Analyse Different Chilli Peppers Biology Essay

Analyse Different Chilli Peppers Biology Essay The purpose of this project was to analyse different chilli peppers and hot sauces for their Capsaicin and Dihydrocapsaicin content but concentration was focused more on the analysis of different chillies than sauces. Samples of chillies were refluxed using Ethanol as an extraction solvent and sauces were untrasonicated using Methanol. The extracts were filtered and analysed via Reverse phase HPLC-UV Vis technique. A number of experiments were performed to optimise the method that has been previously used for analysis of chilli peppers. The first experiment conducted was to optimise the duration of reflux time required to obtain a good yield of Capsaicin. Results showed that 1hr is sufficient for the extraction of Capsaicin. A variety of chilli peppers and sauces were analysed so as to ascertain the hottest pepper. In general all samples had good detection. Different parts of chillies were also examined to establish which part contains the highest concentration of Capsaicin and Endoc arp was found to be the hottest part. The reproducibility of the method was also investigated and the sample showed to have a low RSD value. 1. Introduction Next to Jazz music, theres nothing that lifts the spirit and strengthens the soul more than a good bowl of chillies. Harry James (Late American musician) Loved by millions for their hot and sizzling flavours, the chilli peppers have become very popular over the period of time and are being grown in almost in all parts of the world, with Asia being the biggest producer of chillies followed by Mexico and the U.S. In traditional Indian medical system, chilli is used as way of stimulating the digestion and is also believed to be a natural pain killer. The red chilli peppers are also a source of potassium, magnesium and iron and vitamin C. 1.1 Why Are They So Hot? The heat sensation in chillies is caused by a class of chemicals, called the Capsaicinoids. These compounds are found in members of the capsicum family of plants. Capsaicinoids themselves belong to a group called Vanilloids i.e. containing the Vanillyl group [3dchem.com] Figure 1.1 [ en.wikipedia.org] All Capsaicinoids have same functional groups and differ only in length of hydrocarbon chain. The most common of Capsaicinoids compounds is Capsaicin which is the major constituent of chilli peppers and also responsible for their pungent taste. [3d chem.] 1.2 Structure of Capsaicins: Capsaicin figure 1.2.1 Dihydrocapsaicin figure 1.2.2 Nordihydrocapsaicin figure 1.2.3 Homocapsaicin figure 1.2.4 Homodihydrocapsaicin figure 1.2.5 Out of all the Capsaicins, the capsaicin and dihydrocapsaicin are the major constituents of Capsaicinoids (make up 80-90% of capsaicinoids). 1.3 Cis-trans isomerism in Capsaicin [3dchem] Capsaicin can exhibit cis-trans isomerism due to the presence of C=C bond. The double bond prevents the molecule to rotate freely internally, therefore, giving rise to stereo isomers. Cis isomer of the capsaicin is less stable and has higher energy due to steric hindrance. As the methyl groups are in close proximity to each other it causes repulsion between them and hence making it a less stable arrangement due to this added strain. Trans isomer on the contrary has methyl groups further apart and doesnt have any steric hindrance, making it a more stable/low energy arrangement. Therefore, the Capsaicin is always found in the Trans isomer. Figure 1.3.1 [http://www.homesteadcollective.org/mpg/science/majorcrap5.shtml] 1.4 Scoville Scale The scale for measuring the extent of heat in chillies was first invented by an American Chemist Wilbur Lincoln Scoville in 1912. The test he devised is known as the Scoville Organoleptic test in which he had a group of volunteers to taste the chillies on their own and later diluted them with sugar and water until they didnt have any burning sensation left. The resulting dilution factor was called the Scoville heat value of the sample and a number was then assigned to each sample of chilli i.e. Scoville unit, to ascertain the amount of dilution a chilli needs before its hot flavour dies away. [3d.chem] Table 1.4.1 Scoville heat values for Capsaicinoids [g6csy.net] Molecule Strength /Scoville units Capsaicin 16.1 million Dihydrocapsaicin 16.1 million Nordihydrocapsaicin 9.3 million Homocapsaicin 6.9 million Homodihydrocapsaicin 8.1 million The hottest capsaicin found is in the chilli known as Naga Jolokia, grown in India and has Scoville strength of 855,000-105, 0000 units. The Habanero (Mexican chilli) are the runners-up with Scoville rating range of 200,000-300, 0000. g6csy.net As mentioned earlier, in addition to Capsaicins being used as food additives, they have important medicinal benefits and are known as Phytochemicals.[3dchem.com] Due to having pharmaceutical and antioxidant properties, its widely used in anti-flammatory creams and ointments and also used as a counter irritant in surgical dressings and medicines. Moreover, they are also being used in nutritional supplements for pain relief and Arthritis. [cals.ncsu.edu] 1.5. Extraction Methods for Capsaicin Different methods have been devised as a way of extracting capsaicin from chilies and sauces. The simplest technique is to dissolve chilies in a polar solvent and placing the mixture on hot plates for half an hour and then filtering the extract via simple/vacuum filtration and making up with Methanol (100 ml). The other techniques more efficient for extraction are as follows: 1.5.1 Solid Phase Extraction or SPE This technique is particularly useful in extracting capsaicin as it requires pre-treating the sample prior to analysis. This reduces the amount of unwanted components that may interfere with the analysis. The extraction is completed in 4 steps: Conditioning the cartridge: This involves activating the cartridge by passing the sample through it to achieve same conditions with in the cartridge (e.g. to achieve same pH, composition etc as the sample). Retention: The sample is applied to the cartridge and either contaminants are retained and analyte is flushed through the cartridge. Other way used is holding the analyte with in the column and the unwanted components are passed through the column. Rinsing: The cartridge is then rinsed with distilled water to wash off the impurities. Elution: The last step is to elute the sample with appropriate solvent and the extract can then be used for analysis. 1.5.2 Reflux As demonstrated in this project, this method involves refluxing the chilies in methanol for appropriate duration. The reflux time required can be optimized by refluxing samples for different durations to establish the optimum time required by the capsaicin to leach. 1.5.3 Ultrasonication This method can be used for extracting the capsaicin from sauces or capsaicin based creams in short time. The samples are soaked in Ethanol and placed in the ultrasonic bath for half an hour at high temperature. The ultrasonic vibrations release the capsaicin from the samples. 1.5.4 Supercritical fluid extraction or SCFE In this method, the extraction solvent used is a super critical fluid. A super critical fluid (SCF) is a compound above its critical temperature and pressure. Therefore, an SCF is neither a liquid nor a gas. Hence, a super critical fluid has properties similar to liquids i.e. dissolving compounds and also gas like properties e.g. transportation. SCFE also minimizes the matrix components. Due to these capabilities, this method is more efficient and quicker than the other extraction methods. Carbon dioxide and water are the most commonly used SCF. This technique has a variety of applications in food, petrol, and pharmaceutical industries. 1.6. Previous research on Capsaicin Capsaicin has been widely studied and researched by various organisations and institutions but insufficient literature has been published with respect to the analysis of chilli peppers and sauces. In this section, three articles will be discussed as all three papers deal with the analysis of chillies and sauces relevant to this project. The first literature ¹ investigated the concentration of Capsaicin and Dihydrocapsaicin in the Habanero peppers using Super Critical Fluid Extraction method (SCFE). In addition to the analysis of Habanero whole peppers, different parts of chilli were also examined for their Capsaicin content. The Habanero peppers were obtained from two different locations: Cunningham Research station and Bailey Farm (located in North Carolina, US). The peppers were cut into seeds and shells and prior to extraction, the samples from Bailey farms were prepared fresh, oven dried and freeze dried and samples from Cunningham station were prepared in oven and freeze dried states. The whole peppers and seeds/shells samples were extracted using three polar solvents i.e. Methanol, Acetone and Acetonitrile. The Method for SCFE is as follows as stated in the literature: Fresh, oven and freeze dried preparations (0.5g dry weight) were extracted using a biomass: solvent loading of 15% (w/v) based on the initial moisture of the pepper samples/parts. Sample and solvent mixtures were homogenised in 50ml conical glass tubes and placed in a shaking water bath (50 °C). The extracts (2ml each) were then filtered and stored at -20 °C until the analysis. The preliminary work suggested that 1hr is sufficient to get good yield of capsaicin. The extracts were then analysed using Reverse-phased HPLC with UV VIS Detector. The HPLC was equilibrated with capsaicin standards (10, 30,50ppm). The mobile phase composition was isocratic at 60:40 (Acetonitrile: Water with acetic acid (pH 3)). The researchers of this project compared the capsaicin and Dihydrocapsaicin concentrations from both locations. The results showed that the Cunningham stations peppers had higher concentration of capsaicin in comparison with the Bailey farms chillies but the Bailey Farms pepper had higher amount of Dihydrocapsaicin than the Cunninghams. The results suggested that samples that were oven dried and extracted with Acetone gave maximum yields of the Capsaicinoids. This literature also suggested that regardless of the solvent type and preparation state used, seed has the highest amount of capsaicin. The researchers suggested the reason for differences in capsaicin concentration was due to different environments the fruits are cultivated e.g. chemicals used, weather conditions etc. The second literature ² deals with the analysis of three Capsaicinoids i.e. levels of capsaicin, dihydrocapsaicin and nordihydrocapsaicin in different chillies, sauces and arthritis creams via reverse phased HPLC. This research employed a solvent extraction technique which involved addition of ethanol (extraction solvent) to the samples of ground chillies, sauces and creams and placing the samples on hot plates for 30mins. After cooling and filtration, the extracts were transferred into flasks (100ml) and made up to the mark with Ethanol. 5ml was withdrawn from this sample and filtered again into a syringe filter cartridge (0.45 µm pore size). This aliquot was then used for the analysis. A 1000ppm standard stock solution was used to make standard capsaicin solutions ranging from 1-50ppm and ran through HPLC. The mobile phase in this research was made up of ACN, water and phosphoric acid (0.1%).The UV detector was set at 280nm and 205nm to determine samples responses at different wavelengths. In addition, Isocratic and Gradient elution were used. The findings from this journal suggested that Capsaicinoids present in very little concentrations (e.g. 0.5ppm) were detected better at 205nm wavelength using gradient elution rather than Isocratic method. However, results also indicated that for analysis of Capsaicinoids present in greater concentrations, Isocratic elution and UV wavelength at 280nm. The concentrations of the Capsaicinoids were expressed in terms of the Scoville units and the value calculated for the Habanero peppers (150,000) in this experiment was different to the literature value range (200,000-300,000). The researchers attributed this fact to variations in the environment e.g. weather etc. The third literature ³ determined the capsaicin and dihydrocapsaicin content in chilli peppers. The chillies were grounded for 10mins and Acetonitrile (30ml) was added to the crushed peppers and again grounded for 20mins. The solid residue was filtered and an aliquot (1ml) was made up to the mark with Acetonitrile. (in 10 ml flask). The extraction method used in this work was Solid Phase Extraction (SPE): An SPE cartridge was conditioned with Acetonitrile, methanol and water and the capsaicin extract (10ml) was then applied to the cartridge and the analyte was eluted with methanol (4ml) and then again with 1 ml of methanol (containing 1% acetic acid). The analytes were then run through reverse phase HPLC using UV-VIS detector (at 281nm); mobile phase consisted of 77:33 (Methanol: Water). The HPLC was eluted first with the standards so as to obtain the calibration graphs. The Capsaicinoids concentration of different chillies was expressed in Scoville heat units (similar to literature 2). The results showed that Habanero is the hottest amongst all peppers that were analysed i.e. Scoville heat value of 276,000 which corresponded to the literature value range. The least hot pepper was Jalapeno (41,000 Scoville heat units). 1.6.1 Comparison of three papers: The researches have used different techniques to extract the Capsaicinoids i.e. SCFE and SPE. However, the extraction technique used in this project was Reflux (for chillies) and Ultrasonication (for sauces). The extractions were successful and all samples in general were detected which indicates that more than one method can be employed as a way of extracting the Capsaicinoids from chillies and sauces. Various parameters were manipulated as part of method development in literature 2 e.g. Isocratic/gradient elution and different wavelengths. Similarly in Literature 1 three preparation states and three solvents were used to determine what state/solvent gives maximum yield of Capsaicinoids. Literature 1 also suggested that the hottest part with in the chilli is the seeds, however, the findings of this project have shown that the Endocarp contains the highest amount of capsaicin and dihydrocapsaicin. This is indicative of the fact that the amount of Capsaicinoids can vary even with in d ifferent parts of chilli. However, all researches discussed as well as this project has used polar solvents to extract the analytes and the analytical technique used was reverse phase HPLC which indicates its usefulness in the capsaicin analysis in particular. Once an analyte of interest has been extracted, it can be analysed by a process called Chromatography. 1.7 Chromatography The word Chromatography originates from Greek; Chroma means colour and graphein implies to write. [Skoog West Analytical Chem 7th Edition page 646] The history of this separative technique dates back to early twentieth century when it was developed by a Russian Botanist Mikhail Tswett in 1903[D kealy Instant notes]. He used this method for separation of various plant pigments and samples were passed through a calcium carbonate column. The separated analytes were identified as they left coloured bands on the column. [Skoog West Ana Chem 7th Page 646]. Since its invention by the Russian Scientist, this method has been modified and developed in many forms to give quantitative (amount of the analyte present) and qualitative analysis (identification of the unknowns) of complex mixtures. [d.Kealey instant notes page 119]. Separation in Chromatography is achieved by passing the sample mixture through the stationary phase by continuous flow of a mobile phase. This process is known as Elution. Hence, the chromatographic separation depends on the differences in the distribution ratios of the sample components between the stationary and mobile phase. Therefore, this capability of an analyte to migrate at different rates in both phases gives separation over a period of time and distance travelled. [D Kealey page 120] Kx= Cs/Cm, where kx is the equilibrium partition coefficient and Cs and Cm are molar concentrations of analyte in mobile and stationary phase. There are two types of Chromatography techniques: Year 2 notes page 25-26 1.7.1 Planar Chromatography In this method, the stationary phase is composed of a flat bed of material which is made up of an adsorbed layer distributed evenly over a sheet of glass, plastic or Aluminum (known as Thin Layer Chromatography or TLC) Paper Chromatography is also another type of Planar Chromatography in which the stationary phase is a sheet of cellulose material. 1.7.2 Column Chromatography In this method, the stationary phase is a glass or metal column on to which the stationary phase is tightly packed onto a column where separation takes place. Examples of Column chromatography are Gas Chromatography, High Performance Liquid Chromatography etc. 1.7.3 Chromatogram The plot of detector response Vs elution time is known as the Chromatogram. [Year 2 notes page 25]. Figure 1.7.3.1 [http://www.clu-in.org/characterization/technologies/images/retention.gif] 1.7.4 Retention time In the above figure is a typical chromatogram and term tr is the time taken by the analyte to elute the column, known as Retention time. [year 2 notes page 28] 1.7.4 Dead time [d Kealey page 121] Indicated as tm in the fig is referred to the dead time this is defined as the retention time required by the non retained species (i.e. mobile phase molecule) to pass through the column. A good Chromatogram should have well defined peaks having correct shape and symmetry (i.e. Gaussian shape), eluted in reasonable retention time (tr not too long or too short) and should be separated from the extraneous peaks. [Year 2 notes page 29] 1.8 Describing a Chromatogram There are four parameters used in chromatography that evaluate the quality of a chromatogram. These are: 1.8.1 The Capacity Factor, K It is the amount of mobile phase required to elute a particular peak. The K is calculated for the first and the last peak. This factor is particularly useful when establishing the best mobile phase composition in the HPLC. K can be calculated as following: K= (tr-tm)/tm where tr is the retention time and tm is the dead time. A Chromatogram having well separated peaks in good retention time will have K values between 2-8. 1.8.2 The Selectivity Factor, ÃŽÂ ± This is the ability of a system to separate two analytes (A and B) and is calculated by: ÃŽÂ ±= trb tm/ tra- tm, where trb and tra are the retention times of analytes A and B. A system where peaks are clearly separated has a value of ÃŽÂ ± > 1 1.8.3 The Resolution factor, Rs This determines the ability of a system to resolve two peaks that elute very close to each other. And can be calculated by: Rs = 2 (trb-tra)/Wa+ Wb where tra and trb are retention times and Wa and Wb are the peak widths of analyte A and B. The value of Rs > 1.5 for a good quality chromatogram. 1.8.4 The Efficiency Factor [ D kealey page 126-127] When separation takes place in a column, the chromatographic separation can be evaluated by the resolution factor, Rs or the efficiency factor. The efficiency is defined as the number of theoretical plates in a column. This factor evaluates the extent of band broadening of the analyte peaks. Increasing the number of plates and reducing their heights gives better efficiency and vice versa. The plate height can be calculated using: H= L/N where L is the length of the column (in mm usually) and N is the number of plates. The efficiency factor N is calculated by: N= 16 (tr/W) ² for a peak with a good baseline N= 5.54 (tr/W1/2) ² for a peak with a poor baseline and W1/2 is the width at half the maximum height of the peak. (year 2 notes 42-43) 1.8.5 Band Broadening As an analyte passes down a column, the peaks become shorter and broader due to various factors that cause band broadening. The Van Deemter Equation explains the reason for the band broadening: H= A+ (B/u) + Cu where H is the plate height and u is the linear velocity of the mobile phase. Other variables in the equation are explained below: A- Eddy Diffusion: As the mobile phase carries the sample components through the stationary phase, some components pass through the column in a straight line whilst other may that are retained longer by the stationary phase may deviate from the straight path and cause the peaks or bands to be broader. If evenly sized particles are used for packing the stationary phase, then the Eddy diffusion can be minimized. B- Longitudinal Diffusion: If the mobile phase is travelling at low velocities, then the analyte will spend more time in the column as analytes diffuse into the mobile phase. This longitudinal diffusion contributes towards peak broadening and can be minimized by an increasing the flow rate of the mobile phase. The increased velocity will reduce the retention time resulting in decreased effects caused by this phenomenon. [Veronica HPlc page 17-19] C-Mass transfer: As discussed earlier in this section, the separation depends on the ability of the analyte to distribute itself between the stationary and mobile phase. As the mobile phase is constantly flowing, the true equilibrium distribution of the analyte is never established. This leads to increased retention times and thus resulting in peak broadening. [d kealey page 124] 5. High Performance Liquid Chromatography or HPLC HPLC is a form of liquid chromatography which provides both qualitative and quantitative information about complex mixture samples in short time. The stationary phase in this technique is made up of very small fine particles and the sample is forced through the column by mobile phase solvents under high pressure, hence also bearing the name High Pressure Liquid Chromatography. [page 1 Veronica HPLC]. 5.1 Mobile phase in HPLC The main requirement for this technique is that the analyte must be soluble in the mobile phase as the mobile phase carries the sample mixture through the column where separation takes place. Therefore, if the analyte interacts with the mobile strongly, it will elute the column faster, leading to shorter retention times. [page 66 Chromatographic separations]. The mobile phase can either be a single solvent or different solvents combinations may be used. After suitable mobile phase has been chosen, the system can be set at isocratic or gradient conditions. In Isocratic conditions, the chosen ratio of solvents remains constant throughout the analysis e.g. in this project Isocratic mobile phase used for analysis. In contrast, the gradient mobile phase can be changed over the period of time. [Year 2 notes ]. 1.9 Stationary phase in HPLC The stationary phase in HPLC consists of a solid made out of micro porous material packed into the metal column. Silicas or modified Silicas with nonpolar organic groups attached are commonly used as column packing material. Out of all stationary phases used in HPLC, Octadecyl silica known as ODS or C18 is most extensively used due to its ability to separate the analyte components with high, intermediate and low polarities. Other stationary phases used in HPLC are Aminopropyl, Nitile, Sulphonic acid, quaternary Amines etc [d. kealey 159-161] Elution in HPLC is carried out by determining the extent of interactions of the analyte with the stationary and mobile phases. The degree of separation of the sample components depend on their migration rates and distribution ratios in both phases. 1.9.1 Normal and Reverse phase HPLC In normal phase HPLC (adsorption chromatography), the stationary phase is more polar than the mobile phase which is weakly polar. The separations are based on the relative polarities of the sample components. For instance, if species A is more polar than the species B, A will have strong affinity for the stationary phase and will be held in the column longer. This will result in species A having long retention time compared to B. Thus, in normal phase HPLC, least polar analyte elutes first. In Reverse phase HPLC (bonded phase chromatography), the stationary phase in non polar and the mobile phase solvents used are polar. This phase is governed by the hydrophilic and hydrophobic properties of the analytes. Most polar analyte elutes first and vice versa. As Capsaicinoids are relatively non polar, therefore, if the mobile phase polarity in reverse phase is increased then the analyte will have stronger affinity for the stationary phase and therefore will spend more time in the column, leading to long retention times. The mobile phase solvents used in Reverse phase HPLC are water or aqueous buffer with an organic solvent. The use of protons in mobile phase composition improves the peak shape and travels in column quickly. [http://ionsource.com/tutorial/chromatography/rphplc.htm#Solvents] 1.9.2 HPLC Instrumentation Figure 1.9.2.1 [http://www.youtube.com/watch?v=I-CdTU5X4HA] Pump: In HPLC solvent delivery system, the pump is used to deliver the mobile phase solvents to the column under high pressure. Most commonly used pumps are reciprocating pumps. The pumps used should be free of corrosion, must supply an accurate and controlled flow rate and should be pulse free. The HPLC solvents for mobile phase must be degassed to ensure they are pure and free of any contaminants. This is done by passing an inter gas through the solvent reservoir in vacuum degasser. Injector: The sample is injected through syringe into the injector port. The injection system must not interrupt the flow of mobile phase and should deliver sample into the column in small volumes (5-500 µL). Column: Most commonly used HPLC column is 25cm in length, internal diameter of 4-6mm and particle size of 5 µm. 1.9.3 Detectors in HPLC When the sample components elute the column at different rates, they pass through the detector, and the information form the detector is then displayed in the form of a chromatogram. 1.9.4 UV-Vis Detector The detector is set at a specific wavelength which will be absorbed by the analyte. The degree of absorbance of UV radiation by the analyte is proportional to its concentration. (Beer Lambert law) 1.9.5 Diode array Detector or DAD The Diode Array detector is a type of UV Vis Detector [D Kealey page 162]. When the sample reaches the sensor cells, UV radiation is shone on the analyte. The light source mostly used is a Deuterium lamp. After light passes through the cell, its dispersed onto the photosensitive diodes via diffraction grating or quartz prism. Every diode in the array detects different wavelengths. The measure of differences in extent of absorbance at different wavelengths by the sample components results in their identification and also gives information about the concentration of the analytes. [http://www.chromatography-online.org/topics/diode/array.html] Other detectors used in HPLC are Fluorescence, refractive index, electrochemical detectors etc. [d Kealey 163-165] Figure 1.9.5.1 Experimental Method This project was performed in four different experiments. In all experiments, the methods for extracting the capsaicin from chilli peppers and sauces were the same i.e. reflux and Ultrasonication. However, different masses of chilli peppers, their individual parts were used and solvent volumes were taken in these experiments. (see results) 2.1.1 Preparation of chilli samples and extraction Several chillies were cut into small pieces and weighed into a 250 mL round bottom flasks. Ethanol (75 ml) was then added to chillies. A condenser (fitted with rubber tubing to the water tap) was fixed to the round bottom flask and solution was placed on isomantle (set at 80 °C) and refluxed in fume cupboard. After cooling off, extract was filtered into 100ml flask and made up to the mark with ethanol. A 5ml aliquot was withdrawn into 1.8ml sample vial using a 0.45 µm syringe filter. These aliquots were then analysed through HPLC. 2.1.2 Preparation of chilli sauces and extraction The hot sauces were prepared by dilution of sauce (2g) with ethanol (15ml) in beakers and solutions were places in ultrasonic bath (set at 60 °C) for 30 minutes. The extracts were filtered in the same way as chillies i.e. using 0.45 µl syringe and transferred to 20ml volumetric flasks and bringing up to the mark with methanol. The equipment used was white tiles, knife, weighing boat and weighing scale. 2.1.3 Preparation of glassware In analytical experiments, its of prime importance to ensure the glassware used is clean. Therefore, the glassware used in this project was washed first with deionised water and then with the solvents used. 2.1.4 Preparation of standards for Calibration The standard solutions were prepared from 200ppm stock solution directly into sample vials. Eppendorf pipette was used for accuracy and 0, 20, 40, 60, 80, 100 ppm standards were made with HPLC standard Methanol. Concentration/ppm Volume of 200ppm Capsaicin/ ÃŽÂ ¼L Volume of methanol/ÃŽÂ ¼L 0 0 1000 20 100 900 40 200 800 60 300 700 80 400 600 100 500 500 2.2 HPLC The HPLC used in the laboratory was Agilent 1100 Series Column- ODS hypersil UV VIS Detector- G1315B Diode array detector (set at 280nm) Flow rate- 1.3 ml/min Windows XP CPU Mobile phase- Isocratic at 65:35 Acetonitrile (2% acetic acid): water Particle size- 5 µm, Column dimension- 250mm x 4.6mm 2.3 Determining ÃŽÂ »max for UV- Vis detector The ÃŽÂ »max for the detector was determined by placing methanol blank in UV spectrometer (Perkin Elmer with lambda 40) to calibrate it. After calibration, a 100ppm capsaicin standard was placed in the spectrometer and a graph was obtained with the optimum wavelength (see Appendix 1). This was 280.40nm and the HPLC detector was set at this wavelength. 2.4 Determining optimum mobile phase composition (see Appendix 2) The mobile phase was altered to different ratios to establish what ratio gives the best separation and reasonable retention times (less than 7 minutes). The 80:20 ratio (MeCn: water) gave shorter retention time but the peaks were eluted closer to each other. The 70:30 ratio showed good retention time but peaks were still closer to each other. 50:50 ratio gave long retention time and 60:40 ratio gave good separation but retention time was longer. Therefore ratio of 65:35 was used as this gave the best retention time and separati

Budweiser Paper

MARKETING 474 FINAL: INTEGRATED MARKETING PLAN INSTRUCTIONS: You will complete an Integrated Marketing Plan with your group for Budweiser. As you may know the ailing beer manufacturer is struggling. You will need to do additional research to uncover the issues that are plaguing the brand. The goal of your plan is to drive business. BUDWEISER OVERVIEW: In January, Coors Light surpassed Budweiser to become the #2 selling beer in the U. S. It was a major blow for Budweiser, a brand that has been in decline for the last three decades.Certainly the changing tastes of the American consumers have impacted sales of Anheuser Busch's flagship brand, but marketing decisions have also played a big role in the fall of the iconic brand. Budweiser has tried to be everything from fratty to refined to sporty to hipster. Anheuser-Busch has always put a major focus on marketing. This is the company, after all, that spent $246. 2 million solely on Super-Bowl commercials from 2002 to 2011. Edward McClelland of Salon  says that from its inception, Budweiser was a â€Å"triumph of marketing over quality. The quality was questionable: Adolphus Busch, the company's founder, called his beer â€Å"dot-schlop† and preferred to drink wine and St. Louis drinkers were not fans of the drink either, but the Busch family still bought the licenses and paid rent for bar owners in exchange for serving the product. Budweiser had its glory days in the 1950s when Anheuser-Busch helped strengthen its national brand by sponsoring shows featuring Jackie Gleason, Milton Berle, and Frank Sinatra. It also promoted its beer by sponsoring sporting events and branding stadiums. By the 1980s, Budweiser was synonymous with American culture. â€Å"Budweiser’s kind of a mainstay.It’s a good old American tradition, like going to a baseball game or a college football game,†Ã‚  says Kevin Eichelberger, webmaster of BeerTees. com. But something has happened in the past three decades and, while it still has clout overseas, the Budweiser brand is tired here at home. Undoubtedly, owners A-B InBev will try to save it with marketing, as they have always done. But will that be enough to return Budweiser to the throne and its former glory? Source: businessinsider. comPAPER OUTLINE: Integrated Marketing Campaign Deliverab les: You should outline the following before you begin developing your IMC (Word Times New Roman 12 Font and 1. inch margins):OVERVIEW (2-3 Pages)1. Budweiser Problem2. Company Culture, Values, Mission3. Communication Objective: (e. g. Inform, Persuade, Call to action)4. Brand Positioning and Personality5. Intended Target Audience/Insights CAMPAIGN (At least 3-5 Pages) â€Å"BIG IDEA†: or compelling theme. (e. g. What happens in Vegas stays in Vegas)MESSAGE: Relevant copy and actionable message for your target consumer which includes (e. g. rational or emotional appeal or selling proposition)CHANNELS (examples): a. Print Campaign: Headline, Artwork/Visual and Copy b. Events with Sponsors c.Direct Communication: Web-site, Social Media d. Public RelationsCREATIVE: Headline to capture the target’s attention and photos that capture the essence of the campaignGRADING CRITERIA: 200 Points: Written Strategy-Did you have a compelling strategy for your IMC? Was your strategy al igned to the problems and issues? 100 Points: 5 minute Oral Presentation-How Creative was your IMC presentation? Did you use innovative ways to drive sales that would resonate with the target consumer?Note: Your Oral Presentation should be 4-5 Slides developed from the CAMPAIGN part of the written strategy.

Protecting Interest Of The Minority Shareholders

In Asian countries including Bangladesh, the controlling ownership of public listed companies are dominated by some families. The problem of minority exploitation may arise when the ownership is highly concentrated in any specific group, especially family ownership. One of the consequences of this is the expropriation of minority shareholder rights. Apart from family control another limitation of principles of corporate law is the principle of majority rule, sometimes called the â€Å"supremacy of majority† rule.Those who invested more in the company bear a greater risk in the event of a business failure, but simultaneously they have a greater degree of control over the company. There is certainly a risk that the majority will take advantage of the minority and that a company will be run at the expense of the minority shareholders. Any decision of Annual general meeting (AGM) adapted by majority vote and directors are appointed and may be removed from the office at any time by a simple majority at the general meeting.Thus, the directors are motivated to act in the best interests of the majority who appointed them and who may remove them. Minority shareholder rights expropriation occurred when family ownership directed cash to their own benefit, inefficient projects and connected lending to relatives and friends rather than return it in dividends to minority shareholders. Other expropriation can take the form of profit reallocation, assets misuse, transfer pricing, sell below the market price departments or parts of the firm to other firms that major shareholders own, or acquisition of other firms that major shareholders own at a premium.The majority shareholders treats the company as his own, and acts accordingly, to the detriment of the other shareholders, or where there is a breakdown in the relationship of the shareholders or any of their number, which gives rise to questions about the future ownership and control of the Company. On the other hand, wh ere a single or small number of shareholders hold a substantial block of shares in the company, say, in excess of 25% of the voting rights, securing managerial accountability to the shareholders or at least to the controlling shareholders through the traditional governance mechanisms of company law can dominate the company.In some situation, the ‘non-controlling’ shareholders may collectively hold more voting shares than the ‘controlling’ shareholders. However, if the non-controlling shares are widely dispersed, effective control of the company will lie in the hands of the block-holder, even if that block consists of less than 50% of the voting shares. The shareholder providing the majority of the capital may sometimes not control the company.In such a case the majority shareholder is effectively in a minority position with regard to the exercising of controlling rights. The emergence of such a situations are the principal/agent problem between the controll ing shareholders and the non-controlling ‘minority’ shareholders. The corporate management law and policy must have protection of interest of the minority shareholders. The general purpose of minority protection instruments is to prevent the abuse of power by the major shareholders.There is not an easy solution, to the problem, since the principle of majority rule, in company law and other rules of regulators. It is a long established principle of corporate law that the regulators and courts should not intervene in business decisions due to the nonintervention policy or internal management principle. There is no statutory law of anywhere contains a definition of the minority or majority shareholder. The distinguishing factor between the two is the degree of control over the corporation.The number of shares owned is not  decisive, even a shareholder owning a majority of shares may be a minority shareholder, if other shareholders are well organized and, thus, control th e company. The company must follow the principles ‘partnership’ and consultation aims at balancing the interest between major and minor shareholders, and usually do not infringe minorities rights through guaranteeing at least the following minority rights such as respect of opinion of major shareholders toward minorities, the right of minorities to be heard on regard of business matters and exit rights.The limited Liability Companies, which are, in practical terms, run, as if they were a partnership, between the persons who are shareholders of same, might be regarded by the law, as â€Å"quasi partnership†. The OECD principles on Corporate Governance (2004) provide that: Shareholders, including institutional shareholders, should be allowed to consult with each other on issues concerning their basic shareholder rights as defined in the Principles, subject to exceptions to prevent abuse.The protection comes from better legal protection, stronger structure of the in ternal control mechanisms and more efficient capital markets and market for corporate control. One of the methods to ensure the minority rights is to follow good Corporate Governance principles because there exists a relation between the level of protection of minority shareholders and incorporation of good practices of Corporate Governance. The separation of ownership and control in corporations with dispersed ownership structure highlights the agency issue due to conflict between agents (directors) and principals (shareholders).Due to a different agency problem that arises on account of the conflict between dominant and minority shareholders. The minority shareholders can be empowered by ensuring control over the management and board of directors. The board of directors are accountable to the shareholders as a class is to make it easy for the shareholders to convene meetings to consider the removal of directors, evaluate the board’s performance and remove directors of whom they disapprove.The minority shareholders are afforded the remedies if the majority shareholders, violate a personal right of a minority shareholder, then he can file a personal action against the wrongdoers to rectify such a violation of the articles of association of the Company or of the terms of any shareholder agreement etc. With increasing instances of corporate fraud around the world, another remedy is provisions for class action suits. Class action is a law suit brought by one or more individuals on behalf of a large group of people who have the same complaint.In certain circumstances, minority shareholders may bring a common law derivative action, on behalf of the company, against the wrongdoers, who committed a wrong to the company. Wrongdoers can be shareholders and directors of the company, as well as third parties. In order to be able to proceed with a derivative action at common law, the minority shareholders must have legal options to persuade the courts, that the com pany’s decisions by majority shareholders are not to pursue a remedy for the wrong done to the company which amounts to a â€Å"fraud on the minority† .Another Statutory remedy is of petition to winding up of the company on a just and equitable ground. There is hearsay that few sponsors / families are responsible for share scams causing huge loss of small investors. Security exchange commission (SEC) has such views with perceived experiences of two share market debacles and issued a notification on November 22, 2011 imposing conditions that all sponsors / promoters and directors of a listed company shall jointly hold minimum 30% share of paid up capital of the company. Moreover, each director shall hold minimum 2% of the paid up capital.In case of vacancy of anyone holding 5% share shall be entitled to be directors. The publicly listed companies have usually 15 directors and they will hold 75% of the share and voting rights of the company. This means the companies will gradually go under control of few limited persons who have capacity of investment of sufficient amount. SEC has in mind that, mandatory provision of higher shares will prevent such future stock market debacle. But as per investigation report of Mr Khondaker Ibrahim Khaled, accepted by all, there are many organizations including SEC are jointly responsible for disaster in stock market.The public companies are controlled by few families and the directors are ‘elected’ from same family by rotation and under full control of families. They retire due to compulsion of retirements as per law. Small shareholders are awarded a gift pack and nominal dividends in AGM and have no say against the decision of these controlling families. Companies go for public share to generate fund for investments but shall fail to generate fund with higher investments of sponsors and directors.The over investment of sponsors / directors will not bring sufficient share in the market and the market will remain at the present status of low investment. India has totally different legal framework to safeguard interest of small investors. Indian Companies Act 2013 under section -151. A listed company may have one director elected by such small shareholders in such manner and with such terms and conditions as may be prescribed. For the purposes of this section â€Å"small shareholders† means a shareholder holding shares of nominal value of not more than twenty thousand rupees or such other sum as may be prescribed.There is no policy of a designated directorship of choice of minority shareholder nor there do any provision to control, appoint or remove any director. The global law and policy is to protect the rights of minority shareholders but in contrary Bangladesh SEC make legal provision of make the minority shareholder marginalized and have no option to exercise their rights due to majority rule and lose their voice. The decision of higher investment of directors is not g ood for stock market and should be amended to find way out to safeguard interest of minor shareholders from the proven experience of other markets. Protecting interest of the minority Shareholders In Asian countries including Bangladesh, the controlling ownership of public listed companies are dominated by some families. The problem of minority exploitation may arise when the ownership is highly concentrated in any specific group, especially family ownership. One of the consequences of this is the expropriation of minority shareholder rights.Apart from family control another limitation of principles of corporate law is the principle of majority rule, sometimes called the â€Å"supremacy of majority† rule. Those who invested more in the company bear a greater risk in the event of a business failure, but simultaneously they have a greater degree of control over the company. There is certainly a risk that the majority will take advantage of the minority and that a company will be run at the expense of the minority shareholders.Any decision of Annual general meeting (AGM) adapted by majority vote and directors are appointed and may be removed from the office at any time by a simple majority at the general meeting. Thus, the directors are motivated to act in the best interests of the majority who appointed them and who may remove them.Minority shareholder rights expropriation occurred when family ownership directed cash to their own benefit, inefficient projects and connected lending to relatives and friends rather than return it in dividends to minority shareholders. Other expropriation can take the form of profit  reallocation, assets misuse, transfer pricing, sell below the market price departments or parts of the firm to other firms that major shareholders own, or acquisition of other firms that major shareholders own at a premium. The majority shareholders treats the company as his own, and acts accordingly, to the detriment of the other shareholders, or where there is a breakdown in the relationship of the shareholders or any of their number, which gives rise to questions about the future ownership and control of the Company.On the other hand, where a single or small number of shareholders hold a substantial block of shares in the company, say, in excess of 25% of the voting rights, securing managerial accountability to the shareholders or at least to the controlling shareholders through the traditional governance mechanisms of company law can dominate the company. In some situation, the ‘non-controlling’ shareholders may collectively hold more voting shares than the ‘controlling’ shareholders. However, if the non-controlling shares are widely dispersed, effective control of the company will lie in the hands of the block-holder, even if that block consists of less than 50% of the voting shares.The shareholder providing the majority of the capital may sometimes not control the company. In such a case the majority shareholder is effectively in a minority position with regard to the exercising of controlling rights. The emergence of such a situations are the principal/agent problem between the contr olling shareholders and the non-controlling ‘minority’ shareholders.The corporate management law and policy must have protection of interest of the minority shareholders. The general purpose of minority protection instruments is to prevent the abuse of power by the major shareholders. There is not an easy solution, to the problem, since the principle of majority rule, in company law and other rules of regulators. It is a long established principle of corporate law that the regulators and courts should not intervene in business decisions due to the nonintervention policy or internal management principle.There is no statutory law of anywhere contains a definition of the minority or majority shareholder. The distinguishing factor between the two is the degree of control over the corporation. The number of shares owned is not  decisive, even a shareholder owning a majority of shares may be a minority shareholder, if other shareholders are well organized and, thus, control the company.The company must follow the principles ‘partnership’ and consultation aims at balancing the interest between major and minor shareholders, and usually do not infringe minorities rights through guaranteeing at least the following minority rights such as respect of opinion of major shareholders toward minorities, the right of minorities to be heard on regard of business matters and exit rights. The limited Liability Companies, which are, in practical terms, run, as if they were a partnership, between the persons who are shareholders of same, might be regarded by the law, as â€Å"quasi partnership†.The OECD principles on Corporate Governance (2004) provide that: Shareholders, including institutional shareholders, should be allowed to consult with each other on issues concerning their basic shareholder rights as defined in the Principles, subject to exceptions to prevent abuse.The protection comes from better legal protection, stronger structure of the internal control mechanisms and more efficient capital markets and market for corporate control. One of the methods to ensure the minority rights is to follow good Corporate Governance principles because there exists a relation between the level of protection of minority shareholders and incorporation of good practices of Corporate Governance.The separation of ownership and control in corporations with dispersed ownership structure highlights the agency issue due to conflict between agents (directors) and principals (shareholders). Due to a different agency problem that arises on account of the conflict between dominant and minority shareholders. The minority shareholders can be empowered by ensuring control over the management and board of directors. The board of directors are accountable to the shareholders as a class is to make it easy for the shareholders to convene meetings to consider the removal of directors, evaluate the board’s performance and remove directors of who m they  disapprove.The minority shareholders are afforded the remedies if the majority shareholders, violate a personal right of a minority shareholder, then he can file a personal action against the wrongdoers to rectify such a violation of the articles of association of the Company or of the terms of any shareholder agreement etc. With increasing instances of corporate fraud around the world, another remedy is provisions for class action suits. Class action is a law suit brought by one or more individuals on behalf of a large group of people who have the same complaint. In certain circumstances, minority shareholders may bring a common law derivative action, on behalf of the company, against the wrongdoers, who committed a wrong to the company.Wrongdoers can be shareholders and directors of the company, as well as third parties. In order to be able to proceed with a derivative action at common law, the minority shareholders must have legal options to persuade the courts, that th e company’s decisions by majority shareholders are not to pursue a remedy for the wrong done to the company which amounts to a â€Å"fraud on the minority† . Another Statutory remedy is of petition to winding up of the company on a just and equitable ground. There is hearsay that few sponsors / families are responsible for share scams causing huge loss of small investors.Security exchange commission (SEC) has such views with perceived experiences of two share market debacles and issued a notification on November 22, 2011 imposing conditions that all sponsors / promoters and directors of a listed company shall jointly hold minimum 30% share of paid up capital of the company. Moreover, each director shall hold minimum 2% of the paid up capital. In case of vacancy of anyone holding 5% share shall be entitled to be directors. The publicly listed companies have usually 15 directors and they will hold 75% of the share and voting rights of the company.This means the companies will gradually go under control of few limited persons who have capacity of investment of sufficient amount. SEC has in mind that, mandatory provision of higher shares will prevent such future stock market debacle. But as per investigation report of Mr Khondaker Ibrahim Khaled, accepted by all, there are many organizations including SEC are jointly responsible for disaster in stock market.The public companies are controlled by few families and the directors are ‘elected’ from same family by rotation and under full control of families. They retire due to compulsion of retirements as per law. Small shareholders are awarded a gift pack and nominal dividends in AGM and have no say against the decision of these controlling families. Companies go for public share to generate fund for investments but shall fail to generate fund with higher investments of sponsors and directors. The over investment of sponsors / directors will not bring sufficient share in the market and the m arket will remain at the present status of low investment.India has totally different legal framework to safeguard interest of small investors. Indian Companies Act 2013 under section -151. A listed company may have one director elected by such small shareholders in such manner and with such terms and conditions as may be prescribed. For the purposes of this section â€Å"small shareholders† means a shareholder holding shares of nominal value of not more than twenty thousand rupees or such other sum as may be prescribed.There is no policy of a designated directorship of choice of minority shareholder nor there do any provision to control, appoint or remove any director. The global law and policy is to protect the rights of minority shareholders but in contrary Bangladesh SEC make legal provision of make the minority shareholder marginalized and have no option to exercise their rights due to majority rule and lose their voice.The decision of higher investment of directors is n ot good for stock market and should be amended to find way out to safeguard interest of minor shareholders from the proven experience of other markets.

Attitudes Towards the War in Regeneration and All Quiet...

...no nation is rich enough to pay for both war and civilization. We must make our choice; we cannot have both. -- Abraham Flexner Regeneration is an anti-war novel, reflecting the issues and the concerns in wartime Britain. All Quiet on the Western Front is also an influential anti-war novel and an important chronicle of World War 1. Both are historical fiction set near the end of the war, 1917-1918. The two texts explore similar themes in condemning the war. Remarque’s novel (All Quiet on the Western Front) is a profound statement against war, focusing especially on the ravaging effects of war on the humanity of soldiers. Similarly, Barker (author of Regeneration) offers realistic detail of many abominable war†¦show more content†¦Remarque’s novel dramatizes these aspects of World War 1 and portrays the mind-numbing terror and savagery of war with a relentless focus on the physical and psychological damage that it occasions. At the end of the novel, almost every major character is dead, epitomizing the war’s devastating effect on the generation of young men who were forced to fight it. In its depiction of the horror of the war, All Quiet on the Western Front presents a scathing critique of the idea of nationalism, showing it to be a hollow, hypocritical ideology, a tool used by those in power to control a nation’s populace. Paul and his friends are seduced into joining the army by nationalistic ideas, but the experience of fighting quickly schools them in nationalism’s irrelevance in the face of the war’s horrors. The relative worthlessness on the battlefield of the patriots, Kantorek (former schoolmaster in Paul’s high school) and Himmelstoss (a non-commissioned training officer) accentuates the inappropriateness of outmoded ideals in modern warfare. Remarque illustrates that soldiers on the front fight not for the glory of their nation but rather for their own survival; they kill to keep from being killed. Additionally, Paul and his friends do not consider the opposing armies to be their realShow MoreRelatedAll Quiet On The Western Front982 Words   |  4 Pages There is not as much symbolism shown throughout the text â€Å"All quiet on the western front† to show the effects of war too. However, Kermerich’s boots provide a symbol for the certain attitude a soldier would require – detaching their emotions only to act as an emotionless object that follows orders to reduce the effects of war inflicted on the soldiers. Each Solider (including Kemmerich) who took them from dead air pilot) dies when owning these boots. Therefore, it could be argued that the bootsRead MorePoems: City Planners15330 Words   |  62 Pagesor attitude of the poet / narrator; how Atwoods tone shifts quite noticeably and the effects of this on the reader. Both poems use the word Planners in their titles and both deal with cities as their topic, focussing on the structures and organization of urban spaces. Kim Cheng uses the third person ‘they’ to create a sense of distance - of us and them, whereas Atwood uses the inclusive ‘we’, to suggest that this experience of cities is one that we can all relate to and share. Her attitude - andRead MoreOrganisational Theory230255 Words   |  922 Pagessituates organization theory within the scholarly debates on modernism and postmodernism, and provides an advanced introduction to the heterogeneous study of organizations, including chapters on phenomenology, critical theory and psychoanalysis. Like all good textbooks, the book is accessible, well researched and readers are encouraged to view chapters as a starting point for getting to grips with the field of organization theory. Dr Martin Brigham, Lancaster University, UK McAuley et al. provide aRead MoreStephen P. Robb ins Timothy A. Judge (2011) Organizational Behaviour 15th Edition New Jersey: Prentice Hall393164 Words   |  1573 PagesCredits and acknowledgments borrowed from other sources and reproduced, with permission, in this textbook appear on the appropriate page within text. Copyright  © 2013, 2011, 2009, 2007, 2005 by Pearson Education, Inc., publishing as Prentice Hall. All rights reserved. Manufactured in the United States of America. This publication is protected by Copyright, and permission should be obtained from the publisher prior to any prohibited reproduction, storage in a retrieval system, or transmission in anyRead MoreMarketing Mistakes and Successes175322 Words   |  702 Pagesby Aptara ®, Inc. and printed and bound by Courier/Westford. The cover was printed by Courier/Westford. This book is printed on acid-free paper. Copyright  © 2009, 2006, 2004, 2001, 1998, 1995, 1992, 1989, 1986, 1981, 1976 John Wiley Sons, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107Read MoreStrategic Marketing Management337596 Words   |  1351 PagesStrategic Marketing Management Dedication This book is dedicated to the authors’ wives – Gillian and Rosie – and to Ben Gilligan for their support while it was being written. Acknowledgements Our thanks go to Janice Nunn for all the effort that she put in to the preparation of the manuscript. Strategic Marketing Management Planning, implementation and control Third edition Richard M.S. Wilson Emeritus Professor of Business Administration The Business School Loughborough University Read MoreExploring Corporate Strategy - Case164366 Words   |  658 Pagesand my nice City suits, I was completely naà ¯ve. Just a joke. I found that every Friday and Saturday night my door was taking  £30,000 and the security team was making  £40,000 on Ecstasy. It happens everywhere in the UK leisure business. There are all these fat bastards running chains of discos and bowling alleys, and none of them admits it. We went through a really traumatic time at the club.1 Palumbo changed his security team, bringing in security professionals from the North of England 1