TLC task

Identification of Heteropolysaccharide by TLC

TLC technique and identification
TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. It also permits the optimization of the solvent system for a given

separation problem. In comparison with column chromatography, it only requires small quantities of the compound and is much faster as well. A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action. As the solvent moves past the spot that was applied, equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates. (The plate itself contains a fluor which fluoresces everywhere except where an organic compound is on the plate.)

Spotting a TLC plate with sample:

Running the TLC plate in solvent:

Table 1. Identification of monosaccharide composition of the sample by means of TLC.

Xylose Standard 1 Standard 2 Sample 2 + + + Arabinose + + + + Glucose Mannose + Galactose + Rhamnose + + + GluUA Gal-UA +

Table 2. Identification of monosaccharide composition of the sample by means of TLC.

Xylose Arabinoxylan + Xyloglucan + Xylan + Galactomannan Arabinogalactan Pectin + Xanthan Arabinose + + + + Glucose + + Mannose + + Galactose + + + Rhamnose + GluUA Gal-UA + + + -

As we can see comparing the tables above, the sample 2 coincides with the xyloglucan.

Xyloglucan in Nature
Xyloglucans are members of a group of polysaccharides typically refered to as hemicelluloses. Hemicelluloses are plant cell wall polsaccharides that are not solubilized by water but are solubilized by aqueous alkali (e.g. 1 and 4M KOH). Other hemicellulosic polysaccharides include xylan, glucuronoxylan, arabinoxylan, mannan, glucomannan and galactoglucomannan. Xyloglucan is the quantitatively predominant hemicellulosic polysaccharide in the primary walls of dicots and non-graminaceous monocots. Xyloglucan may account for up to 20% of the dry weight of the primary wall. The cellulose-xylglucan network is believed to be the major loadbearing structure in the primary wall. Xyloglucan coats the surface of the cellulose microfibrils, limiting their aggregation and connecting them via tethers that directly or indirectly regulate the mechanical properties of the wall. The cell wall consists of two independent polysaccharide networks: the cellulosexyloglucan network and the pectic network. The cellulose-xyloglucan network contains a framework of cellulose micro fibrils. These micro fibrils are composed of linear chains of -(1 -4)-linked glucose residues. They are 515nm wide and are spaced 20-40 nm apart. Although the cellulose chains themselves have a length of only -1000 residues, the micro fibrils are much longer since each cellulose molecule starts and ends at a different point in the micro fibrils. The micro fibrils are predominantly interlocked through xyloglucans (length is 30-400 nm), creating a space between microfibrils. The basic repeating unit of the majority of xyloglucans consists of a backbone of four -(1 -4)-linked glucose residues, which is the same as cellulose, and three out of four glucose residues are substituted with a-D-xylose at the O6 position. This is the common structure for all the xyloglucans, but additional residues are attached to xylose, depending on the source of xyloglucan. This variation of the structure dominates the detail of the functionality and physicochemical properties. For instance, the galactose substituted to the xylose dominates the water solubility in the case of xyloglucan extracted from tamarind seed.

One Letter Codes for Xyloglucan Substructure

York et al. performed an exhaustive study of the branching pattern of xyloglucan in relation to the plant species. They found that a branching pattern strongly depends on species.21 For example, the repeating unit of Lactuca sativa, Tanacetum ptarmiciflorum, and Daucus carota is XXXG-type (see Figure 1 for the abbreviation such as X and G) branching pattern, which is a typical xyloglucan repeating unit, but these contain a-D-Xylp, b-D-Galp-(1!2)-a-D-Xylp, and a-L-Fucp-(1!2)-b-DGalp-(1!2)-a-D-Xylp side chains. However, there are atypical species, such as Capsicum annuum (pepper) and Lycopersicon esculentum (tomato), which preclude atypical branching patterns. These have XXGG-type, less-branched type, repeating unit. On the other hand, species such as Ipomoea purpurea (morning glory, order Solanales), Ocimum basilicum (basil, order Lamiales), and Plantago major (plantain, order Lamiales) have an unusual XXGGG-type branching pattern. They have five glucoses in the backbone, although the number of glucose in backbone of the repeating unit is four for most xyloglucans. According to their study, the appearance of this intermediate xyloglucan structure in oleander has implications regarding the

evolutionary development of xyloglucan structure and its role in primary plant cell walls. In the case of xyloglucan as the storage polysaccharide, xyloglucan exists as a polymer in the seeds. The reported molar mass of TSX is 650 000 (gel permeation chromatography; GPC) or 880 000 g/mol (light scattering). A single letter nomenclature is used to simplify the naming of xyloglucan side chain structures. For example, a capital G represents a unbranched Glcp residue. A capital F represents a Glcp residue that is substituted with a fucose-containing trisaccharide.

The chemical structure of the repeating unit of xyloglucan The nomenclature is shown in the figure below:

Examples of biomass sources:

Apple

Tamarind

Bilberry

Olive fruit

Apple xyloglucan oligosaccharides This group of oligosaccharides is produced by extraction from apple and the individual products are purified by HPLC. In some seeds of trees (such as Tamarindus, Impatiens, Annona, Tropaeolum, Hymenaea, and Detarium), xyloglucan polymer also exists as a storage polysaccharide. Seeds of the tamarind tree (Tamarindus indica) contain a xyloglucan. Tamarind seed xyloglucan is used as a food additive in Japan. Comercial use It was proposed to investigate the potential application of thermoreversible gels formed by a xyloglucan polysaccharide derived from tamarind seed for rectal drug delivery.

Potential activities: Xyloglucan is a source of biologically active oligosaccharides. They have been shown to control plant development and metabolism. Additionally, some xyloglucan oligosaccharides were indicated to possess immunomodulator activity. Biosynthesis Xyloglucan is synthesized in Golgi trans cisternae and in the trans Golgi network (TGN) and is transported to the cell membrane by vesicles, where it is expelled and adsorbs on nascent cellulosic microfibrils. Properties of Xyloglucan Xyloglucan is a major structural polysaccharide in the primary cell wall of higher plants. Cell growth and enlargement are controlled by the looseness of a thin net of microfibrils made of cellulose. Xyloglucan cross-links these cellulose microfibrils and provides the flexibility necessary for the microfibrils to slide. Xyloglucan is a neutral polysaccharide with low viscosity and low molecular weight compared to many other polysaccharides, forms a gel at high sugar concentrations and low water activity conditions. Xydroglucan is soluble in water as it suppresses the cellulose-like chain aggregation due to steric hindrance of its side chain. It forms a uniform solution on heating with water while stirring. The maximum viscosity is usually obtained after boiling the dispersion for 2030 min. A thick viscous dispersion is formed that yields strong, transparent, elastic films on evaporation. Xyloglucan can form a gel in cold water only in the presence of alcohol or high levels of sugar.

TSX is widely used in food industries, for instance, as thickener, stabilizer, and/or gelling agent. Aqueous solutions of TSX usually show almost a Newtonian behavior although this depends on the concentration and the molar mass. By adding sugar or alcohol, the viscosity of TSX solution is significantly affected, and in the presence of a large amount of these substances TSX can form a gel. Although aqueous solutions of native TSX alone cannot form a gel, synergistic gel can be formed by mixing with other polysaccharides, or with a dye such as Congo red,or polyphenol, such as epigallocatechin gallate (EGCG). A high water holding capacity with good stability to heat, acids and shear are some unique properties of this particular polysaccharide Aqueous solutions of XG exhibit Newtonian flow behavior at low concentrations (<0.5% w/w). The food industry widely uses XG to improve the rheological and thermal properties of many products. Application of xyloglucan Xyloglucan, a hydrocolloid, has a wide range of applications. There are numerous applications of xyloglucan from thickening, gelling and stabilizing agents in food, to sizing and weaving in textiles, to adhesive and binding agents in industry. Because of their tight binding to cellulose, xyloglucans are used commonly as sizing agents for textiles. A recent application is enzymatic stone washing. The cotton yarn is precoated with xyloglucan before dying, and removal of the xyloglucan by endo-glucanase treatment gives the fabric a used look by partial dye removal. Xyloglucan is used in the print industry in a textile printing method as thickener for printing polyester fabrics with disperses dyes. It is used in the plywood and paper industries as an additive for strengthening these materials and can also be used as a wet-end additive in papermaking. XG can be used as an excellent substitute for fruit pectin and, hence, can be used as an effective remedy against diarrhoea, dysentery and colitis. It is also used in eyebaths and for the treatment of ulcers. XG is used in preparing antiobesity agents, printing compositions, hair dying compositions, preparing starch based compositions, as a viscosifier in ophthalmic solutions and cosmetic and pharmaceutical applications. It is also used in the fabric detergent industry as a conditioning product. It is used as a bulking agent in the food industry. It is also used as a shelf life extender in various food products. XG is also used as a coating composition for frozen desserts. It is used in dental cement composition. It has been used in briquetting of coal powder and pelletizing of minerals. It has also been used for stabilizing soil. In paints, XG can act as a rheology control additive and it can be used in water-based distempers, rubber and synthetic emulsions. Because of its good thermal stability, XG is being evaluated as a thickener in drilling fluids for petroleum oil. XG can be used in cosmetics. Most widely studied xyloglucan is tamarind seed xyloglucan (TSX), because only TSXexisting in the seed of tamarind tree (Tamarindus indica) as a storage polysaccharide has been commercially available.

The various applications of tamarind seed xyloglucan include thickening sauce, ice cream, dressing and processed vegetables. Tamarind seed xyloglucan is expected to find new food applications, serving as a thickener and stabilizer, gelling agent, ice crystal stabilizer and starch modifier, etc. It is called ageing free starch because its property is similar to starch but is more stable. The flow behavior of the solution is very close to Newtonian, and very stable against heat, pH and shear.