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1. IntroductionChitosan is a natural, non-toxic, biodegradable copolymer of (1,4-β-)-2-amino-2-deoxy-D-glucopiranose and (1,4-β-)-2-acetamido-2-deoxy-D-glucopiranose or a homopolymer of (1,4-β-)-2-amino-2-deoxy-D-glucopiranose. It is usually obtained from chitin through chemical deacetylation conducted under heterogeneous conditions. The product of the reaction dissolved in 0.1 M acetic acid is known as chitosan [1]. In addition to organic and non-organic acids, this polymer forms salts [2, 3]. The reactive amino groups present in macromolecule chains are then protonated (NH+3OCOR−).

Chitosan salts, especially those that are water soluble, are one of the forms of the polymer used most frequently in application and implementation studies [3 - 8]. The chemical, physical, and biological properties of the salts can be impacted by the structure of the acid, the conditions under which they were obtained, the degree of deacetylation, and the molecular weight of the chitosan, as well as the nonhomogeneity of the polymer in terms of deacetylation degree and molecular weight [1, 2, 7, 9 - 15]. By determining the dependencies between them, it is possible to establish the optimal quality parameters of salts for potential application.

There is little information in the literature regarding chitosan salts and dicarboxylic acids, especially with reference to those that participate in the metabolic processes of living organisms. This refers primarily to how they are obtained, but only from polymers of a

CHITOSAN DICARBOXYLIC ACID SALTS

Anna Wojtasz-Pająk

Sea Fisheries Institute in Gdynia, ul. Kołłątaja 1, 81-332 Gdynia, Poland

e-mail: biopak@mir.gdynia.pl

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known degree of deacetylation, studies of crystal structures, or the rheological properties of concentrated solutions and gels [3, 12, 16].

The aim of the study presented in this paper was to determine the impact of the properties of the substrates used to obtain chitosan dicarboxylic acids salts on the physicochemical characteristics of these salts.

2. Materials and methods2.1. ChitinThe chitin used in the study was produced from the shells of krill (Euphausia superba) according to technology developed at the Sea Fisheries Institute in Gdynia [17]. The chitin was cleaned of mineral remains with 3% hydrochloric acid (1 : 15 w/v) for 30 minutes at a temperature of about 18°C. The chitin was then rinsed several times with tap water (1 : 10 w/v). In order to obtain chitin with a lower molecular weight than that of the initial polymer, a portion of the wet chitin was treated with 8% hydrochloric acid (1 : 5 w/v) for 70 hours at a temperature of 19 ± 2 °C. Following the degradation of the chitin, it was rinsed several times with tap water (1 : 10 w/v). The samples were held at a temperature of -25 °C until examination.

2.2. ChitosanChitosan was obtained by treating chitin with 50% NaOH (1:10 w/w) for 20 to 90 minutes or 2 × 20 and 2 × 90 minutes at a temperature of 115 - 140 °C. After each step of deacetylation, the chitosan was rinsed exhaustively with hot, distilled water (1 : 10 w/v) and the final product was dried at a temperature of 80 °C for 3.5 hours. Next, it was ground in a Cyclotec 1093 Sample Mill manufactured by Tecator. The polymers were held in a desiccator until examination.

The deacetylation degree (DD) of the chitosan was determined with the potentiometric titration methods [18], while viscosity-average molecular weight ( Mv ) was determined according to Rinaudo et al. [19]. The viscosity measurements were conducted at a temperature of 25 ± 0.01 °C using an AVS-350 (Schott Geräte) with 53210/I Ubbelohde viscometer with a constant K = 0.009852 mm2/s2. The chitosan concentration in 0.3 M acetic acid with 0.2 M sodium acetate was 0.00021 - 0.00095 g/ml of solvent.

2.3. Dicarboxylic acid The succinic and fumaric acids were supplied by Sigma-Aldrich (FCC). Adipic acid was from Merck (DAB, FCC).

2.4. Chitosan salts2.4.1. Preparation of the chitosan salts Salts were obtained from chitosan which the properties described in Table 1.

Chitosan was suspended in hot deionized water and acid was added. The mol ration of acid to amine groups was 1:2, while the final salt concentration was about 2.5%. The sample was

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Chitosan dicarboxylic acid salts

mixed for 240 minutes at room temperature, frozen, and lyophilized in an Alpha 1-4 LCS freeze dryer (Christ). Then it was ground in a Cyclotec 1093 Sample Mill (Tecator). The salts were held in a desiccator until examination.

2.4.2. Determining dry weightThe samples were dried at a temperature of 105 °C to a constant weight.

2.4.3. Determining salt acid contentThe salt (about 0.2 g) was dissolved in 25 ml of cold distilled water and the sample was mixed for 30 minutes. Next, 25 ml of cold distilled water was added to clear samples or 25 ml of hot distilled water was added to turbid samples and then mixed for 60 minutes. The acid content was determined with the potentiometric titration method with a 702 SM Titrino apparatus (Metrohm) using 0.1 N NaOH.

2.4.4. Testing the solubility of salts in waterThe salt was dissolved in cold distilled water (c = 1 g/100 ml) and mixed intensively for an hour. The sample was left for 24 hours at room temperature. It was then mixed for 30 minutes and centrifuged at a temperature of 25 °C for an hour at 10,000 r.p.m. in a MPW 4K15 centrifuge. The salt contents in the supernatant and the precipitate were determined by drying the sample at a temperature of 105 °C to a constant weight.

2.4.5. Viscometric measurementsSalt solutions at a concentration of c = 1g/100 ml were prepared as described in point 2.4.4. The samples were not centrifuged. Turbid solutions were heated intensely and mixed until the salt dissolved and then the sample was left at room temperature to cool.

The shear viscosity was measured at a temperature of 25±0.1°C with a Brookfield DV II viscometer with a SC-4 small adapter working unit using SC4-18/13R and C4-31/13R spindles at a shear rate of 79.2 s-1 (chitosan salts CHN 1, 2) and 20.4 s-1 (chitosan salts CHN 3 - 5, Table 1).

2.4.6. pH measurementsMeasurements of the pH of solutions (c = 1g/100 ml), prepared as in point 2.4.5 were conducted using an 702 SM Titrino apparatus (Metrohm).

Table 1. Chemical and physical properties of chitosan; a deacetylation degree in percentage; b viscosity-average molecular weight.

Sample symbol DDa, % Mv b, kDaDeacetylation reaction parameters

Temperature, °C Time, minCHN 1 69 279 115 20CHN 2 87 282 115 2×20CHN 3 73 67 115 30CHN 4 90 76 140 90CHN 5 97 61 140 2×90

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3. Results and discussion3.1. Chemical properties of the saltsThe chemical properties of the salts obtained during the tests (point 2.4.1) are presented in Tables 2.The salts were characterized by a low water content (not exceed 10%). This is evidenced by the salts dry weight (Tables 2). The determined acid content was similar to the theoretical value, and differences did not exceed 0.5% (Tables 2).

3.2. Physical properties of salts

The examined salts were characterized by various solubilities in water at a temperature of approximately 25 °C. The deacetylation degree and molecular weight of the chitosan and the structure of the acid impacted this (Figure 1).

The salt solutions obtained from chitosan with low deacetylation degrees (69 and 73%) were nearly clear although not homogeneous because there were aggregates and glassy macromolecules that were only swollen and not fully dissolved. In the further part of the paper both types of macromolecules are called regarding to Yanagisawa et al. [20] “aggregates”. Their presence in solutions of chitosan obtained under heterogeneous conditions was described by Yanagisawa et al. [20], Anthonsen et al. [21], and Domard and Rinaudo [22].The molecular weight of the chitosan had the primary impact on the quantity of “aggregates” in the A69/279, B69/279, F69/279, and A73/67, B73/67, F73/67 salt solutions. This was indicated by the solubility of the salts obtained. This increased from about 67 to 96% when the viscosity-average molecular weight was about four fold lower (Figure 1).

Table 2. Chemical properties of chitosan adipate (A), succinate (B) and fumarate (F); a 69 – DD of chitosan, %; b 279 – Mv of chitosan, kDa.

Sample symbol Dry weight, % Determined acid content,%

Theoretical acid content,%

chito

san

ad

ipat

e

A69a/279b 93.5 22.2 22.5A87/282 93.2 28.0 27.7A73/67 94.4 23.7 23.6A90/76 94.3 28.6 28.4A97/61 93.9 30.6 30.3

chito

san

succ

inat

e

B69/279 92.3 19.0 19.0B87/282 91.4 23.9 23.6B73/67 92.9 20.3 19.9B90/76 94.7 24.8 24.3B97/61 93.5 26.2 26.0

chito

san

fu

mar

ate

F69/279 91.1 18.5 18.8F87/282 93.0 23.3 23.3F73/67 93.0 19.9 19.7F90/76 96.2 23.6 24.0F97/61 95.6 25.5 25.7

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At an increased deacetylation degree (from 73 to 97%), only chitosan adipate dissolved nearly entirely in water. The solubility of succinate derived from chitosan of a deacetylation degree of 97% was about 27% lower than the adipate obtained from the same polymer. Succinate B87/282 and adipate A87/282 and B90/76 and A90/76 were characterized by similar solubilities at about 97 and 98%, respectively. These results indicate that a large number of amine groups could cause increase electrostatic interactions between the groups occurring in chitosan and succinic acid. The inter- and intramolecular hydrogen bonds that were created at this time reduced the solubility of the examined salts. The number of hydrogen bonds in chitosan adipate A97/61 was probably lower due to the lack of reactions between carboxylic acid groups. These groups were separated by four methylene groups [23].

The deacetylation degree of the chitosan had the greatest impact on the solubility of its salt with fumaric acid. The solubility of F87/282 salt was about five fold lower than that of succinic B87/282 or of adipic A87/282. Increasing the deacetylation degree from 87 to 97% caused the quantity of salt in the supernatant to fall to 3.5% and was about 21 and 28 times lower than succinic B97/61 or A97/61 (Figure 1). The solutions were turbid, and the undissolved salt particles sedimented.

The results obtained indicate that, in addition to the number of free amine groups in the chitosan, the structure of the acid also had an impact on the solubility of salts. The double bond between the carbon atoms in fumaric acid probably caused the formation of a greater number of intra- and intermolecular hydrogen bonds than occurred in the case of either adipic or succinic acids.

Figure 1. Solubility of chitosan adipate (A), succinate (B), and fumarate (F) at a temperature of about 25 °C.

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The formation of intra- and intermolecular bonds were confirmed by examinations of the impact temperature had on salts solubility. Increases of temperature to about 80 °C caused the hydrogen bonds to break and the salts to dissolve nearly totally in water. The salts did not precipitate when the solution was cooled to a temperature of 25 °C. The increasing pKa of the acids that occurred along with increasing temperature could also have had an impact on the solubility of the salts [3].

The solutions prepared as described above were used to perform determinations of shear viscosity and pH.

The shear viscosity of the salts solutions of adipic, succinic, and fumaric acids was similar when obtained them from chitosan with the same chemical and physical properties (Fig-ures 2 and 3).

The highest shear viscosity was noted in the salts from chitosan with a deacetylation degree of 69% and a viscosity-average molecular weight of 279 kDa (Figure 2). The shear viscosity decreased about five fold when the deacetylation degree increased to 87% while the viscosity-average molecular weight remained practically unchanged. It was comparable to the viscosity-average molecular weight of chitosan with a deacetylation degree of 69% (Figure 2). Such a large differences between the viscosities of salts A69/279, B69/279, F69/279 and A87/282, B87/282, F87/282 indicate that the solutions examined were not homogeneous.

Figure 2. Shear viscosity of chitosan adipate (A), succinate (B), and fumarate (F) obtained from polymers with high viscosity-average molecular weight.

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The “aggregates” that occurred in them meant that salts A69/279, B69/279, F69/279 and A87/282, B87/282 A69/279, F87/282 did not have similar shear viscosities.

“Aggregates” were also present in salt solutions obtained from chitosans that had viscosity-average molecular weights of about four fold less (Figure 3). The shear viscosity of these salts decreased as the deacetylation degree of the chitosan increased. Differences between the viscosities determined were, however, less than in the case of salts obtained from polymers with higher viscosity-average molecular weight. The shear viscosity of salts A90/76, B90/76, and F90/76 was about 1.5 and of salts A97/61, B9/61, and F97/61 about twice as low as salts A73/67, B73/67, and F73/67 (Figure 3).

The pH values of the salt solutions (c = 1g/100 ml) of adipic and succinic acids were similar at 5.4 and 5.5, respectively. The pH of the salt solution of fumaric acid was slightly lower at 4.9. The properties of the chitosan from which the salts were obtained did not impact the pH values. This is indicated by the comparable pH values of salt solutions obtained from the same acid and chitosan of varying degrees of deacetylation and viscosity-average molecular weight (Figure 4).

Figure 3. Shear viscosity of chitosan adipate (A), succinate (B), and fumarate (F) obtained from polymers with low viscosity-average molecular weight.

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4. Conclusionsn It is possible to obtain salts with specified properties from adipic, succinic and fumaric

acids and chitosans of varying degrees of deacetylation and viscosity-average molecular weight.

nThe temperature of the process, the structure of the acid, the degree of deacetylation, and the molecular weight of the chitosans impacted the solubility of the salts.

n The degree of deacetylation of the chitosans of similar molecular weights had an impact on the shear viscosity of the salt solutions.

n The properties of the chitosan did not have an impact on the pH of the salt solutions.

5. References 1. G. A. F. Roberts: Chitosan production routes and their role in determining the structure and

properties of the product. [In:] Advances in Chitin Science, vol. II. A. Domard, G. A. F. Roberts, K. J. Vårum (eds.). Jacques Andre Publisher, Lyon (1997) pp. 22-31.

2. R. A. A. Muzzarelli: Chitin. Pergamon Press, Oxford 1977. 3. M. Hamdine, M. C. Heuzey, A. Bégin: Effect of organic and inorganic acids on concentrated

chitosan solutions and gels. In. J. Biol. Macromol. 37 (2005) pp. 134-142. 4. M. Brzeski, A. Wojtasz-Pajak, A. Ramisz: Implementation of antarctic krill chitosan in veterinary

practice. [In:] Advances in Chitin and Chitosan, Ch. J. Brine, P. A. Sandford, J. P. Zikakis (eds.). Elsevier Applied Science, London and New York (1992) pp. 79-86.

Figure 4. pH of solutions of chitosan adipate (A), succinate (B), and fumarate (F).

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5. G. Borchard, H. L. Lueßen, A. G. de Boer, J. C. Verhoef, C. M. Lehr, H. E. Junginger: The po-tential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. II: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro. J. Contr. Rel. 39 (1996) pp. 131-138.

6. A. Wojtasz-Pająk, A. Ramisz, M. Malesa-Ciećwierz, A. Balicka-Ramisz: Chitosan application in prophylactics and treatment of respiratory system and alimentary canal diseases in farm animals. [In:] Advances in Chitin Science, vol. I. A. Domard, Ch. Jeuniaux, R. Muzzarelli, G. Roberts (eds.). Jacques Andrè Publisher, Lyon (1996) pp. 440-447.

7. M. Dornish, Ø. Skaugrud, L. Illum, S. S. Davis: Nasal drug delivery with ProtasanTM. [In:] Advan-ces in Chitin Science, vol. II. A. Domard, G. A. F. Roberts, K. J. Vårum (eds.). Jacques Andre Publisher, Lyon (1997) pp. 694-697.

8. A. F. Kotzé, H. L. Lueßen, B. J. de Leeuw, B. G. de Boer, J. C. Verhoef, H. E. Junginger: Compa-rison of the effect of different chitosan salts and N-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (Caco-2). J. Contr. Rel. 51 (1998) pp. 35-46.

9. M. Dąbek, A. Wojtasz-Pająk, D. Kruszewska, S. G. Pierzynowski: Effect of chitosan salts on bacterial growth and gut absorption – preliminary results. [In:] Progress on chemistry of chitin and its derivatives, vol. X. H. Struszczyk (ed.). Polish Chitin Society, Łódź (2004) pp. 103-112.

10. H. K. Holme, A. Hagen and M. Dornish: Influence of chitosans with various molecular weight and degrees of deacetylation on the permeability of human intestinal epithelial cells (Caco-2). [In:] Chitosan per os: From Dietary Supplement to Drug Carrier. R. A. A. Muzzarelli (ed.). Atec Edizioni, Grottammare (2000) pp. 127-136.

11. S. Z. Rogovina, G. A. Vikhoreva, T. A. Akopova, I. N. Gorbacheva: Investigation of interaction of chitosan with solid organic acid and anhydrides under conditions of shear deformation. J. Appl. Poly. Scien. 76 (2000) pp. 616-622.

12. J. Kawada, T. Yui, K. Okuyama, K. Ogawa: Crystalline behavior of chitosan organic acid salts. Biosci. Biotechnol. Biochem. 65 (2001) pp. 2542-2547.

13. I. Orienti, T. Cerchiara, B. Luppi, F. Bigucci, G. Zuccari V. Zecchi: Influence of different chitosan salts on the release of sodium diclofenac in colon-specific delivery. Int. J. Pharm. 238 (2002) pp. 51-59.

14. S. Y. Chae, M. Jang, J. Nah: Influence of molecular weight on oral absorption of water soluble chitosans. J. Contr. Rel. 102 (2005) pp. 383-394.

15. M. Rinaudo: Chitin and chitosan: Properties and applications. Prog. Polym. Sci. 31 (2006) pp. 603-632.

16. III Partanin, M. Emmett, II Brode, L. George: Acid decrystallization of aminopolysaccharides and derivatives thereof. United States Patent 4,929, pp. 722 (1990).

17. M. Brzeski, M. Mieczkowska, K. Sowa, H. Stolz, A. Wojtasz-Pająk, W. Neugebauer: Technologia otrzymywania chityny z pancerzy kryla antarktycznego [Technology for obtaining chitin from An-tarctic krill shells]. Studia i Materiały, Ser. S (2). Wydawnictwo Morskiego Instytutu Rybackiego, Gdynia (1985) pp. 13-23.

18. A. Wojtasz-Pająk, I. Kołodziejska, A. Dębogórska, M. Malesa-Ciećwierz: Enzymatic, physical and chemical modification of krill chitin. Bull. Sea Fish. Inst. 1 (143) (1998) pp. 29-39.

19. M. Rinaudo, G. Pavlov, J. Desbrières: Influence of acetic acid concentration on the solubilization of chitosan. Polymer 40 (1999) pp. 7029-7032.

20. Yanagisawa, M., Y. Kato, Y. Yoshida, A. Isogai: SEC-Mals study on aggregates of chitosan molecules in aqueous solvents: Influence of residual N-acetyl groups. Carbohyd. Polym. 66 (2006) pp. 192-198.

21. Anthonsen, M. W., K. M. Vårum, A. M. Hermansson, O. Smidsrød, D. A. Brant: Aggregates in acidic solutions of chitosans detected by static laser light scattering. Carbohyd. Polym. 25 (1994) pp. 13-23.

22. A. Domard, M. Rinaudo: Fully deacetylated chitosan In:] Chitin in Nature and Technology. R. Muzzarelli, C. Jeuniaux, G. W. Gooday (eds..). Plenum Press, New York (1986) pp. 315-316.

23. L. F. Fieser, M. Fieser: Chemia organiczna [Organic Chemistry]. Państwowe Wydawnictwo Naukowe, Warszawa 1958.

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