The states of water in tryptophan grafted hydroxypropyl methylcellulose hydrogels and their effect on the adsorption of methylene blue and rhodamine B

Carbohydrate Polymers 248 (2020) 116765

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

The states of water in tryptophan grafted hydroxypropyl methylcellulose
hydrogels and their effect on the adsorption of methylene blue and
rhodamine B

T

Paulo V.O. Toledoa, Oigres D. Bernardinellib, Edvaldo Sabadinib, Denise F.S. Petria,*
a
b

Fundamental Chemistry Department, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, Brazil
Department of Physicochemistry, Institute of Chemistry, University of Campinas (UNICAMP), 13083-970, Campinas, São Paulo, Brazil

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in the article:
Hydroxypropyl methylcellulose
Citric acid
Tryptophan
Methylene blue
Rhodamine B

Tryptophan (Trp) decorated hydroxypropyl methylcellulose (HPMC) cryogels were prepared by a one-step reaction with citric acid. The increase of Trp content in the 3D network from 0 to 2.18 wt% increased the apparent
density from 0.0267 g.cm−3 to 0.0381 g.cm−3 and the compression modulus from 94 kPa to 201 kPa, due to
hydrophobic interactions between Trp molecules. The increase of Trp content in HPMC-Trp hydrogels increased
the amount of non-freezing water, estimated from differential scanning calorimetry, and the amount of freezing
water, which was determined by time-domain nuclear magnetic resonance. The adsorption capacity of methylene blue (MB) and rhodamine B (RB) on HPMC-Trp hydrogels increased with Trp content and the amount of
freezing water. HPMC-Trp hydrogels could be recycled 6 times keeping the original adsorptive capacity. The
diffusional constants of MB and RB tended to increase with Trp content. RB adsorbed on HPMC-Trp hydrogels
presented a bathochromic shift of fluorescence.

Keywords:
Hydroxypropyl methylcellulose
Tryptophan
States of water
Hydrogels
Cryogels
Adsorption

1. Introduction
Polysaccharide based 3D structures, such as aerogels and cryogels,
are interesting platforms for tissue engineering (Tchobanian, Van
Oosterwyck, & Fardim, 2019), drug delivery (Ulker & Erkey, 2014) and
adsorption of pollutants (Maleki, 2016). Upon contact with aqueous
media, the aerogels or cryogels become hydrogels. Understanding the
structure of water around the polysaccharide chains is important because it might drive the interactions with cells, drugs or pollutants.
Water molecules in hydrogels coexist as three different states: (i) Nonfreezing bound water or non-freezing water (Wnf), which results from
tightly bound water molecules, it is not freezable at 0 °C or below 0 °C,
due to the strong interaction with the polymer chain (ii) freezing bound
water or intermediate water (Wfb), which is not freezable at 0 °C, but it
is freezable below 0 °C because the interactions with polymer chains are
not so strong as in the Wnf, and (iii) free water or freezing water (Wf),

which is freezable at 0 °C, in this case, water molecules hardly interact
with the polymer chains, they are just entrapped in the matrix (Tsuruta,
2010). The amount of water molecules in each state depends

fundamentally on the chemical nature of polymer (Hatakeyama &
Hatakeyama, 2017).
Hydroxypropyl methylcellulose (HPMC) is a family of water-soluble
cellulose ethers widely applied in cosmetics (Lochhead, 2017), pharmaceuticals (Kaur et al., 2018), and food (Burdock, 2007) formulation.
HPMC chains can be crosslinked via esterification with citric acid (CA),
a nontoxic multifunctional acid, enabling the formation of tridimensional structures for drug release (Marani, Bloisi, & Petri, 2015; Reddy
& Yang, 2010) or adsorption of pollutants (Martins, Toledo, & Petri,
2017; Toledo et al., 2019). Cellulose can be modified with amino acids
in order to improve bioaffinity (Kalaskar et al., 2008) or to develop
membrane for methanol fuel cells (Zhao et al., 2019). Synthetic polymers modified with L-tryptophan (Trp), a hydrophobic essential amino
acid (Richard et al., 2009), can be used to produce cryogels with high
affinity for proteins (Türkmen et al., 2015) or DNA (Çorman, Bereli,
Ưzkara, Uzun, & Denizli, 2013). Despite the above-mentioned potential
applications, systematic investigations about cryogels and hydrogels
based on amino acid modified HPMC, the consequences on the mechanical properties, on the states of water in such materials and on their

⁎

Corresponding author.
E-mail addresses: (P.V.O. Toledo), (O.D. Bernardinelli), (E. Sabadini),
(D.F.S. Petri).
/>Received 20 February 2020; Received in revised form 27 June 2020; Accepted 11 July 2020
Available online 25 July 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.

Carbohydrate Polymers 248 (2020) 116765

P.V.O. Toledo, et al.

Fig. 1. Schematic representation of (a) HPMC, (b) CA, (c) Trp, (d) MB and (e) RB.

2. Experimental

Table 1
CA:Trp molar ratios used in the precursor gels to synthesize the HPMC-Trp
cryogels. Nitrogen content determined by elemental analyses (N, %), calculated
Trp content (wt%) and gel content (%).
CA:Trp

N (%)

Trp content (wt%)

Gel content (%)

1.0:0.0
1.0:0.5
1.0:1.0
2.0:0.5
2.0:1.0
2.0:2.0

0
0.10 ± 0.01
0.15 ± 0.01

0.16 ± 0.01
0.19 ± 0.01
0.31 ± 0.01

0
0.73 ± 0.07
1.09 ± 0.07
1.17 ± 0.07
1.38 ± 0.07
2.18 ± 0.07

93 ± 1
89 ± 3
82 ± 1
89 ± 2
85 ± 3
80.5 ± 0.8

2.1. Preparation of Tryptophan decorated cryogels
HPMC E4M (USP HPMC 2910, MS 0.25, DS 1.9) was kindly provided by The Dow Chemical Company (Brazil), GPC measurements
(Supplementary Material SM1) indicated Mn 1.1x105 g. mol−1 and
Mw 2.4x105 g. mol−1. The crosslinking of HPMC chains and the attachment of Trp to the HPMC chains were performed with CA in a onestep reaction. Briefly, aqueous solutions containing HPMC at 2.0 wt%,
CA at 0.10 wt% or 0.20 wt% (CA, LabSynth, Brazil, 192.13 g.mol−1),
sodium hypophosphite monohydrate at 0.05 wt% (HPS, LabSynth,
Brazil, 106.14 g.mol−1) and L-tryptophan (Trp, Sigma-Aldrich, 204.23
g.mol−1, T0254) were stored in the refrigerator at 12 °C for 8 h in order
to achieve complete dissolution of HPMC. The solutions were prepared
at different CA:Trp molar ratios of 1.0:0.0, 1.0:0.5, 1.0:1.0, 2.0:0.5,
2.0:1.0, and 2.0:2.0. The highest concentration of Trp in solution was
2.0 g.L−1, which is well below its solubility at 25 °C of 11.4 g.L−1

[National Center for Biotechnology Information. PubChem Database.
Tryptophan, CID = 6305, />compound/Tryptophan (accessed on Feb. 20, 2020)]. The hydrogels
were poured into molds of different sizes, were frozen in a freezer (-40
°C for 2 h) and freeze-dried (-50 °C, 200 μmHg, for 8–24 hours).
The resulting 3D solid structures were heated at 165 °C for 7 min in
order to promote the reaction between HPMC hydroxyl groups and CA
carboxylic acid groups and/or with Trp carboxylic acid/amine groups.
One should notice that in the absence of CA there is no chemical
crosslinking among the HPMC chains. If CA mass is less than 5 % of
polymer mass, the crosslinking efficiency is very low and the hydrogels
show no mechanical stability. CA mass between 5% and 10 % of
polymer mass yields stable HPMC hydrogels (Marani et al., 2015;
Martins et al., 2017). Ghorpade and co-workers showed that CA mass of
15 % or 20 % of polysaccharide (carboxymethyl cellulose and tamarind
gum) mass is not adequate because the swelling degree of hydrogels
tends to decrease (Mali, Dhawale, Dias, Dhane, & Ghorpade, 2018). For
compressive strength tests, rectangular samples of 13.0 mm x 17.0 mm x
25.0 mm were prepared, whereas for the other analyses the samples
were discs of 2 mm thickness and 35 mm of diameter. The samples were
coded as HPMC-Trp cryogels.

affinity for dyes, are poorly explored. Dyes are environmental pollutants, thus it is important to comprehend the correlation between the
states of water in hydrogels and their adsorption capacity.
Preliminary, HPMC cryogels were prepared with four different
amino acids (AA), namely, tryptophan (Trp, hydrophobic), glutamic
acid (Glu, acid), cysteine (Cys, polar) and histidine (His, basic). Among
all AA modified HPMC cryogels, the HPMC-Trp cryogels presented the
highest compression modulus in comparison to pure HPMC cryogels.
Similarly, the presence of hydrophobic particles, such as lignin, in
cryogels and hydrogels improved their mechanical properties and adsorptive capacity for dyes (Zhang et al., 2019). Based on this, we proceed with the systematic investigation on the (i) crosslinking of HPMC

chains and chemical attachment of Trp moieties to HPMC by esterification in one-step reaction, with different CA:Trp ratios, (ii) the
physicochemical properties of HPMC-Trp cryogels, (iii) the states of
water (Wnf and Wfb) in HPMC-Trp hydrogels and (iv) how the Trp
content and states of water affect the adsorption capacity of HPMC-Trp
hydrogels for methylene blue (MB) and rhodamine B (RB). To the best
of our knowledge, it is the first systematic study about the correlation
between the states of water and the adsorption behavior of dyes involving AA modified polysaccharides.

2


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Fig. 2. (a) Dehydration of citric acid and anhydride formation upon heating. (b) Esterification and crosslinking between two HPMC chains. (c) Esterification of
anhydride and HPMC hydroxyl group followed by attachment of Trp amino group to form an amide linkage.

and HPMC-Trp 2.18 wt% were compressed and decompressed in MilliQ
water up to 5 times; in the first cycle of compression/decompression,
the materials presented larger hysteresis and larger mechanical resistance than in the following cycles due to the presence of air entrapped inside the cryogels, which after the first cycle was expelled by
water. Fourier transform infrared spectroscopy analyses in the attenuated total reflectance mode (FTIR-ATR) were performed in a Perkin
Elmer Frontier equipped with ZnSe crystal, resolution of 4 cm-1 and in
the wavenumber range of 600 cm−1 to 4000 cm−1. Elemental analysis
(CHN) was performed with a Perkin Elmer 2400 Series II equipment.
For the differential scanning calorimetry (DSC TA Instruments Q10),
the samples were swollen in MilliQ water (≈ 5 μS. cm−1) at 20 °C for
12 h, degassed under vacuum pump (10 min at 100 mmHg) and subject
to 2 cycles from – 40 °C to 40 °C, at 5 °C.min−1 rate. Measurements of
TDNMR were performed in a Minispec 20 MHz at 33 °C. T2 relaxation

time measurement was carried out with a standard Carr-PurcellMeiboom-Grill (CPMG) pulse sequence with 30000 echoes and an echo
time of 16 μs (Carr & Purcell, 1954). SKL Neo MultiExp program for
inverse Laplace transform (ILT) was used and Log-Normal distribution
integration aided by an user-guided program called Peakfit 4.00 (Jandel
scientific software); the dried cryogels (≈ 25 mg) were swollen in
MilliQ water (1.0 mL) at 20 °C inside the NMR probe and the results
were acquired every 3 min from the completely dry sample to the absolute swelling that lasted a total of 45 min.

2.2. HPMC-Trp cryogels characterization
For the characterization, all cryogels were rinsed with MilliQ water
until the rinsing water achieved conductivity of ≈ 5 μS. cm−1. This
procedure removed the unreacted molecules, which could be only
physically attached to the samples. After that, they were freeze-dried
and weighed again. The gel content (Gel %) was calculated according to
Eq. (1):

mpol − mdried ⎞ ⎤
⎡
Gel (%) = ⎢1 − ⎜⎛
⎟
⎥ × 100
mpol
⎝
⎠⎦
⎣

(1)

where mpol is the initial mass of HPMC and mdried is the mass of the
freeze-dried sample.

The swelling degree (SD) was determined with a precision tensiometer Krüss K100 at (24 ± 1) °C as the mass of sorbed MilliQ water
(pH 5.5) at equilibrium divided by the mass of dried adsorbent:

SD =

m water
mdried

(2)

The apparent density of HPMC-Trp cryogels was determined at
(24 ± 1) oC by dividing the mass of freeze-dried cryogels by the corresponding volume, which was estimated by their dimensions. The dimensions of seven different samples of the same chemical composition
were measured using a pachometer; the same seven samples were
weighed in an analytical balance. The mean mass divided by the mean
volume determined for seven samples yielded the mean apparent density (ρap) value. SEM analyses were performed for gold-coated (by
sputtering) samples in a Jeol Neoscope microscope JCM 5000, operating at 5 kV voltage. The compressive tests were performed for 10
cryogels (rectangular) samples using an Impac, Digital Dynamometer
IP-90DI, with a 10 N load cell, at the strain rate of 0.01 s−1 and at
(24 ± 1) oC and (70 ± 5) % relative air humidity. The samples HPMC

2.3. Adsorption studies
Prior to the adsorption experiments, all cryogels were rinsed with
MilliQ water until the rinsing water achieved conductivity of ≈ 5 μS.
cm−1. After that, they were freeze-dried and weighed again. For the
adsorption studies, methylene blue (MB, Sigma-Aldrich, 319.81 g.mol1
) dissolved in Tris-HCl 0.05 mol.L-1 buffer at pH 7.0 (Trizma Base,
Sigma-Aldrich, 121.14 g.mol-1) and rhodamine B (RB, Sigma-Aldrich,
3

Carbohydrate Polymers 248 (2020) 116765

P.V.O. Toledo, et al.

Fig. 3. (a) Apparent density (ρap), (b) compression modulus (ε) as a function of Trp content in the HPMC-Trp porous materials, (c) Dependence of ε on ρap, the red line
corresponds to the fit ε = k ρapm, R² = 0.9051, m = 1.8195, and (d) swelling degree (SD). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article).

479.01 g.mol-1) dissolved in Tris-HCl 0.05 mol.L-1 buffer at pH 2.5 were
used as model molecules. Noteworthy, these pH conditions were chosen
because at pH 7 the adsorption of MB on the walls of the vials and selfassociation by π stacking was avoided. At pH higher than three, the
adsorption of RB molecules on the HPMC or HPMC-Trp hydrogels was
too low. All adsorption experiments were performed at (24 ± 1) °C and
contact time of 24 h, in order to assure equilibrium conditions
(Kaewprasit, Hequet, Abidi, & Gourlot, 1998).
The equilibrium adsorption capacity (qe, mg. g−1) of MB or RB was
calculated dividing the concentration of adsorbed MB or RB by the mass
of dried cryogels (m) and multiplying by the solution volume (v):

qe =

C0 − Ce
×v
m

mL) for 15 min. After this period, aliquots of supernatant were withdrawn, and the concentration of released RB was determined by photometry at 553 nm. Then the hydrogels were immersed in the RB solution for 10 min to proceed with next adsorption/desorption cycle.
After this period, aliquots of supernatant were withdrawn, and the
concentration of remaining RB was detected. Then the next desorption
process was conducted.
In order to get insight about the interactions between MB or RB

molecules and Trp moieties bound to HPMC chains, fluorescence was
measured with a Shimadzu RF6000 spectrofluorometer, at (24 ± 1) °C,
at 600 nm.min−1, excitation and emission bandwidth of 5 nm, resolution of 1.0 nm and reproducibility of 0.2 nm. After 48 h adsorption
of MB or RB (C0 = 1.0 mg.L-1) on HPMC-Trp hydrogels, a holder positioned at 45° with respect to the optical axis suspended the swollen
hydrogels in the air. Samples carrying MB or RB were excited at 650 nm
or 540 nm, respectively; the emission spectra were measured in the
range of 670 nm–820 nm or 550 nm–700 nm, respectively.
Fluorescence of MB and RB solutions at 0.33 mg.L-1 in the corresponding buffers was measured in a 10 mm x 10 mm quartz cuvette. All
measurements were performed for at least two samples of the same
composition. For comparison, the intensity values of fluorescence
spectra were normalized with respect to the intensity at the maximum
wavelength. Fig. 1 represents the chemical structures of the main
chemical compounds used in the experiments.

(3)

The concentration of adsorbed MB or RB onto the cryogels was
determined as the difference between the initial concentration (C0) of
MB or RB and the concentration of MB or RB in the supernatants after
24 h contact, or the equilibrium concentration (Ce). First, calibration
curves of absorbance intensity as a function of MB and RB concentration were determined by means of spectrophotometry in a Beckmann
Coulter DU640 spectrophotometer, respectively at 664 nm and 553 nm
(Supplementary Material SM2).
For the adsorption isotherms of MB onto Trp decorated HPMC
cryogels (∼30 mg dry mass), the initial concentration (Ci) of MB
ranged from 0.5 mg.L−1 to 5.0 mg.L−1. For the adsorption isotherms of
RB onto HPMC-Trp cryogels, the initial concentration (Ci) of RB ranged
from 0.5 mg.L−1 to 4.0 mg.L−1. Adsorption/desorption cycles were
carried out by immersing the RB coated samples in MilliQ water (10
4

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P.V.O. Toledo, et al.

Fig. 4. SEM images for (a) pure HPMC cryogels and HPMC-Trp cryogels with (b) 0.73 wt%, (c) 1.09 wt%, (d) 1.17 wt%, (e) 1.38 wt% and (f) 2.18 wt% Trp. Scale bars
correspond to 50 μm.
Table 2
Experimental values of ΔHendo determined for the HPMC-Trp hydrogels by DSC, the Wt , Wnf and (Wf + Wfb) values calculated with Eqs. (4) and (5). Wfb fraction was
determined by TD-NMR measurements (detailed in Section 3.3).
Trp
(wt%)

ΔHendo (J/g)

0
0.73 ± 0.07
1.17 ± 0.07
1.38 ± 0.07
2.18 ± 0.07

267 ± 6
310 ± 2
305 ± 0.7
297 ± 8
301 ± 5

Wt
(%)

Wnf

97.73 ± 0.00
97.18 ± 0.05
97.78 ± 0.01
97.20 ± 0.04
97.9 ± 0.3

18 ± 2
4.4 ± 0.7
6.5 ± 0.2
8±2
8±2

(Wf + Wfb) (%)

Wfb (%) (TD-NMR)

80 ± 2
92.8 ± 0.6
91.2 ± 0.2
89 ± 2
90 ± 1

13.91
65.80
21.80
38.11
92.24

(%)

3. Results and discussion

HPMC and CA carry no N atom in their structures. Considering the N
contents determined from elemental analyses (Table 1) and the N%
content in Trp of 11.75 %, the Trp content in the cryogels was calculated (Table 1). The chemical attachment of Trp to the cryogels was
favored by the increase of CA and Trp concentrations in the precursor
gel; the highest Trp content of 2.18 ± 0.07 wt% was achieved for the
CA:Trp molar ratio of 2.0:2.0. On the other hand, the gel content (Gel
%) was the smallest at the CA:Trp molar ratio of 2.0:2.0, indicating
competition between HPMC hydroxyl groups and Trp amine groups for
the CA carboxylic acid. CA carries three carboxylic acid groups, which
in the presence of HPS and under heating undergoes dehydration and
anhydride formation (Peng, Yang, & Wang, 2012) (Fig. 2a). The anhydride might react with HPMC hydroxyl groups, such esterification
can take place with hydroxyl groups of a second HPMC chain, promoting the crosslinking between HPMC chains (Fig. 2b.) The anhydride
bound to an HPMC chain can also react with Trp amino groups to form
amide groups (Fig. 2c), decreasing the crosslinking between two HPMC
chains. Thus, the increase of CA:Trp ratio increases the competition
between these reactions, decreasing the crosslinking (Gel %) and increasing the attachment of Trp to HPMC chains.
Supplementary Material SM4 provides FTIR-ATR spectra of pure
HPMC and HPMC-Trp in the 4000 to 600 cm−1 range. All samples

3.1. Characterization of HPMC-Trp cryogels
Preliminarily, HPMC cryogels were prepared with four different
amino acids, namely, tryptophan (Trp, hydrophobic), glutamic acid
(Glu, acid), cysteine (Cys, polar) and histidine (His, basic) at CA:AA
molar ratio of 1.0:1.0. The corresponding compression moduli (ε) values followed the sequence: HPMC-Trp > HPMC-His > HPMCCys > HPMC-Glu > pure HPMC; the data are provided as
Supplementary Material SM3. The ε values followed the relative hydrophobicity of amino acids given as Trp > > Cys > His > > Glu

(Wimley & White, 1996), indicating that the inclusion of hydrophobic
amino acid improved the mechanical properties of HPMC cryogels.
Based on this experimental observation and on the lack of reports about
the physicochemical properties of Trp modified HPMC cryogels, Trp
was chosen to proceed with the systematic modification of HPMC
cryogels.
The synthesis of HPMC-Trp cryogels with different CA:Trp molar
ratios led to different contents of N in the HPMC cryogels, as revealed
by CHN analyses (Table 1). The N% content in the samples stems exclusively from the chemical attachment of the Trp to the cryogels, since
5


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Fig. 5. Representation of the T2 decay-time distribution influenced by the pore size schematically indicated in the SEM image, (a) small pores and free “bulk” water,
(b) large pores and free water and (c) small and large pores and free water. (d) T2 decay-time distribution of the hydrogen atoms of water measured as soon as the
HPMC-Trp cryogels were put in contact with water (virtually at 0 min) and after 45 min contact. The Trp content in HPMC-Trp samples varied from 0 wt% to 2.18 wt
%.

presented the characteristic HPMC bands: in the 3500–3200 cm−1 region (OH vibrational stretching); at 2930 cm−1 and 2850 cm−1 (symmetrical and asymmetrical CH stretching); and in the 1200–850 cm−1
region (CO and CCee stretching vibrations of the glucopyranose ring)
(Silverstein, Webster, Kiemle, & Bryce, 2014). The esterification between CA and HPMC was identified by the bands at ≈1730 cm−1 and
1640 cm−1, which were assigned to C]O stretching of ester and acidic
forms, respectively (Bueno, Bentini, Catalani, & Petri, 2013), the bands
at 1458 cm−1, 1408 cm-1, and 1322 cm−1 were assigned to CH2 scissor,
symmetric axial deformation of C]O of esters and stretching of COe of
carboxylate groups. HMPC-Trp presented characteristic amide vibrational bands at 1642 cm−1 and 1312 cm−1, assigned to νC=O amide I
and νC-N amide, respectively (Liu, Shen, Zhou, Wang, & Deng, 2016),

which overlapped other characteristic bands already observed in pure
HPMC cryogels, impairing the identification of Trp by these vibrational
bands. However, HPMC-Trp with 1.38 wt% and 2.18 wt% Trp presented two weak bands at 748 cm−1 and 707 cm−1, which are characteristic of Trp indole ring; these bands did not appear in the spectra of
pure HPMC cryogels and, therefore, indicated the chemical attachment
to the HPMC hydrogels.
Fig. 3a and b shows that the apparent density (ρap) and the

compression modulus (ε) increased considerably with the increase of
Trp content in the HPMC-Trp cryogels, indicating that hydrophobic
interaction between Trp molecules might contributed to the cell wall
structuring. HPMC-Trp 2.18 wt% presented ε value twofold of that
determined for pure HPMC cryogels. For comparison, the ε values of
HPMC cryogels modified with 15 wt% of cellulose nanocrystals increased only 20 % in comparison to pure HPMC cryogels (Toledo et al.,
2019), but the addition of 2 wt% hydrophobic lignin particles to hydrophilic poly(vinyl alcohol), PVA, hydrogels increased fourfold the ε
value over that of pure PVA hydrogels (Bian et al., 2018). Thus, selfassembling of hydrophobic moieties added to hydrophilic cryogels
might improve the mechanical properties of cryogels.
The ε values increased with ρap1.82 (Fig. 3c); the index value of 1.82
is typical for isotropic open-cell structures (Scotti & Dunand, 2018).
Fig. 4 shows the SEM images of pure HPMC and HPMC-Trp with different Trp contents. Regardless of the Trp content, all cryogels presented isotropic open cellular structure, in agreement with the index
value of 1.82. The swelling degree (SD) values (Fig. 3d) presented no
significant dependence on the Trp content. On average, the SD for water
amounted to ≈ 47 g per g of cryogel. This value is ≈ 10 units smaller
than those found for cryogels made of negatively charged
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Fig. 6. Adsorption capacity (qe) of (a) MB at pH 7 and (b) RB at pH 2.5, respectively, onto HPMC-Trp hydrogels (30 ± 5 mg dried basis and 10 mL solution) as a
function of the equilibrium concentration of (Ce), at (24 ± 1) oC. KF values determined for MB and RB as a function of (c) Trp content and (d) freezing bound water
fraction (Wfb).

where msw is the mass of swollen hydrogel at equilibrium.
The ratio of the melting enthalpy values of free water in the hydrogel (ΔHendo) and of ice (ΔHmo = 334 J.g−1) (Bouwstra, Salomon-de
Vries, & van Miltenburg, 1995) was calculated to estimate the fraction
of free water. The DSC curves were provided as Supplementary Material SM6. Table 2 shows the experimental values of ΔHendo determined
for the HPMC-Trp hydrogels by DSC, the Wt , Wnf and (Wf + Wfb) values
calculated with Eqs. (4) and (5). All HPMC-Trp hydrogels presented
lower Wnf values than bare HPMC hydrogels (17.72 %), because the
amount of bound water depends strongly on the polymer hydrophilicity. For instance, the increase of the degree of substitution (DS) of
carboxymethyl cellulose (CMC) from 0.7 to 1.8 increased the Wnf values
from ≈ 75 % to ≈ 90 % (Hatakeyama & Hatakeyama, 2017). However,
among the HPMC-Trp samples, the Wnf values tended to increase with
the Trp content. Higher contents of Trp attached to HPMC were
achieved by higher CA:Trp ratios (2.0:0.5, 2.0:1.0 and 2.0:2.0), so that
not only the Trp content increased but also the number of carboxylate
groups. For those samples, the increase of hydrophobic Trp content
from 1.17 wt% to 2.18 wt% was accompanied by the increase of hydrophilic carboxylate groups stemming from CA.

polysaccharides, like xanthan gum (Toledo, Marques, & Petri, 2019) or
carboxymethyl cellulose (Toledo, Limeira, Siqueira, & Petri, 2019), due
to the absence of charges in the HPMC structure. Fig. Supplementary
Material SM5 shows the compressive stress-strain curves measured in
MilliQ water for HPMC and HPMC-Trp 2.18 wt%. The samples were
compressed and decompressed up to 5 times, but for the sake of clarity,
only the 2nd cycles were presented. Both HPMC and HPMC-Trp 2.18 wt
% hydrogels presented high resilience, small hysteresis and similar
stiffness. This trend is in agreement with the independence of SD values

on Trp contents (Fig. 3d).
3.2. Determination of non-freezing water (Wnf) in HPMC-Trp hydrogels by
means of DSC
HPMC-Trp cryogels become hydrogels upon immersion in water.
The amount of Wnf was calculated with Eq. (4) (Kim, Lee, & Kim, 2004):

ΔHendo ⎞
Wnf (%) = Wt − (Wf + Wfb) = Wt − ⎛⎜
× 100
o ⎟
⎝ ΔHm ⎠

(4)

where Wt is the total water in the hydrogel, the sum Wf and Wfb corresponds to the fraction of free water. One should notice that by DSC it
was not possible to discriminate the Wf and Wfb due to the low polymer
content.
Wt can be determined by Eq. (5):

m − mdried ⎞
× 100
Wt (%) = ⎛ sw
msw
⎠
⎝
⎜

3.3. Investigation of water in HPMC-Trp hydrogels through TD-NMR
Fig. 5 indicates a schematic representation of T2 decay-time distribution of the hydrogen atoms corresponding to the water molecules
that are filling the pores of the cryogels (SEM image) and the excess of

water (free water). The intensity of the signal relates to the number of

⎟

(5)
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Fig. 7. Adsorption capacity qt (mg. g−1) as a function of time t (min) determined for MB at (a) 3.0 mg.L−1 and (b) 5.0 mg.L−1, and for RB at 2.0 mg.L−1 and 4.0
mg.L−1.

remained practically constant, indicating that the majority of the water
was incorporated into the pores of the cryogels very fast (shorter than 3
min). The pattern of the signals for the HPMC-Trp samples differed from
the one for pure HPMC hydrogels. Without HPMC-Trp 0.73 wt%, the
general trend was an increase of Wnf with the increase of Trp content in
the HPMC-Trp hydrogels, as shown in Table 2. The samples with the
highest Trp contents (1.38 wt% and 2.18 wt%) presented the shortest
T2 values and the distributions of the two populations (small and large
pores) became closer. The sample HPMC-Trp 2.18 wt% presented an
interesting redistribution of the free water population after 45 min of
exposition, the band area of free water decreased 2.5 times and the
band area related to water into the small pores increased 1.7 times, in
comparison to the initial contact.

hydrogen nuclei of water molecules inside each pore population, while

the rate of decay is associated with the mobility of the molecules.
Surface relaxation of the wetting phase is strongly dependent on the
wettability environment within the pores space (Câmara et al., 2020;
Schmidt-Rohr & Spiess, 1994; Vidal, Bernardinelli, Paglarini, Sabadini,
& Pollonio, 2019). For this reason, water in small pores provides short
relaxation time (Fig. 5a), while water residing in large pores is related
to longer relaxation times (Fig. 5b). Fig. 5c represents the expected
decay for water in a matrix containing the two pores populations. The
longest relaxation time corresponds to the population of free “bulk”
water. Fig. 5d shows the distribution of T2 obtained for HPMC-Trp
hydrogels with Trp content ranging from 0 wt% to 2.18 wt%.
The corresponding decay curves were previously treated by the ILT,
as shown in the Supplementary Material SM7. For each sample, plots
for water distribution were recorded as soon as water was added to the
cryogel (0’), and after 45 min. The characteristic signal at around
2,500−3,000 ms corresponds to the free water (excess of water) present in the NMR tube and in the hydrogels. Signals at a smaller time
scale were attributed to Wfb. The mean T2 values and the percentage of
water in each population (area of the bands) were analyzed by considering the T2 distribution at 0’ and 45’, as shown in Supplementary
Material Table SM1. The areas corresponding to each band were obtained through the deconvolution of plots by using Log-Normal distribution integration, representing the fraction of water free and present
in the pores (Supplementary Material SM8). The signals below 1 ms,
which might be related to bound water (Wnf) (Wang et al., 2020; Wei
et al., 2018), were too weak to be accurately treated with ILT and appeared only in two samples (Supplementary Material SM9).
Except for HPMC-Trp 2.18 wt%, the population of free water

3.4. Adsorption of MB and RB on HPMC-Trp hydrogels
Fig. 6a and b shows the equilibrium adsorption capacity (qe) of MB
at pH 7 and RB at pH 2.5, respectively, onto HPMC-Trp hydrogels
(30 ± 5 mg dried basis and 10 mL solution) as a function of the equilibrium concentration of (Ce), at (24 ± 1) oC. At pH 7, MB has one
negative charge in the conjugated nitrogen (Flury & Wai, 2003),
whereas at pH 2.5, RB is positively charged (Ramette & Sandell, 1956)

(Supplementary Material SM10). The experimental qe values increased linearly with Ce values. Fittings with Langmuir and Freundlich
models (Supplementary Material SM11) indicated that the adsorption
behavior of MB and RB fitted better the Freundlich model. Moreover,
desorption experiments showed no desorption of MB or RB after 24 h
immersion in the respective solvents, impairing the fitting with the
Langmuir model. The Freundlich model is an empirical model, which
8


Carbohydrate Polymers 248 (2020) 116765

P.V.O. Toledo, et al.

coefficient of the adsorbate. Supplementary Material SM16 provides
typical dependence of qt on t0.5 for MB and RB at 5.0 mg.L−1 and 4.0
mg.L−1, respectively; data for more dilute solutions are available as. In
general, two different slopes were obtained by piecewise linear regression (dot lines), which correspond to fast and slow stages. The
change from the transport rate regime took place after approximately
10 min. Supplementary Material Table SM2 shows that the kintra
values decreased one order of magnitude from the 1st to the 2nd adsorption stage and increased with the increase of adsorbate concentration. There was a general tendency for the increase of kintra values
with the increase of Trp content in the HPMC-Trp hydrogels. The adsorption kinetics was also quantitatively evaluated using the pseudo-1st
order and pseudo-2nd order models, as shown in the Supplementary
Material Table SM3. The adsorption kinetics of MB and RB on HPMCTrp hydrogels fitted better with the pseudo-2nd order equation than
with the pseudo-1st order equation because the fittings were over a
whole time range (30 min) and the calculated qe values were similar to
the experimental data. However, the rate constants presented no general trend regarding the Trp content.
Supplementary Material SM17 shows the normalized fluorescence
spectra obtained for RB (solution), MB (solution), HPMC-Trp hydrogels
with different Trp contents after 48 h RB or MB adsorption, with the
corresponding qe values. The bathochromic shifts of emission maximum

of 8 nm (0 wt% and 0.73 wt% Trp) and 11 nm (1.09 wt% Trp, 1.38 wt%
and 2.18 wt%) indicated specific interactions between RB and Trp
molecules. Trp and dyes might form complexes by van der Waals forces,
leading to bathochromic shifts and quenching effects (Doose,
Neuweiler, & Sauer, 2005). On the other hand, no significant shift in the
emission maximum was observed for MB adsorbed on HPMC-Trp 2.18
wt% hydrogels, only the shoulder at ≈ 750 nm presented redshift to ≈
770 nm.

yields parameters KF and n, the former is related to the adsorptive capacity and the latter to the surface homogeneity (Foo & Hameed, 2010).
Fig. 6c and d shows the KF values determined for MB and RB as a
function of Trp content and freezing bound water fraction (Wfb), respectively. The general trend for MB and RB was an increase of KF
values with Trp content (Fig. 6c) or Wfb (Fig. 6d), indicating that the
adsorption capacity correlated well with intermediate water fraction in
hydrogels. On the other hand, the dependence of KF values on Wnf
showed a maximum at ≈ 8% for both adsorbates (Supplementary
Material SM12), which corresponds to the highest Trp content.
Therefore, the increase of Trp content caused an increase of Wfb, Wnf
and KF values. The n values showed no dependence on Trp content
(Supplementary Material SM13); for MB and RB, the n values
amounted to 1.01 ± 0.05 and 0.92 ± 0.03, indicating similar chemical
homogeneity of HPMC-Trp hydrogels used for MB and RB adsorption.
The qe value of ≈ 1.0 mg.g−1 for MB or RB on HPMC-Trp 2.18 wt%
was similar to those determined for MB on tannin-immobilized cellulose hydrogel (qe = 1,1 mg/g) (Pei et al., 2017) and for RB on carbon
nanospheres (qe = 1,15 mg/g) (Qu, Zhang, Xia, Cong, & Luo, 2015).
The qe value of ≈ 1.0 mg.g−1 for MB or RB on HPMC-Trp 2.18 wt% was
used for estimating the number of adsorbed MB or RB molecules per Trp
molecules chemically attached to the HPMC hydrogels. In one gram of
HPMC-Trp 2.18 wt%, there are ≈ 1 ×10-4 moles Trp, whereas in 1.0 mg
of MB and RB there are ≈ 3 ×10-6 moles MB and ≈ 2 ×10-6 moles MB.

Thus, Trp moieties are in excess in comparison to the adsorbate molecules. In order to achieve complete saturation of Trp adsorbing sites
with MB and RB molecules, the qe maximal values should be ∼ 33
mg.g−1 and 50 mg.g−1, respectively. These values are similar to literature values determined for lignin-based adsorbents (hydrophobic
surfaces) under similar pH and dye dilute range. For instance, the qe
maximal values for MB on organosol lignin (Zhang, Wang, Zhang, Pan,
& Tao, 2016) or blends of lignin and chitosan (Albadarin et al., 2017)
amounted to (∼ 20.6 mg.g−1) and (∼ 36 mg.g−1), respectively. Considering dilute range of RB (> 10 mg/L), the adsorption capacity of
HPMC-Trp (50 mg.g−1) was on the same order of magnitude of that
determined for activated carbons obtained from lignocellulosic waste
(39 mg/g) (da Silva Lacerda et al., 2015).
The reusability of the Trp-HPMC hydrogels for RB was evaluated.
Supplementary Material SM14 shows the removal capacity of RB (Ci
= 1.0 mg/L, 10 mL) by HPMC-Trp 2.18 wt% after six adsorption cycles.
The adsorption time was 10 min, after that the absorbance of supernatant was measured and the hydrogels were immersed in 10 mL MilliQ
water for 15 min. For more concentrated RB solutions (< 2.0 mg/L) the
complete removal was obtained after four consecutive times in contact
with 10 mL of fresh MilliQ water. Regardless the initial RB concentration, the hydrogels were reused six times, keeping the removal efficiency at the original level. The reusability of the HPMC hydrogels for
MB adsorption is also feasible by rinsing with HCl 1.0 mol/L; even after
10 recycles, the adsorption capacity was kept at original level (Toledo,
Martins et al., 2019). However, HPMC-Trp hydrogels are advantageous
over pure HPMC because MB molecules undergo pronounced photofading in the presence of Trp molecules (Knowles and Gurnani, 1972).
Reactions involving singlet oxygen and Trp and triplet state of MB and
Trp cause the photofading of MB (Smith, 1978). Supplementary Material SM15 shows that upon increasing the Trp content in the HPMC
hydrogels the photofading effect becomes more evident.
The adsorption kinetics of MB and RB onto HPMC-Trp hydrogels
was systematically investigated for MB at 3.0 mg.L−1 and 5.0 mg.L−1,
and for RB at 2.0 mg.L−1 and 4.0 mg.L−1, as shown in Fig. 7. The data
were fitted with the intraparticle diffusion model (Weber & Morris,
1963), which has been widely applied for the analysis of mass transfer
from solution to the solid-liquid interface and the diffusion of the adsorbate into the porous media:

q = kintra t 0.5

4. Conclusions
The present study presented the chemical crosslinking and modification of HPMC chains with citric acid (CA) and Trp by one-step
reaction, creating new functional polysaccharides. Upon increasing the
CA:Trp ratio to 2.0:2.0 in the synthesis, the Trp content in the HPMC
cryogels increased to 2.18 wt%, enhancing the compression modulus
from 94 ± 5 kPa (pure HPMC cryogel) to 201 ± 9 kPa. The insertion of
hydrophobic moieties increased the Wnf fraction and the adsorption
capacity for RB and MB. Trp-HPMC hydrogels presented resilience and
reusability. The fluorescence experiments evidenced specific interactions between RB and Trp, but they were absent (or very weak) in the
case of MB. Therefore, the adsorption capacity oh Trp-HPMC hydrogels
was favored not only by π-π interaction among the aromatic rings of
Trp and MB or RB, but also by the increase of Wnf portion, or by the
“cage” water molecules. This finding is of high relevance because it
demonstrated for the first time the interdependence between the adsorption of water-soluble adsorbates (dyes, drugs, cells, DNA, etc.) and
the intermediate water fraction (Wnf), which can be tuned by the degree
of hydrogel modification with hydrophobic moieties (Trp).
CRediT authorship contribution statement
Paulo V.O. Toledo: Methodology, Investigation, Data curation.
Oigres D. Bernardinelli: Methodology, Investigation, Data curation.
Edvaldo Sabadini: Conceptualization, Writing - review & editing.
Denise F.S. Petri: Conceptualization, Writing - review & editing,
Supervision, Funding acquisition, Project administration.
Acknowledgments
Authors gratefully acknowledge financial support from Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq Grant
306848/2017 and 421014/2018) and São Paulo Research Foundation

(6)

where kintra is the diffusion rate, which is proportional to the diffusion
9


Carbohydrate Polymers 248 (2020) 116765

P.V.O. Toledo, et al.

(FAPESP, Grant 2018/13492-2). This study was nanced in part by the
Coordenaỗóo de Aperfeiỗoamento de Pessoal de Nível Superior - Brasil
(CAPES) - Finance Code 001.

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