Biomacromolecules 2004, 5, 458-462
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Laser Photolysis of Carboxymethylated Chitin Derivatives in Aqueous Solution. Part 2. Reaction of OH• and SO4•- Radicals with Carboxymethylated Chitin Derivatives Maolin Zhai,*,† Hisaaki Kudoh,† Radoslaw A Wach,† Guozhong Wu,†,‡ Mingzhang Lin,† Yusa Muroya,† Yosuke Katsumura,† Long Zhao,§ Naotsugu Nagasawa,§ and Fumio Yoshii§ Nuclear Engineering Research Laboratory, School of Engineering, University of Tokyo, Shirakata-Shirane 2-22, Tokai-mura, Naka-gun, Ibaraki-ken, 319-1188 Japan, and Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Watanuki-machi, Takasaki-shi, Gunma-ken, 370-1292 Japan Received August 1, 2003; Revised Manuscript Received November 19, 2003
The reactions of OH• or SO4•- radicals with carboxymethyl chitin (CM-chitin) and its deacetylated product, carboxymethyl chitosan (CM-chitosan), were investigated in aqueous solutions using a laser photolysis technique. The rate constants of the reactions of OH• and SO4•- radicals with CM-chitosan are always higher than those for CM-chitin, indicating that the amino-group could increase the reactivity of carboxymethylated chitin derivatives. The rate of the reactions of CM-chitin and CM-chitosan with OH• radical was found to decrease at lower pH when polymers chains tend to the coiled conformation. In comparison, the rate constant of the reaction of SO4•- radicals with CM-chitin or CM-chitosan decreased with pH, indicating that CM-chitin or CM-chitosan has a higher reactivity with the SO4•- radical at low pH due to the protonation of the amino group. 1. Introduction In our previous report (part 1), the photochemistry of carboxymethylated chitin derivatives, i.e., CM-chitin and CM-chitosan, was studied by the laser photolysis method.1 eaq - and macroradicals have been observed. The formation and decay of eaq - were affected by the presence of carbonyl group (affinity for electron) on CM-chitin or CM-chitosan. Some investigators also have found that CM-chitin and CMchitosan, with amino-, acetamido-, and carboxyl-substituted groups, have obvious different reactivities.2,3 In this part, OH• and SO4•- radicals are selected to further investigate the influence of substituted group on the reactivity of carboxymethylated chitin derivatives. In the photolysis experiment, hydrogen peroxide and persulfate are used frequently as the source of OH• and SO4•radicals to study their reactions with target molecules.4-7 Furthermore, degradation of chitosan by OH• or SO4•produced by thermal dissociation of hydrogen peroxide or potassium persulfate has been also reported.8,9 It was shown that hydrogen peroxide or potassium persulfate could effectively degrade chitosan macromolecules. The mechanism of chitosan degradation by potassium persulfate was explained. SO4•- radicals are attracted to the cationic amino group in the chitosan ring. Subsequently, SO4•- radicals * To whom correspondence should be addressed. Present address: College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 People’s Republic of China. Fax: (+86) 10-62759191. E-mail:
[email protected]. † University of Tokyo. ‡ Present address: Applied Radiation Center, Shanghai Institute of Applied Physics, P.O. Box 800-204, Shanghai 201800 China. § Japan Atomic Energy Research Institute.
attack the C-4 carbon and transfer the radical to the C-4 carbon by subtracting H from it. The presence of a free radical at the C-4 carbon eventually results in the breakage of the glycosidic C-O-C bond in the chitosan main chain. The mechanism of chitosan degradation by hydrogen peroxide was not discussed in detail, but the degradation should occur in a similar manner. A study on the reactions of OH• and SO4•- radicals with carboxymethylated chitin derivatives is very important for both theoretical and practical purposes. 2. Experimental Section Both of CM-chitin and CM-chitosan used in this study were obtained from Koyou Chemical Industrial Co., Ltd., Japan. Their characteristics have been described in part 1.1 Other chemicals were analytical reagents and the solutions were made with Millipore water. The pH was adjusted by adding HClO4 or NaOH. High purity argon was used to remove oxygen if necessary. To determine the rate constants of the reaction of OH• and SO4•- radicals with CM-chitin or CM-chitosan, H2O2 and K2S2O8 were used to generate OH• and SO4•- radicals. The equipment of laser photolysis was described in detail in a previous paper.7 A 248 nm (KrF) excimer laser was used with the maximum energy of 330 mJ per 20 ns pulse. The analyzing light was perpendicular with the laser beam, and the optical length was 15 mm. The batch sample was supplied to the optical cell. 3. Results and Discussion 3.1. Reaction of the OH• Radical with CM-Chitin or CM-Chitosan. When CM-chitosan dilute aqueous solutions
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Figure 1. Transient absorption spectra observed (7 µs) in laser photolysis of 10 mM CM-chitosan solution with or without H2O2 saturated with argon at pH 9.8 (100 mJ/pulse).
containing H2O2 are subjected to a 248 nm laser pulse, the energy is absorbed by CM-chitosan and H2O2. By comparing the optical density data of CM-chitosan with that of H2O2 in the ground state at the same concentration, the later absorbs 75% of the energy deposited on the sample. Therefore, in a solution of 10 mM CM-chitosan containing 5 mM H2O2, the share of the absorbed energy is 45/55 for CM-chitosan/H2O2. The eaq- from photoionization is transformed to the OH• radical by reacting with H2O2 (1). Photodissociation of H2O2 forms directly the OH• radical (2).4 OH• is a strong oxidizing radical that can abstract a H atom from CM-chitin or CM-chitosan, leading to the formation of macroradicals (3) eaq- + H2O2 f •OH + OHhV
H2O2 98 OH• + OH•
(1) (2)
OH• + CM-chitin(CM-chitosan) f •
CM-chitin(CM-chitosan) + H2O (3)
The absorption band of CM-chitosan macroradicals formed by H abstraction is shown in Figure 1. It is similar to that formed by direct irradiation of CM-chitosan or CM-chitin, but the signal is greatly enhanced due to the high OH• yield. 3.1.1. Rate Constant of the Reaction of the OH• Radical with CM-Chitin. Because the OH• radical has no absorption in the visible and near-UV regions, the rate constant of its reaction with CM-chitin was determined by the competition kinetics using KSCN as competitor scavenger. SCN - can be oxidized by the OH• radical to form (SCN)2•- that has a strong absorption at 400-500 nm. It is possible to obtain k4 by following the change of (SCN)2•- absorption with the [CM-chitin]/[SCN-] ratio based on the eq 6 SCN-
OH• + SCN- 98 OH- + (SCN)2•-
(4)
OH• + CM-chitin f •CM-chitin + H2O
(5)
A0/A ) 1 + {k4[CM-chitin]/k3[SCN-]}
(6)
where A0 and A are the transient absorbance of (SCN)2•- at 472 nm in the absence and presence of a polymer, respectively. The concentrations of H2O2 and KSCN were fixed at
Figure 2. Absorption ratio of (SCN)2•- transient in the absence and presence of CM-chitin (A0/A) as a function of the ratio of CM-chitin to SCN- concentration. [KSCN] ) 1.7 mM; [H2O2] ) 8 mM; laser energy, 100 mJ/pulse; Ar-saturated.
8 mM and 1.7 mM, respectively. A was measured by varying the concentration of CM-chitin. Although direct photoionization of SCN- by a 248 nm laser pulse also produces (SCN)2•- in N2O-saturated solution, its contribution (less than 3%) can be neglected in the H2O2-KSCN system. Figure 2 shows a plot of (A0/A) against [CM-chitin]/[KSCN]. As is seen, the line shows a good linear relationship. The k4/k3 value is obtained from the slope of the line, and by substituting k3 of 1.1 × 1010 M-1 s-1,10 k4 is evaluated to be (9.9 ( 0.2) × 108 M-1 s-1. 3.1.2. pH Dependence. CM-chitin is a polyampholyte, contains amino-, acetamido-, and carboxyl- groups, and the pH value of the original 10 mM CM-chitin aqueous solution is 8.9. With the change of pH, there is a change in the conformation of the CM-chitin macromolecular chain. Following the concept of conformation, one expects a rather strong pH dependence of the rate constant of reactions of the polymer with OH• radicals. At an approximate pH of 1.7 ∼ 4.0, most of the ionic groups are absent due to protonation of the carboxyl group and deprotonation of the amino group. The macromolecule in the undissociated state prefers a highly coiled conformation. In the case of low pH ( 4.00), the amino group is protonated or the carboxyl group is unprotonated, respectively. The chains in the dissociated state stretch and form a rodlike conformation (Figure 3). The change of the swelling behavior of CM-chitin gels with pH is shown in Figure 4,3 and it is very obvious that the swelling ratio of CM-chitin gel changes with pH, indicating that the macromolecular chain has different conformations with pH. The rate constant of the reaction of the OH• radical with CM-chitin is shown in Figure 5 and Table 1. The rate constant of the reaction of the OH• radical with CM-chitin exhibits a pH dependence (Figure 5, bars), whereas the absorbance of (SCN)2•- formed in the KSCN + H2O2 system without CM-chitin is pH-independent at a pH range of 1-10. When the pH is higher than 10, the decrease in absorbance is due to the decrease of the effective OH• yield by the reaction OH - + OH• f H2O + O•-. The rate constant is the lowest at pH ) 4.4, near the isoelectric point of CM-
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Zhai et al. Table 1. Rate Constant of the Reaction of the OH• Radical with Carboxymethylated Chitin Derivatives
k (108 M-1s-1)
pH ) 1.0
pH ) 4.4
pH ) 8.1
pH ) 9.8
CM-chitin CM-chitosan
6.0 9.0
5.0
9.9 13.2
9.7 12.9
Figure 3. Schematic illustration of the change of CM-chitin structure with pH.
Figure 6. Schematic illustration of the change of CM-chitosan structure with pH.
Figure 4. (A) Change of swelling behavior of the CM-chitin hydrogels (prepared from 30% aqueous solution at 50 kGy) with pH. (B) Change of swelling behavior of the CM-chitosan hydrogels (prepared from 30% aqueous solution at 75 kGy) with pH.
Figure 5. Bars: Change of rate constant of the CM-chitin + OH• reaction with pH; Curve: the influence of pH on the absorbance of (SCN)2•- formed in the KSCN + H2O2 solution under laser photolysis (without CM-chitin)
chitin. At this conditions, uncharged CM-chitin chains shrink to the coiled state, and the diffusion of OH• radicals through
the solution parts unfilled with polymer is the cause of the rate constant decreasing. The rate constant at pH 1.0 and at pH 8.1 or 9.8 is higher than that at pH 4.4 due to extended conformation of macromolecules, which fill the volume of solution more uniformly than if they are coiled. In the study of the reaction of the OH• radical with poly(acrylic acid), pH dependence of the rate constant has been also observed.11 3.1.3. Rate Constant of the Reaction of the OH• Radical with CM-Chitosan, Effect of the Amino Group. CM-chitosan has a higher degree of deacetylation than CM-chitin. The pH value of the original 10 mM CM-chitosan aqueous solution is 9.8, and there is a difference in the macromolecular structure (higher content of amino- group) and pH dependence of the conformation (Figure 6). Rate constants of the reaction of OH• radical with CM-chitosan at different pH are listed in Table 1. At the same pH, the rate constant of the reaction of the OH• radical with CM-chitosan is higher than that with CM-chitin; that is, it increases with the content of the amino group in chitin derivatives. This is because CMchitosan with a higher amino-group content has a highly extended conformation of macromolecular chain. From Figure 4, it can be found that the swelling ratio of CM-chitosan gels is higher than that of CM-chitin, which indicates that the CM-chitosan macromolecular conformation is more extended, supporting our experimental results. The rate constant of reaction of the OH• radical with CMchitosan in acidic conditions is slightly higher than that with chitosan [Mw ) 4.0 × 105 Da, degree of deacetylation 90.5%, pH ∼ 3, k ) 6.4 × 108 M-1 s-1] reported by Ulanski and von Sonntag who used thymine as the competing OH• scavenger in pulse radiolysis.12 The difference may be due to a lower molecular weight or a different substituted group of CM-chitosan. However, these authors found the value determined using SCN- as the competitor is twice as high as that obtained with the neutral scavenger and they attributed
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Photolysis of CM-Chitin Derivatives. Part 2
Figure 7. Plot of the first-order decay rate constant of SO4•- against CM-chitin concentration after laser pulse of CM-chitin + 5 mM K2S2O8 solution (pH ) 8.1, 80 mJ/pulse)
this difference to the accumulation of SCN- ions in the vicinity of chitosan chains, which leads to a drop in the bulk SCN- concentration. CM-chitosan contains a negatively charged carboxyl group, so the accumulation of SCN- ions in the vicinity of CM-chitosan chains should be very low. From Table 1, it can be found that the rate constant of the reaction of CM-chitosan with the OH• radical at lower pH (amino group is protonated) is smaller. It suggests also that the accumulation of SCN- ions in the vicinity of CMchitosan chains in acid condition (amino group is protonated) is low as well. Therefore, the influence of the KSCN accumulation could be neglected. Since photoionization of thymine may interfere with the results, this scavenger was not used in this work. 3.2. Reaction of the SO4•- Radical with CM-Chitin or CM-Chitosan. Persulfate ions as the photosensitizer are used frequently in photochemistry to study the reactivity of chemicals.5-7 K2S2O8 is irradiated by UV light to form SO4•radical (7). Inversely to the OH• radical, the SO4•- radical is an anion radical, which can also abstract H atoms from CMchitin or CM-chitosan, leading to the formation of macroradicals (8). Reaction of the SO4•- radical with CM-chitin or CM-chitosan will be studied below hυ
S2O82- 98 2SO4•-
(7)
SO4•- + CM-chitin(CM-chitosan) f •
CM-chitin(CM-chitosan) + SO42- + H+ (8)
3.2.1. Rate Constant of the Reaction of the SO4•- Radical with CM-Chitin. Because the SO4•- radical has a strong absorption band with its maximum at 455 nm, the rate constant of the oxidation of CM-chitin or CM-chitosan by SO4•- was measured by a direct following of the decay of SO4•- at 455 nm. A plot of the pseudo-first-order decay constants as a function of CM-chitin concentration gives a straight line (Figure 7). The rate constant of the SO4•- + CM-chitin reaction was calculated from the slope, k ) (4.2 ( 0.1) × 107 M-1 s-1.
Figure 8. Change of the rate constant with pH. Bars: CM-chitin and SO4•- radical reaction; curve: the recombination of SO4•- radical in the solutions without CM-chitin. Table 2. Rate Constant of the Reaction of the SO4•- Radical with Carboxymethylated Chitin Derivatives
k (107 M-1 s-1)
pH ) 1.0
pH ) 4.4
pH ) 8.1
pH ) 9.8
CM-chitin CM-chitosan
9.1 12.1
3.5
4.2 9.5
3.9 6.8
3.2.2. pH Dependence. The rate constants of the SO4•- + CM-chitin reaction at different pH are shown in Figure 8 and Table 2. The rate constant of the reaction of CM-chitin and SO4•- exhibits a pH dependence (Figure 8, bars), whereas the decay of SO4•- for the solution without CM-chitin is pHindependent, (3.0 ( 0.1) × 109 M-1 s-1 from pH 1 to 10 (Figure 8, curve). The change of the rate constant is attributed mainly to the change of CM-chitin molecular structure with pH. At pH 1, the value is (9.1 ( 0.1) × 107 M-1 s-1, and it decreases to (3.5 ( 0.1) × 107 M-1 s-1 at pH 4.4. The value increases slightly (3.9 ( 0.1) × 107 M-1 s-1 at pH 9.8. This change is consistent with the state of NH2 and carboxyl groups. At pH 1.0, the amino group is protonated (NH3+), whereas at pH 9.8, the dominant charge bears the unprotonated carboxyl group. At pH 4.4, near the isoelectric point of CM-chitin, most of the ionic groups disappear, and the CM-chitin chains shrink to the coiled state. In the experiment of chitosan thermal degradation by potassium persulfate, it was considered that SO4•- radicals are attracted to the cationic amino group in the chitosan ring. Subsequently, SO4•- radicals attack the C-4 carbon and transfer the radical to the C-4 carbon by subtracting H from it. The presence of a free radical at the C-4 carbon eventually results in the breakage of the glycosidic C-O-C bond in the chitosan main chain.9 3.2.3. Rate Constant of the Reaction of the SO4•- Radical with CM-Chitosan, Effect of the Amino Group. The rate constants of the reaction of the SO4•- radical with CMchitosan at different pH are listed in Table 2. The pH dependence is like that in the reaction of the OH• radical with chitin derivatives. At a certain pH, the rate constant of the reaction of the SO4•- radical with CM-chitosan is higher than that with CM-chitin; that is, the rate constant of the reaction of the SO4•- radical with polymer increases with the content of amino-group in chitin derivatives.
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4. Conclusions The influence of the substituted group on the reactivity of carboxymethylated chitin derivatives was investigated by reacting them with OH• and SO4•- radicals. The amino group could enhance the reactivity of chitin derivatives with OH• and SO4•- radicals. The change of pH has a different influence on the reactivity of carboxymethylated chitin derivatives with OH• and SO4•- radicals. The reactivity of the OH• radical is lower at low pH due to coiled conformation of chains, but the reactivity of the SO4•- radical is higher at low pH due to the protonation of the amino group. References and Notes (1) Zhai, M. L.; Kudoh, H.; Wu, G. Z.; Wach, R. A.; Muroya, Y.; Katsumura, Y.; Nagasawa, N.; Zhao, L.; Yoshii, F. Biomacromolecules 2003, 5, 453.
Zhai et al. (2) Nordtveit, R. J.; Varum, K. M.; Grasdalen, H.; Tokura, S.; Smidsod, O. Carbohydr. Polym. 1997, 34, 131. (3) Zhao, L.; Mitomo, H.; Nagasawa, N.; Yoshii, F.; Kume, T. Carbohydr. Polym. 2003, 51 (2), 169. (4) Pignatello, J. J.; Liu, D.; Huston, P. EnViron. Sci. Technol. 1999, 33, 1832. (5) Ivanov, K. L.; Glebov, E. M.; Plyusnin, V. F.; Ivanov, Y. V.; Grivin, V. P.; Bazhin, N. M. J. Photochem. Photobio. A 2000, 133, 99. (6) George, C.; Chovelon, J. M. Chemosphere 2002, 47, 385. (7) Zuo, Z. H.; Katsumura, Y.; Ueda, K.; Ishigure, K. J. Chem. Soc., Faraday Trans. 1997, 93 (4), 533. (8) Qin, C. Q.; Du, Y. M.; Xiao, L. Polym. Degrad. Stab. 2002, 76, 211. (9) Hsu, S. C.; Don, T. M.; Chiu, W. Y. Polym. Degrad. Stab. 2002, 75, 73 (10) Elliot, A. J.; Simsons, A. S. Radiat. Phys. Chem. 1984, 24 (2), 229. (11) Behzadi, A.; Chnabel, W. Macromolecules 1973, 6 (6), 824. (12) Ulanski, P.; von Sonntag, C. J. Chem. Soc., Perkin Trans. 2, 2002, 2022.
BM0342739
