9.1.1 Basic conductivity measurement

A solution of chitosan was prepared by dissolving a mass of 150 mg of chitosan in 10 ml of hydrochloric acid (0.1 N) and then the volume was adjusted to 200 ml by addition of distilled water. The prepared solution is titrated with stirring with sodium hydroxide solution (0.1 N). Figure 3A shows the change in the volume of sodium hydroxide as a function of the conductivity of the chitosan solution. The curve has two points of inflection. The difference in the volume of NaOH between these two points corresponds to the amount of HCl required to dissolve the chitosan, which is to say to transform the -NH2 groups into -NH3 + .

The degree of deacetylation (DD) of chitosan is then determined from the following relationship [70]:

$$\text{DD} = 203 \times (\text{V2} - \text{V1}) \times \frac{\text{N}}{\text{m} + 42 \times (\text{V2} - \text{V1}) \times \text{N}} \times 100 \tag{7}$$

where N is the normality of the NaOH solution (mol/l); V2 and V1 are the equivalent volumes of NaOH representing two inflection points, respectively; M is the mass of chitosan; 203 (g/mol) is the molar mass of the acetyl monomer; and 42 (g/mol) is the difference between the molecular weight of the acetyl monomer and the molecular weight of the deacetylated monomer.

$$\text{DD} = 203 \times (9.4 - 2.7).10 - 3 \times \frac{0.1}{0.15 + 42 \times (9.4 - 2.7).10 - 3 \times 0.1} \times 100 \tag{8}$$

The degree of deacetylation according to the conductometric method is: DD ¼ 76:35%:

#### Figure 3.

(A) Variation of the conductivity of the chitosan solution as a function of the volume of the base (B) variation of the conductivity of the chitosan solution as a function of the volume of the acid solution.

irradiation in order to reduce the impact of environmental pollution due to excessive use of chemical treatments [80–82]. The study will examine the effect of chemical addition, reaction time, operating temperature on manufacturing, and chitosan DD under microwave irradiation. These results will be compared to those from conventional heating methods to compare results. As part of this research, they developed the design and manufacture of a proton prototype for the production of chitosan from shrimp shell waste. Research has concluded that microwaves

Degree of deacetylation (DD), molecular weight (mw), and solubility of chitosan samples extracted from

Mean values in the same column bearing the same superscript do not differ significantly.

Chitin treatment NaOH conc. (%) DD% Mw (k Daltons) Solubility (%) 20 mesh <sup>30</sup> 67.58<sup>F</sup> 0.92 2415.09 66.31H 0.35

40 mesh <sup>30</sup> 76.89DE 0.89 866.03 99.05<sup>A</sup> 0.05

60 mesh <sup>30</sup> 88.39<sup>B</sup> 0.49 949.95 83.28<sup>F</sup> 0.87

<sup>40</sup> 75.77<sup>E</sup> 3.54 1476.21 74.94<sup>G</sup> 0.79 <sup>50</sup> 78.83D 1.05 1267.11 96.77BC 0.17

<sup>40</sup> 78.64<sup>D</sup> 0.86 1107.50 92.79<sup>D</sup> 0.01 <sup>50</sup> 83.05<sup>C</sup> 0.29 2160.88 95.60<sup>C</sup> 0.87

<sup>40</sup> 89.17B 0.28 1274.85 85.57<sup>E</sup> 1.37 <sup>50</sup> 95.19A 0.74 4467.05 97.73AB 0.95

85.00<sup>C</sup> 0.66 300 99.00<sup>A</sup> 0.72

The results showed that the demineralization condition of shrimp waste was achieved at the concentration of HCl 3,5 N solution with the weight ratio of shrimp shell waste and HCl solution of 1: 5 (w/v), at a temperature of 50°C during 1 h heating. In those conditions, the ash content was 8.06%. Ash content decreases to 5.4% if the demineralization reaction is carried out under microwave irradiation with 130 watts for 10 min. The optimum condition of the deproteinization process was achieved by heating at a temperature of 70°C for 2 h and at 4% NaOH concentration for shrimp waste ratio: a NaOH solution of 1:5 (w/v). In this condition, they obtained nitrogen levels of 1.882% (11.763% protein content). If the deproteinization reaction was performed under microwave irradiation with 130 W of power for 15 min, the nitro-

gen content obtained was 1.833% (11.461% protein content) (Table 10).

deacetylation (DD) such that: DD = 100 - DA in%.

9. Comparison between IR and other techniques for calculating DD

To differentiate chitin from chitosan, it is necessary to define the degree of acetylation (DA), that is to say the ratio of the number of units comprising an acetyl group on the number of units in the molecule. We can also speak of degree of

The calculation of DD was made according to several methods described in the literature: acid and basic conductometric method, pH-metric method, UV method, and IR spectroscopy method. In a recent study [70], an attempt was made to

will accelerate reaction time.

shrimp wastes using microwave technique [81].

Modern Spectroscopic Techniques and Applications

Commercial chitosan

Data are the mean.

Table 10.

120

### 9.1.2 Acidic conductivity dosing

The determination of the DD by acid conductometric assay is carried out as follows: a mass of 150 mg of chitosan is dispersed in 200 ml of distilled water; while stirring, the mixture is titrated with 0.1 N HCl solution. Figure 3B shows the evolution of the conductivity of the chitosan solution as a function of the volume of HCl poured. The point of inflection corresponds to the amount of HCl consumed by the amine groups of chitosan. The DD will be calculated from the following equation:

$$\text{DD} = (\text{203} \times \text{V} \times \text{N}) / (\text{m} + 4\text{2} \times \text{V} \times \text{N}) \times \text{100} \tag{9}$$

where N is the normality of the HCI solution (mol/l), V is the volume corresponding to the inflection point as shown in Figure 3B, m is the mass of chitosan (g), and 42 (g/mol) is the difference between the molecular weight of the acetylated monomer and the molecular weight of the deacetylated monomer.

DDA calculation gives:

$$\begin{aligned} \text{DD} &= (203 \times 6.8 \times 10 - 3 \times 0.1) / (0.15 + 42 \times 6.8 \times 10 - 3 \times 0.1) \times 100 \\ \text{DD} &= 77.30\%. \end{aligned} \tag{10}$$

#### 9.2 Determination of the degree of deacetylation by pH-metric determination

The determination of the DD by pH-metric assay was carried out according to the method described in the literature [77, 78].

A solution of chitosan was prepared by dissolving a mass of 125 mg of chitosan in a solution of excess HCl (0.1 N) and then neutralizing this solution with sodium hydroxide solution (0.05 N). Figure 4A shows the titration curve of chitosan, and Figure 4B shows the corresponding secondary derivative. From this last curve, the amount of hydrochloric acid necessary to protonate the amine groups is determined. The degree of deacetylation calculated from this method is 77.10%.

The results of the DDA obtained by the different assay methods are shown in Table 11.

The average value of DD calculated for the chitosan prepared during this work is 77.32%. So, IR spectroscopy remains the simplest and most economical technique for calculating DD.

9.3 Comparison to UV: visible

Comparison of DD value measured by different methods [79].

10. Conclusion

123

Table 12.

Table 11.

DD values obtained with different assay methods [70].

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

Another study presented by Xiaofei and al [79] gave a comparison between IR spectroscopy and other techniques and showed the importance of FTIR for calcu-

Dosing method DDA (%) Basic conductivity meter 76.35 Conductometric acid 77.30 pH-metric 77.10 Infrared spectroscopy 78.50 Calculated average 77.32

Quantitative Analysis by IR: Determination of Chitin/Chitosan DD

Method 1 (%) 2 (%) 3 (%) 4 (%) 5 (%) Part-I DDTitration1 90.82 90.57 94.93 93.04 92.48 DDTitration2 92.85 94.62 93.97 95.40 94.99 DDTitration3 93.10 94.45 94.86 94.35 94.66 DD-UV1 95.97 95.96 99.42 98.18 99.34 DD-UV2 94.44 94.87 97.72 97.07 97.00 DD-UV3 95.02 95.36 98.07 97.42 97.99 Part-II DD-UV1 82.00 95.96 97.68 100.00 100.00 DD-UV2 82.27 95.86 97.73 100.00 100.00 DD-IR1658/3450 80.43 94.50 94.60 95.37 96.33 DD-IR1320/1420 87.64 100.00 100.00 100.00 100.00

The calculation of DD and the number of amine present in chitosan by FTIR allows first to follow the transformation reaction of chitin into chitosan and others by finding the optimal conditions for the synthesis of chitosan by studying the effect of different parameters, namely the concentration of the base, the temperature, and the duration of the reaction. The valorization of shrimp exoskeletons by extraction of chitosan according to the hydrothermal-chemical technique proposed in two stages makes it possible to reduce the production time by at least four times compared to the conventional technique in three stages (3–4 days). In addition, the consumption of digestion and energy chemicals is also significantly reduced. The chitosan obtained by the two-step technique is of good quality. Indeed, the degree

lating DD (Table 12). They have been measured for 3 or 2 times.

#### Figure 4.

(A) Variation of the pH according to the volume of soda poured for the solution of the chitosan and the solution of the hydrochloric acid, (B) secondary derivative curve corresponding to the assay.

