2. How to extract chitin

"construction of the molecular edifice." In organic compounds, it allows for example to differentiate the isomers of position (ortho-, meta-, and para-) aromatic hydrocarbons, as well as the cis- and transisomers of olefins. For mineral compounds, the infrared spectrum depends on the symmetry of molecules; it often allows finding the system in which a chemical compound is crystallized. It is also possible, but only in the case of small molecules, to calculate geometric parameters such as moments of inertia. The analysis is done by comparing with reference spectra of which there are several files. Also, FTIR spectroscopy is the method most used for calculating the degree of DD of chitosan. This polymer was discovered in 1859 by C. Rouget by treating chitin with concentrated KOH at elevated temperature. But it was not until 1894 that Hoppe-Seyler gave the "modified chitin" the name chitosan [1]. Chitosan has some advantageous properties, such as biocompatibility, biodegradable polymer of high molecular weight, nontoxic, and antimicrobial activity, that encourage its applications in many fields including agriculture [2, 3], paper industry, food and textile industries, pharmaceutics [4, 5], biochemistry, biotechnology, cosmetics, biomedical applications [6–8], environment, and water treatment [9–13]. The properties of chitin and chitosan depend considerably on the degree of deacetylation (DD), a parameter defined as the mole fraction of deacetylated units in the polymer chain [14, 15]. Therefore, the determination of DD has been one of the interesting parameters to study chitosan preparations. The process of deacetylation involves the removal of acetyl groups from the molecular chain of chitin, leaving behind a complete amino group (-NH2), and chitosan properties are very linked on this high degree of chemical reactive amino groups. Since the degree of deacetylation (DD) depends mainly on the method of purification and reaction conditions, it is therefore essential to characterize chitosan by determining its DD prior to its use. The main parameters involved in the process are temperature, time of reactions, and the concentration of reagents. A simple and nonexpensive chemical treatment of mineral/protein removal from chitin is usually used with HCl/NaOH reagents, respectively, and chitosan is chemically or enzymatically produced. They vary only on the acetyl group container, which is designated by the degree of acetylating (DA) designating the percentage of acetylated units relative to the number of total units. The term chitosan applies to any copolymer whose DD is greater than 50%. Each chitosan is therefore characterized by the fraction of residual N-acetamide groups (DA) or by the relative amount of amino groups of the chitosan molecule (DD = 1-DA) [16]. It is important to distinguish between the degree of acetylation (DA) and the degree of deacetylation (DD). One being the opposite of the other, that is to say that chitosan having an 85% DD, it has

Modern Spectroscopic Techniques and Applications

15% of acetyl groups and 85% of amine groups on its chains. The degree of

108

deacetylation (DD) of chitosan is a dominant structural parameter that significantly influences the physicochemical properties of chitosan such as solubility, overall charge, reactivity, and mechanical properties such as elongation, breaking, and tensile strength. This parameter also influences biological properties [17] such as biocompatibility and biodegradability. For determination of the degree of

deacetylation (DD), several analytical methods have been employed. Infrared spectroscopy [18, 19] and UV spectrophotometry [20] as analytical tools offer advantage over other traditional techniques which are expensive and destructive to the sample. FTIR spectroscopy is a quick technique for a quantitative evaluation of the DD through the determination of absorption ratios. FTIR analysis is attractive due to its nondestructive character, fastness, sensitivity, and suitability for both soluble and nonsoluble samples. Among the solution methods, first-derivative UV spectrophotometry draws attention owing to its simplicity and effectiveness in providing accurate results for highly deacetylated chitin. It was conceived by Muzzarelli and

Chitin is the structural polymer of exoskeletons of all arthropods (crustaceans and insects) and endoskeletons of cephalopods (cuttlefish, squid, etc.). The cuticles of various crustaceans, mainly crabs and shrimp, are the main sources of raw material for the production of chitin (Table 1). Chitin is found as part of a complex network of proteins on which calcium carbonate is deposited to form the rigid shell in crustaceans or more specifically in shellfish. The interaction between chitin and proteins is very intimate and there is also a small fraction of proteins involved in a polysaccharide-protein complex [24]. Thus, the preparation of chitin from shellfish requires the elimination of the two main constituents, namely proteins by deproteinization and calcium carbonate by demineralization, as well as small amounts of pigments and lipids generally removed during the two steps. An additional fading step is applied to remove residual pigments. Many methods have been proposed and used over the years to prepare pure chitin; however, no standard method has been


Table 1. Sources of chitin [25]. adopted. Deproteinization and demineralization can be carried out using chemical or enzymatic treatments. In the case of shrimp, the shell wall is thinner, which facilitates the isolation of chitin compared to other types of shells. The selected shells are then cleaned, dried, and ground into small shell pieces. Shrimp carapaces have the following average mass composition:


We then develop the two essential steps for the preparation of chitin from the carapaces, namely deproteinization and demineralization.

### 2.1 Chemical deproteinization

The deproteinization of chitin consists in eliminating proteins; it is difficult because there is a breakdown of the chemical bonds between chitin and proteins. This is done using basic solutions in a heterogeneous way. Complete protein isolation is particularly important for biomedical applications. A wide range of chemicals have been tested as deproteinization reagents, including NaOH, Na2CO3, NaHCO3, KOH, K2CO3, Ca(OH)2, Na2SO3, NaHSO3, CaHSO3, Na3PO4, and Na2S. The reaction conditions vary considerably in each study. NaOH is the preferred reagent and is applied at a concentration ranging from 0.125 to 12 M, at different temperatures (up to 20°C) and a duration of treatment (from a few minutes to a few days). In addition to deproteinization, the use of NaOH results in partial deacetylation of chitin and hydrolysis of the biopolymer, which decreases its molecular weight.

### 2.2 Chemical demineralization

Demineralization is a necessary step to produce chitosan. It consists of dissolving minerals, mainly calcium carbonate bound to chitin. Demineralization is generally carried out by acid treatment using HCl, HNO3, H2SO4, CH3COOH, and HCOOH [26, 27]. Of these acids, the preferred reagent is dilute hydrochloric acid. Demineralization is an acid–base reaction between carbonate ions and acids in water with the release of carbon dioxide, as indicated in the following equation:

$$\text{2HCl} + \text{CaCO}\_3 \rightarrow \text{CaCl}\_2 + \text{H}\_2\text{O} + \text{CO}\_2 \tag{1}$$

3. How to extract chitosan from chitin

Source CNaOH\* T°C Number of

DOI: http://dx.doi.org/10.5772/intechopen.89708

100 100

90

85

95

Lobster 10% 100 1 2.5 10% HCl

Shrimp 0.125 M

Shrimp 7.5–

0.75 M

12.5 M

Crab 1.25 M 85–

Lobster 5% 80–

Krill 0.875 90–

Chitin extraction conditions.

Crab/ lobster

Table 2.

baths

Quantitative Analysis by IR: Determination of Chitin/Chitosan DD

1 1

lar weight of the chitosan, is also produced.

mass produce [51].

111

Chitosan represents a family of polymers obtained at varying degrees after deacetylation of chitin. In fact, the degree of acetylation (DA), which reflects the balance between the two types of residues (Figure 1), differentiates chitin from chitosan. When the DA (expressed as molar percentage) is less than 50 mol%, the product is called chitosan, and it is characterized by its solubility in acidic solutions [45]. During deacetylation, the amides are protonated and the acetyl groups are removed, but a depolymerization reaction, indicated by the changes in the molecu-

Deproteinization Demineralization

CHCL T°C DURAT-ION

1 30–180 mn 0.3–3.5 M Room 24 [23]

3 24 1.37 M Room 24 [36]

2 0.5 5% 70 4 [40]

1 2 0.6 M Room 2 [42]

Room 18 [41]

90% formic

1.25 M Room 1 [28]

(h)

Duration (h)

> 0.5 0.5

Shrimp 1.25 M 100 1 0.5 1.57 M 20–22 1–3 [29] Shrimp 1% 65 1 1 0.5 M Room — [30] Shrimp 3% 100 1 1 1 M Room 0.5 [31] Shrimp 4% 100 1 1 5% Room — [32]

Shrimp 1 M Room 1 24 h 1 M Room 24 [3] Crab 0.5 M 65 1 2 1.57 M Room 5 [33] Crab 1 M 80 1 3 1 M Room 12 [34] Crab 1 M 100 1 36 2 M Room 48 [28] Crab 1 M 100 3 72 1 M Room — [35]

Crab 1 M 50 1 6 1 M 20 3 [37]

Lobster 1 M 100 5 12 2 M Room 5 [39]

Krill 3.5% 25 1 2 3.5% 20 1.5 [43] Crawfish Room [44]

2.5 M Room 3 72 11 M 20 4 [38]

Chitin can be converted to chitosan by enzymatic preparations [46–49] or by a chemical process [50, 51]. Chemical methods are widely used for commercial purposes for the preparation of chitosan because of their low cost and their ability to

All the other minerals present in the crustacean cuticle react in the same way and give soluble salts in the presence of acid. Then, the salts can be easily separated by filtering the solid phase of chitin, followed by washing with distilled water.

Chemically, a kilogram of fresh shells provides about 40 g of dry chitin. After the chemical treatment, the grinding and sieving processes to obtain a homogeneous chitin cause losses of about 40%. The final yield is 2.5%, 25 g of chitin per kilogram of shell. Table 2 summarizes the operating conditions of chitin extraction according to different sources.

