Eclet. Quim. 49 | e-1485, 2024 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 ISSN 1678-4618 page 1/10 1Federal University of Rio Grande do Norte, Institute of Chemistry, Natal, Brazil. +Corresponding author: Ana Cristina Facundo de Brito Pontes, Phone: +5584987166346, Email address: ana.cristina.pontes@ufrn.br Original Article Influence of chitosan’s purification methodology on the formation of layer-by-layer films Ana Cristina Facundo de Brito Pontes1+ , Luciana Araújo Nascimento1 , Daniel de Lima Pontes1 , Ótom Anselmo de Oliveira1 , Francisco Ordelei da Silva Nascimento1 , Francimar Lopes da Silva Júnior1 Abstract Concern for the environment for the development of new biodegradable materials has been constant in scientific circles. With this in mind, this work proposes a study on the formation of self-assembled thin films using chitosan (Qt), a biodegradable material. This polyelectrolyte has several purification methodologies, but we did not identify any studies on the effect of these methodologies on film formation. Thus, after the purification process and characterization of the three forms of chitosan purification, films were produced using the layer-by-layer (LBL) technique. The growth of the films was monitored using the UV-vis technique. Spectroscopy in the Infrared region showed positions in the main bands present in chitosan and sodium nitroprusside (NP) in the formed films. Two semi-reversible processes were found for the QtN/NP and QtAc/NP films, related to the reduction of iron oxide present in the NP. The effect of pH (4.0, 7.0 and 10) on the electrochemical processes indicated that the charge transfer occurs more efficiently at pH 7.0. Article History Received March 23, 2023 Accepted November 24, 2023 Published April 22, 2024 Keywords 1. biodegradable; 2. self-assembled; 3. sodium nitroprusside. Section Editor Patricia Hatsue Suegama Marcos Carlos de Mattos Highlights The effect of chitosan purification methodology. Monitoring the formation of self-assembled films by UV-Vis. Electrochemical tests indicate different interaction mechanisms between species. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 https://ror.org/04wn09761 mailto:ana.cristina.pontes@ufrn.br https://orcid.org/0000-0003-3895-1808 https://orcid.org/0000-0002-4125-5945 https://orcid.org/0000-0001-7770-0492 https://orcid.org/0000-0003-0409-4000 https://orcid.org/0000-0002-0841-2080 https://orcid.org/0000-0001-9300-8623 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Eclet. Quim. 49 | e-1485, 2024 ISSN 1678-4618 page 2/10 1. Introduction The layer-by-layer (LBL) deposition represents an interesting alternative for the development of films for several applications such as medicine and bio-applications (Ariga et al., 2012; Nesic et al., 2016; Xia et al., 2019) to improve corrosion resistance (Gao et al., 2019) and electrochemical sensing and biosensing (Pannell et al., 2018; Si et al., 2019). LBL method (Crespilho et al., 2006a), has been the main choice for the manufacture of nanostructured films, as they have several advantages, such as low cost, various materials can be used, the film is made under mild conditions and thus, multilayer structures can be constructed to obtain the desired number of bilayers, since apparently there are no limitations on the amount of layers that can be deposited (Cheung et al., 1992; Oliveira Junior et al., 2001). The most common interaction is provided by the electrostatic attraction, the alternative adsorption of opposites charged. Due to their low toxicity, biodegradability, and natural availability (Umoren and Eduok, 2016) polysaccharides can be rather good candidates. Chitosan in an acidic environment presents positive charges due to the protonation of the amino groups (NH3 +), a substrate with a high density of negative sites immersed in this solution will behave as a suitable support for the attraction and subsequent formation of a homogeneous film. Layer-by-layer films with chitosan have been studied as active surfaces with antimicrobial and antioxidant potential (Li and Peng, 2015; Luo et al., 2012). Chitosan is the most well-known natural biopolymer and has a wide range of applications, such as in the health area (Delolo et al., 2014; Lins et al., 2014; Silva et al., 2006; Ungureanu et al., 2015), environment (Marques Neto et al., 2013) and technology (Souza et al., 2015; Vinhola et al., 2012). The properties of chitosan, such as degree of purity, solubility, viscosity, degree of deacetylation (GD), molecular weight and others, are influenced by the methodology used to obtain it, as well as by the purification techniques to which they are subjected (Arrascue et al., 2003; Battisti and Campana-Filho, 2008; Crini and Badot, 2008; Santos et al., 2003; Gonsalves et al., 2011; Kumar, 2000; Laranjeira and Fávere, 2009; Roberts, 1992; Silva et al., 2006). There are several methodologies used for the purification of chitosan, however, these procedures involve the steps of dissolving in an aqueous medium of controlled ionic strength, filtration, precipitation by the addition of non-solvent, washing and drying. This type of purification defines in advance the type of counter ion that will be present in the purified sample. Steps such as dialysis and lyophilization can be added to the procedures (Signini and Campana Filho, 2001). As in the layer-by-layer technique, the production of films is based on the electrostatic interaction between molecules containing ionic groups and having chitosan as the amino group (NH3 +), it is necessary to use another compound with opposite charge. Thinking about the production of films that may have applications as biosensors, NP was used, with molecular formula Na2[Fe(CN)5(NO)]. It has been used for more than 50 years as a vasodilator in cases of emergency in hypertension attacks, blood pressure control in surgeries and the treatment of chronic hypertension as it provides a quick response without the need for overuse (César et al., 2001; Freitas et al., 2012; Sass et al., 2007; Stocche et al., 2003). There are also reports of the use of NP in the determination of sulfur compounds in fresh and saline waters (Sonne and Dasgupta, 1991), as well as in the determination of phenols in aqueous two-phase systems (Rodrigues et al., 2010). The literature reports several types of film formation studies with chitosan, however, they all use it with modifications, whether in the form of Schiff bases or through crosslinking reactions (Amanulla et al., 2017; Lu et al., 2022; Suginta et al., 2013). Therefore, this article makes a study on the effect of chitosan purification on the formation of self-assembled films obtained through electrostatic interaction. 2. Experimental 2.1. Chitosan purification Chitosan of commercial origin (POLYMAR) was purified in three different ways, to obtain a pure and homogeneous material. The methodologies used in the purifications were proposed by Signini and Campana Filho (2001) with modifications and are described below: a. Neutral chitosan (QtNeutral): It is solubilized in acetic acid, precipitated in ammonium hydroxide, filtration, dried, and stored in a dry environment. b. Chitosan acetate (QtAcetate): It is solubilized in acetic acid, precipitated in ethanol, dried, and stored in a dry environment. c. Chitosan hydrochloride (QtHydrochloride): Solubilized in hydrochloric acid, sodium chloride added, precipitated in ethanol, filtered, dried, and stored in a dry environment. 2.2. Chitosan characterization The infrared spectra of chitosan purified in different forms were obtained in the form of a KBr tablet, using a Shimadzu spectrophotometer, model FTIR-8400S, series IRAFFINITY-1, software IRSOLUTION, version 1.60, with scan number equal to 30 and resolution 4. The determination of the degree of deacetylation of the purified chitosan (% GD) was made by conductivity measurement (Raymond et al., 1993), and the viscometrical molar mass was obtained through capillary viscosimetry (Signini and Campana Filho, 1998; 2001). The values of the Huggins constant (K) and α used were 76.0 x 10–5 and 0.76 (Canella and Garcia, 2001). The moisture percentage of chitosan samples was determined by thermogravimetric analysis using a thermogravimetric analyzer unit and simultaneously calorimeter, TA Instruments model SDTQ600 manufacturer. The samples were analyzed in an alumina crucible at a rate of 2.5 °C min–1, heated from 25 °C to 900 °C under a nitrogen atmosphere, with a flow rate of 50 mL min–1. 2.3. Sodium nitroprusside (NP) The Na2[Fe(CN)5(NO)] used was obtained from PROQUÍMIOS and no purification was necessary. For the preparation of the films, a solution of 23 g L–1 in methanol was prepared. 2.4. Layer-by-layer (LBL) fabrication The LBL films were deposited on quartz and ITO glass substrates by alternating immersion into cationic chitosan and anionic NP solutions for 5 min. Chitosan solution (Qt) 5 g L–1 in 1% acetic acid and a solution of sodium https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Eclet. Quim. 49 | e-1485, 2024 ISSN 1678-4618 page 3/10 nitroprusside (NP) 23 g L–1 in methanol. After each dive, the slide was washed with distilled water and dried under nitrogen flow. The films were prepared with (Qt/NP)n, with “n” being the number of layers with n = 2, 4, 18 and 20 layers. 2.5. Characterization of LBL films The growth monitoring of the films was performed using the UV-Vis absorbance spectroscopy technique (Agilent, model 8453) on a quartz plate, the same material used in the IR technique for LBL films using the Shimadzu spectrophotometer, model FTIR-8400S, series IRAFFINITY-1, software IRSOLUTION, version 1.60, with scan number equal to 30 and resolution 4. The electrochemical experiments were performed using an AUTOLAB AUT 85282 system. The film (Qt/NP)20 was deposited on ITO (glass covered with a thin indium-doped tin oxide layer) glass substrates used as a working electrode. The reference electrode was an Ag|AgCl/KCl saturated electrode and the counter electrode was a Pt plate The electrochemical cell used in the cyclic voltammetry measurements had a total volume of 50 mL with a cap with a plug for 3 electrodes and before the measurement, argon gas was bubbled into the solution. The experiments were conducted in a 0.1 mol L–1 KCl at 25 oC. 3. Results and discussion The purification process of chitosan in neutral, acetate and hydrochloride forms yielded 82.5%, 64.1% and 85.6%, respectively. These yield values are due to loss due to handling and, mainly, to the presence of insoluble materials and aggregates present in chitosan that were eliminated during the filtration steps. The presence of insoluble materials in commercial products is reported in the literature (Ottøy et al., 1996). The purification process makes more polar groups available, which increases the ability to interact with other compounds (Signini and Campana Filho, 2001). The differences in purification methodologies resulted in different chitosan molecules in terms of visual aspect and solubility. All the purified samples were soluble in diluted acetic acid solution, the samples purified in the form of acetate and hydrochloride were partially soluble in water. The number of amino groups present in the chitosan polymer chain is an important parameter and is related to electrostatic interactions for the formation of self-assembled films. Its order of magnitude can be determined through the degree of deacetylation (% GD). One of the simplest and most used techniques for this determination is conductometric titration. The data obtained for the different chitosan samples are shown in Table 1. The lower % GD of the QtHydrochloride sample can be explained by the change in inter- and intra-chain relationships, caused by using a strong acid in its purification. The moisture content for the purified chitosan samples in the different forms obtained values lower than that observed in the literature (Signini and Campana Filho, 2001) (23.8 ±0.4) % for the same purification methodology. The difference may be related to the drying step, in the case of literature it was carried out at 25 oC, which does not remove the water present, whereas, in our work, the drying process was used with slight heating. Viscosimetry is one of the most used processes for determining the molar mass of polymers, as it is a simple technique without requiring equipment with high costs. For the three purified chitosan holders, a good correlation index was obtained between the experimental measures with R > 0.99. As can be seen in Table 1, the application used in the purification of chitosan directly affects the viscosity of the polysaccharide and its molar mass, indicating differences in its properties. Table 1. Chitosan properties in its different forms of purification. Sample % GD Ƞ (mL g–1) Mv(g mol–1) Mv (g mol–1)* Moisture content (%) QtNeutral 68 ± 2 273.72 4.79 x 104 16.7 x 104 12.16 QtNeutral 69 ± 5 136.18 1.14 x 104 17.2 x 104 13.49 QtHydrochloride 61 ± 4 44.72 4.54 x 103 16.1 x 104 16.84 Source: Elaborated by the authors using data from Signini and Campana Filho (2001). The main characteristic bands of the infrared absorption spectrum of samples of chitosan in their different purified forms are shown in Table 2. The OH axial stretch band, between 3467 to 3502 cm–1, appears to overlap the band N-H stretch. All bands identified are very similar to those described in the literature (Battisti and Campana-Filho, 2008; Vinhola et al., 2012) and show that the same functional groups are present in all analyzed samples. The band in the 2926 to 2030 cm–1 region is attributed to the C-H stretching of the CH2 groups of pyranoses. It is also possible to observe a peak from 1321 cm–1 to 1381 cm–1 characteristic of the angular deformation of CH2 and CH3. Table 2. Main assignments of the infrared bands for the purified chitosan samples. Sample Infrared band assignments (cm-1) C=O (amide I) N-H (amide II) C-N (amide III) CH COC β-(1-4) CO OH C-H QtNeutral 1664 1556 1381 1423 1156 1078 3467 2878 QtAcetate 1658 1566 1321 1415 1157 1074 3471 2879 Qthydrochloride 1643 1525 1381 1323 1155 1082 3502 2885 Signini and Campana Filho (2001) 1655 1600 1423 - 1153 1031 3450 2904 Note: δ = deformation; ν = Stretch. Source: Elaborated by the authors using data from Signini and Campana Filho (2001). The thermal analysis study for the chitosan samples in their different forms of purification showed two mass losses. The first event is related to the loss of moisture in the material, while the second event is related to the breaking of the polymer bonds, these occurred at 273.8, 260.3 °C and 185.6 °C for Qtneural, QtAcetate and Qt hydrochloride, respectively. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Eclet. Quim. 49 | e-1485, 2024 ISSN 1678-4618 page 4/10 3.2. Characterization of Sodium Nitroprusside (NP) The infrared spectrum for sodium nitroprusside showed bands at 2161, 2158 e 2144 cm–1 related to the stretching vibrations of the cyanide ligand, another characteristic band of this complex is the NO stretch identified at 1942 cm–1. In the region of the infrared spectrum below 700 cm–1, it is possible to identify the signals attributed to the metal, so in 495 and 423 cm–1 bands related to the Fe-CN bond and Fe-C in 466 cm–1. The very intense band at 661cm–1 is attributed to linear deformation Fe-N→O, all these bands follow what has been reported in the literature for complex (Palliani et al., 1971). The electronic spectrum of NP at 23 g L–1 (0.106 mol L–1) in methanol is shown in Fig. 1, where initially only two bands are observed, the first at 208 nm related to the dxy→π*CN transition and the second at 270 nm attributed to dxz, dyz → dz 2. It was necessary to prepare higher concentrations to identify the band related to the dxy→π*NO, transition, identified at 540 nm as can be seen in Fig. 1a. The 270 nm band was used to monitor the growth of the films since it is possible to monitor them at low concentrations (Palliani et al., 1971; Swinehart, 1967). Figure 1. UV-vis absorption spectra for NP 0.03 mg L–1 and (1a) for NP 0.2 mg L–1 in methanol. 3.3. Characterization of LBL films The deposition of materials in the LBL film formation process was investigated by monitoring its increase in absorbance after the preparation of each bilayer. This procedure makes it possible to assess whether the materials are deposited in quantities like each formed bilayer. For all Qt/NP films, prepared with the three purification methodologies, have UV-vis absorption around 270 nm, this band is related to the electronic transition of NP (dxz, dyz → dz 2). The deposition of materials in the process of forming self-assembled films was investigated using its absorbance technique after the preparation of each bilayer, as shown in Fig. 2 for QtNeutral/NP. The thickness of the films can be controlled by the number of bilayers deposited and the polymer used. If linear growth is observed, it indicates that the same amount of material is adsorbed in each deposition step (Eiras et al., 2007). Figure 3 shows the relationship between absorbance and the number of layers formed for the three Qt purification methodologies. There is a linear growth for the three formed films, however with different slopes. The films obtained with Qt in neutral form and acetate obtained better correlation and slope indexes, according to Eiras et al. (2007), greater slopes in these graphs are related to greater affinities between the compounds that form a layer. Thus, we can conclude that among the three methodologies used in the purification, QtAcetate showed a greater affinity with NP for the formation of self-assembled films and QtHydrochloride is the one with less affinity. Figure 2. The electronic absorption spectrum in the UV-vis region of the films self-assembled with an increasing number of layers (QtNeutral/NP) prepared from a solution of Qt 5 g L–1 and NP 23 g L–1. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Eclet. Quim. 49 | e-1485, 2024 ISSN 1678-4618 page 5/10 Figure 3. Increase in absorbance at 270 nm as a function of the number of bilayers of self-assembled films to QtNeutral/NP ( ), QtAcetate/NP ( ) and (3a) QtHydrochloride/NP ( ) prepared from a solution of Qt 5 g L–1 and NP 23 g L–1. The more similar the amounts adsorbed in each adsorption step, the higher the value of the correlation index (R) (Table 3) because the closer the points are to the line. Thus, it can be observed that the film formed from QtAcetate has a linear behavior of deposition of materials on the solid substrate with the number of layers, that is, the same amount of material is deposited in each step (Eiras et al., 2007), for films containing QtNeutral it presented an exponential behavior. Picart et al. (2002) demonstrated that growth can occur linearly or exponentially due to the ability of at least one of the polyelectrolytes to diffuse in and out of the film. This author monitored the growth of films using Poly-L-lysine and identified that it was present in the outermost layer of the film. Regarding the film obtained with QtHydrochloride, an irregular behavior of deposition of the material in the film is noticed. The observed data clearly indicate that the methodology used in the purification of chitosan directly influences the formation of films with the complex. According to Signini and Campana Filho (2001), the chitosan purified in the form of hydrochloride has charges that will alter the inter and intricate interactions, modifying its arrangement, these changes seem to disadvantage the formation of the chitosan film, as it presents a random growth probably caused by the inadequate electrostatic interactions between chitosan and the complex. Table 3. Slope data and correlation indexes of the films of neutral chitosan, acetate, and hydrochloride with NP. Purified form of Qt Inclination Correlation Index QtNeutral 0.01280 0944.06 QtAcetate 0.04034 0.99737 QtHydrochloride 0.00293 0.85615 The Spectroscopy technique in the Infrared region was also used to evidence the species present in the films, Table 4 shows the main attributions for the Qt/NP film with 20 bilayers. In general, the spectra showed displacements, probably caused by the interaction between Qt and NP. The N-H (1566 cm–1) stretch characteristic of chitosan suffered displacements in the films of QtNeutral (1556 cm–1) and QtAcetate (1556 cm–1), respectively. The stretch attributed to NO present in the film was displaced to 1931 cm–1, the Fe-CN deformation also showed a small displacement (Table 4). The characteristic band of the C-H stretching undergoes small shifts compared to the spectrum of purified chitosan, which was expected as it is an indication of a modification in its neighborhood. The results of spectroscopy and UV-vis suggest that both chitosan and NP are being deposited in layers for the formation of the films and that this interleaving is directly related to the type of purification that the chitosan has undergone. Table 4. Band assignments observed in the IR spectrum for chitosan films with NP. Assignment Film QtNeutral/NP Film QtAcetate/NP Film QtHydrochloride/NP OH 3394 3398 3480 C-H 2924 2928 2840 -C≡N 2141 2140 2152 COC β-(1-4) or δCO 1122 1070 1156 δFe-C≡N 482 482 500 NO + 1931 1931 1940 δFe-N→O 471 488 490 The study on the thermal stability of chitosan in different forms of purification and Qt/NP films was carried out, except for the QtHydrochloride/NP film, considering that the results obtained by UV-vis spectroscopy, since it did not show good interaction between chitosan and NP. Figure 4 shows thermogravimetry (TG) and derivative thermogravimetry (DTG)for films Qt/NP, the moisture content in the films was 13.94% for QtNeutral and 16.92% for QtAcetate. A second event is observed in the films at 174 and 182 °C for QtNeutral and QtAcetate, respectively. Osiri et al. (2015) report that there is a loss of mass in coordination compounds containing cyanide and nitrosyl https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Eclet. Quim. 49 | e-1485, 2024 ISSN 1678-4618 page 6/10 as a binder at temperatures close to 190 °C, thus we can infer that the mass losses previously mentioned are related to the decomposition of NP present in the films, coupled with the fact of these losses of mass are not seen in the purified chitosan. Figure 4. TG (solid) and DTG (dot) curves of (a) QtNeutral/NP e (b) QtAcetate/NP analyzed in an alumina crucible at a rate of 2.5 °C min–1, heated from 25 to 900 °C under a nitrogen atmosphere, with a flow rate of 50 mL min–1. A third event is observed at temperatures above 250 °C which is related to the breakdown of glycosidic bonds, followed by the decomposition of the acetylated and deacetylated units (Martínez-Camacho et al., 2010; Martins et al., 2012; Nesic et al., 2016). It was observed that in general the addition of the complex for the formation of self-assembled films decreased the thermal stability of chitosan. It is also possible to identify an event occurring at a temperature of 866 and 820 °C for QtNeutral and QtAcetate, respectively, this event is related to NP degradation. A comparison of the decomposition temperatures for the different samples is shown in Table 5. Table 5. Results of thermogravimetric analysis. 1st mass loss (°C) 2nd mass loss (°C) 3rd mass loss (°C) 4th mass loss (°C) QtNeutral 35.0 - 273.8 - QtNeutral/NP 37.6 174 259.0 886.0 QtAcetate 35.4 - 260.3 - QtAcetate/NP 32.0 181 267.0 821.0 Thus, it is possible to identify that the methodology used in the purification of chitosan plays an important role in the formation of LBL films with NP. 3.4. Cyclic voltammetry of LBL films The cyclic voltammetry study in LBL films (20 layers) of chitosan purified in the form of acetate and neutral with the complete ones deposited on the ITO surface was analyzed in saline medium (KCl 0.1 mol L–1). To evaluate the influence of the chitosan form of purification on the electrochemical response, a study was made of the current variation as a function of the applied potential. The QtHydrochloride sample was not characterized by the cyclic voltammetry technique, since it did not present good results in the deposition for forming the films. The analysis of voltammograms showed that as the scanning speed increases, there is an increase in the value of the anodic and cathodic peak currents. It was observed that at 50 mV s–1 the electrochemical spectrum had higher resolution and the potentials could be determined with greater accuracy, that is, it presented a fast response, without loss of precision in detecting the anodic peak potentials (Epa) and cathodic (Epc), as well as anodic (ipa) and cathodic (ipc) peak currents. Figure 5 shows the voltammetric profiles for the films of QtNeutral/NP and QtAcetate/NP, presenting 2 peaks, one of oxidation and one of reduction related to the redox process (Fe(III)/Fe(II) of the metal present in the complex. For the QtNeutral/NP film the anodic and cathodic peak values were 0.59 and 0.44 V, respectively, whereas for QtAcetate/NP we obtained the values of 0.62 and 0.45 V. Generally, small displacements are observed, for the film formed with the neutral chitosan, a displacement of 0.04 V was observed for the anodic peak when compared to the values obtained for the NP film (oxidation at 0.63 V and reduction at 0.50 V). It is possible to observe a small shift in the anodic peak towards more positive potentials and a shift in the cathodic peak towards more negative potentials; this behavior is characteristic of quasi-reversible processes. Another important aspect is the stability of these electrodes after carrying out several measurements. After electrochemical measurements, the electrolyte solution was analyzed using the UV-vis technique and no band was observed, confirming that there is no migration of the components to the electrolyte and indicating that the films are stable. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Eclet. Quim. 49 | e-1485, 2024 ISSN 1678-4618 page 7/10 (a) (b) Figure 5. Cyclic voltammograms for 20-bilayer (a) QtNeutral/NP and (b) QtAcetate/NP on ITO with multiple scans rates: (––) 5 mV s–1; (––) 10 mV s–1; (––) 25 mV s–1; (––) 50 mV s–1; (––) 75 mV s–1; (––) 100 mV s–1. Electrolyte: KCl 0.1 mol L–1. (a) (b) Figure 6. Monitoring the oxidation peak potential with the scan rate for 20-bilayer (a) QtNeutral/NP and (b) QtAcetate/NP on ITO in KCl 0.1 mol L–1 at 50 mV s–1. The anodic peak potential increases linearly with the scan rate for the QtAcetate/NP film, as can be seen in Fig. 6b, with a correlation index of 0.98807 and for QtNeutral/NP (Fig. 6a) different behavior is observed. Study of chitosan/FeTsPc and chitosan/NiTsPc films (Crespilho et al., 2006b) an increase in anodic potential was observed with the scan rate, even according to the author, this behavior is evidence of a load transport mechanism, like the results obtained for the QtAcetate/NP film. For the films formed by QtNeutral/NP, the diffusion process occurs, since there is no linear behavior with the scan rate. Also, according to Crespilho et al. (2006b), for LBL films formed by chitosan/FeTsPc and chitosan/NiTsPc molecules, the type of mechanism that occurs is electron hopping, since the interaction between species is the ionic interaction between the groups amino of chitosan and the phthalocyanic sulfonic groups present in the complexes. This same type of interaction is proposed for films containing QtAcetate/NP, between the chitosan amino group and the [Fe(CN)₅NO]–2 of the complex. The value of Ep as a function of the scan rate is shown in Fig. 7, in which it is possible to observe that only the QtNeutral/NP film can be considered as a semi-reversible process since the potential values increased with the increase of scan rate. The graphic (Fig. 7) makes clear that the methodology used in the purification of chitosan directly influences the interaction and formation of thin films. Figure 7. Relationship of potential variation (Ep) versus scan rate for QtNeutral/NP (▀) e QtAcetate/NP (•). https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Eclet. Quim. 49 | e-1485, 2024 ISSN 1678-4618 page 8/10 The study of the effect of pH (4.0; 7.0 and 10) on the electrochemical behavior of the QtNeutral/NP and QtAcetate/NP films was carried out and the results are shown in Fig. 8. It is possible to observe that at pH 4, the obtained films did not present a good electrochemical response, making it difficult to identify the anodic process. At pH 10 divergence is observed in the electrochemical behavior for the films purified in different ways, for the QtNeutral/NP film it is not possible to identify the anodic process, different behavior for the QtAcetate/NP film, in which a displacement of the cathodic peaks is observed and anodic for 0.73 V and 0.19 V, respectively. These results indicate that the charge transfer process occurs more efficiently at pH equal to 7.0 for the different forms of purification. (a) (b) Figure 8. Cyclic voltammograms obtained for (a) QtNeutral/NP (b) QtAcetate/NP in pH (––) 4.0; (––) 7.0 e (––) 10 with scan rate de 50 mV s–1. To identify if there was any change in the self-assembled films' thermal stability after the electrochemical study, thermal analysis tests were performed. It was possible to observe that the thermal stability (Table 6) of the films did not undergo any significant change, which may indicate the films are stable after being polarized i.e., after using in the form of electrodes. Table 6. Data thermal analysis after voltammetry. 1st mass loss (°C) 2nd mass loss (°C) 3rd mass loss (°C) 4rd mass loss (°C) QtNeutral/NP 40.0 179 257.8 887.9 QtAcetate/NP 31.7 183.1 263.1 820.0 4. Conclusions The methodology used in the purification of chitosan produced materials with different characteristics such as molar mass, viscosity, humidity, and degree of deacetylation. These characteristics seem to directly interfere with the deposition of layers for the formation of LBL films, as evidenced by UV-vis techniques, in which the chitosan purification methodology in the form of hydrochloride was the one that showed deposition of the material in the film completely random and, therefore, it was not used for the characterization of the electrochemical profile. The Qttneutra/NP film, presented an exponential growth, indicating that one of the polyelectrolytes can diffuse between the layers of the film, as far as the growth of the QtAcetate/NP film was linear. Differences regarding the thermal stability of the films were also identified, in which the film formed by Qttneutra/NP was the one that presented a greater reduction in the decomposition temperature, of approximately 15 °C when compared to purified chitosan. Through this technique it was also possible to identify the loss of mass related to the NP, confirming once again its presence in the film, as also observed by the spectroscopy technique in the infrared region. The electrochemistry tests showed that the QtNeutral/NP and QtAcetate/NP films when used as working electrodes are stable and that there is no migration of their components to the electrolyte. Studies of thermal analysis of the films after electrochemical tests showed changes in relation to its decomposition temperatures. There was a shift towards more positive potentials of the anodic peak when compared to NP under the same conditions for QtNeutral/NP, whereas with respect to the cathodic peak the difference of 0.05 V was the same observed for the two films. There are distinct interactions and behaviors between the two films corroborating the UV-vis results. The QtNeutral/NP film presents a semi-reversible process while the other is reversible, indicating differences in the charge transfer process between the two films. For the QtAcetate/NP film the charge transport occurs, while for the QtNeutral/NP film the diffusion process occurs. In addition, pH equal to 7 proved to be ideal for electrochemical measurements. Authors’ contributions Conceptualization: Pontes, A. C. F. B.; Data curation: Nascimento, L. A.; Silva Júnior, F. L.; Formal Analysis: Nascimento, L. A.; Silva Júnior, F. L.; Pontes, A. C. F. B.; Funding acquisition: Pontes, A. C. F. B.; Pontes, D. L.; Oliveira, O. A.; Nascimento, F. O. S.; Investigation: Nascimento, L. A.; Silva Júnior, F. L.; Pontes, A. C. F. B.; Methodology: Nascimento, L. A.; Pontes, A. C. F. B.; Project administration: Nascimento, L. A.; Silva Júnior, F. L.; Pontes, A. C. F. B.; Pontes, D. L.; Oliveira, O. A.; Resources: Pontes, A. C. F. B.; Pontes, D. L.; Oliveira, O. A.; Nascimento, F. O. S.; Software: Not applicable; Supervision: Pontes, A. C. F. B.; Validation: Not applicable; Visualization: Pontes, A. C. F. B.; Writing – original draft: Pontes, A. C. F. B.; Silva Júnior, F. L.; Pontes, D. L.; Oliveira, O. A.; Nascimento, F. O. S.; Writing – review & editing: Pontes, A. C. F. B. Data availability statement All data sets were generated or analyzed in the current study. https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Original Article https://doi.org/10.26850/1678-4618.eq.v49.2024.e1485 Eclet. Quim. 49 | e-1485, 2024 ISSN 1678-4618 page 9/10 Funding Not applicable. Acknowledgments The Analytical Center of the Chemistry Institute of UFRN for the use of the equipment used in this work. References Amanulla, B.; Palanisamy, S.; Chen, S.-M.; Chiu, T.-W.; Velusamy, V.; Hall, J. M.; Chen, T.-W.; Ramaraj, S. K. 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