**2. Physicochemical properties and gelling characteristics**

Chitin and chitosan are known as secondary abundant polymers obtained from the external skeleton of crustaceans such as crab and shrimp [26]. Apart from this, chitin is also found in and produced from the exoskeleton of insects or the cell walls of fungi and yeast [6, 27, 28]; however, this contribution is much less than that from the marine resources. In recent years, chitin and chitosan have gained attention instead of raw materials of petroleum origin owing to the inevitable depletion of fossil fuels and the prevention of climate change [29]. This section looks at the physicochemical properties of chitosan and focuses on the gelling characteristics to build environmentally-friendly separation media.

#### **2.1 Chemical composition and gelling ability for separation media**

**Figure 1** shows the chemical conversion between chitin and chitosan. The chemical composition of chitin can be described as a long-chain polymer,

*Innovative Separation Technology Utilizing Marine Bioresources: Multifaceted Development… DOI: http://dx.doi.org/10.5772/intechopen.95839*

**Figure 1.** *Chemical conversion among chitin, chitosan, and furthermore functional separation media.*

poly(β-(1 ! 4)-*N*-Acetyl-D-glucosamine). Chitosan, poly(β-(1 ! 4)-Dglucosamine), is obtained mainly by transforming partial deacetylation of chitin in an alkaline condition, such as using sodium hydroxide aqueous solution. It has been reported that chitosan and its oligosaccharides not only possess hydrophilicity, nontoxicity, biodegradability, and biocompatibility, but also possess antimicrobial activity, antioxidant properties, and an affinity for proteins [7, 26].

Chitosan is insoluble in water at neutral pH or in any organic solvent. Consequently, an acidic aqueous solution, such as acetate buffer solution, is usually employed to dissolve chitosan, whereby the acid dissociation constant of chitosan is found as pKa ≈ 6.5 [30]. Chitosan can be dissolved in acidic solutions by protonation of amino groups in glucosamine units.

Deprotonating a chitosan solution through an acid–base neutralization leads to formation of a water-insoluble gel structure without cross-linker due to intermolecular hydrogen bonding [8]. The salt (*e.g.* NaCl) coexisting with chitosan in an acidic solution acts as counter ions and disrupts intramolecular hydrogen bonding, and then the flexibility of chitosan molecular chains increases [31]. In addition, pH neutralization influences the formation of a polymeric network [9]. Therefore, the neutralization condition should be optimized. From the convenient gelling process, chitosan hydrogels have been developed widely as immobilizing matrices, with enzymes, carbon nanotubes, and electroconductive polymers as typical examples [10, 32, 33].

#### **2.2 Deacetylation degree**

Deacetylation degree (*DD*) is the most important factor to regulate physicochemical properties. The deacetylation degree of chitosan samples was determined using the colloidal titration method-based experimental conditions in previous works [34, 35]. We dissolved chitosan powder (0.5 g) in 5% acetic acid solution, and then increased the total weight of chitosan–acetic acid solution to 100.0 g by adding acetic acid. We mixed a 1 g sample of this chitosan–acetic acid solution to 30 ml of deionized water. The titrant was 0.0025 N potassium polyvinyl sulfate (PVS-K), and the indicator was 1% toluidine blue. The terminal point of titration was clearly

as a synthesis process called the "upstream process" [1]. To ensure quality and cost of final products, the separation process is important and has been developed in line with the social demands [2]. For chemical and biochemical industries, the separation process aims to purify objective substances, eliminate undesirable substances, and fractionate each component from their mixture. As environmental awareness around the world increases recently, new separation technologies, such as wastewater treatment [3], advanced desalination [4], air cleaning [5] *etc.*, are in great demand. In addition, materials used in such separation processes are not only expected to be efficient, low cost, easy operation, but also required to be

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

Chitin and chitosan obtained from crustaceans possess sufficient environmental adaptability as well as an attractive potential to build various types of functional media, e.g., membranes [6–10], micro/nanoparticles [11, 12], and nanofibers [13–16]. Many studies have devoted to develop the novel media adapting separation processes using chitosan. The separation performance of such chitosan media should be strongly influenced from chemical properties characterized by

deacetylation degree (*DD*) at amino groups in the chitosan molecular chain. Nevertheless, it was less mentioned that the *DD* could steer not only the structure of the

This chapter describes the preparation and physicochemical properties of novel chitosan-based media and demonstrates the promising ability of chitosan with focus on principal studies for environmentally-friendly separation processes. The essential factors which regulate the performance of separation media prepared from chitosan, such as *DD*, molecular weight, and options of cross-linker, are explained. In particular, the notable impacts of *DD* on the mass transfer mediated by chitosan membrane, the mechanical property, and the antibacterial activity, are introduced based on our previous research [17–19]. Separation media prepared from chitosan are often combined with various adsorbents [20, 21], carbon nanotubes [22, 23], or other functional materials [24, 25]. In such case, the behavior of mass transfer into the chitosan hydrogel is complicated to quantitatively evaluate. The present chapter shows the determination of effective diffusion coefficient of cesium ions in chitosan membrane immobilizing Prussian Blue particles [20]. Furthermore, the chitosan aerogels with macro-porous structure is proposed for selective separation for anionic dye from aqueous phase. Chitosan nanofibers incorporated with polyethylene terephthalate (PET) non–woven are also covered to describe in an application of air filtration.

prepared chitosan gels but also the characteristics as separation media.

**2. Physicochemical properties and gelling characteristics**

**2.1 Chemical composition and gelling ability for separation media**

**Figure 1** shows the chemical conversion between chitin and chitosan. The chemical composition of chitin can be described as a long-chain polymer,

build environmentally-friendly separation media.

**202**

Chitin and chitosan are known as secondary abundant polymers obtained from the external skeleton of crustaceans such as crab and shrimp [26]. Apart from this, chitin is also found in and produced from the exoskeleton of insects or the cell walls of fungi and yeast [6, 27, 28]; however, this contribution is much less than that from the marine resources. In recent years, chitin and chitosan have gained attention instead of raw materials of petroleum origin owing to the inevitable depletion of fossil fuels and the prevention of climate change [29]. This section looks at the physicochemical properties of chitosan and focuses on the gelling characteristics to

environmentally-friendly.

indicated by the color changing from blue to claret. The deacetylation degree was calculated using the following equations.

$$X = f \times 0.0025 \times 10^{-3} \times \nu \times 161 \tag{1}$$

$$Y = \mathbf{0}.\mathbf{5} \times \mathbf{10}^{-2} - X \tag{2}$$

Chitosan membranes with a controlled degree of deacetylation (*DD*) were prepared using a casting method. Changes in the total water content and the pressure driven water flux of the membrane were observed with a change in *DD* (**Figure 3**). The membrane properties were analyzed and evaluated using water permeability measurements, scanning electron microscopy (SEM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). SEM observations indicated that the membrane structure was an individual cellular structure and that this cellular structure grew with decrease in *DD* (**Figure 4**). From XRD measurements, the intensity in the range from 10° to 20° were detected in the chitin (*DD* = 1.1%) and the chitosan membranes (71.3% < *DD* < 92.2%), which indicated that the crystal structure of the membrane was amorphous regardless of *DD*. The free water content (*W*f), the freezable bound water (*W*fb), and the bound water not able to freeze (*W*b) were evaluated by DSC. The total water content and the sum of free water content ratios (*W*<sup>f</sup> + *W*fb) decreased with increasing *DD* whereas *W*<sup>b</sup> gradually increased [18]. That suggests the membrane prepared from lower *DD* chitosan formed remarkable cellular structure. The free water was mainly contained inside of the cellular structure, and resulted in swelling the chitosan membrane (**Figure 5**).

*Innovative Separation Technology Utilizing Marine Bioresources: Multifaceted Development…*

*DOI: http://dx.doi.org/10.5772/intechopen.95839*

*Change in water flux (50 kPa, 298 K, μL = 0.901 mPa s) and total water content of membrane with regulated*

*SEM images observed at cross-section of the membranes prepared from different DD chitosan [18] with permission from Elsevier. (a) and (b) DD 71.3%; (c) and (d) DD 81.8%; (e) and (f) DD 92.2%. The full length of reference scaling measure in (a), (c) and (e) indicates 10 μm. The full length of reference scaling*

DD*. (*●*) water flux,* <sup>J</sup>*<sup>v</sup> and (*○*) total water content,* <sup>W</sup>*<sup>t</sup> [18] with permission from Elsevier.*

**Figure 3.**

**Figure 4.**

**205**

*measure in (b), (d) and (f) indicated 5 μm.*

$$DD\ \left(\%\right) = \frac{X/161}{X/161 + Y/203} \times 100\tag{3}$$

In these equations, *X* is the equivalent mass of glucosamine contained in a 1 g sample of the chitosan–acetic acid solution, *v* [ml] is the volume of 0.0025 N PVS-K solution, and *f* is its concentration factor. Y is the mass of acetyl glucosamine contained in a 1 g sample, calculated as the difference between the mass of the sample and the value of *X*. We evaluated the deacetylation degree of the chitosan samples as a molar fraction of glucosamine [36].

#### *2.2.1 Control of the deacetylation degree*

A chitosan membrane was prepared by the casting method in combination with *N*-acetylation reaction [17]. The deacetylation degree (*DD*) decreased linearly with increasing added amounts of acetic anhydride (**Figure 2**). Stoichiometric control of the deacetylation degree to the desired level was successfully performed. However, gelation reaction due to excess addition of acetic anhydride inhibited formation of the chitosan membrane.

The gelation behavior of chitosan, which has various degrees of acetylation (*DA*) of amino groups, was investigated to ensure preparation of the designed membrane structure [37]. The gelation behavior was evaluated by the gelation time and the quantity of syneresis, and useful information not only for preparing a membrane but also for preparing an immobilized carrier or a chemical reaction system was obtained in this work.

#### *2.2.2 Water permeation mechanism of an N-acetylated chitosan membrane*

A novel model of the water permeation mechanism in an *N*-acetyl-chitosan membrane with a cellular structure was proposed [18]. Although the entire membrane structure has a hydrophilic character, the cellular structure incorporates junction zones that practically prevent water permeation.

#### **Figure 2.**

*Effect of acetic anhydride on* N*-acetylation of chitosan. Acetic anhydride was added to 2 wt.% chitosan solution (50 g) [17] with permission from Elsevier.*

*Innovative Separation Technology Utilizing Marine Bioresources: Multifaceted Development… DOI: http://dx.doi.org/10.5772/intechopen.95839*

Chitosan membranes with a controlled degree of deacetylation (*DD*) were prepared using a casting method. Changes in the total water content and the pressure driven water flux of the membrane were observed with a change in *DD* (**Figure 3**). The membrane properties were analyzed and evaluated using water permeability measurements, scanning electron microscopy (SEM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). SEM observations indicated that the membrane structure was an individual cellular structure and that this cellular structure grew with decrease in *DD* (**Figure 4**). From XRD measurements, the intensity in the range from 10° to 20° were detected in the chitin (*DD* = 1.1%) and the chitosan membranes (71.3% < *DD* < 92.2%), which indicated that the crystal structure of the membrane was amorphous regardless of *DD*. The free water content (*W*f), the freezable bound water (*W*fb), and the bound water not able to freeze (*W*b) were evaluated by DSC. The total water content and the sum of free water content ratios (*W*<sup>f</sup> + *W*fb) decreased with increasing *DD* whereas *W*<sup>b</sup> gradually increased [18]. That suggests the membrane prepared from lower *DD* chitosan formed remarkable cellular structure. The free water was mainly contained inside of the cellular structure, and resulted in swelling the chitosan membrane (**Figure 5**).

#### **Figure 3.**

indicated by the color changing from blue to claret. The deacetylation degree was

*X=*161 *X=*161 þ *Y=*203

In these equations, *X* is the equivalent mass of glucosamine contained in a 1 g sample of the chitosan–acetic acid solution, *v* [ml] is the volume of 0.0025 N PVS-K solution, and *f* is its concentration factor. Y is the mass of acetyl glucosamine contained in a 1 g sample, calculated as the difference between the mass of the sample and the value of *X*. We evaluated the deacetylation degree of the chitosan

A chitosan membrane was prepared by the casting method in combination with *N*-acetylation reaction [17]. The deacetylation degree (*DD*) decreased linearly with increasing added amounts of acetic anhydride (**Figure 2**). Stoichiometric control of the deacetylation degree to the desired level was successfully performed. However, gelation reaction due to excess addition of acetic anhydride inhibited formation of

The gelation behavior of chitosan, which has various degrees of acetylation (*DA*) of amino groups, was investigated to ensure preparation of the designed membrane structure [37]. The gelation behavior was evaluated by the gelation time and the quantity of syneresis, and useful information not only for preparing a membrane but also for preparing an immobilized carrier or a chemical reaction system was

*2.2.2 Water permeation mechanism of an N-acetylated chitosan membrane*

junction zones that practically prevent water permeation.

A novel model of the water permeation mechanism in an *N*-acetyl-chitosan membrane with a cellular structure was proposed [18]. Although the entire membrane structure has a hydrophilic character, the cellular structure incorporates

*Effect of acetic anhydride on* N*-acetylation of chitosan. Acetic anhydride was added to 2 wt.% chitosan solution*

*<sup>X</sup>* <sup>¼</sup> *<sup>f</sup>* � <sup>0</sup>*:*<sup>0025</sup> � <sup>10</sup>�<sup>3</sup> � *<sup>v</sup>* � <sup>161</sup> (1)

*<sup>Y</sup>* <sup>¼</sup> <sup>0</sup>*:*<sup>5</sup> � <sup>10</sup>�<sup>2</sup> � *<sup>X</sup>* (2)

� 100 (3)

calculated using the following equations.

*DD* ð Þ¼ %

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

samples as a molar fraction of glucosamine [36].

*2.2.1 Control of the deacetylation degree*

the chitosan membrane.

obtained in this work.

**Figure 2.**

**204**

*(50 g) [17] with permission from Elsevier.*

*Change in water flux (50 kPa, 298 K, μL = 0.901 mPa s) and total water content of membrane with regulated* DD*. (*●*) water flux,* <sup>J</sup>*<sup>v</sup> and (*○*) total water content,* <sup>W</sup>*<sup>t</sup> [18] with permission from Elsevier.*

#### **Figure 4.**

*SEM images observed at cross-section of the membranes prepared from different DD chitosan [18] with permission from Elsevier. (a) and (b) DD 71.3%; (c) and (d) DD 81.8%; (e) and (f) DD 92.2%. The full length of reference scaling measure in (a), (c) and (e) indicates 10 μm. The full length of reference scaling measure in (b), (d) and (f) indicated 5 μm.*

**2.3 Molecular weight**

sic viscosity (*η* [mL g�<sup>1</sup>

**2.4 Cross-linker**

adsorption [49].

**207**

follows:

**Figure 6**

metric average molecular weight (*M* [g mol�<sup>1</sup>

*DOI: http://dx.doi.org/10.5772/intechopen.95839*

The molecular weight of chitosan also plays a significant role in the properties of a prepared membrane. It was found that the tensile strength and elongation of membranes prepared from high molecular weight chitosan were higher than those prepared from low molecular weight chitosan; however, the permeability of membranes from high molecular weight chitosan is lower than those prepared from low molecular weight ones [38]. For convenient determination, the visco-

*Photographical evidence of antibacterial activity of chitosan membrane immersed in mannitol salt agar culture involved with degree of deacetylation: (a)* DD *92.2%; (b)* DD *90.1%; (c)* DD *88.0%; (d)* DD *83.9%; (e)* DD

*Innovative Separation Technology Utilizing Marine Bioresources: Multifaceted Development…*

For a case, chitosan is dissolved in an acetate buffer composed of 0.2 M acetic acid +0.2 M sodium acetate at 25 ̊C. The parameters in the above relationship were

Various types of cross-linkers are often employed for the fabrication of suitable

chitosan polymer chains *via* the Schiff reaction between aldehyde groups and amino groups to form covalent imine bonds [41–43]; however, GA is also toxic. Genipin, which is produced from the hydrolysis of geniposide extracted from the fruits of *Gardenia jasminoides Ellis*, is attracted as a biocompatible and low biohazardous cross-linker [44, 45]. Tetraethyl orthosilicate (TEOS) is also used as a covalent cross-linker, which serves to cross-link with chitosan polymers at their hydroxyl groups, to immobilize adsorbent particles, and to hydrophobize membrane surfaces

chitosan membranes adapted to separation processes. Glutaraldehyde (GA) is usually used as cross-linker, because it is extremely reactive in cross-linking

[22, 46–48]. Jóźwiak and coworkers widely investigated the effect of the

cross-linker type occurring covalent bond or ionic bond on the chitosan hydrogel prepared for ionic dye adsorption. In case of covalent agents, it is suggested that epoxide functional groups prefer to attack hydroxyl groups of chitosan during cross-linking, and the free amine groups formed are responsible for anionic dye

found as *<sup>K</sup>* = 7.9 � <sup>10</sup>�<sup>2</sup> [mL/g] and *<sup>α</sup>* = 0.796 [�] [27, 39, 40].

*79.7%; (f)* DD *75.5%; (g) PVC; (h) control [19] with permission from Elsevier.*

]) using the Mark-Houwink-Sakurada relationship as

]) can be calculated from the intrin-

½ �¼ <sup>η</sup> *KM<sup>α</sup>* (4)

#### **Figure 5.**

*Schematic illustration of the water permeation mechanism in an* N*-acetyl-chitosan membrane with a cellular structure. (a) the structure of a high* DD *chitosan membrane, (b) the structure of a low* DD *chitosan membrane, and (c) the detailed image of a low DD chitosan membrane with its cellular structure and water channels. The cellular structure illustrated in (b) is composed of immobilized water, the cellular wall, and the junction zone. The structure prevents water flux [18] with permission from Elsevier.*

Pressure driven water flux was measured using the ultrafiltration apparatus; it was dependent on the operational pressure, membrane thickness, and the feed solution viscosity, and obeyed the Hagen-Poiseuille flow. At a higher *DD*, water permeation proceeded due to degradation of the cellular structure; the amount of water in permeation channels was greater than that for lower *DD* membranes even though the total water content in the membrane was less. The water flux of the chitosan membrane was determined by the water content constructing channels through the membrane and not by the total water content in the membrane.

#### *2.2.3 Antibacterial activity*

The antibacterial activity is also explained, because of its long-time practical application. The antibacterial activity of chitosan membranes was investigated by a conductimetric assay using a bactometer [19]. The growth of the gram-positive sample (*S. aureus*) was more strongly inhibited by chitosan than the gram-negative sample (*E. coli*). This inhibitory effect was recognized as a bactericidal effect. Antibacterial activity was also observed and was dependent on the shape and specific surface area of the powdered chitosan membrane. The influence of the *DD* of the chitosan on inhibiting the growth of *S. aureus* was investigated by two methods: incubation using a mannitol salt agar medium and a conductimetric assay (**Figure 6**). In both methods, chitosan with a higher *DD* successfully inhibited growth of *S. aureus*. Our findings regarding the dominant role of the *DD* of chitosan will be useful for designing lasting, hygienic, membrane-based processes.

*Innovative Separation Technology Utilizing Marine Bioresources: Multifaceted Development… DOI: http://dx.doi.org/10.5772/intechopen.95839*

**Figure 6**

*Photographical evidence of antibacterial activity of chitosan membrane immersed in mannitol salt agar culture involved with degree of deacetylation: (a)* DD *92.2%; (b)* DD *90.1%; (c)* DD *88.0%; (d)* DD *83.9%; (e)* DD *79.7%; (f)* DD *75.5%; (g) PVC; (h) control [19] with permission from Elsevier.*

## **2.3 Molecular weight**

The molecular weight of chitosan also plays a significant role in the properties of a prepared membrane. It was found that the tensile strength and elongation of membranes prepared from high molecular weight chitosan were higher than those prepared from low molecular weight chitosan; however, the permeability of membranes from high molecular weight chitosan is lower than those prepared from low molecular weight ones [38]. For convenient determination, the viscometric average molecular weight (*M* [g mol�<sup>1</sup> ]) can be calculated from the intrinsic viscosity (*η* [mL g�<sup>1</sup> ]) using the Mark-Houwink-Sakurada relationship as follows:

$$\left[\eta\right] = K\mathcal{M}^a \tag{4}$$

For a case, chitosan is dissolved in an acetate buffer composed of 0.2 M acetic acid +0.2 M sodium acetate at 25 ̊C. The parameters in the above relationship were found as *<sup>K</sup>* = 7.9 � <sup>10</sup>�<sup>2</sup> [mL/g] and *<sup>α</sup>* = 0.796 [�] [27, 39, 40].

#### **2.4 Cross-linker**

Pressure driven water flux was measured using the ultrafiltration apparatus; it was dependent on the operational pressure, membrane thickness, and the feed solution viscosity, and obeyed the Hagen-Poiseuille flow. At a higher *DD*, water permeation proceeded due to degradation of the cellular structure; the amount of water in permeation channels was greater than that for lower *DD* membranes even though the total water content in the membrane was less. The water flux of the chitosan membrane was determined by the water content constructing channels through the membrane and not by the total water content in the membrane.

*Schematic illustration of the water permeation mechanism in an* N*-acetyl-chitosan membrane with a cellular structure. (a) the structure of a high* DD *chitosan membrane, (b) the structure of a low* DD *chitosan membrane, and (c) the detailed image of a low DD chitosan membrane with its cellular structure and water channels. The cellular structure illustrated in (b) is composed of immobilized water, the cellular wall, and the*

*junction zone. The structure prevents water flux [18] with permission from Elsevier.*

*Chitin and Chitosan - Physicochemical Properties and Industrial Applications*

The antibacterial activity is also explained, because of its long-time practical application. The antibacterial activity of chitosan membranes was investigated by a conductimetric assay using a bactometer [19]. The growth of the gram-positive sample (*S. aureus*) was more strongly inhibited by chitosan than the gram-negative sample (*E. coli*). This inhibitory effect was recognized as a bactericidal effect. Antibacterial activity was also observed and was dependent on the shape and specific surface area of the powdered chitosan membrane. The influence of the *DD* of the chitosan on inhibiting the growth of *S. aureus* was investigated by two methods: incubation using a mannitol salt agar medium and a conductimetric assay (**Figure 6**). In both methods, chitosan with a higher *DD* successfully inhibited growth of *S. aureus*. Our findings regarding the dominant role of the *DD* of chitosan

will be useful for designing lasting, hygienic, membrane-based processes.

*2.2.3 Antibacterial activity*

**Figure 5.**

**206**

Various types of cross-linkers are often employed for the fabrication of suitable chitosan membranes adapted to separation processes. Glutaraldehyde (GA) is usually used as cross-linker, because it is extremely reactive in cross-linking chitosan polymer chains *via* the Schiff reaction between aldehyde groups and amino groups to form covalent imine bonds [41–43]; however, GA is also toxic. Genipin, which is produced from the hydrolysis of geniposide extracted from the fruits of *Gardenia jasminoides Ellis*, is attracted as a biocompatible and low biohazardous cross-linker [44, 45]. Tetraethyl orthosilicate (TEOS) is also used as a covalent cross-linker, which serves to cross-link with chitosan polymers at their hydroxyl groups, to immobilize adsorbent particles, and to hydrophobize membrane surfaces [22, 46–48]. Jóźwiak and coworkers widely investigated the effect of the cross-linker type occurring covalent bond or ionic bond on the chitosan hydrogel prepared for ionic dye adsorption. In case of covalent agents, it is suggested that epoxide functional groups prefer to attack hydroxyl groups of chitosan during cross-linking, and the free amine groups formed are responsible for anionic dye adsorption [49].
