Separation of Tasmania Lanceolata - Essay Example

SEPARATION OF TASMANIA LANCEOLATA USING HIGH PERFOMANCE COUNTER CURRENT CHROMATOGRAPHY AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY Your name Subject Date DISCUSSION Tasmannia Lanceolata is commonly known as Mountain Pepper which comes from the family Winteraceae and is a primitive type of shrub that is tall and evergreen. Tasmannia Lanceolata is considered to have safrole, a carcinogenic compound and is used as a garden plant or for breaking winds. However, it’s most common use is as a food garnish alongside the medicinal properties associated with its essential oil. An unusually fragrant and spicy flavour is usually obtained from the leathery leaves which contain compounds that taste hot, called polygodials. “According to Powell et al (1995”) polygodial has some anti-feed 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 so as to minimize or if possible to totally eliminate the levels of the carcinogenic compound. Concrete Tasmannia Lanceolata can be characterized using Gas Chromatography and Mass spectrometry. In the experiment to extract the compound using the HPCCC, the result showed that components of Tasmannia Lanceolata (concrete) have some retention in the stationary phase, especially for the reversed phase, due to the solubility of sample in non-polar phase. 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.The experiment with HPCCC showed that in normal phase mode using UV visible as the detector, stationary phase is polar and the mobile phase is non-polar. Normal phase was not very useful to separate the whole components of Tasmannia Lanceolata (concrete) because most compounds were eluted very early, indicating low retention in stationary phase. The aim of this experiment was mainly to examine the Extraction of Tasmannia Lanceolata products to confirm the levels of the carcinogenic compound and to find ways of minimizing or if possible total elimination of the carcinogenic compound. This was in order for people to be given the freedom to use the products of Tasmannia Lanceolata plant. Extraction of Tasmannia Lanceolata products has not been allowed despite the plant having an attractive prospect to humans in flavour and the fragrance associated with the plant. This paper has demonstrated that High performance counter current chromatography is high quality chromatographic purifications, which can work at low pressure, low solvent usage and large sample injection loading. High-performance countercurrent chromatography was used also in this project as the sample cannot be lost inside the system as according to , the stationary phase and mobile phase are liquid, so the sample can be totally recovered, high throughput can be achieved due to the use of liquid for both stationary phase and mobile phase, helping sample solubility issues. Consequently, the system is described as high throughput technology, HPCCC is used 10 percent of solvent volume to process equivalent masses of crude samples. Therefore, HPCCC consumes a low solvent and in addition, the duplication of separation
conditions is quite easy due to the reproducibility of column packing at any operation scale. Consequently, HPCCC is described as simple to scale up. Though the use of HPCCC method can be used to generate about 240 g of a product, which is an improvement from the 80 g that is produced by High Speed Counter Current chromatography (HSCCC), and that the sample loadings can be made to low values like few milligrams, 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 (Kusch 2008) During the reverse phase, the aqueous phase is usually mobile and the phase gets pumped as the movable phase while the normal phase involves use of the phase with lower density as the movable phase. Results interpretations and opinions HSCCC Aqueous phase sample Figure 1 which illustrated the results of the aqueous phase of Tasmannia Lanceolata or concrete using HPCCC normal phase presented various results according to the time taken in the experiment. When the aqueous phase of Tasmannia Lanceolata (concrete) was injected, the results gave an appearance of three peaks which indicated a number of unretained compounds followed by two small peaks on the edge of unretained peak. At 2 min, the aqueous phase of Tasmannia Lanceolata was at 1.0.The level then dropped to 0.1 at 4 min and then rose gradually to 0.4 at 9min. from 10 to 30 min, there was constant level of low concentration which was ranging from 0.05 to 0.1. The above results demonstrate that the large peak in the beginning of the graph has high concentration in the mobile phase rather than in stationary phase which leads to a high distribution coefficient. This also meant that a compound of the sample, containing low distribution coefficient, contains higher concentration in the mobile phase than in the stationary phase. This gives the reason why the compound in the sample elutes earlier as demonstrated. Consequently, the component with a high distribution coefficient, obtains a high concentration in the stationary phase and not in the mobile phase making the compounds to be eluted late. The results for figure 1 shows that some fractions of Tasmannia lanceolata sample that were used , gave out identification of various components by their mass spectra . The reason for the compounds to have some retention as indicated in Figure 1 is that in normal phase the stationary phase is water and the mobile phase is heptane when injecting the aqueous phase which contains some compounds partition partially in it. Organic phase The results for organic phase ample gave two peaks which were distinct in their sizes. Figure 2 demonstrated a large unretrained peak at the beginning and a small peak on the edge of the unretained peak, as shown in Figure 2. At 1 min, the peak was at 1.0 where by it dropped drastically to 0.0 at 3.8 up to the end of the experiment duration. These results showed that in the organic phase of Tasmannia Lanceolata (concrete), most compounds are present in the concrete because of it partitions which are more than the aqueous phase. In addition, most compounds of the sample prefer a less polarity index such as Heptane, Hexane and Petroleum Ether to partition in it. These solvents are known to have a 0.1 polarity index. Compounds in figure 2 have been demonstrated to have lesser retention and also elutes very fast as unretained band as demonstrated in the graph. The reason for this is that the organic phase has some compounds partition partially and also compounds partition completely in it. Figure 3gives results for the reversed phase whereby the stationary phase is non-polar while the mobile phase is Polar. This demonstrated the organic phase of Tasmannia Lanceolata (concrete) using reversed phase in HPCCC. The reversed phase helped in separating most components of Tasmannia Lanceolata (concrete) as most compounds were in the organic phase indicating high retention in stationary phase as it is an organic solvent. In figure 3, ten peaks were detected in less than 8 min; the first peak was broad and concentrated, whereas, the other peaks were sharp and small. On the other hand, fluorescence had the same peaks which were same size as in UV visible, but it also had an extra peak at 26 min which was not present in UV visible. This peak has high retention time among other compounds meaning that the last peak from the sample in fluorescence detector is very strong partition in non-polar stationary phase. By using High performance counter current Chromatography, fraction only obtained in a reasonable resolution and then these fractions were had further identification using gas chromatography. UV visible and fluorescence was used in reversed phase assisted in comparing the samples in both detectors whereby these detectors have almost the same chromatogram. Sometime one of the detectors cannot detect the compound but another detector can, so in this project the use of both detectors enable detection of all compounds that have different retention time. Comparison normal phase and reversed phase of Tasmannia Lanceolata using HPCCC In the comparison between normal phase and reversed phase of Tasmannia Lanceolata using HPCCC, we find that these modes have different stationary phases and also have different mobile phases. The normal phase and reversed phase were used as mode for High Performance Counter Current Chromatography to separate Tasmannia Lanceolata (concrete). Figure 3 showed that most compounds can be separated in this mode as the sample prefers the non-polar stationary phase as in reversed phase. However, it is important to note that normal phase is better for compound that partition partially in the aqueous layer as demonstrated in Figure 1. Consequently, normal phase is not good for organic layer because there is no strong retention between organic layer and the polarity of stationary phase as shown in Figure 2. Gas Chromatography Results at Normal phase In the process of examining the Gas Chromatography in the Normal phase, two samples of Tasmannia Lanceolata (concrete), the aqueous phase and the organic phase of Concrete, were analyzed by GC-MS. GC-MS tool is reliable in separating chemical mixtures and in identification of components at a molecular level. The two are analysed by the GC component and the MS component respectively. GC-MS 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. Both had different compounds present in each phase or sometime present with less concentration than in another phase. In the results, these samples were the aqueous phase and the organic phase of Concrete, which have different compounds present in each phase or sometime present with less concentration than in another phase. Table 4 demonstrated the outcome of the compounds Retention time (min) and their Peak Height respectively. Linalool
7.437 29227, Piperitone at 11.486 min had 11899 peak heights, Eugenol at14.253had 46484 peak height Polygodial artifact at26.610 min had 124259; Palmitic acid at 28.268 had 19386, while Polygodial at 29.231had 394200 peak heights. Figure 4 illustrated these results clearly. As indicated earlier, it is important to note that most solids are not volatile at GC temperatures and therefore they have to be treated in a different way. A solution of extracted concrete is first taken through the pyrolysis process, where large molecules of concrete are fragmented to produce small compounds that are volatile and can be analysed by GC. The fragmentation process is reproducible and it produces a chromatogram which is characteristic of the macromolecule that was initially pyrolysed. According to (Menary, 2003), for optimal gas chromatography, the concrete pressure must not be below 10-10 torr (1 torr = 133.322 Pa) and consequently, to analyse compounds at low temperatures, they have to be derivatized chemically .Table 5illustrates the results the Gas Chromatography for the compounds as, Linalool
 at7.437 , Piperitone 11.486, Polygodial artefact 26.610, Polygodial 29.231 and unkowen compound at 29.668. The results showed that the compounds were at between 1000 to 15000 peak heights. According to Hites (2000), GC separates volatile as well as semivolatile compounds in great resolution though 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 together. The samples used are in vapour phase and an incompatibility problem arises because the compound that exits at the GC is about 760 torr while MS operates at about 10-6 to 10-5 torr (Hites 2000). Table 6 demonstrated the results for the Linalool
 at 7.437 and Piperitone at 11.486 which showed the compounds to be at 1000 to 16000 peak heights. Normal phase for organic phase sample results Fractions from normal phase for organic phase sample experiment, gave different levels of various compounds. Table 7 shows the results to be the highest Polygodial at 29.282min having 888996 a peak height. The lowest compound was Elemol at 19.183 min having a 17165peak height. Figure 7 demonstrated these heights. Table 8 Table 8 showed results for C12 Hydrocarbon at 31.485 or 31.548, to be at 23982 or 27277 peak height and , C14 Hydrocarbon 32.892, 28934 , C23 Hydrocarbon 34.678 110749, C26 Hydrocarbon 35.485 37894 respectively. Figure 8 gave an Impression that most compounds of Tasmannia Lanceolata (concrete) were present in fraction 1. The result for this being that there was high concentration of fraction 1 in HPCCC. Fraction 2 has a few of the same compounds as fraction 1, with lesser concentration. It also has high concentration of new compounds not present in fraction 1. The new compounds which are present in fraction two but not in fraction one are long chains of hydrocarbon compounds. Consequentially, compounds of fraction two are retained more in the stationary phase of normal phase than compounds in fraction 1. Aqueous phase vs organic phase of Tasmannia Lanceolata using GC-MS for normal phase While comparing the aqueous phase and organic phase of Tasmannia Lanceolata using GC-MS for normal phase, in the Reversed phase, a sample with some fractions of Tasmannia Lanceolata (concrete) were analysed by GC-MS. Figure 9 gave the results that the Blank was obtained for fractions to exclude any peak present in both the blank and the fraction. The blank was a pure heptane, which was injected in GC-MS using the same conditions as the fractions. Fractions from reversed phase for organic phase sample results The sample used was the organic phase of the Concrete, which have different compounds that present in each fraction or sometimes present with less concentration than in another fraction. According to figure 9, it was noticed that most compounds of Tasmannia Lanceolata (concrete) were present in fraction 1. This was due to the high concentration of fraction 1 in HPCCC. Moreover, fraction 2 has a few of the same compounds as fraction 1, but with less concentration. In table and figure 10, Calamenene, Cadina-1,4-diene, Methyl hinokiate, Polygodial artifact , Palmitic acid Compounds showed 18.552 3881 showed 18.784 1829, 24.752 3376, 26.597 1811, 28.2624751 Retention time (min) and Peak height respectively. Normal phase vs reversed phase of Tasmannia Lanceolata using GC-MS results The interpretation of the mass spectral results for Tasmannia Lanceolata (concrete) showed Compound structures Linalool
,Alpha-cubebene, Beta-cubebene, Eugenol, Beta-caryophyllene, Calamenene , Cadina-1,4-diene, Spathulenol , Methyl hinokiate and Polygodial having Retention time (min) of 7.413, 14.067, 14.775 or 17.791 , 14.244, 15.929, 18.556, 18.778 19.905, 24.751, 29.194 respectively. Production of GC artefact for polygodial The trend in the production of GC artefact for polygodial was demonstrated in in Figure 7 and 9 as a ratio with the main polygodial percentage. The percentage of polygodial breakdown product, eluting approximately 3.2 minutes before the polygodial peak during the chromatography run is shown. Implications of the findings The above finding has implied that the High Performance Counter Current Chromatography (HPCCC) was developed as an improvement of the already existing High Speed Counter Current Chromatography (HSCCC). The improvement was made on the capacity of the process and it was noted that the HSCCC could only generate around 80 g while the HPCCC generates about 240 g of the product. The results also implied that HSCCC can be able to carry small loads of up to few millimetres (Ito 2005). Consequently, it has a High-performance countercurrent chromatography with a revolution speed which can be regulated by speed controller ranging from 0 to 2100 rpm. Chromatography has been the main mode of separation of the components of the plant. Future research Though it is evident that a lot of research has been done on plants and their extracts, there is need for more future research on Tasmannia Lanceolata species. Though research done by Southwell and Brophy (1992) discovered that the plant contained polygodials which is an active secondary metabolite and is found in some plant and animal species, Powell et al. (1995) stated that polygodial has some antifeedant activity in insects and piscicidal properties, Several conclusions have been made by researchers on Tasmannia Lanceolata species. The paper has analysed Tasmannia Lanceolata plant and looked at discussed how to separate concrete from it by the HPCCC process. In addition, the paper has looked at how to characterize concrete using Gas Chromatography and Mass spectrometry (Smith, 2010). It is evident that the Tasmannia Lanceolata solvent extracts chemical composition includes a number of unknown compounds according to the given results. These compounds appear in very small concentrations, which make it difficult to identify every component. The majority of several components was identified and characterized using a range of instruments such as HPCCC and GC-MS. Conclusion In conclusion, Tasmannia Lanceolata contains several compounds, some which are of attractive prospect to humans because of the flavour they contain and the fragrance associated with the plant. However, despite their appealing nature, Tasmannia Lanceolata contains safrole, a carcinogenic compound which is used as a pesticide ad considered to be genotoxic and carcinogenic thus posing some health problems to humans . It 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. This is one of the reasons behind the Extraction process. Despite the fact that these compounds appear in very small concentrations, which make it difficult to identify every component, in this research the majority of obvious components were identified and characterized using a range of instruments such as HPCCC and GC-MS. References 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. Ito, Y. (2005). "Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography." Journal of Chromatography A 1065(2): 145-168. Ltd, D. e. (2004) Chromatography Concepts and Principle. Ltd, D. e. (2007) an introduction to high performance CCC for sample purification. RC Menary, D. V. D. a. S. G. (1999). "Tasmannia Lanceolata Developing a New Commercial Flavour Product." Smith A.C., 2010. Tasmanian Pepper (Tasmannia lanceolata) [Poiret].  Read More

High-performance countercurrent chromatography was used also in this project as the sample cannot be lost inside the system as according to , the stationary phase and mobile phase are liquid, so the sample can be totally recovered, high throughput can be achieved due to the use of liquid for both stationary phase and mobile phase, helping sample solubility issues. Consequently, the system is described as high throughput technology, HPCCC is used 10 percent of solvent volume to process equivalent masses of crude samples.

Therefore, HPCCC consumes a low solvent and in addition, the duplication of separation
conditions is quite easy due to the reproducibility of column packing at any operation scale. Consequently, HPCCC is described as simple to scale up. Though the use of HPCCC method can be used to generate about 240 g of a product, which is an improvement from the 80 g that is produced by High Speed Counter Current chromatography (HSCCC), and that the sample loadings can be made to low values like few milligrams, 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 (Kusch 2008) During the reverse phase, the aqueous phase is usually mobile and the phase gets pumped as the movable phase while the normal phase involves use of the phase with lower density as the movable phase.

Results interpretations and opinions HSCCC Aqueous phase sample Figure 1 which illustrated the results of the aqueous phase of Tasmannia Lanceolata or concrete using HPCCC normal phase presented various results according to the time taken in the experiment. When the aqueous phase of Tasmannia Lanceolata (concrete) was injected, the results gave an appearance of three peaks which indicated a number of unretained compounds followed by two small peaks on the edge of unretained peak. At 2 min, the aqueous phase of Tasmannia Lanceolata was at 1.0.The level then dropped to 0.

1 at 4 min and then rose gradually to 0.4 at 9min. from 10 to 30 min, there was constant level of low concentration which was ranging from 0.05 to 0.1. The above results demonstrate that the large peak in the beginning of the graph has high concentration in the mobile phase rather than in stationary phase which leads to a high distribution coefficient. This also meant that a compound of the sample, containing low distribution coefficient, contains higher concentration in the mobile phase than in the stationary phase.

This gives the reason why the compound in the sample elutes earlier as demonstrated. Consequently, the component with a high distribution coefficient, obtains a high concentration in the stationary phase and not in the mobile phase making the compounds to be eluted late. The results for figure 1 shows that some fractions of Tasmannia lanceolata sample that were used , gave out identification of various components by their mass spectra . The reason for the compounds to have some retention as indicated in Figure 1 is that in normal phase the stationary phase is water and the mobile phase is heptane when injecting the aqueous phase which contains some compounds partition partially in it.

Organic phase The results for organic phase ample gave two peaks which were distinct in their sizes. Figure 2 demonstrated a large unretrained peak at the beginning and a small peak on the edge of the unretained peak, as shown in Figure 2. At 1 min, the peak was at 1.0 where by it dropped drastically to 0.0 at 3.8 up to the end of the experiment duration. These results showed that in the organic phase of Tasmannia Lanceolata (concrete), most compounds are present in the concrete because of it partitions which are more than the aqueous phase.

In addition, most compounds of the sample prefer a less polarity index such as Heptane, Hexane and Petroleum Ether to partition in it. These solvents are known to have a 0.1 polarity index. Compounds in figure 2 have been demonstrated to have lesser retention and also elutes very fast as unretained band as demonstrated in the graph.

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