A dynamic chitosan-based self-healing hydrogel with tunable morphology and its application as an isolating agent

Abstract

A biopolymer chitosan based 3D hydrogel framework (ChF) with good transparency and rapid self-healing activity has been successfully synthesized and utilized to get high purity separation of long-lived fission products ¹⁵²Eu (T_1/2 = 13.33 a) and ¹³⁷Cs (T_1/2 = 30.17 a) employing solid liquid extraction (SLX) technique. The unique properties of covalently cross-linked polymeric ChF have been analyzed through FT-IR, solid-state ¹³CP/MAS NMR, rheological measurement, SEM, AFM, TG-DTG, DSC, XRD and swelling study. Rheological analysis reveals that the maximum mechanical strength of a hydrogel is achieved upon synthesis using 1 : 0.43 chitosan to formaldehyde stoichiometry (ChF3). A plateau of storage modulus (G′), over a wide range of angular frequency of ChF reveals its visco elastic nature. On varying the cross linking of the hydrogel network, we found a precise tuning of morphology and properties of ChF. This ChF was utilized to separate ¹⁵²Eu from a mixture of ¹⁵²Eu and ¹³⁷Cs in aqueous HCl medium (pH 5) using SLX technique and it was observed that ChF specifically adsorbs ¹⁵²Eu without any contamination from ¹³⁷Cs. ¹⁵²Eu was back extracted easily using 1 M aqueous HCl from ¹⁵²Eu-adsorped-ChF. The adsorption of ¹⁵²Eu on ChF is influenced by pH, amount of hydrogel, time of contact, temperature, cross-linking density and dosage of γ-radiation. To establish the self-healing property of the gels, rheological analysis through step-strain measurements (strain = 0.1 to 100%) at 25 °C was monitored. The polymeric hydrogel network displays a reversible sol–gel transition for several cycles due to the dynamic equilibrium between the Schiff base linkage and the aldehyde and amine functional groups.

---

Introduction

Hydrogels are cross-linked viscoelastic solids embedded with highly porous 3D hydrophilic network chains.¹ Generally, hydrogels are synthesised from a material containing natural or synthetic polymeric network chains which in turn is produced by the reaction of one or more co-monomers.^2,3 Among the materials, the biopolymer based hydrogel is very useful in the academic as well as in the industrial world.⁴ Amidst numerous biopolymers, cellulose and chitin are not only abundant but also nontoxic. Chitin and chitosan are co-polymers containing N-acetyl-glucosamine and N-glucosamine units in a random manner. Presence of more than 50% of the N-acetyl-glucoseamine monomer in a biopolymer is termed as chitin, likewise more than 50% presence of N-glucoseamine monomeric units in a biopolymer is termed as chitosan.⁵ Chitosan based hydrogels are very useful for research as their morphology can be tuned depending on pH, cross-linking agent, temperature. They uphold properties like high surface area, highly porous network, transparency in appearance. Furthermore, the dilute acidic solution of chitosan can be easily cast into films and fibers and can be coagulated into well-defined spherical particles by spraying into alkaline solution. Softness, good adsorption capacity, high water retention capacity biocompatibility, biodegradability, high swelling capacity and greater mechanical strength imparts uniqueness to the chitosan based hydrogels.^6,7

Applicability of chitosan based hydrogels are of prime importance in the fields of adsorption,⁸ waste water treatment,^9,10 catalysis,^11,12 agriculture,¹³ sensors,^14,15 biomedical field as tissue engineering,^16,17 self-healing activity,¹⁸ controlled drug delivery¹⁹ and protein chromatography²⁰ etc.

Fission products such as ¹⁵²Eu (T_1/2 = 13.33 a) and ¹³⁷Cs (T_1/2 = 30.17 a) are extremely hazardous if allowed to accumulate in the environment.^21,22 In order to achieve separate confinement of these two long-lived fission products, separation of these products is indispensable. For high selective adsorption and separation of cations (including fission products), hydrogel must have porous network structure and high surface area.^23,24 In the last few decades, researchers has been trying to design such type of hydrogel materials with different cross-linking moiety.²⁵ Chitosan being a polycationic biopolymer, is one of the most well-known precursors in this area.

Among numerous biomedical applications such hydrogels are largely used in self-healing or wound repairing. These self-healing or self-repairing processes of the hydrogels allow the autonomic healing to repair damage without expending any external energy and are sometimes employed via some external stimuli such as pH, temperature, heat, light, or redox.^26,27 Therefore, chitosan based cross-linked hydrogel has been a hot area of research for several years.

We have designed chitosan/formaldehyde based transparent hydrogels by using simple Schiff base formation reaction taking chitosan and formaldehyde as starting materials.²⁸ To know the efficacy of this hydrogel, SLX technique has been utilized to separate long lived fission products such as ¹⁵²Eu (T_1/2 = 13.3 a) and ¹³⁷Cs (T_1/2 = 30.17 a). Reported separation techniques for fission products ¹⁵²Eu and ¹³⁷Cs are many, and a few are listed in this paper. A few such ones are by the use of an amide type open chain crown ether, N,N,N,N-tetraphenyl-3,6-dioxaoctanediamide (TDD).²⁹

Separation of ¹⁵²Eu and ¹³⁴Cs was also reported using inorganic ion exchangers such as zirconium vanadate and ceric vanadate.³⁰ Roy et al. reported the greener analytical technique in which PEG based aqueous biphasic technique is used to study the extraction and separation behaviour of ^152,154Eu and ¹³⁷Cs.³¹ In the present work, SLX technique using dynamic chitosan-based hydrogels as solid phase is employed for specific isolation of ¹⁵²Eu from a mixture of ¹⁵²Eu and ¹³⁷Cs, on the active sites of the hydrogel surface. So, after extraction, the particles which are in the solid phase can be desorbed by using the solvent with greater affinity for the analyte. Adsorption behaviour of ¹⁵²Eu and ¹³⁷Cs using SLX technique are also investigated by varying the pH, amount of hydrogel, time of contact, different cross-linking ratio in ChF, temperature and γ-radiation dosage. Besides, the hydrogel network shows rapid self-healing property.

Experimental

Materials

Starting materials for the synthesis of hydrogel were low molecular weight chitosan (LMWC) (SRL), glacial acetic acid (purity: 99%) (RANKEM), formaldehyde (Sigma-Aldrich) and absolute ethanol (Merck). All other reagents employed for this study are of analytical grade. The radioisotopes, ¹⁵²Eu and ¹³⁷Cs, were procured from Board of Radiation and Isotope Technology (BRIT), Trombay, India. A 2 mL stock solution was prepared by mixing 20 000 dps ¹⁵²Eu and ∼60 000 dps ¹³⁷Cs solution, both individually procured from BRIT. From this stock solution 100 μL were used for each batch of extraction. A CANBERRA made well type HPGe detector of 30% relative efficiency was used for radiometric studies. The energy and efficiency of the detector was calibrated by ¹⁵²Eu and ⁶⁰Co sources of known strength. The photopeaks 121.78 keV and 661.662 keV were monitored for ¹⁵²Eu and ¹³⁷Cs respectively.

Characterization of hydrogels

FTIR, solid-state ¹³C CP-MAS NMR were employed to investigate the chemical structure of a compound. FTIR of the sample was recorded on Fourier-Transform Infrared (FTIR) spectrophotometer, (JASCO, FT/IR-460 PLUS) using KBr pellet method in the range of 500–4000 cm⁻¹.

Solid-state ¹³C CP-MAS NMR spectra were performed on a Bruker spectrometer at 500 MHz equipped with an HP amplifier 1H 500 MHz, 200 W CW and with a pulse amplifier M3205. In this measurement, spin rate, ð/2 pulse, relaxation delay and contact time for the cross-polarization experiment were 8.0 kHz, 3.5 μs, 5 s and 1 ms respectively. Spectra were obtained with 1024 words in the time domain, zero-filled, and Fourier-transformed with a size of 2048 words. For each sample 1000 scans were performed.

To determine storage modulus (G′) and loss modulus (G′′) of the synthesized cross-linked polymeric hydrogel rheological measurement were performed. Strain and frequency-sweep experiments were carried out with an AR-G2 rheometer (TA Instruments) in oscillatory mode using 40 mm parallel plate geometry with a gap size of about 1 mm at 25 °C. The strain and frequency-sweep experiments of the ChF hydrogel were done by varying the angular frequency from 0.1 to 100 rad s⁻¹ with a constant shear strain of 2% which was determined to be within the linear viscoelastic (LVE) regime.

For observing the porosity and surface morphology scanning electron microscopic (SEM), field emission-scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM) studies were carried out. Hitachi S4800 was used for SEM analysis. Completely dry powder of hydrogel was coated with gold using a gold sputter module in a high-vacuum evaporator. The gold coated samples were randomly scanned and then imaged at an acceleration voltage of 15.0 kV. FE-SEM was executed on a Carl-Zeiss Sigma instrument. Before the analysis, dry gel samples were coated with gold–palladium alloy for 1 min under high vacuum. AFM was performed with a semi-contact mode (NT-MDT Solver Next) of the film deposited ChG. Scans were carried out over a 5 × 5 mm area with a speed of 0.5 Hz.

To check the thermal behaviour of the ChG hydrogel, thermogravimetric (TG) analysis and differential (DTG) thermogravimetric analysis were performed Perkin Elmer Diamond TG-DTA (TG/DTA 6300). 3.228 mg of dry powdered sample was taken in an aluminum pan under dynamic nitrogen under a flow rate of 20 mL min⁻¹ atmosphere and heated between 32.78 to 800 °C at a heating rate of 10 °C min⁻¹.

The thermal property of ChG hydrogel was measured by Differential Scanning Calorimetry using DSC Q100 V9.9 Build 303 instrument. About 5 mg of powder form of hydrogel was placed inside an aluminium sample pan. An empty sample pan was used as a reference. The thermal analysis was performed from −50 °C to 150 °C and from 150 °C to −50 °C at the heating rate of 10 °C min⁻¹ under dry nitrogen atmosphere with a flow rate of 50 mL min⁻¹. Thermal Advantage (TA) universal analysis software was used for data analysis. The inflection of the DSC curve was used to determine glass transition temperature (T_g) by TA universal analysis software, and in the DSC curve crystallization peak was referred as the crystallization transition temperature (T_c).

To check the effect of cross-linking on the hydrogel morphology, X-ray diffraction studies of the ChG hydrogel have been carried out by the D8 Advance X-ray diffractometer of BRUKER with a parallel beam optics attachment. The X-ray diffraction study was carried out at a 35 kV voltage and 30 mA current. The X-ray diffraction was employed using Ni-filtered CuKα radiation. The instrument was calibrated with a standard silicon sample. Samples were scanned from 5° to 80° (2θ) in the step scan mode (step size 0.030, preset time 2 s) and the diffraction patterns were recorded using a scintillation scan detector.

pH dependent swelling study

The ChF hydrogel is a cross-linked polymeric hydrogels which is swelled but not dissolve when water or any other solvent enters it. The swelling properties, which usually use to measure the degree of swelling (S_W) to define hydrogels was measured using 0.1 g of hydrogel in 2 mL of a buffer solution of various pH (2.5–8.5) at room temperature. The final weight (W_s) of the hydrogel was calculated after 2 h. The swelling ratio was calculated using the following equation

S_W (%) = [(W_s − W_d)/W_s] × 100

where W_s is the weight of the swollen hydrogel at experimental temperature and W_d is the weight of the dry hydrogel.

Radiochemical separation

Stability of the hydrogel at various pH was investigated for successful separation of ¹⁵²Eu and ¹³⁷Cs. Solutions of varying pH were prepared from HCl and NH₃ solution using proper dilutions. The pH values were measured using a digital pH meter (Thermo Scientific). In this study, 0.1 g of the hydrogel was added to the various concentration of HCl solution and mechanically shaken for 10 min. Then the systems were kept for 10 min and its stability was checked.

Solid liquid extraction was performed for the separation of long-lived radionuclides such as ¹⁵²Eu (T_1/2 = 13.33 a) and ¹³⁷Cs (T_1/2 = 30.17 a) with 3 mL of 10⁻⁶ to 1 M of HCl solution against 0.1 g of hydrogel as solid phase. After that 0.1 mL stock solution containing ¹⁵²Eu and ¹³⁷Cs was added to this system and was mechanically shaken for 10 min. Then system was allowed to stand for 10 min and finally the system was centrifuged at 6000 rpm for 5 min to achieve complete phase separation before collecting 2 mL of each phase for the γ-spectroscopic studies.

Afterwards the solid liquid extraction was carried out using the varying amount weight (0.05 to 0.8 g) of hydrogel as solid phase and 4 mL of 10⁻⁵ M HCl as liquid phase. Similarly shaking and settling time of the system was also varied keeping the amount of hydrogel fixed at 0.5 g.

To see the effect on solid liquid extraction with changing the amount of cross-linking agent i.e. formaldehyde, the hydrogel was prepared using varying amount (1.5, 2, 3, 4, 5 mL) of formaldehyde. Solid liquid extraction was then performed by taking 0.5 g of each dry hydrogel as solid phase and 4 mL 10⁻⁵ M aqueous solution of HCl as liquid phase. Then 0.1 mL stock solution of ¹⁵²Eu and ¹³⁷Cs was added to this mixture and the system was mechanically stirred for 15 min and then the system was allowed to settle for 15 min and finally it was centrifuged at 6000 rpm for 5 min for complete phase separation before collecting 2 mL of each phase for the γ-spectroscopic studies.

Separation experiment were performed by placing 0.5 g of dry ChF and 4 mL aqueous solution of HCl at pH 5 in a series of glass tubes keeping the temperature at 10, 25, 40, 60, 75, 90, 95 °C for 5 min. The tubes were agitated on a shaker for 10 min and then the system was allowed to settle for 15 min and finally it was centrifuged at 6000 rpm for 5 min and each phase was measured by the γ-spectroscopic studies.

Desorption of ¹⁵²Eu from hydrogel phase was carried out with 0.8 M HCl to obtain ¹⁵²Eu in liquid phase. In this study, 4 mL of 0.8 M HCl solution were added to ¹⁵²Eu containing hydrogel phase. Then system was mechanically shaken for 10 min. Then system was kept still for 10 min and finally the system was centrifuged at 6000 rpm for 4 min to achieve complete phase separation before collecting 2 mL of each phase for the γ-spectroscopic studies.

Radiation stability and its effect on the adsorption of ¹⁵²Eu on hydrogel (0.4 g) were measured by gamma irradiation of 0.4 g of hydrogel phase using GAMMA CHAMBER 1200. In this process, hydrogel phase was irradiated with various dosage of γ-radiation ranging from 1 to 15 kGy. After irradiation, SLX technique was carried out using irradiated hydrogel phase. In each set of extraction 3 mL of 10⁻⁴ M of HCl and 0.2 mL of ¹⁵²Eu was added. Then the system was mechanically stirred for 10 min and then the system was allowed to settled for 10 min and finally it was centrifuged at 6000 rpm for 4 min for complete phase separation before collecting 2 mL of each phase for the γ-spectroscopic studies.

Gamma-spectroscopic measurements were carried out using HPGe detector of 2.7 keV resolutions at 1332 keV. The energy as well as efficiency calibration of the detector was carried out with the standard ¹⁵²Eu source of known activity.

Self-healing experiment

ChG were prepared by the aforementioned process (chitosan to formaldehyde ratio 1 : 0.43) and taken in a Petridish. These hydrogels were cut into two half. One half of this hydrogel was adsorbed in a trace amount of rose bengal to show a color difference. The two different colored semicircles were jointly put to form a united disk and at different time intervals, photographs were taken to record the appearance of the united gel.

Rheological analyses were carried out to investigate qualitatively the self-healing process. In brief, a gel was prepared by the process described above and examined for the storage moduli G′ (∼1150 Pa, Fig. 4(a)). The G′ values versus time of the hydrogel were recorded (Fig. 4(b)). The G′ versus shear stress was also performed.

Synthesis of chitosan based hydrogel

The chitosan based hydrogel was prepared by using simple Schiff base condensation reaction.⁵ At first, 0.1 g of chitosan was dissolved in 7 mL 1% aqueous acetic acid. After removal of insoluble impurity this solution of chitosan was stirred and heated to 60 °C. To this mixture, various amounts (1.5, 2, 3, 4, 5 mL) of 5 M ethanolic solution of formaldehyde, used as cross-linking agent, was added drop-wise. The hydrogel became transparent after cross-linking and the time taken for gelation between 3 to 20 min. The hydrogel so formed, was then washed thoroughly with distilled water, followed by ethanol and dried in a freeze drier before further experimental application (Table 1).

Table 1 The composition for synthesis of ChF at 60 °C

Sample no.		Cross-linker (mL)	Chitosan/cross-linker ratio	Gelation time (min)
a Critical gelation concentration.
ChF1	0.1 g chitosan in 7 mL 1% aqueous acetic acid	1.5 (CGC)^a	1 : 0.21	20
ChF2		2	1 : 0.29	12
ChF3		3	1 : 0.43	7
ChF4		4	1 : 0.57	6
ChF5		5	1 : 0.71	3

---

Results and discussion

In the present work we have synthesized ChF using simple Schiff base condensation reaction with cross-linking various amount of formaldehyde and the synthesis is represented in Scheme 1.

Scheme 1 Synthesis of ChF.

Structural evidence for the formation of Schiff base was confirmed by FT-IR and solid state ¹³C CP-MAS NMR spectrum.

FT-IR spectrum chitosan (Fig. S1(a)†) exhibits a characteristics broad band at 3400–3700 cm⁻¹ due to stretching vibration of O–H and N–H groups, amide (amide I) carbonyl groups shows its stretching frequency at 1651.54 cm⁻¹, N–H (amide II) angular deformation at 1553.40 cm⁻¹, C–N stretching vibration at 1417.72 cm⁻¹, symmetrical angular deformation of CH₃ at 1372.48 cm⁻¹ and C–N amino groups stretching frequency at 1320.43 cm⁻¹ and stretching vibration of C–O–C at 1072.10 cm⁻¹. FT-IR spectrum of ChF (Fig. S1(b)†) displays a strong characteristic absorption band at 1590.94 cm⁻¹ due to imine bond and stretching vibration of bridged C–O–C bond at 1072.10 cm⁻¹.^32,33 The IR spectroscopy confirms the Schiff base formation in the hydrogel by the characteristic peak at 1590.94 cm⁻¹. The most significant FT-IR peak of chitosan and ChF is shown in Table S1.†

¹³C NMR spectrum of ChF (Fig. S2(b)†) shows a resonance signal at 104.21 ppm for C₁ carbon of the glucosamine unit, while at 99.68 ppm a much less intense signal of C′₁ of the acetylglucosamine unit is observed, and at 161.19 ppm the most significant peak for C₇ carbon is found which shows the Schiff base formation in the hydrogel.^34,35 The most significant ¹³C NMR spectrum peak of ChF is listed Table S2.†

To understand the mechanical property of a 3D cross-linked polymeric network, hydrogels were synthesized by alternating the stoichiometry of chitosan and formaldehyde. The frequency dependence of the G′ for the polymeric hydrogels was determined in the angular frequency range of 0.1 to 100 rad s⁻¹ and a plateau region of G′ was found at ∼137 Pa for the hydrogel prepared at 1 : 0.21 chitosan to formaldehyde stoichiometry (Fig. 1(b)). By increasing the cross-linking density, the values of G′ increase upto hydrogel ChF3 (∼925 Pa) and then again decreases for hydrogels ChF4 and ChF5 (Fig. 1(b)) respectively. This is because the elastic free energy of a hydrogel network depends upon the number of active polymer chains between the 3D cross-linked polymeric network.³⁶⁻³⁸ For the same reason, the plateau G′ for the polymer gels with 1 : 0.43 chitosan to formaldehyde stoichiometry ratio could be decreased from ∼925 Pa to ∼137 Pa, when the cross-linking density was decreased by changing the stoichiometry of chitosan to formaldehyde from 1 : 0.43 (ChF3) to 1 : 0.21 (ChF1) as shown in Fig. 1(b). Further increase in formaldehyde concentration did not increase G′ value for ChF4 and ChF5.

Fig. 1 (a) Strain sweep (ChF2) (b) frequency sweep plot of various hydrogels with different cross-linking ratios.

Strong elastic polymeric hydrogel with covalent cross-linked network has two distinctive properties: the damping factor (G′/G′′) exceeds by about 2 orders of magnitude and G′ exhibiting a plateau region in wide frequency range, independent of angular frequency.³⁹ Here the damping factor, calculated from the rheological data, was varied between 4 × 10⁻³ and 2.5 × 10⁻¹ and the hydrogel showed a wide plateau region. Hence, ChF are more elastic than it is viscous.

The morphology of the ChF hydrogels has been examined by SEM, FE-SEM and AFM. ChF exhibits rough fiber-like morphology while starting material chitosan has a smooth, dense and flat morphology. The rough surface morphology of ChF hydrogel may be due to chemical cross-linking of formaldehyde with primary amine of chitosan that might induce modifications of chitosan properties. This chemical modification of ChF is favorable for adsorption. The SEM image of ChF (Fig. 2(a), S3(a) and (b)†) was also taken for various hydrogels at different chitosan to formaldehyde ratio. Furthermore, FE-SEM analysis (Fig. 2(b)) reveals the formation of well-defined fibrils by ChF that are entangled with each other upon gel formation. Fig. 2(c), (d) and S3(c–f)† shows the AFM images of the hydrogel formed, which also supports the porous entangled fiber formation in ChF hydrogels.^9,40,41

Fig. 2 (a) SEM (b) FE-SEM, (c) and (d) AFM images of ChF3.

Thermo Gravimetric Analysis (TGA) exhibits thermal stability and the degradation behavior of the polymer matrix for ChF, as displayed in Fig. S4(a).† The thermal degradation of ChF takes place in two steps. The first weight loss of ChF takes place from 39–100 °C with a weight loss of ∼2% which is due to the loss of moisture from the hydrogel. The hydrogel contains many hydrophilic groups that hold water more tightly in the backbone of hydrogel through polar interaction.^42–44

The second stage of degradation displayed a rapid weight loss at 100–450 °C and reached maxima at 170 °C with weight loss of 63%. This phase of the weight loss is due to a series of thermal and oxidative decomposition in the process including the sugar ring dehydration, degradation, molecular chain acetaminophen and N-deacetylation of the cracking unit of chitosan and vaporization and elimination of volatile products. It is further stated that pyrolysis of polysaccharides starts by a random split of the glycosidic bonds, followed by further decomposition forming acetic and butyric acids and a series of lower fatty acids, where C₂, C₃, and C₆ predominate. The DTG curve shows its maxima at about 120 °C.⁴³

DSC thermograms of ChF (Fig. S4(b)†) clearly demonstrates that water evaporation happens during the first DSC run and reveals a prominent endothermic peak at 62.81 °C. This is because that the strong inter- and/or intra-molecular hydrogen bonding is present in hydrogel framework as ChF contains a lot of hydrophilic –OH group and these consequences is confirmed by the previous TGA analysis.^45,46 Glass transition temperature (T_g) and crystallization transition temperature (T_c) can also be obtained from DSC thermograms. These two parameters are controversial aspects because during the course of ChF formation, natural biopolymer chitosan is used and some properties of chitosan such as degree of deacetylation, molecular weight and crystallinity displays extensive variations according to their source and the method of extraction and hence influence the factors. For ChF crystallization transition temperature (T_c) and glass transition temperature (T_g) are 26.47 °C and −18.24 °C respectively which also nicely collaborates with our previous work.⁹

The powder X-ray diffractograms of ChF are employed to analyze the effect of cross-linking on the hydrogel architect. The powder XRD of ChF (Fig. S5†) reveals one of low intensity peaks at 26.17° and a sharp crystalline peak at 11.02°and hence the Crystallization Index (CrI) value of ChF is 17.92. But the starting material chitosan has CrI value 69.58 being a semicrystalline polymer.⁹ Therefore, CrI of ChF is lower than chitosan which is because of incorporation of the cross-linker into chitosan moiety which leads to decrease in intensity and broadening of both the peaks suggesting the amorphous nature of ChF. It results from decrease in the number of free NH₂ groups on chitosan through cross-linking and as a consequence a large number of hydrogen bonds in the chitosan powder are also destroyed. Thus regularity of the packing of the original chitosan is efficiently destroyed which results in the formation of amorphous hydrogel, ChF.^47,48

pH dependent swelling studies

The effect of extent of cross-linking on pH dependent swelling behavior of ChF at room temperature is shown in Fig. 3. The figure shows that maximum swelling was observed for ChF3 and the swelling ratio for ChF1, ChF2, ChF4 and ChF5 were low. For ChF1 and ChF2 was low because the overall cross-linking density in the hydrogel depends on the amount of cross-linking agent, and when the extent of cross-linking in the hydrogel was low, the swelling ratio of the hydrogel was also low. But ChF4 and ChF5 contained excessive amount of cross-linking agent. Higher the density of the cross-linker, greater will be the number of cross-linked points in the ChF network, which results in a decrease in network volume and hence shows low swelling capacity.⁴⁹ In case of ChF3, the amount of cross-linking agent used is neither low nor high, therefore high network volume and hence higher swelling are observed.

Fig. 3 pH dependent swelling studies of ChF of different cross-linking ratios.

ChF also shows various swelling capacity at different pH. From the figure it is clear that ChF has higher swelling capacity at lower pH. At low pH there was an extensive hydrogen bonding, osmosis and charge repulsion in the ChF network because in acidic medium unreacted amino groups of ChF converted into the ammonium ion and thus the ammonium ion would be attached to the hydrogels by ionic bonds and hence, swelling of the hydrogels increased in acidic pH. At higher pH, unreacted amino group in ChF framework remains intact and hence lower swelling ratio. So, greater number unreacted amino groups are an important parameter for higher swelling ratio.^9,50

Application of ChF in radiochemical separation

The ChF is stable within pH range 1 to 6. Extraction behaviour of long-lived fission products such as ¹⁵²Eu and ¹³⁷Cs was studied using SLX technique at the pH range 1 to 6 using aqueous HCl solution as liquid phase against 0.1 g of hydrogel as solid phase.

It was observed from the profile (Fig. 4(a)) that the extraction of ¹⁵²Eu increases with decreasing acidity while that of ¹³⁷Cs was very throughout the entire pH range. Maximum extraction (∼36%) of ¹⁵²Eu was observed at pH 5. The high extraction of Eu was probably due to the formation of [Eu(H₂O)]³⁺ complexes at pH ∼ 5. At lower pH the H⁺ ions compete with the metal ions for occupancy of the active site of the hydrogel.⁵¹ Therefore, the extraction profile of ¹⁵²Eu and ¹³⁷Cs indicating the cation exchange behaviour of the hydrogel at pH ∼ 5. The extraction profile also nicely corroborates with our earlier studies.^9,21,52,53

Fig. 4 SLX profile of ¹⁵²Eu and ¹³⁷Cs in solid phase by varying (a) pH of liquid phase on (ChF) against 0.1 g of ChG, (b) weight of hydrogel, at pH 5.

To see the effect of adsorption of ¹⁵²Eu on solid phase, amount of adsorbent was increased. It was observed that on increasing the amount of hydrogel from 0.1–0.8 g against pH 5, extraction of ¹⁵²Eu was increased upto 0.5 g and then it remains almost constant. However, the extraction of ¹³⁷Cs in solid phase was almost invariant with the increasing amount of hydrogel. Maximum extraction of ¹⁵²Eu was ∼75% at 0.5 g of hydrogel at pH 5 [Fig. 4(b)]. This could be due to the presence of more cation exchange sites for ¹⁵²Eu extraction with increase the amount of hydrogel.

Studies were also carried out by varying the shaking and settling time and results are shown in Fig. S6(a) and (b)† respectively. The plots reveal that the dependency on shaking and settling time was very little. When the shaking and settling time was 15 min, the extraction of ¹⁵²Eu was ∼59% and ¹³⁷Cs was almost 0%.

The amount of the cross-linking agent (i.e. formaldehyde) was varied to see the effect of ¹⁵²Eu extraction on solid phase. It was observed from Fig. 5(a), when ChF3 was used at pH 5 adsorption of ¹⁵²Eu increased to ∼78% (shaking and settling time was 15 min).

Fig. 5 SLX profile of ¹⁵²Eu and ¹³⁷Cs in solid phase by varying (a) amount of crosslinking agent and (b) temperature at pH 5.

The influence of temperature on adsorption of ¹⁵²Eu was monitored by temperature variation study. As shown in the Fig. 5(b), the adsorption of ¹⁵²Eu increases upto 75 °C temperature and then it remain almost same. Maximum adsorption of ¹⁵²Eu was 97% at 75 °C at 0.5 g of ChF at pH 5 without any contamination of ¹³⁷Cs.

The adsorption of ¹⁵²Eu by ChF was increased with increase in temperature. This is because, with increase in temperature interaction between adsorbent and adsorbate increases.⁵⁴ So, the adsorption of ¹⁵²Eu in the active site of ChF increases. With the increase in temperature there is an increase in macromolecular network in the hydrogel surface (Fig. S7(a)–(c)†), is possibly responsible for higher extraction of ¹⁵²Eu in ChF hydrogel phase.⁴³

Distribution ratios (D) and separation factors (S) of ¹⁵²Eu and ¹³⁷Cs at different experimental conditions were given in Table 2. ¹⁵²Eu was back extracted by 4 mL of 0.8 M HCl. About 80% of ¹⁵²Eu was back extracted from solid phase into liquid phase in a single run. After back extraction of ¹⁵²Eu, pure ChF was produced, and it can be reused without loss of its adsorption capacity.

Table 2 Separation factor of ¹⁵²Eu & ¹³⁷Cs

Experimental condition	DEu	DCs	SEu/Cs
Solid phase: 0.1 g ChF, liquid phase: 4 mL of 10^−5 M HCl	0.5523	0.0761	7.26
Solid phase: 0.5 g ChF, liquid phase: 4 mL of 10^−5 M HCl	3.0128	0.0397	76.12
Solid phase: 0.5 g ChF, liquid phase: 4 mL of 10^−5 M HCl, temperature 75 °C	31.4675	0.0236	1333.37

---

Effect of γ-irradiation in radiochemical separation

The extraction behaviour of ¹⁵²Eu and ¹³⁷Cs on gamma irradiated ChF is shown in Fig. S8.† ChF has three-dimensional network structure. Figure shows that the extraction of ¹⁵²Eu on gamma irradiated ChF was almost same upto 8 kGy then decreases with the increase of radiation dose. The maximum extraction of ¹⁵²Eu takes place at 8 kGy radiation and it is ∼80% but ¹³⁷Cs remain almost 0%. This trend is possibly due to the increase of macromolecular network upto 8 kGy then there is a breakdown of the macromolecular network in the ChF.^9,55

Self-healing experiment

Fig. S9,† clearly indicates that the self-healing property is based on the dynamic chemistry. Upon placing two pieces of hydrogels close together remained in air saturated with moisture, they heal automatically within 30 min. As shown in Fig. S9(b),† the two hydrogels merged into a single one with a stronger junction point demonstrating the self-healing ability of the dynamic hydrogel. Since the dynamic imine linkage between aldehyde groups and amino groups of chitosan, the polymer chains on the wound surfaces kept moving and re-cross-linked together, resulting in the recovery of hydrogel network on molecular level. As a result, the dye could spread from one half to the other, blurring the interfaces. Therefore, the hydrogel could heal itself automatically without additional stimuli. In addition, physical cross-linking, mostly due to hydrogen bonds reinforces the network formed by imine bonds.

Thixotropic hydrogels are thick under static conditions but become fluid or quasi-liquid when agitated or stressed.²⁷ This process can be continued for several number of cycles via breaking and reconstructing of the inner structure of the hydrogels. Rheological step-strain measurements under varying strain at room temperature (Fig. 6) were carried out to establish the self-healing property in the ChF hydrogels. At first, the gel was treated under very low strain (0.1%), which is substantially under the deformation limit, because of which G′ (235 Pa) showed higher value than G′′ (42 Pa). But when the strain was increased above the critical region (strain = 100%), the gel network was collapsed to form fluid or quasi-liquid state, thereby losing its mechanical stability, confirmed by the complete inversion of the G′ and G′′ value, where G′′ showed higher value than G′. When the high strain was released, the broken hydrogel network again transformed into stable gel and quickly regained its mechanical strength under the low strain of 0.1%. This self-recovery process was repeated for 3 cycles under varying degrees of strain to prove the self-healing property.⁵⁶ Despite that, self-healing re-established network through the dynamic imine bond formation via successive dissociation and recombination of primary amine and aldehyde functionalities. From here we can infer that some of the functional groups of polymer chains must exist in the hydrogel networks in a form that permits physical interactions or chemical reactions in damaged regions of gels, initiating the self-healing process.

Fig. 6 Deviation of G′ and G′′ during the continuous step strain measurement at alternate 0.1% and 100% strain (frequency = 0.1 rad s⁻¹) with time scale of the self-healing process.

Conclusions

As a deduction, we have designed a covalently cross-linked self-healing polymeric ChF hydrogel with high transparency using Schiff-base condensation reaction in a trivial and a much faster way compared to the other reported methods. The dynamic GhF hydrogel's gelation ability and structural properties have been studied. SEM, AFM and rheological measurements show evidence of typical properties of ChF which vary with the chitosan/cross-linker ratio and temperature. ChF has high mechanical strength and is also fairly stable under radiation. These gels displayed quick self-healing property at room temperature as strain sweep experiments at both low (0.1%) and high strain (100%) indicated the thixotropic property of the gel network. ChF acts as an efficient medium for high purity separation of long lived fission products, ¹⁵²Eu and ¹³⁷Cs. Furthermore, the adsorbed ¹⁵²Eu can be easily removed from the cross-linked hydrogel network and we can get pure ¹⁵²Eu. ChF can be reused without any loss of adsorption capacity. The whole technique is very rapid and environment-friendly. Such dynamic covalently cross-linked hydrogels with high mechanical stability will produce a new insight in the improvement of smart soft materials for the applications in organ repair and efficient delivery media of biologically relevant materials.

Acknowledgements

Part of this work has been carried out under the support of SINP-DAE 12^th five year plan project TULIP. Authors also want to thank stuff of UGC-DAE consortium, Kolkata centre for their help in gamma irradiation studies. Santu Maity is also thankful to University Grant Commission (UGC) for providing UGC-BSR fellowship.

References

1. J. Hao and R. A. Weiss, Macromolecules, 2011, 44, 9390.
2. E. M. Ahmed, J. Adv. Res., 2015, 6, 105.
3. A. Doring, W. Birnbaum and D. Kuckling, Chem. Soc. Rev., 2013, 42, 7391.
4. S. Maity, A. Datta, S. Lahiri and J. Ganguly, RSC Adv., 2015, 5, 89338.
5. M. N. V. R. Kumar, R. A. A. Muzzarelli, C. Muzzarelli, H. Sashiwa and A. J. Domb, Chem. Rev., 2004, 104, 6017.
6. V. G. Kadajji and G. V. Betageri, Polymers, 2011, 3, 1972.
7. M. Rinaudo, Prog. Polym. Sci., 2006, 31, 603.
8. Q. Yu, S. Denga and G. Yu, Water Res., 2008, 42, 3089.
9. Y. H. Gad, Radiat. Phys. Chem., 2008, 77, 1101.
10. T. Jiao, H. Zhao, J. Zhou, Q. Zhang, X. Luo, J. Hu, Q. Peng and X. Yan, ACS Sustainable Chem. Eng., 2015, 3, 3130.
11. F. R. Llansola, J. F. Miravet and B. Escuder, Chem. Commun., 2009, 7303, 7303.
12. M. Maity and U. Maitra, J. Mater. Chem. A, 2014, 2, 18952.
13. W. Wang and A. Wang, Carbohydr. Polym., 2010, 80, 1028.
14. C. Zhang, M. D. Losego and P. V. Braun, Chem. Mater., 2013, 25, 3239.
15. D. Buenger, F. Topuz and J. Groll, Prog. Polym. Sci., 2012, 37, 1678.
16. F. Croisier and C. Jérôme, Eur. Polym. J., 2013, 49, 780.
17. H. Tan, C. R. Chu, K. A. Payne and K. G. Marra, Biomaterials, 2009, 30, 2499.
18. Z. Wei, J. H. Yang, J. Zhou, F. Xu, M. Zrínyi, P. H. Dussault, Y. Osada and Y. M. Chen, Chem. Soc. Rev., 2014, 43, 8114.
19. W. Wu, J. Shen, P. Banerjee and S. Zhou, Biomaterials, 2010, 31, 8371.
20. N. Bhattarai, H. R. Ramay, J. Gunn, F. A. Matsen and M. Zhang, J. Controlled Release, 2005, 103, 609.
21. S. Lahiri, D. Nayak and N. R. Das, Appl. Radiat. Isot., 2000, 52, 1393.
22. S. Dragović, N. Mihailović and B. Gajić, Chemosphere, 2008, 72, 491.
23. I. Tokarev and S. Minko, Adv. Funct. Mater., 2010, 22, 3446.
24. W. Yi, H. Wu, H. Wang and Q. Du, Langmuir, 2016, 32, 982.
25. N. Bhattarai, J. Gunn and M. Zhang, Adv. Drug Delivery Rev., 2010, 62, 83.
26. S. Lu, C. Gao, X. Xu, X. Bai, H. Duan, N. Gao, C. Feng, Y. Xiong and M. Liu, ACS Appl. Mater. Interfaces, 2015, 7, 13029.
27. U. Haldar, K. Bauri, R. Li, R. Faust and P. De, ACS Appl. Mater. Interfaces, 2015, 7, 8779.
28. A. Singh, S. S. Narvi, P. K. Dutta and N. D. Pandey, Bull. Mater. Sci., 2006, 29, 233.
29. Y. H. Wen, S. Lahiri, Z. Quin, X. L. Wu and W. S. Liu, J. Radioanal. Nucl. Chem., 2002, 253, 263.
30. S. Lahiri, K. Roy, S. Bhattacharya, S. Maji and S. Basu, Appl. Radiat. Isot., 2005, 63, 293.
31. K. Roy, R. Paul, B. Banerjee and S. Lahiri, Radiochim. Acta, 2009, 97, 37.
32. E. A. Soliman, S. M. El-Kousy, H. M. Abd-Elbary and A. R. Abou-zeid, Am. J. Food Technol., 2013, 8, 17.
33. S. C. Chen, Y. C. Wu, F. L. Mi, Y. H. Lin, L. C. Yu and H. W. Sung, J. Controlled Release, 2004, 96, 285.
34. L. Yin, L. Fei, F. Cui, C. Tang and C. Yin, Biomaterials, 2007, 28, 1258.
35. J. O. Czubenko and M. G. Druzýnska, Carbohydr. Polym., 2009, 77, 590.
36. S. G. Roy, U. Haldar and P. De, ACS Appl. Mater. Interfaces, 2014, 6, 4233.
37. Y. Tang, Y. Du, X. Hu, X. Shi and J. F. Kennedy, Carbohydr. Polym., 2007, 67, 491.
38. A. Vaish, S. G. Roy and P. De, Polymer, 2015, 58, 1–8.
39. R. Jin, L. S. M. Teixeira, P. J. Dijkstra, M. Karperien, C. A. van Blitterswijk, Z. Y. Zhong and J. Feijen, Biomaterials, 2009, 30, 25441.
40. H. Y. Zhoua, Y. P. Zhang, W. F. Zhang and X. G. Chen, Carbohydr. Polym., 2011, 83, 1643.
41. J. O. Czubenko, M. Gierszewska and M. Pieróg, J. Polym. Res., 2015, 22, 153.
42. E. F. S. Vieira, A. R. Cestari, C. Airoldi and W. Loh, Biomacromolecules, 2008, 9, 1195.
43. C. G. T. Neto, J. A. Giacometti, A. E. Job, F. C. Ferreira, J. L. C. Fonseca and M. R. Pereira, Carbohydr. Polym., 2005, 62, 97.
44. G. Ma, D. Yang, Q. Li, K. Wang, B. Chen, J. F. Kennedy and J. Nie, Carbohydr. Polym., 2010, 79, 620.
45. S. J. Leea, S. S. Kim and Y. M. Lee, Carbohydr. Polym., 2000, 41, 197.
46. G. Sun, X. Z. Zhang and C. C. Chu, J. Mater. Sci.: Mater. Med., 2007, 18, 1563.
47. E. S. Costa-Júnior, E. F. Barbosa-Stancioli, A. A. P. Mansur, W. L. Vasconcelos and H. S. Mansur, Carbohydr. Polym., 2009, 76, 472.
48. J. Wua, W. Wei, L. Y. Wang, Z. G. Sua and G. H. Ma, Biomaterials, 2007, 28, 2220.
49. S. C. Chen, Y. C. Wu, F. L. Mi, Y. H. Lin, L. C. Yu and H. W. Sung, J. Controlled Release, 2004, 96, 285.
50. N. Li and R. Bai, Sep. Purif. Technol., 2005, 42, 237.
51. D. Das and S. Pal, RSC Adv., 2015, 5, 25014.
52. D. Nayak, S. Lahiri, A. Ramaswami, S. B. Manohor and N. R. Das, Appl. Radiat. Isot., 1999, 51, 261.
53. S. Lahiri, K. Mukhopadhyay and D. Nayak, J. Radioanal. Nucl. Chem., 1999, 242, 127.
54. G. Crini and P. M. Badot, Prog. Polym. Sci., 2008, 33, 399.
55. C. Tranquilan-Aranillaa, F. Yoshii, A. M. Dela Rosa and K. Makuuchi, Radiat. Phys. Chem., 1999, 55, 127.
56. Y. Zhang, L. Tao, S. Li and Y. Wei, Biomacromolecules, 2011, 12, 2894.

---

Footnote

† Electronic supplementary information (ESI) available: IR spectra, NMR spectra, SEM, AFM, TGA, DSC, XRD, SLX profile of ¹⁵²Eu and ¹³⁷Cs in solid phase (ChG) by varying shaking time, settling of the medium, γ-irradiate ChF, self-healing activity etc. See DOI: 10.1039/c6ra15138h

---