Supplementary Material

Optimization of dye adsorption capacity and mechanical

strength of chitosan aerogels through crosslinking strategy

and graphene oxide addition

M. Salzano de Lunaa,b,*, C. Ascioneb, C. Santilloc, L. Verdolottic, M. Lavorgnac,*,

G.G. Buonocorec, R. Castaldob, G. Filipponea, H. Xiad, L. Ambrosioc

a Department of Chemical, Materials and Production Engineering (INSTM Consortium – UdR

Naples), University of Naples Federico II, P.le Tecchio 80, 80125 Naples, Italy

b Institute for Polymers, Composites and Biomaterials, National Research Council of Italy, Via

Campi Flegrei 34, 80078 Pozzuoli, Italy

c Institute for Polymers, Composites and Biomaterials, National Research Council of Italy, P.le

E. Fermi 1, 80055 Portici (Naples), Italy

d State Key Laboratory of Polymer Materials Engineering, Polymer Research Institute of

Sichuan University, Chengdu 610065, China

Corresponding authors:

*e-mail: martina.salzanodeluna@unina.it; phone: +39 0817682407 (Martina Salzano de Luna)

*e-mail: mlavorgn@unina.it; phone: +39 0817758838 (Marino Lavorgna)

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S1. Details on the No-GEL and GEL classification of the aerogels

The crosslinking begins soon after the addition of the GA to the CS solution. Therefore, even the

no-GEL samples are actually gelled to some extent before the freeze-drying step. Nonetheless,

this incipient crosslinking is so negligible not to compromise the meaningfulness of the

distinction between the GEL and no-GEL samples. To confirm this aspect, a frozen GEL sample

and a frozen no-GEL one, both at 15 wt.% of GA (i.e. at the highest investigated crosslinker

content, that represents the most critical situation), were soaked in 0.35 M acetic acid solution

under stirring at room temperature for about one week. The evolution of the sample appearance

during this test is shown in Figure S1.

Figure S1. a) No-GEL (left) and GEL (right) samples soon after freezing. b) Vials containing the

no-GEL (left) and GEL (right) samples soon after immersion in the acetic acid solution. c) Same

vials as in b) after one week.

After one week in the acetic acid solution, the no-GEL sample is completely dissolved, while the

GEL sample keeps its integrity. This result confirms the significant difference between the two

sets of investigated samples, in which ice crystals actually form before ("no-GEL") and after

("GEL") the polymer crosslinking step.

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S2. FTIR analyses on CS aerogels after the thermal treatment

At the end of the freeze-drying process, the chitosan (CS) aerogels were thermally treated at

90°C for 60 min and 120 min. FTIR analyses were carried out to evaluate the effect of the

duration of the thermal treatment on the crosslinking reaction between CS and glutaraldehyde

(GA). The FTIR spectra of representative aerogels prepared with low (5 wt.%) and high (15 wt.

%) GA content were compared to not crosslinked CS in Figure S2. For a qualitative comparison,

the spectra were normalized considering the absorption band at 1153 cm-1, attributed to

asymmetric stretching of the C-O-C bridges, as invariant peak.

Figure S2. FTIR spectra of a, c) no-GEL and b, d) GEL CS aerogels crosslinked with ΦGA = a, b)

5 and c, d) 15 wt.% after thermal treatment of different duration: 0, 60, and 120 min. The

spectrum of not crosslinked CS aerogels is also reported as reference.

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The formation of the new peak attributed to imine N=C bonds (1655 cm-1) and the shoulder of

ethylenic C=C bonds (1562 cm-1) can be appreciated in the spectra of the as-prepared (i.e. before

thermal treatment) no-GEL and GEL aerogels at ΦGA = 5 wt.%. Such chemical modifications,

that are the fingerprint of the crosslinking reaction between CS and GA, become more evident in

the spectra of the samples thermally treated at 90°C for 60 min. After longer treatments (up to

120 min), no further chemical modifications take place, confirming that a thermal treatment of

60 min is enough to complete the crosslinking reaction between CS and GA in both no-GEL and

GEL samples. The same considerations also apply for no-GEL and GEL aerogels at ΦGA = 15 wt.

%.

S3. Skeletal density of CS aerogels

The skeletal density of the aerogels, ρS, was determined by means of helium picnometry

(AccuPyc 1340, Micromeritics, USA). Irrespective of the amount of GA and the aerogel

preparation process (i.e. no-GEL or GEL samples), the skeletal density was found to be

essentially the same for all the tested samples, as also reported by Uragami et al. (Uragami,

Matsuda, Okuno, & Miyata, 1994) for GA-crosslinked chitosan membranes. In particular, ρS of

the aerogels varied between 1.38 ± 0.05 and 1.47 ± 0.08 g cm -3, which is in good agreement with

other skeletal density data of chitosan available in the literature (Uragami, Matsuda, Okuno, &

Miyata, 1994; Riegger et al., 2018; López-Iglesias et al., 2019). Finally, no evident trend as a

function of GA content or crosslinking strategy can be appreciated for the values of ρS.

S4. Linear viscoelastic analyses on CS/GA solutions

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The addition of relatively low GA contents (ΦGA ≤2.5 wt.%) did not result in the formation of a

gel, at least in the investigated conditions (Figure S3). For such a sample, indeed, the viscoelastic

moduli exhibit a clear frequency dependence in all the investigated window, with G′ being

always smaller than G′′.

Figure S3. Frequency-dependent storage and loss moduli for CS solution ΦGA = 2.5 wt.% after

24 hours that GA was added. The error bars represent the standard deviation over three

independent measurements.

S5. Evaluation of gel fraction in CS aerogels crosslinked with different amounts of GA

The percentage extent of crosslinked CS in the aerogels was evaluated gravimetrically.

Specifically, dry samples (~15 mg) were immersed in 0.35 M acetic acid solution (15 mL) to

extract soluble (i.e. not chemically crosslinked) fractions. At fixed time intervals, the aerogels

were carefully collected, washed with bi-distilled water and dried at 40°C under vacuum. The

procedure was repeated until reaching a constant value of sample weight. The gel fraction, ,

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was thus determined by the following relationship (Nieto-Suárez, López-Quintela, & Lazzari,

2016):

(S1)

where WI and WF represent the initial weight of the dry sample and the weight at the end of the

washing procedure, respectively. Five aerogels were tested for each set of samples.

The result of the measurements is shown in Figure S4. The gel fraction is over 90% for both the

investigated sets of samples, irrespective of the amount of crosslinker used. The reaction

between CS and GA leads to complete crosslinking of the chitosan chains (as testified by the

non-dissolution of aerogels in acid water) even at the minimum content of GA considered and

independently to the procedure adopted for aerogel preparation.

Figure S4. Gel fraction for no-GEL (red circles) and GEL (blue squares) CS aerogels

crosslinked with different ΦGA. The error bars represent the standard deviation over five

independent measurements

S6. Evaluation of swelling degree of CS aerogels crosslinked with different amounts of GA

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The swelling properties were evaluated by immersion of the dried aerogels in excess bi-distilled

water. The swollen samples were weighted at various time intervals after removing the excess

surface water was removed using filter paper, and the procedure was repeated until reaching a

constant value. The percentage swelling degree was calculated as:

(S2)

where mS and mD represent the mass of the swollen and dried aerogel, respectively.

The result of the measurements is shown in Figure S5.

Figure S5. Swelling degree in water for no-GEL (red bars) and GEL (blue bars) CS aerogels

crosslinked with different ΦGA. The error bars represent the standard deviation over three

independent measurements.

S7. Fitting of the adsorption equilibrium data to the isotherm models for pristine CS

aerogels

Freundlich (Freundlich, 1906) and Langmuir (Langmuir, 1916) isotherm models were exploited

to fit the experimental adsorption data and understand how the pollutant molecules interacted

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with the CS-based adsorbents. The Freundlich model predicts that the equilibrium adsorption

capacity, qe, depends on the equilibrium dye concentration in liquid phase, Ce, as:

(S3)

where kF represents the adsorption capacity and nF is a parameter that accounts for surface

heterogeneity.

Instead, according to the Langmuir isotherm model:

(S4)

where qL is the saturated monolayer adsorption and bL represents the binding energy or affinity

parameter of the sorption system.

The parameters obtained by the best fitting of the experimental adsorption equilibrium curves to

Equations S3 and S4 are reported in Table S1 and Table S2 for pristine no-GEL and GEL CS

aerogels crosslinked with different ΦGA, respectively.

Table S1. Isotherm fitting parameters for no-GEL CS aerogels at different ΦGA.

Freundlich modelΦGA

[wt.%]kF

[(mg g-1) (mg L-1)-1/nF]nF R2

5 168.5 ± 50.7 4.95 ± 1.38 0.8574

7.5 147.1 ± 45.3 4.85 ± 1.34 0.8602

10 125.7 ± 44.8 4.56 ± 1.35 0.8425

12.5 108.2 ± 5.0 4.32 ± 1.28 0.8459

15 100.0 ± 40.3 4.41 ± 1.39 0.8280

Langmuir modelΦGA

[wt.%]qL

[mg g-1]bL

[L mg-1]R2

5 534.4 ± 30.5 0.157 ± 0.016 0.9529

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7.5 484.2 ± 25.5 0.139 ± 0.017 0.9930

10 458.5 ± 16.0 0.099 ± 0.020 0.9805

12.5 431.8 ± 22.1 0.071 ± 0.010 0.9905

15 410.8 ± 18.2 0.069 ± 0.013 0.9849

Table S2. Isotherm fitting parameters for GEL CS aerogels at different ΦGA.

Freundlich modelΦGA

[wt.%]kF

[(mg g-1) (mg L-1)-1/nF]nF R2

5 160.1 ± 34.9 6.80 ± 1.86 0.8690

7.5 128.4 ± 26.0 5.89 ± 1.27 0.9159

10 123.3 ± 30.1 5.91 ± 1.53 0.8773

12.5 115.5 ± 30.7 5.80 ± 1.59 0.8640

15 107.9 ± 29.4 5.98 ± 1.71 0.8505

Langmuir modelΦGA

[wt.%]qL

[mg g-1]bL

[L mg-1]R2

5 365.1 ± 36.6 0.691 ± 0.098 0.9933

7.5 340.4 ± 21.8 0.269 ± 0.065 0.9806

10 330.2 ± 39.3 0.269 ± 0.036 0.9929

12.5 319.0 ± 30.6 0.222 ± 0.038 0.9882

15 292.7 ± 38.3 0.214 ± 0.029 0.9925

Since the Langmuir isotherm equation was the most suitable model to fit the experimental data,

the maximum adsorption capacity of the pristine no-GEL and GEL CS aerogels can be

considered equal to the saturated monolayer coverage of the adsorbent active sites (i.e. the value

of qL obtained through the best fitting procedure).

S8. Insights into dye removal mechanism

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Although all the investigated aerogels significantly swell in aqueous solutions (see Figure S5),

the dye removal process is essentially governed by adsorption phenomena (rather than to

absorption processes. Targeted experiments were performed to prove this aspect. In particular,

the aerogels collected at the end of the adsorption process were immediately soaked in fresh bi-

distilled water. No evident release of dye molecules due to diffusion-related processes can be

appreciated (see the photo in Figure S6). Evidently, if the removal process was merely based on

absorption, the fresh water would be immediately polluted by the dye molecules released from

the aerogel. On the contrary, UV-vis spectroscopy data of the aqueous medium also confirms

that no dye molecules can hardly detected (Figure S6). Note that UV-vis spectroscopy can detect

a concentration of IC as low as 1 mg L-1. The dye molecules are thus firmly anchored to the

surface of the aerogels even when immersed in excess of fresh bi-distilled water, indicating that

the removal process is related to adsorption phenomena.

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Figure S6. Photo of a representative aerogel (no-GEL CS aerogel at ΦGA = 10 wt.%) soaked in

fresh bi-distilled water at the end of the adsorption process of IC (the dye solution obtained at the

end of the adsorption process is shown on the left side as reference). The UV-vis spectra of the

two solutions that are shown in the photo are also reported.

S9. Gelation kinetics of CS/GO solution with GA

The gelation kinetics in the presence of GO nanosheets in the CS solution is slightly faster

(Figure S7). Nonetheless, the gelation time, tGEL, which is identified as the crossover point of G′

and G′′ over time, is still sufficiently long to allow the distinction between no-GEL and GEL

samples.

Figure S7. Time-dependent storage (full symbols) and loss (empty symbols) moduli for CS/GO

solution at ΦGA = 10 wt.% (red squares). The curves for the pristine CS solution at ΦGA = 10 wt.%

(black circles) are reported for comparison.

S10. Fitting of the adsorption equilibrium data to the isotherm models for nanocomposite

CS/GO aerogels

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Freundlich (Freundlich, 1906) and Langmuir (Langmuir, 1916) isotherm models were used to fit

also the dye adsorption data of no-GEL and GEL CS/GO aerogels. The corresponding

mathematical relationships are presented in Section S7. The parameters obtained by the best

fitting of the experimental adsorption equilibrium curves to Equation S3 and S4 are reported in

Table S3 and Table S4 for no-GEL and GEL nanocomposite CS/GO aerogels, respectively.

Table S3. Isotherm fitting parameters for no-GEL CS and CS/GO aerogels for different dyes.Freundlich model

Sample - dyekF

[(mg g-1) (mg L-1)-1/nF]nF R2

CS - IC a) 125.7 ± 44.8 4.56 ± 1.35 0.8425

CS - MB b) - - -

CS/GO - IC 86.7 ± 26.7 4.10 ± 0.92 0.9098

CS/GO - MB 7.5 ± 3.2 2.30± 0.36 0.9516

Langmuir model

Sample - dyeqL

[mg g-1]bL

[L mg-1]R2

CS - IC a) 458.5 ± 16.0 0.099 ± 0.020 0.9805

CS - MB b) - - -

CS/GO - IC 376.8 ± 32.3 0.062 ± 0.010 0.9887

CS/GO - MB 168.6 ± 9.6 0.005 ± 0.001 0.9727a) Same data reported in Table S1 for pristine no-GEL CS aerogel at ΦGA = 10 wt.%. b) The experimental data of qe were too low to be fitted with isotherm models.

Table S4. Isotherm fitting parameters for GEL CS and CS/GO aerogels for different dyes.Freundlich model

Sample - dyekF

[(mg g-1) (mg L-1)-1/nF]nF R2

CS - IC a) 123.3 ± 30.1 5.91 ± 1.54 0.8773

CS - MB b) - - -

CS/GO - IC 96.9 ± 38.2 5.46 ± 2.03 0.7687

CS/GO - MB 2.4 ± 0.6 2.04 ± 0.17 0.9839

Langmuir model

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Sample - dyeqL

[mg g-1]bL

[L mg-1]R2

CS - IC a) 330.2 ± 39.3 0.270 ± 0.036 0.9929

CS - MB b) - - -

CS/GO - IC 300.9 ± 26.9 0.099 ± 0.02 0.9317

CS/GO - MB 87.2 ± 5.2 0.003 ± 0.001 0.9909a) Same data reported in Table S2 for pristine GEL CS aerogel at ΦGA = 10 wt.%.b) The experimental data of qe were too low to be fitted with isotherm models.

REFERENCES

Freundlich, H. M. F. (1906). Over the adsorption in solution. Journal of Physical Chemistry, 57,

1100-1107.

Langmuir I. (1916). The constitution and fundamental properties of solids and liquids. Journal of

the American Chemical Society, 38, 2221–2295.

López-Iglesias, C., Barros, J., Ardao, I., Monteiro, F. J., Alvarez-Lorenzo, C., Gomez-Amoza, J.

L., & García-González, C. A. (2019). Vancomycin-loaded chitosan aerogel particles for

chronic wound applications. Carbohydrate Polymers, 204, 223-231.

Nieto-Suárez, M., López-Quintela, M. A., & Lazzari, M. (2016). Preparation and

characterization of crosslinked chitosan/gelatin scaffolds by ice segregation induced self-

assembly. Carbohydrate Polymers, 141, 175-183.

Uragami, T., Matsuda, T., Okuno, H., & Miyata, T. (1994). Structure of chemically modified

chitosan membranes and their characteristics of permeation and separation of aqueous ethanol

solutions. Journal of Membrane Science, 88, 243-251.

Riegger, B. R., Bäurer, B., Mirzayeva, A., Tovar, G. E., & Bach, M. (2018). A systematic

approach of chitosan nanoparticle preparation via emulsion crosslinking as potential

adsorbent in wastewater treatment. Carbohydrate Polymers, 180, 46-54.

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