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Agriculture and Food Sciences Research 
Vol. 11, No. 2, 187-194, 2024 

ISSN(E) 2411-6653/ ISSN(P) 2518-0193 
DOI: 10.20448/aesr.v11i2.6251 

© 2024 by the authors; licensee Asian Online Journal Publishing Group 

 
 

 
 
 
Preparation of chemically crosslinked chitosan hydrogels with adipic acid and 
EDAC carbodiimide: Tailored biomaterials for multifunctional applications 

 
Talita Martins1   

Ezequiel de Souza Costa Junior2   

Eduardo Henrique Martins Nunes3   
 

 
( Corresponding Author)  

1,3Federal University of Minas Gerais, Belo Horizonte/MG, Brazil. 
1Email: martins.talita2009@gmail.com  
3Email: eduardo.nunes@demet.ufmg.br    
2Federal Center for Technological Education of Minas Gerais, Brazil. 
2Email: escjr50@cefetmg.br  

 
Abstract 

Hydrogels are versatile biomaterials with significant potential in agriculture, food preservation, 
and biomedical applications. Hydrogels are three-dimensional networks of hydrophilic polymers 
that can absorb and retain large amounts of water or biological fluids. Among the polymers 
suitable for hydrogel synthesis, chitosan is particularly notable due to its biocompatibility, 
biodegradability, and antimicrobial properties. This study investigates chitosan characteristics, 
focusing on its various properties and how they influence its potential applications in the synthesis 
of chitosan hydrogels crosslinked with adipic acid using the water-soluble carbodiimide N-(3-
dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) to develop multifunctional 
films for practical applications. The raw materials were characterized using potentiometric 
titration, Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD). The 
hydrogels were analyzed for morphology and crystallinity through Scanning Electron 
Microscopy (SEM), FTIR and XRD, comparing samples with and without the addition of the 
carbodiimide crosslinker. Quantitative analysis of swelling behavior was also conducted using 
standard buffer solutions. The results showed that crosslinking with adipic acid enhanced the 
crystallinity of chitosan, while the inclusion of EDAC reduced crystallinity and the degree of 
swelling, indicating improved crosslinking between polymer chains. These changes in structural 
and functional properties demonstrate the adaptability of these hydrogels for specific applications. 
This study underscores the potential of chitosan-based hydrogels as advanced biomaterials. 

 
Keywords: Adipic acid, Chemical crosslinking, Chitosan, EDAC, Hydrogel, Biomaterials. 

 
Citation | Martins, T., Junior, E. de S. C., & Nunes, E. H. M. 
(2024). Preparation of chemically crosslinked chitosan hydrogels 
with adipic acid and EDAC carbodiimide: Tailored biomaterials for 
multifunctional applications. Agriculture and Food Sciences 
Research, 11(2), 187–194. 10.20448/aesr.v11i2.6251 
History:  
Received: 31 October 2024 
Revised: 29 November 2024 
Accepted: 5 December 2024 
Published: 26 December 2024 
Licensed: This work is licensed under a Creative Commons 

Attribution 4.0 License  
Publisher:  Asian Online Journal Publishing Group 
 

Funding:   This research is supported by Fundação de Amparo à Pesquisa do 
Estado de Minas Gerais (FAPEMIG) (Grant number: BPD-00883-22). 
Institutional Review Board Statement: Not applicable. 
Transparency: The authors confirm that the manuscript is an honest, 
accurate, and transparent account of the study; that no vital features of the 
study have been omitted; and that any discrepancies from the study as planned 
have been explained. This study followed all ethical practices during writing. 
Competing Interests: The authors declare that they have no competing 
interests. 
Authors’ Contributions: All authors contributed equally to the conception 
and design of the study. All authors have read and agreed to the published 
version of the manuscript. 

 
Contents 
1. Introduction .................................................................................................................................................................................... 188 
2. Experimental ................................................................................................................................................................................... 188 
3. Results and Discussion ................................................................................................................................................................. 189 
4. Conclusion ....................................................................................................................................................................................... 193 
References ............................................................................................................................................................................................ 193 
 

 

 

 

mailto:martins.talita2009@gmail.com
mailto:eduardo.nunes@demet.ufmg.br
mailto:escjr50@cefetmg.br
https://creativecommons.org/licenses/by/4.0/
https://creativecommons.org/licenses/by/4.0/
https://www.doi.org/10.20448/aesr.v11i2.6251
https://orcid.org/0000-0002-0309-9849
https://orcid.org/0000-0003-0925-6400
https://orcid.org/0000-0001-6653-5137


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Contribution of this paper to the literature 
This study is original in its investigation of chitosan hydrogels crosslinked with adipic acid 
using EDAC, offering an alternative to commonly used acids. This method enhances properties 
of chitosan, with potential applications in agriculture and food sciences, particularly in 
controlled release and preservation technologies. 

 
1. Introduction 

In the field of biomaterials research, the development of novel hydrogels has been a focal point for several 
years, driven by their potential applications in extending food shelf life, advancing agricultural practices, and 
supporting biomedical innovations. A critical aspect of these studies lies in the selection of polymers for hydrogel 
fabrication. This selection must consider the intrinsic properties of the polymers, ensuring they synergistically 
impart the desired functional characteristics required for the specific target application [1-3]. 

In the focus of this development, biomaterials for use in a multifunctional application such as and agriculture 
and food science represent a major area of research today [1]. In these fields, the compatibility between the 
material and its intended application is crucial for success. Hydrogels exhibit properties that make them highly 
appealing, such as their ability to retain water, hydrophilicity, expandability, selective permeability, soft 
consistency, and low interfacial tension [4, 5]. 

Chitosan (CHI) is one of the most promising polymers for hydrogel applications, as it predominantly consists 
of D-glucosamine units derived from the N-deacetylation of chitin, a natural component of crustacean and insect 
exoskeletons [6, 7]. Chitosan stands out due to its remarkable properties, including biocompatibility, 
biodegradability, antimicrobial activity, and high adhesiveness, largely attributed to its polycationic nature [7].  

Hydrogels are advanced materials characterized by crosslinked polymer networks that form three-dimensional 
structures [8]. The degree of crosslinking is a critical parameter, as it directly impacts the hydrogel's 
physicochemical and mechanical properties. Subtle variations in crosslinking density can alter key attributes such 
as swelling behavior, elasticity, permeability, and structural stability [9]. Therefore, precise control over the 
crosslinking process is essential to tailor hydrogels for specific applications, ensuring optimal performance and 
functionality. 

Crosslinking is a crucial process in hydrogel synthesis, involving the interconnection of polymer chains 
through reactions with low molecular weight crosslinkers containing at least two functional groups. This process 
results in a more robust, three-dimensional polymer network, enhancing the structural integrity and mechanical 
properties of the hydrogel. Importantly, crosslinking agents employed for hydrogels designed as biomaterials must 
be non-toxic to ensure safety and suitability for their intended applications [10, 11]. 

The objective of this work is to evaluate the feasibility of synthesizing chitosan-based hydrogels chemically 
crosslinked with adipic acid through a reaction mediated by the water-soluble carbodiimide N-(3-
dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC). These hydrogels are designed to function as 
biomaterials with versatile and multifunctional applications. 
 

2. Experimental 
2.1. Materials 

Chitosan powder (Cat. # 419419, Sigma-Aldrich Chemical, Milwaukee, Wisconsin, USA), high molecular 
weight, MW= 340.000g/mol, degree of deacetylation, DD= >75%, was used without further purification. Adipic 
acid (Vetec Química). N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) (Fluka). All salts 

and reagents used were of analytical degree and Milli-Q water was used in all solutions (18.0 MΩ). 
 

2.2. Chitosan Hydrogels Preparation 
Chitosan hydrogels were synthesized by dissolving 0.5 g of chitosan in 50 mL of a 1% (w/w) adipic acid 

solution under stirring at 60 °C until complete dissolution. Gradually 4 mL of NaOH (0.1 M) were added with 
continuous stirring, followed by cooling the solution to 40 °C. To initiate the chemical crosslinking, three 
formulations were prepared by varying the addition of EDAC (carbodiimide): sample A (without EDAC), sample B 
(0.06% w/v EDAC), and sample C (0.11% w/v EDAC). Gelation was monitored during the stirring process, and 
the resulting mixtures were uniformly distributed onto Petri dishes. After 7 days at room temperature, the 
hydrogels were dried at 40 °C for 24 hours. The formed films were washed thoroughly with 5% (w/w) acetic acid, 
followed by rinsing with Milli-Q water to remove residual reagents. The films underwent a final drying step in an 
oven at 40 °C for 24 hours to yield the crosslinked chitosan hydrogels.  

 

2.3. Raw Materials Characterization 
The degree of deacetylation (DD) of chitosan was performed by the potentiometric titration method in 

pHmeter DIGIMED D-23 which 0.1 g of chitosan were dissolved in 10 ml of standard solution of 0.1 M HCl for 
protonation of present amino groups. The solution was then introduced in the previously calibrated pHmeter and 
initiated titration with standardized NaOH 0.1 M. A typical potentiometric titration curve was obtained [12]. 

The titration values were applied to Equation 1, where M represents the sample mass, NA and NB are the 
concentrations of the acid and base, respectively, and VA and VB are their corresponding volumes. The molar 
masses of the deacetylated and acetylated monomers were considered as 161 g/mol and 204 g/mol, respectively. 
The calculated degree of deacetylation (DD) was then compared with the reference value provided by the 
manufacturer.        

𝜑 =  𝑁𝐴𝑉𝐴 − 𝑁𝐵𝑉𝐵 / 1000 

𝐷𝐷% = (𝜑 / 𝑀 −
161𝜑

204
 + 𝜑) 𝑥 100 (1) 

The analysis of X-ray diffraction (XRD) of the chitosan and adipic acid was performed on Shimadzu XRD 7000 

from the powdered material with 2θ ranging from 3.0° to 40.0° with a step 0,06°. Chitosan was evaluated based on 



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the monoclinic and orthorhombic structures using it for copper Kα radiation6 the areas under the curve were 
determined using baseline correction and integration, identifying the crystalline fraction (AC) and amorphous 
fraction (AA). These areas were calculated using the Microcal Origin® 8.5 software. The degree of crystallinity 
(GC) was then estimated using Equation 2. 

𝐺𝐶 = 𝐴𝐶/(𝐴𝐶 + 𝐴𝐴) ×  100     (2) 
FTIR spectra of the samples were acquired using a Bruker ALPHA II FT-IR spectrometer, operating at a 

resolution of 4 cm⁻¹ with 32 scans accumulated for each measurement. 
 

2.4. Hydrogels Characterization 
Qualitative visual observations were made taking into account the solubility, miscibility, coloring and phase 

segregation of the films. The average film thickness was measured with a Mitotoyo (Â±10μm) micrometer.  
Morphological analysis of the films was performed in scanning electron microscopy (SEM), equipment 

SHIMADZU SSX-550. The samples were covered with a thin gold layer by "sputtering" and positioned at the the 
maximum distance of the target, thus preventing damage to the films.  

The X-Ray Diffraction (XRD) was performed on SHIMADZU XRD 7000 equipment with 2θ ranging from 3.0 
° to 40.0 ° with step 0.06°.  The degree of crystallinity of the films was calculated using the same methodology as 
applied to the raw materials. 

The swelling degree (SD) determination, the films were carefully weighed and transferred to an Erlenmeyer 
flask containing 50 mL of buffer solution at pH 4 and pH 7 (Hanna Instruments, Brazil). The samples were 
immersed in the solutions for 120 minutes, with swelling measurements taken at regular intervals (30, 60, 90, and 
120 minutes) at ambient temperature. The swelling degree was calculated based on the variation between the initial 
mass (M0) and the final mass (Mf), as described in Equation 3. 

𝑆𝐷 =  𝑀𝑓 𝑥 𝑀0 / 𝑀0 𝑥 100%    (3) 
 

3. Results and Discussion 
Figure 1 shows a typical potentiometric titration curve obtained from the chitosan solution, where the points of 

inflection correspond to neutralization of the excess HCl solution and neutralization of free amino groups present 
in the sample respectively. For better observation Figure 1 shows the values of the first derivative of chitosan’s 
potentiometric titration curve.  

 

 
Figure 1. Potentiometric titration curve of CHI and first derivative curve. 

 
Based on the observed curve and the calculations performed using Equation 1, which relies on the volume of 

NaOH consumed at the second inflection point, the molar quantity of -NH₂ groups in the sample was determined. 
This analysis yielded a degree of deacetylation (DD) of approximately 76% ± 6, aligning closely with the value 
provided by the manufacturer (>75%) for the raw material. 

Figure 2 shows the diffractogram obtained from the sample of chitosan where two characteristic peaks are 
observed, the first of high intensity at 19.8° (d = 0.441 nm) and a lower intensity at 37.83° (d = 0.244 nm). The 
diffractogram highlights the crystalline phase of the sample; however, a broad baseline below the peaks indicates 
the presence of an amorphous phase in the feedstock. The observed angles in the diffractogram can be attributed to 
specific crystallographic planes (2 0 0) for the high-intensity (1 2 4) to the low intensity. From the diffractogram of 
chitosan, it was possible to estimate the crystallinity by 17% [13, 14]. 

Figure 3 shows the diffractogram obtained from the sample of adipic acid where four peaks of highest intensity 
are observed, the first at 12.9° (d = 0.669 nm), the second high intensity at 21.4° (d = 0.411 nm), the third at 25.7° 
(d = 0.345 nm) and fourth peak at 31.2° (d = 0.289 nm) After analysis and calculations it was observed that the 
sample is 100% crystal clear. 

 



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Figure 2. Diffractogram of raw material chitosan. 

 

 
Figure 3. Diffractogram of the raw material adipic acid. 

 

Figure 4 shows the FTIR spectrum of chitosan, highlighting its characteristic functional groups. Key 

absorption bands include the broad band around 3400 cm⁻¹, attributed to O-H and N-H stretching vibrations, and 

peaks near 2900 cm⁻¹ associated with C-H stretching. The amide I band, typically found around 1650 cm⁻¹, and the 

amide II band, near 1550 cm⁻¹, are indicative of residual acetyl groups. Additionally, the peak at approximately 

1375 cm⁻¹ corresponds to the C-H bending of the CH3 groups, while the region around 1150-1025 cm⁻¹ reflects the 
C-O-C stretching of the glycosidic bonds in the polysaccharide backbone. These spectral features confirm the 
molecular structure and degree of deacetylation of the chitosan sample [15, 16]. 
 



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Figure 4. FTIR spectrum of chitosan showing its characteristic functional groups. 

 
Figure 5 presents chitosan and adipic acid films in three conditions: without crosslinking (Film A), crosslinked 

with 0.06% EDAC (Film B), and crosslinked with 0.11% EDAC (Film C). All films exhibit a thickness of 

approximately (75 ± 25μm). 
 

 
Figure 5. Chitosan films with adipic acid were: (A) 0.0%, (B) 0.6% and (C) 0.11% of EDAC. 

 

A qualitative analysis of the films was performed through visual observation, and the results are summarized in 
Table 1. This analysis provides insights into the physical appearance, homogeneity, transparency, and surface 
characteristics of the films under different crosslinking conditions. 

 
Table 1. Evaluation by qualitative observation of the films produced. 

Identification of hydrogels Segregation Opacity Coloring Flexibility 

A Segregated Opaque Yellowish Flexible 

B Segregated Opaque Yellowish Flexible 

C Segregated Transparent portions Yellowish Flexible 

 
Figure 6 shows the SEM images of the films, revealing dendritic formations resulting from the crystallization 

process during film formation. These formations are noticeably reduced with the addition of EDAC, indicating its 
effect on modifying the film structure. Comparing the images of Films A, B, and C, Film C appears to exhibit a 
more amorphous structure, likely due to the increased crosslinking in comparison to Films A and B. 
 

 
Figure 6. Images from films of chitosan and adipic acid with (A) 0.0%, (B) 0.6% and (C) 0.11% EDAC obtained by SEM. 



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In analyzing the diffractogram of the films compared to that of pure chitosan (raw material), it was observed 
that areas of the peaks representing the chitosan showed a relative decrease mainly after the addition of EDAC. 
The relative crystallinity of the films was higher than that of pure chitosan due to the high crystallinity obtained 
with the addition of adipic acid. After the addition of the activator (EDAC), there is a reduction in crystallinity from 
the diffractograms of the films B (48%) and C (45%) compared with the film A (88%) (Figure 7), suggesting that the 
occurrence of crosslinking reduces the mobility of the chains, preventing their crystalline organization. This result 
was anticipated, as the addition of EDAC promotes chemical crosslinking, which takes precedence over the ionic 
crosslinking naturally present in chitosan films when combined with diacids. This shift enhances the structural 
integrity and reduces the crystallization tendencies of the films [9, 17]. 
 

 
Figure 7. Diffractogram of the films A (EDAC 0,00%), B (EDAC 0,06%) and C (EDAC 0,11%). 

 

Figure 8 illustrates the swelling curves of the films at pH 4, while Figure 9 shows the swelling degree of the 
films at pH 7. These figures provide a comparative analysis of the films' responsiveness to different pH conditions, 
highlighting the influence of pH on their swelling behavior. 
 

 
Figure 8. Profile of swelling of the films A (EDAC 0,00%), B (EDAC 0,06%) and C (EDAC 0,11%) at pH 4. 

 



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Figure 9. Profile of swelling of the films A (EDAC 0,00%), B (EDAC 0,06%) and C (EDAC 0,11%) at pH 7. 

 
The swelling process occurs in three main steps: 1) Diffusion of solvent molecules into the polymeric network; 

2) Relaxation of polymer chains as a result of hydration; and 3) Expansion and swelling of the polymeric network, 
also known as matrix swelling. However, as the degree of crosslinking increases, fewer hydrophilic groups are 
exposed to water, reducing their ability to diffuse through the polymer network. Consequently, this results in a 
lower swelling degree for Films B and C compared to Film A, which has a lower degree of crosslinking [18, 19]. 
 

4. Conclusion 
Chitosan films were prepared using adipic acid, with or without the addition of the acid activator, a water-

soluble carbodiimide. The characterization of the raw material not only confirmed the chemical composition of the 
material but also allowed for analysis and comparison with the films produced. The film characterization revealed 
that the use of adipic acid as a crosslinking activator enhances the reactivity of the films due to its functional 
groups, promoting covalent crosslinking over ionic crosslinking. Additionally, changes in the crystallinity pattern 
of the films were observed. The addition of carbodiimide resulted in an increased amorphous portion of the film, 
which correlated with a decrease in the degree of swelling. This reduction in swelling is associated with more 
efficient crosslinking within the films. 
 

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https://doi.org/10.1016/0008-6215(92)80075-c
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