Separation and Characterization of Tasmannia Lanceolata - Coursework Example

Separation and Characterization of Tasmannia Lanceolata (concrete) using high Performance Counter Current Chromatography (HPCCC) and Gas Chromatography-Mass Spectroscopy (GC-MS) Name: Tutor: Course: Date: Introduction A lot of research has been done on plants and their extracts. A research done by Southwell and Brophy (1992) on species of Tasmannia Lanceolata by comparing essential oils and extracts discovered that the plant contained polygodials which is an active secondary metabolite and is found in some plant and animal species. Besides having antimicrobial properties, Powell et al. (1995) state that polygodial has some antifeedal activity in insects and piscicidal properties. It also has a hot taste to humans, alongside having a good fragrance and flavour. It is such features of the plant content that create the drive to extract the plant. Extractions of the plant have been done before but selling of the product has been hindered by lack of registration of the product by an authorized body. Chromatography has been the main mode of separation of the components of the plant. The High Performance Countercurrent Chromatography (HPCCC) was developed as an improvement of the already existing High Speed Countercurrent Chromatography (HSCCC). The improvement was made on the capacity of the process. The HSCCC could only generate about 80 g while the HPCCC generates about 240 g of the product. In addition, it can be able to carry small loads of up to few millimeters. This paper will describe the plant Tasmannia Lanceolata and look at how to separate concrete from it by the HPCC process. It will also look at how to characterize concrete using Gas Chromatography and Mass spectroscopy (Smith, 2010). Tasmannia Lanceolata Tasmannia Lanceolata is commonly known as Mountain Pepper. It comes from the family Winteraceae and is a primitive type of shrub that is tall and evergreen. It has been found to grow to as high as 10 m. The shrub has a straight trunk with several branches that arise at acute angles. Its branches are distinctively red and are thick, hairless in addition to having dark brown and an outer bark that is smooth. It has rough ridges that run down from each leaf’s base. Its under bark is dark brown too and is usually very thin. The leaves of this plant alternate and are attached by small thick stalks without stipules at the base. The thick and narrow leaves have a lance leaf shape, smooth edges and their tips are pointed. They gradually taper at the base of the leaf. The smooth leaves are satiny on each of their sides with no hair and they appear green at the upper side and slightly paler at the lower side. They are usually covered with many fine dots which are oil glands. These oil glands are the ones that when crushed, they produce an aromatic smell and a hot taste on chewing. The flowers of the plant are small in size and yellowy in color. Each of the flowers usually arises from the bud scale axil and they form bunches of about ten flowers resembling a minute terminal umbel. The fruits produced are usually tiny, the size of a pea and are berry-like. They are initially dark red and shinny but turn black when ripe, carrying many seeds (Fitzgerald, 2004). The plant occurs in Victoria, Tasmania and New South Wales. It thrives in habitats that are cool and wet, essentially describing mountainous areas. It could also occur in cool temperate rainforest and open forests that are tall and wet. In some instances, it occurs in subalpine woodland, coniferous shrubberies and subalpine sclerophyll forest. In Tasmania, the plant has been found to occur at altitudes of about 300-1400 m, sea level to alpine places. Tasmannia Lanceolata is a hardy species that survives cold winters and tolerates low temperatures up to -15 ºC. However, it could be damaged in winters that are very cold. Despite the fact that it prefers sheltered sites, it can survive exposed, rocky and windy sites. Mountain pepper has been found to grow best in fertile soils that are lime-free, moist and are well drained. It can also stand clay soils. Although it grows in sunny and sometimes shady positions, its preference is more sheltered places. The plant has separate male and female plants and therefore it requires the presence of both sexes for production of fruits or seeds. Its flowering takes a fairly long duration, usually occurring September to January. This probably explains the broad of altitude that it occupies. As reported, the fruits ripen between March and April and its berries are usually eaten by native birds like currawongs. This plant has aromatic and pungent parts with the wood being very soft because of the conducting tissue’s structure, usually in the stem. This plant has been noted to resist honey fungus although it is vulnerable to Phytophthora cinnamomi. To grow this plant, seeds as well as cuttings can be used. The sex of the plants should first be determined during propagation of cuttings. Sowing of seeds should be done immediately they are ripe because they degrade rapidly. In fact, storing seeds at room temperature keeps them viable for just one year. Before storing or sowing seeds, the flesh must be removed. This plant can be used as a garden plant or for breaking winds. However, its most common use is as a food garnish alongside the medicinal properties associated with its essential oil. An unusually fragrant and spicy flavor is usually obtained from the leathery leaves which contain compounds that taste hot, called polygodials. These compounds are combined together with aromatic compounds from the essential oil. In Australia, the berries and leaves of this plant are used in adding a spicy food flavor to most foods. How HPCCC can be used to separate component of Tasmannia Lanceolata (concrete) using normal phase and reversed phase According to Berthod (2012), Counter Current Chromatography (CCC) refers to a technique in liquid chromatography where the liquid stationary phase used is support free. The liquid system used is biphasic, where one phase is selected as the mobile phase while the other phase is stationary. Maintaining stable volumes of the stationary phase is difficult, and for this reason, centrifugal fields are deployed. Columns of the CCC must have the ability to produce this field. The columns cannot be made of a simple tube having frits at both ends. This feature of centrifugal filed is the one that helps the CCC to retain the liquid phase that is stationary in the column in a way that it will dynamically interact with the mobile phase. The use of HPCCC depends upon the arranging conditions. In this respect, the reaction products, the by-products as well as the reagents that would not have reacted will either have very high or very low partition coefficients. This means that the solutes will be soluble in one of the organic or aqueous phase and insoluble in the other phase. Due to removal of filtration solvent and reaction solvent, the residue is divided in a separating funnel between an organic solvent and an aqueous phase. Low pH results in poor solubility of un-reacted agents like organic acids when in aqueous media. At high pH, the acids get ionized and become polar, thus dissolving in aqueous media. Ionized organic amines are soluble in aqueous media at low pH and less soluble at high pH. Therefore, in carrying out an experiment, the first division would be between an aqueous solution (acidic) and concrete. An amine reactant that is un-reacted and a reagent that is soluble at high pH in concrete will be extracted selectively in the aqueous layer that is removed off the funnel. In HPCCC, KD is the partition coefficient while D is the distribution ratio. They are used to give a relation of concentrations of a solute in the 2 phases that result from a biphasic system that is at equilibrium. A partition coefficient refers to the ratio of the quantity of analyte that is in the equilibrium solvents and it can be related to the affinity of the analyte for one solvent over the other. There values can be given by the following equations:   Or  (Dynamic extractions, 2004). In extracting Tasmannia Lanceolata, its leaves are first dried for about 3 days at temperatures of about 35 ºC. They are then ground by a mortar and pestle. Petroleum ether can then be used as the aqueous solution during extraction. Extraction of concrete solution is done by a high pH solution that extracts acidic solutes from the mountain pepper selectively, leaving a pure product in the layer of concrete (neutral product). The use of HPCCC can be used to generate about 240 g of a product, an improvement from the 80 g that is produced by High Speed Countercurrent chromatography (HSCCC). The sample loadings can be made to low values like few milligrams. According to Kusch (2008), the difference in the two phases (reverse and normal phase) lies in the choice of the phase that is mobile and based on the density of the phase. During the reverse phase, the aqueous phase is usually mobile. In this phase, the phase that is denser gets pumped as the movable phase. On the other hand, the normal phase involves use of the phase with lower density as the movable phase. How Tasmannia Lanceolata (concrete) can be characterized by GC-MS The GC/ MS instrument is used in separation of chemical mixtures and in identification of components at a molecular level. The two are done by the GC component and the MS component respectively. It operates on the principle that a mixture separates to individual components on heating. Heated gases are taken in a column that has an inert gas. The substances that are separated come out of the column and stream into the MS. MS uses mass of the analyte molecule to identify compounds. Most solids are not volatile at GC temperatures and they therefore have to be treated in a different way. A solution of extracted concrete is first taken through the pyrolisis process, where large molecules of concrete are fragmented to produce small compounds that are volatile and can be analyzed by GC. The fragmentation process is reproducible and it produces a chromatogram which is characteristic of the macromolecule that was initially pyrolyzed. For optimal gas chromatography, the concrete pressure must not be below 10-10 torr (1 torr = 133.322 Pa). To analyze compounds at low temperatures, they have to be derivatized chemically (Menary, 2003). The operation of gas chromatography and mass spectrometry is mutual in that they both add to the operation. GC separates volatile as well as semivolatile compounds in great resolution. However, it cannot identify them. On the other hand, MS provides detailed information on structures of the compound. However, it cannot separate them. This is the reason as to why the two have to be used concurrently. The samples used are in vapour phase. However, an icompatibility problem arises because the compound that exits the GC at about 760 torr while MS operates at about 10-6 to 10-5 torr (Hites 2000). The way to make them compatible would entail reducing the pressures of the substance leaving the GC to be almost the same as that of the MS as it goes to the MS. However, this passed most analyte molecules into the MS. One important GC separator that can be used in separating concrete is the jet separator. It uses the difference in diffusibility that exists between the organic compound (in this case, concrete) and the carrier gas. The carrier gas used could be helium or hydrogen since they have small molecules and their diffusion coefficient is high. The molecules of the organic compound have low diffusion coefficients. During operation, the effluent of the GC which contains the carrier gas together with organic analytes gets sprayed via a small nozzle. They are sprayed into an evacuated chamber with about 10-2 torr of pressure. Since helium has a high diffusion coefficient, it is sprayed at a wide solid angle while the organic molecule (concrete) is sprayed over a narrower angle and it goes straight to the region with a partial vacuum. A skimmer is used to collect the middle section of the solid angle and passes it to the MS. This separates the concrete which is higher in molecular weight from the carrier gas and is removed using the vacuum pump. The alignment of the skimmer and the spray orifice should therefore be perfect. For the separation to occur well, the flow rates of the carrier gas should be fast. This method has its drawbacks. GC columns that are packed supply small particles to jet separator. Should any of these particles escape from the column, it gets lodged to the spray orifice and would stop flowing of gas from the GC column to the MS. This can be eliminated by placing a filter the jet separator and the GC column. However, a particle will eventually plug the orifice up. This calls for maintenance of the device. To overcome this, all the carrier gas is flown to the MS ion source (Essential oils of Tasmannia Pty Ltd, 2010). Conclusion Extraction of Tasmannia Lanceolata products has faced a lot of questioning since it is considered to have safrole, a carcinogenic compound. This has led to a lack of recognition by regulatory bodies and it is the reason behind the slow acceptance of the products in the market. However, the compounds are an attractive prospect to humans because of the flavor they contain and the fragrance associated with the plant. Some people will just chew the plant to enjoy the hot taste in the mouth. Examination of the product to confirm the levels of the carcinogenic compound and to find ways of minimizing or if possible total elimination should be done to give people the freedom to enjoy the products of this plant. References Berthod, 2012. Terminology for Countercurrent Chromatography. Université de Lyon, France. Dynamic extractions, 2004. Chromatography Concepts and Principles. UK. Essential oils of Tasmannia Pty Ltd, 2010. Tasmannian Native Oil Pepper. Australia. Fitzgerald Nick, 2004. Tasmannia Lanceolata. Tasmania. Hites A. Ronald, 2000. Gas Chromatography-Mass Spectrometry. Indiana University, USA. Kusch Peter, 2008. Pyrolysis-Gas Chromatography/Mass Spectrometry of Polymeric Materials. Germany. Menary et al, 2003. Tasmannia Lanceolata: Developing a New Commercial Flavour Product; A report for the Rural Industries Research and Development Corporation. University of Tasmania. Powell et al, 1995. Responses of Myzus persicae to the repellant polygodiol in choice and no- choice video assays with young and mature leaf tissue. 74: 91±94. Smith A.C., 2010. Tasmanian Pepper (Tasmannia lanceolata) [Poiret]. Southwell I. A. and Brophy J. J. 1992. Differentiation within the Australian Tasmannia by essential oil comparison. Phytochemistry 31: 3073±3081. Read More

Tasmannia Lanceolata is a hardy species that survives cold winters and tolerates low temperatures up to -15 ºC. However, it could be damaged in winters that are very cold. Despite the fact that it prefers sheltered sites, it can survive exposed, rocky and windy sites. Mountain pepper has been found to grow best in fertile soils that are lime-free, moist and are well drained. It can also stand clay soils. Although it grows in sunny and sometimes shady positions, its preference is more sheltered places.

The plant has separate male and female plants and therefore it requires the presence of both sexes for production of fruits or seeds. Its flowering takes a fairly long duration, usually occurring September to January. This probably explains the broad of altitude that it occupies. As reported, the fruits ripen between March and April and its berries are usually eaten by native birds like currawongs. This plant has aromatic and pungent parts with the wood being very soft because of the conducting tissue’s structure, usually in the stem.

This plant has been noted to resist honey fungus although it is vulnerable to Phytophthora cinnamomi. To grow this plant, seeds as well as cuttings can be used. The sex of the plants should first be determined during propagation of cuttings. Sowing of seeds should be done immediately they are ripe because they degrade rapidly. In fact, storing seeds at room temperature keeps them viable for just one year. Before storing or sowing seeds, the flesh must be removed. This plant can be used as a garden plant or for breaking winds.

However, its most common use is as a food garnish alongside the medicinal properties associated with its essential oil. An unusually fragrant and spicy flavor is usually obtained from the leathery leaves which contain compounds that taste hot, called polygodials. These compounds are combined together with aromatic compounds from the essential oil. In Australia, the berries and leaves of this plant are used in adding a spicy food flavor to most foods. How HPCCC can be used to separate component of Tasmannia Lanceolata (concrete) using normal phase and reversed phase According to Berthod (2012), Counter Current Chromatography (CCC) refers to a technique in liquid chromatography where the liquid stationary phase used is support free.

The liquid system used is biphasic, where one phase is selected as the mobile phase while the other phase is stationary. Maintaining stable volumes of the stationary phase is difficult, and for this reason, centrifugal fields are deployed. Columns of the CCC must have the ability to produce this field. The columns cannot be made of a simple tube having frits at both ends. This feature of centrifugal filed is the one that helps the CCC to retain the liquid phase that is stationary in the column in a way that it will dynamically interact with the mobile phase.

The use of HPCCC depends upon the arranging conditions. In this respect, the reaction products, the by-products as well as the reagents that would not have reacted will either have very high or very low partition coefficients. This means that the solutes will be soluble in one of the organic or aqueous phase and insoluble in the other phase. Due to removal of filtration solvent and reaction solvent, the residue is divided in a separating funnel between an organic solvent and an aqueous phase.

Low pH results in poor solubility of un-reacted agents like organic acids when in aqueous media. At high pH, the acids get ionized and become polar, thus dissolving in aqueous media. Ionized organic amines are soluble in aqueous media at low pH and less soluble at high pH. Therefore, in carrying out an experiment, the first division would be between an aqueous solution (acidic) and concrete. An amine reactant that is un-reacted and a reagent that is soluble at high pH in concrete will be extracted selectively in the aqueous layer that is removed off the funnel.

In HPCCC, KD is the partition coefficient while D is the distribution ratio.

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