Determination of Glucose Concentration Using Trinder Method

Determination of Glucose Concentration Using Trinder Method The Trinder method is used to determine glucose concentration only, (Lott et al, 1975). This method was first described by Trinder in 1969 thus named after him, (Lott et al, 1975). It uses an enzyme glucose oxidase for the first reaction and peroxidase for the second reaction thus the name of the enzyme Glucose oxidase/peroxidase (GODPOD), (Meiattin, 1973). Enzymes are biological or any chemical catalysts that speed up a reaction without it being used up, (Jan, 2010). It functions to catalyse a reaction by lowering the activation energy of the reaction. Activation energy is the energy needed to initiate the reaction. It is a point of high energy and requires more energy than the substrates. An enzyme also contains an active site for the substrate to catalyse the reaction. Its efficiency depends on the concentration of the substrate and conditions like temperature or pH, (Hames et al, 2005). The Trinder method, is based on two sequential enzymatic reactions, the first one involves the oxidation of glucose to gluconic acid and H2O2, (Casabnon et al, 2005). This reaction is catalysed by the enzyme Glucose oxidase. Then, the H2O2produced is quantified by a chromogenic reaction with peroxidase (POD), as the enzyme that catalyses the reaction with the reduced dye, (Casabnon et al, 2005). The oxidised dye changes colour to pink or red according to the glucose concentration. The colour formed is stable at room temperature for at least two hours after development, (Anon, 2010). The main advantage of this method is that it is very specific. It doesnt target other sugars except glucose. It is also simple straight forward and easy to manipulate. Its results are very reliable and specific, (Bauninger, 1974). Its final products are stable as they are not reactive at room temperature. Other colorimetric methods to identify glucose include, oxidation of glucose in the presence of Cu+2 to give Cu2O, and different types of Chromatography, (Casabnon et al, 2005). Glucose can be detected with o-toledine or other amines, (Casabnon et al, 2005). The experiment had to check for the specificity of the assay thus other carbohydrates were assayed. These were galactose, fructose, maltose and ribose. Maltose is a disaccharide which is made up of two glucose molecules joined together by a glycosidic bond. Galactose is a major sugar found in milk, (Hames et al, 2005). Its structure consists of six carbons with a glycosidic bond to join the next glucose molecule, (Berg et al, 2007). Fructose is abundant in fruits. It is a monosaccharide with six carbons as its structure. Ribose is a pentose sugar molecule with 5 carbons. It is mostly abundant in the nucleotides. The aim of the experiment was to determine glucose concentration in different concentrated solutions and unknown solutions. The specificity of the assay was to be determined by application of the assay on different sugar molecules. A standard curve was to be drawn from the absorbencies acquired from the spectrophotometer at 515nm Materials 12 Test tubes 2 Long pipette tips 1ml of 0.5mM Fructose 1ml of 0.5mM Maltose 1ml of 0.5mM Galactose 1ml of Unknown Glucose 1ml of 0.5mM Ribose 5ml of 0, 5mM Glucose 5ml of Distilled water 7ml of 0.1% Phenol 20ml of GODPOD Reagent 9 pipette tips 8 Disposable Cuvettes Black Marker Stop watch P100 Pipette Automatic pipette Recoding paper and pen Spectrophotometer at 515nm Water bath at 37oC Test tubes rack Blotting paper Method The test tubes were marked T1 to T6, for those that had to be inoculated with glucose and S1 to S6, for those that had to be inoculated with different types of sugars. They were arranged in order of concentration on the rack. One row was left out for agitating the test tubes. Inoculation commenced by transferring glucose into different test tubes T1being the least concentrated. 0.5mM of Glucose was transferred using a P100 pipette and not changing the tip. 0.2cm3 was inoculated into T2, 0.4 cm3 into T3, 0.6 cm3 into T4, 0.8cm3 into T5 and 1.0 cm3into T6. Distilled water was then inoculated into the test tubes using a different tip to avoid cross contamination. One centimetre cubed was inoculated into T1, 0.8 cm3 into T2, 0.6 cm3 into T3, 0.4 cm3 into T4 and 0.2 cm3 into T5. There was no water inoculated into the last tube T6. Phenol was then inoculated into all the twelve test tubes. It was transferred using a different tip to avoid cross contamination. One centimetre cubed of different sugars were inoculated in specific S tubes. 0.5mM of Galactose was inoculated into S1, 0.5mM of Glucose was inoculated into S2, Glucose unknown was inoculated into S3, 0.5mM of Fructose was inoculated into S4, 0.5mM of Maltose was inoculated into S5 and then 0.5mM of Ribose was inoculated into S6. These transfers were done with different tips for different sugars. One and half millilitres of GODPOD reagent was then inoculated into all the test tubes using an automatic pipette and a long pipette tip. The test tubes were then agitated on the rack and incubated in the water bath for forty minutes. The temperature was constantly checked during incubation. After forty minutes, the solutions changed colour from colourless to light pink according to the concentration. These different solutions were then read on a spectrophotometer in a cuvette. The spectrophotometer was zeroed at first then absorbencies of Glucose and other sugars were read and recorded. A cuvette was wiped on the soft side to minimize absorbencies caused by contamination. These different absorbencies were recorded on a table. Discussion The reactions of glucose with the GODPOD were slow due to the fact that the enzyme was stored in ice thus it took long for the reaction to take place. The enzymes structure was disrupted because it was kept in cold thus it took time to equilibrate with the conditions. The reason why GODPOD was slow to react was because it is sensitive to its environment, (Teal et al, 1985). Enzymes are sensitive to pressure, temperature and pH. This added to the fact that the colour produced was not very dark because the enzyme was adjusting to the conditions. The enzyme also didnt denature because it was kept in ice at 4oC not in the heat above 40oC. On the graph, the points that are not on the line of best fit might have appeared because there might have been a competitive inhibitor thus the reaction didnt go on well owing to reduced absorbance. The inhibitor might have been so because of cross contamination. The same pipette might have been used to transfer the solutions thus cross contamination. The specificity of the enzymes might have caused the other solutions not to produce reliable results. Enzymes are sensitive to pH, (Jan, 2010). The pH of the test tube might have been so low or higher than the required thus some of the points are not in the line of best fit. This might have been avoided by carrying out the experiment repeatedly and then getting average values. When the enzyme was applied to the other sugars, there was no absorbance at all because the enzyme is specific to one substrate thus it didnt catalyse the reaction of other sugars and GODPOD. There might have no absorbance because these sugars might have their own wavelengths they absorb the light. This might have been avoided by scanning the various wavelengths and determine the exact wavelength. The unknown glucoses were determined by the use of the graph. The line of best fit was used to determine the glucose concentration. An equation was used also to determine the concentrations. The other points not on the line of the best fit might be there because the transitional state might have been great, thus when thirty minutes had passed, the enzyme had not gone past the transitional stage. This might have been characteristic of the colours produced according to the concentration of the different solutions. The unknown glucose solutions showed to have the same concentration as the stock solution. It might have the same compounds and properties as the stock solution. The results of the experiment were according to the literature values. This was highlighted by the absorbencies of stock solutions and different sugars. The specificity of the reaction was achieved. In conclusion, the aims of the experiment were achieved by obtain reliable data and results. The standard curve showed the absorbencies of the different solutions and unknowns.

billy Budd By Herman Melville: Captain Vere Essay -- essays research p

"Billy Budd" by Herman Melville: Captain Vere In the novella "Billy Budd" by Herman Melville, Captain Vere is the â€Å" tragic hero†. he is neither good nor evil, but rather a man whose concept of order, discipline, and legality forces him to obey the codes of an authority higher than himself even though he may be in personal disagreement. Captain Vere is sailor that is distinctive even in a time of renowned sailors. He has noble blood in him, but his advancement through the naval ranks to that of captain is due more to his outstanding service and personal merits than through any connections that he may have had. He was made a post-captain because of his â€Å"gallantry in the West Indian waters as flag-lieutenant under Rodney in that admiral's c... billy Budd By Herman Melville: Captain Vere Essay -- essays research p "Billy Budd" by Herman Melville: Captain Vere In the novella "Billy Budd" by Herman Melville, Captain Vere is the â€Å" tragic hero†. he is neither good nor evil, but rather a man whose concept of order, discipline, and legality forces him to obey the codes of an authority higher than himself even though he may be in personal disagreement. Captain Vere is sailor that is distinctive even in a time of renowned sailors. He has noble blood in him, but his advancement through the naval ranks to that of captain is due more to his outstanding service and personal merits than through any connections that he may have had. He was made a post-captain because of his â€Å"gallantry in the West Indian waters as flag-lieutenant under Rodney in that admiral's c...

The Right to Information Act

The Right to Information Act, 2005 is a landmark legislation in the history of independent India. Before the passing of this historic Act, there was â€Å"The Freedom of Information Act, 2002† But to ensure smoother and greater access to information and to make it more progressive, participatory, and meaningful certain important changes was felt necessary. The Government examined the suggestions made by the National Advisory Council and others and decided to make a number of changes in the said law. The Government in view of the significant changes proposed in the existing Act, decided to repeal the Freedom of Information Act, 2002. As a result, â€Å"The Right to Information Bill, 2005†, passed by Lok Sabha on 11th May, 2005 and by Rajya Sabha on 15th June, 2005 and received the assent of the President on 15th June,2005. The Right to Information Act, 2005 contains VI Chapters and 31 Sections. Some of the important Sections of the RTI Act can be stated as follows—- Under the provisions of the RTI Act, 2005, any citizen can seek information in writing in the form of an application or by e-mail and should be addressed to the Public Information Officer (PIO) or Assistant PIO of the concerned office/public authority either in English Hindi or in the official language of the area. Even if one submits an application for an information to a PIO whose public authority is not in possession of information, has to accept the information and forward the same to the concerned public authority about such transfer [section 6(3)] In such cases, the second public authority becomes responsible for providing the applicant with the information that is requested within prescribed time limit [section 5(5)] Reason for securing information is not required to be stated in the application. Normally the PIO is to provide the information within 30 days. However in case of life and liberty, the PIO is bound to provide information within 48 hours. If the required information is part of a proactive disclosure of section 4 (I) (b), then it should be provided instantly by the PIO without any application fee, except the printing cost of the information. No prescribed format is necessary to apply under RTI Act,2005. An application on a plain paper comprising full postal address of the applicant, PIO or Assistant PIO’s address, details of information required with a statement that the information is sought under RTI Act 2005, are the basic requirements. Application fee of Rs. 10 (in case of Government of Assam office/organization) is required in case of a Government of Indian office/organization. If the applicant does not receive the information or is not satisfied with the information received from the PIO, he/she can file an appeal with the first appellate authority (FAA) under section 19 (I). There is no prescribed format for an appeal. Moreover, neither Govt. of India nor Govt. of Assam prescribed any fees for the first appeal till now. The Act prescribed maximum 45 days time for the disposal of first appeal by the FAA. If an applicant does not receive information within 45 days, then he/she can take the matter forward to the second appeal stage. The RTI Act, 2005 is a historic legislation and no such strong provision has been made in any Act or code in India till date. The right to information covers inspections of works, document, record and its certified copy and information in the form of diskettes, floppies, video cassettes in electronic form, taped or stored information in computers etc. Though certain information are prohibited, every public authority is under obligations to provide information on written request or request by electronic means with payment of fee. The Act vehemently provides for restrictions for third party information. It has been stated earlier that there is the appellate provision in the Act. As such, the appeal against the decision of central information officer and state information officer can be made to a senior officer in rank. The penalty for refusal to receive an application for information or for not providing information is Rs. 250 per day, but the total amount of penalty should not exceed Rs. 25000 The Central Information Commission is to be constituted by the Central Govt. and State Information Commission by the respective state governments. In spite of having the various important and powerful provisions the state Governments are not ready to implement the RTI Act, 2005, in letter and spirit. The case is pertinent in Assam too, which so far has seen the government drag its feet when it comes to implementing the various provisions under the Act. Consequently Assam is among those states where the State Information Commission has been unable to perform to the desired extent, or simply failed to bring in probity and transparency in matters of public finance. When state Government will come forward to implement the provisions of RTI Act then only the true objectives of the Act will be fulfilled. The Act will be able to curb corruption and misuse of taxpayer’s money will be stopped, and can be utilized for the sake of common good and welfare of the state. The posts of Information Commissioner should be filled up by qualified and impartial persons. Free legal education should be provided to the people, to be more conscious about the legal benefit of the Act and help to access the information and exert their rights in true spirit.

What Are the Guidelines for Issuing an AMBER Alert

When children are reported missing, an Amber Alert sometimes is issued, but sometimes its not. Thats because not all missing child cases meet the guidelines necessary for an Amber Alert to be issued. Amber Alerts are issued to call the publics attention to a child who has been abducted and is at risk of being harmed. Information about the child is broadcast throughout the area via news media, on the Internet, and by other means, such as digital highway billboards and signs. Guidelines Although each state has its own guidelines for issuing Amber Alerts, these are the guidelines recommended by the U.S. Department of Justice (DOJ): There is a reasonable belief by law enforcement that an abduction has occurred.The law enforcement agency believes that the child is in imminent danger of serious bodily injury or death.There is enough descriptive information about the victim and the abduction for law enforcement to issue an Amber Alert to assist in the recovery of the child.The abduction is of a child 17 years old or younger.The childs name and other critical data elements have been entered into the National Crime Information Center (NCIC) comptuer system. Runaways This explains why Amber Alerts usually arent issued when children are held past the agreed upon time by noncustodial parents: They are not considered to be at risk for bodily harm. However, if there is evidence that the parent might be a danger to the child, an Amber Alert can be issued. Also, if there is not an adequate description of the child, the suspected abductor, or the vehicle in which the child was abducted, Amber Alerts can be ineffective. Issuing alerts in the absence of significant evidence that an abduction has taken place could lead to abuse of the Amber Alert system and ultimately weaken its effectiveness, according to the DOJ. This is the reason that alerts are not issued for runaways. History On Jan. 13, 1996, a witness saw Amber Hagerman, a 9†year†old Arlington, Texas, girl, being snatched from her bicycle in a parking lot. Four days later, Amber’s body was found 3.2 miles from her home. Among the Dallas†Fort Worth area residents outraged by the abduction was Diana Simone. She suggested that an emergency alert system be implemented to notify residents and allow them to assist in searching for abducted children. Simone asked that such a program by dedicated to Ambers memory. The program, known as America’s Missing: Broadcast Emergency Response plan, or Amber Alert plan, was instituted later that year in the Dallas†Fort Worth area through the National Center for Missing Exploited Children and spread across the country. Statistics According to the U.S. Department of Justice Office of Justice Programs: As of April 2019,  957 children  had been rescued specifically because of Amber Alerts.As of March 2019, there are  83 Amber Plans  throughout the United States.From January 1 to December 31, 2017, 195 Amber Alerts were issued in the U.S. involving 263 children. Of those cases, 193 resulted in a recovery, 39 of which were a direct result of an Amber Alert being issued. Sources Amber Alert Statistics. U.S. Department of Justice Office of Justice Programs.2017 Amber Alert Report. National Center for Missing Exploited Children.