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Two formation mechanisms and renewable antioxidant properties of suspensible chitosan–PPy and chitosan–PPy–BTDA composites
Synthetic Metals 164 (2013) 6–11
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Synthetic Metals journal homepage: www.elsevier.com/locate/synmet
Two formation mechanisms and renewable antioxidant properties of suspensible chitosan–PPy and chitosan–PPy–BTDA composites Rong-Jay Lee a , Tarmo Tamm b , Rauno Temmer b , Alvo Aabloo b , Rudolf Kiefer a,b,∗ a b
Industrial Technology Research Institute (ITRI), Kuang Fu Rd 321, 30011 Hsinchu, Taiwan University of Tartu, Institute of Technology, Intelligent Materials and Systems Lab, Nooruse 1, 50114 Tartu, Estonia
a r t i c l e
i n f o
Article history: Received 25 October 2012 Received in revised form 6 December 2012 Accepted 17 December 2012 Available online 22 January 2013 Keywords: Chitosan–PPy composite Oxidant NaNO2 Porphyrin Chitosan–PPy–BTDA composite Renewable antioxidant property
a b s t r a c t Treatment of chitosan together with pyrrole (Py) by oxidant sodium nitrite (NaNO2 ) forms unique chitosan–polypyrrole composites suspensible in diluted acetic acid. It was discovered that the reactive species polymerizing Py into polypyrrole (PPy) are the aldehydes, formed in situ during the depolymerization reaction of chitosan with NaNO2 . In situ UV–vis and FTIR measurements identify typical porphyrin signals, shedding light to the formation mechanism. To improve the film formation of the composites, the application of additional cross-linking agents such as 3,3 ,4,4 -benzophenone tetracarboxylic dianhydride (BTDA) was investigated. The obtained suspensible chitosan–PPy–BTDA composites showed renewable (by electrochemical means) antioxidant properties comparable in strength to those of natural vitamins. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chitosan, obtained from chitin over N-deacetylation is a natural biopolymer, non-toxic and soluble in light acid, known to show antimicrobial activity [1]. In recent years, many attempts have been made to increase the applicability range of chitosan by grafting polymerization with suitable polymers, producing chitosan–gallic acid [2], carboxymethyl chitosan [3,4], or by cross-linking chitosan with glutardialdehydes [5]. For increasing the solubility of chitosan in aqueous media, certain depolymerization techniques to obtain chain-scission can be applied by using oxidants such as ammonium persulfate (APS) [6], potassium persulfate (PPS) [7], 2,2 -azobis(2-methylpropionitrile) (AIBN) [8] and strong acids [9]. The most common depolymerization agent for chitosan is perhaps sodium nitrite (NaNO2 ) [10]. The use of different depolymerization agents reflects on the endproducts of chitosan which in case of NaNO2 are aldehyde rest products [11]. The polymerization of pyrrole (Py) with oxidant NaNO2 , in light acetic acid conditions forming conductive polypyrrole (PPy) coatings on silica surfaces has been mentioned in two reports [12,13]. The reactive species were identified as in situ formed nitrosonium cations (NO+ ). Whether the reactive species
∗ Corresponding author at: University of Tartu, Institute of Technology, Intelligent Materials and Systems Lab, Nooruse 1, 50114 Tartu, Estonia. Tel.: +372 737 4826; fax: +372 737 4825. E-mail addresses: [email protected] (R.-J. Lee), [email protected] (T. Tamm), [email protected] (R. Temmer), [email protected] (A. Aabloo), [email protected], [email protected] (R. Kiefer). 0379-6779/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.synthmet.2012.12.032
starting the polymerization of Py in depolymerized chitosan are the aldehydes or the nitrosonium, is investigated in this study. Recent attempts of forming composites of chitosan with PPy have been made by applying oxidants such as APS [14] or iron trichloride (FeCl3 ) [15]. The obtained composites did show suspensibility in aqueous or light acid solutions, explained by concentration effects between depolymerized chitosan and the formed PPy [15]. For obtaining conductive materials, chitosan–PPy composites (CPC) can be made; being envisaged for biosensor or biomedical applications [16]. Chemically polymerized PPy in form of powder has been found effective as a radical scavenger material [17–19], as shown by the reaction with di(phenyl)-(2,4,6trinitrophenyl)iminoazanium (DPPH) radicals [20]. Suspensible CPC with antioxidant properties may provide a solution for the shortcoming in processability of conducting polymer powders having rather low solubility. To increase the stability of the composite film, cross-linking agents such as 3,3 ,4,4 -benzophenone tetracarboxylic dianhydride (BTDA) [21] are applied in this study to form chitosan–PPy–BTDA composites (CPBC), and the effect of Py and BTDA concentrations in view of antioxidant properties and film stability are investigated to envisage novel material applicable in food packaging or biosensor functionality. 2. Experimental 2.1. Chemicals Chitosan (middle molecular weight, 103,000 g mol−1 , deacetylation degree 85%), acetic acid (97%), NaNO2 , ammonium
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Scheme 1. Depolymerization of chitosan [11] leads to 2,5-anhydro-d-mannose (P1). P1 and NaNO2 can form P2 (porphyrin) P3 products.
peroxydisulfate ((NH4 )2 S2 O8 , 98%), Py (98%), BTDA, (95%), pentane-1,5-dial (glutardialdehyde, GDA, 25% aqueous solution), DPPH, vitamin E and C were obtained from Sigma–Aldrich. Py was distilled and stored under nitrogen at 4 ◦ C prior to use. 2.2. Suspensible CPC 2.2.1. CPC material 2 g of chitosan was dissolved in 50 ml 1.5% acetic acid to form a 4% solution. The solution was stirred under ice cooling, NaNO2 (25 mg, 7.2 mM) was added in small portions during 30 min, followed by reaction time of 2 h until no N2 gas release was detected. Related to the deacetylation degree of chitosan, the ratio chitosan:NaNO2 was in the range of 1:70. After depolymerization of chitosan, additional NaNO2 (25 mg, 7.2 mM) and Py (0.2 M) were added, and the mixture stirred at 50 ◦ C for 2 h. The CPC compounds were obtained by increasing the pH to 10–11; very fine particles were obtained which were soluble in light acetic acid. 2.2.2. CPBC material Different concentrations of Py (0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M and 1.0 M) with constant concentration of NaNO2 (7.2 mM) and constant concentration of BTDA (1.6 mM) were added to the depolymerized chitosan solution with additional stirring for 3 h at 50 ◦ C. CPBC-1 samples with Py:BTDA ratios 62.5, 125.0, 187.5, 250.0, 312.5, 625.0 were obtained. CPBC-2 samples with different concentrations of BTDA (1.6 mM, 4.7 mM, 14.0 mM, 42.0 mM), constant Py concentration (0.2 M) and constant NaNO2 concentration (7.2 mM) correspond to different Py:BTDA ratios 125.0, 42.5, 14.3, 4.8. To obtain solid composites, the pH value of the solution was increased to pH 10–11 (by adding sodium hydroxide) and the precipitate was washed several time with MilliQ and acetone to remove excess of Py and BTDA, and dried in the oven (40 ◦ C, 24 h, 600 mbar). The solid composites were dissolved in 1.5% acetic acid and in case of CPBC samples, coated on a glass plate to form a film, and dried in the oven.
2.3. Characterization The molecular structure of CPC samples was investigated with Fourier transform infrared (FTIR) spectroscopy (Spectrum One, PerkinElmer) with KBr pressed disks and UV–vis spectroscopy (V550, Jasco, samples dispersed in 1.5% acetic acid). In situ UV–vis was made to investigate the influence of the Py:NaNO2 ratio on Py polymerization in chitosan. In situ FTIR measurements were performed on a KBr plate where chitosan was depolymerized with NaNO2 solution over 2 h and after drying of the probe surface, Py was added and the formation of composites was observed at different time intervals. Cyclic voltammograms (Eco Chemie Autolab, scan rate 10 mV s−1 , ± 0.8 V) of the CPC films (average thickness between 40 and 70 m) were performed in a three electrode cell (working electrodes were CPC or CPBC samples clamped on Ptsheet, counter electrode was Pt-sheet, reference electrode Ag/AgCl (3 M KCl)) in 0.2 M tetramethylammonium chloride (TMACl) electrolyte. The conductivity of the samples (in dry and hydrated state) was studied by means of electrochemical impedance spectroscopy (Eco Chemie Autolab). 2.4. Radical scavenger activity of CPC and CPBC samples Radical scavenger properties of CPC and CPBC (also in form of solid films) samples were determined with a prepared DPPH assay test [18]. The DPPH radical typically extracts a proton from the testing sample forming DPPHH, which can be observed by UV–vis measurements. The solution was shaken and stored for 30 min at room temperature (RT) before the samples were added. The main adsorption peak in UV–vis spectrum for DPPH was observed at 517 nm, the absorbance of this peak was set as the control value (C0 ). The decrease of the 517 nm peak determines the value C1 and with following Eq. (1) the radical scavenger activity (RSA) was calculated [22]. RSA =
C0 − C1 × 100% C0
(1)
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upper bipolaron band of PPy. Ratio 4.0 shows new a peak at 645 nm and a broad wave at 800 nm. The existence of the broad wave between 800 and 900 nm indicates that bipolaron bands of conjugated PPy are formed [27] indicating further oxidation of the PPy chains and the formation of new (more) conjugated structures. With ratios 4.0–21.0, the 527 nm and 645 nm peaks increase in their adsorption. The ratio 4.0 spectrum reminds a typical porphine ring structure with the Soret-band at 416 nm and the Q-bands at 530 nm and 650 nm [28]. In comparison to CPS polymerized using APS, the UV–vis spectra reported by Li et al. [14] show typical signals at 460 nm (–* transition) and 900 nm associated to the bipolaron state of PPy, which are different from those seen in Fig. 1a. The typical chitosan signals (Fig. 1b) in the CPC sample are recorded at 3420, (O H stretching vibration), 1643 ( CONH stretching vibration), 1410 ( CH2 bending), 1257 (C O C valence) and 1153 cm−1 (anti-symmetric stretching of the C O C bridge) [6]. The bands at 1552 cm−1 (2,5-substituted Py) are showing typical PPy ring vibrations [29]. The decrease of the 3420 cm−1 broad peak after certain polymerization time (Fig. 1b, 10 min–2 h) identifies the formation of more PPy; the 1552 cm−1 peak, splitting to two additional peaks at 1541 cm−1 and 1563 cm−1 is assumed to belong to two different substitutes of Py. Porphyrine signals [30] are reported at 3051 (N H), 1594 (C C), 1352 (C N) and 732 cm−1 ( C H, aromatic ring vibration). In CPC composites, out of these signals considering expected shifts, only small peaks at 1659 (CN), 1456 (CC), 1345 (C N), and 722 cm−1 ( C H) could be found [27]. Recent reports discovered that in mild acidic conditions (pH > 4), NO+ ions are formed from nitrites (Eq. (2)) which can initiate the polymerization of Py, leading to a conductive PPy layer on silica surface [12,13]: Fig. 1. (a) In situ UV–vis measurements (270–900 nm) of CPC samples polymerized for 2 h at different Py:NaNO2 ratios: 0.2 (– – ), 0.4 (–. .), 0.6 (. . .), 4.0 (—), 13.0 (. . .) and 21.0 (- - -); (b) in situ FTIR spectra (4000–450 cm−1 ) of CPC (ratio 4.0) measured after 10 min (–. . .), 30 min (. . .), 1 h (- - -) and 2 h (—).
3. Results and discussion
HNO2 + H+ ↔ H2 NO2 + ↔ NO+ + H2 O
(2)
Reaction of Py with NaNO2 (in 1.5% acetic acid) turned the mixture from brown to black after short time, but PPy powder in sufficient amounts could not be obtained. In situ UV–vis measurements were made to investigate which product is obtained at different polymerization conditions (Samples 1–4) (Fig. 2a):
3.1. CPC composite analysis The polymerization procedure to obtain CPC samples requires oxidants which perform two functions: depolymerization of chitosan and polymerization of Py. In case of NaNO2 as oxidant, the free amino groups of chitosan are attacked by nitrous acid HNO2 in 1:1 stoichiometry [10,11] (Scheme 1). The chitosan glycosidic bond is broken and 2,5-anhydro-dmannose (P1) and hydroxyl residual groups are formed [11]. Adding Py without NaNO2 leads to no product, therefore additional NaNO2 as radical starter was included in the reaction. Meso-tetrasubstituted porphyrin was first synthesized by Rothemund [23] by heating mixtures of Py up to 200 ◦ C with an aldehyde in pyridine under anaerobic conditions. The Adler–Longo method [24], based on the reaction at high temperature (200 ◦ C) in acetic or propanoic acid and aerobic condition gives a better yield (10–30%). Depolymerized chitosan contains aldehydes (2,5-anhydro-d-mannose) which can react with Py monomer and additional oxidant NaNO2 in the right stoichiometry [25] to produce P2 (Scheme 1). It is not clear which conditions determine whether porphyrins (P2) or PPy derivates (P3) are formed. In order to identify the reactive species interacting with Py in situ UV–vis and FTIR measurements were performed (Fig. 1) The Py:NaNO2 ratios 0.2, 0.4, and 0.6 with excess of oxidant revealed one strong peak at 416 nm and a weak broad peak at 527 nm (Fig. 1a), which are described in the literature [26] as belonging to constitute transitions from the valence band to the
Sample 1: Chitosan + NaNO2 (reaction 2 h at RT in 1.5% acetic acid) + Py + NaNO2 ; Sample 2: Chitosan + NaNO2 + Py (in 1.5% acetic acid); Sample 3: Pyrrole + NaNO2 (in 1.5% acetic acid); Sample 4: GDA + NaNO2 + Py in (1.5% acetic acid). The effect of higher acetic acid concentration (1.5%, 5%, 10%, and 20%) was studied on samples 1–3. The results of the studies are presented in Fig. 2b. Sample 2 (. . .) and Sample 3 (- - -) in Fig. 2a demonstrate that the reaction without aldehyde formation leads to PPy formation with peaks at 350, 507, and 652 nm, and we assume that the active oxidant is NaNO2 . If GDA is present (Fig. 2a, Sample 4, -·-·), the reactive species oxidizing Py are the aldehydes, and peak positions at 414, 530, and 650 nm are similar to Sample 1 (–). It becomes clear that these new PPy peaks appear due to a different mechanism in contrast to samples 2–3. We assume that for Sample 1 a small amount of porphyrin is formed (Scheme 1, P2), and products of conjugated PPy in combination with chitosan are obtained in the bulk reaction. If the acetic acid concentration is varied (Fig. 2b), the peaks at 414, 511, and 655 nm decrease with increased acid concentration. In case of 20% acetic acid (Fig. 2b, inset), similar peaks for all samples 1–3 appear at 313, 511, and 655 nm. This indicates that for acetic acid concentrations up to 20%, the reactive species polymerizing Py is NaNO2 . At higher acetic acid concentrations, we assume that
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Fig. 2. In situ UV–vis spectra (270–900 nm) of samples with Py:NaNO2 ratio 4.0, (a) chitosan–NaNO2 (2 h depolymerization reaction) + Py (Sample 1, –), chitosan–NaNO2 + Py (Sample 2, . . .), NaNO2 + Py (Sample 3, - - -), and GDA + NaNO2 + Py (Sample 4, -·-·) and (b) Sample 1 polymerized at different acetic acid concentrations (1.5% (. . .), 5% (- - -), 10% (-..), and 20% (–)) with inset: Sample 1 (–), Sample 2 (. . .), and Sample 3 (- - -) at acetic acid concentration 20%.
higher dissociation degree of chitosan [31] inhibits the formation of aldehydes. Earlier investigations of PPy formation with NaNO2 proposed that the oxidant performing the reaction is the in situ formed NO+ cation (Eq. (2)). The corresponding UV–vis measurement shows a broad peak in the range between 350 and 380 nm, identified as conjugated PPy (–* transition) [12,32]. Agreeing with the aforementioned study [32], an adsorption peak at 350 nm (Fig. 2a) is found. Additional peaks at 511 and 655 nm describe the transition state to polaron–bipolaron bands of PPy. Further work is needed to obtain higher yields of porphine ring structures in chitosan with new application envisaged for sensors and electronic wires [33]. 3.2. CPBC analysis To increase the CPC film stability, certain amount of BTDA in function of a cross-linking agent was added during the polymerization procedure. The BTDA reacts with the free amino groups of chitosan, forming imides, leading to the cross-linking of the depolymerized chitosan [21]. CPBC samples are suspensible in acetic acid and could be coated on glass forming a stable film. To investigate the influence of Py concentration on the polymerization process (1.6 mM BTDA, 7.2 mM NaNO2 ), UV–vis and FTIR spectral measurements at different Py:BTDA ratios were performed (Fig. 3). The UV–vis spectrum of CPBC samples (Fig. 3a) with Py:BTDA ratio 62.5 shows two peaks at 470 and 608 nm. With the increasing amount of Py (increasing Py:BTDA ratio) the 470 nm peak decreases
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Fig. 3. (a) UV–vis (350–900 nm) spectra of CPBC-1 samples at different Py:BTDA ratios: 62.5 (—), 125.0 (. . .), 187.5 (- - -), 250.0 (-..), 312.5 (-.-) and 625.0 (- -) and (b) FTIR spectrum (2000–450 cm−1 ) of CPBC-1 sample (Py:BTDA ratio 125.0).
and the 608 nm peak shifts to 635 nm and increases. In comparison to Fig. 2a, the inclusion of BTDA inhibits the formation of the porphyrin, observed as the disappearance of the 414 and 507 nm peaks. The FTIR spectrum (Fig. 3b) of CPBC (Py:BTDA ratio 125.0) shows chitosan signals at 1639 cm−1 ( CONH stretching vibration) and 1055 cm−1 (skeletal vibration involving C O C stretching) [6]. The presence of PPy in CPBC is indicated by peaks at 1537 cm−1 (2,5-substituted Py) and 922 cm−1 ( C H out of plane vibration, indicating polymerization of Py) [34]. BTDA connection can be found as small peaks at 1788, 1741 (symmetrical C O stretching and asymmetric C O stretching of the imide group), and 733 cm−1 (imide ring deformation) [21]. We assume that the BTDA crosslinking agent prevents porphyrin formation due to steric reasons of the depolymerized chitosan chains that are unable to interact with Py in the right position. Therefore the formation of PPy in CPBC samples is caused by in situ formed NO+ (Eq. (2)) [12] leading to a different structure of PPy in comparison to the CPC samples (Fig. 3a vs Fig. 1a). 3.3. Antioxidant characterization of the composites Earlier studies have discovered that polyaniline and PPy possess effective radical scavenger properties, in the latter case 2–4 Py units react with one DPPH radical [18,19]. Small amounts of testing samples were dissolved in 1.5% acetic acid (BTDA in acetone). The decrease of the 517 nm peak (UV–vis analysis) in DPPH standard solution (20 ml methanol, 200 M) determines the radical scavenger properties of the testing samples. The loss of activity of the pure DPPH solution in absence of the testing samples in time was deducted from the measurement results of the
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Fig. 4. Radical scavenger activity (RSA) at different time frames (30 min, 1 h, 2 h, 4 h, 24 h), (a) CPBC-1 from different Py:BTDA ratios (62.5, 125.0, 187.5, 250.0, 312.5, 625.0), inset: DPPH, chitosan, BTDA and CPC and (b) CPBC-2 from different Py:BTDA ratios (125.0, 42.5, 14.3, 4.8), vitamin C (- - -) and vitamin E (. . .).
Fig. 5. (a) Cyclic voltammetry (3rd cycles) with CPBC-2 sample as a working electrode. Samples (length 1.0 cm, width 0.5 cm, thickness 40 m) of CPBC-2 (Py:BTDA ratio 42.5) same film is exposed to DPPH assay (1 mM in 50 ml methanol) reacted for 24 h for repeated measurements during 4 days, while another sample of the same film was not reduced (—), day 1 (. . .), day 2 (- - -), day 3 (-.-), day 4 (-..) and (b) the radical scavenger activity (RSA) of not reduced and reduced samples in repeated experiments of three different films.
DPPH reaction mixtures. The applied sample amounts were: CPC (Py:NaNO2 ratio 4.0, ∼20 mg), CPBC-1 (∼20 mg), CPBC-2 (∼2 mg), chitosan (∼10 mg), BTDA (∼2 mg), vitamin C (∼0.5 mg, ∼150 M) and vitamin E (∼1.3 mg, 150 M). The results (RSA) of the testing samples CPC, CPBC-1 with different Py:BTDA ratios (62.5, 125.0, 187.5, 250.0, 312.5, and 625.0) and CPBC-2 with different Py:BTDA ratios (125.0, 42.5, 14.3, 4.8) are presented in Fig. 4. The radical scavenger activity (RSA) of CPC (Fig. 4a, inset) was found to be in the range of 26% after 24 h. We assume that the lower radical scavenger activity of CPC sample (Fig. 4a, inset) is due to the isolated porphine ring structure in chitosan. CPC samples can be dissolved in light acetic acid but show no film formation if coated on glass. Chitosan shows no antioxidant properties itself but BTDA has some radical scavenger activity [35]. CPBC-1 and CPBC-2 samples were formed by including BTDA in the polymerization. With increasing amount of Py (increasing Py:BTDA ratio, Fig. 4a) the radical scavenger activity of CPBC-1 samples increases up to Py:BTDA ratio 312.5 (RSA = 80%) with hydrated film conductivity in the range of 8 × 10−4 S cm−1 (film thickness 50 m). RSA decrease at Py:BTDA ratio 625.0 (RSA = 50%) is accompanied also with lower conductivity in the range of 1.5 × 10−4 S cm−1 (film thickness 80 m). We assume that at constant oxidant concentration with higher amount of Py, only shorter fragments of conjugated PPy are formed, affecting the conductivity of the sample and leading to the decrease of the radical scavenger activity. Additionally, the radical scavenger properties of samples are lowered by the preparation method
that includes the precipitation of the sample at pH 10–11, causing over-oxidation [36] which reduces the radical scavenger activity [18]. CPBC-2 (Py:BTDA ratio 42.5) sample shows the best antioxidant properties in this study; the measured RSA of 32% is well comparable to vitamin E (29%) and C (69%) at similar concentrations. We assume that in case of CPBC-2 samples (Py:BTDA ratio 42.5), the cross-linking and antioxidant functionality are present in the right proportion. In case of lower Py:BTDA ratios 14.3 and 4.8, the antioxidant properties are reduced, which we assume to be due to the lower stability of the sample. The increased concentration of BTDA in the CPBC-2 film (Py:BTDA ratio 4.8) also adversely effects the conductivity of the sample (thickness 40 m, hydrated, 5 × 10−4 S cm−1 ) as compared to that of CPBC-2 (Py:BTDA ratio 42.5, thickness 60 m, 1.5 × 10−3 S cm−1 ). From the coated CPBC-2 probe (Py:BTDA ratio 42.5) films were obtained from which samples of defined size (length 1.0 cm, width 0.5 cm, thickness 40 m)were cut. One of the samples was electrochemically reduced each day prior to the exposure to DPPH solution. The other sample from the same film was untreated electrochemically, but exposed to the DHHP solution in the same manner. The experiment was repeated three times, the repeatability reporeted as standard deviation values and error bars. The unreduced CPBC-2 sample showed very low electroactivity (Fig. 5a, (–)), and the radical scavenger activity (RSA) was in the range of 18%. With no electrochemical treatment, the RSA
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value decreased continuously, reaching 12% after 4 days of testing (Fig. 5b). The second CPBC-2 sample from the same film underwent redox cycling and was exposed to the DPPH solution in the reduced state every day. The radical scavenger strength for the next 1–4 days was found to be in the range of 50%, the electroactivity of the sample was also increased, accompanied by the appearance of an oxidation peak at 400 mV and reduction peak at 185 mV. The results of this investigation indicate that the antioxidant properties of CPBC samples are activated by redox cycling and the increased activity is renewable. 4. Conclusion NaNO2 was applied as the oxidant in the depolymerization reaction of chitosan, forming chitosan chains with aldehyde residual groups. Chitosan–PPy composites (CPC) were obtained by adding Py to the depolymerized chitosan solution with a small amount of NaNO2 as the radical starter. CPC samples are suspensible in light acetic acid but no film formation was obtained due to poor mechanical properties. UV–vis and FTIR measurements revealed that the reactive species forming CPCs (Py:NaNO2 ratio 4.0–21.0) are the aldehyde residual groups of the chitosan chains, leading to the formation of small amounts of porphine moieties. At higher acetic acid concentrations (20%), the formation of porphyrins is inhibited. Further studies will focus on obtaining higher yield of porphyrin in CPC samples with new application envisaged for sensors and electronic wires. To increase the mechanical properties of CPC, cross-linking agent BTDA was included in the polymerization, also inhibiting the formation of porphyrins, but significantly increasing the stability and antioxidant strength of the composites. Radical scavenger activity of the suspensible CPC (Py:NaNO2 ratio 4.0) and CPBC at different Py:BTDA ratios was demonstrated through their reaction with the DPPH free radicals. The antioxidant properties of the CPBC sample with Py:BTDA ratio 42.5 were found to be comparable to those of vitamin E, while having the advantage of being renewable by means of electrochemical cycling. The electrochemical treatment also further activates the antioxidant activity. Such novel composites could be considered for biomedical or food packaging applications. Acknowledgements The research was supported by the European Union through the European Social Fund (MTT76) and partly through Industrial Technology Research Institute (7301XSY581).
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Contents lists available at SciVerse ScienceDirect
Synthetic Metals journal homepage: www.elsevier.com/locate/synmet
Two formation mechanisms and renewable antioxidant properties of suspensible chitosan–PPy and chitosan–PPy–BTDA composites Rong-Jay Lee a , Tarmo Tamm b , Rauno Temmer b , Alvo Aabloo b , Rudolf Kiefer a,b,∗ a b
Industrial Technology Research Institute (ITRI), Kuang Fu Rd 321, 30011 Hsinchu, Taiwan University of Tartu, Institute of Technology, Intelligent Materials and Systems Lab, Nooruse 1, 50114 Tartu, Estonia
a r t i c l e
i n f o
Article history: Received 25 October 2012 Received in revised form 6 December 2012 Accepted 17 December 2012 Available online 22 January 2013 Keywords: Chitosan–PPy composite Oxidant NaNO2 Porphyrin Chitosan–PPy–BTDA composite Renewable antioxidant property
a b s t r a c t Treatment of chitosan together with pyrrole (Py) by oxidant sodium nitrite (NaNO2 ) forms unique chitosan–polypyrrole composites suspensible in diluted acetic acid. It was discovered that the reactive species polymerizing Py into polypyrrole (PPy) are the aldehydes, formed in situ during the depolymerization reaction of chitosan with NaNO2 . In situ UV–vis and FTIR measurements identify typical porphyrin signals, shedding light to the formation mechanism. To improve the film formation of the composites, the application of additional cross-linking agents such as 3,3 ,4,4 -benzophenone tetracarboxylic dianhydride (BTDA) was investigated. The obtained suspensible chitosan–PPy–BTDA composites showed renewable (by electrochemical means) antioxidant properties comparable in strength to those of natural vitamins. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Chitosan, obtained from chitin over N-deacetylation is a natural biopolymer, non-toxic and soluble in light acid, known to show antimicrobial activity [1]. In recent years, many attempts have been made to increase the applicability range of chitosan by grafting polymerization with suitable polymers, producing chitosan–gallic acid [2], carboxymethyl chitosan [3,4], or by cross-linking chitosan with glutardialdehydes [5]. For increasing the solubility of chitosan in aqueous media, certain depolymerization techniques to obtain chain-scission can be applied by using oxidants such as ammonium persulfate (APS) [6], potassium persulfate (PPS) [7], 2,2 -azobis(2-methylpropionitrile) (AIBN) [8] and strong acids [9]. The most common depolymerization agent for chitosan is perhaps sodium nitrite (NaNO2 ) [10]. The use of different depolymerization agents reflects on the endproducts of chitosan which in case of NaNO2 are aldehyde rest products [11]. The polymerization of pyrrole (Py) with oxidant NaNO2 , in light acetic acid conditions forming conductive polypyrrole (PPy) coatings on silica surfaces has been mentioned in two reports [12,13]. The reactive species were identified as in situ formed nitrosonium cations (NO+ ). Whether the reactive species
∗ Corresponding author at: University of Tartu, Institute of Technology, Intelligent Materials and Systems Lab, Nooruse 1, 50114 Tartu, Estonia. Tel.: +372 737 4826; fax: +372 737 4825. E-mail addresses: [email protected] (R.-J. Lee), [email protected] (T. Tamm), [email protected] (R. Temmer), [email protected] (A. Aabloo), [email protected], [email protected] (R. Kiefer). 0379-6779/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.synthmet.2012.12.032
starting the polymerization of Py in depolymerized chitosan are the aldehydes or the nitrosonium, is investigated in this study. Recent attempts of forming composites of chitosan with PPy have been made by applying oxidants such as APS [14] or iron trichloride (FeCl3 ) [15]. The obtained composites did show suspensibility in aqueous or light acid solutions, explained by concentration effects between depolymerized chitosan and the formed PPy [15]. For obtaining conductive materials, chitosan–PPy composites (CPC) can be made; being envisaged for biosensor or biomedical applications [16]. Chemically polymerized PPy in form of powder has been found effective as a radical scavenger material [17–19], as shown by the reaction with di(phenyl)-(2,4,6trinitrophenyl)iminoazanium (DPPH) radicals [20]. Suspensible CPC with antioxidant properties may provide a solution for the shortcoming in processability of conducting polymer powders having rather low solubility. To increase the stability of the composite film, cross-linking agents such as 3,3 ,4,4 -benzophenone tetracarboxylic dianhydride (BTDA) [21] are applied in this study to form chitosan–PPy–BTDA composites (CPBC), and the effect of Py and BTDA concentrations in view of antioxidant properties and film stability are investigated to envisage novel material applicable in food packaging or biosensor functionality. 2. Experimental 2.1. Chemicals Chitosan (middle molecular weight, 103,000 g mol−1 , deacetylation degree 85%), acetic acid (97%), NaNO2 , ammonium
R.-J. Lee et al. / Synthetic Metals 164 (2013) 6–11
7
Scheme 1. Depolymerization of chitosan [11] leads to 2,5-anhydro-d-mannose (P1). P1 and NaNO2 can form P2 (porphyrin) P3 products.
peroxydisulfate ((NH4 )2 S2 O8 , 98%), Py (98%), BTDA, (95%), pentane-1,5-dial (glutardialdehyde, GDA, 25% aqueous solution), DPPH, vitamin E and C were obtained from Sigma–Aldrich. Py was distilled and stored under nitrogen at 4 ◦ C prior to use. 2.2. Suspensible CPC 2.2.1. CPC material 2 g of chitosan was dissolved in 50 ml 1.5% acetic acid to form a 4% solution. The solution was stirred under ice cooling, NaNO2 (25 mg, 7.2 mM) was added in small portions during 30 min, followed by reaction time of 2 h until no N2 gas release was detected. Related to the deacetylation degree of chitosan, the ratio chitosan:NaNO2 was in the range of 1:70. After depolymerization of chitosan, additional NaNO2 (25 mg, 7.2 mM) and Py (0.2 M) were added, and the mixture stirred at 50 ◦ C for 2 h. The CPC compounds were obtained by increasing the pH to 10–11; very fine particles were obtained which were soluble in light acetic acid. 2.2.2. CPBC material Different concentrations of Py (0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M and 1.0 M) with constant concentration of NaNO2 (7.2 mM) and constant concentration of BTDA (1.6 mM) were added to the depolymerized chitosan solution with additional stirring for 3 h at 50 ◦ C. CPBC-1 samples with Py:BTDA ratios 62.5, 125.0, 187.5, 250.0, 312.5, 625.0 were obtained. CPBC-2 samples with different concentrations of BTDA (1.6 mM, 4.7 mM, 14.0 mM, 42.0 mM), constant Py concentration (0.2 M) and constant NaNO2 concentration (7.2 mM) correspond to different Py:BTDA ratios 125.0, 42.5, 14.3, 4.8. To obtain solid composites, the pH value of the solution was increased to pH 10–11 (by adding sodium hydroxide) and the precipitate was washed several time with MilliQ and acetone to remove excess of Py and BTDA, and dried in the oven (40 ◦ C, 24 h, 600 mbar). The solid composites were dissolved in 1.5% acetic acid and in case of CPBC samples, coated on a glass plate to form a film, and dried in the oven.
2.3. Characterization The molecular structure of CPC samples was investigated with Fourier transform infrared (FTIR) spectroscopy (Spectrum One, PerkinElmer) with KBr pressed disks and UV–vis spectroscopy (V550, Jasco, samples dispersed in 1.5% acetic acid). In situ UV–vis was made to investigate the influence of the Py:NaNO2 ratio on Py polymerization in chitosan. In situ FTIR measurements were performed on a KBr plate where chitosan was depolymerized with NaNO2 solution over 2 h and after drying of the probe surface, Py was added and the formation of composites was observed at different time intervals. Cyclic voltammograms (Eco Chemie Autolab, scan rate 10 mV s−1 , ± 0.8 V) of the CPC films (average thickness between 40 and 70 m) were performed in a three electrode cell (working electrodes were CPC or CPBC samples clamped on Ptsheet, counter electrode was Pt-sheet, reference electrode Ag/AgCl (3 M KCl)) in 0.2 M tetramethylammonium chloride (TMACl) electrolyte. The conductivity of the samples (in dry and hydrated state) was studied by means of electrochemical impedance spectroscopy (Eco Chemie Autolab). 2.4. Radical scavenger activity of CPC and CPBC samples Radical scavenger properties of CPC and CPBC (also in form of solid films) samples were determined with a prepared DPPH assay test [18]. The DPPH radical typically extracts a proton from the testing sample forming DPPHH, which can be observed by UV–vis measurements. The solution was shaken and stored for 30 min at room temperature (RT) before the samples were added. The main adsorption peak in UV–vis spectrum for DPPH was observed at 517 nm, the absorbance of this peak was set as the control value (C0 ). The decrease of the 517 nm peak determines the value C1 and with following Eq. (1) the radical scavenger activity (RSA) was calculated [22]. RSA =
C0 − C1 × 100% C0
(1)
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upper bipolaron band of PPy. Ratio 4.0 shows new a peak at 645 nm and a broad wave at 800 nm. The existence of the broad wave between 800 and 900 nm indicates that bipolaron bands of conjugated PPy are formed [27] indicating further oxidation of the PPy chains and the formation of new (more) conjugated structures. With ratios 4.0–21.0, the 527 nm and 645 nm peaks increase in their adsorption. The ratio 4.0 spectrum reminds a typical porphine ring structure with the Soret-band at 416 nm and the Q-bands at 530 nm and 650 nm [28]. In comparison to CPS polymerized using APS, the UV–vis spectra reported by Li et al. [14] show typical signals at 460 nm (–* transition) and 900 nm associated to the bipolaron state of PPy, which are different from those seen in Fig. 1a. The typical chitosan signals (Fig. 1b) in the CPC sample are recorded at 3420, (O H stretching vibration), 1643 ( CONH stretching vibration), 1410 ( CH2 bending), 1257 (C O C valence) and 1153 cm−1 (anti-symmetric stretching of the C O C bridge) [6]. The bands at 1552 cm−1 (2,5-substituted Py) are showing typical PPy ring vibrations [29]. The decrease of the 3420 cm−1 broad peak after certain polymerization time (Fig. 1b, 10 min–2 h) identifies the formation of more PPy; the 1552 cm−1 peak, splitting to two additional peaks at 1541 cm−1 and 1563 cm−1 is assumed to belong to two different substitutes of Py. Porphyrine signals [30] are reported at 3051 (N H), 1594 (C C), 1352 (C N) and 732 cm−1 ( C H, aromatic ring vibration). In CPC composites, out of these signals considering expected shifts, only small peaks at 1659 (CN), 1456 (CC), 1345 (C N), and 722 cm−1 ( C H) could be found [27]. Recent reports discovered that in mild acidic conditions (pH > 4), NO+ ions are formed from nitrites (Eq. (2)) which can initiate the polymerization of Py, leading to a conductive PPy layer on silica surface [12,13]: Fig. 1. (a) In situ UV–vis measurements (270–900 nm) of CPC samples polymerized for 2 h at different Py:NaNO2 ratios: 0.2 (– – ), 0.4 (–. .), 0.6 (. . .), 4.0 (—), 13.0 (. . .) and 21.0 (- - -); (b) in situ FTIR spectra (4000–450 cm−1 ) of CPC (ratio 4.0) measured after 10 min (–. . .), 30 min (. . .), 1 h (- - -) and 2 h (—).
3. Results and discussion
HNO2 + H+ ↔ H2 NO2 + ↔ NO+ + H2 O
(2)
Reaction of Py with NaNO2 (in 1.5% acetic acid) turned the mixture from brown to black after short time, but PPy powder in sufficient amounts could not be obtained. In situ UV–vis measurements were made to investigate which product is obtained at different polymerization conditions (Samples 1–4) (Fig. 2a):
3.1. CPC composite analysis The polymerization procedure to obtain CPC samples requires oxidants which perform two functions: depolymerization of chitosan and polymerization of Py. In case of NaNO2 as oxidant, the free amino groups of chitosan are attacked by nitrous acid HNO2 in 1:1 stoichiometry [10,11] (Scheme 1). The chitosan glycosidic bond is broken and 2,5-anhydro-dmannose (P1) and hydroxyl residual groups are formed [11]. Adding Py without NaNO2 leads to no product, therefore additional NaNO2 as radical starter was included in the reaction. Meso-tetrasubstituted porphyrin was first synthesized by Rothemund [23] by heating mixtures of Py up to 200 ◦ C with an aldehyde in pyridine under anaerobic conditions. The Adler–Longo method [24], based on the reaction at high temperature (200 ◦ C) in acetic or propanoic acid and aerobic condition gives a better yield (10–30%). Depolymerized chitosan contains aldehydes (2,5-anhydro-d-mannose) which can react with Py monomer and additional oxidant NaNO2 in the right stoichiometry [25] to produce P2 (Scheme 1). It is not clear which conditions determine whether porphyrins (P2) or PPy derivates (P3) are formed. In order to identify the reactive species interacting with Py in situ UV–vis and FTIR measurements were performed (Fig. 1) The Py:NaNO2 ratios 0.2, 0.4, and 0.6 with excess of oxidant revealed one strong peak at 416 nm and a weak broad peak at 527 nm (Fig. 1a), which are described in the literature [26] as belonging to constitute transitions from the valence band to the
Sample 1: Chitosan + NaNO2 (reaction 2 h at RT in 1.5% acetic acid) + Py + NaNO2 ; Sample 2: Chitosan + NaNO2 + Py (in 1.5% acetic acid); Sample 3: Pyrrole + NaNO2 (in 1.5% acetic acid); Sample 4: GDA + NaNO2 + Py in (1.5% acetic acid). The effect of higher acetic acid concentration (1.5%, 5%, 10%, and 20%) was studied on samples 1–3. The results of the studies are presented in Fig. 2b. Sample 2 (. . .) and Sample 3 (- - -) in Fig. 2a demonstrate that the reaction without aldehyde formation leads to PPy formation with peaks at 350, 507, and 652 nm, and we assume that the active oxidant is NaNO2 . If GDA is present (Fig. 2a, Sample 4, -·-·), the reactive species oxidizing Py are the aldehydes, and peak positions at 414, 530, and 650 nm are similar to Sample 1 (–). It becomes clear that these new PPy peaks appear due to a different mechanism in contrast to samples 2–3. We assume that for Sample 1 a small amount of porphyrin is formed (Scheme 1, P2), and products of conjugated PPy in combination with chitosan are obtained in the bulk reaction. If the acetic acid concentration is varied (Fig. 2b), the peaks at 414, 511, and 655 nm decrease with increased acid concentration. In case of 20% acetic acid (Fig. 2b, inset), similar peaks for all samples 1–3 appear at 313, 511, and 655 nm. This indicates that for acetic acid concentrations up to 20%, the reactive species polymerizing Py is NaNO2 . At higher acetic acid concentrations, we assume that
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Fig. 2. In situ UV–vis spectra (270–900 nm) of samples with Py:NaNO2 ratio 4.0, (a) chitosan–NaNO2 (2 h depolymerization reaction) + Py (Sample 1, –), chitosan–NaNO2 + Py (Sample 2, . . .), NaNO2 + Py (Sample 3, - - -), and GDA + NaNO2 + Py (Sample 4, -·-·) and (b) Sample 1 polymerized at different acetic acid concentrations (1.5% (. . .), 5% (- - -), 10% (-..), and 20% (–)) with inset: Sample 1 (–), Sample 2 (. . .), and Sample 3 (- - -) at acetic acid concentration 20%.
higher dissociation degree of chitosan [31] inhibits the formation of aldehydes. Earlier investigations of PPy formation with NaNO2 proposed that the oxidant performing the reaction is the in situ formed NO+ cation (Eq. (2)). The corresponding UV–vis measurement shows a broad peak in the range between 350 and 380 nm, identified as conjugated PPy (–* transition) [12,32]. Agreeing with the aforementioned study [32], an adsorption peak at 350 nm (Fig. 2a) is found. Additional peaks at 511 and 655 nm describe the transition state to polaron–bipolaron bands of PPy. Further work is needed to obtain higher yields of porphine ring structures in chitosan with new application envisaged for sensors and electronic wires [33]. 3.2. CPBC analysis To increase the CPC film stability, certain amount of BTDA in function of a cross-linking agent was added during the polymerization procedure. The BTDA reacts with the free amino groups of chitosan, forming imides, leading to the cross-linking of the depolymerized chitosan [21]. CPBC samples are suspensible in acetic acid and could be coated on glass forming a stable film. To investigate the influence of Py concentration on the polymerization process (1.6 mM BTDA, 7.2 mM NaNO2 ), UV–vis and FTIR spectral measurements at different Py:BTDA ratios were performed (Fig. 3). The UV–vis spectrum of CPBC samples (Fig. 3a) with Py:BTDA ratio 62.5 shows two peaks at 470 and 608 nm. With the increasing amount of Py (increasing Py:BTDA ratio) the 470 nm peak decreases
9
Fig. 3. (a) UV–vis (350–900 nm) spectra of CPBC-1 samples at different Py:BTDA ratios: 62.5 (—), 125.0 (. . .), 187.5 (- - -), 250.0 (-..), 312.5 (-.-) and 625.0 (- -) and (b) FTIR spectrum (2000–450 cm−1 ) of CPBC-1 sample (Py:BTDA ratio 125.0).
and the 608 nm peak shifts to 635 nm and increases. In comparison to Fig. 2a, the inclusion of BTDA inhibits the formation of the porphyrin, observed as the disappearance of the 414 and 507 nm peaks. The FTIR spectrum (Fig. 3b) of CPBC (Py:BTDA ratio 125.0) shows chitosan signals at 1639 cm−1 ( CONH stretching vibration) and 1055 cm−1 (skeletal vibration involving C O C stretching) [6]. The presence of PPy in CPBC is indicated by peaks at 1537 cm−1 (2,5-substituted Py) and 922 cm−1 ( C H out of plane vibration, indicating polymerization of Py) [34]. BTDA connection can be found as small peaks at 1788, 1741 (symmetrical C O stretching and asymmetric C O stretching of the imide group), and 733 cm−1 (imide ring deformation) [21]. We assume that the BTDA crosslinking agent prevents porphyrin formation due to steric reasons of the depolymerized chitosan chains that are unable to interact with Py in the right position. Therefore the formation of PPy in CPBC samples is caused by in situ formed NO+ (Eq. (2)) [12] leading to a different structure of PPy in comparison to the CPC samples (Fig. 3a vs Fig. 1a). 3.3. Antioxidant characterization of the composites Earlier studies have discovered that polyaniline and PPy possess effective radical scavenger properties, in the latter case 2–4 Py units react with one DPPH radical [18,19]. Small amounts of testing samples were dissolved in 1.5% acetic acid (BTDA in acetone). The decrease of the 517 nm peak (UV–vis analysis) in DPPH standard solution (20 ml methanol, 200 M) determines the radical scavenger properties of the testing samples. The loss of activity of the pure DPPH solution in absence of the testing samples in time was deducted from the measurement results of the
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Fig. 4. Radical scavenger activity (RSA) at different time frames (30 min, 1 h, 2 h, 4 h, 24 h), (a) CPBC-1 from different Py:BTDA ratios (62.5, 125.0, 187.5, 250.0, 312.5, 625.0), inset: DPPH, chitosan, BTDA and CPC and (b) CPBC-2 from different Py:BTDA ratios (125.0, 42.5, 14.3, 4.8), vitamin C (- - -) and vitamin E (. . .).
Fig. 5. (a) Cyclic voltammetry (3rd cycles) with CPBC-2 sample as a working electrode. Samples (length 1.0 cm, width 0.5 cm, thickness 40 m) of CPBC-2 (Py:BTDA ratio 42.5) same film is exposed to DPPH assay (1 mM in 50 ml methanol) reacted for 24 h for repeated measurements during 4 days, while another sample of the same film was not reduced (—), day 1 (. . .), day 2 (- - -), day 3 (-.-), day 4 (-..) and (b) the radical scavenger activity (RSA) of not reduced and reduced samples in repeated experiments of three different films.
DPPH reaction mixtures. The applied sample amounts were: CPC (Py:NaNO2 ratio 4.0, ∼20 mg), CPBC-1 (∼20 mg), CPBC-2 (∼2 mg), chitosan (∼10 mg), BTDA (∼2 mg), vitamin C (∼0.5 mg, ∼150 M) and vitamin E (∼1.3 mg, 150 M). The results (RSA) of the testing samples CPC, CPBC-1 with different Py:BTDA ratios (62.5, 125.0, 187.5, 250.0, 312.5, and 625.0) and CPBC-2 with different Py:BTDA ratios (125.0, 42.5, 14.3, 4.8) are presented in Fig. 4. The radical scavenger activity (RSA) of CPC (Fig. 4a, inset) was found to be in the range of 26% after 24 h. We assume that the lower radical scavenger activity of CPC sample (Fig. 4a, inset) is due to the isolated porphine ring structure in chitosan. CPC samples can be dissolved in light acetic acid but show no film formation if coated on glass. Chitosan shows no antioxidant properties itself but BTDA has some radical scavenger activity [35]. CPBC-1 and CPBC-2 samples were formed by including BTDA in the polymerization. With increasing amount of Py (increasing Py:BTDA ratio, Fig. 4a) the radical scavenger activity of CPBC-1 samples increases up to Py:BTDA ratio 312.5 (RSA = 80%) with hydrated film conductivity in the range of 8 × 10−4 S cm−1 (film thickness 50 m). RSA decrease at Py:BTDA ratio 625.0 (RSA = 50%) is accompanied also with lower conductivity in the range of 1.5 × 10−4 S cm−1 (film thickness 80 m). We assume that at constant oxidant concentration with higher amount of Py, only shorter fragments of conjugated PPy are formed, affecting the conductivity of the sample and leading to the decrease of the radical scavenger activity. Additionally, the radical scavenger properties of samples are lowered by the preparation method
that includes the precipitation of the sample at pH 10–11, causing over-oxidation [36] which reduces the radical scavenger activity [18]. CPBC-2 (Py:BTDA ratio 42.5) sample shows the best antioxidant properties in this study; the measured RSA of 32% is well comparable to vitamin E (29%) and C (69%) at similar concentrations. We assume that in case of CPBC-2 samples (Py:BTDA ratio 42.5), the cross-linking and antioxidant functionality are present in the right proportion. In case of lower Py:BTDA ratios 14.3 and 4.8, the antioxidant properties are reduced, which we assume to be due to the lower stability of the sample. The increased concentration of BTDA in the CPBC-2 film (Py:BTDA ratio 4.8) also adversely effects the conductivity of the sample (thickness 40 m, hydrated, 5 × 10−4 S cm−1 ) as compared to that of CPBC-2 (Py:BTDA ratio 42.5, thickness 60 m, 1.5 × 10−3 S cm−1 ). From the coated CPBC-2 probe (Py:BTDA ratio 42.5) films were obtained from which samples of defined size (length 1.0 cm, width 0.5 cm, thickness 40 m)were cut. One of the samples was electrochemically reduced each day prior to the exposure to DPPH solution. The other sample from the same film was untreated electrochemically, but exposed to the DHHP solution in the same manner. The experiment was repeated three times, the repeatability reporeted as standard deviation values and error bars. The unreduced CPBC-2 sample showed very low electroactivity (Fig. 5a, (–)), and the radical scavenger activity (RSA) was in the range of 18%. With no electrochemical treatment, the RSA
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value decreased continuously, reaching 12% after 4 days of testing (Fig. 5b). The second CPBC-2 sample from the same film underwent redox cycling and was exposed to the DPPH solution in the reduced state every day. The radical scavenger strength for the next 1–4 days was found to be in the range of 50%, the electroactivity of the sample was also increased, accompanied by the appearance of an oxidation peak at 400 mV and reduction peak at 185 mV. The results of this investigation indicate that the antioxidant properties of CPBC samples are activated by redox cycling and the increased activity is renewable. 4. Conclusion NaNO2 was applied as the oxidant in the depolymerization reaction of chitosan, forming chitosan chains with aldehyde residual groups. Chitosan–PPy composites (CPC) were obtained by adding Py to the depolymerized chitosan solution with a small amount of NaNO2 as the radical starter. CPC samples are suspensible in light acetic acid but no film formation was obtained due to poor mechanical properties. UV–vis and FTIR measurements revealed that the reactive species forming CPCs (Py:NaNO2 ratio 4.0–21.0) are the aldehyde residual groups of the chitosan chains, leading to the formation of small amounts of porphine moieties. At higher acetic acid concentrations (20%), the formation of porphyrins is inhibited. Further studies will focus on obtaining higher yield of porphyrin in CPC samples with new application envisaged for sensors and electronic wires. To increase the mechanical properties of CPC, cross-linking agent BTDA was included in the polymerization, also inhibiting the formation of porphyrins, but significantly increasing the stability and antioxidant strength of the composites. Radical scavenger activity of the suspensible CPC (Py:NaNO2 ratio 4.0) and CPBC at different Py:BTDA ratios was demonstrated through their reaction with the DPPH free radicals. The antioxidant properties of the CPBC sample with Py:BTDA ratio 42.5 were found to be comparable to those of vitamin E, while having the advantage of being renewable by means of electrochemical cycling. The electrochemical treatment also further activates the antioxidant activity. Such novel composites could be considered for biomedical or food packaging applications. Acknowledgements The research was supported by the European Union through the European Social Fund (MTT76) and partly through Industrial Technology Research Institute (7301XSY581).
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