**5. Experimental section**

182 The Complex World of Polysaccharides

**Figure 16.** Glucose syrup from larch wood CR

hydrolysis of CR and cellulose [118].

We studied the crystallization conditions of glucose using its CC with sodium chloride (C6H12O6)2·NaCl·H2O and examined CC crystals obtained from model mixtures of glucose, sodium chloride and water, as well as from experimental glucose syrups obtained by the

The crystallization of CC was studied using artificial mixtures in which the NaCl content varied from 15% to a twofold excess relative to the glucose content. In all cases, a crystalline phase formed. The composition of the crystalline phase was determined by elemental analysis. We determined that the range of NaCl:glucose ratios from 0.2:1 to 0.7:1 (parts by weight) is optimal for the formation of CC. Similarly, we determined the crystallization conditions for CC in hydrolysates of CR and cellulose. CC crystals were isolated from hydrolysates; the artificial mixtures were colourless and transparent and had well-defined facets. According to the chemical analysis data, they have a composition close to being stoichiometric: C – 32.5%, H – 5.9% and Cl – 9.7%. Theoretically, CC with the general

According to X-ray phase analysis, CC monocrystals synthesized from pure solutions have the unit cell parameters a = b = 16.8, c = 17.0 Å and β = 120°, and represent a hexagonal prism. Based on the symmetry of lauegrams and weissenbergograms, they belong to the diffraction class P 3 m with regular extinctions at 1 ≠ 3n. Therefore, the spatial group of CC

The set of diffraction maxima obtained by X-ray phase analysis of CC crystals and the reference indicates that, under the experimental conditions used, glucose in the presence of

Thus, when studying the crystallization properties of glucose syrups produced by acid hydrolysis of the crystalline glucose of larch wood, we obtained CC glucose crystals with sodium chloride, upon decomposition of which D-glucose is released in crystalline form. In addition, glucose can be directly crystallized from glucose syrups produced by the

formula (C6H12O6)2·NaCl·H2O contains C – 33%, H – 6%, and O – l8.13%.

sodium chloride crystallizes as CC with the formula (C6H12O6)2·NaCl·H2O.

hydrolysis of cellulose with a high factor of merit (more than 85%).

crystals was determined as P31 12 (151) and P32 12 (153).

Arabinogalactan was extracted using technology from *L. sibirica* Ledeb. [76] at the experimental–industrial plant and purified with methods described in [73, 17]. Molecular masses of arabinogalactan were defined with a high-performance liquid chromatography (HPLC) method, assisted by the Agilent Technologies 1260 Infinity chromatographic system using 0.1 M sample solutions of LiNO3 on PL aquagel-OH-40.8 mm, 300x7.5 mm column, with a PL aquagel-OH Guard 8 mm 50x7.5 mm precolumn, standardized according to dextrans with 25, 12 and 5 kDa molecular mass standard solutions and monosaccharides.

IR spectra were registered in KBr tablets on a "Specord 75IR" spectrophotometer with a 500– 4000 cm-1 interval. UV spectra were registered with a "Specord UV-vis" spectrophotometer (10 mm layer thickness). NMR 13C spectra of AG samples were registered with a "Varian VXR 500S" spectrometer with a 125.1 Hz operating frequency; D2O was used as solvent. Deuteroaceton was used as an internal standard. The correlation of galactose and arabinose chains, composed of AG macromolecules, were calculated according to the correlation between integral intensities of carbon galactose anomeral atomic signals and arabinose. The ratio of galactose to arabinose units in AG macromolecules was calculated from the ratio of the intensities of signals from anomeric carbon atoms of glactose and arabinose [10].

Pectin polysaccharides (PS) were extracted from the bark of *L. sibirica* Ledeb. and from *L. gmelini* (Rupr.) Rupr. according to the scheme depicted in Figure 17. Larch air-dried bark (500 g), which was initially ground and treated with ethyl acetate, was extracted using distilled water at 70 oC over 3 h. Raw material residue was poured with a mixture (1:1, v/v) 0.5% of ammonium oxalate water solution and 0.5% oxalic acid water solution and heated at 80 oC for 2 h. The extract was concentrated. Polysaccharides were precipitated with a triple volume of ethyl alcohol or acetone and dried with lyophilization. As a result, we obtained PS. The PS was dissolved in water and aminoacids were detected on ААА339М automatic analyser.

PS (50 mg) was dissolved in 20 ml of water. Pektinaza (2 mg; Sigma, USA) water solution was added. The mixture was temperature-controlled at 37 o C for 3 h. Then, a reaction mixture was heated for 5 min in a water bath at 100 oC. Coagulated protein was separated by centrifugation. The obtained supernatant was concentrated and up to 5 ml 96% ethyl alcohol was added (4 volumes). Deposition was separated with centrifugation. Alcoholic supernatant was concentrated and analysed with the help of PC.

Galacturonic acid content in PS was defined according to the reaction with 3,5-dimethyl phenol in the presence of concentrated H2SO4, protein using the Lowry method [121] and based on the calibrating schedule for a bovine serum albumin 80000 Da. Paper chromatography was carried out on "Filtrak FN-13" paper with a descending method in a nbutanol-pyridine-water system (volume correlations 6:4:3, respectively). To define carbohydrates, aniline phthalate was poured on the paper and heated at 105 oC. Gas–liquid chromatography was carried out using a Hewlett-Packard 4890A (USA) chromatograph equipped with a flame-ionization detector, RTX-1 (0.25 mm x 30 m) capillary column, argon carrier gas, and 1:60 dumping. Temperature rate: 175 oC (1 min)–250 oC (2 min), ∆ 3o/min.

A full acid hydrolysis PS (5 mg) was carried out for the implementation of 2M trifluoroacetic acid (2 ml) which contained *myo-*inositol (1 mg/ml). The mixture was heated in a soldered ampoule for 5 h at 100 oC, and the acid was removed with a repeated dry evaporation with added methanol. As a result, we obtained PVG-1.

Ion-exchange chromatography PS (100 mg) was carried out on a DEAE-cellulose (25x2 cm) column. NaCl solutions were used as an eluent with increasing concentrations (0.01M–1M, 60 ml/h elution speed, fractions selection by 12 ml). Pick correspondent fractions at the output bents were combined, dialysed and lyophilized. As a result, we obtained PS 1-4 fractions. The monosaccharide composition of each fraction was defined with GLC in acetate polyol after preliminary hydrolysis.

In order to obtain acetate polyol, each PS 1-4 fraction was dissolved in a 1M ammonia solution (1 ml) and 5 mg of NaBH4 was added. The mixture was kept for one day at a room temperature. Then, NaBH4 was eliminated by adding 2–3 drops of concentrated acetic acid; 0.2 ml of dry pyridine and acetic anhydride were added to the dry residue. The mixture was acetilized at 100 oC for 1 h. The solution was dry-evaporated until pyridine and acetic anhydride were removed, first by adding 1 ml of toluene and then 1 ml of methanol. The obtained acetate mixture of PS 1-4 polyol fractions was dissolved in 0.2 ml of dry chloroform and moved quantitatively to Appendorf tubes, concentrated up to 0.1–0.2 ml and analysed with the GLC method.

PS (5 mg) partial acidic hydrolysis was carried out using 0.01M TFA (2 ml), which contained *myo-*inositol (1 mg/ml). The mixture was heated in a soldered ampoule at 100 oC for 3 h. The acid was removed using a repeated dry evaporation with added methanol. As a result, we obtained PVG-2.

After extraction of dihydroquercetin, arabinogalactan and resin, the larch chip presented as a cellolignin residue. The chip had the following dimensions: 25x15x5mm, and sawdust fraction, 1x2x2 mm.

Bleached pulp from Baikal Pulp Mill was used for hydrolysis: polymerization degree 573, ash 1.1%, humidity 3%. Cellulose hydrolysis was carried out using 72% sulphuric acid and water in a ratio of 1:3 at room temperature for 1 h. Hydrolysis products—inverted polysaccharides—were precipitated with a five-fold ethanol volume. The precipitate was filtered and washed with alcohol the last washed portion achieved a neutral reaction. The product was dried in the air at up to 6% humidity. Acid content of inverted polysaccharides was defined using 1N HCl titration. Inversion of IPS was carried out in 0.75–1.50% solutions, hydromodulus 1:30, at 100–170 oC, for 0.25–3.0 h inversion duration. The potential content of reducing substances in hydrolyzates was defined by inversion of water-soluble polysaccharides with 5% sulphuric acid. 20% NaOH was used to neutralize the hydrochloric acid. Glucose quantitative content in neutralisate (pH 4–5) was defined by HPLC methodology.

184 The Complex World of Polysaccharides

alcohol was added (4 volumes). Deposition was separated with centrifugation. Alcoholic

Galacturonic acid content in PS was defined according to the reaction with 3,5-dimethyl phenol in the presence of concentrated H2SO4, protein using the Lowry method [121] and based on the calibrating schedule for a bovine serum albumin 80000 Da. Paper chromatography was carried out on "Filtrak FN-13" paper with a descending method in a nbutanol-pyridine-water system (volume correlations 6:4:3, respectively). To define carbohydrates, aniline phthalate was poured on the paper and heated at 105 oC. Gas–liquid chromatography was carried out using a Hewlett-Packard 4890A (USA) chromatograph equipped with a flame-ionization detector, RTX-1 (0.25 mm x 30 m) capillary column, argon carrier gas, and 1:60 dumping. Temperature rate: 175 oC (1 min)–250 oC (2 min), ∆ 3o/min.

A full acid hydrolysis PS (5 mg) was carried out for the implementation of 2M trifluoroacetic acid (2 ml) which contained *myo-*inositol (1 mg/ml). The mixture was heated in a soldered ampoule for 5 h at 100 oC, and the acid was removed with a repeated dry evaporation with

Ion-exchange chromatography PS (100 mg) was carried out on a DEAE-cellulose (25x2 cm) column. NaCl solutions were used as an eluent with increasing concentrations (0.01M–1M, 60 ml/h elution speed, fractions selection by 12 ml). Pick correspondent fractions at the output bents were combined, dialysed and lyophilized. As a result, we obtained PS 1-4 fractions. The monosaccharide composition of each fraction was defined with GLC in

In order to obtain acetate polyol, each PS 1-4 fraction was dissolved in a 1M ammonia solution (1 ml) and 5 mg of NaBH4 was added. The mixture was kept for one day at a room temperature. Then, NaBH4 was eliminated by adding 2–3 drops of concentrated acetic acid; 0.2 ml of dry pyridine and acetic anhydride were added to the dry residue. The mixture was acetilized at 100 oC for 1 h. The solution was dry-evaporated until pyridine and acetic anhydride were removed, first by adding 1 ml of toluene and then 1 ml of methanol. The obtained acetate mixture of PS 1-4 polyol fractions was dissolved in 0.2 ml of dry chloroform and moved quantitatively to Appendorf tubes, concentrated up to 0.1–0.2 ml and analysed

PS (5 mg) partial acidic hydrolysis was carried out using 0.01M TFA (2 ml), which contained *myo-*inositol (1 mg/ml). The mixture was heated in a soldered ampoule at 100 oC for 3 h. The acid was removed using a repeated dry evaporation with added methanol. As a result, we

After extraction of dihydroquercetin, arabinogalactan and resin, the larch chip presented as a cellolignin residue. The chip had the following dimensions: 25x15x5mm, and sawdust

Bleached pulp from Baikal Pulp Mill was used for hydrolysis: polymerization degree 573, ash 1.1%, humidity 3%. Cellulose hydrolysis was carried out using 72% sulphuric acid and water in a ratio of 1:3 at room temperature for 1 h. Hydrolysis products—inverted

supernatant was concentrated and analysed with the help of PC.

added methanol. As a result, we obtained PVG-1.

acetate polyol after preliminary hydrolysis.

with the GLC method.

obtained PVG-2.

fraction, 1x2x2 mm.

**Figure 17.** Extraction scheme of pectin substances from larch bark

Larch cellolignin timber residue with particles having dimensions of 25x15x5 mm was used for explosive autohydrolysis. Autoexplosive hydrolysis was carried out in a special 200 ml

capacity autoclave, which allowed us to conduct a quick decompression of the reactor (steam explosion). Hydrolysis conditions were: 200 and 220 oC, duration 2 and 5min.

The laboratory scheme for obtaining crystalline glucose is depicted in Figure 18.

**Figure 18.** Laboratory scheme for obtaining crystalline glucose

#### **6. Conclusion**

186 The Complex World of Polysaccharides

capacity autoclave, which allowed us to conduct a quick decompression of the reactor

(steam explosion). Hydrolysis conditions were: 200 and 220 oC, duration 2 and 5min.

The laboratory scheme for obtaining crystalline glucose is depicted in Figure 18.

**Figure 18.** Laboratory scheme for obtaining crystalline glucose

This chapter therefore summarizes studies on polysaccharides in the context of the development of technology for 100% processing of larch wood and bark as forestry waste in order to provide new medicines, veterinary drugs, dietary supplements and valuable materials for the cosmetics and agricultural industries. There are data on larch wood and bark extraction by the two-phase solvent system, namely the kinetic study of extraction processes, diffusion constants, mass-transfer coefficients, mechanisms and physicochemical characterization of the transfer process, its mathematical modelling and structural characteristics of the samples isolated. This work aims to support the development of economically and ecologically viable production technology for high-demand products on the basis of renewable raw materials with a 15–20% increase of forestry efficiency due to waste processing. The technology will provide new medicines and food supplements, as well as cheaper, by 40–50%, analogues, to those currently known.

## **Author details**

Natalya Nikolaevna Trofimova, Elena Nikolaevna Medvedeva, Nadezhda Viktorovna Ivanova, Yuriy Alekseevich Malkov and Vasiliy Anatolievich Babkin *Laboratory of Wood Chemistry, Federal Research Budget Institution, A.E. Favorsky Institute of Chemistry, Siberian Branch, Russian Academy of Sciences, Irkutsk, Russia* 

## **Acknowledgement**

This project has been supported by a State Contract with the Department of Industry, Science and Technology of the Russian Federation №43.044.1.1.26.38(2002–2004); by a State Contract with the Agricultural Department of the Government of Irkutsk Oblast №02-66 (2010–2011); and by RAS and SB RAS Grants (2005–2012). The authors are grateful to all their colleagues at the Laboratory of Wood Chemistry of A.E. Favorsky Irkutsk Institute of Chemistry who have contributed to this project.

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