Response surface methodology applied to the study of the microwave-assisted synthesis of quaternized chitosan

A quaternized derivative of chitosan, namely N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride (QCh), was synthesized by reacting glycidyltrimethylammonium chloride (GTMAC) and chitosan (Ch)in acid medium under microwave irradiation.

Carbohydrate Polymers 138 (2016) 317–326 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Response surface methodology applied to the study of the microwave-assisted synthesis of quaternized chitosan Danilo Martins dos Santos, Andrea de Lacerda Bukzem, Sérgio Paulo Campana-Filho ∗ Instituto de Química de São Carlos/Universidade de São Paulo, Av Trabalhador são-carlense, 400-13566-590, São Carlos/SP, Brazil a r t i c l e i n f o Article history: Received 22 July 2015 Received in revised form November 2015 Accepted 24 November 2015 Available online December 2015 Keywords: Chitosan derivatives Quaternization Microwave irradiation Response surface methodology a b s t r a c t A quaternized derivative of chitosan, namely N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride (QCh), was synthesized by reacting glycidyltrimethylammonium chloride (GTMAC) and chitosan (Ch) in acid medium under microwave irradiation Full-factorial 23 central composite design and response surface methodology (RSM) were applied to evaluate the effects of molar ratio GTMAC/Ch, reaction time and temperature on the reaction yield, average degree of quaternization (DQ ) and intrinsic viscosity ([Á]) of QCh The molar ratio GTMAC/Ch was the most important factor affecting the response variables and RSM results showed that highly substituted QCh (DQ = 71.1%) was produced at high yield (164%) when the reaction was carried out for 30 at 85 ◦ C by using molar ratio GTMAC/Ch 6/1 Results showed that microwave-assisted synthesis is much faster (≤30 min.) as compared to conventional reaction procedures (>4 h) carried out in similar conditions except for the use of microwave irradiation © 2015 Elsevier Ltd All rights reserved Introduction Chitosan is a ␤(1 → 4)-linked copolymer of 2-amino-2-deoxy-Dglucopyranose (GlcN) and 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) found as a component of the cell wall of some fungi, however it is generally prepared through the deacetylation of chitin, an abundant polysaccharide present in the exoskeletons of crustaceans, mollusks and insects (Peniche, Argüelles-Monal, & Goycoolea, 2008; Rinaudo, 2006) Due its nontoxic nature and for being biocompatible and biodegradable, a range of applications has been reported for chitosan, including in wound dressing (Mogos¸anu & Grumezescu, 2014), tissue engineering (Muzzarelli, 2009) and drug delivery (Sanyakamdhorn, Agudelo, & Tajmir-Riahi, 2013) Nevertheless, the application of chitosan is often limited by its poor solubility in water at neutral and alkaline pH Thus, several strategies have been adopted for carrying out controlled chemical modifications on chitosan aiming to improve its water solubility and to expand its range of applications In this sense, N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride (QCh), a polycationic derivative of chitosan, is a very interesting alternative as it is soluble in a wider range of pH and it displays improved properties, including antimicrobial activity ∗ Corresponding author Tel.: +55 16 33739929; fax: +55 16 33739952 E-mail addresses: danilomartins 1@hotmail.com (D.M.d Santos), andrea bukzem@hotmail.com (A.d.L Bukzem), scampana@iqsc.usp.br (S.P Campana-Filho) http://dx.doi.org/10.1016/j.carbpol.2015.11.056 0144-8617/© 2015 Elsevier Ltd All rights reserved (Rabea, Badawy, Stevens, Smagghe, & Steurbaut, 2003), mucoadhesivity (Sonia & Sharma, 2011), higroscopicity and moisture retention (Prado & Matulewicz, 2014), as compared to chitosan The synthesis of QCh is usually carried out by reacting chitosan with glycidyltrimethylammonium chloride (GTMAC) in alkaline, neutral or acid medium at relatively high temperature (>70 ◦ C) for long reaction time (4–18 h) (Cho, Grant, Piquette-Miller, & Allen, 2006; Ruihua, Bingchao, Zheng, & Wang, 2012; Wu, Su, & Ma, 2006; Xiao et al., 2012) However, when the synthesis is carried out in neutral or alkaline medium, O-substitution occurs to an appreciable extent (Prado & Matulewicz, 2014; Ruihua et al., 2012) Additionally, the hydrolysis of GTMAC to 2,3dihydroxypropyltrimethylammonium chloride is favored in such reaction media, negatively affecting the reaction yield In contrast, when such a synthesis is carried out in acid medium, highly substituted QCh samples are produced and N-substitution predominates, preventing the formation of undesirable products as compared to procedures carried out in neutral and alkaline media (Cho et al., 2006; Prado & Matulewicz, 2014; Ruihua et al., 2012) Numerous reports have shown that microwave heating has a high potential to accelerate chemical reactions, to increase reaction yield and to enhance product’s purity and material’s properties as compared to conventional experiments in which heating by convection or conduction is used (Caddick & Fitzmaurice, 2009; Gawande, Shelke, Zboril, & Varma, 2014; Moseley & Kappe, 2011; Nuchter, Ondruschka, Bonrath, & Gum, 2004; Zhu & Chen, 2014) The microwave heating involves two main mechanisms, namely dipolar polarization and ionic conduction, and it presents 318 D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 advantages such as rapid heat transfer, volumetric and selective heating Thus, microwave heating has been used in chemical functionalization of polysaccharides, including chitosan (Ge & Luo, 2005; Liu, Wang, Yang, & Sun, 2012; Petit, Reynaud, & Desbrieres, 2015; Singh, Tiwari, Tripathi, & Sanghi, 2006) and cellulose (Biswas, Kim, Selling, & Cheng, 2014; dos Santos, Bukzem, Ascheri, Signini, & de Aquino, 2015) Petit et al (2015) investigated the preparation of amphiphilic derivatives of chitosan by using microwave irradiation and they found that it is possible to obtain modified chitosan at lower reaction time as compared to conventional procedures and without any degradation of the polymer chain Singh et al (2006) described the synthesis of chitosan-g-polyacrylamide by using microwave irradiation and they reported that higher reaction yield was achieved in rather shorter time as compared to the reaction carried out under conventional heating The recent literature also reports on the use of microwave heating in polycondensation reactions (Komorowska-Durka, Dimitrakis, Bogdał, Stankiewicz, & Stefanidis, 2015), ring-opening polymerizations (Hoogenboom & Schubert, 2006) as well as in radical polymerizations (Sugihara, Semsarilar, Perrier, & Zetterlund, 2012) Response surface methodology (RSM) is a set of statistical and mathematical techniques effective for developing, improving, and optimizing processes that involves a response of interest that is influenced by several independent variables (Myers, Montgomery, & Anderson-Cook, 2009) RSM is based on the fit of a polynomial equation to the experimental data that describes the relationship between a dependent variable, or response, and the independent variables as well as the interactions among these latter Simultaneously, this technique allows to optimize the levels of the independent variables to attain the best possible response in a faster and more economical manner when compared to classic onevariable-at-a-time approach (Bezerra, Santelli, Oliveira, Villar, & Escaleira, 2008; Myers et al., 2009) The synthesis of QCh involves reactional parameters such as molar ratio chitosan/GTMAC, reaction time and temperature that influence a series of responses related to the important properties of this chitosan derivative as its degree of quaternization and intrinsic viscosity as well as the reaction yield (Cho et al., 2006; Prado & Matulewicz, 2014; Ruihua et al., 2012) In this context, the RSM can be considered a useful tool to evaluate how the independent variables related to the reaction conditions used to synthesize QCh, as well as the interactions among them, affect the properties of this chitosan derivative Aiming to provide new insights for the preparation of N-(2hydroxy)-propyl-3-trimethylammonium chitosan chloride (QCh), this study focus on the preparation of QCh samples in acid medium under microwave irradiation by using full-factorial 23 central composite design and response surface methodology (RSM) to evaluate the effects of molar ratio GTMAC/Ch, reaction time and temperature on the reaction yield, average degree of quaternization (DQ ) and intrinsic viscosity ([Á]) of QCh The parent chitosan as well as the resulting derivatives are characterized by Fourier transform infrared (FTIR) and H NMR spectroscopy, capillary viscometry, thermogravimetric analysis (TGA), X-ray diffraction and with respect to water-solubility as a function of pH with ethanol/water mixtures of increasing ethanol content (70%, 80%, and 90%) The purified chitosan was dried at 30 ◦ C and named as sample Ch Glycidyltrimethylammonium chloride (GTMAC) was acquired from Sigma-Aldrich (Saint Louis, MO; USA) and its was used as received as well as other reactants and solvents employed in this study 2.2 Synthesis of N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride Purified chitosan (0.5 g) was suspended in 30 mL of deionized water and 150 ␮L of glacial acetic acid were added to the suspension which was kept at constant stirring at room temperature for 10 An aqueous solution of GTMAC was added dropwise to the chitosan suspension which was then submitted to microwave irradiation at a power of 200 W in a monomode microwave reactor (Discover-LabMate, CEM, USA) under constant stirring at the desired temperature and during a given time Following, excess acetone was added to the reaction medium to result in the precipitation of the product which was filtered, thoroughly washed with acetone and dried at 35 ◦ C for 24 h The reaction yield was calculated based on the weights of the parent chitosan and the resulting product 2.3 Experimental design A full-factorial 23 central composite design was used to analyze the main effects and interactions of the reaction variables, namely molar ratio GTMAC/Chitosan, reaction time and temperature, on the average quaternization degree (DQ ) and intrinsic viscosity of QCh and on the reaction yield of the microwave-assisted synthesis of QCh The choice of the parameters and their levels was based on our own previous experimental studies Thus, 11 independent runs of experiments were carried out in duplicate, including 23 orthogonal factorial and six replicate at the center point The independent variables and their levels are shown in Table All the experiments were carried out at random, in order to minimize the effect of unexplained variability in the observed responses due to systematic errors Also, to compare the microwave-assisted synthesis of QCh to reaction performed by using conventional heating, two additional runs were carried out using the same reaction conditions as used to produce samples QCh1 (X1 = 4/1; X2 = 20 min.; X3 = 75 ◦ C) and QCh8 (X1 = 6/1; X2 = 30 and X3 = 85 ◦ C) except for the use of microwave irradiation, resulting in samples QCh1-CH and QCh8CH, respectively Such reactions were carried out in a 100 mL one-necked round bottom flask immersed in a preheated oil bath at the given temperature for the desired time 2.4 Characterization 2.4.1 H NMR spectroscopy The H NMR spectra of the parent chitosan and its derivatives were acquired at 85 ◦ C by using an spectrometer Agilent 400/54 Premium Shielded 9.4 T, operating at 399.8 MHz for H For these analyses, the samples were dissolved in HCl/D2 O 1% (v/v) at a Materials and methods 2.1 Materials Commercial chitosan (Cheng Yue Planting Co Ltd., China) was dissolved in 1% aqueous acetic acid solution to result in Cp = g/L, the resulting solution was filtered through 0.45 ␮m membrane (Millipore® ), and then it was neutralized by addition of mol L−1 NaOH solution to provoke the precipitation of chitosan The solid was thoroughly washed with distilled water and Table Uncoded and coded levels of the independent variables related to the synthesis of N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride Independent variables Symbol GTMAC/Chitosan Time (min) Temperature (◦ C) X1 X2 X3 Levels −1 20 75 25 80 30 85 D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 concentration of 10 mg/mL The average degree of deacetylation (DD) and average degree of quaternization (DQ ) were calculated by treating the H NMR spectra according to Eq (1) (Hirai, Odani, & Nakajima, 1991) and Eq (2) (Desbrières, Martinez, & Rinaudo, 1996), respectively ICH3 /3 DD (%) = 1− DQ (%) = IH1 IH1 + IH1 IH2-H6 /6 × 100 (1) × 100 (2) where, ICH3 is the integral of the signal due to the methyl hydrogens of GlcNAc units (≈2.0 ppm), IH2–H6 is the integral corresponding to the hydrogens H3–H6 from GlcN unit and the hydrogen bonded to C2 of GlcNAc unit (≈3.3–4.0 ppm), IH1 is the integral of the signal due to the anomeric hydrogen bonded to N-substituted GlcN units (≈5.0 ppm) while IH1 is the integral of the signal due to the anomeric hydrogen bonded to unsubstituted GlcN units (≈4.8 ppm) 2.4.2 Conductometric titration The average degree of quaternization (DQ ) of quaternized chitosan was also determined by dosing the counter-ions Cl− ions through titration with standardized 0.017 mol L−1 aqueous AgNO3 solution (Cho et al., 2006) Thus, QCh (0.1 g) was dissolved in deionized water (100 mL) and the conductivity of the solution was measured at 25 ± 0.01 ◦ C as a function of the added volume of aqueous AgNO3 by using a Handylab LF1 conductivimeter (SchottGeräte) The value of DQ of QCh was calculated from the titration curves according to Eq (3) DQ (%) = 1.7 × 10−5 VAgNO3 W (g) − ×100 −5 1.7 × 10 VAgNO3 xMCGTMA / MG xDD + MAG − DD × DD (3) where, VAgNO3 (mL) is the volume of AgNO3 solution added to reach the equivalence point; W (g) is the dry weight of the QCh sample; MGTMAC , MG and MAG are the molar masses (g mol−1 ) of GTMAC, GlcN and GlcNAc units, respectively; DD is the average degree of deacetylation 2.4.3 Fourier transform infrared (FTIR) spectroscopy Infrared spectra were recorded by using a BOMEM MB102 FTIR spectrophotometer Samples were finely ground, mixed with KBr and the mixture was then compressed into pellet form The FTIR spectra were acquired at 400–4000 cm−1 at resolution of cm−1 by accumulating 32 scans 2.4.4 Capillary viscometry The intrinsic viscosities [Á] of the parent chitosan and its derivatives were determined in 0.3 mol L−1 acetic acid/0.2 mol L−1 sodium acetate buffer (pH = 4.5) Thus, the solution of chitosan (or QCh) was prepared by dissolving 50 mg (or 90 mg) in 50 mL of buffer solution, followed by filtration through 0.45 ␮m membrane (Millipore® ) A glass capillary ( = 0.53 mm) containing 15 mL of the polymer solution was immersed in a water bath maintained at 25.00 ± 0.01 ◦ C The viscosity measurements were carried out by using an AVS350 (Schott-Geräte, Germany) viscometer coupled to the AVS-20 automatic burette (Schott-Geräte, Germany) for serial dilution of polymers solutions with buffer solution The relative viscosity (Árel ) of the polymer solutions were in the range 1.2 < Árel < 2.0 and the intrinsic viscosity, [Á], was determined from curves of reduced viscosity (Ásp /C) versus polymer concentration (C) at infinite dilution 319 2.4.5 X-ray diffraction XRD patterns were acquired at room temperature by using a Bruker AXS D8 Advance X-ray diffractometer equipped with CuK␣ ˚ in the scattering range < 2 < 40◦ at scan radiation ( = 1.5406 A) ◦ rate /min The operating voltage was 40 kV, and the current was 40 mA The crystallinity index (CrI) was calculated following the amorphous subtraction method proposed by Osorio-Madrazo et al (2010) by using Eq (4): CrI = Acrist Atotal × 100% (4) where, Acrist expresses the crystalline contribution area obtained by subtracting the amorphous contribution from the total area (Atotal ) of the diffractogram The amorphous contribution was estimated directly from diffractogram using X’pert high score Plus software (2015) 2.4.6 Thermogravimetric analysis (TGA) The thermal stability of the parent chitosan and its derivatives was studied by carrying out TGA measurements using a Shimadzu TGA 50 equipment Thus, the sample (≈8 mg) was heated from room temperature to 700 ◦ C at a heating rate of 10 ◦ C min−1 under nitrogen atmosphere (flow = 50 mL min−1 ), the weight loss being measured as a function of temperature 2.4.7 Water solubility The solubility of the parent chitosan and its derivatives in aqueous medium as a function of pH (2 < pH < 12) was estimated from the measurement of the solutions transmittance Thus, the sample was dissolved in 0.1 mol L−1 HCl to result in Cp = g/L, an aliquot of the solution was poured into a quartz cell (l = cm) and its transmittance was recorded on a UV/vis spectrophotometer (Shimadzu, UV 3600) at = 600 nm The pH of the polymer solution was adjusted by the dropwise addition of a 0.1 mol L−1 NaOH solution A given sample was considered to be insoluble when the transmittance of its solution was lower than 50% as compared to that of a control solution (aqueous 0.1 mol L−1 HCl) 2.5 Statistical analysis The statistical treatment of the experimental data consisted in fitting a polynomial function to the set of experimental data collected from full-factorial 23 central composite design Multiple regression analysis was used to fit Eq (5) to the experimental data by means of the least squares method Y = ˇ0 + ˇ1 X1 + ˇ2 X2 + ˇ3 X3 + ˇ12 X1 X2 ˇ13 + X1 X3 + ˇ23 X2 X3 + ˇ4 X12 + ε (5) where, Y represents the predicted response, ˇ0 , is the model intercept, ˇ1 , ˇ2 , ˇ3 are the coefficients of the linear terms; ˇ12 , ˇ13 and ˇ23 are the interaction coefficients; ˇ4 is the coefficient of the quadratic term; X1 , X2 and X3 are the independent variables and ε corresponds to the model residue The statistical significance of each individual coefficient term was determined by evaluating the p-value and F-value with 95% confidence level obtained from the analysis of variance (ANOVA) The lack of fit of regression model was evaluated with 95% confidence level The extent of fitting of the experimental results to the polynomial model equation was expressed by the coefficient of determination (R2 ) and adjusted coefficient of determination (R2 adj ) Response surface plots were obtained by using the fitted model and by keeping one independent variable constant at zero level while varying the remaining two variables All calculations and graphs were obtained by the Statistica software (Statsoft version 7.0, USA) 320 D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 Results and discussion 3.1 Spectroscopic characterization of chitosan and QCh (d) QCh8-CH Transmittance (%) 1030 1157 1080 1652 1600 1483 2887 3440 The infrared spectrum of chitosan (Fig 1a) exhibited a characteristic intense and broad band centered at 3440 cm−1 due to the axial stretching of O H group, which appears superimposed to the N H stretching band; a weak band at 2877 cm−1 (C H stretch); the bands at 1642, 1600, 1377 and 1258 cm−1 due to the C O stretching, N H bending and NHCO stretching of the amide and C–N stretching, respectively; the bands at 1157, 1080 and 1030 cm−1 attributed to the stretching of C O of GlcN units (Brugnerotto et al., 2001) The same main bands are also observed in the spectra of QCh samples produced by using microwave heating (Fig 1b and c) and conventional heating (Fig 1d, and e), however a new band is observed at 1483 cm−1 , which is attributed to the C H bending of + N(CH3 )3 group (Cho et al., 2006) Additionally, the band corresponding to the primary amine observed at 1600 cm−1 in the spectrum of chitosan is less intense in the spectra of QCh samples and it is shifted to lower wavenumber while that band observed at 1652 cm−1 is more intense in the spectra of QCh samples Such a comparison confirms that the primary amine group of GlcN units of chitosan has been modified to secondary amine group as a consequence of the reaction with GTMAC (Xiao et al., 2012) In contrast, the characteristic bands observed in the range 1157 cm−1 –1030 cm−1 , were not changed, indicating that the reaction has not occurred at the hydroxyl groups bonded to C3 and C6, in agreement with the literature (Huang et al., 2014) Thus, such an analysis highlights the structural changes due to the reaction of chitosan with GTMAC and it indicates the predominant occurrence of N-substitution The structural modifications resulting from the reaction of chitosan with GTMAC can also be evidenced by comparing the H NMR spectra of chitosan (Fig 2a) and quaternized derivatives QCh8 (Fig 2a) and QCh1-CH (Fig 2c) The H NMR spectrum of chitosan exhibited a singlet at 2.0 ppm characteristic of methyl hydrogens of GlcNAc units, a signal at 3.15 ppm related to the hydrogen bonded to C2 of GlcN units, the set of signals in the range of 3.3–4.0 ppm corresponding to the hydrogens H3–H6 from GlcN unit and the hydrogen bonded to C2 of GlcNAc unit, while that signal (d) QCh1-CH (c) QCh8 (b) QCh1 (a) Chitosan 4000 3500 3000 2500 2000 1500 1000 500 -1 Wavenumber (cm ) Fig Infrared spectra of chitosan (a) and quaternized derivatives QCh1 (b), QCh8 (c) QCh1-CH (d) and QCh8-CH (e) occurring at 4.80 ppm is attributed to the hydrogen (H1) bonded to the anomeric carbon (C1) The average degree of deacetylation of chitosan was calculated from its H NMR spectrum by using Eq (1) and resulted in DD = 95% The H NMR spectra of the samples QCh8 and QCh1-CH show additional signals at 3.2 ppm and 3.31 ppm corresponding to the methyl hydrogens of N+ (CH3 )3 and methylene hydrogens of NHCH2 , respectively, which are due to the introduction of the substituent on the chitosan chains In addition, the signal of the hydrogen bonded to the C2 carbon of the GlcN unit shifted from 3.15 ppm to 3.10 ppm upon the chemical modification of chitosan The signals at 4.8 ppm and 5.0 ppm are attributed to the hydrogen bonded to the anomeric carbon of unsubstituted and substituted GlcN units, respectively (Desbrières et al., 1996) The average degree of quaternization (DQ ) of samples QCh8 and QCh1CH was calculated from the corresponding H NMR spectra by using Eq and resulted in DQ = 71.1% and DQ = 15.3%, respectively Indeed, the average degree of quaternization of all QCh samples were also determined from conductometric titration (Eq (3)), the resulting values of DQ showing a good agreement (>93%) with those determined from the H NMR spectra (Table 2) 3.2 Statistical analysis and model fitting Table shows the coded (in parenthesis) and the uncoded values of the independent variables molar ratio GTMAC/chitosan, reaction time and temperature and the experimental values of the response variables DQ and intrinsic viscosity of QCh samples as well as reaction yield The responses ranged as 47% < DQ < 71%, 230 mL g−1 < [Á] < 290 mL g−1 and 120% reaction yield 0.05 In the case of the reaction yield, the linear term molar ratio GTMAC/chitosan (X1 ) was the most important parameter, followed by the linear terms reaction time (X2) and temperature (X3) Besides, the interaction term between time and temperature (X2 X3 ) and the quadratic term of ratio molar GTMAC/chitosan (X1 ) were also significant (p < 0.05) The ANOVA showed that the lack of fit was not significant at 95% confidence level (p-value > 0.05), meaning that the models represented the data satisfactorily In addition, the factors R2 and adjusted R2 were calculated to check the model adequacy Indeed, such an analysis show the close agreement between the experimental results and the theoretical values predicted by these models as high values of R2 (>0.97) and R2 adj (>0.90) were observed for DQ , intrinsic viscosity and reaction yield (Table 3), confirming that the fitted models can satisfactorily explain the total variability of the responses within the range of independent variable studied D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 Fig 321 H NMR spectra of chitosan (a) QCh8 (b) and QCh1-CH (c) in solution D2 O/HCl 1% (v/v) acquired at 85 ◦ C The fitted models for DQ , intrinsic viscosity and yield without insignificants terms and in uncoded form are given in Eqs (6)–(8) DQ (%) = 36.1 − 18.0X1 + 2.6X1 + 0.3X2 + 0.5X3 (6) [Á] = 258 + 67.75X1 − 8.125X1 − 0.875X2 + 0.125X3 (7) Yield (%) = 310 + 32.9167X1 − 4.6667X1 − 8.90X2 + 3.80X3 − 0.10X2 X3 (8) 322 D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 Table Independent variables and experimental values of the response variables for the central composite design related to the synthesis of N-(2-hydroxy)-propyl-3trimethylammonium chitosan chloride Run 10 11 Response variablesa Independent variables GTMAC/Chitosan (mmol/mmol) Time (min) Temperature (◦ C) DQ (−1) (−1) (−1) (−1) (+1) (+1) (+1) (+1) (0) (0) (0) 20 (−1) 20 (−1) 30 (+1) 30 (+1) 20 (−1) 20 (−1) 30 (+1) 30 (+1) 25 (0) 25 (0) 25 (0) 75 (−1) 85 (+1) 75 (−1) 85 (+1) 75 (-1) 85 (+1) 75 (−1) 85 (+1) 80 (0) 80 (0) 80 (0) 47.3 51.7 53.7 57.6 60.5 63.5 67.2 71.1 56.3 56.7 56.8 b (%) DQ ± ± ± ± ± ± ± ± ± ± ± 46.5 52.0 53.3 58.4 60.3 62.4 65.1 69.9 53.3 52.8 53.0 0.4 0.3 0.2 0.1 0.6 0.4 0.8 0.8 0.4 0.2 0.3 c (%) [Á] (mL g−1 ) ± ± ± ± ± ± ± ± ± ± ± 288 277 268 262 258 252 246 233 267 267 270 1.6 0.9 0.3 0.2 1.5 0.5 0.4 0.4 0.2 0.4 0.2 ± ± ± ± ± ± ± ± ± ± ± 3 4 1 Yield (%) 133 125 141 135 150 137 159 164 146 149 148 ± ± ± ± ± ± ± ± ± ± ± 4 3 MeanValues ± SD DQ determined by H NMR c DQ determined by conductometric titration [Á] = intrinsic viscosity in 0.3 mol L−1 acetic acid/0.2 mol L−1 sodium acetate buffer (pH = 4.5) at 25 ◦ C a b Table Analysis of variance (ANOVA) concerning the variable responses degree of quaternization (DQ ), intrinsic viscosity ([Á]) and reaction yield related to the synthesis of N-(2hydroxy)-propyl-3-trimethylammonium chitosan chloride Sourcea X1 X1 X2 X3 X1 X2 X1 X3 X2 X3 Lack of fit R2 R2 adj * a b c d DQ * [Á] Reaction yield F p-value F p-value F p-value 5991.78 255.04 1565.26 474.74 8.22 5.26 0.33 8.22 0.000b 0.004b 0.001b 0.002b 0.103d 0.149d 0.624d 0.103d 477.04 48.01 176.04 51.04 1.04 0.042 0.042 7.042 0.002b 0.020c 0.006b 0.019c 0.415d 0.857d 0.857d 0.118d 309.429 20.364 156.214 25.929 17.357 1.929 21.429 13.714 0.003b 0.046c 0.006b 0.036c 0.053d 0.299d 0.044c 0.066d 0.9987 0.9959 0.9881 0.9605 0.9724 0.9078 DQ determined by H NMR X1 = molar ratio GTMAC/chitosan; X2 = Time (min); X3 = Temperature (◦ C) Significant at 1% probability (p < 0.01) Significant at 5% probability (p < 0.05) Non-significant Table Characteristic temperatures and corresponding weight losses related to the thermal degradation of chitosan and samples QCh1, QCh8, QCh1-CH and QCh8-CH Sample Chitosan QCh QCh QCh1-CH QCh8-CH a b c Stage I Stage II Range TMax (◦ C)a WL (%)b Range Tonset (◦ C)c WL (%) 25–150 25–150 25–150 25–150 25–150 70 60 60 63 60 10 11 220–420 190–390 190–390 190–390 190–390 278 248 246 253 250 42 48 52 44 46 TMax = Temperature of maximum weight loss WL = Weight loss Tonset = Onset temperature Such equations were used to generate three-dimensional surfaces by fixing one independent variable at the zero level while the others are varied within the range of study to further analyze the effects of independent variables on the responses (Fig 3) The response surface plots show that DQ increases as molar ratio GTMAC/Chitosan, reaction time and temperature are increased (Fig 3(a–c)) Such positive effects of the independent variables on DQ can be rationalized as a consequence of the higher excess of GTMAC and longer reaction times, both of them favoring a more complete N-substitution on the chitosan chains In addition, increasing the reaction temperature has a positive effect on DQ as more reactive species have enough energy to overcome the barrier corresponding to the activation energy, resulting in faster and more complete reaction It is noteworthy that the average degree of deacetylation of QCh samples has not been changed as compared to the parent chitosan as evaluated by H NMR spectroscopy Indeed, as it was observed the overlapping of the signals due to H2 and Ha (Fig 2b), the average degree of deacetylation of the QCh samples produced via microwave-assisted reaction was calculated by taking into account the signals due to H1, as proposed by An et al (2009) Thus, by using the same equation to determine the average degree of deacetylation of the parent chitosan and of the QCh samples resulted in DD = 93.2 ± 0.1% and DD = 92.7 ± 0.5% (mean value considering the whole set of QCh samples), respectively D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 323 Fig Response surface plots showing the effect of molar ratio GTMAC/Chitosan, reaction time and temperature on the response variables, namely degree of quaternization (a, b, and c), intrinsic viscosity (d, e, and f) and reaction yield (g, h, and i) The whole set of QCh samples show lower intrinsic viscosity as compared to that of the parent chitosan ([Á]Chitosan = 451 mL g−1 ) Indeed, the response surface plots (Fig 3d–f) also show that increasing the molar ratio GTMAC/Chitosan, reaction time and temperature negatively affected the intrinsic viscosity of the resulting QCh samples, the high the molar ratio GTMAC/chitosan and the reaction temperature and the longer the reaction, the lower the intrinsic viscosity At a first glance, this fact can be attributed to the occurrence of depolymerization, which would be more important when longer reaction time and temperature were used during the derivatization reaction, but one can also consider that the insertion of numerous substituent groups in the chitosan chains can render the interactions polymer/solvent more and more unfavorable, resulting in chain coiling The viscosity average molecular weight (Mv ) of the QCh samples was estimated from the corresponding intrinsic viscosity value by using the Mark–Houwink–Sakurada equation proposed by Yevlampieva, Gubarev, Gorshkova, Okrugin, & Ryumtsev (2015) for quaternized chitosan as determined in 0.3 mol L−1 acetic acid/0.2 mol L−1 sodium acetate buffer at 25 ◦ C The viscosity average degree of polymerization (DPv ) of a given QCh sample was calculated from the ratio between its viscosity average molecular weight (Mv ) and the corresponding average molecular weight of its repeating unit (M0 ), this latter depending on the average degree of quaternization of the sample Thus, it can be clearly seen that the average degree of polymerization of QCh decreases with increasing degree of quaternization (Fig 4) According to Yevlampieva et al (2015) such a decrease of DPv with increasing DQ can be 324 D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 Fig Dependence of the average degree of polymerization of the QCh samples as a function of the degree of quaternization attributed to fact that the solvent used to determine the intrinsic viscosity of QCh is a poor solvent to such a derivative of chitosan as evaluated by static light scattering experiments On the other hand, it is well-known that carrying out chemical modifications on polysaccharides in acid medium favors the occurrence of chain depolymerization, due to the susceptibility of glycosidic bonds to acid hydrolysis Thus, taking into account that the microwaveassisted reaction of chitosan and GTMAC was carried out in aqueous acetic acid and that prolonging the reaction time and increasing the temperature resulted in more substituted QCh samples, it is also probable that such reaction conditions favored the occurrence of depolymerization Indeed, Wasikiewicz & Yeates (2013) studied the degradation of chitosan in 0.1 M aqueous acetic acid under microwave irradiation and an important decrease of molecular weight was observed with increasing irradiation time The effects of molar ratio GTMAC/Chitosan, reaction time and temperature on the reaction yield are shown in Fig 3g–i Thus, it is observed that the reaction yield increases as the molar ratio GTMAC/chitosan is increased, the prolongation of the reaction for longer times also resulting in higher reaction yield (Fig 3g) In addition, the response surface plot concerning the effects of the molar ratio of GTMAC/Ch and reaction temperature on reaction yield (Fig 3h) clearly shows that increasing both variables increase the reaction yield In contrast, the use of low excess of GTMAC and short reaction time result in low reaction yield (Fig 3h and i) Further two synthesis were carried out under conventional heating and employing the same experimental conditions of run (molar ratio GTMAC/chitosan of 4/1; 20 and 75 ◦ C) and run (molar ratio GTMAC/chitosan of 6/1; 30 and 85 ◦ C) to result in samples QCh1-CH and QCh8-CH, respectively Such samples exhibited DQ = 15.3 ± 0.3%, [Á] = 374 ± 11 mg mL−1 (sample QCh1CH) and DQ = 41.4 ± 0.5%, [Á] = 341 ± mg mL−1 (sample QCh8-CH) while samples QCh1 (DQ = 47.3 ± 0.4%, [Á] = 288 ± mg mL−1 ) and QCh8 (DQ = 71.1 ± 0.8%, [Á] = 233 ± mg mL−1 ), both of them prepared under microwave radiation, exhibited much higher average degrees of quaternization but lower intrinsic viscosities Thus, such a comparison shows that microwave-assisted synthesis was much more efficient than conventional heating to promote the substitution reaction on chitosan Also, it is important to highlight that microwave-assisted synthesis as carried out in this study allows the preparation of highly substituted QCh in much shorter time (≤30 min.) as compared to conventional Fig TG (a) and DTG curves (b) of chitosan and samples QCh1, QCh8, QCh1-CH and QCh8-CH synthesis (4–18 h), according to the literature (Cho et al., 2006; Wu et al., 2006; Xiao et al., 2012) 3.3 Thermogravimetric analysis The thermal stability of polymers is affected by the occurrence and extent of substitution reactions and to investigate the effects of the substituents on the thermal behavior of quaternized chitosan samples, TG analyzes were carried out Comparing the TG and DTG curves (Fig 5a and b) reveals that chitosan and samples QCh1, QCh8, QCh1-CH and QCh8-CH display similar thermal behaviors as the same three main events are observed, although the Tonset and weight losses corresponding to the degradation stage II are different (Table 4) The first thermal event (25–150 ◦ C), named as stage I, is attributed to the evaporation of weakly adsorbed and loosely-bound water, the weight loss ranging as 7% - 11% Higher weight losses at the first step were observed in the cases of samples QCh1, QCh8 and QCh8-CH, probably due their higher average degree of substitution, the substituent groups contributing for a higher adsorption of humidity owning to the presence of charges The second thermal event, Stage II, starts at ≈220 ◦ C and extends up to ≈420 ◦ C in the case of chitosan but it occurs in the range 190–390 ◦ C in the cases of the quaternized derivatives Such a thermal event provokes the elimination of volatile products from the decomposition of the substituent groups while D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 325 as compared to the one observed in the diffractogram of chitosan, indicating the loss of order upon the derivatization reaction Indeed, the crystallinity indexes (CrI) of the parent chitosan and samples QCh1, QCh8, QCh1-CH and QCh8-CH were determined from the corresponding XDR patterns according to Osorio-Madrazo et al (2010), resulting in 31.0%, 18.3% 17.5%, 24.2%, and 20.6%, respectively Such results suggest that the arrangement of the polymer chains in the solid state has changed because of the introduction of substituents on the chitosan chains Thus, as a consequence of the presence of charged and bulky substituents on the chains of quaternized chitosan samples, their crystallinity indexes are much lower as compared to chitosan owning the disruption of hydrogen bonding and the occurrence of an important steric hindrance (Xiao et al., 2012) 3.5 Water solubility Aiming to evaluate the effects of substituents on the solubility of quaternized chitosan samples, the absorbance of polymer solutions (Cp = g/L) was measured as a function of the solution pH The comparison of the water solubility of chitosan and its quaternized derivatives as a function pH (Fig 6b) reveals that at pH ≤ 6.0, the polymers are all fully soluble as the transmittances of their solutions were close to 100% However, increasing the pH from 6.0 to 7.0 provoked the occurrence of clouding in the chitosan solution due to precipitation of the polymer as a consequence of the deprotonation of ammonium groups of GlcN units As seen in Fig 5b, the solubility of sample QCh1-CH was high at pH < 6.0 but it dramatically decreased at pH > 7.0 In contrast, as the pH was increased in the range 6.0–12.0, the transmittance of the solutions of samples QCh1, QCh8 and QCh8-CH remained close to 100% Thus, owning to its relatively low average degree of quaternization (DQ = 15.3%), sample QCh1-CH displays a similar solubility as compared to chitosan while samples QCh1, QCh8 and QCh8-CH are fully water soluble at 2.0 < pH < 12.0 due to the high content of charged substituents (DQ > 40%) Fig (a) X-ray diffractograms and (b) water-solubility as a function of pH of chitosan and samples QCh1, QCh8, QCh1-CH and QCh8-CH Conclusions further thermal events leading to the complete thermal degradation of the samples occur at temperatures higher than 420 ◦ C and 390 ◦ C in the cases of chitosan and quaternized chitosan, respectively During Stage II, the weight losses ranged in the interval 42–52%, the higher the average degree of quaternization of the chitosan derivative the higher the corresponding weight loss (Table 5) Also, the values of Tonset (Table 4) show that the quaternized chitosan samples exhibit lower thermal stability as compared to the parent chitosan, in accordance with other studies reporting on the thermal stability of chitosan derivatives (Chethan, Vishalakshi, Sathish, Ananda, & Poojary, 2013; De Britto & Campana-Filho, 2004; Xu, Xin, Li, Huang, & Zhou, 2010) 3.4 X-ray diffraction As the occurrence of ordered/disordered regions strongly depends on intra- and intermolecular interactions, the effects of the substituents on the solid state arrangement of QCh chains was studied by X-ray diffraction (XRD) The XRD pattern of the parent chitosan (Fig 6a) exhibits an intense peak centered at 2 = 20.2◦ which is related to the reflection planes (2 0)h and (0 0)a while the peak occurring at 2 = 10.9◦ corresponds to the plane (0 0)h (Osorio-Madrazo et al., 2010) In the XRD patterns of the quaternized chitosan samples (Fig 6a), the peak at 2 = 10.9◦ is not observed while that peak at 2 = 20.2◦ is significantly less intense The microwave-assisted reaction of chitosan (Ch) with glycidyltrimethylammonium chloride (GTMAC) in acid medium allowed the efficient production of N-(2-hydroxy)-propyl-3trimethylammonium chitosan chloride (QCh) in much shorter time (≤30 min.) as compared to conventional reaction carried out in similar conditions except for the use of microwave radiation Also, the spectroscopic characterization of QCh showed that none other chemical modifications occurred as a consequence of the reaction of chitosan and GTMAC The execution of full-factorial 23 central composite design to study the effects of reaction variables on the variable responses, namely the average degree of quaternization and intrinsic viscosity of QCh and reaction yield, resulted in mathematical equations displaying high determination coefficients and insignificant lack of fit, the molar ratio GTMAC/H displaying the strongest influence followed by reaction time and temperature Thus, using a high molar ratio GTMAC/Ch (6/1) and carrying out the reaction for 30 at 85 ◦ C resulted in a highly substituted QCh sample (DQ = 71.1%) at high reaction yield (164%) The thermal stability and the degree of order of the QCh samples were lower as compared to the parent chitosan while the water solubility was greatly improved as a consequence of the derivatization reaction as samples QCh1 and QCh8 were fully soluble over the range < pH < 12 This study contributes to the improvement of the methodologies aiming the preparation of quaternized chitosan as it highlighted the use of microwave radiation to result in a simple and 326 D.M.d Santos et al / Carbohydrate Polymers 138 (2016) 317–326 fast experimental procedure to produce N-(2-hydroxy)-propyl-3trimethylammonium chitosan chloride (QCh) Acknowledgments The authors are grateful to the agencies Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES 443/2012; Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 142002/2014-3; Brazil), Fundac¸ão de Amparo Pesquisa Estado de São Paulo (FAPESP 2010/02526-1; Brazil) for financial support The authors also address special thanks to Prof Andre L M Porto (University of Sao Paulo—Brazil) for allowing the use of the microwave reactor References An, N T., Thien, D T., Dong, N T., & Dung, P Le (2009) Water-soluble 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higher the average degree of quaternization of the chitosan. .. on the solubility of quaternized chitosan samples, the absorbance of polymer solutions (Cp = g/L) was measured as a function of the solution pH The comparison of the water solubility of chitosan. .. increasing the pH from 6.0 to 7.0 provoked the occurrence of clouding in the chitosan solution due to precipitation of the polymer as a consequence of the deprotonation of ammonium groups of GlcN