Preparation of Biomimetic Materials Made from Polyaspartyl Polymer

Ind. Eng. Chem. Res. 2009, 48, 9823–9829

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Preparation of Biomimetic Materials Made from Polyaspartyl Polymer and Chitosan for Heavy-Metal Removal Bo Sun,*,†,‡ Zhen-Tao Mi,‡ Gang An,† Guozhu Liu,‡ and Ji-Jun Zou‡ School of Chemistry and Chemical Engineering, Tianjin UniVersity of Technology, Tianjin, 300191, P. R. China, and School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin, 300072, P. R. China

By the combination of polyaspartyl polymer and chitosan, a kind of novel environmentally friendly biomimetic blend material with peptide-polysaccharide-resembling structure was synthesized for heavy-metal removal. The resulting blend possesses diversified functional groups and was further modified by introducing a mercapto group through esterification between thioglycollic acid and hydroxyl group in the structure of chitosan. The polyaspartyl polymer-chitosan blend with 2.2 wt % mercapto group content improves the adsorption of organic compounds of mercury and arsenic. The blend exhibits a lower swelling behavior in the presence of water and is verified to have a potential biodegradability by elemental analysis and infrared spectrum (FT-IR). materials such as metal adsorbents, immobilization supports, membrane stuff, and biomaterials.

Introduction Polyaspartate hydrogel has been confirmed to be an excellent binding agent of many heavy metal ions, such as Pb2+, Cd2+, Hg2+, Cr3+, Cu2+, and Mn2+.1,2 But one disadvantage of the gel is its strong water absorbency, which induces obvious volume swelling and poor mechanical properties in an aqueous medium. In order to improve the property of cross-linked polyaspartate and also to develop new kinds of materials, the blend of polyaspartate was taken into consideration. Chitosan was chosen for blending with polyaspartate to ensure biocompatibility of the obtained material. Chitosan is a deacetylated derivative of chitin that is the second most abundant natural biopolymer in crustacean shells, such as shrimps and crabs. The chemical structure of chitosan is similar to that of cellulose, except it is a unique alkaline polysaccharide, having one amino group in addition to the hydroxyl groups in the repeating unit. With active amino and hydroxyl groups in its structure, chitosan is considered as a binding agent for heavy metals. However, the amino group (-NH2) in chitosan can be protonated to -NH3+ in an acid environment, resulting in its dissolution. To overcome this problem, chemical modifications of chitosan have been conducted by many researchers to improve its insolubility in acid media.3-5 Our objective is to combine polyaspartate with chitosan, both of which are environmentally friendly and widely used, attempting to develop a new type of biodegradable polymer. The obtained blend contains -NH2, -OH, -COO-, and -CONHgroups and thus possesses diversified functions. Moreover, because the blend has a similar structure to that of plant cell wall consisting of peptide-polysaccharide, it can be regarded as a biomimetic macromolecule. The synthesized blend was characterized, and its biodegradability was investigated by FTIR and elemental analysis. Then the blend was applied to remove some heavy metals. This blend may find use as functional * To whom correspondence should be addressed. Fax: +86-2227402604. E-mail: [email protected]. † Tianjin University of Technology. ‡ Tianjin University.

Experimental Section Reagents and Materials. Polysuccinimide (PSI) was obtained by acid-catalyzed polycondensation of L-aspartic acid.6 Chitosan powder, with a deacetylation degree of 90-95%, was supplied by Zhejiang Yongyue Ocean Biology Co., Ltd. (Zhejiang Province, China). D418 (chelate ion exchanger), D113 (weakly acidic cationexchanger carboxylic functionality, macroporous type), 110 (weakly acidic cation-exchanger carboxylic functionality, gel type), and D296 (strongly basic anion-exchanger quaternary ammonium functionality, macroporous type) were purchased from the Chemical Plant of NanKai University (Nankai Group, China). Gynostemma pentaphyllum was purchased from the hospital pharmacy. All other reagents were obtained from Tianjin Chemical Reagent Co., Inc. (Tianjin, China) and used as received. Measurements. The total concentration of heavy-metal ion in an aqueous solution was determined with a Vista MPX inductively coupled plasma optical emission spectroscopy (ICPOES) spectrometer (Varian, Inc., Palo Alto, CA). The instrumental operating conditions are shown in Table 1. The swelling ratio of a sample was measured using a gravimetric method. The weighed dry sample was placed into a tea bag (40 mm × 100 mm in size) and then was suspended and fully immersed into deionized water at room temperature (20 °C). After 24 h, the tea bag was hung in the air for 15 min, and then the swelling ratio was calculated as Table 1. Instrumental Parameters and Operating Conditions for ICP-OES radio frequency (RF) power plasma gas flow rate flow rate of argon auxiliary nebulizer gas flow rate precision (general) detection limit Pb element analytical line (λ) Cd element analytical line (λ) Hg element analytical line (λ) As element analytical line (λ)

10.1021/ie900673h CCC: $40.75  2009 American Chemical Society Published on Web 09/23/2009

1.2 kW 15 L/min 1.5 L/min 0.9 L/min 1-3% 0.01 mg/L 220.3 nm 226.5 nm 184.9 nm 193.6 nm

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swelling ratio (wt/wt) )

W t - Ws - Wb Ws

(1)

where Wt is the total weight of the tea bag containing the watersaturated sample, Ws is the weight of the dry sample, and Wb is the weight of the wet tea bag. Fourier transform infrared (FT-IR) spectra were recorded with a Nicolet Magna-560 FTIR spectrometer (Madison, WI) in KBr pellets over a wavenumber range of 4000-400 cm-1 with a 4-cm-1 resolution. Iodimetry is used to measure mercapto group content of the polymer.7 The procedure is as follows: A mixture of 20 mL of 2:1 (v/v) HCl solution and 10.00 mL of 0.1 mol/L iodine was added in a 250-mL ground glass flask. About 0.2000 g of analyte was added, and the flask was stoppered and given a good shake. After 5 min, 100 mL of water was added in, and the solution was titrated with 0.1000 mol/L sodium thiosulfate (Na2S2O3) standard solution. Whenever the color of solution changed to light yellow, 1-2 mL of starch indicator was added in. The titration was continued until the blue color in the tested solution disappeared. The control test was done simultaneously. The mercapto group percentage of analyte can be calculated from the following formula (V0 - V1) × C × 33.07 × 100 2000m 1.6535 × (V0 - V1) × C ) m

SH% )

(2)

where V0 is the volume of Na2S2O3 standard solution consumed in the titration of control test (mL), V1 is the volume of Na2S2O3 standard solution required for the titration of analyte (mL), C is the concentration of Na2S2O3 standard solution (mol/L), m is the weight of analyte (g), and 33.07 is the formula weight of a mercapto group. The elemental analysis of samples was performed using a vario-EL element analyzer (Elementar Analysensysteme GmbH). The estimation of only three elements, that is, carbon, hydrogen, and nitrogen, was undertaken. The precision of the results were estimated to be 0.3%. Preparation of Polyaspartyl Polymer with Amino Side Chain (PAA-A). Polyaspartyl polymer with amino side chain (PAA-A) was prepared by reference to the method reported in the literature.8 The general procedure is as follows: Twenty-five milliliters of dimethyl formamide (DMF) and 4.85 g (0.05 mol) of PSI were charged to a 100-mL flask. The average molecular weight of PSI was 14 000 as measured by gel permeation chromatography (GPC).6 The content in the flask was heated to 50 °C with agitation to ensure total dissolution. A solution of 1.46 g (0.01 mol) of L-lysine and 0.4 g (0.01 mol) of NaOH in an appropriate amount of water was added to the flask at room temperature with stirring. Then, the reaction mixture was quickly placed into a microwave oven and kept at 120 W for about 3 min. After it cooled to about room temperature, the reaction mixture was poured into about 80 mL of methanol with stirring. The precipitate was filtered, washed several times with methanol, and then dried at 40 °C under reduced pressure. About 6.5 g of solid was obtained. This obtained solid (e.g., partially ring-opened polysuccinimide) was treated with alkaline hydrolysis by the addition of 25 mL of 2 mol/L NaOH in water; it was pH-controlled at 11-12 until a clear yellow solution formed. After 80 mL of absolute methanol was poured into the solution, precipitation took place. The precipitate was isolated, washed with methanol,

and then dried in vacuo. About 5.6 g of a brown solid was collected. The synthesis of polyaspartyl polymer with amino side chain is schematically shown in Scheme 1. Preparation of the Blend of Polyaspartyl Polymer and Chitosan in the Form of Granules. The general procedure to prepare the blend of polyaspartyl polymer and chitosan in the form of granules was prepared as follows: A chitosan solution was prepared by dissolving 0.1 g of chitosan powder in a combined solvent of 2.0 mL of 2% (v/v) aqueous acetic acid solution and 10.0 mL of 5% (v/v) HCl aqueous solution until the powder has dissolved. A solution of polyaspartyl polymer with an amino group in the side chain was prepared by dissolving 0.2 g of the polyaspartyl polymer obtained above in 5 mL of deionized water and by acidifying to a pH of approximate 4-5 with dilute hydrochloric acid. Then, 0.2 mL of 50 wt % glutaraldehyde was added in. Two solutions above were mixed thoroughly under vigorous stirring and maintained at room temperature for 15 min, and then 2 mol/L NaOH solution was added dropwise until complete precipitation was achieved and the pH value reached 7-8. The resulting mass was allowed to stand at room temperature overnight. Afterward, the precipitate was isolated, washed several times with deionized water until the pH of washing medium was neutral, and then dried at 40 °C in vacuo to a constant weight. About 0.25 g of an orange-pink material was obtained. The synthesis of the blend of polyaspartyl polymer and chitosan is schematically shown in Scheme 2. Preparation of Polyaspartyl Polymer-Chitosan Blend Containing Mercapto Groups. A mixture of 2 mL of mercaptoacetic acid (thioglycolic acid) and one drop of concentrated sulfuric acid (98%, d ) 1.84 g/mL) was charged in a 100-mL beaker. About 0.1 g of polyaspartyl polymer-chitosan blend obtained above was immersed sufficiently in the solution. Afterward, the beaker was placed into a 1000-mL brown widemouth bottle, and the bottle was kept tightly closed. The bottle was put in a thermostat at 40-45 °C and maintained for 48 h. After reaction was finished, the obtained precipitate was isolated, washed several times with deionized water until the pH of washing medium was neutral, and finally washed with absolute ethanol. Then, it was dried under vacuum in a desiccator and stored in a cool and dark place avoiding light exposure. The mercapto-group content of the product is approximately 2.2 wt %. Preparation of the Aqueous Solution of Gynostemma pentaphyllum. Small pieces of G. pentaphyllum (25 g) were soaked in 500 mL deionized water in a 1000 mL roundbottomed flask equipped with a condenser and allowed to stand at room temperature for 0.5 h. Then, the contents of the flask were heated and maintained at reflux for 1 h. The mixture was cooled and left overnight. The upper liquid layer was carefully decanted and concentrated with a rotary evaporator to a small volume. A certain volume (e.g.10 mL) of herbal solutions in a weighed dish was placed into an oven. The content of the dish was heated and maintained at 80 °C under a reduced pressure to a constant weight. The solid content can be calculated from the difference in weight of the dish before and after baking and the volume of the herbal liquor. Preparation of Polyaspartate Hydrogel (Fe2+-, Fe3+-, or Ca2+-Laden). Approximately 0.3 g of polyaspartate hydrogel (Na type)1 was immersed with three 50-mL portions of 0.1 mol/L FeCl2 (or FeCl3 or CaCl2) aqueous solution. The treated gel was isolated, washed several times with deionized water

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Scheme 1. Synthesis of Polyaspartyl Polymer with Amino Side Chain

Scheme 2. Synthesis of the Blend of Polyaspartyl Polymer and Chitosan

until there were few detectable Cl- ions in the washings, and then dried at 40 °C in vacuo to a constant weight. Results and Discussion Choice of Solvents. Chitosan is soluble in the dilute acids, such as dilute aqueous acetic acid solution and hydrochloric acid, being more dissoluble in the former. However, if only acetic acid was used as the solvent in the preparation of polyaspartyl polymer-chitosan blend, precipitation would occur during the mixing of chitosan with polyaspartyl compound, producing a heterogenetic mixture. While dilute HCl was chosen as a cosolvent, the expected homogeneous mixing of the reactants could be attained. Since chitosan is more soluble in dilute acetic acid than in dilute hydrochloric acid, an appropriate amount of acetic acid was added to promote the dissolution. However, the ratio of the amount of hydrochloric acid to acetic acid should be high enough to ensure a complete solution of chitosan and polyaspartyl polymer with amino group in side chain. The total amount of the solvent should ensure complete dissolution of chitosan and polyaspartyl polymer with amino group in side chain.

Since chitosan is more soluble in dilute acetic acid than in dilute hydrochloric acid, an appropriate amount of acetic acid was added to promote the dissolution. However, it has been found that if the amount of acetic acid was excessive, an unexpected precipitation would be occur during the mixing of chitosan and polyaspartyl polymer, that is, the ratio of amount of hydrochloric acid to acetic acid should be high enough to ensure a complete solution of chitosan and polyaspartyl polymer with amino group in the side chain. By adjusting the ratio of two acids, the 5:1 (v/v) ratio of a dilute hydrochloric acid (36-38% HCl:H2O ) 1:20 v/v) to a dilute acetic acid (2 wt %) was adopted in our experiments. Effect of Temperature. The cross-linking between chitosan and polyaspartyl polymer with an amino group in the side chain by glutaraldehyde can be carried out at room temperature. A higher temperature can cause hydrolysis of polyaspartyl polymer and chitosan, resulting in breakage of chemical bonds, especially in an acidic medium, so an ambient temperature of no more than 30-35 °C was recommended for the preparation of polyaspartyl polymer-chitosan blend.

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Table 2. Effects of Amount of Glutaraldehyde and Ratio of Polyaspartyl Polymer to Chitosan on the Composition of the Blend amount of yield of ratio of PAA-A to chitosana (g · g-1) glutaraldehyde (g) blendb (%) 1:1 2:1 2:1 3.5:1 8:1

0.11 0.11 0.22 0.22 0.44

107.5 81.7 84.0 56.2 27.1

Table 3. Comparison of Polyaspartyl Polymer-Chitosan Blend and Some Adsorbents in Terms of Pb2+ Removal

elemental content in blendc (wt %) N

C

H

6.65 7.95 7.56 7.42 7.31

42.74 40.40 41.57 42.98 41.73

6.31 6.35 6.49 6.64 6.46

a Amount of chitosan is 0.1 g. b Based on the sum of the amount of chitosan and polyaspartyl polymer with amino group in the side chain (PAA-A), not including amount of glutaraldehyde. c Nitrogen content in chitosan used is 7.37% and in polyaspartyl polymer with an amino group in the side chains [PAA-A, n (L-lysine)/n (succinimide) ) 0.25/1] is 8.96%.

Amount of Glutaraldehyde. In preparing the blend of polyaspartyl polymer and chitosan, if the amount of glutaraldehyde is far below the amount needed, polyaspartyl polymer will insufficiently be attached to chitosan. On the contrary, if the dosage of cross-linker is excessive, the NH2 group in chitosan will totally react off, with the blend failing to serve the function of an amino group. Theoretically, the mole of glutaraldehyde should not exceed half the total mole of NH2 group in polyaspartyl polymer and chitosan. The experimental results are given in Table 2. As shown in Table 2, the yield of the prepared blend slightly increases with increasing amount of glutaraldehyde, but Ncontent decreases and C-content increases as the dosage of crosslinker is excessive. This might be attributed to the reaction of redundant glutaraldehyde with the NH2 group in the chitisan structure, since glutaraldehyde has a higher C-content and no N atom. Therefore, the cross-linker should not be overdosed in the preparation of polyaspartyl polymer-chitosan blend. It is also observed from Table 2 that with a increase in the amount of glutaraldehyde from 0.22 to 0.44 g (rows 1-2 from bottom of the table) there is a decrease in % C at ratio 3.5:1 and 8:1, respectively. This might be due to less reaction opportunity between the aldehyde groups in glutaraldehyde and the amine groups in chitosan, compared with that between the aldehyde groups in glutaraldehyde and the amine groups in the side chain of polyaspartyl polymer. Ratio of Polyaspartyl Polymer to Chitosan. From the point of view of strengthening the heavy-metal binding performance, the content of polyaspartyl polymer in the blend may be expected to be high. Alternatively, the ratio of polyaspartyl polymer to chitosan should be high. It can be inferred that if the portion of polyaspartyl polymer in blend were high enough, the performance of the resulting blend would be similar to that of polyaspartyl polymer. However, the blend would be too sticky. Table 2 also shows the effect of this ratio on the composition of resulting blend materials. Note that when the ratio is below 1:1, the N-content in the blend is even lower than that in chitosan. This can also be due to the reaction of surplus glutaraldehyde with chitosan, as mentioned above. The results given in Table 2 show that the greater the ratio of polyaspartyl polymer to chitosan is, the lower the yield of blend (under the condition of sufficient amount of the crosslinker) is, and the N-content in the blend is not increased, suggesting that the amount of polyaspartyl polymer attached to chitosan is limited. If the amount of chitosan is small, the remaining solution after the blend precipitation appears to be orange, the color of polyaspartyl polymer, after the blend was precipitated, implying that a certain amount of polyaspartyl

adsorbentsa polyaspartyl polymerchitosan blendb polyaspartate hydrogel chitosan 110 D113 D418

final concentration Pb2+ of Pb2+ in solution uptakea swelling ratio (mg · L-1) (mg · g-1) (g · g-1, 20 °C) 108

22.5

2.0

50 150 140 150 140

37.5 12.5 15.0 12.5 15.0

15.0 1.6 1.4 1.7

a Amount of adsorbents ) 0.2 g, initial c (Pb2+) ) 200 mg/L, total volume of Pb(NO3)2 solution ) 50 mL, temperature ) 25 °C, pH ) 6, exchange time ) 2 h. b Nitrogen content in blend is 7.95%.

polymer was not attached to chitosan, thus the low yield. This evidence also demonstrated that the amount of polyaspartyl polymer combined to chitosan in the form of a granule is limited in preparing a granular blend. The preparing process can be explained as follows: -NH3+ salt formed by chitosan and acid was deprotonized with the addition of alkaline solution, and then released amine groups react with carboxyl groups in polyaspartyl polymer to form insoluble cross-linked macromolecular complex salt, resulting in the precipitation. Apparently, the greater the amount of polyaspartate combined in, the more soluble the formed macromolecule is in aqueous solution. Thus, the polyaspartyl content in precipitating salt is limited. With more alkaline solution added, polyaspartyl polymer converts to salt with alkali, releasing the -NH2 group in chitosan again. Finally, some of these amine groups react with another aldehyde group of glutaraldehyde in the side chain of polyaspartyl polymer to form the blend of polyaspartyl polymer and chitosan. It can be concluded that the blend composition is nearly the same for the PAA-A/chitosan ratios of 1:1 and 8:1, but the ratio affects the yield of the blend. Comparison of Adsorbing Behavior of Polyaspartyl Polymer-Chitosan Blend, Polyaspartate Hydrogel, and Chitosan. In terms of Pb2+-binding performance, a comparison of polyaspartyl polymer-chitosan blend, polyaspartate hydrogel, chitosan, 110 (polyacrylate cation resin in a gel form), D113 (polyacrylate cation resin in a porous form), and D418 (polystyrene chelating resin in a porous form) for the removal of Pb2+ from an aqueous solution is shown in Table 3. As shown in Table 3, Pb2+ uptake of the blend is lower than that of polyaspartate hydrogel due to the decreased number of -COO- groups, but it is obviously higher than that of chitosan, poly(acrylic acid)-based resins, or polystyrene-based chelating resins. In addition, the water absorbency of the blend is much less than that of polyaspartate hydrogel. This means that the blend has no apparent swelling behavior in aqueous solution, which makes it suitable for bed operation. Characterization of Polyaspartyl Polymer-Chitosan Blend. The polyaspartyl polymer-chitosan blend in granular form is orange in appearance, water-insoluble, and has no apparent water absorbency, distinctly different from polyaspartate hydrogel. A FT-IR spectrum comparison of polyaspartyl polymerchitosan blend, polyaspartate hydrogel, and chitosan is made in Figure 1. The FT-IR spectrum of polyaspartate hydrogel shows strong absorptions at 1646.8 cm-1 (amide-I band, CdO stretching) and at 1398.6 cm-1 (the symmetric stretching of carboxylate anion, Vs,coo-), and a strong, broad band at 3426.8 cm-1 (N-H stretching). IR spectrum of chitosan shows a strong absorption at 3423.88 cm-1, from a combination of O-H and N-H

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Figure 1. FT-IR spectrum comparison of polyaspartyl polymer-chitosan blend, polyaspartate hydrogel, and chitosan. Table 4. Removal of Pb, Cd, and Hg from G. pentaphylluma by Polyaspartyl Polymer-Chitosan Blend with a Mercapto Groupb concentration of M in solution (mg · L-1) initial

final

% M removed

adsorbents

Pb

Cd

Hg

Pb

Cd

Hg

Pb

Cd

Hg

polyaspartyl polymer-chitosan blend polyaspartyl polymer-chitosan blend with -SH group

0.21

0.13

0.10

0.05 0.06