International Journal of Biological Macromolecules 103 (2017) 515–524

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Studies of chitosan/pectin complexes exposed to UV radiation

Jolanta Kowalonek
Nicolaus Copernicus University in Toruń, Faculty of Chemistry, 7 Gagarin St., 87-100, Toruń, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Chitosan and pectin form complexes owing to electrostatic interactions between positively charged
Received 9 February 2017 amino groups in chitosan and negatively charged carboxylate groups in pectin, which was confirmed by
Received in revised form 14 April 2017 ATR-FTIR spectroscopy and contact angle measurements. Moreover, the formation of these complexes
Accepted 15 May 2017
might be associated with the loss of the biopolymers ordering, which resulted in higher surface roughness
Available online 17 May 2017
and lower thermal stability of the complexes in comparison to those of homopolymers.
UV rays, used as a sterilizing agent, caused a moderate increase in the surface polarity of the com-
Keywords:
plexes. Roughness parameters of these samples changed irregularly after irradiation, and their thermal
Chitosan
Pectin stability was slightly affected by UV rays. The results indicated that the complexes studied appeared to
Biopolymer complexes present resistance to UV action higher than homopolymers, which is a desirable property in medical or
UV radiation pharmaceutical applications.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction Both of these biopolymers were mixed to prepare hybrid

Chitosan is a linear polysaccharide composed of (1 → 4)-linked
2-amino-2-deoxy-␤-d-glucopyranose (d-glucosamine) and 2-
acetamido-2-deoxy-␤-d-glucopyranose (N-acetyl-d-glucosamine)
residues. It is a chitin derivative which, after cellulose, is the sec-
ond most common polysaccharide in nature. Chitin is commercially
produced from crustaceans shells that come from food waste [1–5].
Pectins are heteropolysaccharides built of mainly homogalac-
turonan, a linear chain of (1 → 4)-linked ␣-d-galacturonic acid.
Part of carboxyl groups in the chain can be esterified with methyl
alcohol. Apart from homogalacturonan chain, pectins also encom-
pass rhamnogalacturonan I (RG-I) and ramnogalacturonan II (RG-II)
chains. Other saccharides, for example d-galactose and l-arabinose,
constitute a part of the pectin structures. Thus, the pectin structure
is very complex. This polysaccharide is isolated from the cell wall of
plants usually citrus peels, apple pomace, or sugar beet pulp [4–8].
Chitosan and pectin are biomaterials which have found many
applications in medicine due to their biocompatibility and nontox-
icity. In a solution, these biopolymers are weak polyelectrolytes,
chitosan is a weak polybase, and pectin is a weak polyacid. So,
when they are mixed together, they react forming a polyelectrolyte
complex (PEC) owing to the existence of electrostatic attractions
between positively charged amino groups ( NH3 + ) in chitosan and
negatively charged carboxylate groups ( COO− ) in pectin.
E-mail address: jolak@umk.pl
pectin/chitosan nanoparticles by different techniques, i.e. coating
and blending [9], microparticles based on chitosan/pectin/alginate
[10], or beads of pectin coated with chitosan [11]. The aim of the
studies was to design such systems with an ability to deliver drugs,
e.g., proteins, to specific sites in organism. The chitosan/pectin
PEC with entrapped anthocyanin can be used as a pH indicator
in food packaging. [12]. Moreover, a ternary film made of chi-
tosan/poly(vinyl alcohol)/pectin revealed antimicrobial activity,
which is necessary for food packaging applications [13]. Pectin-
chitosan complex can also be applied in a colon-specific capsule
as a drug carrier [14–16]. Instead of using conventional pectin in
the colon drug carrier, amidated pectin might be applied. The mod-
ified pectin is more resistant to variations in pH and calcium level
than the conventional one [17]. Thermoreversible chitosan/pectin
hydrogel was synthesized for its potential usage as a wound heal-
ing bandage [18]. Another nano dressing for wound healing was
made of chitosan/pectin PEC in which titanium dioxide nanoparti-
cles were dispersed [19]. A porous scaffold made of pectin/chitosan
PEC for possible bone tissue engineering was prepared and charac-
terized [20]. Moreover, there is a number of other works dedicated
to studies of the physicochemical properties of chitosan/pectin
complexes [21–28].
Materials based on these biopolymers are intended for medi-
cal applications, so they must be free of contaminants and sterile.
Such requirements can be achieved by different chemical and/or
physical methods [29,30]. For instance, UV radiation is a simple,
inexpensive, and environmentally friendly method of microorgan-
isms killing; however, it may cause damage. Thus, it is important to

http://dx.doi.org/10.1016/j.ijbiomac.2017.05.081
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516 J. Kowalonek / International Journal of Biological Macromolecules 103 (2017) 515–524
check the effects caused by UV rays acting as a potential sterilizing
agent on such biomaterials. Possible changes in surface structure,
morphology, hydrophilicity, and thermal stability of the samples
induced by short-wave radiation can be monitored by infrared
spectroscopy, scanning microscopy, contact angle measurements,
and thermal analysis. A good thermal stability is important if a spe-
cific range of the complexes use and the processing possibility at
higher temperature are considered.
It is known that UV radiation causes oxidation, degradation,
and/or crosslinking. In the first stage, UV rays are absorbed by chro-
mophores present in the polymer backbone or existing as some
external impurities, which can result in free radicals creation. These
radicals react with oxygen molecules from atmosphere yielding
oxidized groups in polymeric chains. Oxidation is accompanied by
degradation reactions during which polymer chains are cut into
shorter fragments. The last stage of photooxidative degradation is
when radicals are joined yielding inactive products also in the form
of crosslinked structures. Thus, after exposition to UV radiation the
polymer structure is altered. New oxidized groups appear and the
molecular weight may either decrease as a result of bond cleav-
ages or increase as a result of crosslinking. Also, volatile products
are created [31]. All these changes influence physicochemical and
utility properties.
The aim of the work was to study the resistance of chi-
tosan/pectin complexes to UV rays emitted by a low pressure
mercury vapour lamp used as a germicidal lamp. Because bioma-
terials are subject to sterilization with UVC radiation, it is vital to
investigate the photochemical processes taking place on the sur-
faces of chitosan/pectin complexes as these processes may differ
from those typical of homopolymers. The influence of UV rays
on chitosan and chitosan-based blends was investigated in the
past [32–37]. Photodegradation of pectin blended with poly(vinyl
pyrrolidone), poly(ethylene oxide) and poly(vinyl alcohol) was also
studied [38–40]. However, the effect of UV radiation on the com-
plexes made of chitosan and pectin has not been tested before.
2. Materials and methods
2.1. Materials
Chitosan was purchased from Sigma-Aldrich. The molecular
weight and degree of deacetylation for chitosan were evalu-
ated in our laboratory. The molecular weight was determined
by viscometric measurements and deacetylation degree, DD, by
UV-spectroscopy. The procedure was described elsewhere [41].
The viscosity-average molecular weight of chitosan was equal to
411,000, and the deacetylation degree was 82% ± 1.7%.
Pectin was also purchased from Sigma-Aldrich. The material
was obtained from citrus fruit, containing 87.6% of polygalac-
turonic acid. The esterification degree, DE, was evaluated by a
spectroscopic method (transmission FTIR and ATR-FTIR) [42,43].
Calculations were conducted for integral intensities (areas) of
bands and absorbance values at band maxima according to the
formula [42,43]:
DE(%) = (A1735 /(A1735 + A1605 )) ∗ 100%
where: A1735 − integral intensity (or absorbance at maximum)
of the band assigned to methyl esterified carboxyl groups, A1605
− integral intensity (or absorbance at maximum) of the band
attributed to non-esterified carboxyl groups. DE calculated from
integral intensities was equal to 46% and from absorbance val-
ues − 56%, which indicates that about half of carboxyl groups was
esterified.
Molecular weight of pectin was determined on the basis of
viscometric measurements using Ubbelohde viscometer. The mea-
surements were performed in 0.1 M NaCl aqueous solution at pH
7, at 25 ◦ C. The viscosity-average molecular weight, Mv , calculated
a
from the Mark-Houwink-Sakurada equation, [␩] = KMv , was equal
to 68,900; where K = 4.36 × 10−2 cm3 /g and a = 0.78 [6,44].
2.2. Preparation of the chitosan/pectin complexes
From these polymers, 2% (m/v) basic solutions were prepared by
dissolving chitosan in 2% aqueous solution of acetic acid and pectin
in distilled water. Then, these solutions were mixed under magnetic
stirring to obtain polymeric complexes of different component
weight ratios. The precipitates (complexes) were filtered out and
rinsed with distilled water. Next, the precipitates were placed on
the leveled microscopic glasses in order to obtain thin polymeric
films which were first dried under ambient conditions and then
in vacuum at 50 ◦ C. The films of the complexes were opaque and
their thickness ranged between 30 ␮m and 70 ␮m. Homopolymers
alone formed transparent films, but the chitosan film was yellow-
ish. The film thickness was measured with a digital micrometer at
a resolution of 0.001 mm (MIB Messzeuge, Germany).
2.3. ATR-FTIR spectroscopy
ATR-FTIR spectra of chitosan/pectin films were recorded with a
FTIR Genesis II (Mattson, USA) spectrophotometer equipped with
ATR (Pike Technologies, Inc.) containing ZnSe crystal (angle of inci-
dence is 45◦ ). The resolution was 4 cm−1 and the number of scans
– 64.
2.4. Contact angle measurements
The contact angle measurements were conducted with a DSA
G10 goniometer (Krüss GmbH, Germany). A drop of glycerol or
diiodomethane was placed on a sample surface with a microsyringe
and the value of contact angle was calculated on the basis of the
obtained liquid drop image. About 8 drops of the liquid were placed
on each sample; the deviation from the average was within ± 2◦ .
The measurements were carried out at room temperature. On the
basis of these measurements, surface free energies as well as polar
and dispersion components were calculated using DSA software.
2.5. Atomic force microscopy (AFM)
AFM images were obtained with a MultiMode NanoScope IIIa
(Veeco Metrology Inc., USA), using a silicon tip (Veeco), operating
in a tapping mode in air and at room temperature. The most typ-
ical images were chosen for presentation; additionally, roughness
parameters, Ra − an arithmetic mean, and Rq − a root mean square,
were calculated for the scan area of 5 ␮m × 5 ␮m.
2.6. Thermogravimetric analysis
Thermogravimetric measurements were conducted on a Ther-
mal Analysis SDT 2960 Simultaneous DSC-TGA analyzer in nitrogen
atmosphere with the heating rate of 10 ◦ C/min to 650 ◦ C. From
thermogravimetric (TG) and derivative thermogravimetric (DTG)
curves characteristic parameters were determined: Tonset (◦ C) −
the temperature at which a process starts, it was determined as the
deviation of the TG curve from baseline, Tmax (◦ C) − the tempera-
ture at the maximum degradation rate (maximum on DTG curve),
m (%) − weight loss, also char residue (%) at 600 ◦ C.
 J. Kowalonek / International Journal of Biological Macromolecules 103 (2017) 515–524 517

0.35

0.3 Chitosan

0.25

0.2 Pectin
A
0.15

0.1

0.05

0
3600 3100 2600 2100 1600 1100 600
Wavenumbers, cm-1

Fig. 1. ATR-FTIR spectra of chitosan and pectin.

Table 1
Maxima of absorption bands in ATR-FTIR spectra and their assignment to the corresponding bond vibrations for chitosan and pectin [33,40,41,45–47].

Chitosan Pectin

Wavenumber (cm−1 ) Bond vibration Wavenumber (cm−1 ) Bond vibration

3350, 3280 OH, NH stretching 3398, 3334, 3266 OH stretching

2924, 2873 CH stretching 2935 CH stretching
1645 C O stretching (amide I) 1735 C O stretching in ester
1552 NH bending (amide II) 1605 COO− stretching
1408 CH2 scissoring 1436, 1409 CH2 scissoring
1380 CH3 bending in amide group 1363 CH wagging
1318 CH wagging 1326 CH wagging
1259 CH2 twisting 1260,1228 CH2 twisting
1150, 1061, 1024 C O C stretching 1142, 1092, 1070, 1010 C O C stretching
895 CH2 rocking 960, 911, 882 CH2 rocking

3. Results and discussion
3.1. ATR-FTIR spectroscopy results
Fig. 1 shows ATR-FTIR spectra of chitosan and pectin films.
In the region of 3000–3700 cm−1 , one can observe a broad band
of hydrogen-bonded OH groups; additionally, in the case of
chitosan, NH stretching vibrations overlap with OH vibrations.
This broad band overlaps the band of CH stretching vibra-
tions at 2800–3000 cm−1 . In the 1500–1800 cm−1 range, amide
I and amide II bands are detected (typical of chitosan) and
also carbonyl and carboxylate bands (typical of pectin). Bands
at 1200–1500 cm−1 correspond to CH deformation vibrations.
Absorption at 950–1200 cm−1 is associated with the stretching
vibration of C O bond. The detailed band assignment to the
bond vibrations of chitosan and pectin can be found in Table 1
[33,40,41,45–47].
Infrared spectroscopy is a useful tool for finding interactions
between components in the blends. It is known that band shifts and
broadenings are indicative of the existence of interactions between
the blend constituents [48,49]. Fig. 2 shows ATR-FTIR spectra of
homopolymers and the complexes in three extended ranges. Fig. 2a
shows the OH vibration region in which one can observe the
broadening and shift of the OH band towards lower wavenumbers
(red shift), which may indicate the formation of hydrogen bonds
between OH/NH groups in chitosan and OH groups in pectin. More-
over, Fig. 2b presents spectra in the range of 1200–1800 cm−1 in
which the main alterations take place. It is seen that the band at
1735 cm−1 , corresponding to the C O vibrations from the ester
groups in pectin, does not change its position, which means that
this group does not participate in interactions. However, the band
of the carboxylate vibrations at 1605 cm−1 from pectin moves to
1598 cm−1 for most of the blends and to 1591 cm−1 for the blend
with 80% of chitosan, which indicates the shift towards lower
wavenumbers. Moreover, this band overlaps amide I and II bands
of chitosan in the complexes. Thus, for most of the samples, one
can observe a broader band with the maximum at 1598 cm−1 and
the shoulder at 1532 cm−1 . The shoulder of the band appears at
lower wavenumbers when compared to that of chitosan for which
the maximum of amide II band is situated at 1552 cm−1 . Only the
spectrum of the complex containing 80% of chitosan is somewhat
different from the other specimens spectra, the maximum of the
carboxylate band is at 1591 cm−1 and the shoulder at 1645 cm−1 .
This shoulder corresponds to the carbonyl group vibrations of chi-
tosan and it is at the same position as for pure chitosan. All these
observations suggest the appearance of electrostatic interactions
between negatively charged carboxyl groups in pectin and posi-
tively charged amino groups in chitosan [9,12,14,20,23], which is
shown in Scheme 1.
No significant changes were observed in the ether range
(800–1200 cm−1 ). The spectra of the complexes with 40–80% of
pectin were very similar to that of pectin. The spectrum of the com-
plex with 80% of chitosan was similar to that of chitosan. This may
suggest that ether bonds do not participate in interactions between
biopolymers in the complexes.
The samples exposed to UV rays in the presence of oxy-
gen undergo photooxidation, the process during which oxygen
atoms are embedded in the polymeric chains. In this process, new
518 J. Kowalonek / International Journal of Biological Macromolecules 103 (2017) 515–524

0.6 OH
A Chit/pec 60/40
Chit/pec 20/80 NHCOCH3
0.5 O
Chit/pec 80/20 O HO O
Chit/pec 50/50 HO O chitosan
0.4 O
Chit NH3
A +
0.3 COO - OH
Chit/pec 40/60 O
O
0.2 Pec COOCH3
HO
O
0.1 HO O pectin
HO
0.0 OH OH O
3800 3600 3400 3200 3000 2800 2600 2400
Wavenumbers, cm-1 NHCOCH3
O O
Chit/pec 60/40 HO O
B Chit/pec 20/80 HO O chitosan
0.30 O
Chit/pec 80/20 NH3
0.25 +

0.20 Scheme 1. Interactions between chitosan and pectin.

A
0.15 increase in this band area was observed, suggesting the creation of
new products bearing hydroxyl groups due to oxidation.
0.10
The changes in the band position and width in infrared spec-
Chit
0.05
tra of the irradiated complexes were also observed in the range
Chit/pec 40/60
Pec of 1480–1690 cm−1 . The band in this region corresponds to the
0.00 vibrations of the carboxylate groups in pectin interacting with pro-
1800 1700 1600 1500 1400 1300 1200
tonated amine groups from chitosan. Under irradiation, this band
Wavenumbers, cm-1 becomes slightly narrower which may suggest the destruction of
0.6
ionic bonds. Moreover, many different competitive photochemi-
C Chit/pec 60/40
cal reactions take place in the samples; hydroxyl/hydroperoxide
0.5 Chit/pec 20/80 groups as well as carbonyl groups can form and decompose. Hence,
Chit/pec 80/20 infrared spectra provide information on average photooxidative
0.4 degradation processes.
Chit/pec 50/50 The appearance of photoproducts containing carbonyl groups
A
0.3
Chit/pec 40/60 results from the photooxidation of the samples; thus, the carbonyl
group vibration region was taken into account. As new carbonyl
0.2 group bands in infrared spectra did not emerge after irradiation,
the changes in the existing bands at 1735 cm−1 and 1605 cm−1 were
0.1 Pec Chit analyzed. To compare the changes in the studied samples quantita-
tively, the maxima of the absorption bands of carbonyl groups were
0.0 normalized in relation to the CH stretching vibrations at 2935 cm−1
1200 1150 1100 1050 1000 950 900 850 800
[32,39,51]. Such a procedure allowed us to compare the results
Wavenumbers, cm-1
obtained for all the samples under the same experimental condi-
Fig. 2. ATR-FTIR spectra of chitosan, pectin, and the complexes of different compo- tions. Although the bands are complex, the same procedure applied
nent ratio in the region of 2350–3850 cm−1 , 1150–1850 cm−1 , 800–1200 cm−1 . for all the studied samples provides a general overview of the mon-
itored process. Fig. 4a presents alterations in the absorbance of the
band at 1735 cm−1 corresponding to the >C O vibrations in the
ester group. Fig. 4b shows changes in absorbance at 1605 cm−1 ,
oxygen-containing groups such as hydroxyl/hydroperoxide, car- attributed to COO− vibrations in pectin or COO− bound ioni-
bonyl, carboxylic acid, aldehyde, ketone, ester are formed [31,50]. cally with NH3 + in chitosan in the complexes. It is noticeable that
Fig. 3 shows examples of infrared spectra of a chitosan/pectin for most samples the amount of carbonyl groups changes insignif-
(50/50) complex. No new absorption bands can be detected in icantly, which may suggest that the formation of carbonyl groups
the spectra; however, alterations within the existing ones can be under UV rays is not a predominant process. The chitosan/pectin
considered. Irregular changes in the range of OH vibrations may (80/20) sample appeared to be most susceptible to carbonyl groups
result from both, photooxidation and the release of water adsorbed formation. In this sample, there might be some excess of chitosan
in the biopolymers. So, in the case of chitosan/pectin complexes, not ionically bonded with pectin. If one takes into account the
the quantitative analysis is complicated in this range, and there- amount of groups capable of forming ionic bonds between chitosan
fore, it has been omitted. However, we can analyze the position and pectin, some excess of chitosan can remain at this composi-
and the width of the band. For pectin and the complexes, the tion ratio and interact with pectin or itself via hydrogen bonds.
OH stretching vibrations band slightly widens and the maximum Probably the higher chitosan content can be responsible for such
moves towards lower wavenumbers indicating the alterations in a behavior as pure chitosan revealed an increase in the absorp-
the hydroxyl groups surroundings and new polar groups formation tion of e.g. hydroxyl and amide bands after exposition to UV rays.
as a result of the polymer oxidation and degradation. For chitosan, The results for chitosan are not shown in Fig. 4 because it does
neither shifts nor broadening of this band was noticed and only an not absorb in the studied range. Anyway, most complexes proved
 J. Kowalonek / International Journal of Biological Macromolecules 103 (2017) 515–524 519

0.5

0h
0.4
6h

0.3
12h 9h
A
0.2

0.1

0
3600 3100 2600 2100 1600 1100 600
Wavenumbers, cm-1

Fig. 3. ATR-FTIR spectra of chitosan/pectin (50/50) complex before and after various irradiation time.

to be more resistant to oxidation than homopolymers, especially
chitosan.
3.2. Results of contact angle measurements
Surface polarity of the studied samples was monitored by
the contact angle measurements with two liquids, glycerol and
diiodomethane. These measurements were used for surface free
energy (␥s ) calculation that is the sum of polar (␥s p ) and disper-
sion (␥s d ) components according to the Owens-Wendt method
[52,53]. The results obtained are presented in Fig. 5 a–c. It is seen
that, before UV treatment, pectin obtains the highest ␥s value
(33.1 mJ/m2 ) (Fig. 5a), the remaining samples reach lower values,
but the differences among them are not large. For chitosan, the ␥s
value was equal to 28.4 mJ/m2 . Fig. 5b shows the dispersion com-
ponent values which are in the range of about 25–28 mJ/m2 for
all the unirradiated samples. These values are much higher than
polar component values, ␥s p , presented in Fig. 5c, which indicates a
rather hydrophobic nature of the surfaces. Fig. 5c shows that pectin
was characterized by the highest ␥s p value before irradiation, but
chitosan presented a lower ␥s p value. Although both homopoly-
mers have a large number of polar functional groups, their surfaces
showed predominantly hydrophobic character, which may result
from the creation of hydrogen bonds hidden beneath the surface.
The complexes, except for the one with 80% of pectin content, show
the lowest ␥s p values indicating that the surfaces in these systems
are most hydrophobic which suggests the existence of interactions
between the blend constituents in the complexes stronger than in
chitosan or pectin molecules separately. For instance, electrostatic
attractions can occur between positively ( NH3 + ) and negatively
( COO− ) charged groups, rendering the complex surfaces more
hydrophobic. The functional groups of the biopolymers, involved
in interactions, tend to be oriented towards the sample interior in
sample oxidation, the polar component was analyzed as the most
representative parameter that reflects polar interactions between
the surface and the testing liquid, i.e. glycerol. As can be seen,
that the most efficient oxidation process was observed for chi-
tosan, and then, for pectin. All the complexes were less sensitive
to polar groups formation than homopolymers, probably owing
to stronger interactions between the blend constituents, which
may indicate mutual stabilizing influence of the blend constituents
on each other. The presence of the interactions makes the struc-
ture stiffer, and the mobility of radicals formed under UV rays is
restricted, which may lead to lower efficiency of the polymer oxi-
dation in the complexes. Moreover, the increase in polar groups
content on the surfaces and hence in the polar component values
may also result from hydrogen bonds or electrostatic interactions
destruction caused by UV radiation, especially in the case of chi-
tosan and pectin for which the growth in the polar component
values was most considerable. The increase in the polar component
values was responsible for the increase in the surface free energy
values as the dispersive component values were altered to a lesser
extent.
3.3. AFM results
Fig. 6 and Table 2 show the results obtained from the atomic
force microscopy (AFM). The left column in Fig. 6 presents the
images of unirradiated sample surfaces, the right column − these
surfaces after 12 h of UV treatment. As can be seen, before irra-
diation the chitosan surface was flat which was reflected in very
Table 2
Roughness parameters (Ra , Rq ) before and after 12 h irradiation for chitosan, pectin
and the complexes for scan area 5 ␮m × 5 ␮m.
Sample Roughness parameters (nm)

order to reach low surface free energy because the surroundings are 0h 12 h
hydrophobic. This confirms the observations made with the use of
Ra Rq Ra Rq
the ATR-FTIR spectroscopy.
UV irradiation of the samples results in oxidation which chitosan 1.34 2.37 1.43 2.12
chitosan/pectin (80/20) 19.58 24.13 12.60 15.34
means that sample surfaces become enriched in oxygen con- chitosan/pectin (60/40) 24.38 35.34 28.17 33.19
taining groups. These different types of polar groups, such as chitosan/pectin (50/50) 12.11 15.95 12.29 16.95
hydroxyl/hydroperoxide, carbonyl groups, make the surfaces more chitosan/pectin (40/60) 12.93 19.30 20.17 24.79
hydrophilic. Consequently, surface free energy values and polar chitosan/pectin (20/80) 22.36 28.18 14.46 19.43
pectin 6.23 9.31 5.94 9.90
component values increase (Fig. 5). To compare the efficiency of the
520 J. Kowalonek / International Journal of Biological Macromolecules 103 (2017) 515–524

A)

pec chit/pec80/20 chit/pec60/40

0.5 chit/pec50/50 chit/pec40/60 chit/pec20/80

0.4

0.3
Δ A 1735

0.2

0.1

-0.1

-0.2
0 5 10 15 20
Irradiation time, h
B)
pec chit/pec80/20 chit/pec60/40
0.16 chit/pec50/50 chit/pec40/60 chit/pec20/80

0.12

0.08
Δ A1598

0.04

-0.04
0 5 10 15 20
Irradiation time, h
Fig. 4. Changes in absorbance A of the bands at: (A) 1735 cm−1 , (B) 1605 cm−1 (B) of pectin and the complexes versus irradiation time; A = At − A0 , At − band absorbance
after t time, A0 –band absorbance before irradiation. The bands were normalized to ACH2 at 2935 cm−1 .

low roughness parameters. But the pectin surface was more rugged
with occasionally occurring high hills, which was reflected in higher
Ra and Rq when compared to chitosan. Both homopolymers present
amorphous nature and the differences in morphology may result
from inter- or intrachain interactions in the polymer chains. As
chitosan revealed lower roughness parameter values and lower
surface polarity than those of pectin, it suggested a greater num-
ber of hydrogen bonds between chitosan molecules, which might
result in a flat surface of this polymer.
However, the chitosan/pectin complexes revealed more folded
surfaces with no significant differences among them (Fig. 6). Such
corrugated surfaces were characterized by higher Ra and Rq val-
ues when compared to the values observed for homopolymers
(Table 2). Both of these biopolymers are polysaccharides with a
huge amount of functional groups able to interact with each other,
which may lead to the changes in polarity and morphology of
the samples. Usually, a higher blends roughness is a result of the
components immiscibility, i.e. attractions between the components
are weak or missing. The presence of interactions between the
blend components, e.g. hydrogen bonds, can yield smooth surface
[39–41]. However, in the case of the complexes, interactions are rel-
atively strong because they are precipitated from a solution. Both
of these polymers are polysaccharides with a similarly stiff main
chain but with different substituents; thus, the mobility and mutual
adjustment of these polymers is limited due to their structures.
The attractions between some groups from polymer chains and
random chains movements associated with this phenomenon may
lead to the alterations in a sample organization causing a growth
in surface roughness. In this case, probably ionic interactions in the
chitosan/pectin complexes are responsible for the rise in the sur-
face roughness and the decrease in the surface polarity of these
samples.
 J. Kowalonek / International Journal of Biological Macromolecules 103 (2017) 515–524 521

50
Surface free energy (γs), mJ/m2 pec chit/pec 20/80 chit/pec 40/60
chit/pec 50/50 chit/pec 60/40 chit/pec 80/20
45 chit

40 A
35

30

25

20
0 2 4 6 8 10 12
Irradiation time, h

40
pec chit/pec 20/80 chit/pec 40/60
Dispersion component (γsd) of

chit/pec 50/50 chit/pec 60/40 chit/pec 80/20

surface free energy, mJ/m2

chit
35
B
30

25

20
0 2 4 6 8 10 12
Irradiation time, h

16
pec chit/pec 20/80 chit/pec 40/60
chit/pec 50/50 chit/pec 60/40 chit/pec 80/20
surface free energy, mJ/m2

14
Polar component (γsp) of

chit
12

10 C
8

0
0 2 4 6 8 10 12
Irradiation time, h

Fig. 5. (A) Surface free energy (SFE), (B) dispersion and (C) polar components of
SFE calculated for chitosan, pectin and chitosan/pectin complexes versus irradiation
time.

The AFM images of irradiated samples are presented in the right

column of Fig. 6. As can be seen, both homopolymers did not experi-
ence significant alterations in topography, which was also reflected
in Ra and Rq values. Irradiated chitosan/pectin complexes showed
a bit fibrous structure, long protruding bands developed on the
surfaces. The Ra and Rq values changed irregularly: for some sam-
ples these values increased but for the others − decreased. Such
behavior might be related to different photochemical processes
and water release. The samples undergo photooxidation which
is accompanied by photodegradation reactions, and consequently,
polymeric chains become oxidized and shorter. Changes in inter-
actions among polymeric macromolecules are also important and
may affect the surface morphology of the complexes. Moreover,
Fig. 6. AFM images of chitosan, pectin, and the complexes before and after 12 h
volatile photoproducts can cause holes in the surface, which is irradiation. Scan area: 5 ␮m × 5 ␮m, z-scale for homopolymers − 100 nm, for the
visible in Fig. 6 for the sample containing 80% of chitosan. This sam- complexes − 200 nm.
ple was most susceptible to oxidation, which was monitored with
infrared spectroscopy and contact angle measurements.
Surface roughness and polarity determine adhesion to other 3.4. Results of thermogravimetric analysis
substances, which can be important in medical applications. Higher
surface roughness and polarity facilitate drug deposition on the Thermal stabilities of the samples were monitored by the
surface or an enzyme immobilization to the surface. The chi- thermogravimetric analysis. Fig. 7a shows the thermogravimetric
tosan/pectin complexes seem suitable for such applications. curves of the studied specimens. It was found that chitosan decom-
522 J. Kowalonek / International Journal of Biological Macromolecules 103 (2017) 515–524

A) is associated with the sample decomposition and the elimination of

volatile products. It was found that chitosan decomposition starts
––––––– pectin with the rupture of glycosidic linkages leading to the formation of
100 –––– chitosan
––––– · chitosan/pectin 40/60 compounds of low molecular weight. Thus, acetic acid, butyric acid,
––– – – chitosan/pectin 50/50
––– ––– chitosan/pectin 60/40 lower fatty acids, acetic anhydride, or acetamide are the main chi-
tosan thermal degradation products [54–56]. The decomposition is
80 not complete since about 26% of the char residue can be found at
Weight (%)

600 ◦ C, which is the result of thermal crosslinking [33].

The thermal stability of pectin is lower if compared to chitosan,
its temperature onset and Tmax are equal to 197 ◦ C and 232 ◦ C,
60
respectively (Table 3). During the main stage of decomposition,
pectin loses about 50% of its weight, which may be related to
depolymerization, bond cleavages along the main chains, abstrac-
40 tions of side substituents and ring opening processes. Volatile
products such as CO2 , CO, CH4 , and organic compounds with
oxygen–containing groups were the main substances, apart from
20
water, detected during pyrolysis [58]. After decomposition, 27% of
0 100 200 300 400 500 600 char residue was found in pectin.
Temperature (°C) Universal V3.0G TA Instruments
The complexes appeared to be less stable than homopoly-
B) mers. Their decomposition process began at about 178–185 ◦ C and
120 1.0
the maximum speed of the process was at about 226 ◦ C (Tab 3).
Lower thermal stability of the complexes may be associated with
0.8 the disturbed organization of these specimens in comparison to
100
Deriv. Weight (%/°C)

homopolymers [12,58]. The weight loss during decomposition was

–––––––
––––
chitosan/pectin 40/60
chitosan/pectin 40/60-UV
in the range of 50–54% for all the complexes. The absence of a
0.6
clear peak from chitosan in thermogravimetric curves may indicate
Weight (%)

80
60
40
20
0 100 200 300 400 500
Temperature (°C) Universal V3.0G TA Instruments
Fig. 7. (A) Thermogravimetric curves of unirradiated chitosan, pectin and chi-
tosan/pectin complexes, (B) TG and DTG curves of chitosan/pectin (40/60) complex
before and after UV-treatment.
poses in three steps, pectin decomposes in two steps, whereas the
complexes undergo decomposition in two stages but in the second
stage a small shoulder can be distinguished (Fig. 7b). The first step
for all the samples is related to the removal of moisture [54–59],
which constitute about 12% of the content and this stage is omitted
in the considerations.
In the case of chitosan, the second step may be associated with
the release of acetic acid residue or water bound with chitosan
molecules more strongly. In this stage, chitosan loses about 10% of
its weight [54]. The third step, related to chitosan decomposition,
began at 230 ◦ C and the temperature at the maximum process rate
(Tmax ) was 290 ◦ C (Table 3). Weight loss during this step, about 42%,
the existence of the chitosan/pectin complexes owing to interac-
0.4 tions between the both components, which was also observed by
Ghaffari et al. [58] and Maciel et al. [12]. During thermal decompo-
sition, the weight loss in the complexes was similar suggesting that
0.2 the composition of these specimens was alike which may indicate
that chitosan and pectin form complexes stoichiometrically. The
formation of the stoichiometric complexes was observed by other
0.0
researchers [14,20,23].
UV rays caused only small changes in the thermal behavior of
600
-0.2
700
the studied samples (Fig. 7b, Table 3). Thus, in the first step, the
weight loss related to water evaporation was slightly smaller when
compared to the unexposed samples, which indicated that water
vaporized during prolonged irradiation. Moreover, the decomposi-
tion of the homopolymers starts at lower temperature. The most
distinct decrease in the decomposition onset temperature was
observed for chitosan. Due to the observation, it can be suggested
that the thermal stability was affected by the presence of oxidized
products. In the case of the complexes, the reduction in this tem-
perature was much smaller if compared to homopolymers, which
indicated that these specimens were characterized by higher resis-
tance to UV radiation. Only the chitosan/pectin 80/20 complex
showed slightly larger drop in the onset temperature. This sample
was more susceptible to oxidation under UV rays when compared
to the remaining specimens, so the higher content of the oxidized
groups which were photoproducts could influence thermal stability
of this sample. A similar situation was found for chitosan.

Table 3
Decomposition temperatures (Tdonset , Tdmax ), weight loss (m) and char residue for chitosan, pectin and the blends before and after 12 h irradiation.

0h 12 h
◦ ◦ ◦
Sample Tdonset ( C) Tdmax ( C) m (%) Residue (%) at 600 C Tdonset (◦ C) Tdmax (◦ C) m (%) Residue (%) at 600 ◦ C

chitosan 230 290 42.4 26.4 190 288 46.7 36.0

chit/pec 80/20 185 231 54.0 27.1 176 229 51.5 29.9
chit/pec 60/40 180 227 51.3 30.0 174 226 52.3 32.5
chit/pec 50/50 178 226 54.6 24.6 172 225 51.1 30.9
chit/pec 40/60 178 225 50.7 30.0 173 225 52.4 31.2
chit/pec 20/80 179 225 50.6 28.8 174 225 52.7 28.9
pectin 197 232 52.9 26.8 183 229 51.1 30.2
 J. Kowalonek / International Journal of Biological Macromolecules 103 (2017) 515–524 523

chitosan OH
NHCOCH3
O
O HO O
HO O
O
NH3
+ side groups abstractions
COO - OH
hν hydrogen abstractions macroradicals O2
O
O chain scission radicals
COOCH3 ring opening
HO
O
OH O
pectin
HO
OH O

peroxyradicals, oxyradicals, recombinations of radicals, inactive photoproducts

oxidation, degradation macroradicals, oxyradicals

Scheme 2. Main photoprocesses in chitosan/pectin complexes.

However, the temperature at the maximum rate of the process
altered insignificantly for all the samples compared to the unirra-
diated ones. Also, the weight loss changed slightly and irregularly.
The lack of profound changes in the thermal stability of the com-
plexes after irradiation confirms the mutual stabilizing effect of the
components in the complexes.
Furthermore, the amount of the residue at 600 ◦ C increased
slightly in the UV-irradiated samples as a result of photocrosslink-
ing reactions. Such a situation can be explained as follows: UV rays
induce the formation of radicals taking part in secondary reactions;
consequently, the exposed sample structure is altered. Also, some
radicals can be trapped in the polymer matrix, and then, they can
crosslink at higher temperature. All these processes can result in
the increased efficiency of thermal crosslinking, and consequently,
a somewhat higher value of char residue.
4. Discussion
UV radiation applied as a sterilizing agent is able to kill
microorganisms effectively but it can also cause damage in poly-
mers leading to changes in the structure and morphology of the
treated materials. The changes are the results of different photo-
chemical processes such as oxidation, degradation, crosslinking,
depolymerization, or etching. Thus, new oxidized groups, shorter
chains, branched, crosslinked structure, or volatile products can
be detected in UV-treated polymers. In the first stage of irradi-
ation, UV rays are absorbed by chromophoric groups present in
polymer chains, usually as structural defects, or by external impu-
rities, e.g. compounds added to polymers during preparation. These
excited individuals are the source of radicals which in the next
stage undergo different secondary reactions, including reactions
with oxygen molecules, which alters the structure of the treated
sample. All the radicals and macroradicals from the both polymers
can interact, which makes the photoprocesses in the mixtures more
complicated. In the last stage, free radicals react with one another
forming inactive photoproducts.
The oxidation process is limited to the surface where the oxygen
concentration is highest. During this process, the polymer radicals
react with oxygen molecules forming oxidized products and using
up the oxygen. The reaction progress into deeper material layers
depends on the rate of oxygen diffusion which is slower when
compared to the surface region. Since the majority of the func-
tional groups in the studied complexes, able to absorb UV radiation,
is bonded via hydrogen bonds and electrostatic interactions and
located beneath the surface, oxygen uptake is reduced and photo-
chemical processes occur to be less efficient in the chitosan/pectin
complexes than in homopolymers. Also changes in ordering the
complexes limits radical mobility and makes photooxidation less
efficient in comparison to that of homopolymers.
The reactions in chitosan/pectin complexes can be presented in
the Scheme 2:
5. Conclusions
Chitosan and pectin form polyelectrolyte complexes due to
the electrostatic attraction between negatively charged carboxyl
groups in pectin and positively charged amino groups in chitosan.
Also the formation of hydrogen bonds was observed. These find-
ings were confirmed by the ATR-FTIR spectroscopy (red shifts of
the absorption bands) and contact angle measurements (surface
hydrophobicity of the complexes higher than that of homopoly-
mers).
A more corrugated surface structure and slightly lower ther-
mal stability of the complexes if compared to homopolymers can
also indicate the occurrence of stronger interactions between blend
components combined with the loss of biopolymers organization
after mixing.
UV radiation induced the oxidative degradation of the samples
studied. This process was more efficient for homopolymers than
for the complexes, which was proved by infrared spectroscopy and
contact angle measurements. Moreover, UV rays did not cause sig-
nificant alterations in the surface morphology and thermal stability
of the complexes. Such a behavior of chitosan/pectin complexes
indicated a mutual stabilizing effect of the components, which may
result from the limited mobility of radicals and more difficult oxy-
gen access owing to the changed structure of the biopolymers in
the complexes.
The chitosan/pectin complexes were relatively resistant to UV
action, which suggests that these materials are suitable for medical
and pharmaceutical applications.
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