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2.1. X-ray spectroscopy

X-ray spectroscopy is unarguably the most versatile and widely used means of characterizing materials of all forms [38]. There are two general types of structural information that can be studied by X-ray spectroscopy: electronic structure (focused on valence and core electrons, which control the chemical and physical properties, among others) and geometric structure (which gives information about the locations of all or a set of atoms in a molecule at an atomic resolution). This method encompasses several spectroscopic techniques for determining the electronic and geometric structures of materials using X-ray excitation: X-ray absorption spectroscopy (XAS), X-ray emission spectroscopy (XES), X-ray photoelectron spectroscopy (XPS) and X-ray Auger spectroscopy. Which type of X-ray spectroscopy is employed depends on whether the target information is electronic, geometric or refers to oxidation states: for instance, XAS (first developed by de Broglie) to probe empty states and the shapes of molecules or local structures [39], and XPS (first developed by Siegbahn) to investigate occupied electronic states [40]. X-ray spectroscopy is thus a powerful and flexible tool and an excellent complement to many structural analysis techniques such as UV-Vis, IR, NMR or Raman.

2.1.1. Typical conditions of X-ray measurements

In an X-ray diffraction measurement, a crystal is mounted on a goniometer and gradually rotated while being bombarded with X-rays, producing a diffraction pattern of regularly spaced spots known as reflections [41]. The two-dimensional images taken at different rotations are converted into a three-dimensional model of the density of electrons within the crystal using the mathematical method of Fourier transforms, combined with the chemical data obtained for the sample. If single crystals of sufficient size cannot be obtained, various other X-ray methods, like fiber and powder diffraction, can be applied to record less detailed information.

Clark and Smith in 1937 were the first to make crystal studies of chitin and chitosan using X-ray diffraction (XRD) [42]. They carried out those investigations using a commercial copper-target diffraction tube operated at 30 kV and 25 mA as the X-ray sources, which generated principally Cu-Kα lines. The diffraction patterns were recorded on a flat film perpendicular to the beam with the sample 5.0 cm from the film. In later research, the conditions for X-ray measurements of chitin and chitosan were mostly modifications of the ones used by Clark and Smith [42]. For instance, X-ray diffraction measurements were done at 100% relative humidity in a helium atmosphere [43] to avoid the X-ray scattering that led to a “dirty” background on the X-ray film. Present-day X-ray analyses of chitin, chitosan and their derivatives are carried out on advanced X-ray diffractometers.

Apart from traditional X-ray diffraction (XRD), other X-ray techniques for determining chitin/chitosan and their derivatives have been applied: X-ray photoelectron spectroscopy (XPS), the second most popular X-ray spectroscopy technique–for determining the bonding energies of C, O and N atoms on the surface of chitosan and its metal chelate, and for other chitin and chitosan investigations [44–49]; X-ray emission spectroscopy (XES)–perfect for studying the chemical bonding in chitosan and cross-linked chitosan derivatives [50]; X-ray absorption spectroscopy (XAS)–for determining the coordination number of Fe atoms in chitosan-metal complexes [51].

2.1.2. X-ray spectra of chitin and chitosan

Numerous X-ray spectroscopic studies of chitin and chitosan have yielded the diffractive patterns of these compounds [52–59]. However, different sources have characterised these patterns with differently indexed crystalline peaks: these can be labeled either using d, the centre-to-centre spacing of the crystallites, or Miller indices, e.g., (020). It is also very common to describe the diffraction pattern using the values of angles.

Typical spectra of chitin fiber and chitosan fiber are shown in Figure 2 [56]. The spectrum of the former exhibits broad peaks at d = 0.34, 0.45, 0.50 and 1.09 nm with a shoulder at 0.71 nm; in the latter the spectral peaks are at d = 0.45, 0.88 and 2.93 nm.

Figure 2

X-ray diffraction spectra of chitin and chitosan fibers. Reprinted from Carbohydrate Polymers 56, 2004, Muzzarelli, C., Francescangeli, O., Tosi, G., Muzzarelli, R.A.A., Susceptibility of dibutyryl chitin and regenerated chitin fibers to deacetylation...

The Miller indices of the diffraction peaks characteristic of chitin were (020), (110), (120), (101) and (130) [57,58], although Muzzarelli et al. reported the lack of a strong (020) peak for chitin. [59]. In turn, the typical chitin diffraction pattern, given in angle form, showed strong reflections at 2θ around 9–10° and 2θ of 20–21° and minor reflections at higher 2θ values, e.g., at 26.4° and higher [60].

2.1.3. X-ray analysis of chitin and chitosan polymorphs

As already mentioned, the crystal structures of chitosan have been examined since the work of Clark and Smith in 1937 [42]. A great number of diffraction experiments have been undertaken in an attempt to elucidate the molecular geometry of chitosan [42,61–71]. The first X-ray studies showed that the chitosan molecule can adopt at least two different conformations in crystals–a 2-fold [42,61–63] and an eight-fold right-handed helical structure [64,65]. Apart from these two helical conformations, other polymorphs of chitosan have been characterized [66–71]. In 1994 Yui et al. determined the detailed crystal structure of the anhydrous form of chitosan by combining X-ray diffraction analysis with a stereochemical model refinement [67]. Chitosan chains crystallized in an orthorhombic unit cell of the following dimensions: a = 0.828 nm, b = 0.862 nm and c (fiber axis) = 1.043 nm. The X-ray diffraction pattern was recorded on the imaging plate, and the intensity of each reflection was estimated by two-dimensional measurement and subsequent background removal. The results showed that the chain conformation of this anhydrous form of chitosan was an extended two-fold helix stabilized by intramolecular O3···O5 hydrogen bonds and that the adjacent parallel chains were connected by O6···N2 hydrogen bonds.

At the same time Mazeau et al. examined the conformation and packing of a chitosan polymorph crystallized at a high temperature on the basis of diffraction data [68]. In this kind of polymorph, the chitosan chains form orthorhombic crystals with a = 0.807 nm, b = 0.844 nm and c (chain axis) = 1.034 nm. This chitosan molecule adopted a two-fold helical conformation stabilized by two intramolecular hydrogen bonds, a strong one between O5′ and O3, and a weaker one between O5′ and O6. These two anhydrous forms of chitosan have relatively similar structures.

In 1997 Yui’s group published another paper on the X-ray fiber diffraction method used to determine the crystal structure of a hydrated form of chitosan [69]. The results indicated that the hydrated form of chitosan molecules had a two-fold helical symmetry reinforced by a O3····O5 hydrogen bond with a repeating period of 10.34 Å; this is a structure typical of β (1→4) linked polysaccharides such as cellulose, mannan and chitin.

The following crystalline polymorphs of chitosan have so far been found using X-ray diffraction measurements: the most abundant “tendon-chitosan” [42,69], as well as the “annealed” [63], “1–2”, “L-2” [66], “form I” and “form II” [61] and “8-fold right-handed” forms [64,65]. Apart from the last one, all the polymorphs of chitosan molecules have the extended 2-fold helix configuration; the 8-fold polymorph is unstable and is easily converted into the 2-fold helix [72].

Depending on the source, chitin can occur in the α-, β- and γ-forms. The differences among them depend on the arrangement of chains in the crystalline regions [52]. The most abundant and stable form is α-chitin, which Minke and Blackwell studied in detail using XRD in 1978 [45]. These authors determined the α-chitin structure on the basis of intensity data from deproteinized lobster tendon. They discovered that α-chitin chains form orthorhombic crystals with a = 0.474 nm, b = 1.886 nm and c (fiber axis) = 1.032 nm. Additionally, the chains form hydrogen-bonded sheets linked by C=O...H–N bonds approximately parallel to the a axis, and each chain is stabilized by an C(3′)O–H····OC(5) intramolecular hydrogen bond, as in cellulose. These data also indicated that a statistical mixture of CH2OH orientations was present, equivalent to half an oxygen on each residue, each forming inter- and intramolecular hydrogen bonds. As a result, Minke and Blackwell found that the α-chitin structure contained two types of amide groups, differing in their hydrogen bonding. In addition, the inability of this polymorph to swell in the presence of water was explained by the extensive intermolecular hydrogen bonding. The modes of hydrogen bonding in α- and β-chitin are illustrated in Figures 3 and ​4 [18].

Figure 3

Modes of hydrogen bonding in α-chitin: (a) intrachain C(3′)OH···OC(5) bond; (b) intrachain C(6′1)OH···O=C(71) bond; (c) interchain C(6′1)O···HOC(6...

Figure 4

Modes of hydrogen bonding in β-chitin: (a) intrachain C(3′)OH···OC(5) bond; (b) interchain C(21)NH···O=C(73) bond and C(6′1)OH···O=C(73) bond (ac plane projection);...

As mentioned above, the differences among chitin polymorphs are due to the arrangement of the chains in the crystalline regions: α-chitin has a structure of antiparallel chains [45], β-chitin has intrasheet hydrogen-bonding by parallel chains [52], and γ-chitin, being a combination of α- and β-chitin [52], has both parallel and antiparallel structures. Because of these differences each chitin polymorph has different properties specific to it. For example, β-chitin is more soluble in and more reactive towards solvents and has a greater affinity towards them; it is also more susceptible to swelling than α-chitin. β-Chitin is also more amenable to N-deacetylation than α-chitin. In this case, comparing the diffraction profiles of α- and β- chitin before and after N-deacetylation using X-ray spectroscopy seems to be a relatively good solution for distinguishing these forms of chitin. Abdou et al. used X-ray diffraction to study two chitin polymorphs, the α- and β-forms, obtained from different sources [60]. The chitin samples were converted into the more soluble chitosan by steeping them in solutions of NaOH of various concentrations and for extended periods of time. The X-ray diffraction patterns of the α-chitin samples and the corresponding hydrolyzed chitosans showed strong reflections at 2θ around 9–10° and 2θ of 20–21° and minor reflections at higher 2θ values at e.g., 26.4° and higher. The chitin bands were sharper than the chitosan bands, even though there was only a slight decrease in the crystallinity percentage. In turn, the X-ray diffraction patterns of the β-chitin samples and their corresponding hydrolyzed chitosans showed that the band at 2θ = 9.9° decreased significantly after deacetylation, and that this was followed by a dramatic decrease in the crystallinity percentage. It was therefore concluded that β-chitin is much more amenable to N-deacetylation than the α-form. The X-ray diffraction patterns of α- and β-chitin and the corresponding hydrolysed chitosans make them easily distinguishable. The crystallinity index (CI) can also be calculated on the basis of X-ray diffractograms. This takes different values for different forms of chitin; for example, Lima showed that CI for α-chitin is 28.3% but that for β-chitin it is 20.8% [73].

Many other studies of chitin polymorphs have revealed differences in crystallinity peaks between α-, β- and γ-chitins obtained from various sources [52–55]. For example, Jang et al. found crystalline peaks at 9.6, 19.6, 21.1 and 23.7° for α-chitin, at 9.1 and 20.3° for β-chitin, and at 9.6 and 19.8° for γ-chitin [52]. Similarly, Cárdenas et al. [53] reported that WAXD patterns of α-chitin (chitins from shrimp, lobster, prawn and king crab) and β-chitin (chitin from squid) exhibited their major characteristic peak at 19.2–19.3° and 18.8° respectively. Kim [54] found that β-chitin from squid pen exhibited crystalline peaks at 9.8° and 19.3°. Yen and Mau [55] found that fungal chitin (γ-chitin) showed two crystalline reflections at 5.4–5.6° and 19.3–19.6°. Irrespective of their origin, the three types of chitin consistently display a major peak at ~19° in their crystallinity structure.

2.1.4. Physicochemical characterization of chitin and chitosan using X-ray diffraction

In 1937, Clark and Smith, in their pioneer X-ray diffraction studies of chitin/chitosan and their derivatives [42], were the first to investigate the physicochemical properties of chitin. They reported the action of hydrochloric acid, lithium thiocyanate and nitric acid on chitin. Their data showed that even at room temperature the ether linkages of chitin were hydrolysed in hydrochloric acid; concurrently, but more slowly, the amide groups were also hydrolysed. In addition, those authors investigated the dispersion of chitin in lithium thiocyanate; at a temperature of 200 °C chitin formed a definite addition compound with lithium thiocyanate, but at lower temperatures only intramicellar swelling was observed. In turn, chitin nitrate was roughly as soluble in hydrochloric acid as the original chitin–no substantial hydrolysis of the acetyl groups had occurred.

Subsequent studies revealed that the properties of chitin and chitosan depended mostly on the degree of N-acetylation, molecular weight, polydispersity and crystallinity [31]. Commonly used to measure crystallinity, XRD is also applied to determine the degree of N-acetylation of chitin and chitosan [74].

In 1990, Focher et al. [75] used XRD to study chitin and postulated the following equation for determining the crystallinity index (CI):

CI(%) = [(I110Iam)/I110] × 100

(1)

where I110 (arbitrary units) is the maximum intensity of the (110) peak at around 2θ = 19°, and Iam (arbitrary units) is the amorphous diffraction at 2θ = 12.6°. This expression had in fact been employed three years earlier by Struszczyk to determine the CI of chitosan [76]. Currently this equation is routinely applied during investigations of chitin, chitosan and their derivatives [73,77,78]. In most cases, CI provides information about the crystal state, but it is also very useful for distinguishing α-chitin from β-chitin [73].

On the basis of X-ray powder diffractograms of chitin and chitosan with different degrees of N-acetylation, Zhang noted two maximum peaks of the following intensities: one at the (020) reflection and the other at the (110) reflection [74]. He therefore postulated a crystallinity index (CI) expressed by two equations:

CI020 = [(I020Iam)/I110] × 100

(2)

CI110 = [(I110Iam)/I110] × 100

(3)

Further chitin and chitosan studies indicated that crystallinity could also be assigned from an X-ray diffractogram by dividing the area of the crystalline peaks by the total area under the curve (background area) [60,79,80]. In these calculations, the crystallinity percentage supplied information on relative crystallinity.

A lot of studies have been carried out in which X-ray measurements were applied to determine which parameters affect the crystalline structure of chitin and chitosan and how they do so. In 1991 Ogawa reported an increase in crystallinity with a decrease in the MW of chitosan [71]. One year later,he also determined how chitosan polymorphism and its crystallinity in the membrane depended on the membrane preparation procedure and the molecular weight of the chitosan [43]. XRD measurements demonstrated that when an acetic acid solution of chitosan was dried in air and then soaked in an alkaline solution, both hydrated and anhydrous polymorphs of chitin were present in the resulting membrane. On the other hand, when a highly concentrated solution of chitosan in aqueous acetic acid was neutralized with an alkaline solution, no anhydrous polymorphs were detected in the membrane because drying was incomplete. In another paper from 1993 [81], Ogawa compared the crystallinities of partially N-deacetylated chitin (PDC) and partially N-acetylated chitosan (PAC) samples with a similar degree of N-acetylation and their behavior by heating them in water. He discovered that the N-acetylation of pure chitosan is far superior to the solid-state N-deacetylation of chitin for producing a less crystalline sample, and in particular, for obtaining a less anhydrous crystal.

The effect of DA on solubility in relation to the crystal structure of deacetylated chitin was also discussed by Cho et al. in 2000 [18]. Wide-angle X-ray diffractometry (WAXD) revealed that chitin with ca 72% DA retained the crystal structure of α-chitin with significantly reduced crystallinity and crystallite perfection. The water-soluble chitin with circa 51% DA had a new crystal structure resembling that of β-chitin rather than that of either α-chitin or chitosan, suggesting that homogeneous deacetylation converted the crystal structure of chitin from the α- to the β-form.

Using X-ray powder diffraction Zhang et al. [74] also tried to look for a relationship between the crystalline state and DA of chitin. Figure 5 presents the XRD patterns of chitin and chitosan with different degrees of N-acetylation [74]. These authors noted that the peak of maximum intensity at the (020) reflection diminished together with the decrease in DA and moved to a higher angle. The second intensive peak at the (110) reflection also diminished with the decrease in DA. Consequently, CI020 decreased linearly with the decrease in DA. This linear relationship between CI020 and the degree of N-acetylation suggested the possible use of XRD for determining DA of macromolecular chitin and chitosan.

Figure 5

Comparison of X-ray powder diffractograms of chitin and chitosan with different degrees of N-acetylation. Figures 0–6 imply different DA (%): 0-83.1, 1-40.6, 2-36.5, 3-41.3, 4-28.6, 5-13.0, 6-7.2. Reprinted from Carbohydrate Research 340, 2005,...

As demonstrated, molecular weight and DA are the major parameters significantly influencing the crystal structure of chitin and chitosan, although it has also been reported that the crystallinity index depends on other factors. For example, Seoudi reported that CI decreased after chitin was treated with HCl and NaOH [82], and Wada and Saito [57] found that when the biopolymer was heated from room temperature to 250 °C the α-chitin remained structurally stable. Moreover, the influence of alkali-freezing treatment on the solid state structure of chitin was examined by Feng [58]. XRD revealed that during this treatment, the crystal space parameters of chitin changed, and the order of the hydrogen bonds in chitin was modified.

X-ray diffraction was used to measure the elastic moduli El of the crystalline regions of α-chitin and chitosan [83]. This parameter provided important information on the molecular conformation in the crystal lattice and the mechanism of deformation in the crystalline regions. The data enabled the elastic moduli El of the crystalline regions in the direction parallel to the chain axis at 20 °C to be assigned as 41 GPa for α-chitin and 65 GPa for chitosan. These El values, which are low compared to those for cellulose I (138 GPa), were due to the contracted skeletons of α-chitin and chitosan in the crystal lattice. In addition, the elastic moduli calculated from X-ray data showed that the molecular chain of α-chitin in the crystal lattice was mechanically stable from − 190 °C to 150 °C.

2.1.5. X-ray analysis of chitosan salts

Chitosan has a regular distribution of aliphatic primary amino groups along its chain. These produce salts when the molecule reacts with inorganic or organic acids. Ogawa and Inukai used XRD to study several crystallized inorganic acid salts of chitosan [64]. On the basis of X-ray fiber diffraction, these authors suggested that chitosan acid salts took up two different conformations. One, which they called “type I salt”, retained the extended two-fold helix of the unreacted chitosan molecule, although they were different crystals. The second one, the “type II salt”, had an eight-fold helical conformation in the crystal. The salts forming with HNO3, HBr and HI took up the former structure, and those with HF, HCl, and H2SO4 the latter one. Crystals of type I salts were anhydrous, whereas those of type II were hydrated. Despite the different anion sizes, all the type II salts gave fiber patterns that were very similar to each other. They crystallized in a monoclinic unit cell with a helical repetition of 4.073 nm. The chirality of the eight-fold helix was right-handed, since their fiber patterns were very similar to those obtained by Cairns et al. [65].

Apart from inorganic salts, chitosan can easily form organic salts, for example, when it reacts with L-ascorbic acid. In 1996 Ogawa carried out an X-ray study of the chain conformation of the ascorbic acid salt of chitosan [84]. He determined that both l- and d-ascorbate chitosan salts retained the extended 2-fold helical conformation of the unreacted chitosan chain and that both crystals were anhydrous. In view of these results, he classified them as type I salts of the chitosan acid salts [64], although during the preparation of the l- and d-ascorbates of chitosan, that author discovered that d-ascorbic acid displayed a higher reactivity towards chitosan than the l-isomer. Those differences in reactivity between l- and d-ascorbic acids could be connected with the optical resolution of ascorbic acid.

Chitosan salts were also examined in 1999 by Kawada et al. [72]. They studied the spontaneous water-removing action of acid by preparing chitosan salts of many different (monocarboxylic, inorganic and organic) acids, and examined their structures using X-ray diffraction. The results indicated that the temperature required for salt formation depended on the hydrophobicity of the acid; for instance, the chitosan formic acid salt could be prepared at room temperature, whereas the formation of the propionic acid salt had to be carried out at 4 °C. Moreover, type II salts of monocarboxylic acids, the hydrated crystals of chitosan, could be dehydrated even at room temperature without any loss of orientation or decomposition of the chitosan specimen.

2.1.6. X-ray analysis of chitosan derivatives

Chitin and chitosan are a family of polymers with highly variable chemical and physical properties. These compounds and their derivatives have at least 200 potential and current applications [32] in the biomedical, food, biotechnological, agricultural and cosmetics industries.

Muzzarelli and co-workers found that chitosan exhibited good adsorption selectivity towards some transition and post-transition metal ions from aqueous solution [85]. A chitosan-metal complex dissociates easily when the pH is lowered; therefore, chitosan is very often used in the recovery of useful transition metals from waste. Ogawa and Oka reported on the behavior of chitosan-metal complex formations examined by X-ray diffraction [86]. The unit cells of all the salt complexes studied were orthorhombic, although the cupric salt complexes showed some unindexed reflections. Their lattice parameters and the number of water molecules in the cell depended on the counteranions of the metal salt and not on the metal ion. The ratio of glucosamine residues to metal salts was 2:1. On the basis of the fiber diffraction patterns of various chitosan-transition metal salt complexes, these authors postulated a coordination mode which they named the “pendant model”. This model had already been put forward by Ogawa et al. back in 1984, but unequivocal experimental evidence to support it was lacking [70]. In this “pendant model” metal anions were coordinated to the amino groups of the chitosan chain like a pendant. Additionaly there was another contrasting model called “the bridge model”, in which metal ions were coordinated with four nitrogen atoms of the intra- and inter-chitosan chains [87]. In spite of these differences, chitosan exhibits a high affinity for metal ions, a property that has been used to recover transition metals from waste water. At present, the adsorption properties of metal ions on chitin and chitosan derivatives are still routinely examined by X-ray diffraction [49,88–90].

The majority of current studies on chitin and chitosan are seeking to discover new derivatives with unusual properties and different potential applications [56,91–97]. X-ray measurements are still very often applied to characterize most of these new derivatives. For example, chitosan-based nanocomposite films, containing chitin nanocrystals as functional components, were successfully prepared and cross-linked using glutaraldehyde [97]. XRD showed that chitin nanocrystals retained their crystalline morphology in the nanocomposites before and after cross-linking, and that chitosan also retained its amorphous characteristics in the nanocomposites. Another example, novel chitosan/gelatin membranes were prepared using a suspension of chitosan hydrogel mixed with gelatin [95]. XRD studies showed that the chitosan and gelatin in these membranes are compatible and interact well with each other. In addition, the incorporation of gelatin reduced the crystallinity of chitosan. Finally, natural rubber/chitosan blends were studied by XRD analysis [91], the measurements indicating that vulcanization enhanced the crystallinity.

2.1.7. Other X-ray techniques used in chitin and chitosan analysis

As mentioned at the beginning of this section, X-ray spectroscopic techniques other than the traditional X-ray diffraction measurements have been successfully used to analyze chitin, chitosan and their derivatives.

X-ray photoelectron spectroscopy (XPS) is usually used to determine the bonding energies of C, O and N atoms on the surface of chitosan and its metal chelates, although this is not its only use [44–49]. For example Matienzo and Winnacker [46] presented high-resolution C1s, N1s and O1s XPS spectra for a chitosan film coated on an Al-silicon surface. The films were treated in either an oxygen plasma environment or under UV/ozone irradiation. XPS data showed that hydroxyl and amine entities participated only minimally in the modification. In addition, deposition of chitosan films onto Al-coated silicon wafers produced films with a more ordered chitosan structure. Surface analysis of modified films by XPS also indicated that neither the hydroxyl groups nor the amine segments appeared to participate in surface degradation reactions by either UV/ozone or oxygen plasma during the exposure times chosen for those studies. XPS also provided information regarding the forms of species absorbed on the polymer [48]. For example, a study of the interactions of Cu2+, Mo4+ and Cr3+ with chitosan beads, cross-linked chitosan beads and chitosan flakes revealed that the adsorption of Mo4+ and Cr3+ on chitosan flakes and beads was followed by reduction of the Mo4+ and Cr3+. Another, example of the use of XPS was presented by Veleshko et al. [47]. They examined the complexation between the uranyl group U(VI) and chitosan by means of X-ray photoelectron spectra. The results showed that the interaction of chitosan with the uranyl group yielded complexes containing the nitrogen atom of the amino group and, very probably, oxygen atoms from the chitosan ring and free hydroxyl groups in the equatorial plane.

X-ray emission spectroscopy (XES), also known as X-ray fluorescence spectroscopy (XFS), is a very sensitive probe for examining the local electronic structure and chemical bonding of the emitting atoms. This X-ray technique was used by Kurmaev et al. in 2002 [98] to study the chemical bonding in chitosan and chitosan cross-linked with ethylene glycol diglycidyl ether (EGDE). These authors concluded that the changes in the width of resonantly excited O Kα XES were due to site-selective excitation of oxygen atoms belonging to different functional groups (OH and –O–). Comparison of the nitrogen Kα spectra of unmodified and cross-linked chitosan proved that the preferred structural model was the one according to which EGDE was linked via the hydroxyl group.

2.2. Infrared spectroscopy

Infrared (IR) spectroscopy is one of the most important and widely used analytical techniques available to scientists working on chitin and chitosan. It is based on the vibrations of the atoms of a molecule. The infrared spectrum is commonly obtained by passing infrared electromagnetic radiation through a sample that possesses a permanent or induced dipole moment and determining what fraction of the incident radiation is absorbed at a particular energy [99]. The energy of each peak in an absorption spectrum corresponds to the frequency of the vibration of a molecule part, thus allowing qualitative identification of certain bond types in the sample. An IR spectrometer usually records the energy of the electromagnetic radiation that is transmitted through a sample as a function of the wavenumber or frequency. Nowadays, the total spectrum is analyzed by an interference process and converted into the frequency or wavenumber range by means of a mathematical process known as the Fourier transform. Fourier-transform infrared (FTIR) spectroscopy has dramatically improved the quality of infrared spectra and minimized the time required to obtain data [99,100]. Progress in modern infrared spectroscopy is reviewed in literature [101,102].

2.2.1. Typical conditions for the FTIR spectroscopic analysis of chitin, chitosan and their derivatives

FTIR spectra are usually recorded in the middle infrared (4000 cm−1 to 400 cm−1) with a resolution of 4 cm−1 in the absorbance mode for 8 to 128 scans at room temperature. The samples for FTIR analysis are prepared by grinding the dry blended powders with powdered KBr, often in the ratio of 1:5 (Sample: KBr) and then compressed to form discs. Spectra are sometimes measured using a deuterated triglycerinesulphate detector (DTGS) with a specific detectivity of 1 × 109 cmHz1/2 w−1 [103] or on films using an attenuated total refraction (ATR) method in an IR spectrometer [104–106]. Diffuse Reflectance Infrared Fourier-Transform (DRIFT) spectroscopic analysis is also applied [107].

2.2.2. Physicochemical characterization of chitin and chitosan using infrared spectroscopy

As already mentioned, natural chitin occurs mainly as α- and β-chitin. The description and interpretation of the infrared spectra of these two forms of chitin have been published by many scientists [108–110]. By way of example, the spectra of α- and β-chitin, and the Ianthella basta scaffold after NaOH treatment and H2O2 purification are shown in Figure 6 [110].

Figure 6

FTIR spectra of β-chitin from T. rotula, α-chitin from crabs and I. basta chitin after NaOH and H2O2 treatment. Dashed vertical lines are drawn to mark characteristic differences between α- and β-chitin. Reprinted from...

The spectra of α- and β-chitin display a series of narrow absorption bands, typical of crystalline polysaccharide samples. The C=O stretching region of the amide moiety, between 1700 and 1500 cm−1, yields different signatures for α- and β-chitin. For α-chitin, the amide I band is split into two components at 1660 and 1630 cm−1 (due to the influence of hydrogen bonding or the presence of an enol form of the amide moiety [109–111]), whereas for β-chitin it is at 1630 cm−1 (Figure 6). The amide II band is observed in both chitin allomorphs: at 1558 cm−1 for α-chitin and 1562 cm−1 for β-chitin [110]. Another characteristic marker is the CH deformation of the β-glycosidic bond. This band shifts from 890 cm−1 in β-chitin to 895 cm−1 in α-chitin. Infrared spectra of β-chitin reveal two additional bands for CHx deformations at about 1455 and 1374 cm−1 and a greater number of narrower bands in the C–O–C and C–O stretching vibration region (1200–950 cm−1) not observed in α-chitin. As shown in Figure 6, the FTIR spectrum of the chitin isolated from I. basta confirmed the finding that this chitin resembles α-chitin more closely than β-chitin [110], demonstrating that FTIR can be used to determine chitin allomorphs.

FTIR spectroscopy has been used to characterize not only isolated chitin [110] but also the source of chitin, e.g., in two species of black coral, Antipathes caribbeana and A. pennacea [112]. Although FTIR absorption spectra of the natural samples (without deproteinization) showed similar distribution patterns for both species of coral, and confirmed the presence of chitin in both species, small differences were observed (e.g., the intensity of the IR absorption bands in A. caribbeana was stronger). The absence of a free hydroxyl in the hydroxymethyl groups CH2OH in A. caribbeana (determined by FTIR analysis) indicated that the chitin chains were organized in sheets, where they were hydrogen-bonded to adjacent chains, a situation that favors a denser fiber packing of chitin. This means that the FTIR measurements permitted an explanation of why natural A. caribbeana coral was harder to pulverize and required a longer deproteinization time than A. pennacea. The presence of chitin in polyplacophoran sclerites was also confirmed by IR [113].

FTIR spectroscopy has also been used to compare the yield and purity of chitin isolated from pupae of the silkworm (Bombyx mori) using two methods of extraction: an open reactor and 1 h of an acidic reaction, and extraction in a closed reactor within 24 h of a basic reaction [114]. The efficiency of chitosan production by the N-deacetylation of chitin was also investigated by IR in this work. During the N-deacetylation of chitin, the band at 1655 cm−1 gradually decreased, while that at 1590 cm−1 increased, indicating the prevalence of NH2 groups. The spectra of chitin and chitosan obtained from the N-deacetylation of chitin using a solution of NaOH (40 wt%) in the presence of NaBH4 for 5 h are presented in Figure 7.

Figure 7

FTIR of chitin (A) and chitosan (B) produced from silkworm pupae; range: 1400–1700 cm−1. Reprinted from Carbohydrate Polymers 64, 2006, Paulino, A.T., Simionato, J.I., Garcia, J.C., Nozaki J., Characterization of chitosan and chitin produced...

The band at 1590 cm−1 displayed a greater intensity than the one at 1655 cm−1 and demonstrated the effective deacetylation of chitin. FTIR analyses were also used to find the optimal conditions for the N-deacetylation of chitin whiskers (the alkali concentration and the treatment time) using a microwave technique [115].

Prashanhi et al. [116] applied IR spectroscopy to observe the changes occurring in the crystallinity and polymorphic nature of chitosan as a function of the N-deacetylation of chitin under different conditions: uncontrolled conditions (chitosan A), under an N2 atmosphere (chitosan B), and with thiophenol (chitosan C). The FTIR spectra of chitosans A, B, and C were similar to each other, but there were subtle differences in the absorption intensities. Apart from the expected decrease in the band at 1665 cm−1 (amide I), the vibrational mode of amide II at 1550 cm−1 for chitin appeared at 1604 cm−1, 1598 cm−1 and 1592 cm−1 for chitosan A, B and C respectively [116]. In none of these spectra were there any sharp absorptions at circa 3500 cm−1, which confirms that the hydroxyl groups in positions C2 and C6 of the chitosans are involved in intra- and intermolecular hydrogen bonds. The region above 3000 cm−1 was centred at 3395 cm−1 in chitosan A, at 3407 cm−1 in chitosan B and at 3419 cm−1 in chitosan C; the shift to the higher frequency demonstrated a higher-order structure for these three chitosans. The CH2 stretching bands of chitosan B around 1425 cm−1 were more intense than those of chitosans A and C. Furthermore, the FTIR spectra exhibited a progressive weakening of the bands at 3265 cm−1 and 3100 cm−1 during N-deacetylation, and the A1382/A2920 cm−1 ratios of 0.65, 0.56 and 0.46 indicated a higher order structure of the chitosans prepared with thiophenol than those prepared under an N2 atmosphere. The ratio of the band intensities at 1379 and 2900 cm−1 was also used to estimate the crystallinity of chitin and chitosan by Focher et al. [75] and Wu et al. [117], whereas Prashanth and Tharanathan used the sharp absorption peak around 618 cm−1 [118].

The impurities in chitosan preparation were determined by FTIR analysis [119]. The FTIR spectra of control chitosan and low-molecular-weight water-soluble chitosan (LMWSC) prepared in this study were compared to establish the contaminants and prove the synthesis of LMWSC. In the LMWSC spectrum the carboxyl group absorption band derived from lactic acid and impurities formed during enzyme degradation disappeared or significantly decreased.

FTIR spectroscopy has been employed to measure the critical concentration of two chitooligosaccharides form a lyotropic liquid crystalline phase in formic acid (C1) [120]. Strong interactions between sugar chains and solvent were revealed by the widening of bands attributed to the –OH, –NH, –NHCO– of the chitooligosaccharide, including the C=O of formic acid. FTIR measurements of the shift of seven bands–1. –NH2, –OH (3390–3418 cm−1), 2. C=O of formic acid (1716–1724 cm−1), 3. amide I (1626–1633 cm−1), 4. amide II (1520–1531 cm−1), 5. 6. C3–OH (double peaks, 1178–1189 cm−1 and 1148–1153 cm−1), 7. C6–OH (1073–1074 cm−1)–enabled the C1 values of these chitooligosaccharides to be established.

2.2.3. Determination of the degree of N-acetylation of chitin and chitosan using infrared spectroscopy

The degree of N-acetylation is one of the most important chemical parameters capable of influencing the performance of chitosan and chitin in many of their applications [2,7,121]. Of the various analytical techniques developed for DA determination [122], infrared spectroscopy is at the centre of attention. A convenient and relatively quick technique, it allows the DA values of chitin/chitosan to be determined on the basis of absorption ratios, also in the solid state [123]. Several procedures using different absorption ratios have already been proposed for determining DA for chitin and chitosan samples [124–130]. A review article summarizing the latest literature information on DA determination by IR spectroscopy for chitin and chitosan was published by Kasaai [131]. In that paper, various IR procedures were compared for their performances and limitations, advantages and disadvantages, and different factors affecting the experimental results were discussed, as were the validity data of DA measurements by FTIR spectroscopy. In view of this, the present review will discuss only general information on DA determination by IR.

DA can be determined by IR techniques in the following ways:

  • Determination of the AM/AR ratio, where AM is the intensity of the characteristic band of N-acetylation, which is a measure of the N-acetyl or amine content, and AR is the intensity of a reference band that does not change with different DA values. The DA parameter of unknown samples can be established by comparing the determined AM/AR values with similar ratios of a few reference samples of known DA.

  • Drawing a calibration curve by plotting the absorption ratio of chitin/chitosan samples of known DA versus their DA as established by IR or a reference method such as 1H NMR spectroscopy. The DA values of unknown samples can then be estimated from the calibration curve.

  • Statistical evaluation of several absorption band ratios [131,132].

IR techniques require choosing an appropriate band measure, an appropriate reference band, and drawing a good base line, necessary for measuring the intensity of absorption. The amide I bands at 1655 cm−1 (sometimes together with the amide I band at 1630 cm−1) or the amide II band at 1560 cm−1 are used as the characteristic band(s) of N-acetylation. Among the postulated internal reference bands are the OH stretching band at 3450 cm−1 [125,133], the C-H stretching band at 2870–2880 cm−1 [134], the –CH2 bending centred at 1420 cm−1 [129], the amide III band at 1315–1320 cm−1 [135], the anti-symmetric stretching of the C-O-C bridge at around 1160 cm−1 [136], the skeletal vibrations involving the C-O-C stretching bands at 1070 or 1030 cm−1 [127,128] and the band at 897 cm−1 (C-O-C bridge as well as glycosidic linkage) [137]. The different baselines suggested in the literature [129] are presented in Figure 8.

Figure 8

IR spectrum of chitin. Representation of the different baselines mentioned in the literature. Reprinted from Polymer 42, 2001, Brugnerotto, J., Lizardi, J., Goycoolea, F.M., Argüelles-Monal, W., Desbrières, J., Rinaudo, M., An infrared...

Many different absorption band ratios, such as A1560/A2875, A1655/A2875, A1655/A3450, A1320/A3450, A1655/A1070, A1655/A1030, A1560/A1160, A1560/A897 and A1320/A1420, have been used to determine DA by FTIR spectroscopy [131]. The different calibration curves proposed in the literature have different baselines and different characteristic bands for measuring the N

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