Water

The American Electrical, Inc. (AEI) is in a middle of an environmental crisis that is likely to impact the listed firm’s stock value, profits, and brand. AEI’s relationships with its clients, employees, and the community in Marietta, Georgia where it is headquartered are also likely to be adversely affected in the face of the looming problem. The purpose of the communication plan, therefore, is to stem the damages that the company will be taking on by outlining and implementing a strategy to transparently communicate several key messages.

These messages include the roles of AEI, Multi-Phase Equipment, Inc. , Wiregrass Disposal, Inc. , Independent Laboratories, Inc. , and Georgia Department of Environmental Protection (GDEP) in the situation; a comprehensive timeline leading to the situation; the tactical response of AEI to the situation; as well as the strategic response of the firm to avoid similar situations from happening in the future. This communication plan is important because it will address all anticipated and possible concerns of AEI’s stakeholders, which include the people in Marietta, customers, employees, government agencies, stockholders, and the press.

Potential Crisis AEI is at the breach of crisis. This is because it is probable that it will be liable for the cleanup of PCBs, compliance with government regulations, and compensation of any damages caused by its and Multi-Phase’s environmental oversight. The situations that contributed to the crisis are the following. First, AEI failed to substantiate the findings of Independent Laboratories that the air, water and soil quality of Multi-Phase Equipment passed quality standards.

Apparently, Independent Laboratories’ samples were not representative and its work did not reveal the fact that illegally buried PCBs were hidden in the site. GDEP excavated in the far northeast corner of the Multi-Phase Equipment site that is adjacent to the WSA treatment plant. It uncovered 200 drums of PCBs, some 25 of these drums have rusted through, that were not supposed to have been buried there. Second, AEI bought Multi-Phase Equipment six months ago for $6. 5million in stock and cash without performing due diligence on its and its contractors’ environmental compliance.

Moreover, it failed to follow up on its possible interests in Wiregrass Disposal when the latter filed for bankruptcy protection. Wiregrass had been responsible for packaging, loading, transporting and properly disposing PCBs as well as maintaining state and federal records for materials removal from the Multi-Phase Equipment site. Third, AEI did not act proactively on possible connections between residential customers’ complaints about the foul taste and smell of water and the company’s resources adjacent to the Water & Sewer Authority treatment plant.

GDEP said that the buried PCBs leaked but the leakage area is contained and is unlikely to be connected to the foul water quality in Marietta. Its hypothesis is that the source of the unpleasant taste of water is algae. Fourth, AEI did not double-check if employees in the plant near where the PCBs were uncovered are indeed safe from health hazards. It must ensure that the employees are actually safe or else it will be liable for paying possible health claims from the workers, an incident that can further hurt AEI’s standing. Contacts

Because of the severity of the situation, the Executive Committee of the AEI must comprise the crisis management team. The Executive Committee can immediately manage corporate time and resources to ensure swift and suitable actions on the part of the company. The first message of the Executive Committee must be the urgent actions that AEI will be taking to remove the PCBs; its coordination with GDEP in terms of holistic environmental regulation compliance; and legal claims that it will might against Wiregrass Disposal and Independent Laboratories.

The second message of the Executive Committee must be the strategic plans and procedures that the company will implement for stringent and uncompromising environmental compliance. The company spokesperson must be Executive Committee member in charge of government regulations compliance. Walter Martinson can release special announcements to public, press, stockholders, customers and employees as necessary.

 This speech was clear and direct to the point, especially both parables David told. He did not force his ideas; he merely explained his realizations and suggested actions. It is how he said it though, that made me want to listen to him more. I sthrongly agree with the suggestion of taking the spotlight away from ourselves and our misfortunes and pointing it towards other things and people we encounter. Some people are really egocentric to care about others and their problems (which are probably worse than theirs).

This type of self-centred thinking affects our mood in a way where it may dampen our happiness. His speech raised my awareness on the arrogance of some people, and of mine too at times. He talked about learning to think, learning to control what you think. I feel that that is a step close to being a more positive and happy person. If you submit yourself to negativity, it will ruin you. We have choices; it’s just a matter of making the right one not only for you but for everyone involved.

What struck me the most was what he called the Capital T– Truth which is what matters — life before death. His message was to not stick to a routine just because you are afraid of change. Do not be dull, be alive. The major lesson I took from his speech was to enjoy every experience life offers us to make the most of our lives instead of living in the fast lane which will cause us to forget what and how things happened. Our lives are limited and so we shouldn’t make the least of it by worrying about mishaps and troubles. If we do, then that is truly a life wasted.

Fluoridation of H2O

Fluoridation of H2O is the controlled add-on of fluoride to a public H2O supply to cut down tooth decay. Fluoridated H2O has fluoride at a degree that is effectual for forestalling pits, this can happen of course or by adding fluoride. This pattern occurs chiefly in English-speaking states, as Continental Europe does non fluoridize public H2O supplies. Fluoridated H2O operates on tooth surfaces: in the oral cavity it creates low degrees of fluoride in spit, which reduces the rate at which tooth enamel demineralises and increases the rate at which it demineralises in the early phases of pits. A fluoridated compound is added to imbibing H2O, a procedure that in the United States costs an norm of about $ 0.94 per person-year. Bottled H2O typically has unknown fluoride degrees, and some domestic H2O filters take some or all fluoride.

Dental pits remain a major public wellness concern in most industrialised states, impacting a bigger per centum of schoolchildren and the huge bulk of grownups. Water fluoridization prevents pits in both kids and grownups, with surveies gauging about 30 per centum decrease in pits when H2O fluoridization is used by kids who already have entree to toothpaste and other beginnings of fluoride. Water fluoridization can do dental fluorosis, which can change the visual aspect of developing dentitions. These are normally non considered to be of aesthetic or public-health concern.

Fluoride ‘s effects

Fluoride ‘s effects depend on the entire day-to-day consumption of fluoride from all beginnings. Drinking H2O is typically the largest beginning ; other methods of fluoride therapy include fluoridization of toothpaste, salt, and milk. Water fluoridization, when executable and culturally acceptable, has significant advantages for subgroups at high hazard. The U.S. Centres for Disease Control listed H2O fluoridization as one of the 10 great public wellness accomplishments of the twentieth century. In contrast to most European states, who experienced significant diminutions in tooth decay without its usage due to the debut of fluoride toothpaste in the seventiess. Fluoridation may be more justified in the U.S. because of socio-economic inequalities in dental wellness and dental attention.

The end of H2O fluoridization is to forestall a chronic disease whose jobs peculiarly fall on kids and on the hapless. The usage of H2O fluoridization creates a struggle between the common good and single rights. Health and dental organisations worldwide have endorsed its safety and effectivity. Its usage began in 1945, following surveies of kids in a part where higher degrees of fluoride take topographic point of course in the H2O. Research workers discovered that moderate fluoridization prevents tooth decay and as of 2004 about 400 million people worldwide received fluoridated H2O.

Fluoridation besides prevents tooth decay by seting the concentration of fluoride in public H2O supplies.

Tooth decay is one of the most prevailing chronic diseases worldwide. Even though it is seldomly dangerous, tooth decay can do ; hurting and impair feeding, speech production, facial visual aspect, and credence into society, it greatly affects the quality of life of kids, peculiarly those of low socio-economic position.

Fluoridation does non impact the visual aspect, gustatory sensation, or odor of imbibing H2O. Normally it is made by adding one of three compounds to the H2O: Na fluoride, fluorosilicic acid, or Na fluorosilicate.

Sodium fluoride ( NaF ) was the first compound used and is the mention criterion. It is a white, odourless pulverization ; the crystalline signifier is preferred if physical handling is used, as it minimizes dust.Fluorosilicic acid ( H2SiF6 ) is an cheap liquid byproduct of phosphate fertiliser industry. It contains so much H2O, transportation can be expensive,Sodium fluorosilicate ( Na2SiF6 ) is a really all right crystal that is easier to transport than fluorosilicic acid.Among the chief benefits of fluoridization are:Community H2O fluoridization is an effectual, safe, and cheap manner to forestall tooth decay. This method of fluoride bringing benefits of all ages and despite socioeconomic position.

Brushing twice a twenty-four hours with a fluoride toothpaste is an easy manner to forestall tooth decay. Fluoridation, which was started in Grand Rapids, Michigan in 1945, has been used successfully in the United States for more than 50 old ages. Fluoride plants by change by reversaling the decay procedure. It keeps tooth enamel strong and solid.A

Community H2O fluoridization is considered one of 10 great public wellness accomplishments of the twentieth century.Of the 50 largest metropoliss in the United States, 42 have community H2O fluoridization. Fluoridation reaches 69 per centum of the population on public H2O supplies this is more than 184 million people.ACommunities with fluoridated imbibing H2O in the United States, Australia, Britain, Canada, Ireland, and New Zealand show dramatic decreases in tooth decay-those with fluoridated imbibing systems have less percent tooth decay.

About all tooth decay can be prevented when fluoridization is combined with dental sealers and other fluoride merchandises, such as toothpaste. Fluoride dietetic addendums can supply fluoride to those who do non hold equal degrees in their imbibing H2O. They are available as tablets, beads, or lozenges. Over-the-counter fluoride dental merchandises such as toothpastes and oral cavity rinses are effectual in forestalling decay.

Merchandises with high concentrations of fluoride that are applied in the dental office or prescribed for place usage offer extra protection for those at increased hazard of tooth decay.

Fluoride will go on to be of import for forestalling tooth decay in this age group. Older Americans are particularly susceptible to tooth decay because of open root surfaces and oral cavity waterlessness that may ensue from many medicines.

The hazards of fluoridization

Fluoride was foremost added to H2O supplies in Grand Rapids, in 1945 to forestall tooth decay. The pattern has had some controversial with some claiming it does small to forestall tooth decay and is unsafe for wellness. Most tooth doctors and public wellness functionaries province that it significantly lowers the rates of tooth decay and presents no of import wellness hazards. A bigger per centum of United States population drinks fluoridated H2O. But on contrary when towns and metropoliss across the state hold elector referenda on fluoridization, its usage has been rejected about half the clip. The long-running argument over fluoride usage and exposure was the topic of a citizens ‘ conference held at St. Lawrence University, on July 28-30. Most research workers report on fluoride did non measure the safety or benefits of H2O fluoridization. But did address specifically the current maximal degree of natural fluoride EPA allows in imbibing H2O and concluded nem con that fluoride at that concentration harms dentitions and castanets. A life-time of fluoride consumption in countries where the H2O is fluoridated at approximately 1 ppm can alter the quality of dentin and bone and may increase break rates for both.

Kidney patients and diabetics are at particular menace from fluoridated H2O because they tend to imbibe more liquid than healthy persons. Bottle-fed babies are besides at particular hazard if expression is assorted with fluoridated H2O. Peoples drink widely different sums of fluoridated H2O and other beginnings of exposure vary as in U.S persons are having immensely different doses of fluoride. Fluoride at degrees found in imbibing H2O affects encephalon map in grownups the research workers report indicates that the receptor cells in the encephalon can change in response to toxins and fluoride. Fluoride impairs the encephalon ‘s ability to transport out signalling maps, with the effect that messages that are passed along the many tracts that are likely to be uncomplete. It does this by interrupting the creative activity and dislocation of neurofilaments in the axons of nerve cells. It besides interferes with both primary and secondary signalling in the nervous system.

Fluoride may besides increase the figure of plaques and tangles in the encephalons of grownups, which could lend to dementia. These surveies were done by Isaacson utilizing rats, chronic exposure to sodium fluoride or aluminum fluoride in imbibing H2O, where by it led to plaques and tangles in the rats ‘ encephalons that are similar to the abnormalcies found in Alzheimer ‘s patients.

It appears furthermore that exposure to fluoride in the uterus and throughout early life lowers intelligence. The Epidemiologic surveies suggest that fluoridization of imbibing H2O decreases the figure of kids at the really bright terminal of the IQ spectrum and increases the figure in the low IQ part the study says.

Moderate fluorosis involves xanthous or brown mottling of dentition from inordinate fluoride exposure, occurs at rates somewhat high per centum in some fluoridated communities. Teeth with moderate fluorosis besides have weaker dentin with increased tubule size, he said. “ This may do grownup teeth break more easy.

Decision

In decision the benefit of H2O fluoridization as reference include ; reduced tooth decay, may merely be a map of delayed tooth eruption. It is known that lasting dentitions take longer to break out in kids who drink fluoridated H2O. Studies comparing decay rates in fluoridated and non-fluoridated communities normally assess the average figure of rotten, losing, and filled surfaces in lasting dentitions of 12-year-old kids. Thus its advisable to take fluoride out of the H2O supply, and utilize the money spent for fluoridization to advance better public wellness.

Blair Company employed over 4000 people with 380 having technical backgrounds and responsibilities Cash Flow: company sales revenue for 1996 would be almost $400 million with an expected profit close to $50 million Annual Growth in sales revenue: averaged 12% for the past 5 years Capital assets: ignored by Chatterjee Sales in the international division: would reach almost $40 million in 1996, about $70 million would come from Latin and south America, $30 million from Europe and $40 million from south Asia and Australia

Materials: ignored in the analyses though it’s important to scan the need materials for operation and theirs availability in the market, he only pointed that importing a few key components would be necessary at the start of the operations The Mckinsey 7 S model must be used here to finalize the step of internal marketing audit and this wasn’t done by chatterjee Mckinsey 7S.By analyzing these 7S we can know the firm’s core competences and it’s competitive advantage, which can be augmented for new market entry. Check List for internal audit:

While doing internal audit for strategic analysis, we can also use the below check list to recognize the organization’s strength and weaknesses and it must be weighted because some weaknesses are of less importance than others , while other strengths are of more importance than others 2-2 external microenvironment analyses: Stakeholders: these were ignored in the analyses although it’s important to scan the stakeholders for the organization Suppliers: again it was ignored in the analyses, our suggestions that the analyses of the suppliers must have be done from the following perspectives:

Their number, their prices, their bargain power (if any), their strategic alliances with competitors (if any present), their distribution channels, their management structure Consumers and demand forecast: Consumer’s analysis is essential for any strategic planning as they are the main concern for the organization and this must be done through: Demand forecasting Consumers (end users needs) First: Demand forecast: Chatterjee estimated the market potential based on collecting unit sales estimates for a 10-year period for 3 similar product categories –vacuum cleaners, sewing machines and colour televisions.

In addition a Delphi based research firm had provided him with estimates of unit sales of Aquagard, the largest selling water purifier in India. Chatterjee had used the data in two forecasting models available at Blair Company along with three subjective scenarios -realistic, optimistic and pessimistic. But it was conservative as they described only first time sales not replacement sales and it only applied to industry sales in larger urban areas which was the present industry focus. Second End Users needs: The target segment was around 40 million households plus those in another 4 million households that share common needs. They valued comfort and product choice. They liked foreign brands and would pay higher price for such brands. One thing that seemed certain was that many Indians felt the need for improved water quality. Folklore, newspapers, consumer’s activities and government officials regularly reinforced this need by describing the poor quality of water. Quality suffered especially during the monsoons and because of numerous leaks and unauthorized withdrawals from the water system

Better educated, wealthier and more health conscious consumers took steps to safeguard their family ‘s health and use water purifiers to purify the water all over the year. This is the target segment for Chatterjee. They are people who value comfort and product choice. They saw consumption of material goods as a way to a higher quality of life. o His analysis was missing forecasting the growth of this segment as his potential market is based on the educated aware segment but he missed forecasting the growth of this segment as the growth of the segment closely relates to the growth of the sales The analysis also missed a deeper look at the target segment. From our point of view, market survey should be conducted to show in details the target segment preferences, their demographics (age, social class, occupation, style of life) whether they like flavours to be added in the water or not, their ethnic direction, even their political views (if they are anti-Americans or not) as these factors may affect potential sales in the short and long terms . Consumers in the target market needs (according to Chatterjee) can be summarized as the following

1. Product performance to remove sediments, bacteria and viruses
2. Purchase price this is only concern for consumers who boiled water who count for 50% of the target market
3. Ease of installation
4. Warranty and availability of financing for purchase

Factors affecting consumer behaviour: We should analyze the uncontrollable and controllable factors affecting the Indian consumer behaviour, the uncontrollable factors are the sum of the macro environment analysis (PEST) but the controllable factors reflect the marketers efforts in designing the 7 PS in a way that make the product convenient for the consumer so from the analysis of the target market done by chatterjee.

Also there are other factors like motivation, life style, consumer perception and attitudes towards the product From the consumer analysis we can summarize that consumers in the target market need product of high quality, medium price (to be able to target a wider segments especially in the rural areas as they count 80% of the population and if they were successfully penetrated this will result in huge sales), ease of installation, warranty is needed, the emphasis on the need of healthy life style and better water will be a motive for consumers to buy the product.

For consumer analysis, we suggest that he should have used also the decision making process to know exactly what are the forces that affect the consumer’s buying behaviour Consumer buying decision process: So according to this model we should analyze how consumers in India become aware of the water problem and so the need for purifiers is recognized, then from where consumers get the information (from magazines, newspapers, reference groups) , then how they evaluate alternatives (based on price or quality or warranty or capacity . Then the purchase and post purchase evaluation Competition analysis:

It is very important for any strategic marketing plan to deeply analyze competition especially when it is done for new market entry Steps of Product /Market analysis

1. Identifying the generic need satisfied by the product categories (The need for improved water quality)
2. Identify the product categories (types / Classification) The need for water purifiers for household
3. Identify the specific product-markets Water purifiers, candle filters
4. Identify the product-variants (brands) competing with each other.

Positioning is determined according to the price strategies that Chatterjee mentioned whether if it skimming or penetration, product design for the skimming strategy would be noticeably superior with higher performance and quality longer warranty period, more features and more attractive appearance than the design of the penetration There are several positioning possibilities performance and taste, value for the money/low price, safety, health, convenience, attractive styling, avoiding diseases and health related bills and superior American technology. The only position he considered taken in the market was that occupied by Aquaguard protect family health and service at your doorstep.

Nineteenth-century America was a time of rapid growth and expansion. The movement of settlers further and further west accompanied by technological advances led to the major growth of cities and industries across the American frontier. However, it was the major innovations of transportation that had the most significant impact on the expansion of Midwestern and western America. The construction of canals and roads led to the increase in the use of stagecoaches, steamboats, and ultimately railroads. Railroads became extremely popular in America in the 1800’s.

The railroad industry itself began to boom; it was supported by its reputation for speed and efficiency. But, along with the booming industry of railroads came the strong debate that plagued Congress for years: should railroads be constructed as the major source of transportation over roads and canals? Although roads had become increasingly popular, the railroad industry was also viewed as being monopolistic, undemocratic, and unsafe. Despite the fact that railroads were sometimes monopolistic and undemocratic in that respect, railroads proved to be most vital in the expansion of the Midwest and western America.

It was the use of railroads over all other methods of transportation that aided in communication, the transportation of goods, and ultimately decreased the costs of transportation, all of which contributed to the expansion of the American frontier. Before the rise of railroads, stagecoaches and steamboats dominated the transportation industry. Stagecoaches were an effective means of transportation, but in the early 1800’s they were accompanied by Robert Fulton’s invention–the steamboat. The steamboat was a “combined river and overland transport system” that proved to be effective in transporting goods and people.

These locomotives were able to travel the rivers from the north and provide military posts and plantations with goods in the south while transporting passengers and immigrants. Prior to railroads, steamboats were the major and often only form of transporting settlers and maintaining communication between certain areas. Yet, the rise of railroads led to the inevitable fall of the steamboats, and the railroads took over as the major method of transportation. Although stagecoaches and steamboats were effective means of transportation, they were soon surpassed by railroads as the preferred method of travel.

Railroads began blanketing America in the 1820’s and by the 1860’s over 30,626 miles where covered by the railroads. This rapid growth of railways was due to the fact that railroads offered better economy, speed, and reliability than other methods of transportation at that time. First, a cheaper alternative to traveling by stage or steamboat was to use the railways. Railroad companies offered low-fare excursion rates that made it more economical to travel. Overall, the cost of transportation was reduced by $150 million by 1859 due to the economic advantages of traveling by rail.

Another advantage the railroads had over stages and steamboats was its high rate of speed. Railroads were two times as fast as stagecoaches and four times as fast as steamboats . Also, the railroads were much more reliable than the roads or canals. Railroads had the ability to travel all year long with few limitations, while both stagecoaches and steamboats had difficulty traveling during the winter months, “at no time… was stagecoach travel more trying and difficult than during cold, winter months” .

With that in mind, steamboats could not function or operate when the canals were frozen over. These difficulties were all contributing factors in the expansion of the railroad industry. With railroads quickly becoming the preferred method of travel, the demand began to rise and railroads were being built in high numbers in order to accommodate the increasing demand. The more railroads that were available meant more and more industries could use them to transport goods. There was better communication between the east and west, and people could also use the railroads to travel throughout the states.

And it was there that railroads began to have a significant impact on the development and expansion of American society. At the peak of expansion in the 1800’s, communication between the east and west was at the forefront of importance. A document from the State of Pennsylvania regarding railways in 1825 remarked that there is extreme “importance and necessity of effecting a communication between the eastern and western divisions of the commonwealth” . The appearance of the railroads in the early 19th century decreased and nearly diminished the communication gap as it provided a reliable and speedy method of communication.

As the railroads became more prominent across the United States, the western states naturally became linked to the Midwestern and eastern states through the Union Pacific Railroad. This railroad running from the east coast to the west coast opened up new doors in trans-continental communication. Businesses between the east and the west were able to communicate more effectively, and thus, trans-continental trade became more defined throughout the United States. The mail services began using railroads as well, which was a large factor in improving communication across the US.

Trans-continental communication was broadened simply by the increased number of people traveling across the country. And, even when the railroads seemed an impractical method of transportation, “they might make possible long lines of communication otherwise impracticable” . So, railroads played an important role in broadening communication between the east and the west. Another factor that was directly benefited from the widespread use of railroads was the transportation of goods between the states.

Before the rise of railroads, interstate trade was limited. Most goods were transported by way of boat, which was a slow, expensive transportation method limited to the water, or by wagons. Also, the types of goods that were transported were limited as well. Perishable items, which were potential victims to bacteria and spoilage, could not be preserved on boats, and therefore could not be traded throughout the states. These were inefficient methods of the transportation of goods. On the other hand, railroads allowed for better inter-continental trade.

The rail cars themselves could carry heavier loads of goods at higher speeds than boats or wagons, which made them extremely efficient methods for trade. Furthermore, goods transported on railroads faced a lower risk of damage or breakage than by boats because land travel is much more uniform in motion than water travel. Oppositely, canals posed a high risk of damage to the transported goods: “much damage has been received by goods from the roughness of the water” . With regards to perishable goods, the railroads offered safer traveling conditions by eliminating the bacteria produced by stagnant water as well as dust particles.

Some rail cars even served as “refrigerators”, so meat could be transported across the country. Of course, the tremendous speed of the railroads contributed to their efficiency in the transportation of goods. Along with communication and trade, the railroads made a significant change in the overall cost of transportation. First, railroads could be constructed across the country at one-third of the expense to build a single canal. This was primarily due to the on land construction, which took one-third of the time to build than canals, so workers were not being paid for such an extended period of time .

The railroads offered greater promise in making more money because they could be used for every season, all year long. The costs decreased even after the construction of railroads. The tolls on canals were expensive while the tolls on railroads were only one-third the cost per ton of that of canals. Also, railroads were preserved and repaired more easily than canals. While the cost to repair canals was extremely expensive, railroads were repaired at one-third the cost. When repairs were needed there was no delay for the passengers or the goods because it was quite customary to transfer from one rail station to another .

The decreased costs to build and repair both contributed to the lower rates of travel for the passengers. Railroad rates were based on cost, determined by weight, distance, and grade, not on profit. These decreased costs were important in promoting the railway industry because they offered such affordable means of traveling for their passengers. Overall, the rise of railroads across the United States in the 1800’s led to great benefits in communication, the transport of goods, and the overall decrease in cost of transportation. These factors all played important roles in the expansion of America.

Breakthroughs in communication and trade led to the rise of major cities and industries. The low rates offered by the railways allowed affordable means of transportation for all people, which brought more and more settlers westward. Oscar Winther relates that “the building of these western railroads had been in a very real sense frontier enterprises; they were, by and large, gigantic thrusts into and across the heretofore unsettled domain” . It was, in fact, the railroads that led to the ultimate expansion of the American frontier.

Materials and Apparatus: Olive oil, 95% ethanol, beaker, 50% sodium hydroxide solution, Boucher alter, hot plate, salt solution, magnetic stirrer, filter funnel, glass rod, filter paper, weighing balance, watch glass and measuring cylinder. From the diagram above, we can clearly see that the ionic (highly polar and charges) natures of salts makes them soluble in water. However, the non- polar hydrocarbon (aliphatic) tail of the soap molecules would cause them to be miscible with non-polar substances and enable them to dissolve in water.

Normally, solid soaps are consist of sodium salts of fatty acids and liquid soaps consist of potassium salts of fatty acids. Soap can be prepared by a process known as specification. Historically, soaps were made by boiling the fat of animal, lye and in a aqueous Noah and KOCH solution containing potash. Of course, this was done long time ago before anyone had any idea of understanding the chemical transformations. Now, we know that this process is a reaction of esters with a strong base such as KOCH or Noah.

The esters, triglycerides are the main constituents of vegetable oil and animal fats. The triglyceride is a trim-ester come along with 3 long fatty acid hydrocarbon chain. When triglyceride reacts with 3 equivalents strong aqueous base solution such as Noah or KOCH, the three ester bonds are hydroxide to yield 3 fatty acid salts (soap molecules) and one glycerol. The specification can also be called “base hydrolysis”. Industrially, soap is made by reaction of Noah together with vegetable fat. It is crucial to utilize enough Noah to make sure a complete specification Of triglycerides.

If there is insufficient base, time and heat provided, then the result would be incomplete specification and the soap product can be very brittle. If the specification is complete, Nasal is added to precipitate the soap. The aqueous portion is decanted off. The remaining substance would be crude soap which contains some impurities such as Nasal, Noah or glycerol. In the lab, we do not have enough time to purify the crude soap. This purification would consist of re-dissolving the soap in boiling water and then cooling and precipitating again by adding Nasal.

The soap in the experiment would be made out from olive oil, the chemical structure of olive oil (C52H9606) would be like this: Procedure: 1 . About ml Olive Oil was added to a mall_ beaker and dissolved it in ml of 95% ethanol. Excessive oil was not used to avoid additional ethanol to dissolve it. 2. When the olive oil was completely dissolved, the beaker was put on the hot plate and the temperature was maintained to about ICC. The flask was swirled to mix the solution. Next, ml of 50% Noah solution was poured down the glass-stirring rod intro the oil solution.

A small amount of the Noah solution was trickled down the rod very slowly and then stopped to stir. Then, a little more Noah was trickled in. The Noah solution was not poured into the oil too quickly. This was due to the reason that pouring the Noah solution too quickly Will cause the oil to separate from the Noah solution, and the final product would be failure. The mixture was stirred until it thickened. We wanted to avoid getting a mixture with 2 distinct layers Noah solution on the bottom and oil on the top.

When 2 distinct layers are forming, it was stirred vigorously using magnetic stirrer until the liquids are well stirred. 3. A small watch glass was placed over the mouth of the flask to minimize evaporation of the alcohol. 4. The oil-alcoholic-Noah solution was allowed to boil for at least 30 minutes. The undisclosed fat in the flask and solved by adding a little more ethanol and stirred. The temperature was kept just hot enough to have controlled boiling of the fat solution. 5. While the specification proceeded, a salt solution was prepared by completely dissolving go Nasal in 1 00 ml distilled water in a mall beaker.

After he salt was completely dissolved, about half of this salt solution was transferred to another beaker and both beakers of salt solution was placed on ice to cool them. 6. After 30 minutes, the fat solution was tested to see if specification was completed by placing a few drops of the solution in a test tube of distilled eater. When the fat droplets floated to the top, the specification was not completed and allowed it to boil for an additional 10 minutes. 7. When the specification was completed, the hot reaction mixture was poured carefully into one beaker of salt solution and stirred for a minute or two.

Green Florescent Protein, abbreviated as GFP, is a protein composed of 238 amino acids that is commonly found in mnemiopsis, comb Jelly. It has a major wavelength at 396 nm and a minor one at 475 nm. GFP is what gives mnemiopsis their bright green florescent glow. ultraviolet light, or blue light, is necessary to see the florescent glow of this protein. GFP is an irregular protein because It Is highly resistant to denaturation by temperature and PH. It can survive In temperatures up to 98 degrees and has a pH of 12. 2 due to Its complex exterior, called the beta barrel. At an pH higher than 12. It denatures. It also has an Isoelectric point at 5. 3. The peripheral beta barrel cannot be digested or broken apart by protease because of the strong bonds holding It together. The beta barrel protects the chromophore, which Is the substance which gives GFP Its green glow. When CFP Is extracted from the plasmid of an E. Coll or from a Jellyfish, It contains an array different contaminants making it difficult for scientist to do experiments with GFP. A procedure in purifying GFP from a crude cell extract to nearly 100% GFP so that it can be analyzed and used in scientific experiments and research is necessary.

The goal is to ptimize each protocol used to purify crude GFP. Methods Ammonium Sulfate Precipitation To purify the crude samples of GFP, the ion exchange method separates substances inside the test tube by similar charge. A sample of crude GFP of 7. 5 mL in a plastic tube was used for the experiment. Knowing that 43. 6 grams of ammonium sulfate in a 100 mL solution yields a 70% percent saturated solution, the proportion 43. 6g 11 00 mL=x/7. 5 mL was used to determine that 3. 27 grams of ammonium sulfate needs to be added to the experimental sample. After adding the ammonium sulfate, the solution was stirred gently to prevent frothing.

Once most of the solution is transferred, the tube was placed on a triple beam balance along with another tube that went through the same process. The centrifuge was set at 15,000 rpms for 15 minutes so that the hydrophobic materials will separate and become the supernatant while the GFP pellet will remain behind. Once the 15 minutes elapsed, a new pipette was used to remove the supernatant, leaving behind the pellet of GFP and hydrophilic contaminates. To remove the hydrophilic substances, 5 mL of 4 molar ammonium sulfate and 15 mL of 10 mL tris at a p of 8 was added Into the oak ridge entrifuge test tube.

The solution Is then put Into the centrifuge at 15,000 rpm for 15 minutes again. Once 15 minutes has passed, the supernatant, containing the GFP, was removed by a pipette and put In a microfuge. Hydrophobic Interaction Hgure yaropnoDlc Interactlon set up One molar ammonium sulfate was added to the column to wash the sample. Adding 1 molar ammonium sulfate washes the sample because a high salt concentration increases hydrophobicity of the GFP and the buffer, causing most of the GFP to be at the very top of the column. Substances that are hydrophilic get flushed out of the olumn while the more hydrophobic substances stay in the container.

After the column has been eluted with 1 molar ammonium sulfate, the tris buffer is added to the ammonium sulfate to dilute it into . 5 molar ammonium sulfate. The volume of 1 molar ammonium sulfate inside the oak ridge centrifuge test tube is the volume of the tris buffer that will be added. After the column chromatography has been flushed with . 5 molar ammonium sulfate, more hydrophobic substances will be flushed out since the hydrophobicity of the tris buffer and the GFP has decreased. This causes the GFP to spread out in the column. Finally the amount of . 5 molar ammonium sulfate is diluted with tris buffer to . 5 molar ammonium which should cause most of the GFP to be flushed out of the column along with other substances that are very hydrophobic. While this experiment is going on the liquid that comes out of the column is collected in multiple test tubes. These test tubes contain GFP and other contaminants. The solutions are than read by a spectrophotometer. Each test tube will be tested by the spectrophotometer so that a graph can be made. Anion Exchange Figure 2: Siphon Bridge set up for Anion Exchange Figure 3: Centricon Test Tube In order to use anion exchange, the starting condition of the sample needs to be in a low salt solution.

However after the GFP had gone through hydrophobic interaction, it was in a high salt solution. Before facing this dilemma, the fractions were pooled by centricon which decreases the overall sample volume by removing some buffer and salt solution. This greatly increases the GFP concentration in the samples. The fractions are placed in the centricon and then into a centrifuge for 25 minutes at 3,000 rpm to be separated by size. The large proteins stay in the entricon while buffer and salt solution goes into the plunger. To reduce the concentration of salt in the GFP sample, the sample is diluted 10 folds.

Since the amount of GFP that was restored was 18 mL, 162 mL of tris buffer needed to be added. The diluted GFP is then put in the chromatography container, containing positively charged DEAE which is attracted to the GFP at a low salt concentration. After the GFP has been completely filled, the column is connected to a beaker that contains a low salt concentration. the low salt concentration beaker is connected to a high salt concentration beaker. As one drop of low salt solution goes into the chromatography column, one drop of high salt solution goes into the low salt solution.

Gradually the salt concentration increases in the low salt beaker and in the column chromatography, causing GFP to spread down the container. The eluted GFP dripped out of the column chromatography to be collected in test tubes. I nree pnase partltlonlng Figure 4: Precipitate of GFP. T-butanol is one top while contaminates are on bottom GFP then went through three-phase partitioning, also known as TTP. The fractions taken after an anion exchange was 15 millilieter. Ten ml of 4 M ammonium sulfate was added to this volume to increase the salt concentration of the solution to 1. M, which is about 40% salt saturation. Twenty-five milliliters of t-butanol was added then added which was the same amount of ammonium sulfate and GFP in the container. The container was then placed in the centrifuge for ten minutes at 4600 RPM, causing the mixture to split into three layers; butanol on top, GFP in solution on the bottom, and precipitated contaminants in-between. The top layer of butanol and disk of precipitate were taken out. The volume of GFP solution was again matched in utanol and the container went into the centrifuge again. An aspirator was used to extract the GFP into a microfuge. . 6M ammonium sulfate was then added to the microfuge and the container was placed in a micro centrifuge for one minute at 13,000 RPM. Butanol and other contaminants that had not been take out previously formed a disc, was then taken out with an aspirator and the remaining GFP was then left in the microfuge. HPLC Figure 5: HPLC basic layout After the sample went through three phase partitioning, it was put through the High Performance Liquid Chromatography for a final purification. First liquid was put into the HPLC to clean out any previous GFP inside the loop of the HPLC and the column of the HPLC.

Then, GFP in the microfuge was sucked into an injector to be put into the HPLC. Pushing the top of the injector slowly, GFP entered into a loop inside the HPLC. Once the GFP was placed in the loop, a knob was turned clockwise to the word lock. The GFP was then sent to the column where it was purified further by size through the minuscule beads. About 6,000 pounds of pressure per square inch was produced by the HPLC to push the GFP through the beads. While this was occurring, a pectrophotometer connected to the HPLC read the wavelengths of substances.

Near the 396 nm wavelength, GFP was collected in a microfuge tube. A UV light was held near tne exlt 0T Results e HPLC to measure tne amount ng sample. Graph 1: Results of the sample after HIC at a wavelength of 395 nm Graph 2: Results of the sample after HIC at a wavelength of 280 nm Graph 3: Results of the sample after HIC of the entire spectrum Seventeen test tubes were received after the HIC purification process. A blank consisting of tris buffer and ammonium sulfate was sampled in the spectrophotometer against liquid from each of the seventeen test tubes.

Graph one represents the sample after HIC at a wavelength of 395 nm while graph two Results shows the results after HIC at a wavelength of 280 nm. After HIC, the fractions 12 to 16 were chosen for their purity and recovery of GFP. Graph one show the amount of GFP in each fraction number while graph two shows the total amount of protein in each fraction number. Graph three shows the spectrum of the entire sample. Graph 4: Results after Anion Exchange at a 397 nm wavelength Graph 5: Results after Anion Exchange at a 280 nm wavelength Graphing 6 Thirteen test tubes were collected from the Anion Exchange purification process.

This time the samples were blanked against tris buffer at 8. 0 pH and 0. 5 molar sodium chloride. Graph four shows results of the Anion Exchange at a 397 nm wavelength and graph five shows the results after Anion Exchange at a 280 nm wavelength. Once again, the graph at a 297 nm wavelength shows the amount of GFP while the graph at a 280 nm wavelength shows the amount of total protein. Graph six represents the results of the entire spectrum. The GFP peak was a lot more visible. Step Iotal sample (mL Abs (280) Total Protein Abs (397) GFP Ratio Crude sample 120 1600 . 25 At-ns042- 20 1 . 61 . 9 118 HIC 18 . 28 . 173 . 618 15 . 126 . 130 1. 03 3 Phase Partitioning . 01 n/a . 75 . 243 . 257 1. 06 Table 1: This is the overall data table. The second column shows the total volume at the start of each purification step. The following two columns are the peaks of the graphs at those wavelengths. The last column represents the ratio of GFP to the total Protein. The most desirable ratio is 1. 25. Dlscusslon The first method in purifying the crude GFP was using the ammonium sulfate precipitate. When ammonium sulfate is placed in water, it dissociates into ammonium (NH4+) and sulfate ions (S042-).

Water, composed of two hydrogen ions and one oxygen ion, is a polar molecule because the oxygen has a high electronegativity. Oxygen has a greater affinity making the oxygen portion of water negative and the hydrogen portion of the water positive. The dissociated positively charged ammonium ion is allured to the negatively charged oxygen while the negatively charged sulfate ions are attracted to the dissociated positively charged hydrogen. The attraction between the ammonium sulfate and the water was so strong that the GFP and other proteins were left unoccupied, causing them to precipitate.

When GFP in the 70% salt solution was placed into the centrifuge, substances such as DNA and RNA was removed because they became part of the supernatant. At a 70% salt concentration, only hydrophilic substances stay in solution while the more hydrophobic substances precipitate. When the GFP in a 25% solution of salt was placed in the centrifuge, the GFP and other substances went back into solution because there not enough water was occupied by the salt. Before the GFP is placed in the centrifuge, it must be balanced with another centrifuge with the same weight and the two containers must be placed across from one another.

This is vital because the centrifuge needs to be balanced when it is rotating at an incredibly fast speed. Failure to have balanced centrifuge containers can result in a broken centrifuge and loud sounds. Also when mixing the GFP with salt, it is important not the shake the container or frothing will occur, making it difficult to transfer the solution in to an oak ridge centrifuge tube. The second purification procedure that GFP underwent was hydrophobic interactions. During this purification, GFP binded to the non-polar Phenyl Sepharose beads because of its non-polar and hydrophobic traits.

However the water in tris buffer is strong enough to separate the attraction between GFP and the Phenyl Sepharose. Therefore a high salt concentration is necessary to occupy the water so that the GFP and the Phenyl Sepharose to be attracted together. At a high salt concentration, GFP with bind easily to the Phenyl Sepharose since very little water molecules would interfere with the attraction and at a low salt concentration, GFP would not bind easily to the Phenyl Sepharose because tnere wlll De a lot 0T unoccuplea water molecules tnat wlll De aDle to InterTere wltn the GFP and Phenyl Sepharose attraction.

Before the experiment, ten millimolar tris buffer at a pH of 8 was used to clean the column in order to keep the pH stable and to wash away the salt, ammonium sulfate, in the column. Removing the salt is vital because the buffer that once surrounds the salt will be allured to the hydrophobic benzene and to the hydrophobic patches on the GFP. Since the hydrophobic patches of the GFP are already filled, they will be flushed out, leaving mostly beads of benzene and the 10 millimolar tris buffer at a pH of 8. Once the column has been clean, it needs to be equilibrated so that the salt concentration is the same through the olumn.

The step gradient used, started ata 1 molar ammonium sulfate concentration and was halved until a . 25 molar concentration to separate substances by hydrophobicity. The third purification procedure was anion exchange. In this procedure, GFP and other contaminants are separated by charge. The beads in the containers are different from the beads from the hydrophobic interaction because on they have a different chemical called DEAE which makes them positively charged. GFP has both protons and electrons on it which is why it was not easily attracted to the DEAE, which is why the GFP is put in a basic solution.

Ata high pH, the amount of negatively charged hydroxide increases and these hydroxides are allured by the protons on the GFP. The protons are than neutralized, making GFP a negatively charged molecule. The isoelectric point of GFP is at a pH of 5. 3. Ata pH higher than 5. 3, it is negatively charged and when it is at a pH lower than 5. 3, it is positively charged. Once the column chromatography is filled with GFP and connected to a beaker of low salt which connected to a beaker of high salt, anion exchange occurs. As the salt concentration increases, the GFP slowly spreads down the column and eventually out f the column into test tubes.

Between the HIC and the Ion exchange chromatography, the sample the fractions were pooled and put in a centricon causing the GFP concentration in the samples to increase. This occurred because the ultrafilter only allowed particles smaller than protein to go in to the pusher. The large proteins stay in the centricon while buffer and salt solution goes into the plunger. The sample of GFP was also diluted 10 folds because the sample needs to be in a low salt solution to use anion exchange and after the GFP had gone through hydrophobic interaction, it was in a high salt solution.

The anion exchange method creates a continuous salt gradient because as one drop of low salt solution goes into the column chromatography, causing GFP to spread down the container. The follow procedure was the three phase partitioning purification. T-butanol and 1. 6 molar ammonium sulfate were essential for this procedure. T-butanol has a low density causing in to stay above the GFP solution. In addition it has an attraction for water and other hydrophobic substances causing 5 mL of water to be drawn out of the GFP sample and precipitated substances to float between the t-butanol and the GFP sample.

Fresh t-butanol is necessary after removing the old t-butanol with the contaminants because at that point, the salt concentration had increased since water was drawn out. was aDle to De preclpltatea Decause 0T tne nlgn salt concentration. The final procedure for purifying GFP was using the HPLC which separated substances by size. The beads used in the HPLC column are miniscule and porous. The pours on the beads give substances of the same size more opportunities to leave the HPLC at the same time. Since the beads are so small, high pressure is needed to push the GFP sample through the beads.

Naturally, smaller substances will exit the HPLC first while larger materials will exist last. In all scientific experiments room for error is unavoidable. During the HIC, IEX, three phase partitioning, and the HPLC, amounts of GFP were lost due to the GFP sticking to a container, a pipette, and even spills. During the HIC some of the GFP was lost due the overflowing the test tubes with liquid exiting the column. During the HPLC some GFP was lost because not all GFP dripping out of the HPLC went in to the microphage. Other errors include letting the column dry because the liquid was not dded to the beaker about the column.

During the spectrophotometer runs, the blank was no inserted correctly causing the reading of the GFP to be incorrect. In addition, the order in which the GFP samples were suppose to be placed in the spectrophotometer was messed up. Judging from the overall purification table, table 1, the purification was quite successful. Originally, the ratio was only . 25, but by the end of all the purification procedures, it obtained a ratio 1. 06. A 1. 25 ratio is most desirable and through the purification, the ratio was nearly reached. The anion exchange, three phase artitioning, and the HPLC purification were the most impacting procedures.

The anion exchange greatly increased the purity of the crude sample compared to the HIC purification. The three phase partitioning and HPLC purified the GFP even more. Some improvements to the protocols would be to start with the anion exchange purification so that overall, the salt solution would go from a low salt concentration to a higher salt concentration. This also eliminates the need to dilute the solution. In addition, an automatic machine could be used to shift the test tubes that collect the iquid exiting the columns to prevent overflowing test tubes and the risk losing GFP.

GFP is unique because of its florescent glow. This glow can be used as a marker or an indicator. If a glowing marker could be placed on infectious cells such as tumor cells or cancerous cells, it would revolutionize the treatment of these diseases because doctors will be able to track where the harmful cells are. In addition, if it is possible to trigger the florescence of GFP with UV light, it can eventually be used in light bulbs to produce light. GFP light bulbs would last for an incredibly long time ince they are very resistant to denaturing.

In addition, in vehicles, GFP can be mixed in the motor oil, transmission oil, power steering oil, air conditioning oil, and other oils so that if a leak occurs in a car, it can easily be spotted by shinning UV light on the car. The purification of GFP can lead to endless new innovations in electrical engineering, automotive repair, and curing deadly diseases.