1. Introduction

Very commonly used in quantitative analysis, infrared spectrometry FTIR has been exploited in many fields. It has been used in mineralogy in addition to chemical analyzes to determine the structure of a rock and to know the bonds between the atoms, in pharmaceutical for the quantitative measurement of the constituents and the thickness of the coating of the tablets, in dairy industries to determine the moisture of milk powders and butters, in the textile industry for fiber sizing and maturity of cottons, in the chemical industry, in agribusiness, and in several other industries. When a specific absorption of a chemical function of a complex molecule is sufficiently isolated in its spectrum, it is always possible to carry out quantitative measurements. There are several major fields of application for infrared spectrophotometry. First, we can quote functional analysis is probably the main application of infrared spectrometry, at least in industry. Then, the structural analysis, infrared spectrometry allows to obtain even more fine information, concerning the

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

Rocchetti [20] and relies on simple reagents and instrumentation. In addition, the results obtained from this method are reasonably independent of protein contamination. Alonso et al. [21] established the possibility to determine the acetylation degree with the use of empirical correlations based on the weight losses associated with the main decomposition peaks. A similar approach has been adopted to investigate if there was any relationship between the weight loss of the sample and its DD. According to Yu et al. [22], the conductometric assay is an adequate and accurate method for determining the degree of deacetylation of chitosan, except for some samples that have a high degree of crystallization. The conductometric method can be carried out in basic and acid medium. Other methods have also been used such as SEM and NMR for magnetic properties of certain atomic nuclei and the

Quantitative Analysis by IR: Determination of Chitin/Chitosan DD

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

The objective of this chapter is to present a bibliographic synthesis on the use of spectroscopy FTIR in order to study and optimize the reaction of deacetylation by calculating the chitosan DD. At the end of the chapter, a simple comparison between IR and other methods of DD determination is presented to find the most

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

Lower plants Annelid Mushrooms Molluscs

Spiders Ants Cockroaches Coleoptera (Order)

Earthworm Leech Cuttlefish Octopus

determination of DD.

reliable formula of DD calculation.

Algae Lichen yeasts Ascomycetes (Class)

Arthropods

Sources of chitin [25].

Lobsters Crabs Shrimps Scampi Krill

Table 1.

109

Penicillium

Crustaceans Arachnids Insects

Octopus Scorpions

Blastocladiales (Family) Chytridiaceae (Family)

2. How to extract chitin

Quantitative Analysis by IR: Determination of Chitin/Chitosan DD DOI: http://dx.doi.org/10.5772/intechopen.89708

Rocchetti [20] and relies on simple reagents and instrumentation. In addition, the results obtained from this method are reasonably independent of protein contamination. Alonso et al. [21] established the possibility to determine the acetylation degree with the use of empirical correlations based on the weight losses associated with the main decomposition peaks. A similar approach has been adopted to investigate if there was any relationship between the weight loss of the sample and its DD. According to Yu et al. [22], the conductometric assay is an adequate and accurate method for determining the degree of deacetylation of chitosan, except for some samples that have a high degree of crystallization. The conductometric method can be carried out in basic and acid medium. Other methods have also been used such as SEM and NMR for magnetic properties of certain atomic nuclei and the determination of DD.

The objective of this chapter is to present a bibliographic synthesis on the use of spectroscopy FTIR in order to study and optimize the reaction of deacetylation by calculating the chitosan DD. At the end of the chapter, a simple comparison between IR and other methods of DD determination is presented to find the most reliable formula of DD calculation.
