Efficient Adsorption and Catalytic Reduction of Phenol Red Dye by Glutaraldehyde Cross-Linked Chitosan and Its Ag-Loaded Catalysts: Materials Synthesis, Characterization and Application

Abstract

In this study, glutaraldehyde cross-linked chitosan support, as well as the catalysts obtained after loading Ag metal (Ag/Chitosan), were synthesised and applied for adsorption and reduction of phenol red dye in an aqueous solution. The Ag/chitosan catalysts were characterised by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis techniques. The catalytic reduction and adsorption performance of phenol red dye with Ag/chitosan and cross-linked chitosan, respectively, was performed at ambient reaction conditions. The reduction of dye was carried out using sodium borohydride (NaBH4) as the reducing agent, while the progress of adsorption and reduction study was monitored with ultraviolet-visible (UV-vis) spectrophotometry technique. The reduction of the phenol red dye varied with the amount of catalyst, the concentration of NaBH4, Ag metal loading, reaction temperature, phenol red dye concentration and initial pH of the dye solution. The dye solution with a nearly-neutral pH (6.4) allowed efficient adsorption of the dye, while acidic (pH = 4) and alkaline (pH = 8, 11, 13.8) solutions showed incomplete or no adsorption of dye. The reusability of the Ag/chitosan catalyst was applied for the complete reduction of the dye, where no significant loss of catalytic activity was observed. Hence, the applicability of cross-linked chitosan and Ag/catalyst was thus proven for both adsorption and reduction of phenol red dye in an aqueous solution and can be applied for industrial wastewater treatment.

Full text

Citation: Siciliano, C.C.; Dinh, V.M.;
Canu, P.; Mikkola, J.-P.; Khokarale,
S.G. Efficient Adsorption and
Catalytic Reduction of Phenol Red
Dye by Glutaraldehyde Cross-Linked
Chitosan and Its Ag-Loaded
Catalysts: Materials Synthesis,
Characterization and Application.
Clean Technol. 2023,5, 466–483.
https://doi.org/10.3390/
cleantechnol5020024
Academic Editor: Susana
Rodriguez-Couto
Received: 25 November 2022
Revised: 9 March 2023
Accepted: 27 March 2023
Published: 6 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
clean
technologies
Article
Efficient Adsorption and Catalytic Reduction of Phenol Red
Dye by Glutaraldehyde Cross-Linked Chitosan and Its
Ag-Loaded Catalysts: Materials Synthesis, Characterization
and Application
Chiara Concetta Siciliano 1, †, Van Minh Dinh 2,† , Paolo Canu 1, Jyri-Pekka Mikkola 2,3
and Santosh Govind Khokarale 2, *
1Department of Industrial Engineering, University of Padova, Via F. Marzolo, 9, 35131 Padova, Italy
2Technical Chemistry, Department of Chemistry, Chemical-Biological Centre, Umeå University,
S-90187 Umeå, Sweden
3Industrial Chemistry & Reaction Engineering, Department of Chemical Engineering, Johan Gadolin Process
Chemistry Centre, Åbo Akademi University, FI-20500, Turku, Finland
*Correspondence: santosh.khokarale@umu.se; Tel.: +46-721262291
† These authors contributed equally to this work.
Abstract:
In this study, glutaraldehyde cross-linked chitosan support, as well as the catalysts obtained
after loading Ag metal (Ag/Chitosan), were synthesised and applied for adsorption and reduction
of phenol red dye in an aqueous solution. The Ag/chitosan catalysts were characterised by X-ray
diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy
(FT-IR) and inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis techniques.
The catalytic reduction and adsorption performance of phenol red dye with Ag/chitosan and cross-
linked chitosan, respectively, was performed at ambient reaction conditions. The reduction of dye
was carried out using sodium borohydride (NaBH
4
) as the reducing agent, while the progress of
adsorption and reduction study was monitored with ultraviolet-visible (UV-vis) spectrophotometry
technique. The reduction of the phenol red dye varied with the amount of catalyst, the concentration
of NaBH
4
, Ag metal loading, reaction temperature, phenol red dye concentration and initial pH of the
dye solution. The dye solution with a nearly-neutral pH (6.4) allowed efficient adsorption of the dye,
while acidic (pH = 4) and alkaline (pH = 8, 11, 13.8) solutions showed incomplete or no adsorption of
dye. The reusability of the Ag/chitosan catalyst was applied for the complete reduction of the dye,
where no significant loss of catalytic activity was observed. Hence, the applicability of cross-linked
chitosan and Ag/catalyst was thus proven for both adsorption and reduction of phenol red dye in an
aqueous solution and can be applied for industrial wastewater treatment.
Keywords:
cross-linkedchitosan; Ag/chitosan catalyst; phenol red dye; adsorption; catalytic reduction;
catalyst reusability
1. Introduction
Organic dyes such as azo dyes are commercially important organic moieties which
are extensively applied in paints, textile, printing, pharmaceutical industry, dyeing, paper
and pulp industries [
1
]. Besides that, these organic dyes are also used as a pH-sensitive
indicator in classical to advanced analytical chemistry applications for qualitative as well
as quantitative measurements [
2
]. The consumption of azo dyes has increased tremen-
dously, especially in textile industries, since global demand for clothes and garments has
surged exponentially in recent decades. The huge amount of these organic dyes and their
derivatives are disposed of in water reservoirs as well as in soil and provide a serious
threat to the aquatic environment and can be responsible for irreversible and unwanted
Clean Technol. 2023,5, 466–483. https://doi.org/10.3390/cleantechnol5020024 https://www.mdpi.com/journal/cleantechnol

Clean Technol. 2023,5467
ecological changes [
3
,
4
]. These dyes are also highly hazardous to human health and ter-
restrial animals, being non-biodegradable as well as carcinogenic and mutagenic [
4
]. To
circumvent this problem, researchers worldwide have focused on finding out effective and
efficient methods to purify the dyes containing industrial effluents either through chemical
as well as physical techniques or combinations thereof. In this regard, physical adsorption,
membrane-based filtration, ion exchange and catalyst-based, as well as microbial degrada-
tion methods, have been fruitfully applied [
4
–
9
]. Adsorption and/or catalytic processing
based on photocatalysis, reduction, degradation and oxidation are more efficient, selective
and economically viable routes for the purification of water contaminated with various
organic dyes since these processes can be carried out under mild reaction conditions as
well as using easily available materials [8,10–12].
Supported nanometal catalysts comprising of metals such as silver, palladium, gold
and platinum stabilised on various heterogeneous supports such as carbonaceous materials,
metal oxides or polymeric supports are widely used for the processing of organic dyes
in aqueous mediums [
13
–
16
]. The supported nanometal catalysts based on silver metal
are considered the preliminary choice of researchers for the processing of organic dyes in
water since the precursors required for catalyst synthesis are relatively cheap and widely
available compared to other precious metals. Besides that, supported silver metal catalysts
still maintain high selectivity and reactivity during the catalytic process in line with other
precious-metal-based catalysts. Silver metal supported on various heterogeneous supports
such as mesoporous carbon, graphene oxide, mesoporous silica, cotton fabric and metal
oxides, etc., is used for the catalytic degradation of organic dyes such as methylene orange,
methylene red, phenol red, crystal violet, Congo red, Chicago Sky Blue and malachite green
dye etc. [
10
–
16
]. Adsorption of the organic dyes from their aqueous solutions over the
active surface of the catalyst is a critical step where adsorption followed by reduction or
degradation of the dye usually takes place during the catalytic process. Like reduction
or degradation approaches, the adsorption of the dyes over the reactive surfaces has also
been well-studied for the purification of a water-containing dye embedded in it. It also
can be considered a cost-effective technique since metal precursors are not required in the
materials synthesis. Besides that, the degradation products which used to remain in the
aqueous medium after catalytic degradation or reduction can be avoided. In this regard,
besides their valuable contribution to the reduction and degradation process, various types
of solid materials, including inorganic oxides, biopolymers or bio-based materials, activated
carbon etc., have also been applied as adsorbent materials for the adsorption of organic
dyes from their aqueous solutions [4,17–19].
Chitosan is a linear and semi-crystalline polysaccharide consisting of repetitive units of
N-acetyl D-glucosamine and D-glucosamine in the polymeric chains. It is a biocompatible
and biodegradable polymer obtained from partial deacetylation of the natural biopolymer
chitin and has emerged in numerous applications as well as the best replacement for fossil-
based synthetic polymers [
20
,
21
]. Besides being a bio-renewable polymer, chitosan has the
ability to perform as a substrate for precious as well as non-precious metal-loaded heteroge-
neous catalysts, which were found highly robust and versatile for various synthetic organic
transformations [
22
–
24
]. Silver metal-loaded chitosan (Ag/chitosan) is also used in various
biomedical applications, such as wound dressing, considering its intrinsic antibacterial
and antimicrobial characteristics in the resultant material [
25
–
27
]. Additionally, chitosan
and Ag/chitosan have also been effectively explored in the processing of impure water
containing organic dyes and other aromatics through adsorption and catalytic reduction
techniques, respectively [28–33].
In this report, glutaraldehyde cross-linked chitosan and its Ag metal loaded analogue,
i.e., Ag/chitosan, was studied for both adsorption as well as reduction of phenol red dye
in its aqueous solution. For the reduction process of dye, NaBH
4
was used as a hydrogen
source. Phenol red is a water-soluble organic dye and used as pH indicator in various
processes such as the development of cell cultures, in-home swimming pool tests, as a
diagnostic aid for the determination of renal function and estrogenic properties, etc. It has

Clean Technol. 2023,5468
been shown that phenol red has a mutagenic effect and causes serious eye damage as well
as skin and respiratory irritation. To the best of our knowledge, a detailed study regarding
the reduction and adsorption of phenol red dye with chitosan as well as Ag/chitosan
catalysts has not been carried out. In this study, initially, the glutaraldehyde cross-linked
chitosan was prepared and used as a substrate for the synthesis of Ag/chitosan catalyst,
and the obtained materials were characterised by various spectroscopic techniques such
as XRD, FTIR and SEM analyses. Further, these chitosan-based materials were used for
the reduction and adsorption of phenol red dye in an aqueous solution, whereupon the
influence of different reaction parameters on the reaction progress was confirmed. The
change in the amount of dye as the reaction proceeded was monitored by a UV-visible
spectrophotometer. Finally, the recyclability of the Ag/chitosan catalyst in the reduction of
the dye was also described.
2. Materials and Methods
2.1. Materials
Chitosan with medium molecular weight and 75–85% degree of deacetylation, phenol
red (powder, ACS reagent) and glutaraldehyde in aqueous solution (50 wt%) were pur-
chased from Sigma Aldrich (Saint Louis, MO, USA). NaBH
4
(powder, 98%) was purchased
from Fisher Scientific; silver nitrate, AgNO
3
(99%, crystal), methanol (100.0%), ethanol
absolute (99.95%), acetone (100.0%), sodium hydroxide and NaOH (98.8%, pellets) were
obtained from VWR chemicals. Acetic acid (glacial, 100%) was purchased from Merck. All
chemicals were used without further purification.
2.2. Instrumentation and Characterisation
The X-ray diffraction (XRD) analysis was obtained on PANalytical X’Pert3 Powder
Diffractometer (Malvern Panalytical, Malvern, Worcs, UK) in the 20 angle range of 10–70
◦
with a scan rate of 1
◦
/min using Cu K
α
radiation. Fourier transform infrared spectroscopy
(FT-IR) analysis of the neat chitosan, cross-linked chitosan with and without adsorbed dye
and Ag metal loaded catalyst were carried out with a Bruker Vertex 80 FTIR spectrometerin
the range between 500 and 4000 cm
−1
. The morphologies of the Ag metal-loaded catalysts
were analysed with scanning electron microscopy (SEM) and energy dispersive X-ray
spectroscopy (EDX) technique using a Zeiss Merlin FEG-SEM instrument (Oberkochen,
Germany) equipped with an in-lens secondary electron detector. The amount of silver metal
on the catalyst was calculated through Agilent 5800 inductively coupled plasma-optical
emission spectrometry (ICP-OES, Agilent Scientific Instruments, Headquarters, Santa Clara,
CA, USA). The UV spectra to evaluate the progress of reactions were obtained using a UV-
3100PC spectrophotometer (VMR International BV, Geldenaaksebaan, Leuven, Belgium).
2.3. Preparation of Cross-Linked Chitosan and Ag/Chitosan Catalysts
2.3.1. Cross-Linked Chitosan Support Preparation
The catalyst was prepared following a previously reported method [
28
]. 0.25 g of chi-
tosan was mixed gently in 12.5 mL of an aqueous solution of acetic acid (2 wt.% CH
3
COOH),
and the mixture was held under stirring for 24 h at room temperature. The amount of
added chitosan was dissolved completely in the acidic solution, and a homogenous, trans-
parent and viscous solution was obtained. The chitosan solution was slowly added to
the previously prepared alkaline water and methanol solution (distilled water, 17 mL;
methanol, 25 mL and NaOH, 5 g) and beads consisting of neutralised chitosan formed
and settled at the bottom of the flask. The alkaline solution with beads was kept at room
temperature for 24 h. The chitosan beads were separated from the alkaline solution by
vacuum filtration and washed with distilled water until neutral pH was reached. The
beads were further exposed for the cross-linking process, where the beads were added
to an alcoholic solution of glutaraldehyde (0.1 mL of 25% of glutaraldehyde solution in
water and 6 mL of methanol), and the reaction mixture was refluxed for 6 h. The beads
were separated from the alcoholic solution by vacuum filtration and washed with 50 mL of

Clean Technol. 2023,5469
ethanol and water mixture (50:50 vol.%). The beads were freeze-dried (Scanvac CoolSafe
Freeze Dryer) overnight after mixing with 10 mL distilled water, and the obtained material
was kept in a desiccator prior to the synthesis of Ag/chitosan catalysts.
2.3.2. Preparation of Ag/Chitosan Catalysts
The Ag/catalyst was prepared from AgNO
3
as a precursor and previously prepared
glutaraldehyde cross-linked chitosan, where the amount of AgNO
3
was varied during
the synthesis. The 0.2 g of freeze-dried chitosan beads were mixed with 10 mL aqueous
solution of AgNO
3
containing either 0.04, 0.02 or 0.005 g of Ag precursor, and the mixture
was heated at 70
◦
C overnight with a reflux condenser. The catalyst beads were filtered
by vacuum filtration and washed with distilled water (3
×
20 mL), and the recovered
solid was further freeze-dried and stored in a desiccator before its characterisation and
catalytic applications. The ICP-OES analysis showed that the catalysts were obtained with
1.4, 4.4 and 6.7 wt.% of Ag metal in their composition after the use of 0.005, 0.02 or 0.04 g of
AgNO
3
, respectively, during the synthesis. The catalysts will be here onwards designated
as 1.4 wt.%Ag/chitosan, 4.4 wt.%Ag/chitosan and 6.7 wt.%Ag/chitosan in upcoming
descriptions regarding their characterisation and application in catalytic measurements.
2.4. Catalytic Phenol Red Dye Reduction
The catalytic reduction of phenol red dye in its aqueous solution was carried out
with Ag/chitosan catalyst in a 5 mL glass vial under various reaction parameters. For
the different concentrations of dye, 6 mg of 6.7 wt.%Ag/chitosan catalyst and 5 mg of
NaBH
4
were mixed with 2 mL of 0.001, 0.003 or 0.005 M solution of phenol red dye and the
reaction mixture was stirred at room temperature (20
◦
C) for the desired reaction time. After
the addition of the catalyst and NaBH
4
, the required 0.1 mL of the reaction mixture was
periodically transferred to a quartz cuvette and diluted with 1 mL of distilled water, where
the absorbance of the resulting solution was measured on the UV-vis spectrophotometer.
The volume of the samples taken from the reaction mixtures varied with the concentration
of phenol red dye solution to maintain the absorbance value near unity. The conversion of
phenol red dye was calculated by the absorbance value obtained with decreasing peak at
571 nm (λmax) and Equation (1).
% Phenol red dye conversion =
A0−At
A0
×100 (1)
where A
0
and A
t
are the absorbance values at time zero and time t, respectively. The A
0
value of the absorbance was measured with the alkaline solution of phenol red dye. In this
case, the solution of dye with various concentrations, such as 0.001, 0.003 or 0.005 M, was
prepared with 0.1 N NaOH solution. In the case of the different amounts of the catalyst, 1,
2, 4 or 6 mg of 6.7 wt.%Ag/chitosan catalyst with 5 mg of NaBH
4
each were exposed to
0.001 M dye solution. For the influence of different amounts of NaBH
4
, 1, 2.5 or 5 mg and
6 mg
of 6.7 wt.%Ag/chitosan catalyst were used with 0.001 M phenol red dye solution. The
influence of the reaction temperature on the reduction of phenol red dye was also examined,
ranging from room temperature (20
◦
C) to 30 and 40
◦
C whereupon a dye solution with
0.001 M concentration, 6 mg Ag/chitosan and 5 mg NaBH4 was applied in the process. For
the influence of the initial pH of the dye solution, the 6 mg 6.7 wt.%Ag/chitosan catalyst
and 5 mg of NaBH
4
were added to solutions at initial pHs of 4–11. Initially, an aqueous
solution with different pHs, such as 4, 8 and 11, was prepared by mixing 0.1 N HCl and
0.1 N NaOH appropriately and thus, obtained solutions were used to prepare 0.001 M
phenol red dye solution. The pH levels of the 0.001 M solution of phenol red dye obtained
with deionised water and 0.1 N NaOH were observed as 6.4 and 13.8, respectively.
2.5. Phenol Red Dye Adsorption
The adsorption of phenol red dye from its aqueous solution with different initial pH
was carried out with cross-linked chitosan support where 6 mg of the support material was

Clean Technol. 2023,5470
mixed with 2 mL of 0.001 M solution of dye. In a separate study, after adsorption of the
dye from its solution with nearly neutral pH, i.e., 6.4, 5 mg of NaBH
4
was added to the
reaction mixture for further reduction of the adsorbed dye. The progress of the adsorption
and reduction of phenol red dye with support material was confirmed by the previously
described method and UV-vis spectrophotometry technique.
2.6. Recyclability of the Ag/Chitosan Catalyst
The 6.7 wt.%Ag/chitosan catalyst (6 mg) was used to reveal the recyclability of the
catalyst for the reduction of phenol red dye. After the complete reduction of the dye,
the catalyst was separated from the reaction mixture by vacuum filtration using a nylon
filter paper. The catalyst was washed several times (10 mL
×
3) with distilled water,
and a moist catalyst, along with a new batch of 5 mg of NaBH
4,
was further mixed with
the 2 mL fresh aqueous solution of phenol red dye (0.001 M). The reaction mixture was
then stirred for
18 min,
and the progress of the reaction and conversion of the dye was
carried out according to the methods described in the previous sections. The catalyst was
recycled additionally four times more for the phenol red dye reduction following a similar
procedure. After the fifth recycle, the recovered catalyst was freeze-dried and the obtained
solid was further analysed by ICP-OES analysis to confirm the Ag metal leaching during
the recyclability study.
3. Results
3.1. Characterisation of Cross-Linked Chitosan and Ag/Chitosan Catalysts
The structural properties of the cross-linked chitosan and Ag/chitosan catalysts were
analysed by powder XRD analysis and the obtained XRD patterns are shown in Figure 1A.
Clean Technol. 2023, 5, FOR PEER REVIEW 5
0.001 M phenol red dye solution. The pH levels of the 0.001 M solution of phenol red dye
obtained with deionised water and 0.1 N NaOH were observed as 6.4 and 13.8, respec-
tively.
2.5. Phenol Red Dye Adsorption
The adsorption of phenol red dye from its aqueous solution with different initial pH
was carried out with cross-linked chitosan support where 6 mg of the support material
was mixed with 2 mL of 0.001 M solution of dye. In a separate study, after adsorption of
the dye from its solution with nearly neutral pH, i.e., 6.4, 5 mg of NaBH
4
was added to the
reaction mixture for further reduction of the adsorbed dye. The progress of the adsorption
and reduction of phenol red dye with support material was confirmed by the previously
described method and UV-vis spectrophotometry technique.
2.6. Recyclability of the Ag/Chitosan Catalyst
The 6.7 wt.%Ag/chitosan catalyst (6 mg) was used to reveal the recyclability of the
catalyst for the reduction of phenol red dye. After the complete reduction of the dye, the
catalyst was separated from the reaction mixture by vacuum filtration using a nylon filter
paper. The catalyst was washed several times (10 mL × 3) with distilled water, and a moist
catalyst, along with a new batch of 5 mg of NaBH
4,
was further mixed with the 2 mL fresh
aqueous solution of phenol red dye (0.001 M). The reaction mixture was then stirred for
18 min, and the progress of the reaction and conversion of the dye was carried out accord-
ing to the methods described in the previous sections. The catalyst was recycled addition-
ally four times more for the phenol red dye reduction following a similar procedure. After
the fifth recycle, the recovered catalyst was freeze-dried and the obtained solid was fur-
ther analysed by ICP-OES analysis to confirm the Ag metal leaching during the recycla-
bility study.
3. Results
3.1. Characterisation of Cross-Linked Chitosan and Ag/Chitosan Catalysts
The structural properties of the cross-linked chitosan and Ag/chitosan catalysts were
analysed by powder XRD analysis and the obtained XRD patterns are shown in Figure
1A.
Figure 1. (A) XRD pattern of cross-linked chitosan (a) cross-linked chitosan, Ag/chitosan catalysts
with (b) 1.4, (c) 4.4 and (d) 6.7 wt.% of Ag. (B) FT-IR spectrum of (a) neat chitosan, (b) cross-linked
chitosan and (c) 6.7 wt.%Ag/chitosan catalyst.
A single characteristic peak assigned to the crystal plane (110) was observed at 2Ɵ
value of 21.8° in cross-linked chitosan, whereas the peak at 11.7° was not observed. The
reason might be the formation of amorphous solids during the acid-base treatment and
Figure 1.
(
A
) XRD pattern of cross-linked chitosan (a) cross-linked chitosan, Ag/chitosan catalysts
with (b) 1.4, (c) 4.4 and (d) 6.7 wt.% of Ag. (
B
) FT-IR spectrum of (a) neat chitosan, (b) cross-linked
chitosan and (c) 6.7 wt.%Ag/chitosan catalyst.
A single characteristic peak assigned to the crystal plane (110) was observed at 2
T
value
of 21.8
◦
in cross-linked chitosan, whereas the peak at 11.7
◦
was not observed. The rea-
son might be the formation of amorphous solids during the acid-base treatment and
glutaraldehyde-induced cross-linking process [
27
]. For Ag/chitosan catalysts, as the
amount of Ag metal increased, the intensity of the peak for the crystalline plane (110)
steadily decreased, perhaps due to the covering by metal particles. For the catalyst with
6.7 wt.%Ag metal loading, a broad and low-intensity peak at 20 value of 21.8
◦
for (110)
plane was obtained while a new characteristic peak corresponding to the crystal plane (111)
for metallic Ag was observed at 37.9
◦
[
27
]. However, the peak for the crystalline phase
assigned to metallic Ag was not observed in the case of catalysts with metal loadings 1.4
and 4.4 wt.%. Considering the probable homogeneous distribution of metal particles over
the support material, the amount of Ag metal may be below the detection limit.

Clean Technol. 2023,5471
The neat and cross-linked chitosan, as well as Ag/chitosan catalyst with 6.4 wt.%
of Ag loading, were also characterised with FT-IR analysis to understand the structural
changes in chitosan after cross-linking and Ag metal loading. As shown in Figure 1B, the
neat and partially deacetylated chitosan displays the characteristic band at 1585 cm
−1
for
N–H stretching vibrations in the amine group as well as bands at 1655 and 1564 cm
−1
belonging to C=O (amide I) and N–H (amide II) bending vibration in the amide group,
respectively [
34
]. The bands for the stretching vibration of the C–N bond were observed
between 1376 and 1255 cm
−1
while the band belonging to 1, 4-glycosidic bond (C–O–C) as
well as C–O bond in secondary (C
3
) and primary (C
6
) –OH groups of chitosan appeared at
1150, 1060 and 1025 cm
−1
, respectively. After cross-linking with glutaraldehyde, the band
attributed to N–H bending vibrations in amine disappeared, and the band belonging to
stretching vibrations in the C=N bond, which formed between the amine group of chitosan
and carbonyl group of glutaraldehyde appeared at 1650 cm
−1
. This newly formed band
overlaps with the band of the amide I functional group (C=O in the amide group); hence
the overall intensity of the band increased [
35
]. After Ag metal loading, significant changes
in the functional groups were not observed except for the increase in the intensity of the
band attributed to C-N stretching vibrations.
The SEM images and SEM-EDX spectrum of the cross-linked chitosan and the Ag/chitosan
catalysts with different Ag metal loadings are shown in Figure 2. The SEM images show that
material with a rough surface was obtained after the cross-linking of the chitosan with glu-
taraldehyde, while materials with identical morphology were obtained after various amounts
of silver loading (Figure 2A). However, the SEM-EDX analysis confirms the deposition of the
Ag metal over the chitosan support, and the intensity of the peak of metallic Ag increased with
increasing metal loading (Figure 2B).
The XRD and SEM-EDX analysis confirmed the formation of Ag nanoparticles on
the cross-linked chitosan support without the use of any additional reducing agents. It
was previously reported that chitosan is considered a non-toxic, mild reducing as well
as stabilising agent in Ag metal nanoparticle synthesis [
36
]. In addition, chitosan also
performs as a capping agent to control the growth of nanoparticles and helps to avoid their
agglomeration. After mixing an aqueous solution of Ag
+
ion salt with chitosan, the oxygen
and/or nitrogen atom of hydroxyl and amine groups in the chitosan chains, respectively,
serve as a ligand and coordinate with the metal ions. Under thermal treatment, hydroxyl
and amine (or amide groups in cross-linked chitosan) groups in chitosan further reduce the
Ag
+
ions and stabilise the resultant Ag metal nanoparticles (Figure 3) [
37
]. In the actual
reduction approach, the oxygen or nitrogen atom may lose their electrons and transform to
their oxidised form upon reduction of the Ag+ions to Ag nanoparticles.
Clean Technol. 2023, 5, FOR PEER REVIEW 6
glutaraldehyde-induced cross-linking process [27]. For Ag/chitosan catalysts, as the
amount of Ag metal increased, the intensity of the peak for the crystalline plane (110)
steadily decreased, perhaps due to the covering by metal particles. For the catalyst with
6.7 wt.%Ag metal loading, a broad and low-intensity peak at 20 value of 21.8° for (110)
plane was obtained while a new characteristic peak corresponding to the crystal plane
(111) for metallic Ag was observed at 37.9° [27]. However, the peak for the crystalline
phase assigned to metallic Ag was not observed in the case of catalysts with metal load-
ings 1.4 and 4.4 wt.%. Considering the probable homogeneous distribution of metal par-
ticles over the support material, the amount of Ag metal may be below the detection limit.
The neat and cross-linked chitosan, as well as Ag/chitosan catalyst with 6.4 wt.% of
Ag loading, were also characterised with FT-IR analysis to understand the structural
changes in chitosan after cross-linking and Ag metal loading. As shown in Figure 1B, the
neat and partially deacetylated chitosan displays the characteristic band at 1585 cm
−1
for
N–H stretching vibrations in the amine group as well as bands at 1655 and 1564 cm
−1
be-
longing to C=O (amide I) and N–H (amide II) bending vibration in the amide group, re-
spectively [34]. The bands for the stretching vibration of the C–N bond were observed
between 1376 and 1255 cm
−1
while the band belonging to 1, 4-glycosidic bond (C–O–C) as
well as C–O bond in secondary (C
3
) and primary (C
6
) –OH groups of chitosan appeared
at 1150, 1060 and 1025 cm
−1
, respectively. After cross-linking with glutaraldehyde, the
band attributed to N–H bending vibrations in amine disappeared, and the band belonging
to stretching vibrations in the C=N bond, which formed between the amine group of chi-
tosan and carbonyl group of glutaraldehyde appeared at 1650 cm
−1
. This newly formed
band overlaps with the band of the amide I functional group (C=O in the amide group);
hence the overall intensity of the band increased [35]. After Ag metal loading, significant
changes in the functional groups were not observed except for the increase in the intensity
of the band attributed to C-N stretching vibrations.
The SEM images and SEM-EDX spectrum of the cross-linked chitosan and the
Ag/chitosan catalysts with different Ag metal loadings are shown in Figure 2. The SEM
images show that material with a rough surface was obtained after the cross-linking of the
chitosan with glutaraldehyde, while materials with identical morphology were obtained
after various amounts of silver loading (Figure 2A). However, the SEM-EDX analysis con-
firms the deposition of the Ag metal over the chitosan support, and the intensity of the
peak of metallic Ag increased with increasing metal loading (Figure 2B).
Figure 2. Cont.

Clean Technol. 2023,5472
Clean Technol. 2023, 5, FOR PEER REVIEW 7
Figure 2. (A) SEM images, and (B) SEM-EDX spectrum of (a) cross-linked chitosan, Ag/chitosan
catalysts with (b) 1.4, (c) 4.4 and (d) 6.7 wt.% of Ag.
The XRD and SEM-EDX analysis confirmed the formation of Ag nanoparticles on the
cross-linked chitosan support without the use of any additional reducing agents. It was
previously reported that chitosan is considered a non-toxic, mild reducing as well as sta-
bilising agent in Ag metal nanoparticle synthesis [36]. In addition, chitosan also performs
as a capping agent to control the growth of nanoparticles and helps to avoid their agglom-
eration. After mixing an aqueous solution of Ag
+
ion salt with chitosan, the oxygen and/or
nitrogen atom of hydroxyl and amine groups in the chitosan chains, respectively, serve as
a ligand and coordinate with the metal ions. Under thermal treatment, hydroxyl and
amine (or amide groups in cross-linked chitosan) groups in chitosan further reduce the
Ag
+
ions and stabilise the resultant Ag metal nanoparticles (Figure 3) [37]. In the actual
reduction approach, the oxygen or nitrogen atom may lose their electrons and transform
to their oxidised form upon reduction of the Ag
+
ions to Ag nanoparticles.
Figure 3. Synthesis of Ag/chitosan catalyst from Ag salt and cross-linked chitosan.
3.2. Reduction of Phenol Red Dye
The catalytic reduction of phenol red dye was carried out with Ag/chitosan catalysts
and NaBH
4
as a hydrogen source. In this case, the catalytic reduction of the phenol red
dye was monitored with a UV-visible spectrophotometer, where the influence of various
reaction parameters on the activity of the catalyst was studied.
3.2.1. Influence of Amount of Catalyst
Initially, the influence of the amount of Ag/chitosan catalyst on the reduction ability
of the catalyst was studied where prior to the experiment, 6 or 2 or 1 mg of 6.7 wt.%Ag/chi-
tosan catalyst and 5 mg of NaBH
4
were added in 2 mL of 0.001 M aqueous solution of
phenol red dye. It was observed that after the addition of Ag/chitosan catalyst and NaBH
4
in the red-orange coloured phenol red dye solution, the colour of the reaction mixture
turned to fuchsia (colour in between pink and violet) (Figure 4A,B). As the reaction
Figure 2.
(
A
) SEM images, and (
B
) SEM-EDX spectrum of (a) cross-linked chitosan, Ag/chitosan
catalysts with (b) 1.4, (c) 4.4 and (d) 6.7 wt.% of Ag.
Clean Technol. 2023, 5, FOR PEER REVIEW 7
Figure 2. (A) SEM images, and (B) SEM-EDX spectrum of (a) cross-linked chitosan, Ag/chitosan
catalysts with (b) 1.4, (c) 4.4 and (d) 6.7 wt.% of Ag.
The XRD and SEM-EDX analysis confirmed the formation of Ag nanoparticles on the
cross-linked chitosan support without the use of any additional reducing agents. It was
previously reported that chitosan is considered a non-toxic, mild reducing as well as sta-
bilising agent in Ag metal nanoparticle synthesis [36]. In addition, chitosan also performs
as a capping agent to control the growth of nanoparticles and helps to avoid their agglom-
eration. After mixing an aqueous solution of Ag
+
ion salt with chitosan, the oxygen and/or
nitrogen atom of hydroxyl and amine groups in the chitosan chains, respectively, serve as
a ligand and coordinate with the metal ions. Under thermal treatment, hydroxyl and
amine (or amide groups in cross-linked chitosan) groups in chitosan further reduce the
Ag
+
ions and stabilise the resultant Ag metal nanoparticles (Figure 3) [37]. In the actual
reduction approach, the oxygen or nitrogen atom may lose their electrons and transform
to their oxidised form upon reduction of the Ag
+
ions to Ag nanoparticles.
Figure 3. Synthesis of Ag/chitosan catalyst from Ag salt and cross-linked chitosan.
3.2. Reduction of Phenol Red Dye
The catalytic reduction of phenol red dye was carried out with Ag/chitosan catalysts
and NaBH
4
as a hydrogen source. In this case, the catalytic reduction of the phenol red
dye was monitored with a UV-visible spectrophotometer, where the influence of various
reaction parameters on the activity of the catalyst was studied.
3.2.1. Influence of Amount of Catalyst
Initially, the influence of the amount of Ag/chitosan catalyst on the reduction ability
of the catalyst was studied where prior to the experiment, 6 or 2 or 1 mg of 6.7 wt.%Ag/chi-
tosan catalyst and 5 mg of NaBH
4
were added in 2 mL of 0.001 M aqueous solution of
phenol red dye. It was observed that after the addition of Ag/chitosan catalyst and NaBH
4
in the red-orange coloured phenol red dye solution, the colour of the reaction mixture
turned to fuchsia (colour in between pink and violet) (Figure 4A,B). As the reaction
Figure 3. Synthesis of Ag/chitosan catalyst from Ag salt and cross-linked chitosan.
3.2. Reduction of Phenol Red Dye
The catalytic reduction of phenol red dye was carried out with Ag/chitosan catalysts
and NaBH
4
as a hydrogen source. In this case, the catalytic reduction of the phenol red
dye was monitored with a UV-visible spectrophotometer, where the influence of various
reaction parameters on the activity of the catalyst was studied.
3.2.1. Influence of Amount of Catalyst
Initially, the influence of the amount of Ag/chitosan catalyst on the reduction ability of
the catalyst was studied where prior to the experiment, 6 or 2 or 1 mg of 6.7 wt.%Ag/chitosan
catalyst and 5 mg of NaBH
4
were added in 2 mL of 0.001 M aqueous solution of phenol
red dye. It was observed that after the addition of Ag/chitosan catalyst and NaBH
4
in the
red-orange coloured phenol red dye solution, the colour of the reaction mixture turned
to fuchsia (colour in between pink and violet) (Figure 4A,B). As the reaction progressed,
the fuchsia colour steadily disappeared with time, and a colourless solution was obtained
(Figure 4C).
Clean Technol. 2023, 5, FOR PEER REVIEW 8
progressed, the fuchsia colour steadily disappeared with time, and a colourless solution
was obtained (Figure 4C).
Figure 4. (A) phenol red dye aqueous solution (0.001 M), the reaction mixture (B) after the addition
of 6 mg of 6.7 wt.%Ag/chitosan catalyst and 5 mg of NaBH
4
and (C) after complete reduction of
phenol red dye.
As shown in Figure 5A, the 0.001 M red-coloured aqueous solution of phenol red dye
gives rise to a peak with a λ
max
value of 436 nm. However, after the addition of the catalyst
and NaBH
4
and followed by a reaction time of 3 min, the peak at 436 nm disappeared,
whereas a peak with a comparatively lower intensity appeared at λ
max
value of 571 nm.
The aqueous solution of phenol red dye is usually pH sensitive in terms of colour and is
frequently used as an indicator in commercial cell culture media. The aqueous solution
phenol red dye shows yellow colour in the acidic pH range, i.e., below pH 6.5, and red-
orange colour in between pH 6.5 and 7.5, while it turns to fuchsia in alkaline pH, i.e., above
pH 7.5 [38]. Figure 6 shows the chemical structures of the phenol red dye in its aqueous
solution with different pH scales, where the dye shows tautomeric and zwitterion form in
the high and moderately acidic pH ranges of the solution, respectively. At a neutral pH,
the phenol red dye transforms to its phenol form, whereas at high pH, the phenate form
of the dye remains dominant [38]. In other words, the phenol red dye forms variable mo-
lecular structures with different charge distributions depending on the pH of the solution
where the –C=C– and –C=O chromophores in these structures impart different colours to
the solution through an extended conjugation. In the case of a catalytic process, since the
colour of the reaction mixture changed to pale fuchsia after the addition of catalysts and
NaBH
4
, the pH of the reaction mixture was over 7.5. The decomposition of NaBH
4
in water
usually increases the pH of the solution, accompanied by the release of H
2
gas (reaction
Equations (2) and (3)).
H
2
O H
+
+ OH
−
(2)
BH
4−
+ H
+
+ 3H
2
O H
3
BO
3
+ 3H
2
(3)
To confirm this, i.e., a change in colour of the reaction mixture with pH, a phenol red
dye solution in 0.1 N NaOH solution was prepared, and it was observed that the resulting
solution became intense fuchsia in colour and a peak with λ
max
value 571 nm was obtained
in UV-vis spectroscopic measurements (Figure 5B). Hence, it was shown that after the
addition of catalysts and NaBH
4
, the tautomeric and zwitterion forms of the phenol red
dye were converted to their phenate analogue in an alkaline medium. As the reaction pro-
gressed, after the addition of the catalyst and NaBH
4
, steady decolouration of the reaction
mixture occurred due to the reduction of the dye with the Ag/chitosan catalyst and re-
leased H
2
and, simultaneously, the intensity of the peak at 571 nm also decreased.
Figure 4.
(
A
) phenol red dye aqueous solution (0.001 M), the reaction mixture (
B
) after the addition
of 6 mg of 6.7 wt.%Ag/chitosan catalyst and 5 mg of NaBH
4
and (
C
) after complete reduction of
phenol red dye.

Clean Technol. 2023,5473
As shown in Figure 5A, the 0.001 M red-coloured aqueous solution of phenol red dye
gives rise to a peak with a
λmax
value of 436 nm. However, after the addition of the catalyst
and NaBH
4
and followed by a reaction time of 3 min, the peak at 436 nm disappeared,
whereas a peak with a comparatively lower intensity appeared at
λmax
value of 571 nm.
The aqueous solution of phenol red dye is usually pH sensitive in terms of colour and is
frequently used as an indicator in commercial cell culture media. The aqueous solution
phenol red dye shows yellow colour in the acidic pH range, i.e., below pH 6.5, and red-
orange colour in between pH 6.5 and 7.5, while it turns to fuchsia in alkaline pH, i.e., above
pH 7.5 [
38
]. Figure 6shows the chemical structures of the phenol red dye in its aqueous
solution with different pH scales, where the dye shows tautomeric and zwitterion form in
the high and moderately acidic pH ranges of the solution, respectively. At a neutral pH, the
phenol red dye transforms to its phenol form, whereas at high pH, the phenate form of the
dye remains dominant [
38
]. In other words, the phenol red dye forms variable molecular
structures with different charge distributions depending on the pH of the solution where
the –C=C– and –C=O chromophores in these structures impart different colours to the
solution through an extended conjugation. In the case of a catalytic process, since the
colour of the reaction mixture changed to pale fuchsia after the addition of catalysts and
NaBH
4
, the pH of the reaction mixture was over 7.5. The decomposition of NaBH
4
in water
usually increases the pH of the solution, accompanied by the release of H
2
gas (reaction
Equations (2) and (3)).
H2OH++ OH−(2)
BH4−+ H++ 3H2OH3BO3+ 3H2(3)
Clean Technol. 2023, 5, FOR PEER REVIEW 9
Figure 5. (A) UV-vis spectra for the reduction of phenol red dye by 6.7 wt.%Ag/chitosan catalyst
and NaBH
4
(B) UV-visible spectra of 0.001M solution of phenol red dye in distilled water and 0.1 N
NaOH solution.
Figure 6. Chemical structures of phenol dye in its aqueous solution with different pH conditions.
Figure 7A shows that the reduction ability of the catalyst steadily decreased with a
decrease in its amount. Further, it was observed that NaBH
4
is also able to reduce the phe-
nol red dye in the absence of a catalyst, but the rate of the reaction was comparatively low
compared to catalyst enhanced process. Since the lower amount of catalyst, i.e., 1 mg, also
gave rise to the complete reduction of the dye; the prepared Ag/chitosan catalysts are
highly efficient in the reduction of phenol red dye.
Figure 7. Reduction of phenol red dye with 6.7 wt.%Ag/chitosan catalyst and NaBH
4
(A) different
amounts of catalyst and 5 mg of NaBH
4
and (B) different amounts of NaBH
4
and 6 mg of 6.7
wt.%Ag/chitosan catalyst.
3.2.2. Influence of Amount of Reducing Agent, NaBH
4
Since NaBH
4
was used as the reducing agent in the reduction process of phenol red
dye, its amount could influence the progress of the reaction in terms of the amount of H
2
released. As shown in Figure 7B, the rate of the reduction of dye was significantly en-
hanced with an increase in the amount of NaBH
4
from 1 mg to 2.5 and 5 mg in the reaction
mixture. With 1 mg of the reducing agent, no complete reduction of dye was observed,
whereupon merely 60% of the phenol red dye was reduced even though the reaction
Figure 5.
(
A
) UV-vis spectra for the reduction of phenol red dye by 6.7 wt.%Ag/chitosan catalyst
and NaBH
4
(
B
) UV-visible spectra of 0.001M solution of phenol red dye in distilled water and 0.1 N
NaOH solution.
Clean Technol. 2023, 5, FOR PEER REVIEW 9
Figure 5. (A) UV-vis spectra for the reduction of phenol red dye by 6.7 wt.%Ag/chitosan catalyst
and NaBH
4
(B) UV-visible spectra of 0.001M solution of phenol red dye in distilled water and 0.1 N
NaOH solution.
Figure 6. Chemical structures of phenol dye in its aqueous solution with different pH conditions.
Figure 7A shows that the reduction ability of the catalyst steadily decreased with a
decrease in its amount. Further, it was observed that NaBH
4
is also able to reduce the phe-
nol red dye in the absence of a catalyst, but the rate of the reaction was comparatively low
compared to catalyst enhanced process. Since the lower amount of catalyst, i.e., 1 mg, also
gave rise to the complete reduction of the dye; the prepared Ag/chitosan catalysts are
highly efficient in the reduction of phenol red dye.
Figure 7. Reduction of phenol red dye with 6.7 wt.%Ag/chitosan catalyst and NaBH
4
(A) different
amounts of catalyst and 5 mg of NaBH
4
and (B) different amounts of NaBH
4
and 6 mg of 6.7
wt.%Ag/chitosan catalyst.
3.2.2. Influence of Amount of Reducing Agent, NaBH
4
Since NaBH
4
was used as the reducing agent in the reduction process of phenol red
dye, its amount could influence the progress of the reaction in terms of the amount of H
2
released. As shown in Figure 7B, the rate of the reduction of dye was significantly en-
hanced with an increase in the amount of NaBH
4
from 1 mg to 2.5 and 5 mg in the reaction
mixture. With 1 mg of the reducing agent, no complete reduction of dye was observed,
whereupon merely 60% of the phenol red dye was reduced even though the reaction
Figure 6. Chemical structures of phenol dye in its aqueous solution with different pH conditions.
To confirm this, i.e., a change in colour of the reaction mixture with pH, a phenol
red dye solution in 0.1 N NaOH solution was prepared, and it was observed that the
resulting solution became intense fuchsia in colour and a peak with
λmax
value 571 nm
was obtained in UV-vis spectroscopic measurements (Figure 5B). Hence, it was shown
that after the addition of catalysts and NaBH
4
, the tautomeric and zwitterion forms of the
phenol red dye were converted to their phenate analogue in an alkaline medium. As the

Clean Technol. 2023,5474
reaction progressed, after the addition of the catalyst and NaBH
4
, steady decolouration of
the reaction mixture occurred due to the reduction of the dye with the Ag/chitosan catalyst
and released H2and, simultaneously, the intensity of the peak at 571 nm also decreased.
Figure 7A shows that the reduction ability of the catalyst steadily decreased with a
decrease in its amount. Further, it was observed that NaBH
4
is also able to reduce the
phenol red dye in the absence of a catalyst, but the rate of the reaction was comparatively
low compared to catalyst enhanced process. Since the lower amount of catalyst, i.e., 1 mg,
also gave rise to the complete reduction of the dye; the prepared Ag/chitosan catalysts are
highly efficient in the reduction of phenol red dye.
Clean Technol. 2023, 5, FOR PEER REVIEW 9
Figure 5. (A) UV-vis spectra for the reduction of phenol red dye by 6.7 wt.%Ag/chitosan catalyst
and NaBH
4
(B) UV-visible spectra of 0.001M solution of phenol red dye in distilled water and 0.1 N
NaOH solution.
Figure 6. Chemical structures of phenol dye in its aqueous solution with different pH conditions.
Figure 7A shows that the reduction ability of the catalyst steadily decreased with a
decrease in its amount. Further, it was observed that NaBH
4
is also able to reduce the phe-
nol red dye in the absence of a catalyst, but the rate of the reaction was comparatively low
compared to catalyst enhanced process. Since the lower amount of catalyst, i.e., 1 mg, also
gave rise to the complete reduction of the dye; the prepared Ag/chitosan catalysts are
highly efficient in the reduction of phenol red dye.
Figure 7. Reduction of phenol red dye with 6.7 wt.%Ag/chitosan catalyst and NaBH
4
(A) different
amounts of catalyst and 5 mg of NaBH
4
and (B) different amounts of NaBH
4
and 6 mg of 6.7
wt.%Ag/chitosan catalyst.
3.2.2. Influence of Amount of Reducing Agent, NaBH
4
Since NaBH
4
was used as the reducing agent in the reduction process of phenol red
dye, its amount could influence the progress of the reaction in terms of the amount of H
2
released. As shown in Figure 7B, the rate of the reduction of dye was significantly en-
hanced with an increase in the amount of NaBH
4
from 1 mg to 2.5 and 5 mg in the reaction
mixture. With 1 mg of the reducing agent, no complete reduction of dye was observed,
whereupon merely 60% of the phenol red dye was reduced even though the reaction
Figure 7.
Reduction of phenol red dye with 6.7 wt.%Ag/chitosan catalyst and NaBH
4
(
A
) differ-
ent amounts of catalyst and 5 mg of NaBH
4
and (
B
) different amounts of NaBH
4
and 6 mg of
6.7 wt.%Ag/chitosan catalyst.
3.2.2. Influence of Amount of Reducing Agent, NaBH4
Since NaBH
4
was used as the reducing agent in the reduction process of phenol red
dye, its amount could influence the progress of the reaction in terms of the amount of H
2
released. As shown in Figure 7B, the rate of the reduction of dye was significantly enhanced
with an increase in the amount of NaBH
4
from 1 mg to 2.5 and 5 mg in the reaction mixture.
With 1 mg of the reducing agent, no complete reduction of dye was observed, whereupon
merely 60% of the phenol red dye was reduced even though the reaction proceeded for
30 min. However, a nearly complete reduction of dye was observed after the use of 2.5
and 5 mg NaBH
4
, within 18 and 9 min, respectively. Surprisingly, without the use of
NaBH
4
, the rate of discolouration of phenol red dye was found to be high compared to
reaction mixtures where a reducing agent was applied, and a colourless reaction mixture
was obtained within 6 min (Figure 8A). As depicted in Figure 8B, a catalyst with violet
colour was obtained when it was separated from the reaction mixture by filtration. After
the addition of NaBH
4
, the dye desorbed from the catalyst, the supernatant solution in the
reaction mixture became pale fuchsia, and it transformed into a colourless liquid in 9 min
of reaction time. A more detailed description of the adsorption of the phenol red dye is
mentioned in the upcoming sections.
Clean Technol. 2023, 5, FOR PEER REVIEW 10
reaction proceeded for 30 min. However, a nearly complete reduction of dye was observed
after the use of 2.5 and 5 mg NaBH
4
, within 18 and 9 min, respectively. Surprisingly,
without the use of NaBH
4
, the rate of discolouration of phenol red dye was found to be
high compared to reaction mixtures where a reducing agent was applied, and a colourless
reaction mixture was
obtained within 6 min (Figure 8A). As depicted in Figure 8B, a
catalyst with violet colour was obtained when it was separated from the reaction mixture
by filtration. After the addition of NaBH
4
, the dye desorbed from the catalyst, the
supernatant solution in the reaction mixture became pale fuchsia, and it transformed into
a colourless liquid in 9 min of reaction time. A more detailed description of the adsorption
of the phenol red dye is mentioned in the upcoming sections.
Figure 8. (A) Addition of 6.7 wt.%Ag/chitosan catalyst in phenol red dye solution, and (B) (a) fresh
Ag/catalyst and (b) catalyst with adsorbed phenol red dye.
3.2.3. Influence of Concentration of Phenol Red Dye Solution
The catalytic activity was further examined upon the reduction of the phenol red dye,
whereupon dye solutions with different concentrations, such as 0.001, 0.003 and 0.005 M,
were used. The dye reduction ability of the catalyst was not considerably varying with
different concentrations containing dye solutions when 5 mg of NaBH
4
was applied
(Figure 9A). However, after reducing the amount of NaBH
4
to 2.5 mg, the rate of the
reduction varied with different concentrations of the phenol red dye solution. As shown
in Figure 9B, the dye conversion rate steadily decreased as its initial concentration in the
aqueous solution increased from 0.001 M to 0.005 M. It indicates that the amount of
evolved H
2
was not sufficient for the reduction of the increased concentration of dye—if
2.5 mg of reducing agent was used.
Figure 9. Reduction of the phenol red dye at different concentrations in aqueous solutions by 6 mg
of 6.7 wt.%Ag/chitosan catalyst and (A) 5 mg or (B) 2.5 mg of NaBH
4.
Figure 8.
(
A
) Addition of 6.7 wt.%Ag/chitosan catalyst in phenol red dye solution, and (
B
) (a) fresh
Ag/catalyst and (b) catalyst with adsorbed phenol red dye.

Clean Technol. 2023,5475
3.2.3. Influence of Concentration of Phenol Red Dye Solution
The catalytic activity was further examined upon the reduction of the phenol red
dye, whereupon dye solutions with different concentrations, such as 0.001, 0.003 and
0.005 M
, were used. The dye reduction ability of the catalyst was not considerably varying
with different concentrations containing dye solutions when 5 mg of NaBH
4
was applied
(Figure 9A). However, after reducing the amount of NaBH
4
to 2.5 mg, the rate of the
reduction varied with different concentrations of the phenol red dye solution. As shown
in Figure 9B, the dye conversion rate steadily decreased as its initial concentration in the
aqueous solution increased from 0.001 M to 0.005 M. It indicates that the amount of evolved
H2was not sufficient for the reduction of the increased concentration of dye—if 2.5 mg of
reducing agent was used.
Clean Technol. 2023, 5, FOR PEER REVIEW 10
proceeded for 30 min. However, a nearly complete reduction of dye was observed after
the use of 2.5 and 5 mg NaBH
4
, within 18 and 9 min, respectively. Surprisingly, without
the use of NaBH
4
, the rate of discolouration of phenol red dye was found to be high com-
pared to reaction mixtures where a reducing agent was applied, and a colourless reaction
mixture was obtained within 6 min (Figure 8A). As depicted in Figure 8B, a catalyst with
violet colour was obtained when it was separated from the reaction mixture by filtration.
After the addition of NaBH
4
, the dye desorbed from the catalyst, the supernatant solution
in the reaction mixture became pale fuchsia, and it transformed into a colourless liquid in
9 min of reaction time. A more detailed description of the adsorption of the phenol red
dye is mentioned in the upcoming sections.
Figure 8. (A) Addition of 6.7 wt.%Ag/chitosan catalyst in phenol red dye solution, and (B) (a) fresh
Ag/catalyst and (b) catalyst with adsorbed phenol red dye.
3.2.3. Influence of Concentration of Phenol Red Dye Solution
The catalytic activity was further examined upon the reduction of the phenol red dye,
whereupon dye solutions with different concentrations, such as 0.001, 0.003 and 0.005 M,
were used. The dye reduction ability of the catalyst was not considerably varying with
different concentrations containing dye solutions when 5 mg of NaBH
4
was applied (Fig-
ure 9A). However, after reducing the amount of NaBH
4
to 2.5 mg, the rate of the reduction
varied with different concentrations of the phenol red dye solution. As shown in Figure
9B, the dye conversion rate steadily decreased as its initial concentration in the aqueous
solution increased from 0.001 M to 0.005 M. It indicates that the amount of evolved H
2
was
not sufficient for the reduction of the increased concentration of dye—if 2.5 mg of reduc-
ing agent was used.
Figure 9. Reduction of the phenol red dye at different concentrations in aqueous solutions by 6 mg
of 6.7 wt.%Ag/chitosan catalyst and (A) 5 mg or (B) 2.5 mg of NaBH
4.
Figure 9.
Reduction of the phenol red dye at different concentrations in aqueous solutions by 6 mg of
6.7 wt.%Ag/chitosan catalyst and (A)5mgor(B) 2.5 mg of NaBH4.
3.2.4. Influence of the Amount of Ag Metal Loadings
In order to understand the influence of Ag metal in the reduction of phenol red dye,
Ag/chitosan catalysts with different Ag metal loadings, such as 1.4 or 4.4 or 6.7 wt.%,
were chosen during the reduction process. As shown in Figure 10A, the rate of reduction
reaction steadily increased with the Ag metal loading on the cross-linked chitosan support.
These observations are in agreement with the studies such as powder XRD and SEM-EDAX
analysis of catalysts with different metal loadings (Figures 1A and 2B). Further, in the case
of cross-linked chitosan, the dye reduced at a slow rate compared to its Ag metal-containing
analogues, as shown in Figure 7A, and possibly mainly NaBH
4
contributed to the reduction
process. Hence, like in the previous study regarding the influence of the catalyst amount,
it again confirmed that the Ag metal is necessary for the reduction of the phenol red dye
since it is not only involved in the activation of H
2
obtained after degradation of NaBH
4
but also facilitates a transfer of electron to dye molecules for their subsequent reduction.
Clean Technol. 2023, 5, FOR PEER REVIEW 11
3.2.4. Influence of the Amount of Ag Metal Loadings
In order to understand the influence of Ag metal in the reduction of phenol red dye,
Ag/chitosan catalysts with different Ag metal loadings, such as 1.4 or 4.4 or 6.7 wt.%, were
chosen during the reduction process. As shown in Figure 10A, the rate of reduction reac-
tion steadily increased with the Ag metal loading on the cross-linked chitosan support.
These observations are in agreement with the studies such as powder XRD and SEM-
EDAX analysis of catalysts with different metal loadings (Figures 1A and 2B). Further, in
the case of cross-linked chitosan, the dye reduced at a slow rate compared to its Ag metal-
containing analogues, as shown in Figure 7A, and possibly mainly NaBH
4
contributed to
the reduction process. Hence, like in the previous study regarding the influence of the
catalyst amount, it again confirmed that the Ag metal is necessary for the reduction of the
phenol red dye since it is not only involved in the activation of H
2
obtained after degra-
dation of NaBH
4
but also facilitates a transfer of electron to dye molecules for their subse-
quent reduction.
Figure 10. (A) Reduction of phenol red dye with 6 mg of Ag/chitosan catalysts with different Ag
metal loadings or cross-linked chitosan and 5 mg of NaBH
4
, (B) reduction of phenol red dye at dif-
ferent temperatures with 6 mg of 6.7 wt.%Ag/chitosan catalyst and 1 mg of NaBH
4
.
3.2.5. Influence of the Reaction Temperature
Reaction temperatures during the reduction process were also varied to understand
their role in the progress of the reaction where the reaction mixture was heated at either
room temperature (20 °C) or 30 or 40 °C. Since the reaction rate was high even at room
temperature with 5 mg of NaBH
4
and 6 mg of 6.7 wt.%Ag/chitosan catalyst, the amount
of NaBH
4
was reduced to 1 mg in the experiments where the reaction temperature was
varied. As shown in Figure 10B, the rate of the reaction increased as the reaction temper-
ature increased from room temperature to 30 and 40 °C.
3.2.6. Influence of Initial pH of the Phenol Red Dye Solution
Lin et al. previously described that the pH of the solution influences the decomposi-
tion of NaBH
4
and subsequent H
2
gas generation during catalytic reduction of p-nitrophe-
nol over Au/Fe
3
O
4
catalyst [39]. In this case, the authors explained that the rate of genera-
tion of H
2
and reduction of p-nitrophenol steadily decreased with an increase in the pH of
the solution, whereas no reduction occurred at pH 11. As shown in the reaction Equations
(2) and (3), the proton (H
+
) released after hydrolysis of water reacts with the hydride (H
−
)
anion of NaBH
4
and generates H
2
gas. The acidic to neutral pH condition facilitates the
hydrolysis of water resulting in the release of a proton, and then H
2
gas generation
through the decomposition of NaBH
4
becomes feasible. On the other hand, under alkaline
conditions, both the rate of hydrolysis of water, as well as decomposition of NaBH4 de-
creases and diminishes the subsequent rate of H
2
generation and reduction of dye. In or-
der to elucidate more about the influence of the initial pH of the phenol red dye solution
in the reduction process, 6.7 wt.%Ag/chitosan catalyst and NaBH
4
was mixed with dye
Figure 10.
(
A
) Reduction of phenol red dye with 6 mg of Ag/chitosan catalysts with different Ag
metal loadings or cross-linked chitosan and 5 mg of NaBH
4
, (
B
) reduction of phenol red dye at
different temperatures with 6 mg of 6.7 wt.%Ag/chitosan catalyst and 1 mg of NaBH4.

Clean Technol. 2023,5476
3.2.5. Influence of the Reaction Temperature
Reaction temperatures during the reduction process were also varied to understand
their role in the progress of the reaction where the reaction mixture was heated at either
room temperature (20
◦
C) or 30 or 40
◦
C. Since the reaction rate was high even at room
temperature with 5 mg of NaBH
4
and 6 mg of 6.7 wt.%Ag/chitosan catalyst, the amount of
NaBH
4
was reduced to 1 mg in the experiments where the reaction temperature was varied.
As shown in Figure 10B, the rate of the reaction increased as the reaction temperature
increased from room temperature to 30 and 40 ◦C.
3.2.6. Influence of Initial pH of the Phenol Red Dye Solution
Lin et al. previously described that the pH of the solution influences the decomposition
of NaBH
4
and subsequent H
2
gas generation during catalytic reduction of p-nitrophenol
over Au/Fe
3
O
4
catalyst [
39
]. In this case, the authors explained that the rate of generation
of H
2
and reduction of p-nitrophenol steadily decreased with an increase in the pH of the
solution, whereas no reduction occurred at pH 11. As shown in the reaction Equations
(2) and (3), the proton (H
+
) released after hydrolysis of water reacts with the hydride
(H
−
) anion of NaBH
4
and generates H
2
gas. The acidic to neutral pH condition facilitates
the hydrolysis of water resulting in the release of a proton, and then H
2
gas generation
through the decomposition of NaBH
4
becomes feasible. On the other hand, under alkaline
conditions, both the rate of hydrolysis of water, as well as decomposition of NaBH4
decreases and diminishes the subsequent rate of H
2
generation and reduction of dye. In
order to elucidate more about the influence of the initial pH of the phenol red dye solution
in the reduction process, 6.7 wt.%Ag/chitosan catalyst and NaBH
4
was mixed with dye
solutions with different initial pH such as 4, 6.4, 8, 11 or 13.8. As shown in Figure 11,
the reduction ability of the catalysts was not significantly influenced by the initial pH of
the solution until it reached 11. For 3 min of reaction time, the reduction rate decreased
somewhat as the pH of the solution increased from 4 to 11, while no significant difference
was observed as the reaction proceeded further. Unlike the previously described reduction
of p-nitrophenol, in this study, the solution with pH 11 also allowed an efficient reduction
of the dye [
39
]. Similarly, Wang et al. showed that Ag nanoparticle-entrapped hydrogel
prepared from chitosan also enabled a complete reduction of methylene blue and Congo
red until pH 11 [
40
]. Furthermore, the reduction of phenol red dye proceeded at a slow rate
when a highly alkaline dye solution with pH 13.8 was applied.
Clean Technol. 2023, 5, FOR PEER REVIEW 12
solutions with different initial pH such as 4, 6.4, 8, 11 or 13.8. As shown in Figure 11, the
reduction ability of the catalysts was not significantly influenced by the initial pH of the
solution until it reached 11. For 3 min of reaction time, the reduction rate decreased some-
what as the pH of the solution increased from 4 to 11, while no significant difference was
observed as the reaction proceeded further. Unlike the previously described reduction of
p-nitrophenol, in this study, the solution with pH 11 also allowed an efficient reduction of
the dye [39]. Similarly, Wang et al. showed that Ag nanoparticle-entrapped hydrogel pre-
pared from chitosan also enabled a complete reduction of methylene blue and Congo red
until pH 11 [40]. Furthermore, the reduction of phenol red dye proceeded at a slow rate
when a highly alkaline dye solution with pH 13.8 was applied.
Figure 11. Reduction of the phenol red dye in an aqueous solution (0.001 M) with different initial
pH in the range of 4–13.8. The amounts of 6.7 wt.%Ag/chitosan catalyst and NaBH
4
are 6 and 5 mg,
respectively.
3.3. Adsorption of Phenol Red Dye with Cross-Linked Chitosan Support
3.3.1. Influence of pH on the Adsorption of Phenol Red Dye
The pH of the dye solution significantly influences its adsorption on the adsorbent
surfaces since a change in pH alters the charges over the adsorbent and also changes the
chemical structure of the dye. Kwok et al. reported that the chitosan surface is sensitive to
the pH of the solution where adsorption of the arsenate ions effectively occurred in a so-
lution within the acidic pH range (below pH 7). However, as the pH value increased above
its neutral value, the rate of adsorption steadily decreased, and the rate of desorption of
the adsorbed species increased [41]. The author also described the adsorption-desorption
phenomenon of the arsenate ions in terms of the point of zero charge (pH
pzc,
the pH value
at which the surface charge is zero) and surface charge density of the chitosan surface
measured by the potentiometric titration method. The pH
pzc
values of the studied chitosan
particles were observed to be around 8 since, above this value, the surface becomes nega-
tively charged. Besides that, the surface charged densities measurements also represent
that the isoelectric point value was observed at 6.38, whereas the surface charge of the
chitosan surface below this value was found to be positively charged. In other words, in
the acidic pH range, the chitosan surface remains positively charged as the negative
charges decrease due to neutralisation which allows efficient adsorption of the arsenate
anion until the neutral pH of the solution. Wang et al. also showed equivalent observation
in the case of adsorption of fulvic acid over a chitosan surface, where the point of zero
charge study shows that the adsorption capacity of the adsorbent was high below pH 9
while its capacity substantially decreased over this value due to reverse of the charges of
the surface from positive to negative [40]. Wahab et al. studied the photodegradation of
the phenol red dye over nanocrystalline titanium oxide (TiO
2
) particles, where it was ob-
served that since the TiO
2
surface remained positively charged under acidic conditions
Figure 11.
Reduction of the phenol red dye in an aqueous solution (0.001 M) with different initial
pH in the range of 4–13.8. The amounts of 6.7 wt.%Ag/chitosan catalyst and NaBH
4
are 6 and 5 mg,
respectively.

Clean Technol. 2023,5477
3.3. Adsorption of Phenol Red Dye with Cross-Linked Chitosan Support
3.3.1. Influence of pH on the Adsorption of Phenol Red Dye
The pH of the dye solution significantly influences its adsorption on the adsorbent
surfaces since a change in pH alters the charges over the adsorbent and also changes the
chemical structure of the dye. Kwok et al. reported that the chitosan surface is sensitive
to the pH of the solution where adsorption of the arsenate ions effectively occurred in a
solution within the acidic pH range (below pH 7). However, as the pH value increased
above its neutral value, the rate of adsorption steadily decreased, and the rate of desorption
of the adsorbed species increased [
41
]. The author also described the adsorption-desorption
phenomenon of the arsenate ions in terms of the point of zero charge (pH
pzc,
the pH
value at which the surface charge is zero) and surface charge density of the chitosan
surface measured by the potentiometric titration method. The pH
pzc
values of the studied
chitosan particles were observed to be around 8 since, above this value, the surface becomes
negatively charged. Besides that, the surface charged densities measurements also represent
that the isoelectric point value was observed at 6.38, whereas the surface charge of the
chitosan surface below this value was found to be positively charged. In other words, in the
acidic pH range, the chitosan surface remains positively charged as the negative charges
decrease due to neutralisation which allows efficient adsorption of the arsenate anion until
the neutral pH of the solution. Wang et al. also showed equivalent observation in the case
of adsorption of fulvic acid over a chitosan surface, where the point of zero charge study
shows that the adsorption capacity of the adsorbent was high below pH 9 while its capacity
substantially decreased over this value due to reverse of the charges of the surface from
positive to negative [
40
]. Wahab et al. studied the photodegradation of the phenol red dye
over nanocrystalline titanium oxide (TiO
2
) particles, where it was observed that since the
TiO
2
surface remained positively charged under acidic conditions (below pH 6.5), the rate
of degradation of the dye was increased and vice versa in case of alkaline medium [
38
].
Further, the authors presumed that from acidic to neutral pH conditions, the phenol red dye
also remained in the zwitterionic form or as a structure with a negatively charged sulfate
group which has an electrostatic attraction towards positively charged surface under a
similar pH range (Figure 5). Ma et al. also applied chitosan as an adsorbent material for the
adsorption of Congo red and methylene blue dye, where the process was studied for a dye
solution with pH 6.5 [5].
In the current study, the influence of the initial pH of the dye solution for its adsorption
on the cross-linked chitosan support was studied, where dye solutions with varying pH
from 4 to 13.8 were applied. As shown in Figure 12A, incomplete adsorption of the dye
was observed in a solution with pH 4, whereupon the peak at 436 nm decreased from its
initial value while peak at 571 nm increased. This suggests that the supernatant solution
became alkaline since the peak assigned to negatively charged species of the dye appeared.
Below pH 6, the surface charge of chitosan became positive due to the protonation of amine
groups (–NH
3+
). As a negatively charged molecule, the phenol red dye eagerly adsorbs on
the chitosan surfaces through its electrostatic interaction with positively charged amine
groups [
42
,
43
]. However, at high concentrations of H
+
, the dye molecule desorbs from the
chitosan surface, which further increases the final pH of the solution [
41
,
44
]. Furthermore,
high adsorption of the dye was observed at pH 6.4, whereby nearly complete adsorption
occurred in 18 min (Figure 12B). In this case, the dye adsorbed through both physical
(hydrogen bonding and van der Waals interaction) as well as electrostatic interaction
with the chitosan surface. In the case of the pH 6.4 solution, also the peak at 571 nm
appeared less intense, suggesting that the reaction mixture became marginally alkaline
after dye adsorption due to continuous protonation and deprotonation of the amine groups
in chitosan support [
41
]. In case of an increase in pH of the solution to 8, the adsorption
capacity of chitosan support for the dye further decreased, and the solution became more
alkaline as the peaks at 436 and 571 nm were highly intense compared to reaction mixtures
with pH 4 and 6.4 (Figure 12C). The negligible adsorption of the phenol red dye observed
at pH 11 and 13.8 was obvious since the peak intensity at 571 nm did not decrease. As

Clean Technol. 2023,5478
described previously, above neutral pH, the surface charge of chitosan becomes negative,
and this disfavoured the adsorption of the negatively charged dye molecules [
38
,
40
]. Hence,
a dye adsorption study on the chitosan surface demonstrated that an aqueous solution
of dye comprised of pH close to neutral facilitates the adsorption contrary to acidic and
alkaline dye solutions.
Clean Technol. 2023, 5, FOR PEER REVIEW 13
(below pH 6.5), the rate of degradation of the dye was increased and vice versa in case of
alkaline medium [38]. Further, the authors presumed that from acidic to neutral pH con-
ditions, the phenol red dye also remained in the zwitterionic form or as a structure with a
negatively charged sulfate group which has an electrostatic attraction towards positively
charged surface under a similar pH range (Figure 5). Ma et al. also applied chitosan as an
adsorbent material for the adsorption of Congo red and methylene blue dye, where the
process was studied for a dye solution with pH 6.5 [5].
In the current study, the influence of the initial pH of the dye solution for its adsorp-
tion on the cross-linked chitosan support was studied, where dye solutions with varying
pH from 4 to 13.8 were applied. As shown in Figure 12A, incomplete adsorption of the
dye was observed in a solution with pH 4, whereupon the peak at 436 nm decreased from
its initial value while peak at 571 nm increased. This suggests that the supernatant solu-
tion became alkaline since the peak assigned to negatively charged species of the dye ap-
peared. Below pH 6, the surface charge of chitosan became positive due to the protonation
of amine groups (–NH
3+
). As a negatively charged molecule, the phenol red dye eagerly
adsorbs on the chitosan surfaces through its electrostatic interaction with positively
charged amine groups [42,43]. However, at high concentrations of H
+
, the dye molecule
desorbs from the chitosan surface, which further increases the final pH of the solution
[41,44]. Furthermore, high adsorption of the dye was observed at pH 6.4, whereby nearly
complete adsorption occurred in 18 min (Figure 12B). In this case, the dye adsorbed
through both physical (hydrogen bonding and van der Waals interaction) as well as elec-
trostatic interaction with the chitosan surface. In the case of the pH 6.4 solution, also the
peak at 571 nm appeared less intense, suggesting that the reaction mixture became mar-
ginally alkaline after dye adsorption due to continuous protonation and deprotonation of
the amine groups in chitosan support [41]. In case of an increase in pH of the solution to
8, the adsorption capacity of chitosan support for the dye further decreased, and the solu-
tion became more alkaline as the peaks at 436 and 571 nm were highly intense compared
to reaction mixtures with pH 4 and 6.4 (Figure 12C). The negligible adsorption of the phe-
nol red dye observed at pH 11 and 13.8 was obvious since the peak intensity at 571 nm did
not decrease. As described previously, above neutral pH, the surface charge of chitosan
becomes negative, and this disfavoured the adsorption of the negatively charged dye mol-
ecules [38,40]. Hence, a dye adsorption study on the chitosan surface demonstrated that
an aqueous solution of dye comprised of pH close to neutral facilitates the adsorption
contrary to acidic and alkaline dye solutions.
Figure 12. Adsorption of phenol red dye by 6 mg of cross-linked chitosan support at various pH (A)
4, (B) 6.4, (C) 8, (D) 11 and (E) 13.8.
Figure 12.
Adsorption of phenol red dye by 6 mg of cross-linked chitosan support at various pH (
A
) 4,
(B) 6.4, (C) 8, (D) 11 and (E) 13.8.
3.3.2. Adsorption Followed by Reduction of Dye with Cross-Linked Chitosan Support
As shown in Figure 12B, the cross-linked chitosan support gave rise to a high adsorp-
tion ability for the phenol red dye over its surface, and the absorbance steadily decreased
as the reaction proceeded. The corresponding changes in the absorbance of the phenol red
dye are shown in Figure 13A. As shown in Figure 14A, the cross-linked chitosan support
displayed yellow colour, which further converted to a violet-coloured solid after the in-
teraction with an orange-red coloured solution of the phenol red dye. Further, the FT-IR
analysis of the cross-linked chitosan support with and without the adsorbed dye was also
carried out, and the obtained spectra were compared to confirm the dye adsorption over
the surface of the support. As shown in Figure 14B, the intensity of the absorption bands
regarding various functional groups, including the –OH group (3400 cm
−1
) in cross-linked
chitosan support, decreased after the adsorption of dye on the surface, and broadened
bands with lower intensity were obtained. Besides that, the characteristic bands attributed
to the benzene ring, –OH, carbonyl (–C=O), C–O and sulphonate (SO
3
−
) groups in phe-
nol red dye molecule also disappeared, and this can be attributed to the homogeneous
distribution of dye over the surface of cross-linked chitosan support [38,45].
To examine the reduction of the adsorbed dye, NaBH
4
was duly added to the reaction
mixture after the adsorption of dye over cross-linked chitosan support. It was observed that
the dye desorbed from the chitosan surface, and the reaction mixture became pale fuchsia
in colour after the addition of the reducing agent. As shown in Figure 13B, the reduction of
the dye proceeded slowly and was mainly due to NaBH
4
since it was previously observed
that the dye reduced at a slow reaction rate with the reducing agent compared to the
Ag/chitosan catalyst involved process (Figure 10A). Here, the adsorption and desorption
of dye before and after the addition of the reducing agent, respectively, can be correlated to
the change in the pH of the reaction mixture since NaBH
4
changes the pH of the solution to
alkaline, which allows the desorption of the dye from the surface of the adsorbent [40,41].

Clean Technol. 2023,5479
Clean Technol. 2023, 5, FOR PEER REVIEW 14
3.3.2. Adsorption Followed by Reduction of Dye with Cross-Linked Chitosan Support
As shown in Figure 12B, the cross-linked chitosan support gave rise to a high adsorp-
tion ability for the phenol red dye over its surface, and the absorbance steadily decreased
as the reaction proceeded. The corresponding changes in the absorbance of the phenol red
dye are shown in Figure 13A. As shown in Figure 14A, the cross-linked chitosan support
displayed yellow colour, which further converted to a violet-coloured solid after the in-
teraction with an orange-red coloured solution of the phenol red dye. Further, the FT-IR
analysis of the cross-linked chitosan support with and without the adsorbed dye was also
carried out, and the obtained spectra were compared to confirm the dye adsorption over
the surface of the support. As shown in Figure 14B, the intensity of the absorption bands
regarding various functional groups, including the –OH group (3400 cm
−1
) in cross-linked
chitosan support, decreased after the adsorption of dye on the surface, and broadened
bands with lower intensity were obtained. Besides that, the characteristic bands attributed
to the benzene ring, –OH, carbonyl (–C=O), C–O and sulphonate (SO
3−
) groups in phenol
red dye molecule also disappeared, and this can be attributed to the homogeneous distri-
bution of dye over the surface of cross-linked chitosan support [38,45].
Figure 13. (A) Adsorption and (B) adsorption followed by reduction of phenol red dye with cross-
linked chitosan support. The amount of chitosan support and NaBH
4
6 and 5 mg, respectively.
Figure 14. (A) Cross-linked chitosan (a) without dye and (b) with adsorbed dye, (B) FT-IR spectra
of (a) pure phenol red dye, cross-linked chitosan (b) without dye and (c) with dye.
To examine the reduction of the adsorbed dye, NaBH
4
was duly added to the reaction
mixture after the adsorption of dye over cross-linked chitosan support. It was observed
that the dye desorbed from the chitosan surface, and the reaction mixture became pale
fuchsia in colour after the addition of the reducing agent. As shown in Figure 13B, the
reduction of the dye proceeded slowly and was mainly due to NaBH
4
since it was previ-
ously observed that the dye reduced at a slow reaction rate with the reducing agent com-
pared to the Ag/chitosan catalyst involved process (Figure 10A). Here, the adsorption and
desorption of dye before and after the addition of the reducing agent, respectively, can be
Figure 13.
(
A
) Adsorption and (
B
) adsorption followed by reduction of phenol red dye with cross-
linked chitosan support. The amount of chitosan support and NaBH46 and 5 mg, respectively.
Clean Technol. 2023, 5, FOR PEER REVIEW 14
3.3.2. Adsorption Followed by Reduction of Dye with Cross-Linked Chitosan Support
As shown in Figure 12B, the cross-linked chitosan support gave rise to a high adsorp-
tion ability for the phenol red dye over its surface, and the absorbance steadily decreased
as the reaction proceeded. The corresponding changes in the absorbance of the phenol red
dye are shown in Figure 13A. As shown in Figure 14A, the cross-linked chitosan support
displayed yellow colour, which further converted to a violet-coloured solid after the in-
teraction with an orange-red coloured solution of the phenol red dye. Further, the FT-IR
analysis of the cross-linked chitosan support with and without the adsorbed dye was also
carried out, and the obtained spectra were compared to confirm the dye adsorption over
the surface of the support. As shown in Figure 14B, the intensity of the absorption bands
regarding various functional groups, including the –OH group (3400 cm
−1
) in cross-linked
chitosan support, decreased after the adsorption of dye on the surface, and broadened
bands with lower intensity were obtained. Besides that, the characteristic bands attributed
to the benzene ring, –OH, carbonyl (–C=O), C–O and sulphonate (SO
3−
) groups in phenol
red dye molecule also disappeared, and this can be attributed to the homogeneous distri-
bution of dye over the surface of cross-linked chitosan support [38,45].
Figure 13. (A) Adsorption and (B) adsorption followed by reduction of phenol red dye with cross-
linked chitosan support. The amount of chitosan support and NaBH
4
6 and 5 mg, respectively.
Figure 14. (A) Cross-linked chitosan (a) without dye and (b) with adsorbed dye, (B) FT-IR spectra
of (a) pure phenol red dye, cross-linked chitosan (b) without dye and (c) with dye.
To examine the reduction of the adsorbed dye, NaBH
4
was duly added to the reaction
mixture after the adsorption of dye over cross-linked chitosan support. It was observed
that the dye desorbed from the chitosan surface, and the reaction mixture became pale
fuchsia in colour after the addition of the reducing agent. As shown in Figure 13B, the
reduction of the dye proceeded slowly and was mainly due to NaBH
4
since it was previ-
ously observed that the dye reduced at a slow reaction rate with the reducing agent com-
pared to the Ag/chitosan catalyst involved process (Figure 10A). Here, the adsorption and
desorption of dye before and after the addition of the reducing agent, respectively, can be
Figure 14.
(
A
) Cross-linked chitosan (a) without dye and (b) with adsorbed dye, (
B
) FT-IR spectra of
(a) pure phenol red dye, cross-linked chitosan (b) without dye and (c) with dye.
3.4. Mechanism for the Phenol Red Dye Reduction with Ag/Chitosan Catalyst
The possible mechanisms for the reduction of phenol red dye over the Ag/chitosan
catalyst with NaBH
4
have been proposed and are shown in Figure 15. As shown in
Figures 7B and 8,
in the absence of the reducing agent, the phenol red dye gets adsorbed
over the catalyst surface. Further, upon the addition of the reducing agent, the adsorbed
dye got reduced and desorbed from the catalyst surface accordingly. It was also observed
that NaBH
4
causes a reduction of the dye at a comparatively slow rate in the absence of
catalysts or Ag metal (Figures 7A and 10A). Hence, Ag/chitosan catalysts facilitate not only
the adsorption of dye but with Ag metal also induce the activation of the H
2
and reduction
of adsorbed dye simultaneously. As shown in the proposed mechanism in Figure 15A,
after the addition of both catalyst and NaBH
4
, the dye gets adsorbed over the catalyst
surface through surface-dye molecule interactions. The Ag metal further activates the
H
2
originating from the decomposition of BH
4−
anion, and the adsorbed dye is reduced
accordingly, where the reduced dye desorbs from the catalyst surface instantly (Figure 14B).
In this case, the Ag metal acts as a relay for the electron (or H
−
), where it is relaying the
electron from BH4−anion to the acceptor dye molecule for subsequent reduction.

Clean Technol. 2023,5480
Clean Technol. 2023, 5, FOR PEER REVIEW 15
correlated to the change in the pH of the reaction mixture since NaBH
4
changes the pH of
the solution to alkaline, which allows the desorption of the dye from the surface of the
adsorbent [40,41].
3.4. Mechanism for the Phenol Red Dye Reduction with Ag/Chitosan Catalyst
The possible mechanisms for the reduction of phenol red dye over the Ag/chitosan
catalyst with NaBH
4
have been proposed and are shown in Figure 15. As shown in Figures
7B and 8, in the absence of the reducing agent, the phenol red dye gets adsorbed over the
catalyst surface. Further, upon the addition of the reducing agent, the adsorbed dye got
reduced and desorbed from the catalyst surface accordingly. It was also observed that
NaBH
4
causes a reduction of the dye at a comparatively slow rate in the absence of cata-
lysts or Ag metal (Figures 7A and 10A). Hence, Ag/chitosan catalysts facilitate not only
the adsorption of dye but with Ag metal also induce the activation of the H
2
and reduction
of adsorbed dye simultaneously. As shown in the proposed mechanism in Figure 15A,
after the addition of both catalyst and NaBH
4
, the dye gets adsorbed over the catalyst
surface through surface-dye molecule interactions. The Ag metal further activates the H
2
originating from the decomposition of BH
4−
anion, and the adsorbed dye is reduced ac-
cordingly, where the reduced dye desorbs from the catalyst surface instantly (Figure 14B).
In this case, the Ag metal acts as a relay for the electron (or H
−
), where it is relaying the
electron from BH
4−
anion to the acceptor dye molecule for subsequent reduction.
Figure 15. Possible mechanisms for the reduction of phenol red dye over Ag/chitosan catalyst (A)
adsorption, and (B) reduction of phenol red dye with NaBH
4.
3.5. Recyclability of Ag/Chitosan Catalyst upon Reduction of Phenol Red Dye
The Ag/chitosan catalyst with 6.7 wt.%Ag metal loading was used for the recyclabil-
ity study in the case of the phenol red dye reduction process. The recyclability of the cat-
alyst was carried out five times, and the obtained recycled catalyst was further studied
with ICP-OES analysis to quantify the loss of Ag metal during the recyclability study.
As shown in Figure 16, a minute decrease in the activity of the recycled catalyst was
observed compared to the fresh catalyst during the recyclability study, whereas the cata-
lyst showed identical activity during all the recycling steps. Further, the ICP-OES analysis
study confirmed that 6.5 wt.% of Ag metal remained in the composition of the catalyst
after the recyclability study. Hence, Ag/chitosan catalyst was found stable during the cat-
alytic reduction of phenol red dye since no significant loss of Ag metal was observed.
Figure 15.
Possible mechanisms for the reduction of phenol red dye over Ag/chitosan catalyst
(A) adsorption, and (B) reduction of phenol red dye with NaBH4.
3.5. Recyclability of Ag/Chitosan Catalyst upon Reduction of Phenol Red Dye
The Ag/chitosan catalyst with 6.7 wt.%Ag metal loading was used for the recyclability
study in the case of the phenol red dye reduction process. The recyclability of the catalyst
was carried out five times, and the obtained recycled catalyst was further studied with
ICP-OES analysis to quantify the loss of Ag metal during the recyclability study.
As shown in Figure 16, a minute decrease in the activity of the recycled catalyst was
observed compared to the fresh catalyst during the recyclability study, whereas the catalyst
showed identical activity during all the recycling steps. Further, the ICP-OES analysis
study confirmed that 6.5 wt.% of Ag metal remained in the composition of the catalyst after
the recyclability study. Hence, Ag/chitosan catalyst was found stable during the catalytic
reduction of phenol red dye since no significant loss of Ag metal was observed.
Clean Technol. 2023, 5, FOR PEER REVIEW 16
Figure 16. Recyclability study of the Ag/chitosan catalyst in catalytic reduction phenol red dye.
Amount of 6.7 wt.%Ag/chitosan and NaBH
4
6 and 5 mg, respectively.
4. Conclusions
The Ag/chitosan catalysts synthesised with cross-linked chitosan and Ag metal were
studied in detail upon adsorption and catalytic reduction of phenol red dye in aqueous
solutions. The Ag/chitosan catalyst prepared without any addition of the reducing agent
showed excellent catalytic activity in phenol red dye reduction in the presence of NaBH
4
.
The activity of the catalyst was increased with increasing catalyst amount, NaBH
4,
Ag
metal loading and applied temperature. The pH of the reaction mixture in the range of 4–
11 did not significantly influence the outcome as a complete reduction of the dye was
observed and high catalytic activity. However, the dye reduction rate decreased exces-
sively in a highly alkaline reaction mixture with pH 13.8, and no complete reduction of
the dye was observed under such reaction conditions. The adsorption of the dye on the
cross-linked chitosan support varied with the pH of the dye solution, where incomplete
adsorption of dye was observed at pH 4 and 8 while a solution with nearly neutral pH,
i.e., 6.4, facilitated efficient dye adsorption. No adsorption of the dye was observed in an
alkaline dye solution with pH 11 and 13.8. The 6.7 wt.%Ag/chitosan catalyst was recycled
five times in the dye reduction process, where identical catalyst activity was observed,
and no significant loss of Ag metal was observed. Hence, in this report, a study regarding
the adsorption as well as catalytic reduction of phenol red dye with cross-linked chitosan
and Ag/chitosan catalyst, respectively, were studied where both processes were carried
out at mild and economically feasible reaction conditions. Upon industrial-scale water
purification, the concept might be feasible considering the low cost and easy availability
of the Ag metal precursor as well as the use of renewable support material.
Author Contributions: C.C.S.: conceptualisation, visualisation, formal analysis, validation and writ-
ing-reviewing and editing; V.M.D.: resources, visualisation, formal analysis and validation; P.C.:
supervision, writing-reviewing and editing, project administration and funding acquisition. J.-P.M.:
supervision, writing-reviewing and editing, project administration and funding acquisition. S.G.K.:
supervision, conceptualisation, investigation, resources, visualisation, validation, writing-original
draft, project administration, writing-reviewing and editing. All authors have read and agreed to
the published version of the manuscript.
Funding: This work is part of the activities by the Swedish Bio4Energy program and the Wallenberg
Wood Science Center under the auspices of the Alice and Knut Wallenberg Foundation.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created.
Figure 16.
Recyclability study of the Ag/chitosan catalyst in catalytic reduction phenol red dye.
Amount of 6.7 wt.%Ag/chitosan and NaBH46 and 5 mg, respectively.
4. Conclusions
The Ag/chitosan catalysts synthesised with cross-linked chitosan and Ag metal were
studied in detail upon adsorption and catalytic reduction of phenol red dye in aqueous
solutions. The Ag/chitosan catalyst prepared without any addition of the reducing agent
showed excellent catalytic activity in phenol red dye reduction in the presence of NaBH
4
.
The activity of the catalyst was increased with increasing catalyst amount, NaBH
4,
Ag
metal loading and applied temperature. The pH of the reaction mixture in the range of

Clean Technol. 2023,5481
4–11 did not significantly influence the outcome as a complete reduction of the dye was
observed and high catalytic activity. However, the dye reduction rate decreased excessively
in a highly alkaline reaction mixture with pH 13.8, and no complete reduction of the dye
was observed under such reaction conditions. The adsorption of the dye on the cross-linked
chitosan support varied with the pH of the dye solution, where incomplete adsorption of
dye was observed at pH 4 and 8 while a solution with nearly neutral pH, i.e., 6.4, facilitated
efficient dye adsorption. No adsorption of the dye was observed in an alkaline dye solution
with pH 11 and 13.8. The 6.7 wt.%Ag/chitosan catalyst was recycled five times in the dye
reduction process, where identical catalyst activity was observed, and no significant loss of
Ag metal was observed. Hence, in this report, a study regarding the adsorption as well as
catalytic reduction of phenol red dye with cross-linked chitosan and Ag/chitosan catalyst,
respectively, were studied where both processes were carried out at mild and economically
feasible reaction conditions. Upon industrial-scale water purification, the concept might be
feasible considering the low cost and easy availability of the Ag metal precursor as well as
the use of renewable support material.
Author Contributions:
C.C.S.: conceptualisation, visualisation, formal analysis, validation and
writing-reviewing and editing; V.M.D.: resources, visualisation, formal analysis and validation; P.C.:
supervision, writing-reviewing and editing, project administration and funding acquisition. J.-P.M.:
supervision, writing-reviewing and editing, project administration and funding acquisition. S.G.K.:
supervision, conceptualisation, investigation, resources, visualisation, validation, writing-original
draft, project administration, writing-reviewing and editing. All authors have read and agreed to the
published version of the manuscript.
Funding:
This work is part of the activities by the Swedish Bio4Energy program and the Wallenberg
Wood Science Center under the auspices of the Alice and Knut Wallenberg Foundation.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created.
Acknowledgments:
This work is part of activities of the Technical Chemistry, Department of Chem-
istry, Chemical-Biological Centre, Umeå University, Sweden. The Swedish Bio4Energy program and
the Wallenberg Wood Science Center under the auspices of the Alice and Knut Wallenberg Foundation
are gratefully acknowledged. This work is also a part of the activities of the Johan Gadolin Process
Chemistry Centre at Åbo Akademi University.
Conflicts of Interest: The authors declare no conflict of interest.
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