Cellulose

https://doi.org/10.1007/s10570-018-1986-x (0123456789().,-volV)
(0123456789().,-volV)

ORIGINAL PAPER

Comparison between solid and liquid acids for production

of low molecular weight chitosan using systematic DOE-
based approach
Saurabh Patil . R. Akhil Krishnan . Shashank Bhangde . Prajakta Dandekar .
Ratnesh Jain

Received: 10 January 2018 / Accepted: 9 August 2018

Ó Springer Nature B.V. 2018

Abstract Chitosan is an abundantly available permeation chromatography. Furthermore, spray dry-

biodegradable polymer with diverse applications in
various chemical and biomedical industries. However,
it has remained underutilized due to its bulky and
intractable nature, limiting its application in various
fields. The major drawback, however, is poor aqueous
solubility and thus preparation of low molecular
weight derivatives of chitosan (LMWC) is an ideal
solution to overcome the drawbacks associated with
the parent polymer. The present study reports a
sustainable method to produce LMWC, using H-b
Zeolite as a green catalyst in comparison to the
traditional depolymerization methods, based on
nitrous acid. Successful decrease in molecular weight
from 1157 to 175 kDa was achieved as studied by gel
ing assisted in achieving uniform sized particles of
LMWC of size less than 100 lm. Integration of
response surface methodology for process optimiza-
tion led to the negligible formation of by-products like
5-hydroxymethylfurfural. Comparative investigations
confirmed the consistency of the developed method,
simultaneously presenting additional advantages such
as catalyst reusability, process reproducibility and
process safety. Our findings demonstrate a greener
approach for preparation of consistent quality LMWC,
by implementing systemic stratagem of design of
experiments.

Electronic supplementary material The online version of

this article (https://doi.org/10.1007/s10570-018-1986-x) con-
tains supplementary material, which is available to authorized
users.

S. Patil  R. Jain (&)

Department of Chemical Engineering, Institute of
Chemical Technology, Nathalal Parekh Marg, Matunga,
Mumbai 400 019, India
e-mail: rd.jain@ictmumbai.edu.in

R. A. Krishnan  S. Bhangde  P. Dandekar (&)

Department of Pharmaceutical Sciences and Technology,
Institute of Chemical Technology, Nathalal Parekh Marg,
Matunga, Mumbai 400 019, India
e-mail: pd.jain@ictmumbai.edu.in

123

Graphical abstract
Keywords Chitosan  H-b Zeolite  Solid acid
catalysis  Response surface methodology  Low
molecular weight chitosan
Introduction
Chitosan is a naturally occurring polymer consisting
of b-1,4-linked D-glucosamine units, with varying
degrees of N-acetylated units. It is primarily obtained
by deacetylation of chitin, originally extracted from
crustacean shells that constitute an abundant marine
waste (Kumar 2000). Over the last few decades,
chitosan has proven its importance in diverse fields,
owing to a remarkable combination of its biological,
mechanical and physical characteristics (Kumar et al.
2004). Nevertheless, its widespread employment has
been compromised due to its inherent insolubility in
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Cellulose
water, high viscosity, low bulk density and polydis-
perse nature (Rabea et al. 2003). More specifically, in
key areas such as food and medicine the use of low
molecular weight variant (LMWC) is preferred owing
their versatile properties such as biocompatibility,
biodegradability and low toxicity. For instance, Li
et al. (2005) suggest the superior digestion of LMWC
in the gastrointestinal tract in both, humans and
animals, due to their smaller molecular size. This is
due to the presence of suitable degrading enzymes for
LMWC. Apart from these, it has been found that the
efficiency of LMWC is majorly driven by their
molecular weight, with polymers of certain molecular
weight range being more suitable for specific appli-
cations (Allan and Peyron 1995). For instance,
immunostimulatory activity of a 3 kDa LMWC was
found to be higher than a 50 kDa LMWC (Zheng et al.
2016). 20 kDa LMWC effectively inhibited Bacillus
cereus when added to raw rice before steam cooking
Cellulose
(Tsai et al. 2006). Thus, it becomes very important to
process chitosan to LMWC before using them espe-
cially in food and pharmaceutical applications. In the
past decade, LMWC has been explored tremendously
in various avenues including pharmaceutical, biomed-
ical and biotechnological applications (Mao et al.
2004). Thus, efforts are being made to establish a
simple, reproducible and scalable method for the
production of LMWC.
Traditionally, low molecular weight derivatives of
chitosan have been prepared by physical, chemical and
enzymatic hydrolyses. However, underdeveloped pro-
cesses, high installation costs of the required equip-
ment and uncontrolled breakdown, yielding furfural-
based side-products has limited the use and industrial
scalability of the physical methods (Moura et al.
2015). On the other hand, higher costs of hydrolytic
enzymes and their unstable nature reduce the effec-
tiveness and commercial feasibility of enzymatic
methods (Kim and Rajapakse 2005). Chemical meth-
ods involve use of nitrous acid, hydrochloric acid,
phosphoric acid etc., which require tedious processes
for separation or eventual destruction of catalysts
(Prashanth and Tharanathan 2005). These methods
create devastating impact on environment owing to the
usage of harsh and corrosive chemicals. Amongst all
the chemical methods, the use of nitrous acid has been
preferentially adopted owing to its simplicity and
reproducibility. However, acute and chronic toxicity
of nitrous acid cannot be neglected while establishing
process safety (van Strien et al. 2004). Since all of
these methods are impractical in terms of sustainabil-
ity and industrial feasibility, extensive research is
being carried out to seek alternate methods for
chitosan hydrolysis. Here, use of heterogeneous
catalysts (solid acids) is looked upon as a prospective
solution for efficient depolymerization of chitosan into
LMWC. One such catalyst is zeolite, a solid acid
composed of alumino-silicate building blocks, form-
ing a tetrahedral geometry. These solid acids have
been exploited in petrochemical industries, for fluid
catalytic cracking since the last three decades
(Weitkamp 2000). Various types of zeolites has been
investigated for their efficiency to hydrolyze cellulose
into glucose and 5-hydroxymethylfurfural (5-HMF)
(Nikolla et al. 2011; Onda et al. 2008; Rinaldi et al.
2008). Onda et al. reported the selectivity of zeolites to
cleave the b-1,4-glycosidic linkages, to yield glucose
of the desired quality. Considering the structural
similarity of chitosan with cellulose, zeolites were
employed in the present work to depolymerize
chitosan. The idea was to exploit their unique features
such as acid strength, porous surface, reusability and
tunable properties, for the depolymerization reaction
conducted herein. H-b Zeolite (HbZ) is one such
catalytic material that possesses loosely bound H?
ions on their surfaces and pores. These H? ions can
release into solution and under appropriate conditions,
can catalyse conversion of chitosan into LMWC
(Huang and Fu 2013).
In the present study, HbZ (solid acid) was evaluated
and compared with nitrous acid (liquid acid), with
respect to the quality of final product, process efficacy
and process safety. We have utilized design of
experiments (DOE) strategy that minimizes experi-
mental trials and enables appropriate interpretation of
results, to utilise them for industrial applications.
Conventionally, influence of a particular variable on
the end product is done using one variable at a time
(OVAT) methodology. However, this methodology is
material and time consuming, and also incapable of
optimizing the process or studying the interaction
between process variables (Frey et al. 2003). Conse-
quently, a multivariate statistical system was devel-
oped with the help of DOE approach. This stratagem
combines statistical and mathematical techniques to
generate empirical models for experimental data,
which is further processed and presented graphically
using response surface models (Bezerra et al. 2008). It
was found that the catalytic activity of HbZ can
breakdown chitosan to LMWC and has the breakdown
potential similar to nitrous acid. The results obtained
suggest additional advantages of HbZ, such as ease of
catalyst separation and reusability, which makes the
process green, sustainable and industrially feasible.
Materials and methods
Materials
Chitosan (degree of de-acetylation: 83%,
Mw = 1157.13 kDa, PDI of 2.12) was purchased from
Pelican Biotech and Chemical Labs Pvt. Ltd., Alap-
puzha, India. HbZ was provided as a gift sample by
Nanoxpert Technologies, Mumbai, India. Hydrother-
mal glass reactor was fabricated and procured from
Komal Scientific Co., Mumbai, India. The reactor was
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made up of borosilicate glass tube of dimension 15 cm
(length) 9 2.7 cm (outer diameter) 9 2.1 cm (inner
diameter). Glacial acetic acid and sodium nitrite were
purchased from S.D. Fine Chemicals, Mumbai, India.
5-HMF was a kind gift from AVA Biochem, Muttenz,
Switzerland. Deionized water from Milli-Q Plus
system (Millipore, Massachusetts, USA) was utilized
in all experiments. All the other chemicals used were
of analytical grade.
Pre-treatment and characterization of HbZ
Pellets of HbZ were micronized and the resulting
material was sifted through 40# sieve, as per ASTM
standards, to yield catalyst particles of uniform size.
Surface area of HbZ was determined by nitrogen (N2)
adsorption and desorption, at - 196 °C, using a BET
instrument (Porous Materials Inc., New York, USA).
150 mg of the sample was weighed and N2 adsorption
and desorption cycles were carried out to measure the
surface area. Mean pore diameter of HbZ was
calculated on the basis of Pierce adsorption and
desorption.
The acidity of HbZ was determined by temperature
programmed desorption of ammonia method, using
TPD RO 1100 (Thermo scientific, Massachusetts,
USA). Approximately 100 mg of the catalyst sample
was heated from 30 to 1000 °C, with linear ramping,
maintaining a 5 min hold-time, in a flowing helium
gas stream. The desorbed ammonia was analyzed to
calculate acidic sites present on the HbZ.
Morphology and elemental composition of HbZ
were determined by scanning electron microscopy
coupled with an energy-dispersive X-ray spectroscopy
(SEM-EDAX), on JEOL-JSM 6380 LA scanning
electron microscope (JEOL, Tokyo, Japan). 2 mg of
sample was placed on a carbon tape and was sputter
coated with platinum, for 30 s. Measurements were
conducted at an accelerating voltage of 20 kV. A
small region of the image obtained at 1000 9 mag-
nification was used to determine the mass percentage
of the elements present in the sample.
Pre-treatment of chitosan
Commercially purchased chitosan was pre-treated by
solubilizing in 1% acetic acid (100 mL), filtered
through muslin cloth and finally precipitated at pH
10.0, using 1 N NaOH (30 mL). The precipitate was
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Cellulose
centrifuged and washed with water till pH of the
washing was neutral and then dried in a vacuum oven
(ThermoFisher, Massachusetts, USA), at 50 °C, for
45 min. Molecular weight of the polymers was
analysed using Gel Permeation Chromatography
(Malvern Instruments Ltd., Malvern, UK), using a
Viscotek cationic (MBMMW) column. 5% acetic acid
was used as the mobile phase/eluent, at a flow rate of
0.7 mL/min, and the instrument was calibrated using
Pullulan (Mw = 122,267 Da, Mn = 114,678 Da) and
dextran (Mw = 73,227 Da, Mn = 56,115 Da), as nar-
row and broad molecular weight standards. Molecular
weights of both, the pre-treated chitosan and the
products obtained after hydrolysis, were determined
using this technique. Further, in order to understand
the structural integrity of chitosan and the respective
reaction products, FTIR analysis was carried out on an
IR Affinity-1 FTIR spectrophotometer (Shimadzu,
Kyoto, Japan). KBr discs of the sample were prepared
by compressing their homogenous mixture in a
hydraulic pellet press and the discs were scanned over
the range of 4000–400 cm-1.
Optimization of depolymerisation of chitosan
using DOE approach
The experimental design and data analysis for liquid
and solid acid catalysis (LA & SA) were conducted
using Design expertÒ software, version 10.0.6 M/s
(Stat-Ease, Inc, Minneapolis, USA). Chitosan to
catalyst ratio, as well as reaction processing parame-
ters were regarded as the crucial factors for production
of LMWC. Based on the number of factors and their
level, user defined response surface methodology was
employed, using multiple regression statistics. A full
quadratic model was fitted to the collected responses.
P values for each of the factors were used to determine
their significance during the production of LMWC.
The responses were set considering the quality of
target product and controlled breakdown of chitosan,
in order to minimize side-products like 5-HMF, which
can also be produced from chitosan (Lee and Jeong
2015).
The effect of independent variables [amount of
catalyst (A), reaction time (B) and reaction tempera-
ture (C)] on dependent variables [molecular weight
(Y1) and 5-HMF content (Y2)] was studied, as shown
in Table 1. The linear equation of the model is stated
in Eq. 1.
Cellulose

Table 1 Experimental design for solid acid catalysis

Batch code Catalyst (A) (mg) Time (B) (Min) Temperature (C) (°C) Molecular weight (Y1) (Da) 5-HMF (Y2) (lg/10 mL)

SA 1 25 30 100 635,129 ± 2379 0.19 ± 0.014

SA 2 62.5 30 125 241,865 ± 3215 0.81 ± 0.018
SA 3 62.5 60 100 256,981 ± 4592 0.77 ± 0.021
SA 4 25 90 100 370,562 ± 3356 0.56 ± 0.014
SA 5 100 30 100 267,895 ± 9328 0.75 ± 0.02
SA 6 100 30 125 123,058 ± 1064 3.18 ± 0.11
SA 7 62.5 60 150 167,259 ± 6722 2.1 ± 0.086
SA 8 25 90 125 304,527 ± 5681 0.69 ± 0.024
SA 9 25 30 150 354,682 ± 6354 0.62 ± 0.02
SA 10 100 90 125 99,975 ± 895 6.94 ± 0.12
SA 11 25 60 150 588,214 ± 1245 0.33 ± 0.016
SA 12 62.5 60 125 175,125 ± 3990 0.98 ± 0.022
SA 13 25 60 100 632,015 ± 1078 0.20 ± 0.01
SA 14 100 60 100 190,874 ± 6288 1.32 ± 0.024
SA 15 62.5 90 100 210,546 ± 7564 1.08 ± 0.033
SA 16 100 30 150 264,863 ± 4325 0.76 ± 0.042
SA 17 100 60 125 152,864 ± 4484 2.70 ± 0.089
SA 18 100 90 100 145,283 ± 2653 2.40 ± 0.091
SA 19 25 90 150 137,895 ± 1991 3.02 ± 0.10
SA 20 62.5 30 100 250,654 ± 1235 0.64 ± 0.05
SA 21 100 90 150 50,452 ± 869 9.86 ± 0.29
SA 22 25 60 125 384,562 ± 3941 0.5 ± 0.029
SA 23 62.5 90 125 125,897 ± 4275 3.22 ± 0.16
SA 24 100 60 150 95,654 ± 1604 7.23 ± 0.32
SA 25 62.5 30 150 184,569 ± 1533 1.05 ± 0.08
SA 26 62.5 90 150 89,356 ± 1926 6.92 ± 0.21
SA 27 25 30 125 531,589 ± 8749 0.47 ± 0.06

Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b4 X4 þ    Procedure for de-polymerization of chitosan

þ bnXn
ð1Þ
The Stat-Ease software designed twenty seven and
nine experimental runs for solid acid and liquid acid
respectively, as mentioned in Tables 1 and 2. Multi-
linear regression analysis and multiple ANOVA were
performed to evaluate the factor coefficients and
significance of the model. In order to validate the
model and to check its reproducibility, batches
predicted by software were performed and analyzed
in triplicates.
Stock solution of chitosan was prepared by dissolving
the pre-treated chitosan in 1% v/v acetic acid, to make
a 1% w/v solution. De-polymerization of chitosan was
performed by two different methods, solid acid (SA)
and liquid acid (LA) catalyses. To optimize the
reaction conditions DOE stratagem was used, as
described in ‘‘Optimization of depolymerisation of
chitosan using DOE approach’’ section.
For SA approach, 10 mL of the chitosan stock
solution was combined with different ratios of HbZ in
a hydrothermal glass reactor. HbZ was separated from
the reaction mixture by centrifugation and the super-
natant was precipitated using 0.1 N NaOH. Similarly,

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 Cellulose

Table 2 Experimental design for liquid acid catalysis

Batch code Catalyst (A) (mg) Time (B) (Min) Molecular weight (Y1) (Da) 5-HMF (Y2) (lg/10 mL)

LA 1 5 150 399,254 ± 8925 0.19 ± 0.012

LA 2 10 30 356,943 ± 7221 0.28 ± 0.019
LA 3 10 150 185,625 ± 1047 0.92 ± 0.05
LA 4 15 90 62,269 ± 940 1.83 ± 0.072
LA 5 5 90 402,155 ± 8259 0.13 ± 0.010
LA 6 15 150 34,751 ± 2106 2.97 ± 0.11
LA 7 10 90 219,382 ± 3792 0.76 ± 0.059
LA 8 5 30 439,821 ± 5617 0.11 ± 0.01
LA 9 15 30 154,789 ± 2396 1.21 ± 0.047

in the LA approach, 50 mL of chitosan solution was
combined with different ratios of nitrous acid, in a
glass beaker, and the resultant reaction mixtures were
neutralized and precipitated using 0.1 N NaOH. The
precipitates of LMWC obtained from both, SA and LA
were then washed with water and solubilized in 0.1 N
HCl. The precipitates were further washed with
acetone, till the pH of the washings reached neutrality.
These neutralized solutions were subjected to spray
drying to obtain powdered form of LMWC.
Spray drying of LMWC
The neutralized solutions obtained from LMWC-SA
and LMWC-LA were spray dried using a two-fluid
nozzle spray dryer (Hemraj India Pvt Ltd, Mumbai,
India), fabricated and customized as per the product
requirement. Spray drying of LMWC was carried out
at an inlet temperature of 190–200 °C and outlet
temperature of 80–90 °C. The atomization, aspiration
and feed rate were set at 1.5 kg/cm2, 2100 rpm,
20 mL/min respectively. The spray dried LMWC was
collected and percent yield was determined. It was
immediately stored in vacuum desiccator to prevent
moisture absorption.
Characterization of spray dried LMWC
Moisture content of powdered LMWC produced by
SA and LA was studied using moisture analyzer
(Citizen Scale Pvt. Ltd, Mumbai, India). Particle size
and morphology of the spray dried particles was
studied using SEM, as described in ‘‘Pre-treatment and
characterization of HbZ’’ section. GPC analysis was
conducted under similar conditions as the parent
chitosan (described in ‘‘Pre-treatment of chitosan’’
section), to understand the molecular weight of the
products.
Solubility analysis of LMWC
Water solubility of the product was assessed using
Zetasizer Nano ZSP (Malvern Instruments Ltd.,
Malvern, UK), based on a modified method (Krishnan
et al. 2016). Intensity of scattered light generated due
to the particles in Brownian motion, which is
measured in terms of count rate (kcps) was considered
for assessment of solubility. The count rate of parent
chitosan and both the LMWC products were measured
after standing the solutions for 30 min and filtering
them, to ascertain the presence of dissolved matter in
solution.
Quantitative determination of 5-HMF
Side products/formation of other derivatives is a
common setback associated with any reaction. 5-
HMF is an important side-product, which may be
formed during de-polymerization of polysaccharides
at high temperatures (Moura et al. 2015). The quan-
tification method for 5-HMF formed during reaction
was developed using UV–Visible spectrophotometer
DeNovix DS-11 (DeNovix Inc., Delaware, USA).
Standard calibration curve was plotted by dissolving
5-HMF standard in DI water and measuring its
absorbance at 283 nm. The unknown concentration

123
Cellulose
of 5-HMF produced in all batches of SA and LA
(Tables 1, 2) was determined by using the standard
plot.
Differential scanning calorimetry (DSC)
DSC analysis was performed, using Pyris-6 DSC
(Perkin Elmer, Massachusetts, USA), to ascertain the
de-polymerization of chitosan. Approximately 10 mg
of each sample were weighed and sealed hermetically
in aluminum pan. Samples were heated from 35 to
300 °C, at the rate of 5 °C/min, under an inert
environment of nitrogen gas, at a flow rate of
20 mL/min. An empty and sealed aluminum pan was
used as the reference. The exothermic and endother-
mic peaks observed in the thermograms of LMWC-SA
& LMWC-LA were compared with the peaks of the
parent chitosan.
Degree of de-acetylation (DD) of chitosan
LMWC produced using SA and LA and the parent
chitosan were analysed by FTIR to compare their
functional groups, as discussed in ‘‘Pre-treatment of
chitosan’’ section. The DD was calculated using the
method developed by Sabnis & Block, based on
infrared absorption spectroscopy. The absorbance
ratio of A1655/A3450 was utilized in determining the
DD using the following formula stated in Eq. 2
(Sabnis and Block 1997):
DD ¼ 97:67  ½24:486  ðA1655 =A3450 Þ: ð2Þ
Catalyst reusability and recyclability
After the de-polymerization reaction, in the reactions
catalysed by SA, HbZ was recovered by centrifugation
of the reaction mixture in a REMI CPR 24 Plus
centrifuge (REMI Instruments, Mumbai, India), at
12,000 rpm for 15 min. The catalyst was washed with
1% acetic acid solution, followed by washing thrice
with water. The separated HbZ was dried in hot air
oven, at 60 °C, for 3 h. The dried catalyst was
characterized for its crystallinity using a Bruker
X-ray diffractometer (Bruker, Massachusetts, USA).
Sufficient amount of the powdered samples were
uniformly spread on the sample chamber and analysed
between a 2-theta of 5°–80°, to understand changes in
crystallinity, if any. In case of LA, since it was a
homogenous catalyst, the acid was neutralised and
hence could not be recovered for reuse.
Results and discussion
Pre-treatment and characterization of HbZ
The micronization process led to decrease in particle
size of HbZ up to 100 lm, as depicted in SEM
photomicrograph (Supporting Information Fig S1).
This reduction in particle size exposes more number of
proton donating sites that can participate in the
catalytic reaction. Surface area of HbZ was found to
be 437.4 m2/g, as determined by BET instrument
(Supporting Information Fig S2). Mean pore diame-
ters on the basis of Pierce adsorption and desorption
were found to be 61.2 Å and 80.1 Å, respectively.
Presence of acidic sites was confirmed by ammonia
TPD method and it was found to be 7.6 mmol/g. SEM
analysis of solid acid showed micron-sized, uneven-
shaped particles, supporting the high surface area
obtained by BET analysis. Elemental distribution of
HbZ suggested a SiO2/Al2O3 ratio of 3 (Supporting
Information Fig S3), signifying a good number of acid
sites on the catalyst (Ward 1970).
Pre-treatment of chitosan
Raw chitosan was purified before the reaction. Filtra-
tion process aided in the removal of any insoluble
material, while precipitation using NaOH eliminated
the dissolved impurities retained during extraction
process of chitosan. Molecular weight of chitosan, as
determined by GPC, was found to be 1157.13 kDa,
with a PDI of 2.12.
Optimization of chitosan de-polymerization using
DOE approach
Design of experiments (response surface
methodology)
The experimental runs designed by Stat-Ease software
were conducted for methods involving both, SA and
LA, in order to understand the influence of reaction
processing parameters on the extent of de-polymer-
ization of chitosan (Y1) and formation of 5-HMF
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 Cellulose

residues (Y2). Maximum breakdown of chitosan, with
probable formation of less than 1 lg of 5-HMF as the
by-product, was the key objective behind implemen-
tation of RSM. Three levels were selected for each
independent variable in order to perceive the interac-
tion of variables to yield responses (Nobandegani et al.
2016). Response surface methodology was employed
successfully for optimization of de-polymerization
reaction using SA and LA.
Fitting of model and statistical analysis
Results obtained from experimental runs were pro-
cessed to confirm their statistical significance. In case
of a model fitting both responses (molecular weight
and 5-HMF content) should fit in a linear pattern.
However, linear fitting does not consider the curvature
of responses as well as the interaction between factors.
In order to fit the responses accurately, quadratic
model was utilized to study the impact of each factor
alone and in combination (Paulo and Santos 2017). It
was found that proposed quadratic model was statis-
tically significant. f values (Degrees of freedom) and
P values (Probability) of less than 0.05 indicate the
significance of the response surface quadratic model,
as depicted in ANOVA (stated in Table 3). Further-
more, correlation coefficient for the regression equa-
tion (R2) was found to be 0.87 and 0.98, for SA and
LA, respectively. This implied that the experimental
values showed minor deviation from the values
predicted by the Design expert software (Chopra
et al. 2007). Adeq precision is the measure of signal to
noise ratio and it was found to be 12.07 for SA and
16.61 for LA, respectively. Usually, Adeq precision of
more than 4 is desirable to denote an ideal model (Liu
et al. 2017). Data generated by implementing multiple
ANOVA showed good model fitting and was used for
construction of the design space.
Effect of variables on responses was stated using
polynomial equations, wherein the impact of individ-
ual factor and the interaction between factors are
studied using ANOVA. The final mathematical model,
in terms of coded factors, for both the reactions is
stated in Eqs. 3 and 4 for SA and Eqs. 5 and 6 for LA,
respectively.
For solid acid catalysis
Mol wt ðY1Þ ¼ þ 1:997E þ 005  1:416E þ 005
 A  73;322:83  B  57;055:28  C
þ 29;025:83  AB þ 30;319:33  AC
 8260:33  BC þ 1:070E þ 005  A2
 49;905:50  B2 þ 34;108:83 C2
ð3Þ
% 5-HMF ðY2Þ ¼ þ1:52 þ 1:59  A
þ 1:46  B þ 1:33  C þ 0 :96  AB þ 0:86
 AC þ 1:24  BC þ 0:37  A2
þ 0:61  B2 þ 0:046  C2 :
ð4Þ
For liquid acid catalysis
Mol wt ðY1Þ ¼ þ 2:314E þ 005  1:6499E
þ 005  A  55;320  B  19;867  AB
 5144  A2 þ 33;928  B2
ð5Þ
5-HMF ðY2Þ ¼ 0:63 þ 0:93  A þ 0:41  B
þ 0:42  AB þ 0:42  A2
þ 0:040  B2 :
ð6Þ
The polynomial equation represents coefficient of
intercept, first order main effect, interaction terms and
higher order effects. The positive sign in polynomial
equation specifies the synergistic effect in favour of a
particular response, whereas negative sign indicates
the inverse effect on that response (Motwani et al.
2008). Mathematical data generated using multiple
linear regression analysis and polynomial equation
was employed to produce 3D response surface graphs

Table 3 ANOVA for Model Molecular weight reduction (Y1) % 5-HMF formation (Y2)
response surface quadratic
model Mean square f value P value Mean square f value P value

Solid acid (SA) 6.985E?010 11.67 \ 0.0001 17.44 19.76 \ 0.0001

Liquid acid (LA) 3.709E?010 35.21 0.0073 1.46 51.16 0.0042

123
Cellulose

123

b Fig. 1 Response surface 3D plots for SA showing the effect of
amount of catalyst, time and temperature on molecular weight
(a–c) and formation of 5-HMF (d–f)
(Figs. 1, 2) and their Contour plots (Supporting
Information Fig. S4 and Fig. S5).
3D response surface graphs are plotted by varying
two factors while keeping one factor constant. It can
be seen from these graphs that increasing the amount
of catalyst enhances the breakdown of chitosan,
concurrently promoting the formation of 5-HMF. It
is known that the strength of HbZ depends on the
acidic sites present on it. Higher amount of catalyst
provides more catalytic sites for cleavage of the
glycosidic bonds present in chitosan (Zhou et al.
2016). According to the polynomial Eq. 3, the catalyst
concentration, time and temperature individually
showed negligible effect on de-polymerization. How-
ever, it was observed that temperature, in synergy with
HbZ, showed a positive effect on de-polymerization.
Temperature higher than 130 °C, in the presence of
HbZ, for longer time, potentiates 5-HMF formation.
This has also been observed during catalytic conver-
sion of fructose to 5-HMF (Wang et al. 2011). HbZ
showed temperature-based catalytic performance due
to the presence of Bronsted acidic sites, which donated
protons at elevated temperature, making the environ-
ment more acidic and hence facilitating the reaction
Fig. 2 Response surface 3D plots for LA showing the effect of amount of catalyst and time on molecular weight (a) and formation of
5-HMF (b)
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Cellulose
rates (Xu et al. 2006). Interaction between time and
temperature showed antagonistic effect on LMWC
production. Thus, it can be affirmed that the presence
of catalyst is mandatory for the de-polymerization
reaction, in association with the appropriate time and
temperature. Equation generated for response (Y2)
showed that all three factors, individually and in
combination, favour 5-HMF formation, beyond a
particular level. Considering the contribution of all
factors and their limitations, the S12 batch (62.5 mg of
HbZ, at 125 °C, for 60 min) was found to be the
optimum for catalytic breakdown of chitosan up to 6
folds, with 0.98 lg of side-product being formed
(0.98 lg amounts to less than 1% of the total reaction
mixture).
Resemblance was noted for the models developed
by LA and SA with respect to the patterns of de-
polymerization. Amount of sodium nitrite and time did
not exhibit any significant impact on responses, when
considered individually. On the contrary, both factors,
in unison, showed excellent reduction in molecular
weight. Mao et al. demonstrated that the first hour of
the reaction is crucial for de-polymerization. More-
over, beyond this time point, the amount of un-reacted
nitrous acid was found to reduce subsequently,
thereby, slowing down the rate of the reaction (Mao
et al. 2004). Equation 6 demonstrates that the forma-
tion of 5-HMF increased when the concentration of
Cellulose
nitrous acid and reaction time were increased. More-
over, the post-reaction stability of LMWC was found
to be less when the polymer was not neutralized. This
may happen due to rapid formation of 5-HMF in the
reaction mixture (Rosatella et al. 2011).
Validation of response surface model
Validation of the response surface model was carried
out by comparing the experimental results with the
predicted responses designed by Stat-Ease software.
The target allotted was maximum reduction in
molecular weight (less than 200 kDa), while control-
ling the excess 5-HMF production (less than 1 lg)
throughout the reaction. Based on this, the software
predicted values, also known as ‘‘points’’, for the
independent variables. Points were selected to assure
experimental reproducibility by considering desirabil-
ity factor close to one. 5% deviations from the
predicted values were permitted during validation.
Point confirmation (indicated in Table 4) showed that
the experimental values were in compliance with
predicted values, for both the responses. Apart from
response confirmation, multiple ANOVA and poly-
nomial regression equation strengthened the reliability
of the quadratic response surface model.
Molecular weight analysis of LMWC
Reduction in molecular weight is a key indicator to
ensure the conversion of chitosan to LMWC. Low
molecular weight derivatives produced after the
reaction, by using SA as well as LA, were character-
ized for their molecular weight by GPC (depicted in
Fig. 3). Decrease in molecular weight was observed to
some extent in all the batches. For SA, batches were
Table 4 Point prediction and confirmation table for validation
of model
Parameters Solid acid Liquid acid
Amount of catalyst (mg) 62.5 11.53
Time (min) 90 95
Temperature (°C) 100 –
Predicted Molwt (kDa) 175,000 175,039
Observed Molwt (kDa) 173,058 ± 4754 173,024 ± 5766
Predicted 5-HMF (%) 1.067 1.109
Observed 5-HMF (%) 0.97 ± 0.015 1.06 ± 0.06
designed by DOE software with different combina-
tions of factors such as amount of catalyst
(25–100 mg), time (30–90 min) and temperature
(100–150 °C), as stated in Table 1. Maximum con-
version of chitosan to LMWC, with minimal forma-
tion of 5-HMF, was observed using 62.5 mg of
catalyst, reaction time of 60 min and reaction temper-
ature of 125 °C. This batch (SA 12) yielded LMWC
with a molecular weight of 175,125 Da and resulted in
0.98 lg of by-product (5-HMF) formation. Nitrous
acid based catalysis (LA) of chitosan was optimized
by varying the time (30–90 min) and amount of
sodium nitrite (5–10 mg). Since liquid acid based
catalytic reactions are not governed by any particular
temperature, it was not included as an independent
factor while designing the batches. Among all the
batches, LA 3 was found to be the most effective for
LMWC production. Treatment with 10 mg of sodium
nitrite, for 150 min of reaction time, resulted in de-
polymerization of chitosan to yield LMWC, with a
molecular weight of 185,625 Da and resulted in
0.92 lg of 5-HMF.
Characterization of LMWC
Characterization of spray dried LMWC powder
The practical yield of spray dried LMWC (obtained
using both, SA and LA) was found to be 85 ± 3%.
Some amount of LMWC was thought to be lost due to
the air flow generated by the blower of instrument,
Fig. 3 GPC chromatograph of a parent chitosan, b LMWC-SA
and c LMWC-LA
123

during the drying procedure, as well as deposition of
the fine powder on the walls of the spray dryer. In
addition to this, some amount of LMWC was predicted
to be lost during the processing steps such as
precipitation and washing. This may also be consid-
ered as a probable reason for the overall loss in the
final product. LMWC was found to be heteroge-
neously distributed, with a red blood cell-like mor-
phology and with diameter ranging between 1 and
10 lm (depicted in Fig. 4). Similar morphology has
been reported for chitosan hydrochloride salt, wherein
the spherical particles had smooth external surface,
with concave cores, after spray drying (He et al. 1999).
Percent moisture content for parent chitosan, LMWC-
SA and LMWC-LA was found to be 10.1 ± 0.7,
9.8 ± 0.4 and 9.5 ± 0.5, respectively. Moisture anal-
ysis of LMWC revealed that spray drying is capable of
removing adequate moisture from the product, while
maintaining the moisture content similar to that of the
parent chitosan. It was also found that the LMWC-SA
and LMWC-LA demonstrated excellent stability even
after 180 days (Supporting Information Fig S7, S8 &
Table S1). Such LMWC, in micro-particulate form, is
of immense importance in pharmaceuticals for encap-
sulation and controlled release of drugs. In biophar-
maceuticals, it finds application in enzyme
immobilization, cells encapsulation and protection of
proteins and amino acids (Kas 1997).
Solubility of LMWC
Aqueous solubility is an important factor for applica-
tion of LMWC in key areas of pharmaceuticals. For
Fig. 4 SEM photomicrograph of LMWC (Scale bar: 10 lm)
123
Cellulose
instance, DNA, peptide or proteins are unstable at
acidic pH and hence aqueous solubility becomes a
primary requisite (Kim 2010) Zetasizer is an instru-
ment typically used to measure the size of particles in
solution based on the principle of batch-mode light
scattering. However, in this study the solubility was
determined based on the ‘Count rate or kcps value’,
and not the particle size. Count rate or kcps value is
indicative of the matter dispersed or solubilized in a
particular solvent. For example, deionized (DI) water
shows a count rate of 17.86 ± 3.2 kcps. With increase
in concentration of parent chitosan in DI water, the
count rate was observed to increase up to
60.5 ± 5.96 kcps, for 5 mg of the polymer (Fig. 5).
This increase in count rate was attributed to the low
density of the parent chitosan, responsible for its
buoyancy on water surface. This was confirmed when
the buoyant chitosan was filtered and the filtrate was
re-analysed for the presence of soluble matter. As
anticipated, the count-rate was found to be the same as
that of the DI water, thus confirming that the parent
chitosan was insoluble. On the other hand, when
LMWC, produced using solid and liquid acid, were
added to the DI water, in steps of 3 mg/mL, the
LMWC solubilised completely in water till
* 18–20 mg/mL. Both the LMWC, thus, gave a
stable count rate of 179.4 ± 17.33 and
178.0 ± 16.65, respectively, post 30 min before and
after filtration, thus confirming that the count rate
obtained was only due to the presence of soluble
matter. Results from solubility determination have
been depicted in Fig. 5. The pH of the solution for SA
and LA was found to be 6.2 ± 0.2 and 5.9 ± 0.18,
Fig. 5 Count rate of deionized water, parent chitosan without
filtration, LMWC-LA and LMWC-SA
Cellulose
respectively. Moreover, spray drying generated amor-
phous microstructures, by reducing its particle size
and concurrent increase in the surface area of the
polymers. Thus, particle drying technique contributed
to solubility enhancement of the final product (Paudel
et al. 2013).
Quantitative determination of 5-HMF
UV spectroscopy was used for the quantification of
5-HMF based on its simplicity and reproducibility
(Supporting Information Fig. S6) in supplementary
information shows UV spectra of parent chitosan,
5-HMF and LMWC. Parent chitosan showed a broad
peak at 218 nm due to the presence of far-UV
chromophoric groups, N-acetylglucosamine and glu-
cosamine. Since decrease in molecular weight of
chitosan did not affect the UV absorbance of these two
groups, LMWC retained this peak and the overall
absorbance (Kumirska et al. 2010). Shoulder peak at
283 nm was observed in both LMWC-SA and
LMWC-LA, which denoted the formation of
5-HMF, the side-product in the reaction. Ascertain-
ment of its presence was further confirmed by
comparison of the peak with that of the peak of
5-HMF standard. Amounts of 5-HMF, formed during
individual reactions, was quantified using UV spec-
troscopic technique and have been listed in Table 1.
Differential scanning calorimetry (DSC)
DSC thermograms, depicted in Fig. 6, showed
endothermic peaks at 75 °C due to the evaporation
of water present in the samples. The exothermic peaks,
between 220 and 300 °C, represented degradation of
chitosan due to the decomposition of glucosamine
units (Guinesi and Cavalheiro 2006; Kittur et al.
2002). Chitosan with higher molecular weight of
1157 kDa showed degradation at 301 °C, whereas
LMWC obtained by SA and LA, having molecular
weight of 175 kDa, degraded at 275 and 266 °C,
respectively. Thus, molecular weight dependant
degradation was observed in chitosan and this is in
accordance with the data obtained by GPC.
Degree of deacetylation (DD) of chitosan
FTIR analysis was conducted to study the conforma-
tional changes in the structure of LMWC after the
Fig. 6 DSC thermogram of a parent chitosan, b LMWC-SA,
c LMWC-LA
depolymerization reaction. Comparative FTIR spectra
depicted in Fig. 7 showed the presence of peaks at
3400–3450 cm-1 (OH stretching combined with the
N–H stretching), 2920–2935 cm-1 (CH2 stretching),
1640–1650 cm-1 (C=O stretching of amide I group),
1545–1590 cm-1 (deformation of amide II),
1410–1425 cm-1 (deformation of CH2 group),
1070–1080 cm-1 (O bridge stretching of the glu-
cosamine residue), * 650 cm-1 (CH2 group defor-
mation for glycosidic bond; Kumirska et al. 2010;
Vasilieva et al. 2017). The spectra of LMWC-SA and
LMWC-LA samples exhibited no significant differ-
ence when compared with the spectrum of the parent
chitosan. Resemblance in spectra indicated structural
integrity of chitosan after the catalysis reaction. Slight
broadening at 3400–3450 cm-1 was observed in case
of LMWC, which may be attributed to rearrangement
of hydrogen bonding during the depolymerization
process (Focher et al. 1992). The amide I band
signified the amount of CO–NH residual groups and
was utilized for determination of acetyl content in the
products. The decrease in intensity of the amide band
suggested that there was a reduction in the acetyl
content of LMWC (Domard and Rinaudo 1983;
Kumar et al. 2007). This was verified by estimating
% DD of chitosan, by using the formula developed by
Sabnis and Block (Sabnis and Block 1997). % DD of
LMWC-SA was found to be slightly higher (85%) than
parent chitosan (83%), whereas LMWC-LA showed
83% DD, confirming no effect of depolymerization on
DD.
123
 Cellulose

Catalyst reusability and recyclability

Catalyst reusability and recyclability are the vital

parameters, which signify the sustainability of devel-
oped method. For LA, the reactions were terminated
by neutralizing nitrous acid using ammonium hydrox-
ide solution. Heterogeneous catalysis exhibited a
crucial advantage over the nitrous acid method,
wherein HbZ could be reused 2–3 times, without
compromising the quality of the product as shown in
Fig. 8. Also, the catalyst was found to be easily
separable after the reaction. In case of HbZ, two cycles Fig. 8 Molecular weight of LMWC-SA showing catalyst
of reaction showed similar molecular weight decrease, reusability
up to 170,000 Da, with a consistent PDI of around 1.2.
However, the catalytic performance of HbZ was found The XRD diffractogram showed the appearance of
to decrease during the third reaction. Acidity results sharp peaks at 7.7° and 22.5°, which are the charac-
obtained by ammonia TPD method implied that the teristic peaks for crystalline b zeolites (Liu et al.
acidity of HbZ reduced from 7.6 to 5.01 mmol/g, 1991). Comparative results from the analysis of HbZ
justifying the decreased catalytic efficiency. Catalyst and recovered HbZ are shown in Fig. 9. Recovered
coking could be the reason for lower catalytic activity. HbZ exhibited a decrease in peak intensity, indicating
Coke molecules generated from chitosan might have a decrease in crystallinity. Crystal size of catalyst was
been retained in the pores as well as on outer surfaces estimated by Scherrer’s equation and was found to be
of zeolite, resulting in less number of active sites 105 Å, whereas after the reaction, the crystal size
available for cleavage of chitosan (Guisnet and reduced up to 87 Å. It was thus inferred that
Magnoux 1997). hydrothermal exposure reduced the particle size as

Fig. 7 FTIR spectra of

a parent chitosan, b LMWC-
SA and c LMWC-LA

123
Cellulose
Fig. 9 X-ray diffractogram of a HbZ and b recovered HbZ
well as the relative crystallinity of HbZ. These
changes may have occurred due to the splitting of
Al–O–Si and Si–O-Si bonds, under the influence of
protons and hydroxyl groups (Dimitrijevic et al. 2006).
The same has been reiterated by other scientists where
they have reported that excess and continuous
hydrothermal treatment destroys the framework of
zeolites and turns them to amorphous alumina silica
(Camblor et al. 1998; Ding et al. 2007).
Conclusion
Our study reports the development of an alternative,
sustainable method for the production of LMWC using
solid acid (HbZ) and compares with a well known
liquid acid (nitrous acid). The results revealed that the
solid acid HbZ exhibited catalytic potential equivalent
to conventional liquid acid, nitrous acid. More than
sixfold reduction in molecular weight was obtained
from both the methods. Approximately 85% of
LMWC was obtained from both the methods after
spray drying, and both products demonstrated excel-
lent water solubility (18–20 mg/mL). Spray drying
technique was used owing to its stability, scale-up
ability and to impart quality to the final product
obtained from the processes utilizing both HbZ and
nitrous acid. Implementation of DOE strategy allowed
systematic planning of experiments, by reducing time
and resources involved, and reducing the side-product
(5-HMF) formation to less than 1% in both SA and
LA. RSM showed the influence of each factor on the
responses and the influence of their interaction, on the
quality of the final product; thus proving to be a
valuable tool for optimization of polymer hydrolytic
reactions.
Acknowledgments The authors are thankful to University
Grants Commission for the UGC-SAP fellowship [F.25-1/2014-
15 (BSR)/No. F.8-10/2007(BSR)] and [F.4-1/2006 (BSR)/8-10/
2007(BSR)] for financial support. The authors would like to
acknowledge DST-Nanomission (Sanction No. SR/NM/NS-
1145/2012) for the zetasizer instrument and DST-FIST
(Sanction No. SR/FST/ETII-058/2013 (C)) for Gel Permeation
Chromatography. The authors are also very thankful to Nano-
xpert technologies for technical support and providing gift
sample of solid acid. Special thanks to Tejal Pant and Manish
Gore for assistance in drafting the manuscript.
References
Allan GG, Peyron M (1995) Molecular weight manipulation of
chitosan II: prediction and control of extent of depoly-
merization by nitrous acid. Carbohydr Res 277:273–282
Bezerra MA, Santelli RE, Oliveira EP, Villar LS, Escaleira LA
(2008) Response surface methodology (RSM) as a tool for
optimization in analytical chemistry. Talanta 76:965–977
Camblor M, Corma A, Valencia S (1998) Characterization of
nanocrystalline zeolite Beta. Microporous Mesoporous
Mater 25:59–74
Chopra S, Patil GV, Motwani SK (2007) Release modulating
hydrophilic matrix systems of losartan potassium: opti-
mization of formulation using statistical experimental
design. Eur J Pharm Biopharm 66:73–82
Dimitrijevic R, Lutz W, Ritzmann A (2006) Hydrothermal
stability of zeolites: determination of extra-framework
species of HY faujasite-type steamed zeolite. J Phys Chem
Solids 67:1741–1748
Ding L, Zheng Y, Hong Y, Ring Z (2007) Effect of particle size
on the hydrothermal stability of zeolite beta. Microporous
Mesoporous Mater 101:432–439
Domard A, Rinaudo M (1983) Preparation and characterization
of fully deacetylated chitosan. Int J Biol Macromol
5:49–52
Focher B, Naggi A, Torri G, Cosani A, Terbojevich M (1992)
Chitosans from Euphausia superba. 2: characterization of
solid state structure. Carbohydr Polym 18:43–49
Frey DD, Engelhardt F, Greitzer EM (2003) A role for ‘‘one-
factor-at-a-time’’ experimentation in parameter design.
Res Eng Des 14:65–74
Guinesi LS, Cavalheiro ETG (2006) The use of DSC curves to
determine the acetylation degree of chitin/chitosan sam-
ples. Thermochim Acta 444:128–133
Guisnet M, Magnoux P (1997) Deactivation by coking of zeolite
catalysts. Prevention of deactivation. Optimal conditions
for regeneration. Catal Today 36:477–483
He P, Davis SS, Illum L (1999) Chitosan microspheres prepared
by spray drying. Int J Pharm 187:53–65
Huang Y-B, Fu Y (2013) Hydrolysis of cellulose to glucose by
solid acid catalysts. Green Chem 15:1095–1111
Kas HS (1997) Chitosan: properties, preparations and applica-
tion to microparticulate systems. J Microencapsul
14:689–711
Kim S-K (2010) Chitin, chitosan, oligosaccharides and their
derivatives: biological activities and applications. CRC
Press, Boca Raton
123

Kim S-K, Rajapakse N (2005) Enzymatic production and bio-
logical activities of chitosan oligosaccharides (COS): a
review. Carbohydr Polym 62:357–368
Kittur F, Prashanth KH, Sankar KU, Tharanathan R (2002)
Characterization of chitin, chitosan and their car-
boxymethyl derivatives by differential scanning
calorimetry. Carbohydr Polym 49:185–193
Krishnan RA et al (2016) Proton play in the formation of low
molecular weight chitosan (LWCS) by hydrolyzing chi-
tosan with a carbon based solid acid. Carbohydr Polym
151:417–425
Kumar MNR (2000) A review of chitin and chitosan applica-
tions. React Funct Polym 46:1–27
Kumar MR, Muzzarelli RA, Muzzarelli C, Sashiwa H, Domb A
(2004) Chitosan chemistry and pharmaceutical perspec-
tives. Chem Rev 104:6017–6084
Kumar ABV, Varadaraj MC, Gowda LR, Tharanathan RN
(2007) Low molecular weight chitosans—preparation with
the aid of pronase, characterization and their bactericidal
activity towards Bacillus cereus and Escherichia coli.
Biochim Biophys Acta (BBA) Gen Subj 1770:495–505
Kumirska J, Czerwicka M, Kaczyński Z, Bychowska A, Brzo-
zowski K, Thöming J, Stepnowski P (2010) Application of
spectroscopic methods for structural analysis of chitin and
chitosan. Mar Drugs 8:1567–1636
Lee S-B, Jeong G-T (2015) Catalytic conversion of chitosan to
5-hydroxymethylfurfural under low temperature
hydrothermal process. Appl Biochem Biotechnol
176:1151–1161
Li J, Du Y, Yang J, Feng T, Li A, Chen P (2005) Preparation and
characterisation of low molecular weight chitosan and
chito-oligomers by a commercial enzyme. Polym Degrad
Stab 87:441–448
Liu S-b, Wu J-F, Ma L-J, Tsai T-C, Wang I (1991) On the
thermal stability of zeolite beta. J Catal 132:432–439
Liu L, Wang X, Zou H, Yu M, Xie W (2017) Optimizing syn-
thesis parameters of short carbon fiber reinforced poly-
sulfonamide composites by using response surface
methodology. Polym Test 59:355–361
Mao S, Shuai X, Unger F, Simon M, Bi D, Kissel T (2004) The
depolymerization of chitosan: effects on physicochemical
and biological properties. Int J Pharm 281:45–54
Motwani SK, Chopra S, Talegaonkar S, Kohli K, Ahmad FJ,
Khar RK (2008) Chitosan–sodium alginate nanoparticles
as submicroscopic reservoirs for ocular delivery: formu-
lation, optimisation and in vitro characterisation. Eur J
Pharm Biopharm 68:513–525
Moura FA, Macagnan FT, Silva LP (2015) Oligosaccharide
production by hydrolysis of polysaccharides: a review. Int J
Food Sci Technol 50:275–281
Nikolla E, Román-Leshkov Y, Moliner M, Davis ME (2011)
‘‘One-pot’’ synthesis of 5-(hydroxymethyl) furfural from
carbohydrates using tin-beta zeolite. Acs Catal 1:408–410
Nobandegani MS, Birjandi MRS, Darbandi T, Khalilipour MM,
Shahraki F, Mohebbi-Kalhori D (2016) An industrial
Steam Methane Reformer optimization using response
surface methodology. J Nat Gas Sci Eng 36:540–549
Onda A, Ochi T, Yanagisawa K (2008) Selective hydrolysis of
cellulose into glucose over solid acid catalysts. Green
Chem 10:1033–1037
123
Cellulose
Paudel A, Worku ZA, Meeus J, Guns S, Van den Mooter G
(2013) Manufacturing of solid dispersions of poorly water
soluble drugs by spray drying: formulation and process
considerations. Int J Pharm 453:253–284
Paulo F, Santos L (2017) Design of experiments for microen-
capsulation applications: a review. Mater Sci Eng C
77:1327–1340
Prashanth KH, Tharanathan R (2005) Depolymerized products
of chitosan as potent inhibitors of tumor-induced angio-
genesis. Biochim Biophys Acta (BBA) Gen Subj
1722:22–29
Rabea EI, Badawy ME-T, Stevens CV, Smagghe G, Steurbaut
W (2003) Chitosan as antimicrobial agent: applications and
mode of action. Biomacromolecules 4:1457–1465
Rinaldi R, Palkovits R, Schüth F (2008) Depolymerization of
cellulose using solid catalysts in ionic liquids. Angew
Chem Int Ed 47:8047–8050
Rosatella AA, Simeonov SP, Frade RF, Afonso CA (2011)
5-Hydroxymethylfurfural (HMF) as a building block
platform: biological properties, synthesis and synthetic
applications. Green Chem 13:754–793
Sabnis S, Block LH (1997) Improved infrared spectroscopic
method for the analysis of degree of N-deacetylation of
chitosan. Polym Bull 39:67–71
Tsai G-J, Tsai M-T, Lee J-M, Zhong M-Z (2006) Effects of
chitosan and a low-molecular-weight chitosan on Bacillus
cereus and application in the preservation of cooked rice.
J Food Prot 69:2168–2175
van Strien RT, Gent JF, Belanger K, Triche E, Bracken MB,
Leaderer BP (2004) Exposure to NO2 and nitrous acid and
respiratory symptoms in the first year of life. Epidemiology
15:471–478
Vasilieva T et al (2017) Formation of low molecular weight
oligomers from chitin and chitosan stimulated by plasma-
assisted processes. Carbohydr Polym 163:54–61
Wang J, Xu W, Ren J, Liu X, Lu G, Wang Y (2011) Efficient
catalytic conversion of fructose into hydroxymethylfur-
fural by a novel carbon-based solid acid. Green Chem
13:2678–2681
Ward JW (1970) The nature of active sites on zeolites. J Catal
17:355–358. https://doi.org/10.1016/0021-
9517(70)90111-9
Weitkamp J (2000) Zeolites and catalysis. Solid State Ion
131:175–188
Xu B, Sievers C, Hong SB, Prins R, van Bokhoven JA (2006)
Catalytic activity of Brønsted acid sites in zeolites:
intrinsic activity, rate-limiting step, and influence of the
local structure of the acid sites. J Catal 244:163–168
Zheng B, Wen Z-S, Huang Y-J, Xia M-S, Xiang X-W, Qu Y-L
(2016) Molecular weight-dependent immunostimulative
activity of low molecular weight chitosan via regulating
NF-jB and AP-1 signaling pathways in RAW264. 7
macrophages. Mar Drugs 14:169
Zhou L, Liu Z, Bai Y, Lu T, Yang X, Xu J (2016) Hydrolysis of
cellobiose catalyzed by zeolites—the role of acidity and
micropore structure. J Energy Chem 25:141–145