4. How to follow the transformation reaction of chitin into chitosan (deacetylation) and calculate DD by IR spectroscopy

The determination of average DD for chitosan may be performed by different techniques: infrared spectroscopy, elementary analysis, potentiometric titration,


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

Table 3.

3.1 Chemical deacetylation

Equation of chitin conversion to chitosan by deacetylation.

Modern Spectroscopic Techniques and Applications

Figure 1.

112

3.2 Enzymatic deacetylation

To deacetylate chitin chemically, acids or bases are used. However, glycosidic bonds are very sensitive to acid treatment [51, 52]; therefore, alkaline deacetylation is often used. The deacetylation reaction is heterogeneous [53] or homogeneous [54]. Generally, in the heterogeneous process, chitin is treated with hot concentrated NaOH solution for a few hours and chitosan is produced as an 85–99% deacetylated insoluble residue. According to the homogeneous method, the alkaline chitin is prepared after dispersion of the chitin in concentrated NaOH (30 g NaOH/45 g H2O/3 g Chitin) at 25°C for 3 h or more and then dissolved in crushed ice around 0°C. This method gives a soluble chitosan with an average degree of acetylation of 48–55% [50]. This process produces a deacetylation with acetyl groups uniformly distributed in the

The details of the process parameters employed by various researchers that include the number of demineralization, deproteinization, and deacetylation step

The main disadvantages of chemical deacetylation are energy consumption, waste concentrated alkaline solutions and thus increased environmental pollution. In order to avoid these disadvantages, an alternative enzymatic method exploiting chitin deacetylases has been explored. The use of chitin deacetylase offers the possibility of a nondegradable controlled process, leading to the production of welldefined chitosan [56]. This method is especially used to prepare chitosan oligomers. Chitin deacetylase catalyzes the hydrolysis of N-acetamido linkages in chitin to produce chitosan. The presence of this enzymatic activity has been reported in several fungi [57, 58] and insect species [59]. The most studied enzymes are those extracted from the mushrooms Mucor rouxii [46, 56, 57], Absidia coerulea [60, 61],

lindemuthianum [64, 65]. All enzymes are glycoproteins and are secreted either in the periplasmic region or in the culture medium. In addition, all the enzymes exhibit remarkable thermal stability at their optimum temperature (50°C) and a

4. How to follow the transformation reaction of chitin into chitosan

The determination of average DD for chitosan may be performed by different techniques: infrared spectroscopy, elementary analysis, potentiometric titration,

chains, for example chitosan with DA = 10% after 580 h at 25°C [54].

and Aspergillus nidulans [62, 63], and two strains of Colletotrichum

very high specificity for the bound N-acetyl-D-glucosamine polymers.

(deacetylation) and calculate DD by IR spectroscopy

have been reviewed and summarized in Table 3.

Comparison of chitosan production from different sources according to Tolaimate et al. [55].

and <sup>1</sup> H liquid state and solid state 13C-NMR. But FTIR technique has specifically proved to be useful for the analysis of chitin due to its limited solubility in most of the solvents. Nevertheless, FTIR needs a calibration versus an absolute technique like nuclear magnetic resonance (NMR). Identifying the right combination of bands and baselines required a lot of effort, which led the authors to preface a large number of methods in the literature. In fact, several methods have been tried to determine the degree of deacetylation (DD) by FTIR [18, 66–69]. Below is a description of the major three types used to calculate DD by FTIR.

#### 4.1 Formula 1

El ouahli [70] calculated from the absorption bands at 1320 and 1420 cm�<sup>1</sup> . The first band is characteristic of the acetylated amine or amide function, while the second band is chosen as the reference band. The following equation is used to determine DD of chitosan:

$$\text{DD\%} = 100 - \left(\frac{\text{A1320}}{\text{A1420}} - 0.3822\right) \* 1/0.03133 \tag{2}$$

#### 4.2 Formula 2

This IR characterization formula for chitosan is based on the relationship between the absorbance (A) value of the primary amide at 1655 cm�<sup>1</sup> and that of the hydroxyl at 3450 cm�<sup>1</sup> . The degree of deacetylation (DD) was calculated by Eq. (3), [66, 69]:

$$\text{DD\%} = \mathbf{100} - \left[ \left( \frac{\mathbf{A\_{1655}}}{\mathbf{A\_{3450}}} \right) \* \mathbf{115} \right] \tag{3}$$
