the increase of irradiation dose, but remains at about 20–40. Crosslinked material after use and disposal undergoes biodegradation by the action of naturally occurring microorganisms. It is the additional benefit of employing cellulose ethers as a material for hydrogel production by radiation technique. According to their good swelling properties in connection with biodegradability, these hydrogels may be useful as superabsorbent materials for agriculture as controlled release systems. Acknowledgements The authors thanks to Professor J.M. Rosiak from Institute of Applied Radiation Chemistry, Lodz, Poland, for his valuable comments and discussion of the results.
References [1] A. Charlesby, Atomic Radiation and Polymers, Pergamon Press, Oxford, 1960.
543
[2] D. Darwis, H. Mitomo, F. Yoshii, Polym. Degr. Stab. 65 (1998) 279. [3] J.M. Rosiak, P. Ulanski, L.A. Pajewski, F. Yoshii, K. Makuuchi, Radiat. Phys. Chem. 46 (1995) 161. [4] N.A. Peppas (Ed.), Hydrogels in Medicine and Pharmacy, Vols. I–III, CRC Press, Boca Raton, FL, 1986–1987. [5] E. Marsano, S. Gagliardi, F. Ghioni, E. Bianchi, Polymer 41 (2000) 7691. [6] F. Madsen, N.A. Peppas, Biomaterials 20 (2000) 1701. [7] H. Kaur, P.R. Chatterji, Macromolecules 23 (1990) 4868. [8] L. Relleve, F. Yoshii, A. dela Rosa, T. Kume, Angew. Makromol. Chem. 273 (1999) 63. [9] F. Esposito, M.A. del Nobile, G. Mensitieri, L. Nicolais, J. Appl. Polym. Sci. 60 (1996) 2403. [10] U. Anbergen, W. Oppermann, Polymer 31 (1990) 1854. [11] A. Charlesby, J. Polym. Sci. 15 (1995) 263. [12] C. von Sonntag, Adv. Carbohydr. Chem. Biochem. 37 (1980) 7. [13] B.G. Ershov, A.S. Klimentov, Rus. Chem. Rev. 53 (1984) 1195. [14] P. Ulanski, C. von Sonntag, J. Chem. Soc., Perkin Trans. 2000 (2) (2000) 2022. [15] F.C. Leavitt, J. Polym. Sci. 45 (1960) 536. [16] F.C. Leavitt, J. Polym. Sci. 51 (1961) 349. [17] Y. Ikada, T. Mita, F. Horii, I. Sakurada, M. Hatada, Radiat. Phys. Chem. 9 (1977) 633. [18] W.B. Neely, J. Polym. Sci., Part A 1 (1963) 311. [19] W. Brown, D. Henley, J. Ohman, Macromol. Chemie 63 (1963) 49. [20] Japanese Patent Application No. 09-293597 (1997), Publication No. JP, 11-11 35 95, A (1999). [21] F. Bin, R.A. Wach, H. Mitomo, F. Yoshii, T. Kume, J. Appl. Polym. Sci. 78 (2000) 278. [22] R.A. Wach, H. Mitomo, F. Yoshii, T. Kume, Macromol. Mater. Eng. 287 (2002) 285. [23] R.A Wach, H. Mitomo, F. Yoshii, T. Kume, J. Appl. Polym. Sci. 81 (2001) 3030. [24] R.A. Wach, Radiation-Induced Crosslinking of Cellulose Ethers in Water, Ph.D. Dissertation, Gunma University, Japan, 2000, Chapter 5, p. 107. [25] D.W. Clegg, A.A. Collyer (Eds.), Irradiation Effects on Polymers, Elsevier, London, NY, 1991. [26] P. Ulanski, E. Bothe, J.M. Rosiak, C. von Sonntag, Macromol. Chem. Phys. 195 (1994) 1443. [27] A. Chapiro, Radiation Chemistry of Polymeric Systems, John Wiley & Sons, NY, 1962, p. 1. [28] J.E. Wilson, Radiation Chemistry of Monomers, Polymers and Plastics, Marcel Dekker Inc., NY, 1974. [29] J.M. Rosiak, J. Olejniczak, A. Charlesby, Radiat. Phys. Chem. 32 (1988) 691. [30] J. Olejniczak, J.M. Rosiak, A. Charlesby, Radiat. Phys. Chem. 37 (1991) 499. [31] J.M. Rosiak, Radiat. Phys. Chem. 51 (1998) 13. [32] J. Simon, H.P. Muller, R. Koch, V. Muller, Polym. Degr. Stab. 59 (1998) 107. [33] H. Vink, Macromol. Chem. 94 (1966) 1.
544
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
[34] R.G.H. Siu, R.T. Darby, P.R. Burkholder, E.S. Barghoorn, Textile Res. 19 (1949) 484. [35] S. Ardica, E. Calderaro, C. Cappadona, Rad. Phys. Chem. 13 (1984) 719.
[36] S. Ardica, E. Calderaro, C. Cappadona, Rad. Phys. Chem. 26 (1985) 701. [37] E.T. Reese, Ind. Eng. Chem. 49 (1957) 89. [38] W. Brown, Ark. Kemi 18 (1961) 227.
Radiation crosslinking of methylcellulose and hydroxyethylcellulose in concentrated aqueous solutions Radoslaw A. Wach b
a,*
, Hiroshi Mitomo a, Naotsugu Nagasawa b, Fumio Yoshii
b
a Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu 376-8515, Japan Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki 370-1292, Japan
Received 24 February 2003; received in revised form 19 May 2003
Abstract The effects of ionizing radiation on aqueous solutions of cellulose ethers, methylcellulose (MC) and hydroxyethylcellulose (HEC) were investigated. The well-established knowledge states that cellulose and its derivatives belong to degrading type of polymers. However, in our study intermolecular crosslinking initiated by gamma rays or electron beam leaded to the formation of insoluble gel. This is an opposite effect of irradiation to the degradation. Paste-like form of the initial specimen, i.e. concentration 20–30%, when water plasticizes the bulk of polymer; and a high dose rate were favorable for hydrogel formation. Gel fraction up to 60% and 70% was obtained from solutions of HEC and MC, respectively. Produced hydrogels swell markedly in aqueous media by imbibing and holding the solvent. Radiation parameters of irradiation, such as yields of degradation and crosslinking and the gelation dose, were evaluated by sol–gel analysis on the basis of Charlesby–Rosiak equation. Despite of the crosslinked structure, obtained hydrogels can be included into the group of biodegradable materials. They undergo decomposition by the action of cellulase enzyme or microorganisms from compost. 2003 Elsevier B.V. All rights reserved. PACS: 61.25.H; 61.82.P; 81.05; 82.70.G Keywords: Hydrogels; Methylcellulose; Hydroxyethylcellulose; Crosslinking; Radiation yield; Biodegradation
1. Introduction Irradiation of polymeric materials generates some effects depending on the kind of polymer, parameters of irradiation, the state of the material under processing, etc. The two main reactions, which determine the final properties of the poly* Corresponding author. Present address: Nuclear Engineering Research Laboratory, The University of Tokyo, 2-22 Shirakata-Shirane, Tokai-mura, 319-1188 Ibaraki-ken, Japan. Tel.: +81-29-287-8984; fax: +81-29-287-8488. E-mail address: [email protected] (R.A. Wach).
mer, include (a) scission of main chain, leading to the diminishing of the molecular weight of macromolecules and (b) crosslinking, the opposite process ending with macroscopic, insoluble material [1]. The yields of scission and crosslinking, or more precisely the mutual ratio of these two parameters, determine the outcome. To achieve the desired aim, irradiation of polymeric material can be conducted under special conditions. Particularly, the state of material under processing is of a great significance. Thus, an elevated temperature, the polymer is in molten state, is often use [2]. Dose rate can be the variable; irradiation with EB
0168-583X/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-583X(03)01513-1
534
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
gives higher local concentration of radicals. Furthermore, irradiation of water-soluble polymers in aqueous solution, due to the high yield of radicals, is often use for formation of hydrogels [3]. Gamma or EB irradiation has been regarded as a very useful method in order to get macroscopic threedimensional able to swell lattice. Polymeric network, which exhibits the ability to absorb and retain a significant amount of water within its structure, but which does not dissolve is defined as hydrogel. One can regard hydrogel-like material possessing both liquid-like and solid-like properties. The former one results from the fact that the main constituent is water. Thus, penetration of solvent or low molecular weight solutes throughout the bulk of gel is possible. The later one is due to the difference in osmotic pressure (Donnan equilibrium), elastic strength of the network maintains the shape of gel but allows for increasing its dimensions [4]. Hydrophilicity of the polymer to be used to the formation of hydrogel is of great importance for the final properties of the product. To change hydrogel water uptake an addition of hydrophobic segments can be applicable as accompanying copolymer or grafted chain [5,6], or as in interpenetrating polymer network by incorporation of another polymer, which creates independent lattice in the matrix [7,8]. Crosslinking density, the average molecular mass between the two junction points, affects water absorption characteristic of the hydrogel. In the synthesis of gels by chemical method, i.e. by bi/ multifunctional monomers, crosslinking density is controlled by the concentration of this crosslinker, reaction time, temperature and others. While, for radiation method it is determined by the absorbed dose, which means by the irradiation time. Moreover, crosslinking by the chemical method can be performed only in liquid state, which is in distinct contrast with the later one. Since the ionizing radiation is highly penetrating, it is possible to initiate chemical reactions in liquid or in solid state. In cellulose, the most abundant renewable raw material, the presence of active hydroxyl groups has been utilized in a variety of chemical modifications to produce derivatives. Among them cellulose ethers are commercially important products due to
their superior properties with respect to the native material. Chemically crosslinked gels of hydroxyethylcellulose (HEC) with carboxymethylcellulose (CMC) have been synthesized by divinylsulphone crosslinker. The chemical composition and manufacturing procedures have the influence on equilibrium water content [9]. Swelling ability of products enhanced with an increase of CMC content in the blend. It has been proposed that the crosslinker reacted with the unsubstituted groups on the cellulose ethers [10]. Polysaccharides and their derivatives exposed to ionizing radiation, gamma rays or electron beam (EB) undergo degradation [11]. It is apparent that polymers of natural origin, irradiated in solid state suffer scission of acetal linkages in main chain. Some mechanisms of the processes have been proposed [12–14]. Even after the first successful attempts of Leavitt to crosslink cellulose ethers by ionizing radiation only a little interest of researchers in this subject had been reported [15,16]. Possibility of radiation-induced crosslinking of methylcellulose (MC) was mentioned by the occasion of hydrogel fabrication from synthetic polymers [17]. The authors achieved a swellable gel by EB irradiation of MC aqueous solution (10% w/w). The degree of swelling equilibrium of the resulted hydrogel was about 20 g of water per gram of polymer. The key factor for successful intermolecular bonding of MC and HEC, according to the results, was a high dose rate of electron radiation [16]. The aim of this study was to provide more systematic data concerning crosslinking of cellulose derivatives, MC and HEC in order to fill the gap in knowledge on radiation processing of cellulosics. In addition some results on hydrogels degradation by the action of enzyme and naturally occurring microorganisms are reported.
2. Experimental 2.1. Materials Ethers o cellulose: HEC obtained from Sumitomo Seika Co. Ltd. Japan and MC from Shin Etsu Co. Ltd. Japan were used in our experiments.
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544 Table 1 Characteristic of cellulose ethers Sample
Degree of substitution (DS)
Intrinsic viscosity (dL/g)
Weight-average molecular weight
MC5 MC6 HEC7 HEC8 HEC9
1.8 1.8 2.0 2.0 2.0
1.96 7.58 3.72 5.39 9.35
1.20 · 105 14.0 · 105 2.02 · 105 3.09 · 105 5.82 · 105
Scheme 1. Chemical structure of 2 repeating units of cellulose, MC and HEC connected by glycosidic linkage at 1 and 4 positions with b configuration.
Characteristic of these polymers is summarized in Table 1 and the structure is presented in Scheme 1. Average molecular weights were determined by measuring intrinsic viscosity, see below. All chemicals used were analytical grade. 2.2. Molecular weight of polymers Weight-average molecular weights of the polymers were determined from intrinsic viscosity on the basis of the Mark–Houwink equation ½g ¼ K DPa for HEC or ½g ¼ K Mwa for MC, where K and a are constants, DP is the weightaverage degree of polymerization and Mw is the weight-average molecular weight. The viscosity of polymer solution was measured by an Ubbelohde viscometer in water at 25 C. The intrinsic viscosity was found by plotting the obtained reduced viscosity gsp =c and lnðg=gsp Þ=c against concentration (in g dl1 ) and extrapolation to zero concentration.
535
For this polymers and measurement conditions constant K equals to 3.16 · 103 , 1.1 · 102 and a to 0.55, 0.87 for MC [18] and HEC [19], respectively. 2.3. Sample preparation and irradiation Polymer was mixed with an appropriate amount of deionized water and the material was kept for 7 days at room temperature to ensure complete dissolution and homogeneity of the specimen. Higher concentrated solutions had a tixotropic paste-like properties, while those of lower concentrations were extremely viscous however, exhibited slow gravity flow. Irradiation was conducted with gamma rays generated from 60 Co source at a dose rate of 10 or 1 kGy/h at inert temperature. For irradiation by high-energy electrons, the 2 MeV accelerator was used at the following irradiation parameters: current 1 mA, voltage 1 MeV and the dose per pass 10 kGy. Irradiation was carried out in polyethylene bag. Alternatively, for air-free irradiation, to avoid the penetration of additional oxygen during irradiation, specimens were sealed in poly(vinylidene chloride) bag after removal of the air by a vacuum pump. 2.4. Gel content and swelling properties of hydrogel Amount of gel content and swelling properties of resulted hydrogels were determined gravimetrically. Gel content in the dried hydrogels was estimated by measuring insoluble part after extraction in deionized water for 7 days at room temperature. Gel fraction was calculated as follows: Gel fraction ½% ¼ ðGd =Gi Þ 100;
ð1Þ
where Gi is the initial weight of polymer, Gd is the weight of the dried insoluble part after extraction. Hydrogels were swollen in deionized water, NaCl solution or buffers of certain pH value. An equilibrium water uptake was reached, when the mass of two consecutive weighing did not differ more than 0.5%. The surface of hydrogel was blotted by filtration paper prior to weighing. If Gs is the weight of hydrogel in swollen state, swelling is presented as follows:
536
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
Swelling ½grams of water=1 g of dried gel ¼ ðGs Gd Þ=Gd :
ð2Þ
2.5. Biodegradation Enzymatic degradation was carried out in an acetic acid–NaOH buffer, at pH 5.0, by cellulase enzyme, from penicillium funiculosum. About 10 mg of dried film of gel, with a thickness about 0.3 mm, was immersed in 2.5 ml of the enzyme solution for given time. The concentration of the cellulase enzyme was 0.1 mg ml1 . Tests were performed at the most appropriate temperature for the enzyme activity, 37 C, with shaking. After incubation, the samples were washed and kept in an excess of distilled water to wash away the degraded polymer then, dried at 35 C under vacuum. The result of enzymatic degradation is expressed as percentage of the initial sample weight: Degradation ð%Þ ¼ Ge =Gd 100%;
ð3Þ
where Ge and Gd are the weights of films after and before enzymatic degradation, respectively. Microbial degradability of MC and HEC in soil under controlled conditions was evaluated by measuring CO2 production. Especially designed apparatus – Microbial Oxidative Degradation Analyzer (MODA) [20], comprised of 4 independent lines of columns, was used. 10 grams of the sample was mixed with rinsed sea sand – 450 g and compost – 130 g and then placed in a heated reactor. Inside the column monitored temperature was 35 C and the flow in of the carbon dioxidefree but moisturized air was 30 ml min1 . After flowing through the sample inside the reactor, the air carrying formed due to polymer disintegration CO2 was passing series of columns filled in turn with silica gel, calcium chloride, soda lime and calcium chloride. Ammonia, which could be formed from the decomposing sample, was trapped in sulfuric acid solution, and water vapor was absorbed into first two columns (silica gel and calcium chloride). The CO2 was collected quantitatively by soda lime and water produced during the reaction was caught in the last CaCl2 column.
Thus, the mass of produced carbon dioxide was calculated as a difference in the weight of two last columns (containing soda lime and calcium chloride) at the beginning and during the test. Pure compost mixed with sea sand was used as a blank and cellulose powder as a reference sample.
3. Results and discussion 3.1. Radiation crosslinking Examined polymers exposed on c-rays in solid state and in aqueous solutions of low concentrations undergo degradation, in the atmosphere of air as well as in the absence of oxygen. The results of degradation are closely similar to those reported previously for another cellulose derivatives CMC and HPC thus, there is no reason to present them in the present paper [21,22]. It just has to be mentioned that the main reason of the low yield of the intermolecular recombination in diluted solutions of concentration less than 10% is caused by insufficient number of chains in close vicinity one to each other. In the solid state, extremely limited mobility of chain segments bearing radicals prevents their mutual recombination. In both cases scission is the predominant reaction leading to the diminishing of degree of polymerization of cellulose ethers. Irradiation of MC and HEC at moderate or high concentrations ends with the formation of insoluble gel by intermolecular crosslinking. The relation of gel fraction to irradiation dose of hydrogel produced from 20% HEC aqueous solution initiated by EB and gamma rays is presented in Fig. 1. For all samples processes by EB the gel fraction increases with absorbed dose and then stabilize at the maximum value. Gelation dose, at which first insoluble material appears, and the gel fraction depend on the initial molecular weight of HEC. Gel fraction reaches about 58%, 53% and 49% for HPC9, HPC8 and HPC7, respectively. Since crosslinking and scission takes place simultaneously, macromolecules of the longest chains are more prone to bond each other before they undergo substantial degradation. Even if scission undergoes with the same yield for every molecular
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544 60 HEC 9 HEC 8 HEC 7 HEC 9 HEC 8
Gel Fraction [%]
50 40 30 20 10 0
0
50
100
150
200
Dose [kGy] Fig. 1. Influence of irradiation dose on the resulted gel fraction of HEC hydrogels. Hydrogels formed by c-rays (solid points) and EB (open points) irradiation of HEC 20% aqueous solution in air-free atmosphere.
weight fraction, to reduce the size of the biggest molecules, it is necessary more scission events to occur than for the degradation of short chains. For HEC9 and HEC8 irradiated by gamma rays without access of oxygen at dose rate of 10 kGy/h the maximum gel fraction is significantly lower than that for EB irradiation. Moreover, the gel part of HEC8 reaches the maximum and then diminishes – apparently, for higher doses scission prevails over intermolecular crosslinking. In Fig. 2, there is presented data concerning MC. The distinction between the two molecular
weight samples irradiated by gamma rays is the most pronounced in the gelation dose and the value of gel fraction. The gel starts to arise at a dose lower than 10 kGy for MC6, while for MC5 gelation begins at the dose as high as 70 kGy. Irradiation by EB results in higher gel fraction than that by gamma rays. For MC6 it reaches about 60% in comparison to less than 40% obtained by gamma irradiation. During all irradiation process, up to 200 kGy, crosslinking dominates over cleavage of mainchain bonds, and no deterioration of the gel is observed. Influence of dose rate, irradiation at 1 kGy/h, and air atmosphere on the gel fraction is depicted in Fig. 3. Diminishing of the gel fraction takes place for HEC9 from 30% (10 kGy/h) to 20% (1 kGy/h). No apparent change in gelation of MC6 is observed as a function of the dose rate. MC resembles the behavior of another cellulose ether, CMC examined previously, for which also no evident diversification was found [23]. Probably, the lifetime of radicals plays an important role in the share of crosslinking – scission reactions. Radicals formed on MC macromolecules seem to be more stable than those on HEC chains and in consequence, there is a higher probability for their recombination with the other fitting macroradical, before their transformation with the rupture of mainchain. Radicals on MC and HEC was
60
70
Gel Fraction [%]
Gel Fraction [%]
60 50 40 30 20 MC6 MC5
10 0
0
20
40
60
80 100 120 140 160 180 200
537
1kGy/h MC 6 HEC 9 Air MC 6 MC 5
50 40 30 20 10 0
0
50
100
150
200
Dose [kGy]
Dose [kGy] Fig. 2. Influence of irradiation dose on the resulted gel fraction of MC hydrogels. Irradiation in air-free atmosphere by c-rays, MC 25% aqueous solution (solid points); and EB irradiation, MC 20% aqueous solution (open points).
Fig. 3. Influence of irradiation conditions (c-rays) on the resulted gel fraction of hydrogels. Irradiation of 20% aqueous solutions of MC6 and HEC9; dose rate – 1 kGy/h; air-free atmosphere. Irradiation of 25% MC aqueous solutions; dose rate – 10 kGy/h; air atmosphere.
538
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
investigated by ESR technique, described elsewhere [24]. Ionizing radiation in means of electromagnetic and charged-particle radiation interacts with matter by distinctly different physical processes, although the final overall effects – generation of a mixture of excited and ionized species – are qualitatively similar [25]. Beam of ionizing radiation, independently on its origin, causes the cascade of primary and later secondary electrons. Such electrons have the same character for all kinds of ionizing radiation. However, they may differ in their kinetic energy. The next steps of the attenuation are in turn: (a) formation of positive ions, excited molecules and thermal electrons, (b) formation of free radicals and (c) chemical effects. The number of created radicals is two orders of magnitude higher for EB than for gamma irradiation at dose rate 10 kGy/h. Thus, if the polymer concentration is high enough, much higher density of macroradicals exist in the same time, and intermolecular recombination is favored over chain scission. According to this, the dose rate has a significant influence on the gel fraction, and in general, on overall gelation process. Another important factor, which affects the hydrogel formation, is presence of oxygen in the solution during irradiation. In the above investigations the poly(vinylidene chloride) bags were used which, is assumed to protect against penetration of air into the bulk during irradiation. Similarly to irradiation without air the gel fraction of MC increases throughout the applied dose range, but is greatly reduced. It corresponds to the results obtained for CMC [21]. It is due to the reaction of oxygen with macroradicals, which transform to peroxy-macroradicals. Subsequently, formed unstable compounds decompose and affect the main chain of polymer causing rupture of glycosidic linkages [26]. The shear of scission reaction greatly increases. 3.2. Radiation yield of crosslinking and scission The main chemical effects on polymers subjected to ionizing irradiation are crosslinking and chain scission. Both processes occur simultaneously and their yields determine the final results
of irradiation [27]. If crosslinking predominates over scission, an insoluble macroscopic gel is formed. In such case, the sol–gel analysis allows to estimate parameters of the irradiation process [1]. The yield value (G) is a number of chemical events or molecules changed, e.g. crosslink bonds or scission acts, per 100 eV of absorbed energy [28]. Deviations of the initial molecular weight distribution of irradiated polymers from the random one can affect calculated yields values. Thus, in the present study, to avoid an inaccuracy resulting from unknown molecular weight distribution of used polymers the Charlesby–Rosiak equation (4) was used [29]. This equation allows for estimation of radiation parameters of linear polymers of any initial weight distribution as well as is applicable to systems when an initial material is monomer or branched polymer. pffiffi p0 p0 Dv þ Dg sþ s¼ þ 2 ; ð4Þ q0 q0 D v þ D GðxÞ ¼
4:8 105 ; M w0 Dg
GðsÞ=GðxÞ ¼ 2p0 =q0 :
ð5Þ ð6Þ
In the above equations s is the sol fraction, p0 is the degradation density, q0 is the crosslinking density, D is the absorbed dose, Dg is gelation dose and Dv is the virtual dose – a dose required to change the distribution of molecular weight of the certain polymer in such a way that the relation between weightaverage and number-average molecular weight would be equal to 2 (for detailed explanation see [30,31]). The data for examined polymers irradiated under selected conditions is listed in Table 2. According to calculated results, gelation dose (Dg ), a dose when the first insoluble gel appears, diminishes for higher molecular weight fractions of the same cellulose ether. Dg is significantly lower – gelation starts earlier – also for material irradiated by EB. Degradation to crosslinking density, p0 =q0 corresponds directly to the obtained gel fraction. If the parameter decreases, the crosslinking takes greater share; one can expect higher part of gel after irradiation. It is evident for the example of HEC9, for which Dg and p0 =q0 enhance greatly for gamma processing in contrast to EB irradiation.
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
539
Table 2 Radiation parameters of irradiated polymers, evaluated on the basis of sol–gel analysis No.
Sample/concentration
Irradiation and conditions a
Dgb [kGy]
p0 =q0 b
GðxÞb [1/100 eV]
GðsÞb [1/100 eV]
1 2 3 4 5 6 7 8 9 10 11
MC5/20% MC5/25% MC5/25% MC6/20% MC6/25% MC6/25% MC6/20% HEC7/20% HEC8/20% HEC9/20% HEC9/20%
EB c, vacuum c, air EB c, vacuum c, air c, vacuum (1 kGy/h) EB EB EB c, vacuum
41.3 76 148 4.5 7.1 24.2 6.6 21 9.6 3.56 9.1
0.98 1.02 1.85 0.80 0.96 1.38 1.02 1.00 1.08 1.07 1.54
0.01 0.05 0.03 0.08 0.05 0.014 0.05 0.11 0.16 0.23 0.09
0.19 0.11 0.1 0.12 0.09 0.04 0.11 0.22 0.35 0.5 0.28
The influence of oxygen during irradiation on the obtained results is apparent after comparison of p0 =q0 for MC5 and MC6 (compare rows 2 and 3; 5 and 6). The share of degradation increases considerably. Gelation dose doubles for samples irradiated in air. The comparison of gelation parameters indicates that gelation occurs more effectively by EB processing. No significant diverse between gamma irradiation of MC with the dose rates 1 and 10 kGy/h was observed, row 5 and 7. The gelation process of these two samples proceeds similarly with only a little advantage of maximum gel fraction of the irradiation at 10 kGy/h. 3.3. Swelling of hydrogels The main feature of hydrogel is its ability to absorb and hold in its structure an amount of solvent. Swelling, usually presented as weight of solvent absorbed per 1 g of dried gel, depends on the hydrophilicity of the polymer, density of intermolecular links i.e. molecular weight of chain part between crosslinks and others. The important factor is also a composition of a solvent if it is not distilled water. Presence of low molecular weight solutes, e.g. inorganic salt, as well as pH of the solvent can affect greatly the swelling of hydrogel. Fig. 4 shows swelling of HEC hydrogel made from 20% aqueous solution by EB as a function of
Swelling [g water / g dry gel]
p0 =q0 – degradation to crosslinking density, Dg – gelation dose, GðxÞ, GðsÞ – radiation yield of crosslinking and radiation yield of scission, respectively. a If not indicated, the dose rate of gamma radiation was 10 kGy/h. b Values calculated by Charlesby–Rosiak (4), and Eqs. (5), (6), indicated in text.
175 HEC 9 HEC 8 HEC 7
150 125 100 75 50 25 0
0
50
100
150
200
Dose [kGy] Fig. 4. Swelling in distilled water of hydrogels formed by EB irradiation of 20% HEC aqueous solutions.
absorbed dose. Obtained shape of swelling curves is common for gels formed by ionizing radiation. Swelling is the highest just after the dose oversteps gelation point. To form a gel, statistically one crosslink per chain is necessary to form an insoluble macroscopic gel [1]. Then, the network is weak and susceptible to break, but because of a relatively low number of intermolecular bonds, more solvent can penetrate inside the crosslinked polymer matrix. With continuous increasing of the density of crosslinks, due to further irradiation the absorption ability of the gel decreases, voids accessible for
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544 500
Swelling [g water / g dry gel]
water shrink. The structure of hydrogel becomes more firmly connected and rigid. For HEC swelling drops sharply with the dose and finally remains at the level of 20–40, for samples irradiated at 200 kGy. Obviously, higher initial molecular weight fraction is characterized by lower swelling after crosslinking. Changes in crosslinks density for crosslinking type polymers, are reflected by the extent to which the material is swollen by soaking in a compatible solvent – this is determined by comparison the weight of the solvent swelled material with its dry weight. During irradiation the density of crosslinks increases consequently leading to a decrease in the solvent absorption ability of hydrogel. When the scission prevails, opposite process occurs. The percentage of crosslinked polymer that is insoluble in the appropriate solvent may decrease. The process is presented schematically in Fig. 5. An increase of soluble fraction – sol – reflects degradation, whereas a reduced soluble fraction indicates crosslinking. Swelling of hydrogels formed in air-free atmosphere by c-rays at a dose rate of 1 kGy/h from 20% MC and HEC aqueous solutions is presented in Fig. 6. The amount of solvent imbibed by MC6 hydrogel decreases gradually with dose increase. It is in accordance with the fact that the gel fraction does not decrease with the dose. Thus, it can be concluded that throughout all irradiation process the reaction of intermolecular crosslinking overweighs over scission and the density of crosslinks increase or, at least does not reduce. The process stops at the B step from Fig. 5. Swelling of HEC9 reveals different characteristic in response to irradiation with a low dose rate, 1 kGy/h from that observed for MC6. It is apparent that HEC9 degrade slightly with continuous irradiation
HEC 9 MC 6
400 300 200 100 0
10
20
30
40
50
60
70
Dose [kGy] Fig. 6. Swelling of hydrogels formed by c-rays irradiation at low dose rate. Conditions: MC6 and HEC9 20% aqueous solution; 1 kGy/h; air-free atmosphere.
(compare Fig. 3). Breaking of chains inside the gel is directly reflected in swelling. Evidently, swelling starts to increase at higher doses, over 30 kGy. The C step from Fig. 5 is reached. Equilibrium swelling of hydrogels in NaCl aqueous solutions of various ionic strengths and of several pH values is presented in Fig. 7. Gels were prepared from 20% aqueous solutions in air-free
pH of the solution 40
Swelling [g water / g dry gel]
540
0
2
4
6
8
10
12
35 30 25 20 15 10 5 0.0
0.2
0.4
0.6
0.8
1.0
Ionic strength [M]
Fig. 5. Schematic representation of intermolecular crosslinking of polymer chains and their degradation with continuous irradiation.
Fig. 7. Equilibrium swelling of hydrogels in aqueous solutions of various ionic strength and pH. Gels were prepared from 20% aqueous solutions of HEC9 (solid points) and MC6 (open points) by EB irradiation in air-free atmosphere at 50 and 20 kGy, respectively.
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
3.4. Biodegradation of hydrogels Biodegradable polymers can be converted, to carbon dioxide and water as end products, with releasing of energy. Degradation is a desirable feature of hydrogels utilized in medicine, agriculture and other fields. However, most hydrogels with high water absorption capacity prepared from synthetic polymers are tough to decompose naturally. It is well known that cellulose and its derivatives disintegrate in soil and under controlled
100
80
Degradation [%]
atmosphere by an EB at doses 50 kGy (HEC9) or 20 kGy (MC6). HEC9 hydrogel reveals continuous deswelling with the increase of solutions pH from 38 at pH 1.7 to about 30 at pH 7, and then the gel volume increases slightly at basic pH. The solution uptake is independent on the presence of NaCl at various ionic strengths. Hydrogel of MC6 presents similar character of swelling as a function of solution pH, but much less pronounced. On the contrary, the uptake decreases with an increase in ionic strength. This diverse swelling manner, despite that both polymers are nonionic, results from different hydrophilic/hydrophobic character of their functional groups. MC is more hydrophobic in nature than HEC because of its pendant methylene groups. MC swelling is less responsible to the pH, but displays dependence on the ionic strength. This could be due to the fact that salt competes with the polymer for water molecules, which cause easier hydrophobic aggregation of chains [18]. As a hydrophobic and nonionic gel, MC is hardly affected by changes of pH value of the solution. HEC provides numerous sites for hydrogen bonding, thereby facilitating the formation of highly organized structure consisting of a layer of water molecules around the polymer backbone and the ethylene glycol side chains. Obviously these hydrogen bonding occur in neutral pH, resulting in contraction of the gel. In acidic and basic media the presence of hydrogen cations or hydroxyl anions is evidently conducive to higher swelling. It might be due to disruption of hydrogen bonds structure and, for low pH values, formation of –OHþ 2 ions, which induces osmotic swelling pressure and imbibes the solution.
541
HEC 20 kGy HEC 50 kGy HEC 100 kGy MC 20 kGy MC 50 kGy
60
40
20
0
0
20
40
60
80
Time [h] Fig. 8. Biodegradation of hydrogels by the cellulase enzyme C0901 from P. funiculosum in an acetic acid – NaOH buffer (pH 5.0). Hydrogels were prepared by EB irradiation of 20% MC6 and HEC8 aqueous solutions in air-free atmosphere. Degradation of the hydrogels did not exceed 5% for blank tests (without the enzyme).
conditions by using enzymes or by means of acidic hydrolysis by cleavage of glycosidic linkages [32,33]. Therefore, especially suitable for those purposes are cellulose ethers. Results of degradation tests carried out by cellulase enzyme, in acetic acid–NaOH buffer of pH 5.0, are presented in Fig. 8. Hydrogels were prepared by EB irradiation of MC or HEC in 20% aqueous solution and only gel part, after extraction of sol, was subjected to the enzymatic hydrolysis. Blank test in buffer solution without the enzyme diminished the mass of each gel by less than 5%. Taking into consideration the applied dose of irradiation, a tendency of decomposition of the both polymers is identical. Hydrogel of lower crosslinking density degrades faster than that crosslinked more tightly at higher dose. It is because of greater number of intermolecular bonds in the latter. MC6 and HEC8 formed at 20 kGy posses relatively high gel fraction, 60% and 30%, respectively. However, they degrade completely at less than 24 of incubation. Those irradiated to 50 kGy become fully soluble before 72 h of enzymolysis. For HEC sample irradiated at 100 kGy the degradation is 15% (85% of remained weight) after 72 h of incubation. Similar rate of enzymatic degradation was achieved for MC and HEC samples instead of that
542
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
MC6 had the initial molecular weight more that four times higher that HEC8. Obviously, size and the number of substituent group as well as its kind played the crucial role in the degradation mechanism. The higher DS hinders biodegradation [34]. Access of the enzyme in to the proximity of glycosidic bond was not hampered by a spatial hindrance of such small group as –CH3 , but it was balanced by a relatively high DS, 1.8. Thus, the degradation rate of these two hydrogels (MC and HEC) is comparable. Microbial degradability was evaluated by assessment of carbon dioxide produced from the decomposition of the cellulosic material. Samples of MC6 and HEC8, also cellulose as a reference material, were investigated and the results are presented in Table 3. Cellulose undergoes the fastest degradation, which reaches 80% after one month (after subtraction of the carbon dioxide produced in blank test, the sample comprised of only compost). Cellulose ethers decompose much slower. Radiation processing of MC polymer improves its susceptibility for bacterial digestion comparing to initial MC powder. MC6 irradiated in 20% at 50 kGy yields faster decomposition in spite of a number of introduced crosslinks. The gel fraction of the tested hydrogels was about 65%. It was reported that treatment by ionizing radiation of cellulose before its enzymatic hydrolysis induces faster degradation [35]. At the dose as low as 10 kGy, HEC8 irradiated in 20% aqueous solution does not form a rigid gel. After placing the gel into excess of water, the system had a form of viscous slurry without any specific shape. Apparently, the gelation process has started, but scission has not
occurred. This restrained susceptibility to biodegradation. It was reported that enzymatic hydrolysis of cellulose of various origin yields glucose, which is a measure of degradation, in smaller amount after radiation treatment at low doses than without irradiation or after irradiation at high dose [36]. Comparing the data collected by the two methods it can be noticed that the degradation rate of individual material by enzymatic test and in composted soil varies. It may be due to the difference in the entity to be determined. In the case of enzymolysis, a decreasing weight of the insoluble fraction was measured. Thus, one scission event of the end linked chain or two places of degradation in the chain between the two closest joint points resulted in significant mass reduction of the specimen. On the other hand, for CO2 evolution a total disintegration of molecules is essential. In the former case, cellulase enzyme was employed but degradation in composted soil engaged number of natural microorganisms, which activities toward certain cellulose ether could differ. It was proposed that the cleavage of main chain bonds is the fastest between two adjacent unsubstituted anhydroglucose units [37]. The lower probability of an enzyme attack and slower degradation rate was postulated to occur next to a single, isolated unsubstituted ring. Possibly, degradation also takes place at linkages involving single, unsubstituted unit at one end of the chain. Thus, the higher DS reduces the probability for the presence of unsubstituted glucose unit, and consequently obstructs degradation. Lower rate of microbial degradation was detected for MC6, in
Table 3 Microbial oxidative degradation of cellulose derivatives Sample
Irradiation
Time [days]
CO2 [g]
Degradation [%]
Control (compost) Control (compost) Cellulose MC6 MC6 HEC8 HEC8
– – Unirradiated Unirradiated 20%, EB, 50 kGy Unirradiated 20%, EB, 10 kGy
30 20 30 30 30 20 20
0.90–0.93 0.59–0.61 13.4 2.0 2.4 6.5 5.32
– – 80 5.9 8.1 33.5 26.8
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
which every average anhydroglucose repeating unit was substituted directly in the number of 1.8 replacements. Such high DS hampers biodegradation. In the case of HEC, which is characterized by MS 2.0, its average DS might vary in the range 0.8–1.2 [38]. Hence, in HEC the access of microorganisms in the vicinity of glycosidic bond and the glucose ring is unrestrained in such an extent like for MC. 4. Conclusion Formation of hydrogels from polysaccharides of two popular kinds, MC and HEC was achieved by radiation-induced crosslinking. Hydrogels were chemically pure, since neither crosslinking agents nor monomers were used. It is a significant advantage of the product for applications in the biomedical field. The most effective gelation occurred by irradiation of highly concentrated aqueous solutions of the polymers. Swelling of gels in distilled water was the highest for low irradiation doses and the amount of imbibed solvent decreased with the increase of irradiation dose, but remains at about 20–40. Crosslinked material after use and disposal undergoes biodegradation by the action of naturally occurring microorganisms. It is the additional benefit of employing cellulose ethers as a material for hydrogel production by radiation technique. According to their good swelling properties in connection with biodegradability, these hydrogels may be useful as superabsorbent materials for agriculture as controlled release systems. Acknowledgements The authors thanks to Professor J.M. Rosiak from Institute of Applied Radiation Chemistry, Lodz, Poland, for his valuable comments and discussion of the results.
References [1] A. Charlesby, Atomic Radiation and Polymers, Pergamon Press, Oxford, 1960.
543
[2] D. Darwis, H. Mitomo, F. Yoshii, Polym. Degr. Stab. 65 (1998) 279. [3] J.M. Rosiak, P. Ulanski, L.A. Pajewski, F. Yoshii, K. Makuuchi, Radiat. Phys. Chem. 46 (1995) 161. [4] N.A. Peppas (Ed.), Hydrogels in Medicine and Pharmacy, Vols. I–III, CRC Press, Boca Raton, FL, 1986–1987. [5] E. Marsano, S. Gagliardi, F. Ghioni, E. Bianchi, Polymer 41 (2000) 7691. [6] F. Madsen, N.A. Peppas, Biomaterials 20 (2000) 1701. [7] H. Kaur, P.R. Chatterji, Macromolecules 23 (1990) 4868. [8] L. Relleve, F. Yoshii, A. dela Rosa, T. Kume, Angew. Makromol. Chem. 273 (1999) 63. [9] F. Esposito, M.A. del Nobile, G. Mensitieri, L. Nicolais, J. Appl. Polym. Sci. 60 (1996) 2403. [10] U. Anbergen, W. Oppermann, Polymer 31 (1990) 1854. [11] A. Charlesby, J. Polym. Sci. 15 (1995) 263. [12] C. von Sonntag, Adv. Carbohydr. Chem. Biochem. 37 (1980) 7. [13] B.G. Ershov, A.S. Klimentov, Rus. Chem. Rev. 53 (1984) 1195. [14] P. Ulanski, C. von Sonntag, J. Chem. Soc., Perkin Trans. 2000 (2) (2000) 2022. [15] F.C. Leavitt, J. Polym. Sci. 45 (1960) 536. [16] F.C. Leavitt, J. Polym. Sci. 51 (1961) 349. [17] Y. Ikada, T. Mita, F. Horii, I. Sakurada, M. Hatada, Radiat. Phys. Chem. 9 (1977) 633. [18] W.B. Neely, J. Polym. Sci., Part A 1 (1963) 311. [19] W. Brown, D. Henley, J. Ohman, Macromol. Chemie 63 (1963) 49. [20] Japanese Patent Application No. 09-293597 (1997), Publication No. JP, 11-11 35 95, A (1999). [21] F. Bin, R.A. Wach, H. Mitomo, F. Yoshii, T. Kume, J. Appl. Polym. Sci. 78 (2000) 278. [22] R.A. Wach, H. Mitomo, F. Yoshii, T. Kume, Macromol. Mater. Eng. 287 (2002) 285. [23] R.A Wach, H. Mitomo, F. Yoshii, T. Kume, J. Appl. Polym. Sci. 81 (2001) 3030. [24] R.A. Wach, Radiation-Induced Crosslinking of Cellulose Ethers in Water, Ph.D. Dissertation, Gunma University, Japan, 2000, Chapter 5, p. 107. [25] D.W. Clegg, A.A. Collyer (Eds.), Irradiation Effects on Polymers, Elsevier, London, NY, 1991. [26] P. Ulanski, E. Bothe, J.M. Rosiak, C. von Sonntag, Macromol. Chem. Phys. 195 (1994) 1443. [27] A. Chapiro, Radiation Chemistry of Polymeric Systems, John Wiley & Sons, NY, 1962, p. 1. [28] J.E. Wilson, Radiation Chemistry of Monomers, Polymers and Plastics, Marcel Dekker Inc., NY, 1974. [29] J.M. Rosiak, J. Olejniczak, A. Charlesby, Radiat. Phys. Chem. 32 (1988) 691. [30] J. Olejniczak, J.M. Rosiak, A. Charlesby, Radiat. Phys. Chem. 37 (1991) 499. [31] J.M. Rosiak, Radiat. Phys. Chem. 51 (1998) 13. [32] J. Simon, H.P. Muller, R. Koch, V. Muller, Polym. Degr. Stab. 59 (1998) 107. [33] H. Vink, Macromol. Chem. 94 (1966) 1.
544
R.A. Wach et al. / Nucl. Instr. and Meth. in Phys. Res. B 211 (2003) 533–544
[34] R.G.H. Siu, R.T. Darby, P.R. Burkholder, E.S. Barghoorn, Textile Res. 19 (1949) 484. [35] S. Ardica, E. Calderaro, C. Cappadona, Rad. Phys. Chem. 13 (1984) 719.
[36] S. Ardica, E. Calderaro, C. Cappadona, Rad. Phys. Chem. 26 (1985) 701. [37] E.T. Reese, Ind. Eng. Chem. 49 (1957) 89. [38] W. Brown, Ark. Kemi 18 (1961) 227.