Chitosan Film Containing an Iron Complex: Synthesis and Prospects

Departamento de Quı́mica Analı́tica e Fı́sico-Quı́mica, Universidade Federal do Ceará, 60440-900 Fortaleza, Ceará, Brazil. J. Agric. Food Ch...
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Chitosan Film Containing an Iron Complex: Synthesis and Prospects for Heterocyclic Aromatic Amines (HAAs) Recognition Maria Aparecida S. da Silva,† Dieric S. Abreu,† Leandro A. Costa,† Natanna de A. Aguiar,† Tércio F. Paulo,† Elisane Longhinotti,‡ and Izaura C. N. Diógenes*,† †

Departamento de Quı ́mica Orgânica e Inorgânica, Universidade Federal do Ceará, Cx. Postal 6021, 60455-760 Fortaleza, Ceará, Brazil ‡ Departamento de Quı ́mica Analı ́tica e Fı ́sico-Quı ́mica, Universidade Federal do Ceará, 60440-900 Fortaleza, Ceará, Brazil S Supporting Information *

ABSTRACT: Hybrid organic−inorganic materials have been seen as a promising approach to produce sensors for the detection and/or recognition of heterocyclic aromatic amines (HAAs). This work shows the synthesis of a hybrid film as a result of the incorporation of [Fe(CN)5(NH3)]3− into chitosan (CS); CS[(CN)5Fe(NH3)]3−. The sensitivity of CS[(CN)5Fe(NH3)]3− toward HAA-like species was evaluated by using pyrazine (pz) as probe molecule in vapor phase by means of electrochemistry and spectroscopic techniques. The crystallinity (SEM-EDS and XRD) decrease of CS[(CN)5Fe(NH3)]3− in comparison to CS was assigned to the disturbance of the hydrogen bond network within the polymer. Such conclusion was reinforced by the water contact angle measurements. The results presented in this work indicate physical and intermolecular interactions, mostly hydrogen bond, between [Fe(CN)5(NH3)]3− and CS, where the complex is likely trapped in the polymer with its sixth coordination site available for substitution reactions. KEYWORDS: chitosan, coordination compound, hybrid materials, heterocyclic aromatic amines, pyrazine

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INTRODUCTION The human diet may contain potentially harmful substances such as microbial contaminants, toxins, or undesirable chemicals. A group of toxic chemicals that are currently under scrutiny are the heterocyclic aromatic amines (HAAs). Such compounds are usually formed during cooking of proteinrich foods at temperatures >150 °C.1,2 Several HAAs have been found to possess very potent mutagenic activity in the Ames Salmonella assay3 and are multisite carcinogens in rodents and in non-human primates.4,5 Epidemiological investigations have shown the correlation of HAA intake with the increased risk of human cancers such as colon, prostate, and mammary gland.6−11 Moreover, DNA adducts of HAAs have been detected in human tissues, demonstrating that HAAs induce genetic damage even at concentrations as low as the parts per billion (ppb) range.3 More than 25 different HAAs have been isolated and identified as potent mutagens. All of these heterocyclic amines contain from two to five (generally three) condensed aromatic cycles with one nitrogen atom or more in their ring system and, usually, one exocyclic amino group.12 For such reasons, the International Agency of Research on Cancer (IARC) recommends limiting daily intake of HAAs.13 Given the toxicology results reported on the consumption of food containing HAAs, there has been a growing demand for methods of identification and quantification of these types of xenobiotics in different foods.14−23 Currently, HAAs have been detected by using gas chromatography (GC), mass spectrometry (MS), ultraperformance liquid chromatography (UPLC), and synchronous fluorescence spectroscopy (SFS).14−23 Therefore, further investigations are needed aiming to develop effective fast and low-cost methods © 2017 American Chemical Society

for the determination of HAAs. The combination of organic and inorganic domains in a hybrid material is a promising approach toward new functionalities, which may arise either from the synergistic interaction between the different domains or from the simple addition of the properties of both domains in a single matrix, such as chitosan.24−30 The biopolymer chitosan (CS) presents the additional advantage of easily forming thin films whose chemical structure includes numerous oxygen- and nitrogen-based groups for incorporating functional substances through covalent and/or physical modifications, making CS films suitable materials for the construction of analytical sensors.31,32 Coordination compounds are known as colored species due to the electronic transitions that are dependent on the Lewis bases (ligands) directly bonded to the metal center. This feature has been used, for instance, to build hybrid organic−inorganic structures by mixing cyanoferrate complexes with organic polymers.33−43 Getting together the known kinetic lability of the sixth ligand (L = NH3 or H2O) in pentacyanoferrate type complexes, [Fe(CN)5(L)]3−, with the ability of chitosan to easily form thin films, we carried out the incorporation of the [Fe(CN)5(NH3)]3− ion complex in the CS matrix. The sensitivity of the produced hybrid material in electrochemical and nakedeye colorimetric recognition of HAA-like species was evaluated by using pyrazine (pz) as probe molecule. Received: Revised: Accepted: Published: 1387

August 20, 2016 January 26, 2017 January 26, 2017 January 26, 2017 DOI: 10.1021/acs.jafc.6b03742 J. Agric. Food Chem. 2017, 65, 1387−1394

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Journal of Agricultural and Food Chemistry

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PINAACLE 900T. X-ray diffraction (XRD) data were recorded on a DMAXB Rigaku diffractometer using Cu Kα radiation (1.5418 Å), operated at 40 kV and 30 mA in the 2θ range from 5 to 90°. All samples were measured at 298 K. The static contact angles for the film samples were measured at ambient condition using a contact angle instrument model GBX Instrumentation Scientifique, within 10 s, after placement of a 15 μL drop of deionized water on the film surface. For each sample, contact angles of five different positions on the surface were measured and calculated by sessile drop method by using Visiodrop software considering the average value. Cyclic voltammetry measurements were carried out on a computer-controlled EC Epsilon Potentiostat (BAS − Bioanalytical Systems, Inc., West Lafayette, IN, USA) by using a conventional three-electrode glass cell with a platinum wire as an auxiliary electrode, Ag/AgCl electrode as reference (saturated KCl) and laboratory-made PGE (A = 0.1 cm2), CS−PGE, and CS[(CN)5Fe(L)]3−−PGE as working electrodes. A 0.1 mol L−1 aqueous solution (pH 7.0) of NaCF3COO was used as supporting electrolyte. Electrolyte solution was deoxygenated by bubbling argon for 30 min before the measurements. Absorption spectra in the ultraviolet and visible (UV−vis) regions were taken for the samples dispersed in KBr powder and films on a Varian Cary 5000 UV−visNIR spectrophotometer. Fourier transform infrared (FTIR) spectra of the solid samples dispersed in KBr powder and of the films were carried out by using a FT-IR ABB Bomen FTLA 2000-102 spectrometer at a resolution of 4 cm−1 in a frequency range of 4000−400 cm−1. Computational Details. Density functional theory (DFT) calculations, at the B3LYP/Lanl2dz/6-311++g(d,p) level of theory, were performed to analyze which linkage isomer is most stable for the [Fe(CN)5IQx]3− ion complex, where IQx = quinoxaline derivatives. Structural optimization of the complexes was performed using the Gaussian 09 package,47 revision A.02 (Gaussian Inc., Wallingford, CT, USA), adopting the exchange-correlation B3LYP functional48−50 and the basis sets 6-311++G(d,p) (for C, H, and N) and LANL2DZ (for Ru). The tight criterion was used for a more stringent optimization.

EXPERIMENTAL PROCEDURES

Materials. All reagents were used as received from commercial sources, with no further purification. Chitosan (deacetylation degree of 85%), pyrazine (≥99%), CH3COOH (≥99.7%), and CF3COOH (99%) were purchased from Sigma-Aldrich, and HNO3 (70%) was from J. T. Baker. Sodium nitroprusside, Na2[Fe(CN)5(NO)]·2H2O (99%), was obtained from Merck. Organic solvents and other chemicals were all of analytical grade or comparable purity. Aqueous solutions were prepared using Millipore ultrapure water of at least 18 MΩcm resistance at 25 °C. Na3[Fe(CN)5(NH3)]·3H2O complex was synthesized by using Na2[Fe(CN)5(NO)]·2H2O in concentrated NH4OH, according to the literature.44 Na3[Fe(CN)5(pz)]·3H2O complex was also prepared to serve as a reference compound for the pz coordination to the pentacyanoferrate immobilized in CS matrix. Such a procedure was carried out by the addition of an excess amount of pz ligand on an aqueous solution of Na3[Fe(CN)5(NH3)]·3H2O complex, as reported in the literature.45 Modification of Chitosan Film with [Fe(CN)5(NH3)]3−. Film Preparation. A chitosan aqueous solution of 2% (w/v) was prepared by dissolving 2 g of chitosan powder in 100 mL of acetic acid solution 1% (v/v) under vigorous stirring for 2 h followed by filtering by vacuum to remove any undissolved impurities. Afterward, 5 mL of the freshly prepared CS solutions was reacted with aqueous solutions of [Fe(CN)5(NH3)]3− (2.0 mmol L−1) in CS:complex molar ratios of 1:1 and 1:10. The mixtures were stirred continuously for 3 h, protected from light and at room temperature. The pure CS and CS[(CN)5Fe(NH3)]3− solutions were applied on the surfaces of Teflon plates and air-dried at room temperature for 96 h, thus forming films that were removed from the plates. The stability of the CS[(CN)5Fe(NH3)]3− film under basic, neutral, and acidic conditions, mainly the leaching process of the complex from the polymeric matrix, was investigated by incubating CS[(CN)5Fe(NH3)]3− (30 mg) in solutions of different pH values (5, 7, and 9) at room temperature within a period of 24h. Knowing that the [Fe(CN)5(NH3)]3− complex presents an absorption at ca. 230 nm in the electronic spectrum in the ultraviolet and visible (UV−vis) regions,45 we used such a technique to analyze the solutions as well as the films after the incubation times. Preparation of the Modified Pyrolytic Graphite Electrode (PGE). Previous to the modification step, a pyrolytic graphite electrode (PGE) was polished with a 0.05 μm alumina slurry on a polishing cloth, rinsed thoroughly with water, and sonicated in water for 2 min. The modified PGE was prepared by casting a 300 μL aliquot of CS[(CN)5Fe(NH3)]3− solution on the electrode surface and allowing the solvent to evaporate at room temperature. A PGE coated with pure CS was also obtained, in a similar way, for comparative analysis. The CS and CS[(CN)5Fe(NH3)]3−-modified PGEs were employed as working electrodes in the cyclic voltammetry (CV) experiments. Application of the CS[(CN)5Fe(NH3)]3− Film as a Sensor for Pyrazine. To evaluate the reactivity of CS[(CN)5Fe(NH3)]3− toward the detection of HAA-like contaminants, the CS[(CN)5Fe(NH3)]3− film and a PGE electrode modified with CS[(CN)5Fe(NH3)]3− were placed in sealed vessels containing solid pz, and the substitution reaction was monitored by UV−vis and CV, respectively. All of the measurements were also performed for CS and CS[(CN)5Fe(NH3)]3− films before pz adsorption. For comparative purposes, the substitution reaction was also carried out by immersing the CS[(CN)5Fe(NH3)]3− film in a 0.1 mol L−1 aqueous solution of pyrazine for 3 h. After the incubation time, the films were removed from the solution, washed with water, and dried under reduced pressure. Characterization and Measurements. The morphology of the film surface and the elemental composition of microstructures were investigated by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) using a DSEM962-ZEISS/Inspect 50FEI scanning electron microscope operating at 10−20 kV. Before examination, the samples were coated with gold to make them conductive. The amount of Fe(II) complex within the CS film, previously digested by nitric acid, was determined by atomic absorption spectroscopy (AAS) using a PerkinElmer model

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RESULTS AND DISCUSSION Chitosan-Modified Film: CS[(CN)5Fe(NH3)]3−. Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy. SEM analysis was employed to investigate the morphological changes on the film surfaces after the immobilization of [Fe(CN)5(NH3)]3− into the CS matrix. The SEM micrographs of the nonmodified CS film exhibited a homogeneous, smooth, and nonporous surface with no gross defects, as seen in Figure 1A, which is consistent with the literature.46 In contrast, the micrographs obtained for the CS[(CN)5Fe(NH3)]3− film, Figure 1B, showed a heteroge-

Figure 1. SEM micrographs of the surface of the (A) CS and (B) CS[(CN)5Fe(NH3)]3−; EDS mapping micrographs for Fe Kα spectral line of (C) CS and (D) CS[(CN)5Fe(NH3)]3− films; EDS spectra for (E) CS and (F) CS[(CN)5Fe(NH3)]3−. (Insets) Expanded view of the region from 6 to 10 keV. 1388

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Figure 2. (A) UV−vis spectra and (B) cyclic voltammograms at 0.1 V s−1 of (i) CS, (ii) CS[(CN)5Fe(NH3)]3−, and (iii) CS[(CN)5Fe---pz]3− films. The CVs of the films were obtained on a PGE electrode (see Experimental Procedures for details). Electrolyte solution: 0.1 mol L−1 NaCF3COO (pH 7.0).

neous and irregular surface with flat lamellar phases on which are evident a large number of protruding microfibrils. The different surface morphologies between pure CS and the CS[(CN)5Fe(NH3)]3− films indicate that CS was successfully modified by the iron complex ion. In comparison with the EDS elemental mapping of CS (Figure 1C), the elemental spots seen in the image of CS[(CN)5Fe(NH3)]3−, Figure 1D, indicate not only the presence of Fe atoms but also an almost even distribution of the complex ions within the CS film. This image profile is attributed to the existence of interaction and good compatibility between the CS polymer and [Fe(CN)5(NH3)]3− complex ions.51,52 The X-ray emission peaks observed in the region from 6.0 to 8.0 keV in the EDS spectrum of the CS[(CN)5Fe(NH3)]3− film (Figure 1F) are assigned to Fe Kα and Fe Kβ. In comparison to the spectrum of CS (Figure 1E), where no Fe peaks are seen, and assuming the immobilized cyanoferrate complex is the only source of Fe atoms, the EDS spectra confirm the incorporation of [Fe(CN)5(NH3)]3− into the CS polymer. In addition to gold from the conductive layer, other major elements such as carbon, oxygen, and calcium were also detected in the EDS spectra being derived from the CS matrix. Atomic Absorption Spectroscopy (AAS). For the 1(CS):1(complex) molar ratio used in the synthetic procedure, the AAS analysis of CS[(CN)5Fe(NH3)]3− resulted in 0.083 wt % of Fe2+ ions, indicating an incorporation of 0.48 wt % (14.9 μmol/g) of [Fe(CN)5(NH3)]3− into the CS matrix, consistent with the initial concentration used in the film preparation process. For the molar ratio of 1(CS):10(complex), an incorporation of 0.63% (19.3 μmol/g) of the complex in the matrix was observed, that is, an increase of only 1.31 in relation to the 1:1 ratio, indicating a saturation limit for the incorporation of [Fe(CN)5(NH3)]3− within the CS polymer. In fact, previous studies have shown the production of materials with a low content of cyanoferrate complexes when chitosan and its derivatives are used as sorbent matrices.42,43 It was noted that the quantity of sorbed pentacyanoferrate (II) did not exceed 25 μmol/g when sodium aminoprusside interacts with CS polymer under static sorption conditions.43 Leaching Tests on CS[(CN)5Fe(NH3)]3−. It is wellknown that free −NH2 groups of glucosamine units of CS are protonated (pKa ≈ 6−6.5) at low pH, making the polymer behave as a soluble cationic polyelectrolyte.53 On the other hand, at high pH values, there is an increase in the negative charge density of the polymer matrix.54 Therefore, whereas in

the former situation, high concentrations of protons would compete with the iron complex for the CS amine groups, at high pH values the negative charge density of the polymer would repel the anionic complex. Both situations would imply the release of the complex from the polymer matrix. The stability of CS[(CN)5Fe(NH3)]3− regarding the release of the complex from the polymer matrix was evaluated by leaching analysis in solutions of different pH values. Upon immersion of three samples of CS[(CN)5Fe(NH3)]3− for 24 h in aqueous solutions of pH values 5, 7, and 9, the films, which were removed and dried under vacuum, were analyzed by UV−vis spectroscopy. The spectra thus obtained showed the characteristic bands of [Fe(CN)5(NH3)]3−, suggesting the leaching process is not taking place in the experimental conditions used. Also, the spectra of the working solutions presented no absorption that could be assigned to the [Fe(CN)5(NH3)]3− complex, thus reinforcing the previous conclusion. Figure S1 of the Supporting Information shows the spectra obtained for the films and solutions after the leaching analysis. Application of CS−[(CN)5Fe(NH3)]3− as a Sensor for Pyrazine. Electronic UV−Vis and CV Measurements. The reactivity of CS[(CN)5Fe(NH3)]3− toward pyrazine in the vapor phase was evaluated by using electronic spectra in the UV−vis regions and cyclic voltammetry techniques. Figure 2 shows the electronic UV−vis spectra and cyclic voltammograms (CVs) of the blank CS and CS[(CN)5Fe(NH3)]3− samples before and after exposure to pz atmosphere (CS[(CN)5Fe--pz]3−). By analogy to the reactivity of [Fe(CN)5(L)]3− type complexes in solution, it is expected that the replacement of NH3 by pz in [Fe(CN)5(NH3)]3− takes place through a substitution reaction, thus forming [Fe(CN)5(pz)]3−.40,55,56 It is worth mentioning that in aqueous medium this substitution reaction involves, at first, an aquation reaction producing [Fe(CN)5(H2O)]3− which, in the presence of pyrazine, forms [Fe(CN)5(pz)]3−. The ligand exchange reaction causes a color change going from pale yellow (CS[(CN)5Fe(NH3)]3−) to pink (CS[(CN)5Fe---pz]3−), indicating the pz coordination to FeII center, as shown in Figure 3. The pale yellow and pink colors seen in the photographs illustrated in Figure 3 are due to metal-to-ligand charge-transfer (MLCT) transitions45 from the dπ orbitals of FeII to π* orbitals of CN− and pyrazine moieties at 230 and 490 nm, respectively. Whereas the spectra recorded for CS and CS[(CN)5Fe(NH3)]3− samples exhibited no absorption bands over the 1389

DOI: 10.1021/acs.jafc.6b03742 J. Agric. Food Chem. 2017, 65, 1387−1394

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0.41 V vs Ag/AgCl, respectively. On the basis of the E1/2 value of the nonimmobilized [Fe(CN) 5 (pz)] 3− complex in NaCF3COO solution (0.39 V vs Ag/AgCl; Table 1), the potential shift observed upon exposure to pz atmosphere indicates the coordination of this ligand to the iron metal center. Upon coordination of pz, the reduced state of the metal, Fe(II), is thermodynamically stabilized due to the π-backbonding interaction. As a consequence, the oxidation of the metal center is made more difficult, thus explaining the shift of the oxidation potential to more positive values. In comparison to the nonimmobilized [Fe(CN)5(NH3)]3− complex, a potential shift of 64 mV is observed, indicating a stability enhancement upon trapping in the polymer matrix. Assuming that positive potential shifts are assigned to the stabilization of the reduced state of the metal, the additional stabilization observed upon immobilization of [Fe(CN)5(NH3)]3− into CS is believed to be due to hydrogen bonds formed between the cyanide ligands of the complex ion and the free hydroxyl and amine groups of CS.33,40 In fact, such intermolecular interactions enhance the electron-withdrawing capability of cyanide ligands, bringing about the stabilization of the lowest oxidation state. For CS[(CN)5Fe---pz]3−, a lower potential shift is observed as indicated by the value of 20 mV in Table 1. We hypothesize the π-back-bonding interaction to pyrazine is too strong to make possible additional stabilization due to the delocalization of electron density as a consequence of intermolecular interactions with the CS polymer. Accordingly, the UV−vis and CV results indicate that the incorporation of [Fe(CN)5(NH3)]3− ion complex into the CS matrix occurs through physical (electrostatic) and intermolecular interactions, mostly hydrogen bonds. The complex, therefore, is likely trapped in the polymer matrix with its sixth coordination site available for substitution reactions, as seen by the coordination of pz molecules. X-ray Diffraction. Aiming to study the structural changes of the CS polymer induced by the incorporation of [Fe(CN)5(NH3)]3− before and after the coordination of pyrazine, XRD measurements were carried out. Figure 4 depicts XRD patterns obtained for CS, CS[(CN)5Fe(NH3)]3−, and CS[(CN)5Fe---pz]3− films showing the effect of the

Figure 3. Photographs of CS[(CN)5Fe(NH3)]3− (A) before and (B) after exposure to pz atmosphere.

range from 350 to 800 nm, the spectrum of the film that was left for 3 h under exposure to pyrazine atmosphere, CS[(CN)5Fe---pz]3−, shows a new absorption with maximum at 490 nm assigned to the MLCT transition (pπ*(pz) ← dπ(FeII)), Figure 2A. This assignment is reinforced by the observation of an absorption at the same wavelength in the spectrum of the nonimmobilized [Fe(CN)5(pz)]3− complex dispersed in KBr (Figure S2 of the Supporting Information). UV−vis spectroscopy was also used in this work as a nondestructive technique to evaluate the recognition capability and the detection range of the CS[(CN)5Fe(NH3)]3− sample toward pyrazine. At this point, it should be made clear that it is not the intention of this work to compete with any chromatographic method due to the comparatively low detection limit of UV−vis spectroscopy. In fact, as shown in Table S1 of the Supporting Information, the magnitude of the detection limit of the chromatographic methods is as low as ng/ mL.16−23 Figure S3 of the Supporting Information shows the electronic UV−vis spectra of the CS[(CN)5Fe(NH3)]3− sample upon immersion in aqueous solutions containing pyrazine in a concentration range from 1.0 × 10−1 to 1.0 × 10−5 mol L−1. From the set of the acquired spectra, it is reasonable to suggest the concentration of 1.0 × 10−5 mol L−1 as a virtual limit of detection. A validation study using electronic spectroscopy and electrochemistry is underway as well. Cyclic voltammograms obtained for PGE modified with CS and CS[(CN)5Fe(NH3)]3− films before and after exposure to pz atmosphere are shown in Figure 2B. Table 1 displays the half-wave potentials (E1/2) that were calculated as the mean value of anodic (Ea) and cathodic (Ec) peak potentials from the CV curves. Table 1. Half-Wave Potentials (E1/2) of CS[(CN)5Fe(NH3)]3− Immobilized on PGE before and after Exposure to Pyrazine Atmospherea E1/2, V vs Ag/AgCl compound

free

film

ΔE1/2, mV

[Fe(CN)5(NH3)]3− [Fe(CN)5(pz)]3−

0.17 0.39

0.23 0.41

64 20

a

E1/2 values for the complexes in solution are also displayed for comparative purposes.

As can be ascertained from Figure 2B, the cyclic voltammogram obtained for the PGE coated with CS (black line) showed no redox process in the potential window studied in this work. In contrast, the CV curves recorded for PGE modified with CS[(CN)5Fe(NH3)]3− before (red line) and after (blue line) exposure to pyrazine atmosphere present redox processes assigned to the FeII/III redox pair with E1/2 values of 0.23 and

Figure 4. X-ray diffraction patterns showing the influence of concentration of [Fe(CN)5(NH3)]3− (A) and exposure to pz atmosphere (B) on the CS, CS[(CN)5 Fe(NH3 )] 3− (1:1), CS[(CN)5Fe(NH3)]3− (1:10), and CS[(CN)5Fe---pz]3− films, as indicated by colors. 1390

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Journal of Agricultural and Food Chemistry concentration of [Fe(CN)5(NH3)]3− (A) as well as the coordination of pyrazine (B). The XRD profile of CS (black line) exhibits two peaks at around 12° and 21°, which, according to the literature,57−59 are attributed to the crystal forms I and II, respectively. These two phases correspond to crystalline (less hydrated and harder) zones dispersed in an amorphous (more hydrated and softer) zone and describe the development of crystallinity in chitosan matrices.60 This is because plenty of hydroxyl and amino groups of the CS structure form strong intermolecular and intramolecular hydrogen bonds, and the structure of CS molecules has certain regularity, thus forming crystalline regions very easily.61 As can be ascertained from Figure 4, the XRD patterns of the CS[(CN)5Fe(NH3)]3− films (red and blue lines) are similar to that of pure CS. However, an intensity decrease of the peaks with the increase of the molar fraction of the complex within the CS matrix can be seen, Figure 4A. As suggested previously, the complex ions interact with CS via the −NH2 and −OH binding sites, reducing the number of hydrogen bonds in CS and effectively destroying the regular structure of the CS polymer chains responsible for its partially crystalline nature. Such results are in agreement with the modified film microstructure observed in SEM image after incorporation of [Fe(CN)5(NH3)]3− ions into CS structure, Figure 1. Thus, the pure CS film shows a smooth surface appearance due to the ordered packing of polymer chains. The CS[(CN)5Fe(NH3)]3− films, in turn, present appreciable irregularities that are assigned to a more disordered network in consequence of the partial destruction of the intermolecular hydrogen bonds of CS. Moreover, it is remarkable that the coordination of pyrazine molecules to the CS[(CN)5Fe(NH3)]3− film modifies even more its crystallinity by creating steric hindrances for hydrogen bond formation between CS chains, as can be seen in Figure 4B, line green. Water Contact Angle (WCA) Measurements. The changes in the surface properties of the CS-modified films were evaluated by the measurements of the water droplet contact angle. Figure 5 shows the mean WCAs on the surface of CS, CS[(CN)5Fe(NH3)]3−, and CS[(CN)5Fe---pz]3− and the corresponding photographs of the water droplets.

CS is responsible for the hydrophilic character and wettability of the CS polymer,62 so the decrease of this parameter corroborates the previous assignment of physical and intermolecular interactions, mainly hydrogen bonds, between [Fe(CN)5(NH3)]3− and CS via −NH2 and −OH fragments.63 The increase of the surface tension and the contact angle were indeed expected due to the appearance of an irregular and rough surface, as seen in the SEM image, Figure 1. Upon exposure to pz atmosphere, the CS[(CN)5Fe---pz]3− film presented a value of 102.20 ± 3.16° for the water contact angle. In comparison to the CS[(CN)5Fe(NH3)]3− film, the hydrophobicity decrease is assigned to the incorporation of pyrazine in the backbone structure of the biopolymer, which brings back more −NH2 and −OH fragments available to form hydrogen bonds.64 FTIR Spectroscopy. Figure 6 shows the FTIR spectra acquired for the CS and CS[(CN)5Fe(NH3)]3− films before

Figure 6. FTIR spectra of (A) CS, (B) CS[(CN)5Fe(NH3)]3− (1:1), and (C) CS[(CN)5Fe---pz]3− films. (Inset) FTIR spectrum of CS[(CN)5Fe(NH3)]3− (1:10) in the ν(CN) region.

and after exposure to pz atmosphere. Full FTIR spectra of the films are presented in Figure S4 of the Supporting Information together with the spectrum of [Fe(CN)5(pz)]3− for comparative purpose. The FTIR spectra presented in Figure 6 are dominated by the bands typically seen in the vibrational spectra of the CS polymer,57,65−68 namely, CO stretching (amide I band) at 1648 cm−1, NH bending (amide II band) at 1583 cm−1, C N stretching (amide III band) at 1320 cm−1, and asymmetric CH bending of the CH3 group at 1378 cm−1. Such vibrations are associated with remaining acetamide groups in CS, indicating that polymer was not totally desacetylated. The spectrum of CS[(CN)5Fe(NH3)]3− formed with 1:1 molar ratio of CS:complex shows, in addition to the CS absorption bands, the vibrational absorptions at ca. 2050 cm −1 attributed24,42,43,69 to the vibrational stretching modes of the CN bonding (νCN). Such vibrational modes are better resolved in the spectrum obtained for the CS[(CN)5Fe(NH3)]3− formed with 1:10 molar ratio of CS:complex, which is shown as an inset in Figure 6. Indeed, not only the equatorial and axial vibrational stretching modes of the CN bonding are

Figure 5. Mean static contact angles between water and the surfaces of CS, CS[(CN)5Fe(NH3)]3− (1:1), and CS[(CN)5Fe---pz]3− films.

The immobilization of [Fe(CN)5(NH3)]3− into CS matrix induces an increase in the WCA from 76.27 ± 0.35 to 123.00 ± 4.17°, indicating an increase in the surface tension and confirming the successful modification of the polymer. Moreover, the increase in the water angle indicates the decrease in the hydrophilic character and wettability of the surface of the CS-modified film. The presence of −OH and −NH2 groups in 1391

DOI: 10.1021/acs.jafc.6b03742 J. Agric. Food Chem. 2017, 65, 1387−1394

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Journal of Agricultural and Food Chemistry

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seen but also it is observed an intensity increase. The spectrum obtained for the CS[(CN)5Fe---pz]3− film presents a similar profile in comparison to that shown for CS[(CN)5Fe(NH3)]3− film. Although the vibrational modes of the pyrazine ring are probably covered by those of the polymer matrix, the intensity increase observed in the region from 1500 to 1700 cm−1 is assigned70 to the contribution of the vibrational stretching modes of CC and CN bondings of the pyrazine moiety. Theoretical Comments. Computational calculations were performed by using different quinoxaline (IQx) derivatives aiming to determine the coordination site of this class of Lewis bases toward the [Fe(CN)5]3− metal center. Three coordination sites were considered: the NH2 fragment and the nitrogen atoms of the pyrazine and imidazole rings. As illustrated in Figure S5, only the calculation performed by considering the nitrogen atom of the pyrazine ring resulted in a bonded final configuration. For the other conditions, the states of lower energies are those in which the molecules are not bonded, that is, there is no coordination to the metal center when the NH2 group or the nitrogen atom of imidazole ring is chosen as the coordination site. The theoretical data, therefore, support the suggestion of the synthesized hybrid organic−inorganic material as a potential sensor for pyrazine derivatives such as HAA species.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b03742. Figures S1−S5 and Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(I.C.N.D.) E-mail: [email protected]. Phone: +55 (85) 3366 9166. Fax: +55 (85) 3366 9977. ORCID

Izaura C. N. Diógenes: 0000-0002-2765-4982 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS I.C.N.D. (304285/2014-5) and E.L. (306305/2015-1) and D.S.A. and M.A.S.S. are thankful to CNPq and CAPES for grants and financial support. The authors thank CENAPADUFC for the use of the GAUSSIAN 09 software package and for computational facilities.

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REFERENCES

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