Accepted Manuscript Title: Synthesis and characterization of water soluble O-carboxymethyl chitosan Schiff bases and Cu(II) complexes Author: Talat Baran Ayfer Mentes¸ H¨ulya Arslan PII: DOI: Reference:
S0141-8130(14)00504-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.07.029 BIOMAC 4495
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
7-4-2014 7-7-2014 9-7-2014
Please cite this article as: T. Baran, A. Mentes¸, H. Arslan, Synthesis and characterization of water soluble O-carboxymethyl chitosan Schiff bases and Cu(II) complexes, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and characterization of water soluble O-carboxymethyl chitosan Schiff bases and Cu(II) complexes
Department of Chemistry, Faculty of Science and Letters, Aksaray University, Aksaray,
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1
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Talat Baran1, Ayfer Menteş1*, Hülya Arslan2
Turkey
Department of Chemistry, Faculty of Science and Letters, Bülent Ecevit University,
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2
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Zonguldak, Turkey
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Corresponding author:
Address: Department of Chemistry, Faculty of Science and Letters, Aksaray University,
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Aksaray, Turkey, 68100
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Tel/fax: +903822882141, E-mail address: [email protected]
Abstract
In this study, mono-imine was synthesized (3a and 4a) via a condensation reaction between 2,4-pentadion and aminobenzoic acid (ortho or meta) in alcohol (1:1). The second-imine (CS3a and CS-4a) was obtained as a result of the reaction of the free oxo groups of mono-imine (3a and 4a) with the amino groups on the chitosan (CS). Their structures were characterized with FTIR and
13
C CP-MAS. Then, the water soluble forms of CS-3a and CS-4a were
obtained through oxidation of the hydroxide groups on the chitosan to carboxymethyl groups using monochloracetic acid ([O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O). Thus, the solubility problem of chitosan in an aqueous media was overcome and Cu(II) complexes 2 Page 1 of 32
could be synthesized more easily. Characterization of the synthesized O-carboxymethyl chitosan Schiff base derivatives and their metal complexes, [O-CMCS-3a-Cu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O, was conducted using FTIR, UV-Vis, TG/DTA, XRD,
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SEM, elemental analysis, conductivities and magnetic susceptibility measurements.
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Keywords: Water soluble O-carboxymethyl chitosan, Schiff base, 13C CP-MAS NMR
1. Introduction
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Chitin is the second most abundant biopolymer in nature after cellulose [1], and its primary sources are the exoskeletons of insects and crustaceans, as well as the cell walls of yeast [2,
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3]. Chitosan, the most important product of chitin, is obtained via the deacetylation of chitin in an alkaline media [4]. Chitosan is one of the few cationic polysaccharides, and because of
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its unique physicochemical and biological characteristics, its derivatives are utilized in the
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removal of metals, arsenic, radioactive elements and textile coloring materials from
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wastewater. Applications of chitosan have been reported in many areas, such as medicine, food, agriculture, cosmetics, biotechnology and pharmaceuticals [5-8]. Problems related to dissolving chitosan in neutral or basic media restricts its usage in many fields. To enhance the solubility of chitosan in aqueous solutions, water-soluble chitosan derivatives are synthesized via the reactive hydroxyl and amino groups that are already present on the chitosan polymer [9-13]. One of the most important chitosan derivatives is Ocarboxymethyl chitosan (O-CMCS), which is obtained from the reaction of chitosan with monochloroacetic acid [14, 15]. The diverse applications of O-CMCS in various areas like biomedical materials, water treatment and the textiles sector have been reported by other studies in the literature [16-18].
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Various chemical modifications of chitosan (such as acylation, carboxylation, enzymatic substitution, sulfation, metal chelation, nitration, phosphorylation and cyanoethylation) are possible due to the reactive amine group of the chitosan polymer chain [19-22]. One of the
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most important modifications is related to Schiff bases, which are formed from the condensation of amino groups on the chitosan with aldehyde or ketone. Schiff bases are
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described as a compound containing an HC=N group. There has been growing demand for Schiff bases synthesized with β-diketones (like 2,4-pentanedione) as they are easily
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synthesized in high yield. Their productions are economically feasible and widely used in
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coordination chemistry [23, 24]. Schiff bases and their metal complexes have gained attention because of their potential utility as catalyses and their various biological applications as
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antibacterial, antifungal, anticancer and antiviral agents [25, 26]. Many studies have been reported in literatures regarding Schiff bases of chitosan and its derivatives. A study by Guo
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et al. [27] showed that Schiff bases of chitosan and O-carboxmethly chitosan exhibited
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antioxidant activity depending on the concentration of active hydroxyl and amine groups on the polymer chain. Additionally, Leonhardt et al. [28] synthesized Schiff bases of chitosan
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from salicylaldehyde and 2-pyridinecarboxaldehyde. They obtained palladium complexes of these Schiff bases and investigated their catalytic activity for Suzuki and Heck reactions. In the present study, for the purpose of eliminating the solubility problem of chitosan, and thereby extending its usability, two new water-soluble O-carboxymethyl chitosan Schiff bases and their copper complexes were synthesized. The chemical structures of the Ocarboxymethyl chitosan Schiff bases and their metal complexes were illuminated using the techniques of FTIR, 1H-NMR,
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C CP-MAS, UV-Vis, TG/DTG, XRD, SEM, elemental
analysis, conductivities and magnetic susceptibility measurements. 2. Materials and Method
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Chitosan (low molecular weight, 75-85% deacetylated, code: 448869), monochloroacetic acid, copper(II) acetate, 2,4-pentadion, m-amino-benzoic acid, p-amino-benzoic acid, acetic acid, sodium hydroxide, methanol, ethanol, and diethyl ether were purchased from either
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Merck or Sigma-Aldrich.
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2.1. Infrared analysis (FTIR)
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IR spectra were analyzed at 4000-625 cm-1 using a Perkin Elmer Spectrum 100 FTIR spectrophotometer.
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2.2. Thermogravimetric Analysis (TGA)
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The thermogravimetric analysis was conducted using an EXSTAR S11 7300. The TG/DTG curves of the chitosan, water soluble O-carboxymethyl chitosan Schiff bases and their copper
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complexes were carried out under a nitrogen atmosphere and at a sample heating rate of 10 °C
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min−1.
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2.3. 1H NMR, 13C NMR and 13C CP-MAS NMR H and
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C NMR spectra were recorded on a Varian Gemini 200 spectrometer and Varian
Mercury 400 spectrometer respectively. The 13C CP-MAS NMR spectra were measured using a Bruker Superconducting FT.NMR Spectrometer Advance TM 300 MHz WB. 2.4. UV-Vis
The electronic spectra of the complexes in the UV–Vis region were obtained in H2O solutions using a Shimadzu UV-1240 spectrophotometer in the range of 200–800 nm. 2.5. Scanning electron microscopy (SEM)
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The surface morphologies of the chitosan, the O-carboxymethyl chitosan Schiff bases and their complexes were determined using an EVO LS 10 ZEISS scanning electron microscope. The samples were coated with gold before the SEM analysis with a Sputter Coater
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(Cressingto Auto 108).
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2.6. X-ray diffraction (XRD)
X-ray diffraction profiles were obtained at 40 kV, 30 mA and 2θ with a scan angle from 5º to
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50º using a Rigaku D max 2000 system.
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2.7. Calculation of yield (%)
The yield of water soluble O-carboxymethyl chitosan Schiff bases and their Cu(II) complexes
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[(Product weight / Reactant) x100]
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were calculated using the following formula:
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2.8. Elemental analysis
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Elemental analysis was performed by Thermo Scientific FLASH-2000. 2.9. Magnetic moments
The magnetic susceptibility values of the complexes were measured by using a Sherwood magnetic susceptibility balance.
2.10. Measurements of conductivity Conductivity measurement of the copper(II) complexes was performed with four-point probe technique by using Keithley 2400 Electrometer. To press the pellets, a hydraulic press was used under pressure of 1700 kg/cm2. 3. Experimental 6 Page 5 of 32
3.1. Synthesis mono-imine ligands (3a, 4a) Ligand 3a (3-{[(1Z)-1-methyl-3-oxobut-1-en-1-yl]amino}benzoic acid) and 4a (4-{[(1Z)-1methyl-3-oxobut-1-en-1-yl]amino}benzoic acid) were synthesized by refluxing a mixture of
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amino benzoic acid (1.0 g, 7.29 mmol) and 2,4-pentadion (0.73 g, 7.29 mmol) in ethanol (50 mL). The mixture was heated under reflux for 72 h. The reaction was followed by FTIR. After
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the reaction was completed, the solvent (3/2 ratio) was removed by rotary. The yellow
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crystalline solid which were obtained was then filtered and washed with cold ethanol, methanol and diethyl ether several times, and dried over P2O5 in a vacuum (Yield 3a, 82%,
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4a, 78%).
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3.2. Preparation of completely deacetylated chitosan (CS)
Commercial chitosan (5 g) was refluxed in 60% NaOH for 5 h. Then, the mixture was cooled
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to RT, filtered and rinsed with deionized water until a neutral pH was reached. Completely
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deacetylated chitosan was obtained and dried at 60 °C for 48 h in an oven (yield 95%).
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3.3. Synthesis of water soluble O-carboxymethyl chitosan Schiff bases: [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O
[O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O were synthesized following the similar procedure:
CS (0.5 g) was dissolved in a solution of 2% acetic acid (25 mL) in a three-necked flask. Then, 20 mL of anhydrous ethanol was added to the solution and stirred for 1 h. After 1 h, the compound of 3a or 4a was added dropwise (drop by drop) in small portions (3 fold of CS) and dissolved in 50 mL anhydrous ethanol, and the mixture was heated at 60 °C (CS-3a: 12 h; CS4a: 8 h). Then, 2.5 g of monochloroacetic acid in 10 mL of ethanol was added dropwise into the reaction mixture and stirred at 60 °C ([O-CMCS-3a].2H2O: 5 day; ([O-CMCS-4a].2H2O: 7 Page 6 of 32
8 day). At the end of the reaction, a solid orange product was filtered and washed with hot ethanol to remove the unreacted 3a or 4a compound. The resulting solid ([O-CMCS3a].2H2O) or ([O-CMCS-4a].2H2O) was dried at 50 °C for 6 h in an oven (yield 52%, 65%
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respectively).
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3.4. Synthesis of Cu(II) complexes
Copper(II) complexes were synthesized using the following procedure. O-carboxymethyl
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chitosan Schiff bases ([O-CMCS-3a].2H2O) or [O-CMCS-4a].2H2O) (0.3g) were dissolved in water (20 mL). Then a water solution of 0.5 g of Cu(CH3COO)2.H2O was added and stirred at
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70 °C for 6 h. At the end of the reaction, the solution was cooled to RT and the resulting dark green precipitate was filtered out. The solid of ([O-CMCS-3a-Cu(OAc)2].2H2O) and [O-
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CMCS-4a-Cu(OAc)2].2H2O) were washed with water several times and dried at 50 °C for 6 h,
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and the product yield was 42% and 45%, respectively.
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4. Results and discussion
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Scheme 1
A condensation reaction between 2,4-pentadion with amino-benzoic acid (meta or para) in alcohol (1:1) produced a high-yield of a mono-imin Schiff base, Fig. 1. Schiff base ligand 3a and 4a are not dissolved in water in spite of their solubility in polar organic solvents such as methanol, ethanol, DMF and DMSO. O-Carboxymethyl chitosan Schiff bases [O-CMCS3a].2H2O and [O-CMCS-4a].2H2O are completely soluble in water but they partially are dissolved or swelled in polar organic solvents such as methanol and ethanol. Copper complexes are dissolved in hot water but they are less soluble in polar organic solvents (e.g. methanol or ethanol). 4.1. FTIR
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Fig. 1 shows the FTIR spectra of chitosan (a, CS), Schiff base 3a (b, 3a), O-carboxymethyl chitosan-Schiff base 3a (c, [O-CMCS-3a].2H2O) and O-carboxymethyl chitosan-Schiff base Cu(II) complex (d, [O-CMCS-3a-Cu(OAc)2].2H2O). Fig. 2 shows the FTIR spectra of
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chitosan (a, CS), Schiff base 4a (b, 4a), O-carboxymethyl chitosan-Shiff base 4a (c, [OCMCS-4a].2H2O) and O-carboxymethyl chitosan-Schiff base Cu(II) complex (d, O-CMCS-
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4a-Cu(OAc)2].2H2O).
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The FTIR spectra of 3a and 4a were analyzed. Normally free C=O stretching vibrations are expected at 1710 cm-1, but in our case, due to the hydrogen bond of the keto-amine form, the
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stretching of the C=O was observed shifting to 1705 cm-1 for 3a (Fig. 1b) and 1694 cm-1 for 4a (Fig. 2b) [29]. For both 3a and 4a, the stretching vibrations of the O-H and N-H were
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observed at 3385 cm-1 at a moderate magnitude. A sharp absorbance band was observed at 1283 cm-1 and 1285 cm-1 for 3a and 4a, respectively. These bands can be attributed to the O-H
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groups in the ring system. The band at 1488 cm-1 for 3a and the one at 1497 cm-1 for 4a
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indicate the presence of C-N stretching.
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In the FTIR spectrum of the commercial chitosan, there are two characteristic bands NHCOCH3 (acetyl) units (with C = O stretching) and N-H bending, which are observed at 1651 cm-1 and 1586 cm-1 respectively [30-32]. Infrared spectrum of deacetyled chitosan (CS) was examined in this study, it was seen that magnitude of band at 1651 cm-1 decreased. Additionally, the band at 1586 cm-1 was shifted to 1590 cm-1 and its magnitude increased. These observations proved the almost complete deacetylation of chitosan. (Fig. 1a) [33]. The other important bands; the bands at 3360 cm-1
and 3430 cm-1, can be attributed to
symmetrical and asymmetrical (NH2) stretching, respectively, C-N amino groups axial deformation at 1324 cm–1, –C–O asymmetric stretching vibration at 1078 cm-1, primary hydroxyl group (characteristic peak of CH2-OH in primary alcohols, C-O stretching) at 1029 cm-1. The infrared spectrum of [O-CMCS-3a].2H2O is presented in Fig. 1c. The appearance of 9 Page 8 of 32
a new signal at 1625 cm-1 and the disappearance of the band at 1705 cm-1 belonging to the C=O stretching of 3a verified the condensation of 3a onto CS. Also, the C=O stretching of carboxymethly was observed at 1731 cm-1. Asymmetric and symmetric stretching of the C=O
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appeared at 1611 cm-1 and 1525 cm-1, respectively. Also, peak at 3350 cm–1 corresponds to the stretching vibration of N–H and O–H bonds, at 2938 cm–1 to C-H stretching; at 1374 cm–1 to
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stretching C-N and the peak nearly at 750 cm-1 shows the vibration of -CH of the aromatic ring. The disappearance of the band at 1029 cm-1 (C6-OH of CS) as a result of the formation
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of [O-CMCS-3a].2H2O was an indication of O-carboxymethylation (Fig. 1c) [34, 35]. Fig. 2c shows the infrared spectrum of [O-CMCS-4a].2H2O. A new signal at 1635 cm-1 and the
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disappearance of the band at 1694 cm-1 belonging to C=O stretching of 4a was proof of the condensation of 4a onto CS. C=O stretching of carboxymethyl was observed at 1717 cm-1.
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Asymmetric and symmetric stretching of the C=O appeared at 1605 cm-1 and 1515 cm-1,
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respectively. The band at 3350 cm–1 is referred to the stretching vibration of N–H and O–H
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bonds, at 2930 cm–1 to stretching of C-H, at 1378 cm–1 to stretching of C-N and the bands between 700 and 900 cm-1 belongs to vibration of -CH of the aromatic ring. The
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disappearance of the band at 1029 cm-1 (C6-OH of CS) as a result of the formation of [OCMCS-4a].2H2O was an indication of O-carboxymethylation (Fig. 2 c) [34, 35]. The FTIR spectrum of [O-CMCS-3a-Cu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O are given in Fig. 1d and 2d respectively. The peak at 1617cm-1 in the spectrum of [O-CMCS3a-Cu(OAc)2].2H2O and at 1622 cm-1 in the spectrum of [O-CMCS-4a-Cu(OAc)2].2H2O was attributed to imine (C=N) vibration. When the FTIR spectra of copper complexes were compared to that of [O-CMCS-3a].2H2O and [O-CMCS-3a].2H2O, the shifting of the imine stretching to a lower wave number showed the coordination of Cu(II) ions with the azomethine imine [36, 37]. In addition, two signals were observed at 1567 cm-1 and 1380 cm-1 for [O-CMCS-3a-Cu(OAc)2].2H2O, and at 1575 cm-1 and 1387 cm-1 for [O-CMCS-4a10 Page 9 of 32
Cu(OAc)2].2H2O. These signals were considered to be asymmetric and symmetric stretching of the acetate (CH3COO-) carbonyl groups [38, 39].
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Figure 1 Figure 2
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4.2. 1H NMR, 13 C NMR and 13C CP-MAS NMR
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The NMR spectra 3a and 4a were examined, and it was determined that the structures belonged to the keto-amine tautomerization. 1H NMR 3a: δ 2.25, s, 3H; 2.50, s, 3H; 5.3, s, H; 13
C NMR 3a: δ 166.78
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5.8, s, NH; 7.47, t, H; 7.51, d, H; 7.64, d, H; 7.75, s, H; 12.5, s, H.
C(1); 132.16 C(2); 127.75 C(3); 129.85 C(4); 125.86 C(5); 123.76 C(6); 138.87 C(7); 159.22
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C(8); 98.87 C(9); 195.17 C(10); 28.13 C(11); 19.27 C(12). 1H NMR 4a: δ 1.88, s, 3H; 2.17, s, 3H; 5.2, s, H; 5.5, s, NH; 7.2, d, 2H; 8.1, d, 2, H; 12.7, s, H.
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C NMR 4a: δ 170.13 C(1);
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131.65 C(2); 125.12 C(3); 122.87 C(4); 144.16 C(5); 20.14 C(6); 158.45 C(7); 99.08 C(8);
C CP-MAS spectra of chitosan have been examined in previous studies, and seen that the
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13
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196.66 C(9); 29.18 C(10) (Supporting information, Figure 1S, Figure 2S and Table 1S). The
peaks at 23.50 and 173.90 ppm belonged to the –CH3 and -C=O of the acetyl groups [40, 41]. In the present study on the
13
C CP-MAS NMR spectrum of CS, peaks were not observed at
23.50 ppm and 173.90 ppm (Fig. 3a), which indicated that the chitosan was almost 100% deacetyled. 13C CP-MAS, CS: δ 100.18 C(1); 52.11 C(2); 70.57 C(3); 81.14 C(4); 76.47 C(5); 56.16 C(6). In the spectrum, the peak at 195 ppm of 3a disappeared and a new signal at 164.13 ppm was observed, which was attributed to the C=N. This signal indicated the condensation of CS within 3a (Fig. 3b). 13C CP-MAS, CS-3a: δ 101 C(1); 56.60 C(2); 75.06 C(3); 91.85 C(4); 80.06 C(5); 71.21 C(6); 22.90 C(7); 164.13 C(8); 110.90 C(9); 143.07 C(10); 15.26 C(11); 137.41 C(12); 125.14 C(13); 114.50 C(4); 117.60 C(15); 133.11 C(16); 127.64 C(17); 190.83 C(18). The 13C CP-MAS NMR spectrum of CS-4a was examined (Fig. 11 Page 10 of 32
3c), and a new signal belonging to the imine carbon (C=N) at 163.79 ppm was observed. This signal showed condensation of CS with 4a. 13C CP-MAS, CS-4a: δ 101.14 C(1); 56.65 C(2); 75.08 C(3); 92.06 (C4); 79.53 C(5); 71.63 C(6); 23.14 C(7); 163.79 C(8); 109.36 C(9); 148.31
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C(10); 15.20 C(11); 128.14 C(12); 111.55 C(13); 113.89 C(14); 126.50 C(15); 190.85 C(16)
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4.3. TG-DTG
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Figure 3
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(Supporting information, Table 2S).
In this examination of the TG-DTG curve of chitosan (Fig. 4a), there was a 9.07% mass loss
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in the first stage between 0-150 °C that was due to the evaporation of the absorbed water [42]. In the second stage, the 51.94% loss of mass between 151-450 °C was related to the
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decomposition of the acetylated and deacetylated units of the chitosan and saccharides that
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construct the molecule. In the last stage, between 451-1000 °C, 11.82% of the mass was lost
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due to the thermal decay of the glucosamine residuals [43]. The main thermal decay temperature of CS was determined to be 302 °C. The main thermal decay temperature was 198 °C for [O-CMCS-3a].2H2O and 240 °C for [OCMCS-4a].2H2O (Fig. 4b and 4c). This decrease in the thermal decay temperature was related to the reduction in the number of amine groups on the chitosan upon modification of CS and the lower crystallinity of the resulting polymer [44]. The TG-DTG curve of [O-CMCS-3a].2H2O was examined (Fig. 4b), and three stages of mass were observed. The 6.1% loss in the first stage between 0-152 °C corresponded to 2 moles of crystal water per Schiff base (calculated: 5.69). The 35.74% mass loss in the second stage between 153-301 °C was attributed to the O-CMCS units (C7H11NO6) (calculated: 32.45). 12 Page 11 of 32
Finally, the 30.8% mass loss in the third stage between 301-1000 °C belonged to the 3a (C12H13NO2) unit (calculated: 32.13). The TG-DTG curve of the [O-CMCS-3a-Cu(OAc)2].2H2O complex (Fig. 4d) was examined,
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and the 4.9% mass loss between 0-125 °C corresponded to 2 moles of crystal water (calculated: 4.42), the 38.5 mass loss between 126-354 °C was due to the O-CMCS units
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355-1250 °C was from the 3a (C12H13NO2) (calculated: 24.96).
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(C7H11NO6) and coordinated acetate units (calculated: 39.71) and the 25.2% mass loss at
The TG-DTG curve of [O-CMCS-4a].2H2O was examined (Fig. 4c), and three mass loss
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stages were observed. The 6% loss in the first stage between 0-150 °C belonged to 2 moles of crystal water (calculated: 5.69), the 34.21% mass loss in the second stage between 151-310
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°C was due to the O-CMCS units (C7H11NO6) (calculated: 32.45) and the 31.90% mass loss in
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the third stage between 311-1000 °C was due to the 3a (C12H13NO2) unit (calculated: 32.13).
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The TG-DTG curve of [O-CMCS-4a-Cu(OAc)2].2H2O (Fig. 4e) was examined, and the 5% mass loss between 0-125 °C was attributed to 2 moles of crystal water (calculated: 4.42), the
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37.6% mass loss between 126-354 °C was due to the O-CMCS units (C7H11NO6) and coordinated acetate units (calculated: 39.71) and the 25.50% mass loss between 355-1250 °C was due to the 4a (C12H13NO2) (calculated: 24.96). It was observed that these findings were consistent with theoretically calculated values (Supporting information, Table 3S). In addition, the remaining amounts of the substance after thermal decomposition in both complexes of [O-CMCS-3a-Cu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O were more than those from the compounds of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O. These increases in the amounts of the substance can be explained by the formation of an undecayed metal oxide (CuO) [44]. Figure 4 13 Page 12 of 32
4.4. Electronic spectra (UV-Vis) The UV spectrum of chitosan was examined in this study. There are no reports of any transitions between 300-800 nm in the literature [45]. However, we observed that there were
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two transitions at 286 nm and 313 nm for [O-CMCS-3a].2H2O; at 266 nm and 305 nm for [OCMCS-4a].2H2O. These transitions were attributed to the π-π* (benzene) and n-π* (imine,
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C=N) [46]. In the metal complexes π-π* and n-π*, the transitions were shifted to a higher
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wavelength. These changes indicated that copper ions coordinated with the ligand by virtue of the nitrogen atom of the azomethine [47]. Additionally, transitions at 730 nm for [O-CMCS-
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3a-Cu(OAc)2].2H2O and at 680 nm for [O-CMCS-4a-Cu(OAc)2].2H2O can be regarded as metal ion d-d transitions [44, 47].
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4.5. SEM
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The surface morphology analysis of CS with SEM showed a smooth surface (Fig. 5a), but in
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the case of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O, some differences in both form and size of surface structures were observed (Fig. 5b and 5c). The roughness of the surface may
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be explained by the formation of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O [48]. However, when the surface morphologies of [O-CMCS-3a-Cu(OAc)2].2H2O and [O-CMCS4a-Cu(OAc)2].2H2O were examined (Fig. 5d and 5e) it was found that the roughness of these surfaces was more obvious than those of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O. These differences are thought to be a result of the coordination of the metal with the ligands [44, 46, 47].
Figure 5 4.6. XRD
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Chitosan has more crystalline character than other natural carbohydrate polymers because of its strong intermolecular hydrogen bonds [49]. In the literature, chitosan characterization studies with XRD have shown that it has two characteristic peaks at 2θ = 10.40 and 19.80
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[50]. Here we report that the X-ray diffraction spectrum of the CS shows two peaks: 2θ = 10.44 and 19.86 (Fig. 6a). Moreover, peaks at 2θ = 9, 13, 23 and 26 were not observed in the
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spectrum of CS, and this demonstrates that the chitosan has been almost deacetylated [51]. In the spectra of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O (Fig. 6b and 6c) it was seen that
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the magnitude of the peak at 2θ = 19.8 decreased and was hardly observed. This can be
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explained by the decrease in the crystallinity of the polymer resulting from the reduction in the number of amino groups and the deformation of hydrogen bonds [46, 47].
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As a result of the o-carboxymethylation in the spectra, these peaks were also observed for [OCMCS-3a].2H2O at 12.28, 24.28, 27.12, 31.22, 34.08 and 35.96 (Fig. 6b) and for [O-CMCS-
d
4a].2H2O at 12.30, 25.30, 27.16, 31.44, 34.84 and 35.84 (Fig. 6c). In the case of copper
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complexes of the polymer, there is a decrease in the crystallinity as well as a widening and a decrease in the magnitude of the peaks (Fig. 6d and 6e).These indications prove that the
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copper complexes are more amorphous than the CS, [O-CMCS-3a].2H2O and [O-CMCS4a].2H2O [52].
The values of the CS, [O-CMCS-3a].2H2O, [O-CMCS-4a].2H2O and their copper complexes were calculated using the following equation [53, 54]. Crystalline index (%) = [(I110− Iam)/I110] × 100
(1)
Where I110 is crystallite index the maximum intensity at ~ 20° and Iam is the intensity of the amorphous diffraction at 16°. The calculated crystalline index order of the compounds is as follows:
15 Page 14 of 32
CS > [O-CMCS-3a].2H2O > [O-CMCS-4a].2H2O > [O-CMCS-3a-Cu(OAc)2].2H2O > [OCMCS-4a-Cu(OAc)2].2H2O Differences in the crystalline indexes may originate from 1) meta or para position of carboxyl
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groups of Schiff bases (3a, 4a), 2) steric hindrance, 3) hydrophobic interactions, 4) π-π
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stacking [55].
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Figure 6 4.7. Elemental analysis
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Elemental analysis data of CS, O-carboxymethyl chitosan Schiff bases and their copper complexes are given in Table 1. Degree of deacetylation (DD) of CS was calculated using
(2)
d
DD = [1-(C/N-5.145)/(6.861-5.145)]x100
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equation given below [53] and it was determined to be 98.38%.
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Degree of substitution (DS) of O-carboxymethyl chitosan Schiff bases were estimated
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employing the following formula [56, 57]: DS = [(aC/N)m – (C/N)o]/n
(3)
where (C/N)m is the C/N of the modified chitosan ([O-CMCS-3a].2H2O and [O-CMCS4a].2H2O), (C/N)o is the C/N of the CS, and a and n are the number of nitrogen and carbon introduced after CS modification, respectively. Degree of substitution of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O was 0.36 and 0.34, respectively. It was found that elemental analysis data of all the compounds were consistent with calculated and theoretical values. In addition considering both TG and elemental analysis data, we may conclude that O-carboxymethyl chitosan Schiff bases and their complexes have crystalline water. Table 1 16 Page 15 of 32
4.8. Magnetic moments and conductivity Magnetic moments measurement performed at room temperature showed that [O-CMCS-3aCu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O had 1.81 BM and 1.78 BM. These
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values suggested that the d9 complexes had square planar geometry [55]. Conductivity of [OCMCS-3a-Cu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O was determined to be
cr
2.3x10-13 S/cm and 1.55x10-12 S/cm, indicating that the complexes were not conductive.
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Acknowledgements
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The authors thank The Scientific and Technological Research Council of Turkey (TUBITAK) (Project Number: 113Z296), Aksaray University Scientific Research Projects Coordination
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for their financial support (Project Number: 2012-20) and Aksaray University, Science and
5. Conclusions
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Technology Application and Research Center, Turkey.
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It is known that chitosan is not soluble in aqueous solutions, and that this restricts its
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application in many areas. In this present study, to overcome this restriction, we synthesized two soluble derivatives of chitosan. These two derivatives are newly produced Ocarboxymethyl chitosan Schiff bases. Characterization studies of the synthesized compounds were carried out employing FTIR, 1H-NMR,
13
C CP-MAS, UV-Vis, TG/DTA, XRD, SEM,
elemental analysis, conductivities and magnetic susceptibility measurements. It was found that the thermal stability and crystallinity of the O-carboxymethyl chitosan Schiff bases and their copper complexes were lower than that of CS.
17 Page 16 of 32
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te
d
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an
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Scheme 1. Synthesis pathway for water soluble O-carboxymethyl chitosan Schiff bases and
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their Cu(II) complexes Figure captions
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Fig.1 FTIR spectra of CS (a), 3a (b), [O-CMCS-3a].2H2O (c), [O-CMCS-3a-Cu(OAc)2].2H2O
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(d)
Fig.2 FTIR spectra of CS (a), 4a (b), [O-CMCS-4a].2H2O (c), [O-CMCS-4a-Cu(OAc)2].2H2O
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(d)
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Fig. 3 13C CP-MAS NMR spectra of CS (a), CS-3a (b), CS-4a (c)
Fig. 4 TG-DTG spectra of CS (a), [O-CMCS-3a].2H2O (b), [O-CMCS-4a].2H2O (c), [O-
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d
CMCS-3a-Cu(OAc)2].2H2O (d), [O-CMCS-4a-Cu(OAc)2].2H2O (e) Fig. 5 Scanning electron microscopy (SEM) images of CS (a), [O-CMCS-3a].2H2O (b), [O-
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CMCS-4a].2H2O (c), [O-CMCS-3a-Cu(OAc)2].2H2O (d), [O-CMCS-4a-Cu(OAc)2].2H2O (e) Fig. 6 XRD patterns of CS (a), [O-CMCS-3a].2H2O (b), [O-CMCS-4a].2H2O (c), [O-CMCS3a-Cu(OAc)2].2H2O (d), [O-CMCS-4a-Cu(OAc)2].2H2O (e)
25 Page 24 of 32
Elemental analysis data Compound
Found (%)
Calculated (%)
H
N
C
H
N
CS
43.14
6.78
8.34
44.72
6.88
8.69
[O-CMCS-3a].2H2O
50.65
6.90
5.29
53.24
7.18
6.65
[O-CMCS-4a].2H2O
53.57
7.10
5.76
53.24
7.18
6.65
[O-CMCS-3a-Cu(OAc)2].2H2O
45.87
6.20
5.09
47.26
6.32
5.17
[O-CMCS-4a-Cu(OAc)2].2H2O
45.9
5.98
5.02
47.26
6.32
5.17
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C
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Table 1. Elemental analysis (C, H, N) data of the compounds
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Figure 1
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Figure 2
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Figure 3
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Scheme 1
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S0141-8130(14)00504-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.07.029 BIOMAC 4495
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
7-4-2014 7-7-2014 9-7-2014
Please cite this article as: T. Baran, A. Mentes¸, H. Arslan, Synthesis and characterization of water soluble O-carboxymethyl chitosan Schiff bases and Cu(II) complexes, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and characterization of water soluble O-carboxymethyl chitosan Schiff bases and Cu(II) complexes
Department of Chemistry, Faculty of Science and Letters, Aksaray University, Aksaray,
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1
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Talat Baran1, Ayfer Menteş1*, Hülya Arslan2
Turkey
Department of Chemistry, Faculty of Science and Letters, Bülent Ecevit University,
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2
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Zonguldak, Turkey
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Corresponding author:
Address: Department of Chemistry, Faculty of Science and Letters, Aksaray University,
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Aksaray, Turkey, 68100
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Tel/fax: +903822882141, E-mail address: [email protected]
Abstract
In this study, mono-imine was synthesized (3a and 4a) via a condensation reaction between 2,4-pentadion and aminobenzoic acid (ortho or meta) in alcohol (1:1). The second-imine (CS3a and CS-4a) was obtained as a result of the reaction of the free oxo groups of mono-imine (3a and 4a) with the amino groups on the chitosan (CS). Their structures were characterized with FTIR and
13
C CP-MAS. Then, the water soluble forms of CS-3a and CS-4a were
obtained through oxidation of the hydroxide groups on the chitosan to carboxymethyl groups using monochloracetic acid ([O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O). Thus, the solubility problem of chitosan in an aqueous media was overcome and Cu(II) complexes 2 Page 1 of 32
could be synthesized more easily. Characterization of the synthesized O-carboxymethyl chitosan Schiff base derivatives and their metal complexes, [O-CMCS-3a-Cu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O, was conducted using FTIR, UV-Vis, TG/DTA, XRD,
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SEM, elemental analysis, conductivities and magnetic susceptibility measurements.
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Keywords: Water soluble O-carboxymethyl chitosan, Schiff base, 13C CP-MAS NMR
1. Introduction
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Chitin is the second most abundant biopolymer in nature after cellulose [1], and its primary sources are the exoskeletons of insects and crustaceans, as well as the cell walls of yeast [2,
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3]. Chitosan, the most important product of chitin, is obtained via the deacetylation of chitin in an alkaline media [4]. Chitosan is one of the few cationic polysaccharides, and because of
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its unique physicochemical and biological characteristics, its derivatives are utilized in the
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removal of metals, arsenic, radioactive elements and textile coloring materials from
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wastewater. Applications of chitosan have been reported in many areas, such as medicine, food, agriculture, cosmetics, biotechnology and pharmaceuticals [5-8]. Problems related to dissolving chitosan in neutral or basic media restricts its usage in many fields. To enhance the solubility of chitosan in aqueous solutions, water-soluble chitosan derivatives are synthesized via the reactive hydroxyl and amino groups that are already present on the chitosan polymer [9-13]. One of the most important chitosan derivatives is Ocarboxymethyl chitosan (O-CMCS), which is obtained from the reaction of chitosan with monochloroacetic acid [14, 15]. The diverse applications of O-CMCS in various areas like biomedical materials, water treatment and the textiles sector have been reported by other studies in the literature [16-18].
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Various chemical modifications of chitosan (such as acylation, carboxylation, enzymatic substitution, sulfation, metal chelation, nitration, phosphorylation and cyanoethylation) are possible due to the reactive amine group of the chitosan polymer chain [19-22]. One of the
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most important modifications is related to Schiff bases, which are formed from the condensation of amino groups on the chitosan with aldehyde or ketone. Schiff bases are
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described as a compound containing an HC=N group. There has been growing demand for Schiff bases synthesized with β-diketones (like 2,4-pentanedione) as they are easily
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synthesized in high yield. Their productions are economically feasible and widely used in
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coordination chemistry [23, 24]. Schiff bases and their metal complexes have gained attention because of their potential utility as catalyses and their various biological applications as
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antibacterial, antifungal, anticancer and antiviral agents [25, 26]. Many studies have been reported in literatures regarding Schiff bases of chitosan and its derivatives. A study by Guo
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et al. [27] showed that Schiff bases of chitosan and O-carboxmethly chitosan exhibited
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antioxidant activity depending on the concentration of active hydroxyl and amine groups on the polymer chain. Additionally, Leonhardt et al. [28] synthesized Schiff bases of chitosan
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from salicylaldehyde and 2-pyridinecarboxaldehyde. They obtained palladium complexes of these Schiff bases and investigated their catalytic activity for Suzuki and Heck reactions. In the present study, for the purpose of eliminating the solubility problem of chitosan, and thereby extending its usability, two new water-soluble O-carboxymethyl chitosan Schiff bases and their copper complexes were synthesized. The chemical structures of the Ocarboxymethyl chitosan Schiff bases and their metal complexes were illuminated using the techniques of FTIR, 1H-NMR,
13
C CP-MAS, UV-Vis, TG/DTG, XRD, SEM, elemental
analysis, conductivities and magnetic susceptibility measurements. 2. Materials and Method
4 Page 3 of 32
Chitosan (low molecular weight, 75-85% deacetylated, code: 448869), monochloroacetic acid, copper(II) acetate, 2,4-pentadion, m-amino-benzoic acid, p-amino-benzoic acid, acetic acid, sodium hydroxide, methanol, ethanol, and diethyl ether were purchased from either
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Merck or Sigma-Aldrich.
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2.1. Infrared analysis (FTIR)
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IR spectra were analyzed at 4000-625 cm-1 using a Perkin Elmer Spectrum 100 FTIR spectrophotometer.
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2.2. Thermogravimetric Analysis (TGA)
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The thermogravimetric analysis was conducted using an EXSTAR S11 7300. The TG/DTG curves of the chitosan, water soluble O-carboxymethyl chitosan Schiff bases and their copper
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complexes were carried out under a nitrogen atmosphere and at a sample heating rate of 10 °C
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min−1.
1
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2.3. 1H NMR, 13C NMR and 13C CP-MAS NMR H and
13
C NMR spectra were recorded on a Varian Gemini 200 spectrometer and Varian
Mercury 400 spectrometer respectively. The 13C CP-MAS NMR spectra were measured using a Bruker Superconducting FT.NMR Spectrometer Advance TM 300 MHz WB. 2.4. UV-Vis
The electronic spectra of the complexes in the UV–Vis region were obtained in H2O solutions using a Shimadzu UV-1240 spectrophotometer in the range of 200–800 nm. 2.5. Scanning electron microscopy (SEM)
5 Page 4 of 32
The surface morphologies of the chitosan, the O-carboxymethyl chitosan Schiff bases and their complexes were determined using an EVO LS 10 ZEISS scanning electron microscope. The samples were coated with gold before the SEM analysis with a Sputter Coater
ip t
(Cressingto Auto 108).
cr
2.6. X-ray diffraction (XRD)
X-ray diffraction profiles were obtained at 40 kV, 30 mA and 2θ with a scan angle from 5º to
us
50º using a Rigaku D max 2000 system.
an
2.7. Calculation of yield (%)
The yield of water soluble O-carboxymethyl chitosan Schiff bases and their Cu(II) complexes
d
[(Product weight / Reactant) x100]
M
were calculated using the following formula:
te
2.8. Elemental analysis
Ac ce p
Elemental analysis was performed by Thermo Scientific FLASH-2000. 2.9. Magnetic moments
The magnetic susceptibility values of the complexes were measured by using a Sherwood magnetic susceptibility balance.
2.10. Measurements of conductivity Conductivity measurement of the copper(II) complexes was performed with four-point probe technique by using Keithley 2400 Electrometer. To press the pellets, a hydraulic press was used under pressure of 1700 kg/cm2. 3. Experimental 6 Page 5 of 32
3.1. Synthesis mono-imine ligands (3a, 4a) Ligand 3a (3-{[(1Z)-1-methyl-3-oxobut-1-en-1-yl]amino}benzoic acid) and 4a (4-{[(1Z)-1methyl-3-oxobut-1-en-1-yl]amino}benzoic acid) were synthesized by refluxing a mixture of
ip t
amino benzoic acid (1.0 g, 7.29 mmol) and 2,4-pentadion (0.73 g, 7.29 mmol) in ethanol (50 mL). The mixture was heated under reflux for 72 h. The reaction was followed by FTIR. After
cr
the reaction was completed, the solvent (3/2 ratio) was removed by rotary. The yellow
us
crystalline solid which were obtained was then filtered and washed with cold ethanol, methanol and diethyl ether several times, and dried over P2O5 in a vacuum (Yield 3a, 82%,
an
4a, 78%).
M
3.2. Preparation of completely deacetylated chitosan (CS)
Commercial chitosan (5 g) was refluxed in 60% NaOH for 5 h. Then, the mixture was cooled
d
to RT, filtered and rinsed with deionized water until a neutral pH was reached. Completely
te
deacetylated chitosan was obtained and dried at 60 °C for 48 h in an oven (yield 95%).
Ac ce p
3.3. Synthesis of water soluble O-carboxymethyl chitosan Schiff bases: [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O
[O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O were synthesized following the similar procedure:
CS (0.5 g) was dissolved in a solution of 2% acetic acid (25 mL) in a three-necked flask. Then, 20 mL of anhydrous ethanol was added to the solution and stirred for 1 h. After 1 h, the compound of 3a or 4a was added dropwise (drop by drop) in small portions (3 fold of CS) and dissolved in 50 mL anhydrous ethanol, and the mixture was heated at 60 °C (CS-3a: 12 h; CS4a: 8 h). Then, 2.5 g of monochloroacetic acid in 10 mL of ethanol was added dropwise into the reaction mixture and stirred at 60 °C ([O-CMCS-3a].2H2O: 5 day; ([O-CMCS-4a].2H2O: 7 Page 6 of 32
8 day). At the end of the reaction, a solid orange product was filtered and washed with hot ethanol to remove the unreacted 3a or 4a compound. The resulting solid ([O-CMCS3a].2H2O) or ([O-CMCS-4a].2H2O) was dried at 50 °C for 6 h in an oven (yield 52%, 65%
ip t
respectively).
cr
3.4. Synthesis of Cu(II) complexes
Copper(II) complexes were synthesized using the following procedure. O-carboxymethyl
us
chitosan Schiff bases ([O-CMCS-3a].2H2O) or [O-CMCS-4a].2H2O) (0.3g) were dissolved in water (20 mL). Then a water solution of 0.5 g of Cu(CH3COO)2.H2O was added and stirred at
an
70 °C for 6 h. At the end of the reaction, the solution was cooled to RT and the resulting dark green precipitate was filtered out. The solid of ([O-CMCS-3a-Cu(OAc)2].2H2O) and [O-
M
CMCS-4a-Cu(OAc)2].2H2O) were washed with water several times and dried at 50 °C for 6 h,
d
and the product yield was 42% and 45%, respectively.
Ac ce p
4. Results and discussion
te
Scheme 1
A condensation reaction between 2,4-pentadion with amino-benzoic acid (meta or para) in alcohol (1:1) produced a high-yield of a mono-imin Schiff base, Fig. 1. Schiff base ligand 3a and 4a are not dissolved in water in spite of their solubility in polar organic solvents such as methanol, ethanol, DMF and DMSO. O-Carboxymethyl chitosan Schiff bases [O-CMCS3a].2H2O and [O-CMCS-4a].2H2O are completely soluble in water but they partially are dissolved or swelled in polar organic solvents such as methanol and ethanol. Copper complexes are dissolved in hot water but they are less soluble in polar organic solvents (e.g. methanol or ethanol). 4.1. FTIR
8 Page 7 of 32
Fig. 1 shows the FTIR spectra of chitosan (a, CS), Schiff base 3a (b, 3a), O-carboxymethyl chitosan-Schiff base 3a (c, [O-CMCS-3a].2H2O) and O-carboxymethyl chitosan-Schiff base Cu(II) complex (d, [O-CMCS-3a-Cu(OAc)2].2H2O). Fig. 2 shows the FTIR spectra of
ip t
chitosan (a, CS), Schiff base 4a (b, 4a), O-carboxymethyl chitosan-Shiff base 4a (c, [OCMCS-4a].2H2O) and O-carboxymethyl chitosan-Schiff base Cu(II) complex (d, O-CMCS-
cr
4a-Cu(OAc)2].2H2O).
us
The FTIR spectra of 3a and 4a were analyzed. Normally free C=O stretching vibrations are expected at 1710 cm-1, but in our case, due to the hydrogen bond of the keto-amine form, the
an
stretching of the C=O was observed shifting to 1705 cm-1 for 3a (Fig. 1b) and 1694 cm-1 for 4a (Fig. 2b) [29]. For both 3a and 4a, the stretching vibrations of the O-H and N-H were
M
observed at 3385 cm-1 at a moderate magnitude. A sharp absorbance band was observed at 1283 cm-1 and 1285 cm-1 for 3a and 4a, respectively. These bands can be attributed to the O-H
d
groups in the ring system. The band at 1488 cm-1 for 3a and the one at 1497 cm-1 for 4a
te
indicate the presence of C-N stretching.
Ac ce p
In the FTIR spectrum of the commercial chitosan, there are two characteristic bands NHCOCH3 (acetyl) units (with C = O stretching) and N-H bending, which are observed at 1651 cm-1 and 1586 cm-1 respectively [30-32]. Infrared spectrum of deacetyled chitosan (CS) was examined in this study, it was seen that magnitude of band at 1651 cm-1 decreased. Additionally, the band at 1586 cm-1 was shifted to 1590 cm-1 and its magnitude increased. These observations proved the almost complete deacetylation of chitosan. (Fig. 1a) [33]. The other important bands; the bands at 3360 cm-1
and 3430 cm-1, can be attributed to
symmetrical and asymmetrical (NH2) stretching, respectively, C-N amino groups axial deformation at 1324 cm–1, –C–O asymmetric stretching vibration at 1078 cm-1, primary hydroxyl group (characteristic peak of CH2-OH in primary alcohols, C-O stretching) at 1029 cm-1. The infrared spectrum of [O-CMCS-3a].2H2O is presented in Fig. 1c. The appearance of 9 Page 8 of 32
a new signal at 1625 cm-1 and the disappearance of the band at 1705 cm-1 belonging to the C=O stretching of 3a verified the condensation of 3a onto CS. Also, the C=O stretching of carboxymethly was observed at 1731 cm-1. Asymmetric and symmetric stretching of the C=O
ip t
appeared at 1611 cm-1 and 1525 cm-1, respectively. Also, peak at 3350 cm–1 corresponds to the stretching vibration of N–H and O–H bonds, at 2938 cm–1 to C-H stretching; at 1374 cm–1 to
cr
stretching C-N and the peak nearly at 750 cm-1 shows the vibration of -CH of the aromatic ring. The disappearance of the band at 1029 cm-1 (C6-OH of CS) as a result of the formation
us
of [O-CMCS-3a].2H2O was an indication of O-carboxymethylation (Fig. 1c) [34, 35]. Fig. 2c shows the infrared spectrum of [O-CMCS-4a].2H2O. A new signal at 1635 cm-1 and the
an
disappearance of the band at 1694 cm-1 belonging to C=O stretching of 4a was proof of the condensation of 4a onto CS. C=O stretching of carboxymethyl was observed at 1717 cm-1.
M
Asymmetric and symmetric stretching of the C=O appeared at 1605 cm-1 and 1515 cm-1,
d
respectively. The band at 3350 cm–1 is referred to the stretching vibration of N–H and O–H
te
bonds, at 2930 cm–1 to stretching of C-H, at 1378 cm–1 to stretching of C-N and the bands between 700 and 900 cm-1 belongs to vibration of -CH of the aromatic ring. The
Ac ce p
disappearance of the band at 1029 cm-1 (C6-OH of CS) as a result of the formation of [OCMCS-4a].2H2O was an indication of O-carboxymethylation (Fig. 2 c) [34, 35]. The FTIR spectrum of [O-CMCS-3a-Cu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O are given in Fig. 1d and 2d respectively. The peak at 1617cm-1 in the spectrum of [O-CMCS3a-Cu(OAc)2].2H2O and at 1622 cm-1 in the spectrum of [O-CMCS-4a-Cu(OAc)2].2H2O was attributed to imine (C=N) vibration. When the FTIR spectra of copper complexes were compared to that of [O-CMCS-3a].2H2O and [O-CMCS-3a].2H2O, the shifting of the imine stretching to a lower wave number showed the coordination of Cu(II) ions with the azomethine imine [36, 37]. In addition, two signals were observed at 1567 cm-1 and 1380 cm-1 for [O-CMCS-3a-Cu(OAc)2].2H2O, and at 1575 cm-1 and 1387 cm-1 for [O-CMCS-4a10 Page 9 of 32
Cu(OAc)2].2H2O. These signals were considered to be asymmetric and symmetric stretching of the acetate (CH3COO-) carbonyl groups [38, 39].
ip t
Figure 1 Figure 2
cr
4.2. 1H NMR, 13 C NMR and 13C CP-MAS NMR
us
The NMR spectra 3a and 4a were examined, and it was determined that the structures belonged to the keto-amine tautomerization. 1H NMR 3a: δ 2.25, s, 3H; 2.50, s, 3H; 5.3, s, H; 13
C NMR 3a: δ 166.78
an
5.8, s, NH; 7.47, t, H; 7.51, d, H; 7.64, d, H; 7.75, s, H; 12.5, s, H.
C(1); 132.16 C(2); 127.75 C(3); 129.85 C(4); 125.86 C(5); 123.76 C(6); 138.87 C(7); 159.22
M
C(8); 98.87 C(9); 195.17 C(10); 28.13 C(11); 19.27 C(12). 1H NMR 4a: δ 1.88, s, 3H; 2.17, s, 3H; 5.2, s, H; 5.5, s, NH; 7.2, d, 2H; 8.1, d, 2, H; 12.7, s, H.
13
C NMR 4a: δ 170.13 C(1);
d
131.65 C(2); 125.12 C(3); 122.87 C(4); 144.16 C(5); 20.14 C(6); 158.45 C(7); 99.08 C(8);
C CP-MAS spectra of chitosan have been examined in previous studies, and seen that the
Ac ce p
13
te
196.66 C(9); 29.18 C(10) (Supporting information, Figure 1S, Figure 2S and Table 1S). The
peaks at 23.50 and 173.90 ppm belonged to the –CH3 and -C=O of the acetyl groups [40, 41]. In the present study on the
13
C CP-MAS NMR spectrum of CS, peaks were not observed at
23.50 ppm and 173.90 ppm (Fig. 3a), which indicated that the chitosan was almost 100% deacetyled. 13C CP-MAS, CS: δ 100.18 C(1); 52.11 C(2); 70.57 C(3); 81.14 C(4); 76.47 C(5); 56.16 C(6). In the spectrum, the peak at 195 ppm of 3a disappeared and a new signal at 164.13 ppm was observed, which was attributed to the C=N. This signal indicated the condensation of CS within 3a (Fig. 3b). 13C CP-MAS, CS-3a: δ 101 C(1); 56.60 C(2); 75.06 C(3); 91.85 C(4); 80.06 C(5); 71.21 C(6); 22.90 C(7); 164.13 C(8); 110.90 C(9); 143.07 C(10); 15.26 C(11); 137.41 C(12); 125.14 C(13); 114.50 C(4); 117.60 C(15); 133.11 C(16); 127.64 C(17); 190.83 C(18). The 13C CP-MAS NMR spectrum of CS-4a was examined (Fig. 11 Page 10 of 32
3c), and a new signal belonging to the imine carbon (C=N) at 163.79 ppm was observed. This signal showed condensation of CS with 4a. 13C CP-MAS, CS-4a: δ 101.14 C(1); 56.65 C(2); 75.08 C(3); 92.06 (C4); 79.53 C(5); 71.63 C(6); 23.14 C(7); 163.79 C(8); 109.36 C(9); 148.31
ip t
C(10); 15.20 C(11); 128.14 C(12); 111.55 C(13); 113.89 C(14); 126.50 C(15); 190.85 C(16)
an
4.3. TG-DTG
us
Figure 3
cr
(Supporting information, Table 2S).
In this examination of the TG-DTG curve of chitosan (Fig. 4a), there was a 9.07% mass loss
M
in the first stage between 0-150 °C that was due to the evaporation of the absorbed water [42]. In the second stage, the 51.94% loss of mass between 151-450 °C was related to the
d
decomposition of the acetylated and deacetylated units of the chitosan and saccharides that
te
construct the molecule. In the last stage, between 451-1000 °C, 11.82% of the mass was lost
Ac ce p
due to the thermal decay of the glucosamine residuals [43]. The main thermal decay temperature of CS was determined to be 302 °C. The main thermal decay temperature was 198 °C for [O-CMCS-3a].2H2O and 240 °C for [OCMCS-4a].2H2O (Fig. 4b and 4c). This decrease in the thermal decay temperature was related to the reduction in the number of amine groups on the chitosan upon modification of CS and the lower crystallinity of the resulting polymer [44]. The TG-DTG curve of [O-CMCS-3a].2H2O was examined (Fig. 4b), and three stages of mass were observed. The 6.1% loss in the first stage between 0-152 °C corresponded to 2 moles of crystal water per Schiff base (calculated: 5.69). The 35.74% mass loss in the second stage between 153-301 °C was attributed to the O-CMCS units (C7H11NO6) (calculated: 32.45). 12 Page 11 of 32
Finally, the 30.8% mass loss in the third stage between 301-1000 °C belonged to the 3a (C12H13NO2) unit (calculated: 32.13). The TG-DTG curve of the [O-CMCS-3a-Cu(OAc)2].2H2O complex (Fig. 4d) was examined,
ip t
and the 4.9% mass loss between 0-125 °C corresponded to 2 moles of crystal water (calculated: 4.42), the 38.5 mass loss between 126-354 °C was due to the O-CMCS units
us
355-1250 °C was from the 3a (C12H13NO2) (calculated: 24.96).
cr
(C7H11NO6) and coordinated acetate units (calculated: 39.71) and the 25.2% mass loss at
The TG-DTG curve of [O-CMCS-4a].2H2O was examined (Fig. 4c), and three mass loss
an
stages were observed. The 6% loss in the first stage between 0-150 °C belonged to 2 moles of crystal water (calculated: 5.69), the 34.21% mass loss in the second stage between 151-310
M
°C was due to the O-CMCS units (C7H11NO6) (calculated: 32.45) and the 31.90% mass loss in
d
the third stage between 311-1000 °C was due to the 3a (C12H13NO2) unit (calculated: 32.13).
te
The TG-DTG curve of [O-CMCS-4a-Cu(OAc)2].2H2O (Fig. 4e) was examined, and the 5% mass loss between 0-125 °C was attributed to 2 moles of crystal water (calculated: 4.42), the
Ac ce p
37.6% mass loss between 126-354 °C was due to the O-CMCS units (C7H11NO6) and coordinated acetate units (calculated: 39.71) and the 25.50% mass loss between 355-1250 °C was due to the 4a (C12H13NO2) (calculated: 24.96). It was observed that these findings were consistent with theoretically calculated values (Supporting information, Table 3S). In addition, the remaining amounts of the substance after thermal decomposition in both complexes of [O-CMCS-3a-Cu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O were more than those from the compounds of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O. These increases in the amounts of the substance can be explained by the formation of an undecayed metal oxide (CuO) [44]. Figure 4 13 Page 12 of 32
4.4. Electronic spectra (UV-Vis) The UV spectrum of chitosan was examined in this study. There are no reports of any transitions between 300-800 nm in the literature [45]. However, we observed that there were
ip t
two transitions at 286 nm and 313 nm for [O-CMCS-3a].2H2O; at 266 nm and 305 nm for [OCMCS-4a].2H2O. These transitions were attributed to the π-π* (benzene) and n-π* (imine,
cr
C=N) [46]. In the metal complexes π-π* and n-π*, the transitions were shifted to a higher
us
wavelength. These changes indicated that copper ions coordinated with the ligand by virtue of the nitrogen atom of the azomethine [47]. Additionally, transitions at 730 nm for [O-CMCS-
an
3a-Cu(OAc)2].2H2O and at 680 nm for [O-CMCS-4a-Cu(OAc)2].2H2O can be regarded as metal ion d-d transitions [44, 47].
M
4.5. SEM
d
The surface morphology analysis of CS with SEM showed a smooth surface (Fig. 5a), but in
te
the case of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O, some differences in both form and size of surface structures were observed (Fig. 5b and 5c). The roughness of the surface may
Ac ce p
be explained by the formation of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O [48]. However, when the surface morphologies of [O-CMCS-3a-Cu(OAc)2].2H2O and [O-CMCS4a-Cu(OAc)2].2H2O were examined (Fig. 5d and 5e) it was found that the roughness of these surfaces was more obvious than those of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O. These differences are thought to be a result of the coordination of the metal with the ligands [44, 46, 47].
Figure 5 4.6. XRD
14 Page 13 of 32
Chitosan has more crystalline character than other natural carbohydrate polymers because of its strong intermolecular hydrogen bonds [49]. In the literature, chitosan characterization studies with XRD have shown that it has two characteristic peaks at 2θ = 10.40 and 19.80
ip t
[50]. Here we report that the X-ray diffraction spectrum of the CS shows two peaks: 2θ = 10.44 and 19.86 (Fig. 6a). Moreover, peaks at 2θ = 9, 13, 23 and 26 were not observed in the
cr
spectrum of CS, and this demonstrates that the chitosan has been almost deacetylated [51]. In the spectra of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O (Fig. 6b and 6c) it was seen that
us
the magnitude of the peak at 2θ = 19.8 decreased and was hardly observed. This can be
an
explained by the decrease in the crystallinity of the polymer resulting from the reduction in the number of amino groups and the deformation of hydrogen bonds [46, 47].
M
As a result of the o-carboxymethylation in the spectra, these peaks were also observed for [OCMCS-3a].2H2O at 12.28, 24.28, 27.12, 31.22, 34.08 and 35.96 (Fig. 6b) and for [O-CMCS-
d
4a].2H2O at 12.30, 25.30, 27.16, 31.44, 34.84 and 35.84 (Fig. 6c). In the case of copper
te
complexes of the polymer, there is a decrease in the crystallinity as well as a widening and a decrease in the magnitude of the peaks (Fig. 6d and 6e).These indications prove that the
Ac ce p
copper complexes are more amorphous than the CS, [O-CMCS-3a].2H2O and [O-CMCS4a].2H2O [52].
The values of the CS, [O-CMCS-3a].2H2O, [O-CMCS-4a].2H2O and their copper complexes were calculated using the following equation [53, 54]. Crystalline index (%) = [(I110− Iam)/I110] × 100
(1)
Where I110 is crystallite index the maximum intensity at ~ 20° and Iam is the intensity of the amorphous diffraction at 16°. The calculated crystalline index order of the compounds is as follows:
15 Page 14 of 32
CS > [O-CMCS-3a].2H2O > [O-CMCS-4a].2H2O > [O-CMCS-3a-Cu(OAc)2].2H2O > [OCMCS-4a-Cu(OAc)2].2H2O Differences in the crystalline indexes may originate from 1) meta or para position of carboxyl
ip t
groups of Schiff bases (3a, 4a), 2) steric hindrance, 3) hydrophobic interactions, 4) π-π
cr
stacking [55].
us
Figure 6 4.7. Elemental analysis
an
Elemental analysis data of CS, O-carboxymethyl chitosan Schiff bases and their copper complexes are given in Table 1. Degree of deacetylation (DD) of CS was calculated using
(2)
d
DD = [1-(C/N-5.145)/(6.861-5.145)]x100
M
equation given below [53] and it was determined to be 98.38%.
te
Degree of substitution (DS) of O-carboxymethyl chitosan Schiff bases were estimated
Ac ce p
employing the following formula [56, 57]: DS = [(aC/N)m – (C/N)o]/n
(3)
where (C/N)m is the C/N of the modified chitosan ([O-CMCS-3a].2H2O and [O-CMCS4a].2H2O), (C/N)o is the C/N of the CS, and a and n are the number of nitrogen and carbon introduced after CS modification, respectively. Degree of substitution of [O-CMCS-3a].2H2O and [O-CMCS-4a].2H2O was 0.36 and 0.34, respectively. It was found that elemental analysis data of all the compounds were consistent with calculated and theoretical values. In addition considering both TG and elemental analysis data, we may conclude that O-carboxymethyl chitosan Schiff bases and their complexes have crystalline water. Table 1 16 Page 15 of 32
4.8. Magnetic moments and conductivity Magnetic moments measurement performed at room temperature showed that [O-CMCS-3aCu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O had 1.81 BM and 1.78 BM. These
ip t
values suggested that the d9 complexes had square planar geometry [55]. Conductivity of [OCMCS-3a-Cu(OAc)2].2H2O and [O-CMCS-4a-Cu(OAc)2].2H2O was determined to be
cr
2.3x10-13 S/cm and 1.55x10-12 S/cm, indicating that the complexes were not conductive.
us
Acknowledgements
an
The authors thank The Scientific and Technological Research Council of Turkey (TUBITAK) (Project Number: 113Z296), Aksaray University Scientific Research Projects Coordination
M
for their financial support (Project Number: 2012-20) and Aksaray University, Science and
5. Conclusions
d
Technology Application and Research Center, Turkey.
te
It is known that chitosan is not soluble in aqueous solutions, and that this restricts its
Ac ce p
application in many areas. In this present study, to overcome this restriction, we synthesized two soluble derivatives of chitosan. These two derivatives are newly produced Ocarboxymethyl chitosan Schiff bases. Characterization studies of the synthesized compounds were carried out employing FTIR, 1H-NMR,
13
C CP-MAS, UV-Vis, TG/DTA, XRD, SEM,
elemental analysis, conductivities and magnetic susceptibility measurements. It was found that the thermal stability and crystallinity of the O-carboxymethyl chitosan Schiff bases and their copper complexes were lower than that of CS.
17 Page 16 of 32
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ip t
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[14] H. Bidgoli, A. Zamani, M.J. Taherzadeh, Effect of carboxymethylation conditions on the
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[16] M.L. Zheng, B.Q. Han, Y. Yang, W.S. Liu, Synthesis, characterization and biological safety of o-carboxymethyl chitosan used to treat sarcoma 180 tumor, Carbohyd. Polym. 86
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Scheme 1. Synthesis pathway for water soluble O-carboxymethyl chitosan Schiff bases and
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their Cu(II) complexes Figure captions
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Fig.1 FTIR spectra of CS (a), 3a (b), [O-CMCS-3a].2H2O (c), [O-CMCS-3a-Cu(OAc)2].2H2O
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(d)
Fig.2 FTIR spectra of CS (a), 4a (b), [O-CMCS-4a].2H2O (c), [O-CMCS-4a-Cu(OAc)2].2H2O
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(d)
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Fig. 3 13C CP-MAS NMR spectra of CS (a), CS-3a (b), CS-4a (c)
Fig. 4 TG-DTG spectra of CS (a), [O-CMCS-3a].2H2O (b), [O-CMCS-4a].2H2O (c), [O-
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CMCS-3a-Cu(OAc)2].2H2O (d), [O-CMCS-4a-Cu(OAc)2].2H2O (e) Fig. 5 Scanning electron microscopy (SEM) images of CS (a), [O-CMCS-3a].2H2O (b), [O-
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CMCS-4a].2H2O (c), [O-CMCS-3a-Cu(OAc)2].2H2O (d), [O-CMCS-4a-Cu(OAc)2].2H2O (e) Fig. 6 XRD patterns of CS (a), [O-CMCS-3a].2H2O (b), [O-CMCS-4a].2H2O (c), [O-CMCS3a-Cu(OAc)2].2H2O (d), [O-CMCS-4a-Cu(OAc)2].2H2O (e)
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Elemental analysis data Compound
Found (%)
Calculated (%)
H
N
C
H
N
CS
43.14
6.78
8.34
44.72
6.88
8.69
[O-CMCS-3a].2H2O
50.65
6.90
5.29
53.24
7.18
6.65
[O-CMCS-4a].2H2O
53.57
7.10
5.76
53.24
7.18
6.65
[O-CMCS-3a-Cu(OAc)2].2H2O
45.87
6.20
5.09
47.26
6.32
5.17
[O-CMCS-4a-Cu(OAc)2].2H2O
45.9
5.98
5.02
47.26
6.32
5.17
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Table 1. Elemental analysis (C, H, N) data of the compounds
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Scheme 1
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