**Nanostructured Chitosan**/**Maghemite Composites Thin Film for Potential Optical Detection of Mercury Ion by Surface Plasmon Resonance Investigation**

**Nurul Illya Muhamad Fauzi 1, Yap Wing Fen 1,2,\* , Nur Alia Sheh Omar 2, Silvan Saleviter <sup>2</sup> , Wan Mohd Ebtisyam Mustaqim Mohd Daniyal 2, Hazwani Suhaila Hashim <sup>1</sup> and Mohd Nasrullah <sup>3</sup>**


Received: 26 May 2020; Accepted: 12 June 2020; Published: 4 July 2020

**Abstract:** In this study, synthesis and characterization of chitosan/maghemite (Cs/Fe2O3) composites thin film has been described. Its properties were characterized using Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM) and ultraviolet-visible spectroscopy (UV-Vis). FTIR confirmed the existence of Fe–O bond, C–N bond, C–C bond, C–O bond, O=C=O bond and O–H bond in Cs/Fe2O3 thin film. The surface morphology of the thin film indicated the relatively smooth and homogenous thin film, and also confirmed the interaction of Fe2O3 with the chitosan. Next, the UV-Vis result showed high absorbance value with an optical band gap of 4.013 eV. The incorporation of this Cs/Fe2O3 thin film with an optical-based method, i.e., surface plasmon resonance spectroscopy showed positive response where mercury ion (Hg2<sup>+</sup>) can be detected down to 0.01 ppm (49.9 nM). These results validate the potential of Cs/Fe2O3 thin film for optical sensing applications in Hg2<sup>+</sup> detection.

**Keywords:** chitosan; maghemite; optical; mercury ion; surface plasmon resonance

#### **1. Introduction**

Organic polymeric materials made up of many repeating monomer units have made a significant impact on biological and biomedical research activities because of the flexibility and the ease of fabrication [1]. One of the well-known organic polymeric materials is chitosan, easily derived from partial deacetylation of chitin with a degree of 50% or greater [2–4]. To be more specific, chitosan is a family of linear polysaccharide as a part of glucosamine and N-acetyl glucosamine units linked via β-1,4 glucosidic bonds [5,6]. Chitosan contains three types of reactive functional groups, primary amine groups and primary and secondary hydroxyl groups, respectively, at positions C-2, C-3 and C-6. Among the three types of functional groups, the primary amine groups at C-2 positions are the most favorable sites interacting with the biological molecules, metal ions and organic halogen substances. Taking the advantages of chitosan with high absorption capacity and high biocompatibility, chitosan is known as an ideal substrate for enzyme immobilization [7]. Other excellent advantages of chitosan including non-toxicity, great film-forming ability, powerful adhesion property and high mechanical strength, offers great room for sensor applications [8–10]. However, the problem of poor stableness

of chitosan because of the hydrophilic character and pH sensitivity restricts its application [11,12]. Previous reports showed that the stability of chitosan could be improved by combining with oxide or metal oxides and the product can be effectively used as recognition elements for chemical sensors and biosensors [13–15].

Iron (III) oxide or ferric oxide is the inorganic compound with the Fe2O3 formula, which varies in color depending on its phase [16]. Fe2O3 materials have four polymorphs phases such as α-Fe2O3 (hematite), β-Fe2O3, γ-Fe2O3 (maghemite) and ε-Fe2O3 [17,18]. The differences of the phases are known from their originality, for examples, hematite and maghemite are naturally obtained and the other two of phases are synthesized in laboratory [19,20]. Among the phases, γ-Fe2O3 is one of the chief interests. It is the second most common sustainable form of Fe2O3, known as completely oxidized magnetite. Maghemite has a high curie temperature, but has a lower saturation magnetization at room temperature and a supermagnetism property that makes it quite efficient in removing heavy metal pollutants from water [21,22]. Moreover, it is believed that Fe2O3 can improve and provide better mechanical properties to chitosan [23].

Accumulation of heavy metals in water and food production, primarily mercury (Hg) is the most hazardous heavy-metal pollutants even at a very low concentration. The most toxic chemical forms of Hg are ionic Hg (Hg2+), causes serious damage to human health such as brain damage, immune dysfunction and paralysis [24–26]. Therefore, the removal and detection of Hg2<sup>+</sup> in the aqueous environment are of great significance [27–31]. Among the existing optical techniques to detect Hg2<sup>+</sup> are colorimetric, fluorescent, chemosensor, electrochemiluminescence (ECL) and photoluminescent (PL) [32–34]. Though these techniques are widely used, they encounter from many drawbacks, such as high instrument operating costs, repetitive pretreatment procedures and long initiation times [35].

Corresponding to the previous methods, surface plasmon resonance (SPR) proposed a cost-effective, label-free detection method for convenient usage, rapid detection and excellent sensitivity and selectivity to heavy metal ions [36–40]. Since enormous efforts devoted to creating sensors with high sensitivity to Hg2<sup>+</sup> are greatly needed currently, selection of the metallic layer such as the gold layer is an important aid in producing higher sensor sensitivity in SPR [41]. Over the last decade, the surface SPR technique has emerged as an effective optical technique for various applications including detection of heavy metal ions [42–51]. Unfortunately, the main problem to detect optically the heavy metal ions solution is the similar refractive indices of heavy metal ions for lowest concentration, which eventually becomes the goal of researchers. Hence, many researchers have dedicated their time to develop chitosan-based materials onto SPR interfaces in lowering the detection limit of Hg2+, specifically [52–54]. A recent study documented the utilization of polypyrrole-chitosan/nickel-ferrite nanoparticles as an active layer to a prism-based on SPR technique for Hg2<sup>+</sup> sensing, which reached a limit of detection (LOD) as low as 1.94 μM [54]. Other recent studies using chitosan-based materials as sensing layers for the detection of Hg2<sup>+</sup> by SPR are summarized in Table 1. It is of interest to further improve the LOD using chitosan-based SPR sensor.


**Table 1.** Chitosan based material by surface plasmon resonance (SPR) for the detection of Hg2<sup>+</sup>.

Ref.: reference. LOD: limit of detection.

To the best of our knowledge, the study for Cs/γ-Fe2O3 composite to detect Hg2<sup>+</sup> using the SPR technique is not reported yet. There is also a lack of studies on the structural and optical properties of these composites. Therefore, an effort was made to apply the chitosan/γ-Fe2O3 thin film onto a thin gold surface, as a novel active layer for the SPR technique in sensing Hg2<sup>+</sup> as low as nanomolar. Besides, the studies of structural and optical properties of Cs/γ-Fe2O3 thin film on the gold surface are also reported and explored.

#### **2. Materials and Methods**

#### *2.1. Reagent and Materials*

The Fe2O3 was purchased from R&M Marketing, Essex, U.K. The medium molecular weight chitosan and acetic acid were purchased from Aldrich (Saint louis, MO, USA). Standard solution of Hg2<sup>+</sup> with concentration of 1000 ppm was purchased from Merck (Darmstatd, Germany).

#### *2.2. Preparation of Chemical*

Firstly, 50 mL distilled water was added into Fe2O3 (4 mg/mL). Then 10 mL of NH3 (25%) and 0.615 mg of ethylenediaminetetra acetic acid (EDTA) was added as precipitation agent and as capping agent to the solution with stirring respectively. The reaction was allowed to proceed for 1 h at 50 ◦C with constant stirring. Finally, the black precipitate of nano-Fe2O3-EDTA formed and it was rinsed with distilled water and left to dry 80 ◦C for 3 h. For chitosan preparation, 1% acetic acid was prepped by diluting stock 1 mL acetic acid with deionized water in 100 mL volumetric flask. Next, 400 mg medium molecular weight chitosan that was acquired from Aldrich was dissolved in 50 mL of 1% aqueous acetic acid and the solution vigorously stirring to ensure powder chitosan dissolved completely. To produce the nanostructured chitosan/maghemite (Cs/Fe2O3) composites, 30 mg Fe2O3 capped EDTA was dispersed in 10 mL of 0.1% in chitosan solution and sonicated in room temperature for 15 min. The Hg2<sup>+</sup> standard solution with a concentration of 1000 ppm was diluted with deionized water to produce Hg2<sup>+</sup> solutions with concentrations of 0.01, 0.05, 0.08, 0.1 and 0.5 ppm [55,56].

#### *2.3. Preparation of Thin Film*

To begin, glass slips (24 mm × 24 mm × 0.1 mm, Menzel-Glaser, Braunschweig, Germany), as a substrate, were coated with a thin layer of gold with thickness 50 nm using SC7640 sputter coater [57]. Next, approximately 0.55 mL of the chitosan, Fe2O3 and Cs/Fe2O3 composites solution was set separately on the surface of the gold coated glass slip. Then the glass slips were spun at 6000 rev min for 30 s using the Specialty Coating System, P-6708D (Inc. Medical Devices, Indianapolis, IN, USA) to produce the chitosan, Fe2O3 and Cs/Fe2O3 composites thin films.

#### *2.4. Instrumental*

Fourier transform infrared (FTIR) spectra for each surface modification of thin films were recorded in the transmittance mode using a Perkin-Elmer spectrophotometer (Waltham, MA, USA) under the wavelength range 400–4000 cm−1. The absorbance spectra of the films were recorded from 200 to 500 nm using UV-Vis-NIR spectroscopy (UV-3600 Shimadzu, Kyoto, Japan). The optical band gap energy was calculated using the data obtained. Atomic force microscopy (AFM) analysis was carried out using Qscope 250, Qesant Instrument Corporation (Quesant, CA, USA) in intermittent mode to study the topography and height of Cs/Fe2O3 thin film. An optical-based sensing method based on surface plasmon resonance (SPR) was designed to identify the potential of the Cs/Fe2O3 thin film to detect Hg2<sup>+</sup>. Figure 1 shows the schematic diagram of the SPR instrument setup [58–61]. The SPR experiment was carried out by inserting Hg2<sup>+</sup> solutions with different concentration varied from 0.01 to 0.5 ppm. It was injected one after another into the cell to bind with Cs/Fe2O3 thin film coated onto gold surface thin film. The SPR curve and resonance angle for all concentrations was monitored and recorded.

**Figure 1.** Optical setup of surface plasmon resonance spectroscopy.

#### **3. Results and Discussion**

#### *3.1. FTIR Analysis*

FTIR spectroscopy was used to identify the functional groups existed in Cs/Fe2O3 thin film. The spectrum of chitosan, Fe2O3 and Cs/Fe2O3 thin films in the range of 450–4000 cm−<sup>1</sup> are represented in Figure 2. From the FTIR spectrum of chitosan thin film, the broad absorption band at 3386.43 cm−<sup>1</sup> can be appointed to the stretching vibration of O–H. A weaker band found at 2901.26 cm−<sup>1</sup> can be attributed to C–H stretching in chitosan. Another absorption band at 1655.48 cm−<sup>1</sup> was associated with the presence of the C=O stretching bond. There is an absorption peak at 1084.47 cm−<sup>1</sup> that corresponds to the C–O group, which indicates the presence of the –COOH group in chitosan thin film. Two more bands at 500.76 cm−<sup>1</sup> and 458.22 cm−<sup>1</sup> were assigned to the C–C bond and C–N bond respectively. This finding is well aligned to the previous study by Anas et al. [62].

**Figure 2.** FTIR spectrum for chitosan, Fe2O3 and Cs/Fe2O3 thin films.

Next, a particular major peak in the Fe2O3 thin film was identified with the degree of cation vacancy, ordering between octahedral Fe cation and O atoms [63]. The absorption peak at 789.63 cm−<sup>1</sup> is a characteristic of maghemite Fe–O stretching vibrations particles. This peak is solely attributed to the high degree of cationic vacancy ordering [64]. The broad band characteristic for bending vibration of water adsorbed on the maghemite's surface is at 2078.99 cm<sup>−</sup>1. The intense bands at 1642.79 cm−<sup>1</sup> and 3153.55 cm−<sup>1</sup> were then assigned to CO2 vibration and O–H vibrations, respectively, ratifying the presence of surface γ-Fe2O3 hydroxyl groups.

In the spectrum of Cs/Fe2O3, the chitosan does not provide clear absorption bands at a lower wavenumber. This is due to the low percentage of chitosan compared to maghemite in the synthesization process. However, the presence of chitosan can be observed based on the intensity peak. The peak intensity of Cs/Fe2O3 clearly increased after the sorption of chitosan and Fe2O3, i.e., at C–H stretching (458.22 cm−1), C–C bond (611.23 cm−1) and O=C=O stretching (1630.85 cm−1). An increase in the peak intensity usually indicates an increase in the sum of the functional group (per unit volume) associated with the molecular bond [65]. On the other hand, a strong absorption band was observed at 789.63 cm<sup>−</sup>1, confirmed the presence of Fe-O as the main phase of the Fe2O3 and a band at 3110.26 cm−<sup>1</sup> that appointed to the O-H vibration of surface maghemite hydroxyl groups. Overall, the FTIR results showed the increasing peak intensity of Cs/Fe2O3, which confirmed the physical interaction of chitosan and γ-Fe2O3 in those composites.

#### *3.2. Surface Morphology*

The in situ atomic force microscopy (AFM) measurements enable the chitosan, Fe2O3 and Cs/Fe2O3 adsorption on thin films to be visualized in real time. The AFM images illustrate the topographical in the thin films as shown in Figures 3–5. The topographical can be observed by various parameters that exist to quantify the root mean square (rms) roughness of a surface. The RMS roughness value can be calculated from the cross-sectional profile or a surface area [66]. The RMS roughness obtained by chitosan, Fe2O3 and Cs/Fe2O3 thin film were 1.4 nm, 47 nm and 37.3 nm, respectively. The magnitude decreased in RMS roughness of Cs/Fe2O3 thin film compared to Fe2O3 thin film attributable to the association of two materials, which are chitosan and Fe2O3. The roughness implies that a smoothening mechanism by surface diffusion [67]. This result indicates that the presence of chitosan can enhance the surface of the thin film. The roughness introduced in the nanostructured maghemite in chitosan thin film intended appropriate form to enhance the thin film as sensing element [68]. This result is in line with the FTIR data, proving the presence of maghemite and chitosan in the Cs/Fe2O3 thin film based on the RMS roughness.

**Figure 3.** Atomic force microscopy (AFM) image of chitosan thin film.

**Figure 4.** AFM image of Fe2O3 thin film.

**Figure 5.** AFM image of Cs/Fe2O3 thin film.

#### *3.3. Optical Studies*

For the optical properties, the absorbance spectrum of the thin films was observed and measured at wavelength from 250 to 500 nm. The UV-Vis results of chitosan, Fe2O3 and Cs/Fe2O3 thin films are shown in Figure 6 it can be spotted that all of the thin film has diverse value of absorbance. From the graph, the absorbance spectra of Cs and Fe2O3 thin films were slightly higher as compared to the Cs/Fe2O3 thin film. The maximum absorption wavelength can be observed at 260–300 nm. The absorption peak about 300 nm corresponds to π→π\* transitions of C=O [69,70].

**Figure 6.** Absorbance spectrum chitosan, Fe2O3 and Cs/Fe2O3 thin films.

The UV-Vis absorbance spectrum was then quantitative analyzed based on the Beer–Lambert law theory. This law refers to a relation between the attenuation of light by a material and its properties, which the monochromatic light (single wavelength) is travelling. Since the amount of the emitted radiation intensity is only dependent on the thickness, *t* and concentration of the solution, the absorbance, *A* of the samples can be collected at a single wavelength, as follows [62]:

$$A = \log\_{10} \frac{I\_o}{I\_t} \tag{1}$$

The transmittance, *T* of sample is given by the ratio of intensities of the presence *It* and the absence *Io* of the sample:

$$T = \frac{I\_t}{I\_o} \tag{2}$$

Thus, the absorbance and transmittance can be related by:

$$A = -\log\_{10} T \tag{3}$$

Apart from the absorbance, absorbance coefficient is a useful parameter to compare samples with varying thickness. The sample thickness was obtained by using atomic force microscopy. The absorbance coefficient, α (in unit of m<sup>−</sup>1) is given by:

$$
\alpha = 2.303 \frac{A}{t} \tag{4}
$$

where *t* is the thickness of sample in unit of m. The absorbance coefficient and optical band gap can be related by:

$$\alpha = \frac{k(hv - E\_{\mathcal{S}})^n}{hv} \tag{5}$$

Rearranging Equation (5) gives:

$$(\alpha lv)^{1/n} = k(lv - E\_{\mathcal{S}}) \tag{6}$$

where *hv* is the photon energy, *h* is Plank's constant, *Eg* is the optical band gap, *k* is constant and *n* is the transition states, i.e., direct or indirect transitions. Direct transition is transition in which a photon excites an electron from the valence band to the conduction band directly if the momentum of electrons and holes is the same in both bands (conduction and valence). On the other hand, indirect transition is a photon cannot be emitted because the electron must pass through an intermediate state and transfer momentum to the crystal lattice. From these, it can be concluded that the absorption in the thin films corresponds to a direct energy gap. For direct transition, *n* = 1/2 and this value is substituted in Equation (6) and becomes:

$$(\alpha lv)^{1/n} = k(lv - E\_{\mathcal{S}}) \tag{7}$$

To evaluate the optical band gap, *Eg* of the chitosan, Fe2O3 and Cs/Fe2O3 thin films, the graphs of (α*hv*) <sup>2</sup> against hv are plotted as shown in Figures 7–9, respectively. As a result, the intersection of straight line on the edge was obtained, indicating the direct transition of the optical band gap [71]. The calculated values of the optical band gap were 4.073 eV, 4.078 eV and 4.013 eV for chitosan, Fe2O3 and Cs/Fe2O3 thin films respectively (with the corresponding error of ±0.001 eV) [72,73]. This result indicated the maghemite had a band gap energy of 4.078 eV, which was higher than to the 2 eV bulk [64]. This might be due to the structure defects, that have changed the phase, strain and size of nanoparticles during heat treatment that led to the increase of band gap [74]. When Fe2O3 added on chitosan, the band gap became lower as compared to the individual band gap. It can be due to the increased of crystallite size attributed to the confinement effects that related to the rise amount of orbitals participating in the formation of valence bands and covalent bands through orbital overlap [75]. Thus, this showed that defects and confinement effects have a huge impact on the optical properties of a composite.

**Figure 7.** Optical band gap for chitosan thin film.

**Figure 8.** Optical band gap for Fe2O3 thin film.

**Figure 9.** Optical band gap for Cs/Fe2O3 thin film.

#### *3.4. Optical-Based Sensing of Hg2*<sup>+</sup>

The optical sensing based on surface plasmon resonance (SPR) phenomenon was conducted by using Cs/Fe2O3 thin film to identify the SPR angle for deionized water as a control experiment. The SPR angle of 55.225◦ was further applied to compare the SPR angle for different concentration of Hg2<sup>+</sup> solution ranged from 0.01 to 0.5 ppm. The SPR reflectively curves for Cs/Fe2O3 thin film in contact with the different concentration of Hg2<sup>+</sup> are shown in Figure 10. It can be seen that the SPR curves of Hg2<sup>+</sup> solution shifted from 0 to 0.5 ppm as compared with the deionized water SPR curve. The SPR angle for 0.01, 0.05, 0.08, 0.1 and 0.5 ppm of Hg2<sup>+</sup> were 54.615◦, 54.398◦, 54.212◦, 54.027◦ and 53.836◦, respectively, with the corresponding error of ±0.001◦ (the resolution of the stepping motor of the SPR). Overall, it was observed that the SPR shifted to the left with increasing concentration of Hg2<sup>+</sup> solution. This finding can be attributed to the increase in binding between analyte–ligand, which resulted in the change of refractive index as well as the thickness of the Cs/Fe2O3 sensing layer [76–79]. Hence it is confirmed that Cs/Fe2O3 thin film has an affinity with Hg2<sup>+</sup> and can be integrated with SPR optical-based sensing method for detection of Hg2+.

**Figure 10.** SPR curves for Cs/Fe2O3 thin film in contact with deionized water and Hg2<sup>+</sup> solution with a concentration of 0.01–0.5 ppm.

#### **4. Conclusions**

In this study, a Cs/Fe2O3 thin film was successfully developed using the spin coating technique. The functional groups analysis from the FTIR results confirmed the correlation between chitosan and γ-Fe2O3, with the peak intensity of Cs/Fe2O3 clearly increasing after the sorption of chitosan and Fe2O3 at C–H stretching, C–C bond and O=C=O stretching. Next, the AFM result showed that the thin film was homogenous when the surface of chitosan on the thin film was covered by Fe2O3. Besides, the UV-Vis results confirmed that the Cs/Fe2O3 thin film had the lowest absorbance value compared its individual thin films with an optical band gap of 4.013 eV. The incorporation Cs/Fe2O3 thin film with the optical-based sensing method using the surface plasmon resonance technique provided positive response to the Hg2<sup>+</sup> solution of different concentrations. This result demonstrated the enormous ability of Cs/Fe2O3 thin film for optical sensing of Hg2<sup>+</sup> as low as 0.01 ppm.

**Author Contributions:** Conceptualization, methodology, writing—original draft preparation, N.I.M.F.; validation, supervision, writing—review and editing, funding acquisition, Y.W.F.; investigation, formal analysis, N.A.S.O.; software, S.S.; visualization, W.M.E.M.M.D. and H.S.H.; resources, M.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded and supported by the Ministry of Education Malaysia through the Fundamental Research Grant Scheme (FRGS) (FRGS/1/2019/STG02/UPM/02/1) and Putra Grant Universiti Putra Malaysia.

**Acknowledgments:** The authors acknowledged the laboratory facilities provided by the Institute of Advanced Technology, Department of Physics, and Department of Chemistry, Universiti Putra Malaysia.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Dynamics and Rheological Behavior of Chitosan-Grafted-Polyacrylamide in Aqueous Solution upon Heating**

#### **Mengjie Wang, Yonggang Shangguan \* and Qiang Zheng**

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China; mengjiewang@zju.edu.cn (M.W.); zhengqiang@zju.edu.cn (Q.Z.)

**\*** Correspondence: shangguan@zju.edu.cn

Received: 11 March 2020; Accepted: 13 April 2020; Published: 15 April 2020

**Abstract:** In this work, the transformation of chitosan-grafted-polyacrylamide (GPAM) aggregates in aqueous solution upon heating was explored by cryo-electron microscope (cryo-TEM) and dynamic light scattering (DLS), and larger aggregates were formed in GPAM aqueous solution upon heating, which were responsible for the thermo-thickening behavior of GPAM aqueous solution during the heating process. The heating initiates a transformation from H-bonding aggregates to a large-sized cluster formed by self-assembled hydrophobic chitosan backbones. The acetic acid (HAc) concentration has a significant effect on the thermo-thickening behavior of GPAM aqueous solution; there is a critical value of the concentration (>0.005 M) for the thermo-thickening of 10 mg/mL GPAM solution. The concentration of HAc will affect the protonation degree of GPAM, and affect the strength of the electrostatic repulsion between GPAM molecular segments, which will have a significant effect on the state of the aggregates in solution. Other factors that have an influence on the thermo-thickening behavior of GPAM aqueous solution upon heating were investigated and discussed in detail, including the heating rate and shear rate.

**Keywords:** chitosan-grafted-polyacrylamide; thermo-thickening; rheological; dynamic light scattering; cryo-electron microscope

#### **1. Introduction**

Thermo-responsive polymers have been extensively studied over the past few decades owing to their industrial and biomedical applications [1–7]. Among them, thermo-thickening has received much attention from academic and industrial fields because of its huge application potential in many fields [3,7–12], especially in oil recovery [9,11]. It is always a great challenge in oil recovery to keep high viscosity of polymer displacement agent at a high temperature because the viscosity of most current oil displacement agents will decrease upon heating or in a high-temperature environment [9,11]. Differing from most of the commonly used polymers, which can hardly solve this problem [7,13], the thermo-thickening polymers whose viscosity can be enhanced during heating process have great potential to be used in enhanced oil recovery as well as water treatment and paper manufacturing [14]. In addition, because the thermo-thickening polymers with high concentration usually present a sol–gel transition temperature, they also have great potential in controlled permeation [7], tissue engineering [13], drug delivery [2,6], and so on.

Chitosan (CS) has received tremendous interest owing to its intrinsic properties, biocompatibility, nontoxicity, amphipathic, accessibility, and abundance in nature [15,16]. It is the precursor for other applications such as drug delivery [17–19], emulsifier [20], water treatment [21–24], and so on. Furthermore, it was found that CS modified by chemical or physical methods [25,26] could present thermo-thickening behavior and can be roughly divided into two kinds: CS complexes [27–29] and the derivatives of CS [30–32]. For CS complexes, thermo-thickening behaviors were less reported thus far except chitosan/poly(vinyl alcohol) (CS/PVA) [27] and chitosan/glycerophosphate (CS/β-GP) [29]. For CS derivatives, modification by poly-*N*-isopropylacrylamide (PNIPAM) is a common way to realize thermo-thickening [13,31], and recently, the carboxymethyl chitin also shows a novel thermo-thickening, which is pH- and temperature-dependent [33]. As there are many kinds of molecular interactions involved in these aqueous solutions mentioned above, it is difficult to elucidate the molecular mechanism of the thermo-thickening behavior of these complex systems. For CS/PVA, Schuetz et al. [27] thought that CS linked with PVA through hydrogen bonds at a low temperature. With temperature increasing, hydrogen bonds were broken and hydrophobic association among hydrophobic segments of CS chains gradually increased, as a result the gel-like structure formed. In CS/β-GP, there is no clear graph presented just excluding the influence of the hydrogen bond [29]. Among CS derivatives, the mechanism of thermo-thickening for modification by PNIPAM has been acknowledged [13,31,34] universally. Owing to the lower critical solution temperature (LCST) of PNIAPM, a phase separation will happen once the temperature rises to ~33 ◦C. Recently, the carboxymethyl chitin also showed a novel thermo-thickening associated with pH-dependence [33]. However, the mechanism is also not clear. By far, except CS-*g*-PNIPAM, there is no straight evidence to illustrate their assumption or figure out the mechanism.

Chitosan-grafted-polyacrylamide (GPAM) is one of the CS derivatives that has been reported to be a high-efficiency flocculating agent [35–38] and a potential oil-displacing agent [39]. Recently, we observed the thermo-thickening behavior of GPAM aqueous solution and proposed a preliminary outline of the molecular mechanism based on nuclear magnetic resonance (NMR) and transmission electron microscope (TEM) results. It was found the transformation from a hydrogen bonding (H-bonding) aggregate to a hydrophobic aggregate upon heating was responsible for the thermo-thickening [12]. However, some details during the thermo-thickening of GPAM solution are still unclear, such as the effect of acids on thermo-thickening, among others. In this work, we focus on the dynamics and rheological behavior of GPAM aqueous solution upon heating. GPAM samples with various grafting ratios, as shown in Table S1, are used to explore the thermo-induced structure in aqueous solutions using dynamic light scattering (DLS) and cryo-electron microscope (cryo-TEM). The influences of acid concentration, ramp rate, and shear rate on thermo-thickening of GPAM aqueous solutions are addressed.

#### **2. Experimental Section**

#### *2.1. Materials*

Chitosan (CS) powder and acetic acid-d4 (99.9%) were purchased from Sigma and Aldrich, Shanghai, China. Acetone (99.5%) and sodium hydroxide (96.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Ammonium ceric nitrate (CAN, 99.0%), acetic acid (98.0%), acrylamide (99.0%), and deuterium oxide (D2O, 99.0%) were purchased from Aladdin, Shanghai, China. All chemicals and reagents were used without further purification.

#### *2.2. Sample Preparation*

Highly deacetylated chitosan was obtained by intermittent alkali treatment. Twenty (20) grams (g) of CS with a deacetylation of 78.9% was added into a mixture of distilled water (600 mL) and sodium hydroxide (300 g), which was stirred for 20 min at 110 ◦C. Then, the mixture was heated to 110 ◦C with mechanical agitation. After 1 h of mechanical agitation, the mixture was cooled down to room temperature and the precipitates were filtrated and washed by ultrapure water to neutrality. The chitosan, after being washed in water, was treated again in the alkaline solution for further deacetylation. The collected precipitate was dried at 50 ◦C until reaching a constant weight. The deacetylation degree of CS was increased by the intermittent alkali treatment to 98.0%, as determined by proton nuclear magnetic resonance (1H-NMR) using a Bruker 500 spectrometer (500 MHz) (Bruker, Karlsruhe, Germany) at room temperature (see Figure S1 in Supporting Information), and the calculation method of the degree of deacetylation is shown in Equation S1 in Supporting Information. The synthesis of chitosan-*g*-polyacrylamide (GPAM) has been presented in the previous work [12]. The graft ratio of GPAM was characterized by 1H-NMR (see Figure S2 in Supporting Information), and the calculation method of the graft ratio is shown in Equation S2. In this work, we synthesized GPAM samples with three kinds of grafting rate, as shown in Table S1.

#### *2.3. Rheological Measurements*

Rheological experiments were performed on a stress-controlled rotational rheometer, Discovery Hybrid Rheometer-2 (DHR, TA Instruments, Newcastle, DE, USA). A 40 mm cone-plate geometry with a 2◦ cone angle and a 50 mm gap size was chosen for the steady shear tests. A 40 mm parallel plate geometry with a 500 mm gap size was chosen for all of the dynamic rheological tests. A defined amount of sample solution was directly poured very slowly onto the Peltier region in order to avoid the shear thinning effect and small air bubbles caused by using pipettes. Liquid paraffin was coasted around the margin of the solution sample to prevent the evaporation of solvent. Oscillatory temperature sweep tests were carried out using a strain amplitude of 0.2% (within the linear viscoelastic region, LVR), an oscillatory frequency of 6.283 rad·s<sup>−</sup>1, and a heating rate of 5 ◦C/min, unless otherwise stated.

#### *2.4. Dynamic Light Scattering (DLS) and Ultraviolet and Visible Spectrum (UV)*

The hydrodynamic radius *R*<sup>h</sup> and size distribution were measured by dynamic light scattering (DLS) at the scattering angle of 90◦ using a 90 plus particle size analyzer (Brookhaven Instruments Corp., Holtsville, NY, USA) The wavelength of laser light was 635 nm. The CONTIN program was used for the analysis of dynamic light scattering data. The sample was filtered through the Millipore filters of 1 μm and 0.45 μm, successively, which was repeated at least three times.

The transmittance of GPAM solution was analyzed by a Lambda 35 UV/vis absorption spectrometer (PerkinElmer, Waltham, MA, USA) at 20 ◦C.

#### **3. Results and Discussion**

#### *3.1. Transformation of GPAM Aggregates upon Heating*

The structural transformation of GPAM in the solution sample upon heating was investigated by TEM and DLS in our previous work [12]. However, the microstructures of GPAM aqueous solution observed by TEM, which were obtained from dried solution samples, could not be the same as those in water. In addition, the concentration of GPAM used for DLS in the previous work is too low to observe the thermo-thickening process, because DLS is not appropriate to investigate high concentration polymer solution samples. Considering the possible impact of the above facts, in this work, we made appropriate improvements to investigate the macromolecular mechanism of thermo-thickening. Compared with ordinary TEM, the sample preparation method for cryo-TEM is to rapidly freeze the solution sample with liquid ethane, and then observe the frozen sample under low temperature and vacuum conditions [40,41]; as a result, the true structural form of GPAM in solution is preserved to the greatest extent. So, we used cryo-TEM instead of TEM to examine the structure of GPAM in aqueous solution. In addition, the transformation of GPAM aggregates in solution upon the heating process was investigated by increasing the concentration of GPAM solution used for DLS measurement while guaranteeing the existence of the thermo-thickening phenomenon.

As the viscosity of the GPAM solution may start to increase when the temperature is higher than 20 ◦C, as reported previously [12], here, we investigate the macromolecular architecture in solution at 10 ◦C. To understand the evolution of the macromolecular architecture of GPAM upon heating, a thermal treatment at 40 ◦C for 10 min was applied to the solution sample. In addition,

the thermo-thickening curve of the GPAM solution sample held at 40 ◦C was also investigated for comparison, as shown in the inset of Figure 1a. It can be observed that the viscosity of the solution sample basically tends to constant value after 10 min at 40 ◦C. As no thermo-thickening appears at 10 ◦C, the size distribution of GPAM is shown in Figure 1a. Only a single peak at about 200 nm appears for the original GPAM solution at 10 ◦C, which should be attributed to the aggregations of serval macromolecules rather than a single chain. After the sample was heated and held at 40 ◦C for 10 min, a bimodal distribution arises at 10 ◦C; a peak of about 400 nm and a peak of about several thousand nm. These results suggest that the aggregates with larger size form upon heating compared with the original sample.

**Figure 1.** (**a**) Size distribution at 10 ◦C of 3 mg/mL chitosan−grafted−polyacrylamide 1 (GPAM1) solutions (0.01 M HAc) without and with a thermal treatment of holding at 40 ◦C for 3 min; inset displays the viscosity evolution of 3 mg/mL GPAM1 solutions with 0.01 M HAc at 40 ◦C. (**b**) Dependence of size distribution for GPAM1 solutions with 0.01 M HAc on concentration at 10 ◦C.

It should be pointed out that the size distribution of GPAM aggregates in the initial sample measured here is about several hundred nanometers, and more aggregations with larger size appear during the thermo-thickening process. These results are because of the fact that we use a higher solution concentration of 3 mg/mL, which is closer to the truth of the GPAM molecular mechanism of the thermo-thickening process, rather than using a lower concentration solution to demonstrate the conformation and aggregation changes of GPAM molecules in the previous report [12]. Figure 1b gives the aggregation information of GPAM at different concentrations. When the concentration is low enough, about 0.5 mg/mL, it shows a bimodal distribution. The peak with a smaller size distribution, about ~80 nm, corresponds to single chain conformation, and the peak with a larger size, about ~700 nm, corresponds to macromolecular aggregates. In the cellulose solution and other solutions, the double peaks distribution was also reported [42]. With the increase of concentration, the single chain conformation disappears and the size of aggregations becomes smaller.

The size evolution of GPAM in aqueous solution at 40 ◦C is given in detail in Figure 2. The size distribution of GPAM always presents a single peak, while the size and peak width increase gradually until 6 min, indicating the formation of larger aggregations. After 11 min, the peak gradually evolves into a bimodal distribution; a larger single peak at about 3000 nm and a small peak at about 350 nm. The result is in accordance with Figure 1a. As time goes by, another smaller single peak at about 70 nm appears at 16 min and 21min, which corresponds to the single chain size. This is a very interesting result, because it means that some molecules not only do not contribute to thickening, but exist in the solution as single molecules. This indicated that the newborn lager aggregations upon heating were unstable with the increasing thermal treatment time; when the size of the association continues to increase, a small number of GPAM molecules are separated from the aggregates owing to the damage of hydrogen bonding, and exist as single molecules When the time reaches 21 min, there are even three

peaks in the GPAM solution, indicating the macromolecular do exist in complex and heterogeneous forms during thermo-thickening.

**Figure 2.** Size distribution of 3 mg/mL GPAM1 solutions with 0.01 M HAc at different times at 40 ◦C.

During ordinary TEM sample preparation, there is a process of volatilization of the solvent and the solute in the solution will accumulate, so it is necessary to use a lower concentration sample for observation. The GPAM samples for cryo-TEM observation are obtained by rapidly freezing with liquid ethane, so it could realistically show the morphological feature of GPAM aggregates in solution. When the sample concentration is very low, the target content in the observation field is so low that it is difficult to observe. When the GPAM concentration reaches 10 mg/mL, the viscosity is relatively large, which is not conducive for sample preparation; therefore, we selected 6 mg/mL GPAM aqueous solution with 0.02 M HAc for cryo-TEM observation.

Figure 3a,b are the cryo-TEM images of the samples of GPAM solutions aged for 30 min at 10 ◦C and 40 ◦C respectively. The cryo-TEM images exhibit a dark domain and a sparse dark region. We simply consider the dark domain as aggregates and the sparse dark region as loose structures. Compared with Figure 3a, these aggregates gathered together and formed a much larger cluster structure. This result is consistent with the results measured by DLS above and confirms the formation of larger-size aggregates in GPAM solution during the heating process.

**Figure 3.** Cryo−electron microscope (cryo−TEM) observations of 6 mg/mL GPAM2 in 0.02 M HAc solution at (**a**) 10 ◦C and (**b**) 40 ◦C.

#### *3.2. E*ff*ects of HAc on GPAM Solution*

As GPAM aqueous solution in the presence of HAc, the effect of HAc on the existence of GPAM must be considered. When GPAM is dispersed in acetic acid solution at different concentrations, its ionization in water will obey the following law:

$$\text{GPAM} - \text{NH}\_2 + \text{HAc} \xrightarrow{\text{K}} \text{GPAM} - \text{NH}\_3^+ + \text{Ac}^- \tag{1}$$

Usually, the pKa value of CS is about 6.5 [43], and the pka value of HAc is about 4.76 [44]:

$$K = \frac{\left[-NH\_3^+\right][Ac^-]}{\left[-NH\_2\right][HAc]} = \frac{[Ac^-][H^+]}{\left[HAc\right]} \times \frac{\left[-NH\_3^+\right]}{\left[-NH\_2\right][H^+]} = \frac{Ka\_{HAc}}{Ka\_{CS}}\tag{2}$$

According to Equation (1) and (2), the relationship between the degree of protonation of the amino group and the concentration of HAc can be obtained, as shown in Figure S3 in Supporting Information. As the concentration of HAc increases, the degree of protonation of the amino group increases.

Figure 4 gives the transparency for the GPAM solution with the increasing HAc concentration. It is stable when the concentration of HAc is higher than 0.035 M. The macromolecules and aggregates in GPAM solution decrease gradually with the increase of HAc concentration, as shown in the inset of Figure 4. This is because the protonation of –NH2 is enhanced with the increasing HAc; the electrostatic repulsion between GPAM molecular segments increases accordingly; and, consequently, the hydrogen bond interaction among aggregations is gradually weakened, resulting in a decrease of the aggregation's size.

**Figure 4.** Optical transmittance at 600 nm for 10 mg/mL GPAM2 aqueous solutions as a function of HAc concentration at 20 ◦C. Inset displays the particle size of 10 mg/mL GPAM2 aqueous solutions with the HAc concentration of (a) 0.00175 M, (b) 0.0105 M, (c) 0.0175 M, (d) 0.0351 M, (e) 0.0702 M, and (f) 0.175 M.

Figure 5a gives the steady flow results of GPAM at different HAc concentrations. All samples show obvious shear thinning. With the increase of HAc, the viscosity of GPAM solutions decreases, especially at a low shear rate. All GPAM solution samples present a constant viscosity in the low shear rate region, so zero-shear-rate viscosity (η0) can be obtained from steady flow curves, as listed in Table 1. η<sup>0</sup> decrease with the increasing concentration of HAc indicates the decrease of the aggregation's size, which is in accordance with the above results. However, irreversible thermo-thickening can be found in the GPAM solution with 0.01 M HAc after a thermal treatment (holding at 60 ◦C for 15 min) rather than the GPAM solutions without HAc. As shown in Figure 5b, the GPAM solution presents a slight

increase of viscosity at a low shear rate after thermal treatment, while viscosity for the GPAM solution with 0.01 M HAc increases sharply at a low shear rate. Those results indicate that HAc plays an important role in the thermo-thickening behavior of the GPAM solution.

**Figure 5.** Steady flow curves of 10 mg/mL GPAM2 solutions (**a**) with different HAc concentration and (**b**) with and without a thermal treatment (holding at 60 ◦C for 15 min). All tests were conducted at 10 ◦C.

**Table 1.** η<sup>0</sup> of 10 mg/mL chitosan-grafted-polyacrylamide 2 (GPAM2) solutions with different HAc concentration at 10 ◦C.


To further investigated the effect of acid on the structure evolution of the GPAM solution during the above thermo-cycle, frequency sweeps for GPAM solutions subjected to thermal cycle were conducted at 10 ◦C. In Figure 6a, moduli for the two GPAM solutions without HAc have the similar tendency: *G* lower than *G*" in the low frequency regime and *G* larger than *G*" in the high frequency regime, while their G" are close in the whole investigation range. In general, *G* ~ ω<sup>2</sup> and *G*" ~ ω mean the existence of a homogenous structure; the decreased exponent indicates the existence of a physical network or aggregations [27]. In Figure 6a, the exponent is smaller than the theoretical value, indicating the existence of aggregations in the GPAM solution without HAc in a sense. Figure 6b gives the frequency sweep results for solution samples containing HAc. When the sample is subjected to thermo-cycle, *G* is larger than *G"* in the low frequency regime and the modules present a weak frequency dependency. This suggested a sol-to-gel transformation had taken place. On the contrary, the GPAM solutions without thermo-cycle also seem sol-like. Therefore, GPAM solution containing HAc presents an irreversible structure evolution from aggregations to a more structured fluid in the thermo-cycle for GPAM solutions.

**Figure 6.** Frequency sweeps of 10 mg/mL GPAM2 solutions (**a**) without HAc and (**b**) with 0.01 M HAc subjected to thermo−cycle (temperature increases from 10 ◦C to 60 ◦C, then decreases to 10 ◦C, ramp rate: 5 ◦C /min) at 10 ◦C.

The thermo-thickening process of GPAM solutions was investigated as a function of HAc concentration; Figure 7 gives the temperature sweep results of GPAM solution samples with different HAc concentrations and the mass concentration of the GPAM solution is fixed at 10 mg/mL. The different HAc concentrations determine the content of the protonation of –NH2, that is, in the range of the HAc concentration of 0.00175 M to 0.175 M, the larger the concentration of HAc, the larger amount of the protonation of –NH2 of the moiety. As shown in the inset of Figure 7, the onset temperature (the critical temperature at the onset of an increase in viscosity) increases with the concentration of HAc. This may be attributed to stronger hydrophilicity and electrostatic repulsion induced by more protonation of –NH2, whichs inhibit the hydrophobic aggregation. The onset temperature obviously decreases when the concentration of HAc decreases to 0.01 M. Moreover, both GPAM solutions containing 0.0017 M and 0.0052 M HAc present little thermo-thickening, indicating that there is a critical value of the concentration (>0.005 M) for the thermo-thickening of 10 mg/mL GPAM solution. This is concentration of HAc may be too low, leading to the protonation of GPAM being significantly less and a correspondingly small amount of positive charges, so the electrostatic repulsive force between GPAM molecules is very weak and the water solubility is poor, which leads to the shrinkage of molecular segments, and the molecules are tightly bonded through hydrogen bonding. These densely structured hydrogen-bonded associations cannot be destroyed easily during the heating process, so GPAM molecules cannot be reorganized in large-sized association structures through hydrophobic association, and the thermo-thickening behavior cannot be exhibited. When the HAc concentration reaches a certain value, the water solubility of GPAM increases and the electrostatic repulsion will destroy some of the intermolecular hydrogen bonds, so the structure of the GPAM association will be loose in the aqueous solution. The molecular chain can be extracted from the hydrogen-bonded association and form a large-sized association structure through hydrophobic association, thereby exhibiting thermo-thickening behavior. It is worth mentioning that when the HAc concentration is 0.01 M, the onset temperature of thermo-thickening is the lowest, which means the thermo-thickening structures can be formed easily; therefore, we chose the GPAM aqueous solution with 0.01 M HAc for the following rheological experiments.

**Figure 7.** Influence of HAc concentration on thermo−thickening for the GPAM aqueous solution with 10 mg/mL concentration. Inset displays the relationship between onset temperature and concentration of HAc in the 10 mg/mL GPAM aqueous solution. The strain is 0.2% and the oscillatory frequency is 6.283 rad·s<sup>−</sup>1. The heating rate of the samples is 5 ◦C/min.

#### *3.3. Influence of Heating Rate and Shear Rate*

Figure 8a gives the influences of heating rate on the thermo-thickening behavior. The sample presents a lower *T*trans (the temperature for *G* –*G*" crossover) at a slow heating rate, indicating *T*trans has a dependence of heating rate. When the heating rate was 0.5 ◦C, *G* and G" of the GPAM aqueous solution after thermo-cycle treatment were significantly greater than those of 5 ◦C and 10 ◦C. These results indicate that a slower heating rate is conducive to the formation of thermo-thickening structures. The thermo-thickening behavior of the GPAM aqueous solution is dependent on the heating time and temperature; the thermo-thickening structure of the GPAM aqueous solution will be more complete at a longer heating time or a higher temperature [12]. When the heating rate is lower, it means that the heating time and the residence time in the high temperature region are longer, so the *T*trans will be lower and G , G" will be greater. Furthermore, it can be found from Figure 8a,b that modulus variation with temperature and ω were consistent with each other for 5 ◦C/min and 10 ◦C/min. The possible reason may lie in that structure evolution cannot keep up with the ramp rate to cause no apparent discrepancy in a higher ramp rate.

In order to study the shear-resistance of GPAM after thermo-thickening, a serious of shear recover experiments were carried out by fixing different maximal shear rates. As shown in Figure 9, the increased viscosity of the GPAM solution after thermo-treatment can remain. Furthermore, with the increase of shear rate, the viscosity shear thinning occurs, while its viscosity can recover well as the shear rate decreases in 0.1~0.001 s−<sup>1</sup> (Figure 9a). With the shear rate range increasing, the viscosity cannot recover in time as the shear rate decreases in 10~0.001 s<sup>−</sup>1, meaning that the damaged associated structure of GPAM in solution needs more time to achieve complete recovery (Figure 9b). When the maximum shear rate reached 100 s−<sup>1</sup> or 1000<sup>−</sup>1, the structure was broken thoroughly and came back to the original structure without thermal treatment (Figure 8c,d).

**Figure 8.** (**a**) Temperature sweeps of 10 mg/mL GPAM2 solutions with 0.01 M HAc during the heating process at different ramp rates; (**b**) frequency sweeps at 10 ◦C for 10 mg/mL GPAM2 solutions with 0.01 M HAc after a thermal cycle with different ramp rates (from 10 to 60 to 10 ◦C).

**Figure 9.** Shear and recovery rates of 10 mg/mL GPAM2 solutions with 0.01 M HAc in different limits of the upper shear rate of (**a**) 0.1 s<sup>−</sup>1, (**b**) 10 s<sup>−</sup>1, (**c**) 100 s<sup>−</sup>1, and (**d**) 1000 s<sup>−</sup>1, with a thermal treatment (holding at 60 ◦C for 15 min). All tests were conducted at 10 ◦C.

#### **4. Conclusions**

During the heating process, large-size aggregates were formed in the GPAM aqueous solution through hydrophobic association from the hydrophobic groups on GPAM, which were responsible for the thermo-thickening of the GPAM aqueous solution. As the concentration of HAc in the GPAM aqueous solution increased, the protonation degree of GPAM increased and the electrostatic repulsive force between GPAM molecules would gradually increase, so the size of the GPAM aggregates in the solution gradually decreased. When the concentration of HAc was less than 0.05 M, the protonation degree of GPAM was very low and the solubility was very poor, and then a dense hydrogen bonding association was formed in the solution, which cannot be destroyed during the heating process. As a result, the thermo-thickening behavior disappeared. Furthermore, a higher HAc concentration leads to stronger hydrophilicity and electrostatic repulsion induced by more protonation of –NH2, which inhibit the hydrophobic aggregation. In addition, a slower heating rate is conducive to the formation of thermo-thickening structures and a strong shear rate will destroy the thermo-thickening structure of GPAM.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4360/12/4/916/s1, Figure S1: 1H-NMR spectrums of 0.5 wt.% CS solution in an acetic acid-d4/water-d2 mixture at room temperature (the DDA value of samples of (a) and (b) are 79% and 98% respectively). Figure S2: 1H-NMR spectrums of CS and GPAM in an acetic acid-d4/water-d2 mixture at room temperature. Figure S3: Relationship between the degree of protonation of the amino group and the concentration. Table S1: Molecular parameters of GPAM.

**Author Contributions:** Conceptualization, M.W.; methodology, M.W.; software, M.W.; validation, M.W.; formal analysis, M.W.; investigation, M.W.; resources, Y.S.; data curation, M.W.; writing—original draft preparation, M.W.; writing—review and editing, Q.Z.; visualization, M.W.; supervision, Y.S.; project administration, Y.S.; funding acquisition, Y.S. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** This work was supported by the National Nature Science Foundation of China (Grant No. 51473145, 51773174, 51973467) and Zhejiang Provincial Natural Science Foundation of China (No. LR16E030002).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Antioxidant and Moisturizing Properties of Carboxymethyl Chitosan with Di**ff**erent Molecular Weights**

**Nareekan Chaiwong 1, Pimporn Leelapornpisid 2, Kittisak Jantanasakulwong 1,3, Pornchai Rachtanapun 1,3,4 , Phisit Seesuriyachan 1,3, Vinyoo Sakdatorn 1, Noppol Leksawasdi 1,3 and Yuthana Phimolsiripol 1,3,\***


Received: 3 June 2020; Accepted: 23 June 2020; Published: 28 June 2020

**Abstract:** This research aimed to synthesize carboxymethyl chitosan (CMCH) from different molecular weights of chitosan including low MW (L, 50–190 kDa), medium MW (M, 210–300 kDa) and high MW (H, 310–375 kDa) on the antioxidant and moisturizing properties. The L-CMCH, M-CMCH and H-CMCH improved the water solubility by about 96%, 90% and 89%, respectively when compared to native chitosan. Higher MW resulted in more viscous of CMCH. For antioxidant properties, IC50 values of DPPH and ABTS radical scavenging activity for L-CMCH were 1.70 and 1.37 mg/mL, respectively. The L-CMCH had higher antioxidant properties by DPPH and ABTS radical scavenging assay and FRAP. The moisturizing properties on pig skin using a Corneometer® showed that 0.5% H-CMCH significantly presented (*p* ≤ 0.05) greater moisturizing effect than that of untreated-skin, distilled water, propylene glycol and pure chitosan from three molecular weights.

**Keywords:** chitosan; carboxymethyl chitosan; molecular weight; antioxidant properties; skin moisturizing

#### **1. Introduction**

Chitosan was generally considered in the way that it has low toxicity, biodegradable, accelerates wound-healing, antibacterial properties and gel-forming properties [1]. Chitosan is cheap and inexhaustible material with numerous applications in cosmetics, pharmaceuticals, nourishment science and biotechnology [2,3]. The uses of chitosan are restricted because of its insolubility at neutral or basic region. Hence the solubility of chitosan must be improved. Carboxymethylation is a chemical modification which can improve water solubility. The water solubility properties and applications of carboxymethyl chitosan (CMCH) strongly depended on its structural characteristics, the average degree of substitution (DS), the position of the carboxymethylation (grafting to amino or hydroxyl groups) and the average number of hydroxyl groups substituted by carboxymethyl groups [4]. The CMCH is prepared by the replacement of –OH groups of chitosan with –CH2COOH groups with the alternative functional groups such as *O*-, *N*- and *N,O*- carboxymethyl chitosan [2]. Substitution of *N*- and *O*-carboxymethyl chitosan derivatives take place when chitosan reacts with monohalocarboxylic acids using different reaction conditions to control the selectivity of reaction such as temperature and ratios

of chitosan, pH as well as monochloroacetic acid. Promotion of *O*-substitution occurs when the reaction is carried out at low temperature such as 0–10 ◦C [5,6], but *N*-substitution is dominated at high temperature [7]. The optimal reaction temperature of N-CMCH synthesized from chitosan was 90 ◦C [8]. The solubility of cellulose derivatives did not only depend on the DS, but also on the distribution of the substituents for glucose units along the cellulose chain [9]. The –COOH and–NH2 groups replacement indicate capacity of the chemical modifications to improve their physical properties [3]. CMCH is dissolvable in a wide pH range with several advantages and low harmfulness [10]. CMCH not only has a good solubility in water, but also has unique chemical, physical and biologic properties such as high viscosity, large hydrodynamic volume, biocompatibility, good ability to form films, fibers and hydrogels [11–13]. Hence, it was widely utilized in numerous biomedical fields, for example, a moisture-retention agent, wound dressing agent, artificial bone and skin, blood anticoagulant and as a component in different drug delivery [14]. Chitosan and CMCH were investigated for coating and film forming abilities to extend product shelf life. The effects of different chitosan types and molecular sizes on properties of CMCH films to plastic replacement were also studied [15]. Zhang et al. [16] found that chitosan modification could improve the antioxidant activity by addition of quaternium on amino groups. Ying et al. [17] prepared various Schiff base typed chitosan saccharide derivatives to enhance the ability of DPPH scavenging radical and also water solubility in comparison to native chitosan. Moreover, antioxidant activities of *N*-carboxymethylchitosan oligosaccharides with different DS (0.28, 0.41 and 0.54) were also evaluated by the scavenging of DPPH radical, superoxide anion and the determination of reducing power. The increase in DS of *N*-CMCH resulted in decreased DPPH radical scavenging activity with increased reducing power [18].

The antioxidant activities of chitosan and CMCH are evidence that the active hydroxyl and amino groups within the polymer chains may participate in free radical scavenging which were varied with MW [17]. Zhao et al. [19] reported that CMCH was a better antioxidant than native chitosan, especially in terms of its reducing power, scavenging ability towards DPPH and superoxide radicals as well as chelating ability of ferrous ions. In case of native chitosan, the moisture-absorption and moisture-retention capacities of chitosan depended on the MW and DS. The ability to absorb moisture increased when the MW was decreased [20]. Humectant property of chitosan improved with increasing MW. For the CMCH, Jimtaisong et al. [21] reported that MW and DS could also affect the exhibitions of the moisture-retention capacity of CMCH. Water-holding capacity of CMCH is related to the presence of positive electrical charges and high molecular weight that facilitate adherence onto the skin when implemented as a skin moisturizer. Muzzarelli et al. [22] revealed that 0.25% CMCH solution was comparable with 20% propylene glycol in terms of moisture-retention capacity with equivalent viscosity to hyaluronic acid (HA), a compound with excellent moisture-retention property. Furthermore, moisture absorption and moisture retention capacities of CMCH could also be significantly improved by utilizing higher MW with the presence of intermolecular hydrogen bonds within molecular chains. Gel formation resulting from addition of CMCH as hydrating agent in cosmetics is also ideal for the skin as it asserts positive feeling of the customers. In cosmetic products, humectants are used to increase the amount of water in the top layers of the skin. The activity of humectant polymers depends on cationic charges, molecular weight and hydrophobicity of polymer. The positively charged ions facilitate neutralization of negatively charged ions on the skin [23].

However, the antioxidant and moisturizing properties of CMCH prepared from different molecular weights of chitosan have not yet been investigated. Therefore, this research aimed to synthesize CMCH from different molecular weights of chitosan including low MW (L, 50–190 kDa), medium MW (M, 210–300 kDa) and high MW (H, 310–375 kDa) and characterized their respective antioxidant and moisturizing properties.

#### **2. Materials and Methods**

#### *2.1. Materials*

Three different molecular weights of chitosan including low MW (L, 50–190 kDa), medium MW (M, 210–300 kDa) and high MW (H, 310–375 kDa) with degree of deacetylation above 90% were obtained from Ta Ming Enterprises Co., Ltd.; Samutsakon, Thailand. Ethanol, methanol, isopropanol, sodium hydroxide and glacial acetic acid were purchased from RCI Labscan (Bangkok, Thailand). Monochloroacetic acid was obtained from Sigma-Aldrich (Darmstadt, Germany). All other reagents were of analytical grade.

#### *2.2. Synthesis of CMCH*

CMCH was synthesized by following method of Tantala et al. [14]. Chitosan flake was grounded and sieved to obtain particle size under 60-mesh (Endecotts, UK). The chitosan (25 g) was suspended in 50% (*w*/*v*) sodium hydroxide solution (400 mL) and 100 mL of isopropanol was added and mixed well at 50 ◦C for 1 h. Monochloroacetic acid (50 g) was dissolved in isopropanol (50 mL), gradually dropped into the reaction for 30 min and the system was allowed to continuously react at 50 ◦C for 4 h. The reaction was stopped by adding 70% (*v*/*v*) methanol. The pH of the sample was later adjusted to 7.0 by 1% (*v*/*v*) glacial acetic acid. From that point, the solid was separated and washed in 70–90% ethanol for desalting and dried in a hot air oven (Binder, Germany) at 80 ◦C for 12 h. The mass yield of CMCH was calculated using Equation (1).

$$\text{Yield } \left( \% \right) = \frac{\text{chitosan } \left( \text{g} \right) - \text{CMCH } \left( \text{g} \right)}{\text{chitosan } \left( \text{g} \right)} \times 100 \tag{1}$$

#### *2.3. Moisture Content, pH and Viscosity Measurement*

Moisture content was determined according to the Association of Official Analytical Chemists (AOAC) standard method no. 930.15 [24]. The pH values were measured by a pH meter (FiveEasy F20, Metter Toledo, Switzerland). The viscosity of 1% (*w*/*v*) solution of chitosan and CMCH were estimated by Brookfield viscometer (DV-II, Brookfield Engineering Labs Inc., Stoughton, MA, USA) using a spindle No. 28 at 100 rpm.

#### *2.4. Water Solubility of CMCH*

The water solubility of CMCH samples at 25 ◦C was tested by using the method of Rachtanapun et al. [15]. After addition of 0.3 g samples (initial dried weight) into 10 mL water (3% w/v), the solutions were filtered with Whatman filter paper No. 4 (Sigma-Aldrich, Germany) which was previously dried at 105 ◦C for 24 h before use. The mass of dried CMCH residues was obtained by weight difference to obtain final dry weight. The tests were performed in triplicate to detect random error and the solubility was determined using Equation (2).

$$\text{Water solubility (\%)} = \frac{\text{initial dried weight of CMCH (g)} - \text{final dried weight of CMCH (g)}}{\text{initial dried weight of CMCH (g)}} \times 100\tag{2}$$

#### *2.5. FTIR Analysis*

The FTIR spectra of chitosan and CMCH were obtained using a Fourier transform infrared spectrometer (Frontier, PerkinElmer, Waltham, MA, USA). All spectra were recorded in the range of 500–4000 cm−<sup>1</sup> as described by Surin et al. [25].

#### *2.6. Antioxidant Properties*

The stock solution of L, M and H (stock 5-mg/mL in 0.2% (*v*/*v*) acetic acid), L-CMCH, M-CMCH and H-CMCH (stock 5-mg/mL in distilled water) at different concentrations of 1, 2, 3, 4 and 5-mg/mL were prepared and used for DPPH, ABTS and FRAP assays.

#### 2.6.1. DPPH Radical Scavenging Assay

The ability of antioxidants to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was completed by modified method of Hu et al. [26]. After that, 100 μL of the stock samples (as described above) were blended with 100 μL of 0.2-mM DPPH reagent (Sigma-Aldrich, Singapore) and incubated at 25 ◦C for 30 min in the dark. Absorbance was measured at 517 nm in a 96-wells microplate reader (SpectraMax® i3x, Molecular Devices, San Jose, CA, USA). The radical scavenging activity of the sample was calculated based on the gallic acid (Sigma-Aldrich, Schnelldorf, Germany). Results were expressed as milligram gallic equivalent per gram of sample (mgGAE/g sample). The percentage of DPPH radical scavenging activity can be calculated as shown in Equation (3) before plotting of IC50 against respective concentration.

DPPH radical scavenging activity (%) = [(A517 control − A517 sample)/A517 control] × 100 (3)

#### 2.6.2. ABTS Radical Scavenging Assay

The 2,2-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity was conducted according to method described by Xie et al. [27]. ABTS (Sigma-Aldrich, Singapore) reagent was freshly prepared by mixing 8 mL of 7-mM ABTS stock solution with 12.5 mL of 2.45-mM potassium persulfate (Sigma-Aldrich, Singapore). ABTS powder and potassium persulfate powder were individually dissolved with water to the required concentration and then combined together in a bottle. After 16 h of incubation in the dark at 25 ◦C, the resultant dark blue color of ABTS reagent solution was diluted with ethanol until the absorbance reading reached 0.7 ± 0.2. The solution of L, M, H, L-CMCH, M-CMCH and H-CMCH were prepared as described previously in Section 2.6.1. Each sample solution (0.5 mL) was mixed with 1.0 mL of ABTS stock solution and incubated for 6 min in the dark. Absorbance was measured at 734 nm in the 96-well microplate reader. The ABTS radical scavenging activity was expressed as milligram gallic equivalent per gram of sample (mgGAE/g sample). The percentage of ABTS radical scavenging activity can be calculated as shown in Equation (4) with plotting of IC50 against respective concentration.

ABTS radical scavenging activity (%) = [(A734 control − A734 sample)/A734 control] × 100 (4)

#### 2.6.3. FRAP Assay

The ferric reducing antioxidant power (FRAP) assay was carried out according to the technique of Woranuch et al. [28]. The FRAP reagent was prepared by mixing 25 mL of 0.3-M acetate buffer (pH 3.6), 2.5 mL of 4,6-tripyridyl-s-triazine (TPTZ) (Sigma-Aldrich, Schnelldorf, Germany) solution in 40-mM HCl (RCI Labscan, Bangkok, Thailand) and 2.5 mL of 20-mM ferrous sulfate (Loba Chemie, India). Thus, 50 μL of samples were mixed with 950 μL of FRAP reagent and incubated in dark for 30 min. Absorbance was measured at 593 nm in 96-well microplate. The ferric reducing antioxidant power of sample was determined based on the ferrous sulfate (Merck, Darmstadt, Germany). Results were expressed as ferrous sulfate equivalent antioxidant capacity, with μmol Fe<sup>2</sup>+/g sample.

#### *2.7. Moisturizing Properties on Pork Skin*

The skin moisturizing of the 0.5% (w/v) L, M, H, L-CMCH, M-CMCH and H-CMCH solutions were examined on pork skin and compared with untreated-skin, water and propylene glycol. The pork skins were prepared from back side of the pig ear obtained from three different market sources including the Mae Hia fresh market, the Ton Payom fresh market and the Hangdong fresh market (Chiang Mai, Thailand). The samples were washed and cleaned with removal of the fat layer prior to cutting into 3 × 3 cm. Each sample (100 μL) was applied on the skin surface. The skin without any substance was used as a control. The skin moisturizing was measured before applying on samples and after application at 0, 15 and 30 min intervals using Corneometer® (Courage + Khazaka Electronic, Germany). Before applying the sample and recording the parameter, the pig skins were kept at 25 ◦C for 30 min. This method was adapted from Kassakul et al. [29]. The degree of skin moisturizing (%) was tested in triplicate to detect random error and calculated using Equation (5).

$$\text{Degree of skin moistureizing} \left( \% \right) = \frac{\text{after applying} - \text{before applying}}{\text{before applying}} \times 100 \tag{5}$$

#### *2.8. Statistical Analysis*

All data were analyzed by one-way ANOVA. Mean separation was performed by Duncan's multiple range tests with significance level (*p* ≤ 0.05). Statistical analyses were performed with the SPSS 17.0 (SPSS, Inc.; IBM Corp.; Chicago, IL, USA).

#### **3. Results and Discussion**

#### *3.1. E*ff*ect of CMCH Synthesis*

CMCH was prepared at three different molecular weights of chitosan (L, M and H). The yield, moisture content, water solubility, viscosity and pH of chitosan products (L-CMCH, M-CMCH and H-CMCH) were reported in Table 1. L-CMCH had the highest yield, water solubility and viscosity, while moisture content and pH of L-CMCH, M-CMCH and H-CMCH were not significantly different (*p* > 0.05) among the range of 6.36–6.87% and 7.27–7.33%, respectively. The solubility is a significant property of CMCH that measures their resistance to water. Table 1 shows water solubility of the L-CMCH M-CMCH and H-CMCH which indicates the significant effect (*p* ≤ 0.05) of larger MW on decreased water solubility. The decreasing trend was 96.87% for L-CMCH, 90.06% for M-CMCH and 89.49% for H-CMCH compared to the L, M and H. The solubility and conformation of CMCH happens from the deacetylation, pH and MW of native chitosan. The solubilization process of CMCH related to functionalized polymers, different types of chemical and physical interactions such as hydrogen bonds, hydrophobic interactions and van der Waals forces. high water solubility suggests that CMCH is moisture absorption and more helpful ability to bind with water than chitosan. higher solubility is due to forming hydrogen bonding with carboxylic groups of CMCH with water molecules. This causes the hydrated water molecules that around the chain of CMCH are more than that surrounding the chitosan chains, resulting in higher water solubility [30]. This results also are consistent with the report of Siahaan et al. [31] who found that the temperature and NaOH concentration affected to CMCH synthesis. The interactions between NaOH and monochloroacetic acid resulted in reduced CMCH forming and lower solubility. The mitigation in solubility may stem from the loss of free amino-functional groups that enhance hydrophobic nature of the compounds [32]. The greater solubility also corresponded to the decrease in viscosity L-CMCH and M-CMCH are slightly different, but H-CMCH requires significantly higher viscosity. This could be explained that CMCHs with chains longer or higher MW were contributing to the gel.

FTIR spectra of chitosan and CMCH are presented in Figure 1. The essential characteristic peaks of chitosan are at 3288 cm−<sup>1</sup> (O–H stretch), 2875 cm−<sup>1</sup> (C–H stretch), 1591–1645 cm−<sup>1</sup> (N–H bend), 1059 cm−<sup>1</sup> (bridge-O stretch) and 1023 cm−<sup>1</sup> (C–O stretch) [2]. For CMCH, the spectrum was different from the spectrum of chitosan (Figure 1 and Table 2). The IR spectrum of CMCH showed the intrinsic peak at 1747 cm<sup>−</sup>1, the most visible difference was the appearance of a new peak which belonged to C = O stretching vibration (amide I) Putra et al. [33] identified C = O peak on CMCH whose wave number could be 1600–1850 cm−1, 1660–1680 cm−<sup>1</sup> and also 1606 cm−1. The CMCH showed the

disappearance of the–NH2 associated band at 1647 cm−<sup>1</sup> which could be associated with characteristic vibration deformation of the primary amine N–H and the combination N–H peak with new peak at 1583. The appearance of some new intensive peaks at 2922–2853 and 1583 cm−<sup>1</sup> could be attributed to the methyl groups and the long carbon segment of the quaternary ammonium salt [34]. Compared to the peaks of chitosan, the new bands at 1583 cm−<sup>1</sup> and 1411 cm−<sup>1</sup> corresponded to the carboxy group (overlapped with N–H bending) and–CH2COOH group, respectively. The intense spectrum of CMCH indicating carboxymethylation on both the amino and hydroxyl groups of chitosan [2]. Characteristic peaks of the first C–O and the second C–O groups between 1052 and 1024 cm−<sup>1</sup> (C–O stretch) did not change. It was confirmed that chitosan was converted to CMCH by new transmission peaks of -COO groups at 1583 and 1747 cm<sup>−</sup>1. These new -COO groups enhanced hydrophilic properties of the CMCH which enhanced solubility of the compound.

**Figure 1.** *Cont*.

**Figure 1.** FT-IR spectra of (**a**) L and L-CMCH; (**b**) M and M-CMCH; (**c**) H and H-CMCH.



\* Water solubility (%) of L-CMCH, M-CMCH and H-CMCH indicates the comparison to native chitosan. Different letters (a–c) in each column indicate significant differences (*p* ≤ 0.05). ns means no significant difference.


**Table 2.** Functional groups and wave number (cm<sup>−</sup>1) of chitosan and CMCH.

#### *3.2. Antioxidant Properties*

The results from DPPH assay of L, M, H, L-CMCH, M-CMCH and H-CMCH are shown in Figure 2. L-CMCH showed the highest (*p*≤0.05) scavenging activity. The DPPH scavenging activities of L-CMCH, M-CMCH and H-CMCH were higher than those of L, M and H. IC50 values of DPPH and ABTS radical scavenging activities of L-CMCH were 1.70 and 1.37 mg/mL, respectively. However, no significant differences in DPPH scavenging potential were found among the L, M and H. Younes et al. [35] also found that IC50 value was determined between 1.62- 2.20 mg/mL for shrimp chitosan (*Metapenaeus monoceros*) at different concentrations (0–5 mg/mL). The DPPH radicals scavenging ability of chitosan and its derivatives (CMCH) increased as the concentration increased [36]. This is probably due to the relatively poor hydrogen-donating ability of chitosan that prevent chain breaking [37]. Some studies suggested that DPPH radical scavenging of chitosan increased with decreasing MW [38]. Again,

it is confirmed that the CMCH have strong antioxidant activity, which is dependent on the particle size [34]. In addition, chitosan chains possess active hydroxyl and amino groups that can react with free radicals [39]. Scavenging activity of chitosan is related to the extent of reaction between free radicals and protonated amino groups [18]. Our results indicated that the DPPH scavenging ability of CMCH synthesized with low, medium or high MW was higher than that of pure chitosan from three MW. Elbarbary & Mostafa [40] also confirmed that the antioxidant activities of CMCH could be enhanced by decreasing MW of CMCH. high MW can contribute to a more compact structure and relatively stronger effect of intramolecular hydrogen bond. The antioxidant activity for ABTS radical was similar to those of DPPH assay even though ABTS radicals are more reactive than DPPH radicals [19]. Hence, these showed that antioxidant activity is expanded with decreasing MW with L-CMCH, M-CMCH and H-CMCH, respectively, compared to the L, M and H Figure 3.

**Figure 2.** (**a**) DPPH radical scavenging activity (%) and (**b**) IC50 of L, M, H, L-CMCH, M-CMCH and H-CMCH. Different letters (a–d) indicate significant difference between treatments (*p* ≤ 0.05).

**Figure 3.** (**a**) ABTS radical scavenging activity (%) and (**b**) IC50 of L, M, H, L-CMCH, M-CMCH and H-CMCH. Different letters (a–d) indicate significant difference between treatments (*p* ≤ 0.05).

FRAP assay in Figure 4 revealed the variation of antioxidant capacity with corresponding concentration levels [41]. In similar manner of DPPH assay, control could slightly reduce ferric to ferrous ions. In this assay, L-CMCH had the highest (*p* ≤ 0.05) ability to reduce ferric to ferrous ion [42], followed by M-CMCH and H-CMCH, while L, M and H showed the lowest ability (*p* ≤ 0.05). The replacement of -NH groups by -COO groups in the CMCH structure was previously reported to be beneficial not only to level of solubility, but also antioxidant activities [43]. Although some studies suggested the effects of molecular weights to FRAP antioxidant activities [44], such effect was not evident in current study.

**Figure 4.** Ferric reducing antioxidant power (FRAP) of L, M, H, L-CMCH, M-CMCH and H-CMCH. Different letters (a–c) indicate significant difference between treatments (*p* ≤ 0.05).

#### *3.3. Skin Moisturizing Properties*

The degree of skin moisturizing indicates the water-holding capacity of the skin which can be tested by the Corneometer method. The Corneometer® measures the changes of electrical capacitance related to the moisture contents of the skin before and after applying the solutions [29]. The degree of skin moisturizing of the L, M, H, L-CMCH, M-CMCH and H-CMCH solutions were examined on pork skin and compared with untreated skin, water and propylene glycol at 15 and 30 min as presented in Figure 5. The effect degree of moisturizing on time at 15 and 30 min showed that the degree of skin moisturizing of solutions decreased with increasing time after applying solutions, except 0.5% H-CMCH. The degree of skin moisturizing of H-CMCH had no significant difference after applying between 15 and 30 min. Applying H-CMCH solution for 15 and 30 min were the highest degree of skin moisturizing, showing high moisturizing effect (more than 200%). While the degree of skin moisturizing of untreated skin, water propylene glycol, L, M, H, L-CMCH and M-CMCH solutions applying on pork skin for 30 min were significantly decreased from 15 min. This confirms that the H-CMCH solution provided a good moisture absorption. In fact, the skin moisturizing effects appeared to decrease with increasing time due to lack of mechanisms to maintain skin moisturizing and dryness of pork skin cells [45]. The higher molecular weight CMCH also had the superior moisture retention capacity. Kassakul et al. [29] found that 0.2% *Hibiscus rosa sinensis* mucilage as natural ingredient provided good results of skin moisturizing after applying for 30 min by about 130%. The results showed that moisturizing products could increase the water content of the skin while maintaining softness and smoothness [20]. After applying solutions containing different MW of water-soluble CMCH (L-CMCH, M-CMCH, H-CMCH), the moisture content of the skin increased. The mechanism of moisturizing effect is based on the formation of water film of skin surface after dissolution of CMCH and subsequent stage of water evaporation could further prevent water evaporation from the skin [46]. Positive electrical charges and relatively high MW facilitates prolong skin adherence [21]. Our results also showed that H-CMCH decreased the loss of water while elevating skin humidity. The higher apparent viscosity of H-CMCH can improve the stability and enhance skin hydration. In fact, 0.5% H-CMCH was superior to untreated skin, water and propylene glycol in terms of degree of skin moisturizing effect. The higher MW of CMCH also indicates potential for film forming and coating to multilayer of the skin. Subsequently, it could be used in cosmetic preparation with suggested further studies of the testing skin irritation in human subjects.

**Figure 5.** Degree of skin moisturizing (%) as affected by time (15 and 30 min) and different treatments (skin, DI, PG, L, M, H, L-CMCH, M-CMCH and H-CMCH) on pork skin Different lowercase letters (a–g) indicate significant differences between solutions at 15 min and different uppercase letters (A–G) indicate significant differences between solutions at 30 min.

#### **4. Conclusions**

Carboxymethyl chitosan (CMCH) was effectively synthesized and characterized by FTIR. The modifications in biologic properties including water solubility, antioxidant properties as well as efficacy of moisturizing property of CMCH were evident. It is clearly seen that higher MW of chitosan and CMCH resulted in lower antioxidant properties but provided greater moisturizing property. The H-CMCH improved the water solubility by about 89%, when compared to chitosan. The higher levels of DPPH, ABTS and FRAP were also detected. The moisturizing effect was at the highest level when 0.5% H-CMCH was applied to pig skin. H-CMCH is an effective water-soluble polymer with high viscosity which could be successfully utilized in pharmaceuticals and cosmetics as emulsion stabilizers and thickening agents. Future work is required to investigate this biopolymer for skin irritation in human subjects.

**Author Contributions:** N.C. designed and performed the experiments and participated in the interpretation of the results and the writing of the paper. P.L., K.J., P.R., P.S., V.S. and N.L. interpreted the results and edited the manuscript. Y.P. supervised and discussed the research and edited the manuscript. All the authors contributed to the realization of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** The authors acknowledge the financial support provided by the National Research Council of Thailand (NRCT) and Thailand Institute of Scientific and Technological Research (TISTR) for microbial strains support for this project. We wish to thank Center of Excellence in Materials Science and Technology, Chiang Mai University for financial support under the administration of Materials Science Research Center, Faculty of Science, Chiang Mai University. This research work and APC was also partially supported by Chiang Mai University under the Cluster of Agro Bio-Circular-Green Industry under Grant number CMU-8392(10)/W.152-12032020.

**Conflicts of Interest:** The authors declare no conflict of interest.

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