Preparation of Water-Soluble Chitosan Derivatives ...

ISSN 1070-4272, Russian Journal of Applied Chemistry, 2012, Vol. 85, No. 1, pp. 120−127. © Pleiades Publishing, Ltd., 2012. Original Russian Text © A.B. Shipovskaya, V.I. Fomina, N.A. Solonina, K.A. Yusupova, 2012, published in Zhurnal Prikladnoi Khimii, 2012, Vol. 85, No. 1, pp. 126−133.

MACROMOLECULAR COMPOUNDS AND POLYMERIC MATERIALS

Preparation of Water-Soluble Chitosan Derivatives by Modification of the Polymer in Vapors of Monobasic Acids A. B. Shipovskaya, V. I. Fomina, N. A. Solonina, and K. A. Yusupova Chernyshevsky Saratov State University, State Educational Institution of Higher Professional Education, Saratov, Russia Received February 11, 2011

Abstract—Sorption of vapors of monobasic acids and of vapors over their solutions of various concentrations by high-molecular-weight chitosan was studied. The dependence of the degree of the vapor sorption by the polymer on the acid solution concentration, chitosan molecular weight, and sorption temperature was determined. Watersoluble chitosan derivatives were obtained. The hydrodynamic properties of aqueous solutions of the modified samples were examined. DOI: 10.1134/S1070427212010235

Chitosan, a biologically active polymer, is widely used today in the development of multipurpose pharmaceutical forms (powder, gel, capsules, films, sponges) for use as wound coatings, biomatrices, and containers for the delivery of drugs to the intended localization site. As a rule, water-insoluble chitosan samples are used for these purposes, which restricts the polymer application field and complicates the reprocessing. The solubility of chitosan in water depends on its molecular weight M¯ and degree of deacetylation DD. Oligochitosans (M¯ = 2–16 kDa) and low-molecularweight fractions (M¯ ≤ 25 kDa, DD ≥ 70 mol %) of chitosan are soluble in neutral media [1]. These samples are prepared by deep depolymerization of chitosan macromolecules as a result of oxidative, acid, and enzymatic degradation [2–5]. The molecular weight of the initial chitosan recovered from crustacean shell chitin can reach 1000 kDa. Such polymer is insoluble in water and shows only limited swelling. The following approaches are used to prepare water-soluble derivatives of high-molecular-weight chitosan. The most facile route to water-soluble chitosan derivatives is dissolution of the polymer in inorganic or organic acids and isolation of the polymeric salt from the

solution [6–10]. Chitosan salts with monobasic inorganic and monocarboxylic acids are well soluble in water [6]. The solubility of the salt form of chitosan in aqueous medium depends on the procedure and conditions for the salt isolation from the solution [6, 10]. In addition, the solubility of chitosan salts in water depends on pKa and structure of the acid. In some cases, to obtain the salt form, the initial chitosan should be preliminarily activated in a cavitation or shear field [7, 9]. The chitosan solubility in water can also be improved by developing formulations or polyelectrolytic complexes of chitosan with natural or synthetic polymers [11–13]. Polymer-analogous transformations are also widely used for preparing water-soluble N- and О-substituted chitosan derivatives [14–17]. Of particular interest is graft copolymerization [18–20]. By varying the structure and type of grafted pendant chains, it is possible to prepare water-soluble chitosan derivatives not only with preset properties, but also with new functions. To prepare water-soluble chitosan derivatives, we used in this study a new approach: imparting new properties to a polymer by treating it with vapors of appropriate solvents. This approach was successfully tested with vegetable polysaccharides, cellulose esters [21, 22]. It consists in modification of a polymer with 120

[α PREPARATION OF WATER-SOLUBLE CHITOSAN DERIVATIVES

121

Table 1. Molecular parameters of chitosan samples prepared from crab shell Producer

Application field

– Мη, kDa

DD

[α]20°C , deg ml dm–1 g–1 578 nm

Bioprogress Private Joint-Stock Company, Shchelkovo

Food industry

640

0.83

–21.3

Sonat Private Joint-Stock Company, Moscow

''

520 275

0.80 0.81

–21.5 –21.7

Japan

Medicine

90

0.95

–21.5

vapors over solutions of mineral or monocarboxylic acids used for chitosan dissolution [23, 24]. The goal of this study is modification of chitosan in vapors over solutions of formic (FA), acetic (AA), and hydrochloric acids with the aim to obtain water-soluble derivatives of high-molecular chitosan and examination of the hydrodynamic properties of their aqueous solution. EXPERIMENTAL We examined the effect of the vapor phase over aqueous acid solutions on the chitosan properties. In so doing, we paid major attention to the chitosan solubility in water. The investigation objects were commercial chitosan samples produced in Russia and other countries from crab shells (Table 1). The viscosity-average molecular weight M¯η of chitosan was determined by viscometry. The constants of the Mark–Kuhn– Houwink equation were taken from [25]. The degree of deacetylation was determined by potentiometry. The specific optical rotation [α]20°C , of the solution was 578 nm

evaluated with an SPU-E automatic spectropolarimeter equipped with a DRSh-250 high-pressure mercury lamp as a light source, at 20°C and λ = 578 nm. We also used a sample reprecipitated from a commercial chitosan sample with M¯η = 640 kDa. The polymer was reprecipitated from its 0.5 g dl–1 solution in 0.1 M HCl into a 0.02 M NaOH solution (volume ratio 1 : 5). The resulting precipitate was washed to fully remove low-molecular-weight substances and was dried in a vacuum oven at 50°С and 0.05 MPa for 11 h. Solutions of concentrations с = 0.05 and 0.5 g dl–1 were prepared by dissolving a weighed portion of the initial chitosan in hydrochloric and acetic acids of concentrations сa = 0.01–0.1 and 0.17–2.0 M,

respectively, in an acetate buffer solution (0.33 M CH3COOH + 0.2 M CH3COONa) with рН 4.4, and in a mixture of the above buffer with 8 M urea at 20 ± 2°С for 24 h. The chitosan modification was performed under isobaric-isothermal conditions in the temperature interval 4–98°С [23, 24]. A weighed portion of the polymer was kept in sorbate vapor in a sealed vessel filled to 1/25 with an acid solution of fixed concentration сa. The vapor phase over glacial acetic acid, formic acid, and hydrochloric acid (chemically pure grade) and over their aqueous solutions served as sorbate. In some experiments, we used vapor over distilled water as sorbate. The sorbed vapor amount was determined gravimetrically, by weighing a polymer sample before and after vapor sorption using an analytical balance with an accuracy of ±0.0001 g. The samples were weighed in an open system at 20 ± 2°С and normal atmospheric pressure. The process was characterized by the degree of vapor sorption Сs = [(m – m0)/m0] × 100 (wt %), where m0 and m are the weights of the air-dry and swollen polymer. To evaluate the solubility of modified chitosan samples, we prepared their aqueous solutions of concentration с = 0.1–1.0 g dl–1 at 20°С. The solubility in water was monitored visually, by measuring pH, and by gravimetric and turbidimetric methods. Hydrodynamic characteristics of the control and modified chitosan samples were determined with an Ubbelohde viscometer with a capillary diameter of 0.54 mm at 25°С. In the experiments we used both the initial solutions and those kept under static conditions at 20 ± 2°С for 26 days. Turbidimetric parameters were determined with a KFK-2 photocolorimeter, and the pH values, with an EV-74 universal pH meter. A study of sorption of active medium vapor by chitosan at various temperatures (4–98°С) showed that

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the sorption at 20°С was the most efficient. Figure 1 shows the kinetic curves of the sorption by chitosan-640 [here and hereinafter, the numerals denote M¯η (kDa) of the polymer] of vapor over 2% AA solution at various temperatures. As can be seen, the degree of swelling of the polymer in the active medium vapor increases with temperature. However, swelling of the polymer at ~90°С and higher temperatures is accompanied by a change in the sample color from light beige to dark brown and black, observed already within 2–4 h. Special experiments showed that the sample degraded under these conditions. In the subsequent experiments, the polymer modification was performed at 20 ± 2°С. The kinetic curves of sorption by chitosan of water, acetic acid, and formic acid vapors and of vapors over aqueous solutions of carboxylic acids and hydrochloric acid of various concentrations are shown in Fig. 2. As can be seen, the polymer shows limiting swelling in vapor of water and 96% acetic acid and in vapor over 5% HCl solution, with the degree of sorption Сs reaching a limiting value. In formic acid vapor and in vapors over soluitons of monocarboxylic acids and hydrochloric acid (сa = 20%), Сs does not reach an equilibrium value even in 30 days. In these experiemnts, there is a clear trend toward unlimited sorption of vapors of water–acid binary mixtures. Vapors over 75% solutions of monocarboxylic acids show the highest affinity for chitosan (Fig. 2a, curve 6; Fig. 2b, curve 4). Hence, the dependence of the degree of the vapor absorption on the monocarboxylic acid concentration at a fixed treatment time τ is described by a curve with a maximum. This fact may be due to

a specific structure of 75% solutions of these acids, in which the water to acid ratio is approximately equimolar [26]. A study of the sorption kinetics showed that, starting from a certain value of Сs, the samples kept in vapor start to dissolve in water [23]. The characteristics of some samples after absorption of different amounts of sorbate vapors are given in Table 2. Thus, by treatment of a powdered chitosan sample with vapors over acid solutions (except vapor over 5% HCl solution), we obtained water-soluble forms of chitosan. The minimal degree of sorption of water–acid vapor Сs,min ensuring the polymer solubility in water was 80 ± 5, 120 ± 5, and 180 ± 5 wt % for the vapors over hydrochloric, formic, and acetic acids, respectively (a) Cs, wt %

τ, h Cs, wt %

(b)

Cs, wt % (c)

τ, h

Cs, wt %

τ, h

τ, h Fig. 1. Kinetic curves of sorption by chitosan-640 of vapor over 2% AA solution at (1) 4, (2) 20, (3) 35, (4) 94, and (5) 98°С. (Сs) Degree of sorption and (τ) time; the same for Figs. 2 and 3.

Fig. 2. Kinetic curves of sorption by chitosan-640 of (1) water vapor and (2–7) vapor over solutions of (a) AA, (b) FA, and (c) hydrochloric acid, Т = 20°С. Concentration, wt %: AA: (2) 2, (3) 4, (4) 10, (5) 48, (6) 75, and (7) 96; FA: (1) 4, (2) 1, (3) 20, (4) 75, and (5) 100; hydrochloric acid: (1) 5 and (2) 20. The horizontal line corresponds to Сs,min.


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– Table 2. Characteristics of modified chitosan samples with Mη = 640 kDa, Т = 20°С Vapor medium

Acid concentration ca, %

Water

0

Acetic acid

2 4 10 48 75 96

Formic acid

4 10 20 75 100

Hydrochloric acid

5 20

Degree of sorption of sorbate vapor Cs, wt %/Sorption time τ, h water-insoluble 42 7 62 28 73 45 78 9 82 6 54 2 65 2 52 7 48 5 50 4 63 3 45 3 20 2 44 100

(Fig. 2, Table 2). The straight line corresponding to Сs,min is shown in Fig. 2 for clearness. As can be seen, the time in which Сs,min is attained depends on the acid concentration. Prolonged (τ up to 30 days) treatment of the samples with water vapor (Fig. 2a, curve 1) does not make them water-soluble (Table 2). The data obtained (Fig. 2, Table 2) show that it is most appropriate from technological and economical viewpoints to perform modification of chitosan with vapor over acetic acid of concentration no higher than 48%. Therefore, for further studies, in particular, for finding conditions of vapor modification of chitosan samples of different molecular weights, we used the water–acetic acid system (сa = 2–48%).

water-soluble 59 240 140 115 114 72 126 22 120 25 115 7 102 6 83 24 75 7 71 7 98 4 72 5 37 5 62 255

–

–

178 145 182 145 184 50 183 40 180 15 175 28 117 48 115 25 116 25 118 4 122 7 –

270 240 214 170 240 100 330 120 508 140 22 150 190 143 118 26 205 72 360 25 208 25 –

76 280

80 300

The kinetic curves of swelling of chitosan samples with different M¯η in vapors over acetic acid solutions of different concentrations are shown in Fig. 3. The vapor over 48% acetic acid solution shows the highest affinity for the samples tested, which is consistent with the data of Fig. 2a, curve 5. The vapor absorption rate is higher for lower-molecular-weight chitosan-90, irrespective of the concentration of the acid solution used in the vapor sorption experiments. The preparation conditions and characteristics of water-soluble chitosan samples of different molecular weights, modified in vapor over acetic acid solutions, are given in Table 3. Taking into account that pH of the aqueous solutions is below 5, it can be assumed that the solubility of the samples studied (with the degree of


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Cs, wt %

(a)

τ, h Cs, wt %

(b)

τ, h Fig. 3. Kinetic curves of swelling of (2, 2′, 2′′, 2′′′) chitosan-640, (3) chitosan-275, and (1, 1′, 1′′) chitosan-90 in vapors over AA solutions of concentrations (1, 2) 48, (1′, 2′) 10, (1′′, 2′′) 2, and (2′′′, 3) 4 wt %; Т = 20°С.

swelling Сs ≥ Сs,min) is caused by the formation of the salt form of chitosan due to protonation of the amino groups of the D-glucosamine units. Figure 4b and Table 3 show that treatment of chitosan with acetic acid solution vapor affects M¯η of the samples

insignificantly, because the intrinsic viscosity [η] of solutions of the water-soluble polymer forms in acetate buffer solution differs from that of solutions of the initial chitosan in the same solvent insignificantly. The similar behavior of [η] is observed for the samples modified with formic acid vapor. Treatment with vapor over 20% HCl solution is apparently accompanied by the sample degradation, because [η] decreases by a factor of approximately 3. It should be noted, however, that M¯η = 150 kDa of this sample is still sufficiently high, so that it cannot be considered as low-molecular-weight polymer. We also found that aqueous solutions of modified samples exhibit a polyelectrolytic effect: the reduced viscosity ηsp/c of solutions increases on their dilution (Fig. 4a). The dependence ηsp/с = f(с) passes through a maximum (Fig. 4a, curves 1–4) and has a descending τ, h branch (curves 5–7). This effect is caused by polyelectrolytic swelling of the macroion: On dilution, the shielding of the fixed charges decreases and their mutual repulsion increases, which leads to an increase in ηsp/с. Because swelling of the macroion ultimately reaches the upper limit, the decrease in ηsp/с observed on further dilution reflects a decrease in the interaction of the swollen macroions [27]. The intrinsic viscosity [η] of aqueous solutions of modified chitosan was calculated by the Fuoss equation according to which the quantity (ηsp/с)–1 is proportional to c1/2. For example, depending on the degree of sorption of water–acid vapor, [η] of aqueous solutions of modified chitosan-640 varies within 100 ± 30 dl g–1 (Table 3). In an acetate buffer solution containing CH3COONa,

(a)

ηsp/c, dl g–1

ηsp/c, dl g–1

(b)

c, g dl–1

c, g dl–1

Fig. 4. Reduced viscosity ηsp/с of solutions of chitosan-640 (a) in water and (b) in acetate buffer solution after treatment of the polymer in vapors over AA solutions as a function of polymer concentration с. AA concentration, wt %: (1) 2, (2) 4, (3) 10, (4) 20, (5) 48, (6) 75, and (7) 96; (8) reduced viscosity of a solution of chitosan-640 in acetate buffer solution after treatment of the polymer with water vapor for ~30 days. RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 85 No. 1 2012


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Table 3. Characteristics of water-soluble forms of chitosan modified in vapor over acetic acid solutions Sorption conditions Sample

a

сa, % τ, h

Сs, wt %

Parameters of aqueous solution

Intrinsic viscosity [η], dl g–1

Chitosan solubility in watera

c,g dl–1

рН

turbidity τλ=490 nm, cm–1

in water

in acetate buffer solution

Chitosan-640

0 2 4 10 48 75 96

240 240 170 100 120 140 150

59 270 214 240 330 510 220

is s s s s s s

− 0.1 0.1 0.1 0.1 0.1 0.1

− 4.9 4.9 4.6 4.4 4.3 4.4

− 0.02 0.02 0.03 0.02 0.02 0.01

− 139 130 125 70 87 100

9.1 10.0 9.4 8.5 8.2 9.9 10.3

Chitosan-275

0 4 4 4

240 72 170 530

40 340 340 580

is s s s

− 0.5 1.0 0.13

− 4.8 4.5 4.8

− 0.01 0.02 0

− 32.5 − −

5.8 5.5 − −

Chitosan-90

0 2 10 48

– 74 70 73

− 170 230 370

is is s s

− 0.27 0.27 0.27

− 5.2 4.5 3.7

− − 0.05 0.06

− − 26.3 20.0

2.15 − − −

(is) Insoluble and (s) soluble.

ηsp/c, dl g–1

[η] decreases (Fig. 4b), suggesting a decrease in the macroion volume due to an increase in the solution ionic strength. At isoionic dilution, the dependence ηsp/с = f(с) is linear, which is similar to the dependence for uncharged polymers. For comparison, we present the concentration dependence of the reduced viscosity of chitosan-520 solutions in acetic and hydrochloric

c, g dl–1 Fig. 5. Reduced viscosity ηsp/с of solutions of chitosan-520 in (1–4) AA and (5, 6) hydrochloric acid as a function of the polymer concentration с. pH of AA solutoins: (1) 4.5, (2) 4.0, (3) 3.5, and (4) 3.0. Acid concentration, M: (1) 0.17, (2) 0.5, (3) 1.0, (4) 1.5, (5) 0.01, and (6) 0.1.

acids of various concentrations (Fig. 5). Notably, the dependence ηsp/с = f(с), as at isoionic dilution, is linear at any acid concentration in the examined interval. However, in view of the fact that the slope of the straight lines and the quantity [η] regularly decrease with an increase in the acid concentration, it can be stated that chitosan macromolecules behave as polyelectrolytes, and addition of an acid leads, along with protonation of amino groups in the polymer chain, also to a change in the ionic strength of the aqueous acid solutions. Only in 0.1 M HCl, as in acetate buffer solution, [η] = 10 dl g–1 and the plot of ηsp/с = f(с) is a straight line parallel to the abscissa. It should be noted that [η] of an equimolar aqueous solution of high-molecular-weight chitosan modified with acetic acid vapor to Сs = 240 wt % is 125 dl g–1, exceeding by a factor of ~3 the intrinsic viscosity [η] of a 0.17 M acetic acid solution of the initial sample, at equal pH values of the solutions (рН 4.55 ± 0.05). Another specific feature of aqueous solutions of modified chitosan samples is higher pH of these solutions compared to equiconcentrated (with respect to the polymer) solutions of the initial sample at equimolar ratio of the amino groups to the acid. This fact suggests


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ηsp/c, dl g–1

used for dissolving the polymer can be considered as a procedure alternative to the preparation of chitosan salts by their isolation from solutions. CONCLUSIONS

c, g dl–1 Fig. 6. Reduced viscosity η sp/с (dl g –1) of solutions of chitosan-640, (1–5) initial and (1'–5') reprecipitated, in acetate buffer solution with the addition of 8 M urea. Solution storage time τ, days: (1, 1') 0, (2, 2') 5, (3, 3') 13, (4, 4') 20, and (5, 5') 26.

(1) Modification of chitosan with vapors of water and carboxylic acids and with vapors over solutions of carboxylic acids and hydrochloric acid of various concentrations was performed. (2) The optimal modification conditions were found: concentration 2–48%, temperature 20 ± 2°С; the minimal degree of sorption of water–acid vapors by the polymer, controlling the solubility of chitosan in water, was determined. (3) Aqueous solutions of the modified samples exhibit polyelectrolytic properties. REFERENCES

that the vapor also affects the structure of the polymer matrix. To confirm this assumption, we examined the effect of a powerful hydrogen bond acceptor such as urea on the hydrodynamic parameters of the chitosan solutions. Introduction of 8 M urea into an aqueous-salt solution of the initial and reprecipitated chitosan-640 leads to an increase in ηsp/с on dilution of the system (Fig. 6). The polyelectrolytic effect is manifested to a greater extent in dilution of solutions of a reprecipitated chitosan-640 sample and of stored solutions. Under the action of 8 M urea, [η] of solutions of the initial and reprecipitated samples increases by 30 and 40%, respectively. The whole set of the above data confirms the presence of intra- and intermolecular bonds in chitosan and the effect of reprecipitation and storage on these bonds. This fact suggests that the solubility of chitosan derivatives in water after modification of the initial sample with solvent vapor is apparently due not only to formation of chitosan salts, but also to the effect of the excess amount of the sorbed water–acid vapor on the supramolecular structure of chitosan. To conclude, modification of chitosan by keeping a polymer powder in vapors of monobasic acid solutions is simple in implementation, environmentally safe (as performed in a closed system), and economically advantageous owing to the use of only one reagent, monobasic acid. Preparation of water-soluble chitosan derivatives by treatment with vapors of acids traditionally

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