**2. Experimental**

212 Nitroxides – Theory, Experiment and Applications

for the oxidation of catecholamines (Paradossi et al., 1998).

and as pervaporation membranes (Varghese et al., 2010).

(Zakharova et al., 2005).

aqueous media.

Chitosan, poly-D-β-glucosamine, is a commercially available amino polymer that is a perfect complexing agent, due to the strong donor properties of both the amino and hydroxyl groups (Varma et al., 2004). Chitosan is thus widely used in obtaining various catalytic materials, including those containing Au0 that are used in the hydroamination of alkenes (Corma et al., 2007); Pd0 used for the reduction of ketones (Yin et al., 1999); the Pd0–Ni0 bimetallic system, used for carbonylation (Zhang & Xia, 2003); Os (VIII), used for hydroxylation (Huang et al., 2003); Co2+, used for hydration (Xue et al., 2004); and Cu2+, used

SiO2 is usually used as the inorganic component for these systems. The obtained hybrid materials are used to create sorbents of 3d-metal ions (Liu et al., 2002); to immobilize enzymes (Airoldi & Monteiro, 2000); as a solid phase for the liquid chromatography of organic compounds (Budanova et al., 2001), including enantiomers (Senso et al., 1999); and to improve the mechanical properties of other polymers (Yeh et al., 2007). Other oxides in combination with chitosan allow us to obtain biosensors based on ZnO substrate (Khan et al., 2008), selective sorbents of fluoride ions based on Al2O3 substrate (Viswanathana & Meenakshib, 2010) and magnetic materials based on Fe3O4 substrate (Li et al., 2008). Using an organic polymer (e.g., cellulose) as a substrate also has advantages in the sorption of metal ions (Corma et al., 2007). Metal-containing hybrid organo–inorganic materials can also be used as antibacterial composites (Mei et al., 2009), as sorbents of proteins (Shi et al., 2003),

Nanostructured metal oxides, which are distinguished by extremely developed surface and porosity of particles, are new promising materials for different elds of science and technology, especially, for heterogeneous catalysis and chemistry of adsorption phenomena

Many sorption and catalytic processes are pH-dependent. Therefore, the determination of acidity and other acid–base characteristics in pores of inorganic, organo-inorganic materials is of great practical interest, since the catalytic and adsorption properties of solid-phase objects are affected by not only the chemical nature of solutions, but also specic conditions inside pores and on the surface of these materials. The mobility of liquid molecules in pores of inorganic sorbents was investigated by some authors using the spin probe method (Borbat et al., 1990; Martini et al., 1985 ). Recently, a new method was developed for the determination of medium acidity in pores of solids (pHint) by means of pH-sensitive nitroxide radicals (NRs) as spin probes (Molochnikov et al., 1996 ; Zamaraev et al., 1995). In recent years, this method was used to measure pHint in micropores of various cross-linked organic polyelectrolytes (ion-exchange resins and lms) (Molochnikov et al., 1996, 2004) and in pores of some zeolites and kaolin (Zamaraev et al., 1995). We found that pHint inside sorbents differ from the pH of external solutions by 0.8–2.1 units (Molochnikov et al., 1996). The method developed allowed us to study the processes of sorption and hydrolysis in ionexchange resins and the catalytic properties of Cu2+- containing carboxyl cation exchangers (Kovaleva et al., 2000), to determine ionization constants of functional groups and to give a critical estimation to the regularities previously found for the behavior of adsorbents in

### **2.1. Objects of study**

**-Al2O3 (basic aluminum oxide), γ-Al2O3 and its acid-modified (HF and H2SO4) derivatives** were supplied by A. M. Volodin. **The IK-02-200 type γ- Al2O3** was synthesized by the calcination of aluminum hydroxide at 600°C. γ-Al2O3 was modied through the sample impregnation with acids followed by calcination at 600°C that resulted in changes in the acidic properties of its surface (the phase composition and specic surface area of the samples remained unchanged). **-Al2O3** was prepared by the long-term heating of **γ-Al2O3** to 1300°C. **γ- Al2O3** and **-Al2O3** had specic surface areas of 220 and 145 m2/g, and an average pore diameter of 6 nm, respectively. The structural characteristics of the matrices were determined from the isotherms of nitrogen adsorption at 77 K measured on a Micromeritics ASAP 1400 volumetric setup and by mercury porosimetry with a Micromeritics Pore Size 9300 setup in the Institute of Catalysis, Siberian Division, Russian Academy of Sciences.

The technique of synthesis of **TiO2 hydrogel** through the hydrolysis of a tetrabutyl titanate solution in methanol with water at room temperature and under intensive agitation is given in (Shishmakov et al., 2003). The precipitate was washed out with water until no butanol in washing water was observed and heated to 100°C. The resulting product was **TiO2 xerogel.**

**SiO2 hydrogel** was synthesised by dissolving 10 ml of Na2SiO3 (ТU 6-15-433-92) in 30 ml of H2O. Then the hydrolysis of Na2SiO3 solution in 30 ml of 10% HCl solution was pursued under intensive agitation. During the reaction of condensation a gelatinous SiO2 gel and NaCl are formed. The precipitate was filtered off until no chlorine ions in washing water was observed, and dried at 100 C during 24h until it attained a constant weight. The resulting product was **SiO2 xerogel.**

**Powder cellulose (PC)** was obtained by hydrolysis of cellulose sulfate (Baikal Cellulose plant, TU OP 13-02794 88-08-91) in 2.5 N hydrochloric acid at 100°C. The hydrolysis was carried out for 2 h. The resulting product was washed on a filter with distilled water to the neutral pH of the washing water and dried at 100°C.

Composite materials (CMs) based on nanostructured TiO2 and PC called as TiO2–PC xerogels of 70, 53 and 43 % wt. TiO2 were prepared by diluting 3 g of tetrabutoxytitanium and 0.5 ; 1 and 1.5 g of PC, respectively, in 3 mL of methanol. The hydrolysis was pursued in 10 mL of water at 20°C under intensive agitation, resulting in the condension of TiO2 (PC didn't participate in condensation). The TiO2 particles formed were deposited on a surface of PC.

**Composite materials (CMs) based on nanostructured SiO2 and PC called as SiO2–PC xerogels of 68, 52 and 35% wt.** were prepared .from the solutions of 10 ; 5 ; 5 ml of Na2SiO3 and 30 ; 15 ; 15 ml of H2O which were modified by introducing 2 ; 2 ; 4 g of PC, respectively. The hydrolysis of the first solution were peformed in 30 mL of 10 %HCL, and that of other ones was done in 15 mL of 10% HCL.

The precipitates of the CMs prepared were washed out with hot water, filtered and dried at 100 C during 24h until they attained a constant weight.

The specific surface (Ssp) of the synthesized samples was measured using a SORBIMS instrument (ZAO Meta, Novosibirsk) and calculated by the BET procedure. The data are given in Table 1 (Parshina et al., 2011).


**Table 1.** The specific surface of the pure xerogels of TiO2 and SiO2 and the composites with different percentage of PC

Since the specific surface of powder cellulose did not exceed 1 m2/g , the growth of Ssp was caused by the fragmentation of TiO2 and SiO2 particles deposited on the PC surface during the synthesis of CMs. According to the absolute values of Ssp, the procedure used for the preparation of CMs afforded dioxides with a high degree of dispersity.

Powdered samples **nanostructured TiO2** were prepared through heating a sol for 1 h at 200°C followed by washing with distilled water to remove residual acid used for the sol stabilization and drying at room temperature. The specic surface area of the samples was 240 m2/g, and the average particle diameter ranged from 4 to 5 nm (Poznyak et al., 1999).

Microcrystalline cellulose (MCC) with an ash content of 0.16% and a humidity of 1.1% produced by JSC Polyex; Basic aluminum oxide; BS-50 silica and chitosan produced by JSC Sonat (Moscow) were used to obtain chitosan-containing ahybride organo-inorganic systems. The degree of deacetylation of chitosan (DD) determined by 1H NMR spectroscopy, its molecular weght as determined by viscosimetry and the ash content were found to be 0.84, 250 kDa and 0.19%, respectively (Mechaev et al., 2011a). The BS-50 type silica had a specic surface area of 45 m2/g and an average diameter of pores of 15 nm (Mekhaev et al., 2011a).

**The hybrid chitosan–(SiO2, Al2O3, cellulose) systems** were obtained by depositing chitosan on the support surface.

0.3 g (1.8 mmol) of citosan was dissolved in 14.5 ml of water containing 0.22 ml (3.84 mmol) of acetic acid with constant stirring. The substrate in quantities of 3 g was then added, and the solution was stirred for 30 min more.

1 M NaOH solution was added to the suspension under stirring until the pH value reached 13. The precipitate was filtered, washed until the pH value was 7, and dried at 60°С until it attained a constant weight.

CHN analysis was performed using an automatic analyzer PerkinElmer, Inc. The data are given in Table 1. IR spectra of diffuse reflection were recorded using the PerkinElmer Spectrum One spectrometer.


**Table 2.** Composition (%) of hybrid systems (calculated values are shown in brackets )

The surface area of the samples was determined by nitrogen adsorption in accordance with the BET method using a TriStar 3000 V.6.03A instrument. The instrumental error was 0.1 m2/g. The size of particles was estimated under the assumption that the particles were spherical.

The surface area (Ssp) and the diameter of particles (D) were found to be 28.9 m2/g and 47 nm ; 123.9 m2/g and 7 nm ; 2.4 m2/g and 818 nm for chitosan-SiO2 , chitosan-Al2O3 and chitosan-MCC hybride systems, respectively.

## **2.2. Saturation of samples with Cu2+ and Co2+ ions**

### *2.2.1. Saturation of samples with Cu2+ Ions*

214 Nitroxides – Theory, Experiment and Applications

ones was done in 15 mL of 10% HCL.

given in Table 1 (Parshina et al., 2011).

percentage of PC

neutral pH of the washing water and dried at 100°C.

100 C during 24h until they attained a constant weight.

**Powder cellulose (PC)** was obtained by hydrolysis of cellulose sulfate (Baikal Cellulose plant, TU OP 13-02794 88-08-91) in 2.5 N hydrochloric acid at 100°C. The hydrolysis was carried out for 2 h. The resulting product was washed on a filter with distilled water to the

Composite materials (CMs) based on nanostructured TiO2 and PC called as TiO2–PC xerogels of 70, 53 and 43 % wt. TiO2 were prepared by diluting 3 g of tetrabutoxytitanium and 0.5 ; 1 and 1.5 g of PC, respectively, in 3 mL of methanol. The hydrolysis was pursued in 10 mL of water at 20°C under intensive agitation, resulting in the condension of TiO2 (PC didn't participate in condensation). The TiO2 particles formed were deposited on a surface of PC.

**Composite materials (CMs) based on nanostructured SiO2 and PC called as SiO2–PC xerogels of 68, 52 and 35% wt.** were prepared .from the solutions of 10 ; 5 ; 5 ml of Na2SiO3 and 30 ; 15 ; 15 ml of H2O which were modified by introducing 2 ; 2 ; 4 g of PC, respectively. The hydrolysis of the first solution were peformed in 30 mL of 10 %HCL, and that of other

The precipitates of the CMs prepared were washed out with hot water, filtered and dried at

The specific surface (Ssp) of the synthesized samples was measured using a SORBIMS instrument (ZAO Meta, Novosibirsk) and calculated by the BET procedure. The data are

**PC, %** 0 30 47 57 0 32 65 **Ssp,m2/g** 66 177 226.4 261.9 29.5 145 239 **Table 1.** The specific surface of the pure xerogels of TiO2 and SiO2 and the composites with different

Since the specific surface of powder cellulose did not exceed 1 m2/g , the growth of Ssp was caused by the fragmentation of TiO2 and SiO2 particles deposited on the PC surface during the synthesis of CMs. According to the absolute values of Ssp, the procedure used for the

Powdered samples **nanostructured TiO2** were prepared through heating a sol for 1 h at 200°C followed by washing with distilled water to remove residual acid used for the sol stabilization and drying at room temperature. The specic surface area of the samples was 240 m2/g, and the average particle diameter ranged from 4 to 5 nm (Poznyak et al., 1999).

Microcrystalline cellulose (MCC) with an ash content of 0.16% and a humidity of 1.1% produced by JSC Polyex; Basic aluminum oxide; BS-50 silica and chitosan produced by JSC Sonat (Moscow) were used to obtain chitosan-containing ahybride organo-inorganic systems. The degree of deacetylation of chitosan (DD) determined by 1H NMR spectroscopy, its molecular weght as determined by viscosimetry and the ash content were found to be 0.84, 250 kDa and 0.19%, respectively (Mechaev et al., 2011a). The BS-50 type silica had a specic surface

area of 45 m2/g and an average diameter of pores of 15 nm (Mekhaev et al., 2011a).

preparation of CMs afforded dioxides with a high degree of dispersity.

**TiO2 SiO2** 

A 0.1 M NaNO3 solution (10 ml) was added into weighed samlpes (200 mg) of nanostructured TiO2, and the samples were kept for one week at a constant solution pH (5.5) held by adding dilute NaOH and HNO3 solutions. The sorption of Cu2+ ions on nanostructured TiO2 was performed by exposing samples in Cu(NO3)2 solutions (10 ml)

with concentrations of 10–4, 10–3, and 10−2 mol/L and ionic strength (µ) of 0.1, which was adjusted using NaNO3. Solution pH equal to 4.3 was maintained by the titration with small volumes of NaOH and HNO3 solutions. After the equilibrium was established, the residual amount of Cu2+ ions the equilibrium solutions was measured to determine the amount of sorbed Cu2+. Then, TiO2 was separated from the solutions by centrifugation. The samples were washed twice with a 0.1 M NaNO3 solution (pH 4.3) to remove adsorbed Cu2+ -ions.

Cu2+ ions were sorbed on TiO2 hydrogel from CuCl2 and Cu(NO3)2 with subsequent its removal by ltration and drying at 20°C for 3 days upto constant weights of the precipitates. A volume of solution and a mass of hydrogel were changed to vary the content of Cu2+ ions in the phase of the studied TiO2 hydrogel, which was determined by the atomic absorption method on a Perkin Elmer 403 spectrometer. As the ESR spectra of Cu2+-containing hydrated gels are difcult to record, hydrogel samples ltered and dried at room temperature were used. Preliminary experiments were performed to select the sample drying conditions preventing the structural changes of the complexes formed.

**Cu2+ - containing composites based nanostructured SiO2, TiO2, and cellulose powder** were prepared by sorption of Cu2+ ions on a hydrogel from an aqueous solution of CuCl2 2H2O. The volumes of hydrogels of the samples studied were calculated from the masses of xerogels obtained by hydrogels drying. For preparation of Cu2+-containing TiO2 and SiO2 xerogels and the related CMs, a 0.01 g CuCl2 2H2O containing 0.059 mmol of Cu2+ ions and 2 ml of H2O and the calculated volumes of hydrogels prepared were added into flasks. The samples were kept in the contact with a Cu2+-containing solution about 24 h upto the equilibrium was established. Then, the residual amounts of Cu2+ ions in the equilibrium solutions was measured to determine the amount of sorbed Cu2+ ions. The initial and residual amounts of Cu2+ -ions in a solution were measured using colorimeter KFK-2MP.

### *2.2.2. Synthesis and characterization of the Cobalt-Containing Chitosan hybrid systems*

**Cobalt-Containing Chitosan–Supported Systems** were synthesised through stirring a mixture containing 0.24 g of CoCl2 6H2O, 2 g of chitosan–supported hybrid system and 20 ml of ethanol under reflux condenser for 24 h. The obtained cake was filtered off, rinsed with ethanol (15 ml × 3 times) and dried at room temperature until the weight became constant. The elemental compositions of the hybrid system surfaces were determined using an analytical setup based on a VEGA II LMH scanning electron microscope and an INCA ENERGY energy dispersive microanalysis system (Mekhaev et al., 2011a, 2011b). The data are shown in Table 2.

### **2.3. pH probes**

The pH values of solutions inside pores and near the surface of the studied inorganic and organo-inorganic materials were determined using spin probes, namely, pH-sensitive NRs of the imidazoline (R1, R2) and imidazolidine (R3) types (Table 3), which were synthesized at the Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences (Volodarskii et al., 1988; Khramtsov et al., 1998; Kirilyuk et al., 2005).

### **2.4. Recording and processing of the ESR spectra of NR**

216 Nitroxides – Theory, Experiment and Applications

are shown in Table 2.

**2.3. pH probes** 

with concentrations of 10–4, 10–3, and 10−2 mol/L and ionic strength (µ) of 0.1, which was adjusted using NaNO3. Solution pH equal to 4.3 was maintained by the titration with small volumes of NaOH and HNO3 solutions. After the equilibrium was established, the residual amount of Cu2+ ions the equilibrium solutions was measured to determine the amount of sorbed Cu2+. Then, TiO2 was separated from the solutions by centrifugation. The samples were washed twice with a 0.1 M NaNO3 solution (pH 4.3) to remove adsorbed Cu2+ -ions.

Cu2+ ions were sorbed on TiO2 hydrogel from CuCl2 and Cu(NO3)2 with subsequent its removal by ltration and drying at 20°C for 3 days upto constant weights of the precipitates. A volume of solution and a mass of hydrogel were changed to vary the content of Cu2+ ions in the phase of the studied TiO2 hydrogel, which was determined by the atomic absorption method on a Perkin Elmer 403 spectrometer. As the ESR spectra of Cu2+-containing hydrated gels are difcult to record, hydrogel samples ltered and dried at room temperature were used. Preliminary experiments were performed to select the sample drying conditions

**Cu2+ - containing composites based nanostructured SiO2, TiO2, and cellulose powder** were prepared by sorption of Cu2+ ions on a hydrogel from an aqueous solution of CuCl2 2H2O. The volumes of hydrogels of the samples studied were calculated from the masses of xerogels obtained by hydrogels drying. For preparation of Cu2+-containing TiO2 and SiO2 xerogels and the related CMs, a 0.01 g CuCl2 2H2O containing 0.059 mmol of Cu2+ ions and 2 ml of H2O and the calculated volumes of hydrogels prepared were added into flasks. The samples were kept in the contact with a Cu2+-containing solution about 24 h upto the equilibrium was established. Then, the residual amounts of Cu2+ ions in the equilibrium solutions was measured to determine the amount of sorbed Cu2+ ions. The initial and residual amounts of Cu2+ -ions in a solution were measured using colorimeter KFK-2MP.

*2.2.2. Synthesis and characterization of the Cobalt-Containing Chitosan hybrid systems* 

**Cobalt-Containing Chitosan–Supported Systems** were synthesised through stirring a mixture containing 0.24 g of CoCl2 6H2O, 2 g of chitosan–supported hybrid system and 20 ml of ethanol under reflux condenser for 24 h. The obtained cake was filtered off, rinsed with ethanol (15 ml × 3 times) and dried at room temperature until the weight became constant. The elemental compositions of the hybrid system surfaces were determined using an analytical setup based on a VEGA II LMH scanning electron microscope and an INCA ENERGY energy dispersive microanalysis system (Mekhaev et al., 2011a, 2011b). The data

The pH values of solutions inside pores and near the surface of the studied inorganic and organo-inorganic materials were determined using spin probes, namely, pH-sensitive NRs of the imidazoline (R1, R2) and imidazolidine (R3) types (Table 3), which were synthesized at the Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of

Sciences (Volodarskii et al., 1988; Khramtsov et al., 1998; Kirilyuk et al., 2005).

preventing the structural changes of the complexes formed.

The ESR spectra were recorded on a PS 100.X ESR spectrometer (ADANI, Belarus) in a three-centimeter (X) wavelength range at room temperature. Quartz sample holders with an internal diameter of 3.5 mm were used for solid samples. Solution spectra were recorded using quartz capillaries.

Figure 1 shows characteristic ESR spectra of the pH-sensitive NR in aqueous solutions. According to the ESR theory, isotropic signals are induced by the fast-motioned NR molecules (correlation times of 10–10 s and less) and present the triplet of fine lines**.** Depending on solution pH, NR can be in protonated (RH+), deprotonated (R), or intermediate (mixed) forms. Because hyperne splitting constants aN for RH+ and R forms of the radicals are different (Table 3), in their ESR spectra, the distance *a* between the low and central-field component of the triplet increases gradually with pH of a solution, from the values characteristic of the RH+ form to those typical of the R form (Fig. 1). This characteristic is a superposition of hyperfine splitting constants (aN) characterizing protonated and deprotonated forms of the nitroxide radical. From the results of measuring *a* values during titration, the calibration curves reecting the dependences *a* vs. pH were plotted for each NR used (for example, see Fig. 2, 3 curve 1). In order to plot the calibration curves, NR solutions (10–4 mol/l ; µ = 0.1 ) were titrated either with dilute HCl and KOH solutions (used for all the samples, excepting hybride systems)( Molochnikov et al.,2007 ; Parshina et al., 2011 ; Shishmakov et al., 2010) or citrate-phoshate (pH 3.5-7.8) and citrate-salt (pH 1.6-4.8) buffer solutions (used for hybride systems) (Mekhaev et al., 2011a, 2011b). to vary the pH within the range of NR sensitivity of 2.5 – 7.5.

**Figure 1.** The ESR spectra of the aqueous solution of NR R3 at different pH in the X range of wavelengths at 293K. IRH+ and IR are the intensities of ESR peaks for RH+ and R forms of the radical, respectively

**Table 3.** ESR paramters and pKa values of nitroxide radicals used

R1

R2

H

R3

N

<sup>N</sup> <sup>N</sup>

**Table 3.** ESR paramters and pKa values of nitroxide radicals used

.

N

O

.

N

N

O

N

N

O

H N NH2

C

.

Radical pKa

( 0.1)

3.15

g-factor ( 0.0001)

4.89 2.0048 2.0051 1.520 1.390

3.55 2.0048 2.0051 1.590 1.515

4.70 2.0048 2.0051 1.590 1.485

aN ( 0.006 mT)

R RH+ R RH+

**Figure 2.** Titration curves for NR R1 in bulk aqueous solution (calibration curve) (1), -Al2O3 (2), the BS-50 SiO2 (3) and γ- Al2O3 (4). a, % = ((a-aNRH+)/(aNR-aNRH+))×100%

### **2.5. Determination of pH in the pore and near the sample surface using pHsensitive spin probes**

An aqueous KCl solution (10 ml) with an ionic strength of 0.1 was added to an oxide sample (200 mg) and the mixture was allowed to stand for a preset time. Then, the solution was thoroughly decanted and an NR solution (10–4 mol/L, µ = 0.1) was added to the sample. In some cases, required initial pH values of radical solution were obtained by preliminary mixing of HCl and KOH solutions. After the equilibrium was established, the suspension was titrated with HCl and KOH (HNO3 and NaOH) solutions to plot the titration curve for the NR present in the sample.

For chitosan cobalt-containing hybrid systems and solid-phase composites based on SiO2, TiO2 and cellulose powder the method of multiply batches was used: 0.05 g of sample was kept in 5 ml of buffer aqueous solution containing nitroxide radicals for 2 days (established experimentally). The solution was then decanted.

The pH values of the equilibrium solutions (pHext) over the samples were measured using a Mettler Toledo pH meter (Switzerland) with an accuracy of 0.01 units. The samples separated from the solutions by centrifugation or ltration were placed into unsealed quartz ampules and their ESR spectra were recorded. After measuring the *a* distances in the ESR spectra of corresponding radicals located in the samples (Fig. 4), the pHint values of the studied materials were determined using the calibration curves (Fig. 2,3).

**Figure 3.** Titration curves for NR R1 in bulk aqueous solution (calibration curve) (1), PC (2), SiO2 (3) and TiO2 (4) xerogels. a, % = ((a-aNRH+)/(aNR-aNRH+))×100%

As can be seen from Fig. 4, the ESR spectra of NR in the samples studied represent the superpositions of three components of an isotropic signal of the probes in aqueous solutions inside pores and a spectrum of the probes immobilized on the surface of the objects studied. For determination of pHint values, only the isotropic signals in the ESR spectra were used.

I, II,II – components of an isotropic spectrum ; IV, V, VI – components of the spectrum of the immobilized probes **Figure 4.** The ESR spectra of NR R1 in the samples of CMs SiO2-PC (68% wt.SiO2) at pH 7.8 (1) and pH 3.7(2)
