Open access peer-reviewed chapter

Quantitative Analysis by IR: Determination of Chitin/Chitosan DD

By Boukhlifi Fatima

Submitted: April 21st 2019Reviewed: September 13th 2019Published: March 4th 2020

DOI: 10.5772/intechopen.89708

Downloaded: 340

Abstract

FTIR spectroscopy has been widely used to quantitatively study the parameters of the chitin deacetylation. A new research on a Canadian chitin has shown that a degree of deacetylation (DD) of 90% has been reached with a base concentration of 12.5 M, a reaction time of 120 min, and a temperature of 110°C. In parallel, our study on Moroccan chitin allowed to reach 75%. A degree of deacetylation of 75% was obtained at T = 120°C and at CNaOH = 12 N in a single step for 6 hours. Another study followed by IR prepared the chitosan under pressure or under irradiation. Firstly, the compression method was used for preparing 100% deacetylated chitosan with less environmental pollution. The 100% fully deacetylated chitosan is produced in low-concentration alkali and high-pressure conditions under 0.11–0.12 MPa for 120 min. Secondly, microwave deacetylation showed that a degree of deacetylation of 95.19% was achieved after irradiating chitin at 60 meshes with 50% NaOH solution in a microwave for 10 min at 1400-W power. To find these results, the authors used different formulas to calculate DD by FTIRM, but the most used and reliable formula is that which calculates DD of chitosan by the report of absorbance of amide at 1655 cm−1 that measures the acetyl group and absorbance at 3430 cm−1 relating to the hydroxyl group.

Keywords

  • chitin
  • chitosan
  • DD
  • FTIR
  • optimization
  • synthesis

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, 7, 8], environment, and water treatment [9, 10, 11, 12, 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 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.

2. How to extract chitin

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 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:

  • 75% water

  • 12% protein

  • 9% mineral salts

  • 4% chitin

  • traces of lipids and organic pigments

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:

2HCl+CaCO3CaCl2+H2O+CO2E1

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.

Lower plantsAnnelidMushroomsMolluscs
Algae Lichen yeastsAscomycetes (Class)
Penicillium
Blastocladiales (Family)
Chytridiaceae (Family)
Earthworm LeechCuttlefish Octopus
Arthropods
CrustaceansArachnidsInsects
Lobsters
Crabs
Shrimps
Scampi
Krill
Octopus
Scorpions
Spiders
Ants
Cockroaches
Coleoptera (Order)

Table 1.

Sources of chitin [25].

DeproteinizationDemineralization
SourceCNaOH*T°CNumber of bathsDuration(h)CHCLT°CDURAT-ION (h)
Shrimp0.125 M
0.75 M
100
100
1
1
0.5
0.5
1.25 MRoom1[28]
Shrimp1.25 M10010.51.57 M20–221–3[29]
Shrimp1%65110.5 MRoom[30]
Shrimp3%100111 MRoom0.5[31]
Shrimp4%100115%Room[32]
Shrimp7.5–12.5 M130–180 mn0.3–3.5 MRoom24[23]
Shrimp1 MRoom124 h1 MRoom24[3]
Crab0.5 M65121.57 MRoom5[33]
Crab1 M80131 MRoom12[34]
Crab1 M1001362 MRoom48[28]
Crab1 M1003721 MRoom[35]
Crab1.25 M85–903241.37 MRoom24[36]
Crab1 M50161 M203[37]
Crab/lobster2.5 MRoom37211 M−204[38]
Lobster1 M1005122 MRoom5[39]
Lobster5%80–8520.55%704[40]
Lobster10%10012.510% HCl
90% formic
Room18[41]
Krill0.87590–95120.6 MRoom2[42]
Krill3.5%25123.5%201.5[43]
CrawfishRoom[44]

Table 2.

Chitin extraction conditions.

3. How to extract chitosan from chitin

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 molecular weight of the chitosan, is also produced.

Figure 1.

Equation of chitin conversion to chitosan by deacetylation.

Chitin can be converted to chitosan by enzymatic preparations [46, 47, 48, 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 mass produce [51].

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

The details of the process parameters employed by various researchers that include the number of demineralization, deproteinization, and deacetylation step have been reviewed and summarized in Table 3.

SourceNumber of deproteinization bathsNumber of demineralization bathsDA
0.3 M; NaOH 80°C; 1 h0.55 M HCl; 25°C; 2 h
CirripediaAnatife4251
ReptantiaRed crab3548
BrachyuraMarbled crab3350
ReptantiaSpider crab3347
MacruraLobster33
NatantiaCrayfish7351
Slipper lobster32
Freshwater cayfish32
Pink shrimp3351
Gray shrimp2251
StomatopodaSquilla3351
CephalopodaSquid2251

Table 3.

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

3.2 Enzymatic deacetylation

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 well-defined 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], and Aspergillus nidulans [62, 63], and two strains of Colletotrichum 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 very high specificity for the bound N-acetyl-D-glucosamine polymers.

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, and 1H 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, 67, 68, 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−1. 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:

DD%=100A1320A14200.38221/0.03133E2

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−1 and that of the hydroxyl at 3450 cm−1. The degree of deacetylation (DD) was calculated by Eq. (3), [66, 69]:

DD%=100A1655A3450115E3

4.3 Formula 3

In our study, a ratio had been already proposed [68, 71, 72]. However, choosing an appropriate calculation procedure was not an easy task since the choice of the baseline on FTIR spectra, reference, and the probe bands was difficult. In our work, the average DD was determined by the following formula:

DD%=100A1655A34501001.33E4

where A1655 is the absorbance at 1655 cm−1 of the amide I band as a measure of the N-acetyl group content and A3430 is the absorbance at 3430 cm−1 due to hydroxyl group as an internal standard. The value 1.33 represents the ratio of this absorbance for a fully acetylated compound. An appropriate baseline in each spectrum was determined by using origin software.

The baseline problem for reference peaks has been studied and summarized in Table 4 and Figure 2. Table 4 shows that there are other formulas for calculating chitosan DD by FTIR and that these formulas have been approved by other techniques such as NMR.

DA calibration curveMethod for DA standardRef.
1. DA = (A1655/A3450) × 155Titration[66]
2. (A1320/A1420) = 0.3822 + 0.03133 × DA1H NMR, 13C NMR[67]
3. (A1320/A3450) = 0.03146 + 0.00226 × DA1H NMR, 13C NMR[67]
4. (A1560/A2875) = 0.2 + 0.0125 × DAElemental analysis[71]

Table 4.

Calibration curves from absorption ratios versus standard DA values [73].

Figure 2.

FTIR spectrum of chitin and the baseline [73].

A synthesis of the study on the effect of the operating conditions to know temperature concentration of the base and the time on the reaction of deacetylation will be presented using IR.

5. Optimization of chitosan extraction by IR

5.1 IR study of the effect of alkaline concentration on the chitin deacetylation

In this deacetylation reaction, it is an interrelation between the following variables: the concentration of the basic solution, the temperature, and the reaction time. The deacetylation reaction in basic medium is summarized in Eq. (5):

RNHCOCH3+OHRNH2+CH3COOE5

For CNaOH = 12 N, the DD values calculated from the FTIR shown in Table 5 do not exceed 55% and stabilize after a critical time t < 60 min. At a low concentration of NaOH, the equilibrium time became higher, which shows that the chitosan DD is low, which means that the deacetylation reaction is highly dependent on the concentration of NaOH, due to the inaccessibility of acetamide groups in the polymer chain. This type of behavior, observed by other authors, is explained firstly by the fact that the N-deacetylation occurs preferably at the level of the amorphous region of chitin and then passes from the edge to the interior of the crystalline region [26, 27]. The second reason concerns the equilibrium of the reaction and the degradation of chitosan. Other authors [10, 22, 28] have assumed that it can be controlled by both reaction and diffusion. The low deacetylation of chitin has also been attributed to the rearrangement of acetyl groups in the monomer unit with respect to the OH hydroxyl group [9]. Analysis of the results of the measurement of the DD (Table 5) shows that this increased by approximately 8% (66–74% DD), at a high concentration of NaOH (10 M) and with a thermal increase of 20°C (100–120°C). This variation of DD seems less important, only +2% (26–28% DD) at low concentration of NaOH (7.5 M). This increase in DD is particularly significant (13%) with a base concentration increase of 2.5 M (10 M to 12.5 M). Thus, the variation in NaOH concentration more significantly influences the reaction rate of DD than temperature.

Temperature of DDConcentration of the base (M)Time (mn)DD
25°C818038
1012045
126055
80°C43835
83852
103855
123863
120°C850 min40
1250 min50
8300max65
12300max70

Table 5.

Evolution of DD as a function of the concentration of the base at different temperatures [3].

5.2 IR study of the effect of temperature on the chitin deacetylation

Table 6 shows the effect of temperature on deacetylation; the more the temperature increases, the more the deacetylation increases, but the total conversion of chitin to chitosan is only carried out at the temperature of 80°C and for high concentrations of the base [23]. Analysis of the results of the measurement of DD shows that it has increased approximately 8% (66–74% DD), at high concentration of NaOH (10 M) and a thermal increase of 20°C (100–120°C). This variation of DD seems less important, only +2% (26–28% DD) at low concentration of NaOH (7.5 M). This discrepancy can be considered as an error of analysis. However, this increase in DD is particularly significant (13%) with a base concentration increase of 2.5 M (10–12.5 M). The temperature in this step is a factor that weakens the binding of the acetyl groups and accelerates the deacetylation reaction. Ahlafi and al [3] also found that the DDs increase as the NaOH concentration and temperature increase as the time of reaction increases. These reached a maximum of 63% and 71% for CNaOH = 12 N at T = 80 and 120°C, respectively. On the other hand, it can be observed that the critical time decreases when the temperature and the concentration of NaOH increase. It can be seen in the case of CNaOH = 12 N and T = 120°C. The increase of DD values with these parameters can be attributed to the change of the structure during deacetylation of chitin at height temperature, as confirmed by FTIR studies. These results confirm the hypothesis that the interaction of concentration and thermal energy are the main criteria to be taken into account for the deacetylation reaction.

Ref. [23]Ref. [3]
T°C/CNaOH mol/l7.51012.5CNaOH mol/lT°CDD
4012818038
601014201012045
80246067126055
100266878818038
1202874901012045

Table 6.

Evolution of DD in relation to temperature at different concentrations of the base [3, 23].

5.3 FTIR study of the effect of time on the chitin deacetylation

The analysis of the results (Table 7) shows that the best deacetylation started after 60 min, for the three concentrations of NaOH studied. At this time, the activation energy for deacetylation can be reached and the chemical bond break occurs. However, this breakdown of the acetyl groups is significant only at the level of the high alkaline concentration (> 10 M). Diffusion of the alkaline solution on the shell substrate increases with time. These results confirm the hypothesis that there is an interaction between the concentration of NaOH and the reaction time on DD.

[23]120 °C [3]
C mol/l/t mn8.751011.2512.5C mol/l/t mn812
308234245504050
60136070781004852.5
90187581861505354
120238286912005567
15026.58488922506468
180308690933006570

Table 7.

Evolution of DD as a function of time at different concentrations of the base [3, 23].

At the lowest NaOH concentration (7.5 M), the percentage of deacetylation increases from about 60°C but remains low even at 120°C (Table 7). The extracted product remains predominantly in the form of chitin. The deacetylation is terminated after a period ranging from 90 to 120min depending on the concentration of NaOH. Moreover, for a lower concentration of NaOH (8.75 M), this reaction is incomplete even after 3 h for different authors [74]. Therefore, in the case of a low concentration of NaOH (less than 10 M), the deacetylation does not occur completely. Thus, to obtain a good-quality chitosan, it is necessary to resume the treatment several times using NaOH solution [75, 76]. This reprocessing scheme is technically not easy and economically unviable on a large scale. So, for better deacetylation, 1 h is sufficient for concentrations greater than 10 M.

The rate of deacetylation therefore depends not only on the concentration of NaOH used but also on the temperature for a breakdown of the acetyl bonds. However, a high temperature leads to a degradation of chitosan, which causes the viscosity η to drop and the molecular weight M (η = kM a) to drop, which affects the solubility of chitosan. The variation of the concentration of NaOH does not affect its molecular weight [76].

5.4 Determination of apparent rate constant k and energetic activation (Ea)

The chitin deacetylation process followed the pseudo–first-order kinetics for all the temperatures studied (25, 80, and 120°C) and at the same alkaline concentration (12 N) [3]. The Table 8 showed that the values decreased as a function of temperature. This indicates that the speed of the deacetylation reaction is faster at the beginning of the reaction t < 60 min, but it was very slow at the end of reaction.

T(°C)2580120
k2 (min−1) 2nd step1.70 × 10−52.23 × 10−422 × 10−4

Table 8.

Apparent rate constant [3].

The apparent activation energy was estimated at about 48.76 kJ/mol from the straight line of the Arrhenius plot (in k vs. (1/T)) [3]. This value is in the same order of magnitude as that found by other authors for heterogeneous N-deacetylation performed between 80 and 120°C [10, 12, 22, 26]. The concentration of NaOH significantly influences the variation of the reaction temperature. Rinaud and al. [14] mention that at a NaOH concentration of 10–15 M, the energetic activation (Ea) of the deacetylation is, respectively, about 22–50 kJ/mol, which makes it possible to increase the degree of deacetylation. The results indicated that the reaction at higher concentration and temperature proceeded easier than that at their lower values.

5.5 Mechanism of the chitin deacetylation

The reagent diffusion mechanism represents the second step in deacetylation of chitin. Recently, Sarhan et al. [28] have proposed a mechanism in which heterogeneous N-deacetylation is controlled by both reaction and diffusion: the first step involves the reaction of the onium salt, designated (Q+ X), with NaOH to give the corresponding onium hydroxide (Q+ OH) capable of diffusing from the aqueous phase to the organic phase to start the deacetylation process by attacking the C = O of the acetyl group and then at the end of the reaction of hydrolysis, the resulting onium acetate (CH3COOQ+) will diffuse into the aqueous phase to be regenerated to a new onium hydroxide by the reaction with NaOH. From our results, the DD/CNaOH ratios remain constant at each temperature at longer deacetylation times, which means that the deacetylation reaction is complete.

6. IR statistic study for optimization of deacetylation conditions

The results obtained under various conditions were analyzed statistically using multilinear regression analysis (uncertainty value =0.05) [23]. The NaOH concentration, temperature, and reaction time were chosen as independent parameters for the three-variable and three-level (maximum, mean, and minimum) factorial design. The best equation obtained for the chitosan extraction process in this study is Eq. (6). Shrimp at a higher temperature is not reliable. In addition, the production of chitosan at high temperature causes degradation of the container and therefore involves manipulations more difficult to adapt for industry. To ensure the quality of the chitosan product, the digestion temperature must be kept constant at 110°C.

%DD=72,56+5,6NaOH+3,78temperature+24,86temps11,92tempsE6

The values of this equation indicate that the effect of three factors studied influences DD in the following order: reaction time (24.86) > NaOH concentration (5.6) > reaction temperature (3.78). Further, a reaction time of 152 min (≈2.5 h) is predicted from Eq. (5) to reach 90% DD using a NaOH concentration of 12.5 M and a temperature set at 110°C. This time predicted by the factorial plane seems consistent with the actual deacetylation reaction time (120 min) with an error of 27%.

The production of chitosan from dried shrimp exoskeletons can be done in 1 day instead of the 3 days required by the conventional method. The results obtained show that the extracted chitosan has a DD greater than 90% under optimal conditions by the hydrothermal-chemical technique in two stages at 110°C, that is, 11.25 M NaOH for 3 h or 12.5 M NaOH during 2 h. For the extraction of chitosan at an SD of 85% in the context of water treatment, the transformation procedure must be carried out under the following optimal conditions:

  • Demineralization at 50°C for 2.5 h in 2 M HCl

  • Deproteinization and deacetylation at 110°C with 11.25 M NaOH for 2 h

7. Deacetylation of chitin by compression method study by FTIR

A new method of producing very high DD content chitosan under low concentration alkaline conditions has been introduced by Xiaofei et al. [79]. The synthesis was produced at high temperatures and pressures. The results in Table 9 were deduced from IR spectra. Compared to traditional methods, low concentration alkaline and short reaction time are excellent benefits. In addition, compared to the enzymatic and organic solvent treatment method, the pressure method was inexpensive and convenient without further purification. Excellent repeatability and simplified operation increased its availability in production and large application chitosan scale with large quantity production. In order to produce chitosan with a very high content of DD by chitin in a more efficient and more environmentally friendly way, Xiaofei et al. [79] have changed the pressure and have used the multistep method for the deacetylation of chitin in alkaline at low concentration. They adopted the alkaline recycling model using alkaline waste in the next deacetylated step. In this model, they added an alkali according to the different demands of each phase and eliminated acetyl in a timely manner, in case the high concentration of the acetyl group would inhibit the deacetylation. In addition, the use of the base in several steps will be effective not only to control the degree of deacetylation but also to control the molecular weight of the resulting product.

C/%CT:NaOHRT/minT/CProductivity/%DD/%
51:2012012088.95A
51:2024012090.48A
101:2012012088.41A
101:2024012090.88A
151:2012012086.76A
151:2024012088.73A
201:2012012087.66A
201:2024012086.94A
251:2012012085.69A
251:2024012085.88A
301:2012012085.19A
301:2024012086.53A
301:2036012085.44A
351:2012012084.1785.05
401:2012012082.0090.13
401:2012012081.2794.26
401:2012012081.9389.19

Table 9.

The effect of different reaction conditions on DD value of chitosan [79].

A: Undissolved in HCl.

Working under high pressure and with low concentrations of the base, they were able to extract chitosan with DD of 100%. In fact, only 15% of alkali solution and a ratio of 1:10 chitosan powder to NaOH solution and to a pressure of less than 0.11–0.12 MPa for 120 min lead to 100% DD. When the alkali concentration varies from 5–15%, the very high value DD chitosan (up to 95%) is produced. The method under pressure to prepare 100% deacetylated chitosan with less environmental pollution is very interesting.

8. Deacetylation of chitin under microwave irradiation effect

To extract a large quantity of chitosan, industrialists need high temperatures and chemicals in large quantities. In addition, the conventional process requires a lot of time and consumes a lot of energy, which would harm the environment. Recently, microwave irradiation has been used as an unconventional energy source in chemical reactions. The objective of this study is to synthesis chitosan under microwave irradiation in order to reduce the impact of environmental pollution due to excessive use of chemical treatments [80, 81, 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 will accelerate reaction time.

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

Chitin treatmentNaOH conc. (%)DD%Mw (k Daltons)Solubility (%)
20 mesh3067.58F ± 0.922415.0966.31H ± 0.35
4075.77E ± 3.541476.2174.94G ± 0.79
5078.83D ± 1.051267.1196.77BC ± 0.17
40 mesh3076.89DE ± 0.89866.0399.05A ± 0.05
4078.64D ± 0.861107.5092.79D ± 0.01
5083.05C ± 0.292160.8895.60C ± 0.87
60 mesh3088.39B ± 0.49949.9583.28F ± 0.87
4089.17B ± 0.281274.8585.57E ± 1.37
5095.19A ± 0.744467.0597.73AB ± 0.95
Commercial chitosan
85.00C ± 0.6630099.00A ± 0.72

Table 10.

Degree of deacetylation (DD), molecular weight (mw), and solubility of chitosan samples extracted from shrimp wastes using microwave technique [81].

Data are the mean.

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

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

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 compare IR and other analytical methods to compute DD. The analysis was performed by acidic conductometric assay. Basic conductometric dosing and pH-metric dosing techniques will be compared to IR.

9.1 Determination of the degree of deacetylation by conductometric assay

According to Yu et al. [22], the conductometric assay is an adequate and accurate method to determine the degree of deacetylation of chitosan. It was carried out in basic and acid medium.

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+.

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.

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

DD=203×V2V1×Nm+42×V2V1×N×100E7

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.

DD=203×9.42.7.103×0.10.15+42×9.42.7.103×0.1×100E8

The degree of deacetylation according to the conductometric method is: DD=76.35%.

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:

DD=203×V×N/m+42×V×N×100E9

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:

DD=203×6.8×103×0.1/0.15+42×6.8×103×0.1×100DD=77.30%.E10

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%.

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.

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

Dosing methodDDA (%)
Basic conductivity meter76.35
Conductometric acid77.30
pH-metric77.10
Infrared spectroscopy78.50
Calculated average77.32

Table 11.

DD values obtained with different assay methods [70].

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

Another study presented by Xiaofei and al [79] gave a comparison between IR spectroscopy and other techniques and showed the importance of FTIR for calculating DD (Table 12). They have been measured for 3 or 2 times.

Method1 (%)2 (%)3 (%)4 (%)5 (%)
Part-I
DDTitration190.8290.5794.9393.0492.48
DDTitration292.8594.6293.9795.4094.99
DDTitration393.1094.4594.8694.3594.66
DD-UV195.9795.9699.4298.1899.34
DD-UV294.4494.8797.7297.0797.00
DD-UV395.0295.3698.0797.4297.99
Part-II
DD-UV182.0095.9697.68100.00100.00
DD-UV282.2795.8697.73100.00100.00
DD-IR1658/345080.4394.5094.6095.3796.33
DD-IR1320/142087.64100.00100.00100.00100.00

Table 12.

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

10. Conclusion

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 of deacetylation is greater than 90% under the optimal conditions for the simultaneous deproteinization and deacetylation.

The compression method for preparing 100% deacetylated chitosan with less environmental pollution was studied by FTIR. The 100% fully deacetylated chitosan was produced in low-concentration alkali and high-pressure conditions, which only requires 15% alkali solution and 1:10 chitosan powder to NaOH solution ratio under 0.11–0.12 MPa for 120 min. When the alkali concentration varied from 5–15%, the chitosan with ultra-high DD value (up to 95%) is produced.

In parallel, the FTIR calculation was also used to show that the microwave could be used in the extraction step of chitosan from chitin. From these results, it could be concluded that shrimp waste is an excellent source for chitin, and the yields of chitosan increased with decreasing the chitin particle size and increasing the concentration of NaOH solution used in deacetylation step. The highest degree of deacetylation was obtained from chitin samples at particle size of 60 mesh deacetylated by 50% NaOH solution, and it was 95.19% compared with 85% for commercial chitosan. Based on this synthesis, it is concluded that FTIR is an effective and reliable technique for the determination of DD and the study of the deacetylation reaction of chitin. All the formulas quoted are valid for calculating DD of chitosan, but the most reliable formula and the formula most approved by other techniques is formula 3 quoted in our works.

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Boukhlifi Fatima (March 4th 2020). Quantitative Analysis by IR: Determination of Chitin/Chitosan DD, Modern Spectroscopic Techniques and Applications, Maaz Khan, Gustavo Morari do Nascimento and Marwa El-Azazy, IntechOpen, DOI: 10.5772/intechopen.89708. Available from:

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