Cellulose (2015) 22:23912407

DOI 10.1007/s10570-015-0673-4

ORIGINAL PAPER

Study of chitosans interaction with Cu(II)

Received: 18 February 2015 / Accepted: 28 May 2015 / Published online: 4 June 2015
Springer Science+Business Media Dordrecht 2015

Abstract The interaction of copper ions with chi- application studies. This result was supported by
tosan and three synthesized derivatives with different Raman spectroscopy results in which vibrations of
chelating centers, N-benzylidene chitosan, N-benzyl O=S=O groups were observed. X-ray diffraction
chitosan and poly-N-(4-(4-R-methoxyphenyl)diazenyl)- patterns showed that the chemical modification of
benzyl-chitosan using CuSO45H2O and CuCl22H2O chitosan and formation of complexes resulted in the
salts was studied. The content of Cu2? in the complexes decrease of crystallinity. Electron paramagnetic reso-
was determined by atomic absorption spectrometry nance was used to investigate structural aspects of the
and the results showed that chitosan exhibited higher materials complexed with Cu2? ions in the solid state.
chelating capacity for both salts. Morphological
changes of derivatives and complexes were demon- Keywords Chitosan  Derivatives  Copper 
strated by SEMEDS. In addition, the presence of Complexes
some crystals attributed to copper sulfate adsorbed on
the polymer surface was also observed, which indicates
that part of the metal content is in the salt adsorbed and Introduction
might influence in the use of the materials for further
In recent years, chitosan has become a very attractive
biopolymer due to its physicochemical and biological
F. S. Pereira  E. R. P. Gonzalez (&)
Laboratorio de Qumica Organica Fina, Departamento de properties, such as biocompatibility, biodegradability
Fsica, Qumica e Biologia, Univ Estadual Paulista, and non-toxicity. For this reason, chitosan and its
Campus de Presidente Prudente, derivatives have been explored for different uses in
C.P. 467, Presidente Prudente, SP 19060-900, Brazil several fields. The OH and NH2 groups on the
e-mail: [email protected]
skeleton of chitosan can bind transition metal ions
G. G. de Souza  P. G. P. Moraes  S. Lanfredi  either by chelation or by physical adsorption to form
H. M. Gomes complexes. Thus, chitosan has demonstrated greater
Departamento de Fsica, Qumica e Biologia, Univ chelating ability than chitin.
Estadual Paulista, Campus de Presidente Prudente,
C.P. 467, Presidente Prudente, SP 19060-900, Brazil Copper complexes were found to be important
materials in medicinal procedures as they have shown
R. P. Barroso  A. J. Costa-Filho potential use as antimicrobial, antiviral, anti-inflamma-
Laboratorio de Biofsica Molecular, Departamento de tory and antitumor agents (Giovagnini et al. 2008;
Fsica, Faculdade de Filosofia Ciencias e Letras de
Ribeirao Preto, Universidade de Sao Paulo, Dulcevscaia et al. 2013; Facchin et al. 2009; Urquiola
Ribeirao Preto, SP, Brazil et al. 2008; Torre et al. 2005) and enzyme inhibitors

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2392 Cellulose (2015) 22:23912407

(Iakovidis et al. 2011). The chelation of copper ions 2004). Rhazi et al. (2002) have reported that the
with chitosan has received considerable attention due to optimal pH for the fixation of cupric ions by chitosan is
its applications in waste water treatment (Domard 1987; in the range of 57 (Rhazi et al. 2002). In acidic
Huang et al. 1996; Bassi et al. 2000), food industries solutions, the protonation of amine groups gives the
and as catalysts in a variety of reaction systems (Guibal polymer the potential for attracting metal anions, such
2005; Calo et al. 2004; Adlim et al. 2004; Wang et al. as molybdate, vanadate, chromate and arsenate (Chas-
2005). Chitosan Cu2? complexes have also been sary et al. 2004). Sorption studies of carboxymethyl
reported for their antibacterial and antitumor properties and carboxybutyl chitosans have shown that both
(Zheng et al. 2006). Results indicated that the inhibitory amino and carboxylic groups are involved in the metal
effects of chitosanmetal complexes can depend on the chelation. However, at low pH media, the binding
property of metal ions, the molecular weight and degree ability of N-carboxymethyl chitosan towards Cu(II)
of deacetylation of the polymer and environmental pH and Pb(II) ions decreased (Delben et al. 1992).
values (Wang et al. 2005). Furthermore, azobenzene chitosan derivatives can
Some works have given attention to understand be studied as metal chelating materials. Wang et al.
more about the structure of chitosan copper ions. reported the synthesis, characterization and applica-
X-ray studies of chitosan transition metal complexes tion of 4-amino-40 -nitro azobenzene modified cross-
demonstrated that a metal salt is coordinated with an linked chitosan for the adsorption of Au(III) and Pd(II)
amino group of a dimer residue of chitosan by a from aqueous samples (Wang et al. 2010) and the
pendant model (Ogawa and Oka 1993; Ogawa et al. adsorption capacity of 30 -nitro-4-amino azobenzene
1984). In agreement with this result, Schlick (1986) modified chitosan toward Pd(II) and Pt(IV) (Wang
reported that ESR parameters suggest that chitosan is et al. 2011).
bonded through four nitrogen ligands to Cu2? ions in a In the present manuscript, we report the interaction
square-planar geometry (Schlick 1986). Other ESR of copper ions from aqueous solutions of two different
studies showed that copper ions are trapped in a great salts, CuSO45H2O and CuCl22H2O, with chitosan and
amount when the polymer exhibits a lower crys- its derivatives N-benzylidene chitosan (CTB), N-benzyl
tallinity index and a higher deacetylation degree chitosan (NBC) and poly-(4-(4-methoxyphenyl)di-
increases the retention capacity (Valdes and Trivino azenyl)-N-benzyl chitosan (Azo-NBC). N-azobenzyl
2009). Cardenas et al. (2003) reported that EDX chitosan derivatives have been previously prepared and
studies revealed the highest retention of copper metal characterized by thermal and spectroscopy (Pereira
ions by chitosan of 1.2 9 10-3 eq/g, which is in et al. 2013, 2014). The series of chitosan derivatives
agreement with the electron micrograph that exhibit a were employed in this work to study the influence of
smooth surface (Cardenas et al. 2003). different chelating centers for Cu(II). The biological
On the other hand, chemical modification of activity of complexes can be related to the amount of
chitosan can change the polymer properties and ions coordinated and the coordination mode of the
demonstrate different interaction capacity with metal- metal with specific centers, in which can present
lic ions (Jiao et al. 2001; Rodrigues et al. 1998. different crystallinity, bond lengths and bond angles
Copper(II) complexes of Schiff-bases, for example, (Haas and Franz 2009). In addition, for the use of metal
are becoming relevant due to the wide applications in complexes in catalytic systems, it is of great importance
several fields (Genin et al. 2000; Suslick and Reinert to investigate the most active form of the complex in
1985; Katwal et al. 2013). relation to its coordination center (Das et al. 2014).
The sorptivity capacity of N-(2-carboxyethyl) chi- Another point for studying these materials is their lack
tosan was studied using a mixture of the appropriate of solubility in the majority of common solvents tested,
sulfates of Zn2?, Cu2?, Co2?, Ni2? at pH 4.5. The which make them excellent candidates for use as
highest capacity and selectivity for Cu(II) reached the heterogeneous catalysts due to their recovery and
maximum capacity up to 3.7 mmol/g; corresponding to reusability (Anastas et al. 2001).
80 %. It was found that anions of the absorbed solution The influence of both salts on the copper complex-
are involved in the sorption (Dobetti and Delben 1992). ation reaction was analyzed by AAS and the com-
Considering the pH media, the sorption of metal plexes with higher Cu2? content were characterized by
cations can be studied at pH close to neutral (Guibal SEMEDS, XRD, Raman and EPR.

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Materials and methods solutions and the mixture was stirred for 30 min at
05 C. The dark brown product was filtered off,
Materials washed with ethanol and dried for 3 h at 40 C. The
product showed solubility in dimethyl sulfoxide
Chitosan from crab shells was purchased from Sigma- (DMSO). The DS determined by 1H-NMR (Sajom-
Aldrich, lot # SLBC2867V and cat # 417963, humid- sang et al. 2006) was 46 %.
ity: 11.8 %, and was used without further purification.
The deacetylation degree of chitosan was 78 %. The Preparation of chitosan and derivatives copper
other chemicals used were of analytical grade. Ben- complexes
zaldehyde, sodium cyanoborohydride, copper(II) sul-
fate pentahydrate and copper(II) chloride dihydrate The complexes were prepared by adding 0.1 g of dry
were purchased from Sigma-Aldrich and used without chitosan or derivatives (CTB, NBC and Azo-NBC) to
further purification. 10 mL of CuSO45H2O or CuCl22H2O aqueous
solution (0.01 mol/L) with stirring at room tempera-
N-alkylation of chitosan ture. In order to investigate the influence of the
reaction time, the formation of metal complex was
Chitosan derivatives were prepared according to the studied from 30 min to 48 h. After stirring during the
literature (Borch et al. 1971). For the preparation of the corresponding time, the mixture was filtered off and
Schiff-base, 0.5 g of chitosan was dissolved in 15 mL the precipitate was dried in an oven at 60 C for 4 h.
of acetic acid 1 % and the pH was adjusted to 45. The obtained complexes were denominated Chitosan
This solution was diluted with 30 mL of ethanol and Cu, CTBCu, NBCCu and Azo-NBCCu.
stirred for 30 min to form a colorless gel. Then,
2 mmol of benzaldehyde dissolved in 10 mL of Measurements
ethanol (99 %) was added to the solution and the
mixture was stirred for 8 h at 60 C. After that, the Cu(II) ions content of the remaining solution was
solvent was evaporated under reduced pressure and the measured by A Model Analyst 200 PerkinElmer Atomic
product was washed with 15 mL of aqueous NaHCO3 Absorption Spectrometer in the optimal conditions
(5 %). The product was filtered off and washed with recommended by the supplier for this quantification.
distilled water and ethanol to remove the excess of The electron micrograph studies were carried out in
reagents. The resulting yellowish powder was dried Carls Zeiss EVO LS15 Model, with secondary elec-
for 4 h at 60 C and yielded 0.65 g. The degree of trons detector and high vacuum. Sputter Coater
substitution (DS) determined by 1H-NMR (Sajomsang Quorum Model Q 150R ES was used to metalize the
et al. 2006) was 52 %. samples.
The Schiff base was reduced with sodium X-ray diffractograms on powder samples were
cyanoborohydride (0.12 g, 2 mmol) dissolved in obtained using a SHIMADZU (model XRD-6000)
10 mL of ethanol (99 %) and the mixture was stirred diffractometer, with Cu-Ka1 radiation (k =
for 24 h at room temperature. The white precipitate 1.54056 A) and a graphite monochromator, at 40 kV
was filtered off and washed with water and ethanol to and a current of 30 mA. Measurements were carried
remove the excess of reagents. Afterwards, the out over an angular range of 5 B 2h B 40 with a
material was dried for 4 h at 60 C, yielding 0.7 g of scanning step of 0.02 and a fixed counting time of
product. The DS determined by 1H-NMR (Sajomsang 10 s. Divergence, scattered and receiving radiation
et al. 2006) was 50 %. slits were 1, 1 and 0.2 mm respectively.
The Raman spectra were obtained using a micro-
Synthesis of the N-azobenzyl derivative (Azo- Raman spectrograph Renishaw model inVia, equipped
NBC) with an electrically cooled CCD camera, with laser
lines at 532, 633 or 785 nm, and diffraction grades
1 mmol of p-methoxybenzenediazonium tetrafluorbo- with 1800 and 1200 L/mm, 1 accumulation, 10 s of
rate (0.19 g) was dissolved in 10 mL of acetonitrile. acquisition and the laser power was adjusted to best
N-benzyl chitosan (0.5 g) was slowly added to the relation signal/noise.

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EPR spectra were recorded using a JEOL, Model were carried out at the time previously determined,
FA-200 (JEOL Ltd., Tokyo, Japan) at X-band. The 24 h for chitosan and 36 h for the derivatives. The
EPR spectra were measured at the microwave power chelation capacities of these materials were studied by
of 10 mW and amplitude of modulation 8 mT, 2 scans AAS and are listed in Table 2. The copper content
with the sweep time of 4 min. chelated by the compounds using CuCl22H2O salt is
lower when compared to the results observed for the
reaction with CuSO45H2O. However, part of the
Results and discussion copper amount present in the polymeric compounds is
in the form of copper sulfate salt adsorbed on the
The Schiff base has been synthesized by the conden- surface of chitosan and derivatives, as evidenced by
sation reaction of chitosan and benzaldehyde. Subse- SEMEDS analysis. On the other hand, no
quently, this compound was reduced with NaBH3CN CuCl22H2O salt was observed on the polymeric
to yield N-benzyl chitosan. N-azobenzyl chitosan surface, therefore, the complexation reaction with this
derivative was prepared via azo-coupling of N-benzyl salt was more efficient as the great majority of Cu2?
chitosan and the p-methoxybenzenediazonium ions, in this case, are chelated/adsorbed by such
tetrafluoroborate salt, Scheme 1. compounds.
For the subsequent studies, it was chosen the copper
Atomic absorption spectrometry (AAS) complexes with the highest Cu2? content, for both
copper sulfate and copper chloride salts, as determined
Table 1 shows the Cu2? content, from CuSO45H2O by AAS.
solution, adsorbed by chitosan and derivatives deter-
mined by AAS in different reaction times. The results
showed that the maximum chelating capacity of Scanning electron microscopy (SEM) and energy-
chitosan was when the reaction occurred during dispersive X-ray spectrometer (EDS) studies
24 h. For the derivatives, the higher concentration of
copper ions chelated was obtained with 36 h of The scanning electron micrograph of pure chitosan is
reaction time. The results also show that chitosan is shown in Fig. 1a. It exhibits smooth and compact
the most efficient chelating agent, adsorbing 120 mg morphology with some particle sizes. These surface
of Cu(II) ions by gram of polymer, which corresponds morphology changes for ChitosanCu complex,
to 75 % of adsorption. As the chemical modification Fig. 1b, due to the coordination of copper ion to the
occurs in the polymer chain the adsorption capacity active sites of the biopolymer. The Schiff base
decreases, probably due to the decrease of free amino presents irregular surface with agglomerates, as shown
groups, one of the chelating sites. Another reason in Fig. 1c. The SEM images of CTBCu, Fig. 1d,
might be the steric hindrance of the new groups revealed the presence of some crystals attributed to the
bonded to chitosan. Therefore, for chitosan deriva- presence of copper sulfate adsorbed on the polymer
tives, Cu2? ions might be coordinated by the amino surface, confirmed by EDS analyses (Fig. 2b). The
groups which are not chemically modified. The best sample presented in the image was washed with
results are shown in bold in Table 1. distilled water at 50 C three times in order to remove
The pH of Cu2? solutions before the complexation the copper sulfate adsorbed, however, the crystals
reaction was 6.5, as the optimum pH range reported by were still observed in the image. The SEM images of
Rhazi et al. (2002). After the reaction time, the pH was CTBCu sample after being washed is shown in the
measured and the obtained value was 5.2 for all inset of Fig. 1d. Figure 1e shows the surface mor-
compounds. The formation of the complexes may be phology of NBC. It can also be seen a heterogeneous
accompanied by the release of a proton for the surface with small particle sizes accompanied with
coordination, and consequently, the decrease of the pores. The SEM image of NBCCu (Fig. 1f) also
pH (Rhazi et al. 2002). presented copper sulfate adsorbed on the surface. The
The formation of chitosan and derivatives Cu(II) morphology of the azo compound, Fig. 1g, presents
complexes was also investigated using CuCl22H2O roughness and pores in the surface. In the SEM image
salt as the precursor of copper ions. The reactions of Azo-NBCCu, the crystals of copper sulfate were

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Scheme 1 Synthesis of chitosan compounds

Table 1 Concentration (mg/g) and percentage of Cu2? ions Table 2 Concentration (mg/g) and percentage of Cu2? ions
chelated by chitosan and derivatives at different reaction times, chelated by chitosan and derivatives at 25 C using CuCl2-
25 C, using CuSO45H2O salt (0.01 mol/L) 2H2O salt (0.01 mol/L)
Compound 30 min 24 h 36 h 48 h Compounds Reaction time (h) mg Cu2?/g % of Cu2?
mg/g % mg/g % mg/g % mg/g % Chitosan 24 96 60
Chitosan 73 45 120 75 120 75 120 75 CTB 36 43 27
CTB 24 15 73 45 86 54 86 54 NBC 36 14 9
NBC 8 5 23 14 52 32 40 25 Azo-NBC 36 18 12
Azo-NBC 5 3 15 10 25 16 25 16

not observed due to the fact that this compound has the The composition of Cu(II) complexes of chitosan
lowest capacity of chelating Cu(II), as displayed in and derivatives from CuSO45H2O salt was defined by
Fig. 1h and confirmed by AAS studies. energy-dispersive X-ray spectrometer (EDS) as shown

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 Cellulose (2015) 22:23912407 2397

b Fig. 1 SEM images of a Chitosan, b ChitosanCu, c CTB, in Fig. 2. The results confirmed the existence of
d CTBCu with inset of the sample after being washed with carbon, nitrogen and oxygen as well as copper and
distilled water, e NBC, f NBCCu, g Azo-NBC and h Azo-
NBCCu, from CuSO45H2O salt. The red arrow shows the sulfur elements in all complexes. The presence of S
copper sulfate crystals. (Color figure online) corroborates with the SEM images in which were
observed crystals on the surface of the complexes

Fig. 2 EDS spectra of

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corresponding to copper sulfate. The results also presence of crystals, which suggests that this salt was
indicated that Cu content is higher than S, which not adsorbed on the polymers surface. The agglomer-
suggests that apart from copper sulfate adsorbed in the ates observed on NBCCu complex, Fig. 3c, were
surface of the polymer, there are copper ions coordi- verified by EDS and the results indicated that they
nated with the reactive sites of chitosan and deriva- were not salt adsorbed by the compound, and were
tives. The EDS results for CTBCu after being washed formed after chemical modification of chitosan. The
with distilled water was the same for CTBCu before EDS studies indicated a content of chlorine atom
being washed, which indicates that this process did not present in the complexes, Fig. 4. It might be due to the
remove the copper salt adsorbed by the polymer. fact that this atom may take part in the coordination
Moreover, it can be seen that the weight percent (%) bond with the metal ion.
of C element increases with the chemical modification The reason why copper sulfate is adsorbed but not
of chitosan, as the weight percent (%) of N increases copper chloride can be associated to the stronger
for Azo-NBC when compared to chitosan, CTB and electric field in the divalent ion sulfate and the great
NBC, confirming the formation of the azo derivative. distance of the hydration water from the ion center
The SEM images of ChitosanCu, CTBCu, NBC which can lead to a greater release of water molecules,
Cu and Azo-NBCCu, from CuCl22H2O salt, are resulting in an endothermic contribution for the salt
displayed in Fig. 3ad. The samples did not show any adsorption on the polymer surface (Jiang et al. 2005).

Fig. 3 SEM images of a ChitosanCu, b CTBCu, c NBCCu and d Azo-NBCCu, from CuCl22H2O salt

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Fig. 4 EDS spectra of a ChitosanCu, b CTBCu, c NBCCu and d Azo-NBCCu, from CuCl22H2O salt

X-ray powder diffraction (XRD) Fig. 5a, showed the characteristic peaks at 2h = 9.5
and 2h = 20, which suggest the formation of inter-
Figure 5 shows the X-ray diffraction patterns of and intra-molecular hydrogen bonds in chitosan (Haas
chitosan, chitosan derivatives and the Cu2? complexes and Franz 2009). The XRD patterns of its copper
from CuSO45H2O salt. The pattern of chitosan, complex showed a decrease in the intensity of the

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Fig. 5 X-ray diffraction patterns of a Chitosan and ChitosanCu, b CTB and CTBCu, c NBC and NBCCu and d Azo-NBC and Azo-
NBCCu, from CuSO45H2O salt

peaks and they were more broadened than those is adsorbed on the polymer surface, as confirmed by
observed for pure chitosan, indicating a low crys- SEM images.
tallinity of the complex (Anan et al. 2011). X-ray diffraction patterns of Azo-NBCCu,
In Fig. 5b, the XRD patterns of the Schiff base CTB Fig. 5d, did not show any significant difference when
showed a peak at 2h = 6.7 and a weak reflection at compared to the X-ray patterns of Azo-NBC due to the
2h = 14 due to the introduction of new groups in the fact that this compound demonstrated lower capacity
side chain of chitosan (Li-xia et al. 2010). The peak at of metal chelation, as observed in AAS analysis.The
2h = 20 is still observed for this compound. The XRD crystallinity of the compounds was calculated using
pattern of Schiff base Cu(II) complex, Fig. 5b, was the following Eq. (1):
appreciably different from CTB. It showed a remarkable
Crystallinity index % I110  Iam = I110  100
decrease in crystallinity with a broad peak at 2h = 14
and 20, due to the coordination with copper ions. 1
The X-ray diffraction patterns of NBC copper where I110 is the maximum intensity at *20 and Iam
complex, Fig. 5c, showed a wider peak 2h = 20, is the intensity of amorphous diffraction at *10
evidencing a reduction in crystallinity as presented by (Focher et al. 1990).
the other complexes. Moreover, some smaller peaks Table 3 shows the calculated crystalline index
that did not appear in the X-ray patterns of NBC are values for all compounds. The crystallinity of chitosan
observed. It is characteristic of pure copper sulfate that derivatives decreased significantly, CTB 31 %, NBC

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Table 3 Crystalline index of chitosan and derivatives and illustrates the Raman spectra of Chitosan and Chi-
their respective Cu(II) complexes tosanCu. The characteristic peaks of chitosan are
Compound Crystallinity index (%) observed at 2936 and 2884 cm-1 attributed to the
stretching vibrations of CH groups, at 1661 cm-1
Chitosan 61 ascribed to amide I and at 1588 cm-1 due to the NH
ChitosanCu 28 deformation of amino groups. In addition, the peaks at
CTB 31 1456 and 1376 cm-1 are attributed to asymmetrical
CTBCu 14 CH bending of the CH2 group, and the peak at
NBC 13 1256 cm-1 is assigned to OH deformation vibration
NBCCu 27 of the CH2-OH group. The peaks at 1108 and
Azo-NBC 48 896 cm-1 are derived from the symmetric and asym-
Azo-NBCCu 52 metric stretching vibrations of glycosidic bonds,
respectively (Schrader 1995).
In the Raman spectrum of ChitosanCu, the signal
13 % and Azo-NBC 48 %, when compared to that of at 1661 shifts to 1650 cm-1 and at 1588 to 1598 cm-1.
pure chitosan, 61 %. The change of crystallinity is This result indicates the participation of these groups
mainly dependent on the presence of new groups in the coordination bond with the metal. A new peak at
bonded in the polymeric side chain and some other 973 cm-1 is observed in the spectrum and attributed to
factors, such as spacial hindrance and hydrophobic stretching vibrations of O=S=O groups (Pye and
force (Jeon and Holl 2003). The low crystallinity of Rudolph 2001; Rudolph et al. 2003; Hug 1997) due to
chitosan derivatives indicates that they are more the presence of CuSO45H2O salt adsorbed on the
amorphous than the polymer. After coordinating with polymer surface. Bands related to metal and ligand
copper ion, chitosan and CTB have shown higher vibrations, like CuO and CuN, could not be
reduction of crystallinity noted by the less intense and identified.
wider diffraction peaks, being 28 and 14 %, respec- In comparison with chitosan spectrum, CTB Raman
tively. This reduction was resulted from the deforma- spectrum, Fig. 7, shows additional peaks at 1641 cm-1
tion of strong hydrogen bonds (Yi et al. 2006) because due to C=N of imine group and at 3068, 1603 and
copper ions may be coordinated with the nitrogen 1000 cm-1 attributed to aromatic ring vibrations
atoms and hydroxyl groups of the polymers. However, (Socrates 2001) The peak at 1583 cm-1 indicates that
the crystallinity index values for NBCCu, 27 %, and free NH2 groups are still present in the compound and
Azo-NBCCu, 52 %, increased when compared to were not chemically modified. The Raman spectrum of
pure NBC and Azo-NBC. In the case of NBCCu, the CTBCu presents the peak at 970 cm-1 due to
increase in crystallinity is associated with the great stretching vibrations of O=S=O groups (Pye and
amount of copper sulfate adsorbed on the derivative Rudolph 2001; Rudolph et al. 2003; Hug 1997)
surface, as observed in the SEM image (Fig. 1f). This because of the salt adsorbed on the polymer surface.
is also in agreement with the appearance of some The Raman spectrum of NBC is presented in Fig. 8.
peaks in the X-ray diffraction patterns of NBCCu, The characteristic absorptions peaks are shown at
Fig. 5c. For Azo-NBCCu, the slight increase of 1585 cm-1 ascribed to the NH deformation and at
crystallinity index can be due to the different coordi- 3072, 1603 and 1000 cm-1 assigned to aromatic ring
nation bond established by the azo-compound and the vibrations (Socrates 2001). In addition, the peak of the
Cu2? ions with participation of N=N groups. In imine group at 1641 cm-1 disappears, which indicates
addition, the lower concentration of copper ions the successful reduction of these groups. The Raman
coordinated with both compounds also generates a spectrum of NBCCu also presents the peak at
lower system disorder. 977 cm-1 due to stretching vibrations of O=S=O
groups (Pye and Rudolph 2001; Rudolph et al. 2003;
Raman spectroscopy Hug 1997) because of the salt adsorbed on the
derivative surface.
The Cu2? complexes formed from CuSO45H2O salt For the N-Azobenzyl compound, Azo-NBC, the
was also studied by Raman spectroscopy. Figure 6 Raman spectrum, Fig. 9, shows the characteristic peak

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Fig. 6 Raman spectra of

Fig. 7 Raman spectra of

Fig. 8 Raman spectra of

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Cellulose (2015) 22:23912407 2403

Fig. 9 Raman spectra of

at 1503 cm-1 due to the N=N stretching vibration and those values are listed as g\ in Table 4. The g\
(Socrates 2001). In addition, the peaks at 3060, 1605 parameter for ChitosanCu was not calculated
and 1000 cm-1 are assigned to aromatic ring vibra- because the HFS was not resolved for the perpendic-
tions. In the Raman spectrum of Azo-NBCCu, the ular orientation (Molochnikov et al. 2008).
stretching vibrations of O=S=O groups appear with a One interesting feature observed in the spectra of
lower intensity at 974 cm-1 when compared to the CTBCu, NBCCu, and Azo-NBCCu is the com-
other complexes. It might be due to lower capacity that plete change in lineshape when compared to Chi-
this derivative presented to adsorb copper ions. tosanCu. All three spectra from the former
The presence of new peaks observed in the Raman complexes are very similar to each other and present
spectra suggests the successful formation of the only minor peaks with the parallel hyperfine structure
derivatives. Furthermore, the results of Cu2? com- visible. This suggests that the spectra seen in
plexes corroborate with the information obtained by Fig. 10bd contains two spectral components: one
the SEMEDS images which showed the presence of minor contribution, which would arise from copper
CuSO45H2O salt on the polymer surface. centers similar to those found in ChitosanCu, and a
major component coming from either dimeric or
Electron paramagnetic resonance (EPR) polymeric copper centers, where the copper center
coupling would vanish the hyperfine interaction. This
The EPR spectra of chitosan and derivatives Cu(II) result corroborates with the large number of copper
complexes, from copper sulfate salt, are represented in sulfate crystals observed in the SEMEDS images.
Fig. 10 and the values of A//, g// and g\ are listed in Figure 11 shows the EPR spectra of chitosan and
Table 4. The results indicated the presence of copper derivatives Cu(II) complexes, from copper chloride
ions in the polymers. The spectrum of ChitosanCu is salt. The first observation that can be made is the
typical of a monomeric copper center with resolved presence of the well-resolved hyperfine structure as
hyperfine structure in the so-called parallel direction, noticed above for ChitosanCu. This indicates that, for
whose peak position and separation lead to the values the complexes from copper chloride salt and unlike
of g// and A//, respectively. The resolved parallel those from copper sulfate, the great majority of copper
hyperfine structure vanishes upon complexation in centers are monomeric and probably incorporated in
CTBCu, NBCCu, and Azo-NBCCu, which hinders the polymer. This result is in agreement with the
the determination of the respective values for those images obtained by SEMEDS analysis, in which
complexes. In these latter cases, only an average great part of the copper retained from copper sulfate is
g-value can be determined directly from the spectra in the salt adsorbed on the polymers surface.

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Fig. 10 EPR spectra of a ChitosanCu, b CTBCu, c NBCCu and d Azo-NBCCu, from CuSO45H2O salt

Table 4 EPR parameters Compounds A|| (9104 cm-1) g|| g\ g||/A|| (cm)
of Cu2? complexes from
CuSO45H2O salt ChitosanCu 173.58 2.1947 126
CTBCu 2.06590
NBCCu 2.08643
Azo-NBCCu 2.08956

For the four complexes, g// [ g\ [ 2.05, which is tetrahedral distortion of a square-planar geometry is
suggestive of distorted tetragonal, square-pyramidal observed when A// reduces and g// increases. Thus,
or square-planar geometry (Valdes and Trivino 2009). from the results in Table 5, the four compounds have a
Sakaguchi and Addison (1979) reported that the g/// slightly tetrahedral distortion. The increase of g//
A// ratio can be used as a convenient empirical values and decrease of A// values also show that the
measure of tetrahedral distortion in Cu(II) complexes. ligand field strength decreases in these compounds in
This value ranges from 105 to 135 cm for square- the same order. Therefore, the complex ChitosanCu
planar structure, and this quotient increases on the has a stronger ligand field than CTBCu, NBCCu and
introduction of tetrahedral distortion. Furthermore, Azo-NBCCu.

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Cellulose (2015) 22:23912407 2405

Fig. 11 EPR spectra of a ChitosanCu, b CTBCu, c NBCCu and d Azo-NBCCu, from CuCl42H2O salt

Table 5 EPR parameters of Cu2? complexes from CuCl2- which indicates that part of the metal content deter-
2H2O salt mined by AAS is in the salt adsorbed and might
Compounds A|| (9104 cm-1) g|| g\ g||/A|| (cm)
influence in the use of the materials for further
application studies, for instance, it can promote
ChitosanCu 174.77 2.1637 124 variation on reproducibility of catalysis experiments
CTBCu 173.58 2.1808 126 or compromise their biological activity. The presence
NBCCu 172.39 2.1825 126 of the salt was confirmed by Raman spectroscopy.
Azo-NBCCu 169.99 2.1910 128 X-ray diffraction patterns showed that the chemical
modification of chitosan and formation of complexes
resulted in the decrease of crystallinity. This decrease
is mainly attributed to the spatial hindrance and
Conclusions hydrophobic forces in the aromatic substituent groups
and to the deformation of strong hydrogen bonds when
Chitosan compounds and their copper complexes have Cu2? is coordinated to the compounds. EPR spectra of
been synthesized and characterized by several tech- the complexes from copper chloride salt showed that
niques. The morphology studies by SEMEDS exhib- the great majority of copper centers are monomeric
ited copper sulfate adsorbed on the polymers surface, and probably incorporated in the polymer, unlike those

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2406 Cellulose (2015) 22:23912407

from copper sulfate. EPR parameters also suggested Dulcevscaia GM et al (2013) New copper(II) complexes with
that the Cu2? complexes present a square-planar isoconazole: synthesis, structures and biological proper-
ties. Polyhedron 52:106114
geometry. Facchin G, Kremer E, Barrio DA, Etcheverry SB, Costa-Filho
AJ, Torre MH (2009) Interaction of Cudipeptide com-
Acknowledgments The authors gratefully thank Fundacao de plexes with Calf Thymus DNA and antiproliferative
Apoio a Pesquisa do Estado de Sao Paulo (FAPESP), activity of [Cu(ala-phe)] in osteosarcoma-derived cells.
2012/13901-3, Programa de Pos-graduacao em Ciencia e Polyhedron 28:23292334
Tecnologia de Materiais (POSMAT) and Coordenacao de Focher B, Beltrame PL, Naggi A, Torri G (1990) Alkaline
Aperfeicoamento de Pessoal de Nvel Superior (CAPES) for N-deacetylation of chitin enhanced by flash treatments.
financial support and post-graduation fellowship. The authors Reaction kinetics and structure modification. Carbohydr
thank Leonardo Negri Furini (UNESPPresidente Prudente, Polym 12:405418
SP) and Dr. Jose Vicente Garca Ramos (CSICMadrid, Spain) Genin JM, Biles C, Keiser JB, Poppe SM, Swaney SM, Tarapley
for Raman Spectroscopy analyses. WG, Romero DL, Yagi Y (2000) Novel 1,5-
diphenylpyrazole nonnucleoside HIV-1 reverse transcrip-
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