**3. Cobalt(II)‐dextran biocomplexes**

A lot of investigations in the field of coordination chemistry are based on synthesis and charac‐ terizations of different biocomplexes present in the biological systems. Synthetic ligands, which can serve as model molecules for complex biomolecular structures, are also investigated [19]. Bioligands or synthetic ligands are mostly natural macromolecular compounds. These prod‐ ucts of special importance mostly represent complexes of different metals (Fe, Co, Cu, Zn) with ligands of polysaccharide type (such as pullulan, inulin, dextran) [43–45]. However, the native polysaccharide possessing antigen characteristics wherefore is not of pharmaceutical impor‐ tance [18]. Depolymerization of raw polysaccharides, trying to get products with adequate molar masses distribution for commercial purposes, has been done. Dextran, is a well‐known, extracellular, water‐soluble neutral polysaccharide with α‐(1–6)‐linked d‐glucopyranose unit chain, with a wide range of applications. Dextran gained from *Leuconostoc mesenteroides* B‐512(F) is composed of α‐(1–6)‐linked glucan with side chains attached to C3‐positions of backbone glucopyranose units. Various biometal ions (Fe, Co, Cu, Ca, Zn, Mg, etc.) are included in the complexation with dextran in alkaline solutions. Complexes of iron [17, 46] and copper [47–49] with polysaccharides have a great significance, and they have been described in detail. The con‐ tent of metals and solution composition is pH dependent [17, 48]. Cobalt preparations, based on carbohydrates and its derivatives, are used in both human and veterinary medicine [50, 51]. The different FTIR spectroscopic methods (such as microspectroscopy, attenuated total reflection) are commonly used for the characterization of these complexes [52, 53]. This section applies to some spectroscopic examination of dextran complexes with cobalt ions.

### **3.1. Complex synthesis**

The cobalt complexes with reduced low‐molar dextran as ligand (Co(II)‐RLMD) were syn‐ thesized in water solutions, at different pH values (7.5‐13.5) and different temperatures (298–373 K), using CoCl2 × 6H2 O and RLMD (5000 g/mol). The details of synthesis have been described [54, 55]. The complexes were isolated in the solid state. For further structural exami‐ nation, the samples of Co(II)–RLMD were deuterated (D2 O, Merck) for 2 h, at room tempera‐ ture, in vacuum.

### **3.2. FTIR study**

KBr pastille method has been used for sample preparation. The FTIR spectra have been recorded at room temperature, as an average value of 40 scans (resolution of 2 cm−1) on a Bomem MB‐100 FTIR spectrometer (Hartmann & Braun, Canada) coupled with a DTGS/KBr detector. Spectra‐ structural correlation of dextran by FTIR spectroscopy has been the subject of attention of many researchers [30, 31, 56–58]. It was shown that by studying the individual spectral areas, the information on linearity [content of α‐(1–6) bond], crystallinity, conformation, conforma‐ tional transitions, and changes in the structure of differently treated dextrans can be obtained. The FTIR spectrum of reduced low‐molar dextran is shown in **Figure 11a**. Bands at 765 and 916 cm−1 are indicating the presence of the α‐(1–6) glycosidic bonds, and the estimated content of these bonds is greater than 96% that indicates a high linearity of polysaccharide. The pres‐ ence of these bands as those at the 845 cm−1 indicates a C1 conformation of glucopyranose units (eq‐ax‐ax‐ax‐ax arrangement of adjacent C–H groups). There is an intense broad band whose centroid is at about 3400 cm−1, in the area of stretching OH vibrations. Summary intensity of this band comes from the ν(O‐H) vibrations of hydroxyl groups involved in the formation of several by the strength of hydrogen bonds, but also from the H2 O molecule whose presence is confirmed by the band at 1640 cm−1, which is result of δ(HOH) vibrations [54].

**3. Cobalt(II)‐dextran biocomplexes**

162 Fourier Transforms - High-tech Application and Current Trends

**3.1. Complex synthesis**

ture, in vacuum.

**3.2. FTIR study**

(298–373 K), using CoCl2 × 6H2

nation, the samples of Co(II)–RLMD were deuterated (D2

ence of these bands as those at the 845 cm−1 indicates a C1

A lot of investigations in the field of coordination chemistry are based on synthesis and charac‐ terizations of different biocomplexes present in the biological systems. Synthetic ligands, which can serve as model molecules for complex biomolecular structures, are also investigated [19]. Bioligands or synthetic ligands are mostly natural macromolecular compounds. These prod‐ ucts of special importance mostly represent complexes of different metals (Fe, Co, Cu, Zn) with ligands of polysaccharide type (such as pullulan, inulin, dextran) [43–45]. However, the native polysaccharide possessing antigen characteristics wherefore is not of pharmaceutical impor‐ tance [18]. Depolymerization of raw polysaccharides, trying to get products with adequate molar masses distribution for commercial purposes, has been done. Dextran, is a well‐known, extracellular, water‐soluble neutral polysaccharide with α‐(1–6)‐linked d‐glucopyranose unit chain, with a wide range of applications. Dextran gained from *Leuconostoc mesenteroides* B‐512(F) is composed of α‐(1–6)‐linked glucan with side chains attached to C3‐positions of backbone glucopyranose units. Various biometal ions (Fe, Co, Cu, Ca, Zn, Mg, etc.) are included in the complexation with dextran in alkaline solutions. Complexes of iron [17, 46] and copper [47–49] with polysaccharides have a great significance, and they have been described in detail. The con‐ tent of metals and solution composition is pH dependent [17, 48]. Cobalt preparations, based on carbohydrates and its derivatives, are used in both human and veterinary medicine [50, 51]. The different FTIR spectroscopic methods (such as microspectroscopy, attenuated total reflection) are commonly used for the characterization of these complexes [52, 53]. This section

applies to some spectroscopic examination of dextran complexes with cobalt ions.

The cobalt complexes with reduced low‐molar dextran as ligand (Co(II)‐RLMD) were syn‐ thesized in water solutions, at different pH values (7.5‐13.5) and different temperatures

described [54, 55]. The complexes were isolated in the solid state. For further structural exami‐

KBr pastille method has been used for sample preparation. The FTIR spectra have been recorded at room temperature, as an average value of 40 scans (resolution of 2 cm−1) on a Bomem MB‐100 FTIR spectrometer (Hartmann & Braun, Canada) coupled with a DTGS/KBr detector. Spectra‐ structural correlation of dextran by FTIR spectroscopy has been the subject of attention of many researchers [30, 31, 56–58]. It was shown that by studying the individual spectral areas, the information on linearity [content of α‐(1–6) bond], crystallinity, conformation, conforma‐ tional transitions, and changes in the structure of differently treated dextrans can be obtained. The FTIR spectrum of reduced low‐molar dextran is shown in **Figure 11a**. Bands at 765 and 916 cm−1 are indicating the presence of the α‐(1–6) glycosidic bonds, and the estimated content of these bonds is greater than 96% that indicates a high linearity of polysaccharide. The pres‐

O and RLMD (5000 g/mol). The details of synthesis have been

O, Merck) for 2 h, at room tempera‐

conformation of glucopyranose units

**Figure 10.** The FTIR spectra of RLMD (a) and Co(II)‐RLMD complexes synthesized on the boiling temperature and different pH: 7.5 (b) and 13.0 (c).

**Figure 11.** FTIR spectral segments of dextran (a), partially deuterated (b), and fullydeuterated (c) RLMD analogs in ν(O‐ H), ν(O‐D), and δ(HOH) vibrations.

The FTIR spectra of synthesized Co(II)‐RLMD complex, which were obtained under various reaction conditions, are presented in **Figure 10**. The FTIR spectra of RLMD and its Co(II)‐ RLMD complex are basically similar. In the FTIR spectra of synthesized complex, there are the differences in the area of O–H vibrations. In this area, there is a large, complex band approxi‐ mately 3390 cm−1 of ν(O–H), which is likely due to the stretching vibrations of polysaccha‐ ride OH groups. The characteristic IR band of δ(HOH) at about 1645 cm−1 in the spectra of synthesized complexes, as well as in the spectrum of RLMD, as noted above, indicates the presence of crystal water in the structure [54, 55]. By analyzing the low‐frequency part of the RLMD spectrum (γ(C‐H) vibrations, **Figure 10a**), the FTIR spectra of Co(II)‐RLMD complex (**Figure 10b** and **c**), and the presence of bands at about 915 and 845 cm−1, 4 C1 conformation of glucopyranose units, which indicates that the complexation with Co(II) ions does not lead to conformational changes in glucopyranose units, can be determined. In accordance with this is the change in the intensity of IR band in the area approximately 1350 cm−1 that originates from δ(C‐H) and δ(O‐H) vibrations. With an increase in the pH synthesis (from 7.5 to 13.5), the band intensity of ν(O‐H) vibration increases, and the frequency of ν(O‐H) vibration band at lower pH (7.5–8.5) stays almost unchanged and then increases with increasing pH (11–13.5). If the complexation with Co(II) ions takes place via OH groups at the C–2, C–3, or C–4 carbon atoms of dextran glucopyranose units (involved in the formation of various by the strength of hydrogen bonds in dextran), hydrogen bonds disappear by complexation, so the bands are expected at the higher frequencies. In complexes with the highest metal content (10.07% Co), which were synthesized at pH 12, a set of IR bands in this area is close to that at starting RLMD. In the complex which was synthesized at pH 13 with a minimum cobalt content of 1.89% in the IR spectrum (**Figure 10c**), there are intense bands at 3400 cm−1 to the binder of low‐frequency side in this region. This could indicate that the structure of this complex differs slightly from the structure of other Co(II)‐RLMD complex, which were synthesized under different reaction conditions. In the low‐frequency area (<800 cm−1) of the FTIR spectra of RLMD and Co(II)‐ RLMD complex, there are some differences. In this region of the IR spectrum, in addition to the band of ν(Co‐O), the bands of deformation γ(O‐H) vibrations of polysaccharide as well as the deformation vibrations of glucopyranose ring are expected (**Figure 10**). Wide band of medium intensity in the FTIR spectra of Co(II)‐RLMD complex at about 450 cm−1 shows a fine structure.

#### **3.3. Isotopic D2 O exchange study**

Isotopic substitution of hydrogen atoms by deuterium, connected with FTIR spectroscopy, has an important role in determining the structure of dextran. Isotopic exchange results indicate that dextran and its Co(II)‐RLMD complex are crystal hydrates (probably one type of water molecules) [59]. Structural changes in the process have been detected by absorption bands in the area of 3600–3000 cm−1, caused by ν(O‐H) vibrations. In the case of isotopic exchanges of O‐H to O‐D group, the frequency of stretching vibration is reduced to √2, and it is located in the area of 2700–2300 cm−1. Deuteration is a very sensitive method to assess the environ‐ ment of OH groups, which is associated with the intensity generated by hydrogen bonds. The degree of crystallinity of the polysaccharide can be determined by FTIR spectroscopy method with deuteration. Crystallinity is a part of the regulated saccharide area in which the macromolecules are connected with parallel hydrogen bonds. In processing the sample with D2 O, usually OH groups in less regulated or amorphous regions were rapidly converted into OD groups. Conversion of OH groups in the crystal areas is very slow. Thus, the degree of crystallinity has been determined by the change in intensity of asymmetrical ν(O‐H) band vibrations and by the appearance of new bands of ν(O‐D) vibration. The relations of band intensity at 1429 and 893 cm−1 were taken as empirical indicators of the degree of crystallin‐ ity of samples. With decrease in the crystallinity, the band at about 1430 cm−1 disappears and comes to an increase in the intensity of the band at approximately 900 cm−1, typical for the amorphousness. Even better relationship can be seen at the band at approximately 1370 and 2900 cm−1. Namely, in the spectrum of partially deuterated analogs of dextran (**Figure 11**) in the ν(O‐D) area of the vibration of HDO molecules, there is a single band at about 2495 cm−1. Partners of these vibrations would be expected at about 3400 cm−1 in the ν(O–H) areas (taking into account the displacement factor of 1.35).

(**Figure 10b** and **c**), and the presence of bands at about 915 and 845 cm−1, 4

164 Fourier Transforms - High-tech Application and Current Trends

**3.3. Isotopic D2**

D2

**O exchange study**

glucopyranose units, which indicates that the complexation with Co(II) ions does not lead to conformational changes in glucopyranose units, can be determined. In accordance with this is the change in the intensity of IR band in the area approximately 1350 cm−1 that originates from δ(C‐H) and δ(O‐H) vibrations. With an increase in the pH synthesis (from 7.5 to 13.5), the band intensity of ν(O‐H) vibration increases, and the frequency of ν(O‐H) vibration band at lower pH (7.5–8.5) stays almost unchanged and then increases with increasing pH (11–13.5). If the complexation with Co(II) ions takes place via OH groups at the C–2, C–3, or C–4 carbon atoms of dextran glucopyranose units (involved in the formation of various by the strength of hydrogen bonds in dextran), hydrogen bonds disappear by complexation, so the bands are expected at the higher frequencies. In complexes with the highest metal content (10.07% Co), which were synthesized at pH 12, a set of IR bands in this area is close to that at starting RLMD. In the complex which was synthesized at pH 13 with a minimum cobalt content of 1.89% in the IR spectrum (**Figure 10c**), there are intense bands at 3400 cm−1 to the binder of low‐frequency side in this region. This could indicate that the structure of this complex differs slightly from the structure of other Co(II)‐RLMD complex, which were synthesized under different reaction conditions. In the low‐frequency area (<800 cm−1) of the FTIR spectra of RLMD and Co(II)‐ RLMD complex, there are some differences. In this region of the IR spectrum, in addition to the band of ν(Co‐O), the bands of deformation γ(O‐H) vibrations of polysaccharide as well as the deformation vibrations of glucopyranose ring are expected (**Figure 10**). Wide band of medium intensity in the FTIR spectra of Co(II)‐RLMD complex at about 450 cm−1 shows a fine structure.

Isotopic substitution of hydrogen atoms by deuterium, connected with FTIR spectroscopy, has an important role in determining the structure of dextran. Isotopic exchange results indicate that dextran and its Co(II)‐RLMD complex are crystal hydrates (probably one type of water molecules) [59]. Structural changes in the process have been detected by absorption bands in the area of 3600–3000 cm−1, caused by ν(O‐H) vibrations. In the case of isotopic exchanges of O‐H to O‐D group, the frequency of stretching vibration is reduced to √2, and it is located in the area of 2700–2300 cm−1. Deuteration is a very sensitive method to assess the environ‐ ment of OH groups, which is associated with the intensity generated by hydrogen bonds. The degree of crystallinity of the polysaccharide can be determined by FTIR spectroscopy method with deuteration. Crystallinity is a part of the regulated saccharide area in which the macromolecules are connected with parallel hydrogen bonds. In processing the sample with

O, usually OH groups in less regulated or amorphous regions were rapidly converted into OD groups. Conversion of OH groups in the crystal areas is very slow. Thus, the degree of crystallinity has been determined by the change in intensity of asymmetrical ν(O‐H) band vibrations and by the appearance of new bands of ν(O‐D) vibration. The relations of band intensity at 1429 and 893 cm−1 were taken as empirical indicators of the degree of crystallin‐ ity of samples. With decrease in the crystallinity, the band at about 1430 cm−1 disappears and comes to an increase in the intensity of the band at approximately 900 cm−1, typical for the amorphousness. Even better relationship can be seen at the band at approximately 1370 and 2900 cm−1. Namely, in the spectrum of partially deuterated analogs of dextran (**Figure 11**) in

C1

conformation of

The FTIR spectra of Co(II)‐RLMD complex (a) and its deuterated analog (b), which was syn‐ thesized at pH 13, are shown in **Figure 12**. In the FTIR spectrum of Co(II)‐RLMD complex (**Figure 12b**), in the area of ν(O‐D) vibrations of HDO molecules, there is a single band at about 2483 cm**<sup>−</sup>**<sup>1</sup> in the corresponding complexes with crossfold on the high‐frequency side. Partners of these vibrations would be expected at about 3400 cm**<sup>−</sup>**<sup>1</sup> in ν(O‐H) area. Results of partial deuteration indicate that the band at about 3400 cm**<sup>−</sup>**<sup>1</sup> is sensitive to isotopic substitu‐ tion, in both cases (RLMD and Co(II)‐RLMD complexes). Reducing the intensity of this band by deuteration demonstrates that the ν(O‐H) vibrations of water molecules are its part. This fact indicates that both compounds contain crystal water in their structure. Confirmation of this conclusion is that in the spectra of deuterated analogs of both compounds (**Figures 11** and **12**), an intense band near 1645 cm**<sup>−</sup>**<sup>1</sup> is also highly sensitive to isotopic substitution and is to be attributed to the HOH deformation vibration of the crystal water.

**Figure 12.** FTIR spectra of Co(II)‐RLMD complex (a) and its deuterated analog (b) synthesized at pH 13.

As known, Seidl et al. [60] proposed criteria according to which, based on the study of spectra of protonated, partially and fully deuterated hydrate, it is possible to determine the number of types of H2 O molecules (n) and the number of nonequivalent OH groups (m). By the spectra appearances, in the stretching OD area of HDO molecules, and the appearance of a band, whose intensity increases monotonically with increasing degree of deuteration when the frequency does not change, it can be concluded from the above criteria [60] that in the structure of dextran and its complexes with Co(II) ions is present one crystallographic type of water molecule (n = 1). On the basis of Berglund correlation [61], from equation (3), Ow…O distances are estimated at 283.1 pm for dextran and 281.8 pm for Co(II)‐RLMD complex:

$$\mathbf{v}\left(\mathbf{OD}\right) = \mathbf{2727} - 8.97 \times 10^6 \times \mathbf{e}^{-3.73 \times \text{R} \left(\text{W} \times \times \times \text{O}\right)}.\tag{3}$$

Water protons are involved in the formation of relatively weak hydrogen bonds (m = 1). In bending area of HDO and D2 O, in the spectrum of deuterated analogs of the complex, there are bands around 1315 and 1070 cm−1, which confirm the previously disclosed consideration of the water binding. From FTIR spectrum shown in **Figure 12b**, decrease in the intensity of the band around 1430 cm−1 and an increase in the band intensity at approximately 910 cm−1 can be observed, which is a characteristic for amorphous character. An even better relation‐ ship can be observed in the FTIR spectrum of **Figure 12**, with the band of about 1370 and 2900 cm−1. Based on the results of FTIR spectroscopy, an amorphous structure of the synthesized Co(II)‐RLMD complex can be assumed.

### **3.4. ATR‐FTIR microscopy study**

The ATR‐FTIR spectral analysis has been performed by microspectroscopy ATR‐FTIR system (Bruker, Tensor‐27). Within this system, FTIR spectroscope is connected to a microscope (15× objective) (Bruker, Hyperion‐1000/2000) and a computer system capable of microanalysis by using a liquid‐nitrogen‐cooled (250 μm) MCT detector (GMBH, Germany). The ATR‐FTIR spectra (Kubelka‐Munk option) have been recorded in the range of 4000–400 cm−1, with 4 cm−1 resolution and 260 scans. The newly formed FTIR vibrational microspectroscopy can provide information on the sample at the molecular level, with high spatial resolution at the micro‐ scopic level. Small sample can be analyzed by both nondestructive vibrational spectroscopic techniques (Raman, IR) [62–67]. Spectra can be recorded continuously in different parts of the microsample in order to obtain appropriate databases. **Figures 13** and **14** show the absorp‐ tion ATR‐FTIR spectra of Co(II)‐RLMD complex, which were obtained under various reaction conditions.

**Figure 13.** ATR‐FTIR spectra of Co(II)‐RLMD complex synthesizedat the boiling temperature and pH values in the range of 7–11.

The wavenumber values of characteristic IR bands in the ATR‐FTIR spectra of Co(II)‐RLMD complex are given in **Table 4**.

Absorption bands corresponding to the specific chemical components can be represented as a map. ATR‐FTIR spectra, presented in **Figures 13** and **14**, correspond to the different Study of Green Nanoparticles and Biocomplexes Based on Exopolysaccharide by Modern Fourier Transform Spectroscopy http://dx.doi.org/10.5772/64611 167

of the water binding. From FTIR spectrum shown in **Figure 12b**, decrease in the intensity of the band around 1430 cm−1 and an increase in the band intensity at approximately 910 cm−1 can be observed, which is a characteristic for amorphous character. An even better relation‐ ship can be observed in the FTIR spectrum of **Figure 12**, with the band of about 1370 and 2900 cm−1. Based on the results of FTIR spectroscopy, an amorphous structure of the synthesized

The ATR‐FTIR spectral analysis has been performed by microspectroscopy ATR‐FTIR system (Bruker, Tensor‐27). Within this system, FTIR spectroscope is connected to a microscope (15× objective) (Bruker, Hyperion‐1000/2000) and a computer system capable of microanalysis by using a liquid‐nitrogen‐cooled (250 μm) MCT detector (GMBH, Germany). The ATR‐FTIR spectra (Kubelka‐Munk option) have been recorded in the range of 4000–400 cm−1, with 4 cm−1 resolution and 260 scans. The newly formed FTIR vibrational microspectroscopy can provide information on the sample at the molecular level, with high spatial resolution at the micro‐ scopic level. Small sample can be analyzed by both nondestructive vibrational spectroscopic techniques (Raman, IR) [62–67]. Spectra can be recorded continuously in different parts of the microsample in order to obtain appropriate databases. **Figures 13** and **14** show the absorp‐ tion ATR‐FTIR spectra of Co(II)‐RLMD complex, which were obtained under various reaction

The wavenumber values of characteristic IR bands in the ATR‐FTIR spectra of Co(II)‐RLMD

**Figure 13.** ATR‐FTIR spectra of Co(II)‐RLMD complex synthesizedat the boiling temperature and pH values in the range

Absorption bands corresponding to the specific chemical components can be represented as a map. ATR‐FTIR spectra, presented in **Figures 13** and **14**, correspond to the different

Co(II)‐RLMD complex can be assumed.

166 Fourier Transforms - High-tech Application and Current Trends

**3.4. ATR‐FTIR microscopy study**

conditions.

of 7–11.

complex are given in **Table 4**.

**Figure 14.** ATR‐FTIR spectra of Co(II)‐RLMD complex synthesized at the boiling temperature and pH values in the range of 12–14.


**Table 4.** Assignment of characteristic IR bands of RLMD and the synthesized Co(II)‐RLMD complexes.

parts of the sample of Co(II)‐RLMD complex, which show a homogeneity of the samples. A new way of visualization shows the capability of visualization not only of heterogeneous region of the samples, but also at the same time provides microspectroscopic spatial infor‐ mation. The visualization of different concentrations of components and presentation as 3D maps is also enabled. Application of ATR‐FTIR microscopy to Co(II)‐RLMD complex, which were synthesized under different reaction conditions, is shown in **Figure 15**. The changes in color contours at certain parts of the image indicate the content and distribu‐ tion of cobalt and polysaccharides in Co(II)‐RLMD samples. ATR‐FTIR microspectroscopic data show a high homogeneity of the samples, and the presence of Co(II) ions (the results obtained by other spectroscopic techniques) has been confirmed by the color of Co(II)‐ RLMD complex.
