Bioanalytical Technique Practical

Bioanalytical Technique Practical Introduction Improvement in technology has widened the domain of bioanalytics, reliable and reproducible data can be obtained from several instruments and protocols. The drug arena has become really competitive and it is thus imperative that an understanding of the different techniques is crucial to the isolation and analysis of biomolecules. This report is focused on the BCA assay for protein estimation and data analysis of SEC using a UPLC system. The BCA assay is a modified assay that is used for the detection and quantification of total protein in a given sample. The assay generates a purple colour which is as a result of the chelation reaction of bicinchoninic acid with cuprous ions. The complex formed as a result of the reaction is known to exhibit a very strong absorbance at a wavelength of 562nm and this shows an increasing linearity with the amount of protein in a given sample. Two main components make up the assay; the standard curve and the unknown protein sample. The BCA assay is widely used because of its sensitivity and compatibility with detergents and several other buffer types. The drawback however with the assay is that it is not as rapid as some other estimation method such as the Bradford due the incubation time required and moreover it is not an endpoint reaction as colour continues to develop even af ter incubation. The second part of this report is concerned with running a system suitability test on the waters BEH200 SEC UPLC instrument. The American and European Pharmacopeia specifically mentioned that the requirements for a system suitability testing on the day of analysis showing that it is fit for its intended use. It is worth mentioning that this has no bearing with the qualification of the instrument. Failure of any of the parameters simply means that an assay cannot commence. This testing is concerned more about the method on the day of analysis rather than the instrument per se. 1.1 Materials: Pipettes and appropriate tips Microcentrifuge tubes Microwell Plates HPLC Vials Bovine Serum Albumin Protein 2mg/ml BCA Reagent Deionised Water Perkin Elmer Plate Reader 100Mm sodium Phosphate Buffer Waters Aquity H Class Bio UPLC instrument Waters BEH200 SEC UPLC Column 1.2 Preparation of Standards: Standards were prepared as per instruction manual Table 1: Preparation of Protein Standards Calculations: Dilution factor = concentration of stock solution / concentration of diluted solution Volume of stock to add to water = Required volume of diluted solution/ Dilution factor Volume of water to add = required final volume / Volume of stock required 1.3 Preparation of Sample: The sample was prepared as per the instructions on the practical manual. Table 2: Test Sample Dilution 1.4 Preparation of BCA Reagent and Well: The BCA reagent was prepared and the 96 –well microplate was prepared and read in the [emailprotected] 562nm as per the instruction manual. 1.5 Data Analysis: Calculation: Equation of the linear least square fit can be represented as outlined below. Y = 0.0008 (X) – 0.0051 0.035691= 0.0008 (X) – 0.0051 X = 0.035691 + 0.0051/0.0008 X = 0.040791/0.0008 X = 50.98875 Taking the dilution factor into account we multiply by 5 The protein concentration is thus 50.98875 x 5 = 254.94mg/ml Discussion: The sample data had an anomaly, showing a negative reading on the third well. This is suggestive of contamination. The possibility of interference from the reagent can be ruled out because the standard was treated the same way and also taking into cognisance the fact that the experiment was not carried out under a non-denaturing condition. The likely cause could be due to dirt on the Microwell thus blocking out the necessary wavelength for the absorbance reading or the sampling pipette not delivering the right amount of reagent. The intensity of the colour change for the third well was observed to be less than the other two wells. There is also the possibility of the sample not being vortexed properly or sample settling to the bottom of tube. As mentioned earlier there seem to be an anomaly with our absorbance reading and this can be validated from our standard curve as it is not quite linear and on this basis we cannot absolutely rely on the result of the experiment. 2.1 Size Exclusion Chromatography Experiment The priming and purging of the UPLC instrument was carried out by the trainer as per the instruction manual. System suitability testing was then carried out to ensure that it is fit for purpose. The test serves to assure the reproducibility of the instrument and the method. It is a regulatory requirement which was mentioned in both the EU and US pharmacopoeias. The testing is important as it can allow for critical factors that could affect the performance of the instrument to be adjusted to meet the test criteria. Parameters such as the resolution, efficiency of the column, tailing factors, relative standard deviation etc. are used as criteria for comparison with regards to standards and test samples. The table below details the results obtained from the system suitability testing, reference standard and our test sample. The UPLC system used in our experiment can be said to be fit for purpose taking into consideration, the system suitability test. The results obtained were within our test criteria. The resolution of the peak and standard deviation of the different retention time was less than 1 which as a rule of the thumb is quite acceptable. Comparing the test sample to the reference standard one would not fail to notice that the first peak in the reference standard was a dimer while the second peak was a monomer but in our test sample the retention time of the first peak was really short showing evidence of a high molecular weight aggregation .Also from our result the second peak was our product dimer while the third peak was our product monomer. This result serves to highlight the mechanism of protein aggregation and the reason why it should be minimised as it impacts on the yield of the product and moreover it can affect the potency and therapeutic potential of the parenteral. It is also worth mentioning that sometimes early elution may not necessarily mean that there is aggregation, it could be for the simple reason that sometimes intrinsically unstructured proteins can elute so fast that they tend to behave like aggregates. A molecular weight comparison testing can be used to differentiate them. From our experiment the test sample showed increase aggregation and this can be explained given the fact that the experiment was not carried out under a non-denaturing environment. The possibility of column contamination or buffer contamination can help to encourage aggregation. It is also important that samples should be free of extraneous particles during injection as this can also be a determining factor. The changing environment of the mobile phase can also be construed as a possible cause of the aggregation observed. The temperature of the instrument is another factor that can play a role and as we all know that the Arrhenius theory of a 10Â °C increase in temperature speeding up a reaction does not relate to proteins as it rather opens up the pathway of denaturation and aggregation. Questions: Estimation of protein concentration is important as we have to know the amount of protein in our final product after fermentation to know if the bioprocess has to be optimised with regards to the expected titre value. The concentration of the protein can also allow for the portioning of the product into the right dosage formulation, certain therapeutic proteins are required in a very high dosage form and their production can be sometimes targeted at a particular section of the population e.g. during an epidemic outbreak to ensure potency and biological activity. The knowledge of the concentration of proteins can also allow us to work out the economy of scale with regards to the profit margin taking into account, the expense incurred in research and development and other aspect of the production process. It is also important to estimate the amount of protein in our biomass so as to be able to optimise our subsequent purification steps. The estimation of the protein concentration can a lso give us an idea of product related impurities and those associated with the process. Proteins are very complex molecules and are prone to several types of condition than cause instability from the starting stage of production to the end of their shelf life. Aggregation can be described in a layman’s term as the propensity for proteins to stick together under conditions such as a slight increase in temperature, pH, shear force, ionic strength of the solution they are contained in etc. Aggregation have been seen to cause delay in several novel biologics due to the debilitating effect on the health of the population that the drug is directed at and also in the context of compliance to regulatory authority as there is a specification to the amount of aggregates that can be allowed. It is extremely difficult if not impossible to totally eradicate aggregation from the process. It is worth mentioning however that the mechanism of aggregation is still subject to debate as it has not been fully understood. Aggregation can be reversible or irreversible depending on the stage it has attained as can be loss of primary structure. The potency of biologics as we all know are normally related to them being in their native structure, in most instances aggregation leads to the loss of activity and moreover the overall yield of the biotherapeutic is greatly affected. Aggregation has also been known to spur immune response in patients that have been administered with protein therapeutics affected by aggregation this could be by way of the neutralisation of antibodies that helps to ensure the effectiveness of the drug. In a worst case scenario the immunogenic reaction can lead to incurable conditions such as seen in patients with pure red cell aplasia where the red blood cells are attacked and blood transfusion is needed for life. The route of administration of biologics is intravenous and the presence of aggregation especially those of very high molecular size can result in the blockage of blood vessels. It is thus very important that at each stage of our production testing should be carried out to check for aggrega tion. Size exclusion chromatography is a purification system that exploits the molecular size of the compound of interest. Simply put it works just like a molecular sieve, smaller particles passes through the sieve which is the stationary phase and could be a bead coupled to a resin. The pore size of the beads are defined and on this basis it will only allow certain particle sizes to pass through while excluding those that are too large for the pore. The larger particles because they are not passing through the beads are thus excluded quickly, their retention time is thus said to be short. The smaller particles are retained longer while the larger particles earlier mentioned are eluted through the void volume. Different gels in use would typically have different pore sizes and can be used to determine the size of the molecules to be separated. Despite all the numerous advantages of size exclusion chromatography which has made it the gold standard over the years for analysing protein aggregation there are still some limitation associated this method. The possibility of the stationary phase and the analyte reacting together can be sometimes rife thus leading to a longer retention time which serves to mimic the compound as being of low molecular size. The cost associated with running this type of separation technique can also be enormous due to the fact that large columns and eluents are required and this serves to add to the overall cost of the unit operation. In comparison to other modes of separation, size exclusion chromatography can be said to have an inherent low resolution as there is a limited range of molecular weight that can be separated as a result of dependence on the pore size of the beads in use. There is also the possibility of proteolytic degradation as the protein of interest can become targets for proteolyt ic enzymes still present in solution. The accuracy of this technique can sometimes come into question due to the fact some aggregates will remain in solution and as such would not be detected. Also taking into consideration the fact that larger molecular aggregate leaves the column through the void volume, there is also the possibility. The possibility of the polymer in use to degrade is also a drawback as this can occur at a very high flow rate. The high flow rate as mentioned earlier can degrade the polymer and it also has the ability of altering the geometry of the beads in use making the separation technique inefficient

Philippine Literature Essay

Multilingualism became one of the key factors why unity cannot be thoroughly assessed in a specific country. It became the basis here in our country that having many languages implies a rich culture, diverse culture but carries with it the brand that national unity might be generally impossible due to regionalistic factors. Our country experienced so many shifts in language policy. Colonizers from Spain and America became the key contributors for it to happen. Looking back on the time of Spanish regime. Not all people can speak Spanish so the Spaniards made serious efforts to use Spanish to teach and speak it and make it as a national language. During that time, increasing number of tagalong users were growing. Slowly, it established the title as one of the major local language and until now, was strengthened and included as part of the school curriculum. To counter the growing number of Filipinos using other foreign languages, local literatures emerged to battle out the western culture. Filipinos during that time made use of their local language that is commonly spoken on that specific place and then use this language to write literary pieces like poets, production of drama; and sometimes as a form of entertainment during fiestas, other writers create a play using the foreign language emphasizing how it is awkward to speak the foreign language using ridicule included in the play. Though Filipinos made serious efforts to preserve the culture, some are slowly being converted to use the foreign language, and that approach is in the form of a religion. During the Spanish regime, it became a must to use Spanish as a medium of speaking while inside the church. And many people was moved that if they will not do church work, they will not go to heaven, they became a â€Å"panatiko† without realizing that religion; Roman Catholicism; became the way for the Spaniards totally colonize the culture and imply the Spanish culture.

Topics for a Rogerian Argument Essay Explained

Topics for a Rogerian Argument Essay Explained The Unexpected Truth About Topics for a Rogerian Argument Essay A statement form creates a speaker defend their point, even though the ultimate aim is to obtain a consensus. Although the aim isn't necessarily to change the person who you are speaking to's mind, the Rogerian technique is still considered a sort of argumentation because you are persuading your opponent to at least respect your viewpoint. Balanced Your primary goal is to be utterly objective and present your ideas and ideas of your opponents equally. Add support and the primary reasons to back up your position. One of the greatest approaches to change anybody's mind is with an emotional investment. Finding the compromises can help you in your life. If you're still searching for assistance with your Rogerian essay, there are many places you may turn to. Be attentive, as this sort of academic paper differs a great deal from the others. What You Need to Know About Topics for a Rogerian Argument Essay A Rogerian essay is intended to help students understand issues from two distinct perspectives to produce an agreeable solution. It is not aimed at changing someone else's mind, so forget about aggressive strategies and look for reasonable dialogue. An argumentative essay requires you to choose a topic and have a position on it. The expression Rogerian essay can throw off lots of individuals. Quite frequently, the very best topic is one which you truly care about, but you also will need to get well prepared to research it. Selecting an emotional topic is also a superb idea. For the aims of this essay, this issue of Hiroshima and Nagasaki Bombing is going to be discussed. Having a gun by every citizen is good for the society. It's possible to decrease the range of accidental deaths brought on by guns without banning them completely. Some feel it's extremely easy for just about anyone to acquire access to a handgun. As stated by the law, marijuana ought to be legal. Let's see the way the Rogerian essay may look on a concise example to comprehend the structure. Thus, let's see what you want to do in order to compose a great Rogerian essay within the next section. To compose a fantastic Rogerian essay you should pass through the next six steps. Consider that assignments generally don't call for a title to every paragraph. In addition, it helps simplify the writing process as you can develop an outline for your paper and quickly produce the ideas you would like to have in your article. E-learning is an obligatory part of contemporary education. Apps utilizing evidence-based practices have the capability to transform a patient-provider relationship. Produce and download marriage biodata online, Bio data format is similar to a resume used for matrimonial purposes which assists people to acquire a summary of the other individual. Furthermore, you ought to be precise and utilize impartial language. A terrifying truth is that the use of cellular phones only increases, and the variety of accidents increases as well. It's very difficult to acknowledge that a very simple phone call can end a person's life. A cell phone is regarded to be a di stractive aspect which disturbs a man who drives a vehicle. It becomes an additional item in the driver's hand, so that the driver has only one hand to control the vehicle. How to Choose Topics for a Rogerian Argument Essay Needless to say, your imaginary adversary doesn't have an actual possibility to speak to you, but you need to remember that any Rogerian essay may function as a foundation for a possible future dialogue. In fact, Rogerian rhetoric can be thought to be a peculiar variant of the democratic debate. It isn't only convincing but in addition drives to persuasion. Don't criticize or utilize bias. When writing a Rogerian paper, it's fundamental to remember the essential points together with the Rogerian argument ideas. The point is that, by finding common ground, you are more inclined to be in a position to locate a way to solve the argument or disagreement. There is almost always a golden middle or common ground for unique beliefs. To write a fantastic argumentative essay the students first must investigate several sides of the argument, which permits them to make an educated stance. Whatever They Told You About Topics for a Rogerian Argument Essay Is Dead Wrong...And Here's Why It is essential that the author recognize the simple fact that individuals may not agree with his view. An individual has to be sure that they don't force the reader to accept their views whatsoever. Give background information on the matter and add as many details as you are able to in order to earn a complete picture of the discussed issue. You will need to back up your viewpoint with well- researched facts and data too.

Interior Design At The Present Time - 1886 Words

Introduction: This essay aims to look at ethics within interior design at the present time. I will research the most common ethics related to interior design and gather information from different research methods Methodology: The three main research methods used to gather information on Ethics within interior design at the present time was a literature review, looking into the main ethics within interior design currently, The second research method was a survey that looked at the consumers thoughts on these ethics, asking them about there knowledge of Environmentally friendly alternatives and there own choices for there homes The third research method used was a case study of a construction project in Seattle, where architecture firm Case Design, designed and built two ECO friendly homes. It looks at the environmentally friendly features used and also the benefits that these ECO homes had to the local area. Literature review: One of the research methods used was a literature review looking into ethics within interior design. It will look at the main ethics at the present time. When researching into the main ethics in current design the main topic found was sustainable and environmentally friendly design. There were many articles talking about the need for sustainable design but also the need for a fare trade of these sustainable materials. When looking into the ecological side of ethics in design the first article, named Ethical House, was written by ReidShow MoreRelatedWhat Factors That Designers From Other Professions?1525 Words   |  7 PagesWhat factors distinguish interior designers from other professions? Creating and designing a room full of ideas and textures with hints of different personalities is the real work of interior designers. In interior design, an empty room and a head full of imaginations is all it takes to be a professional. This profession requires an imaginative mindset that most professionals do not have. Interior design not only requires this unique mindset, but also requires many individual skills and a strongRead MoreAdvantage Of Modern Architecture1148 Words   |  5 Pagesmight come to people’s mind when they hear the word â€Å"Modern?† How about for Modern †Architecture?† Based on Merriam-Webster’s â€Å"Modern† term, Modern means; â€Å"relatin g to, or characteristic of a period extending from a relevant remote past to the present time.† On the other hand, â€Å"Modern Architecture† article also defines Modern architecture as; â€Å"A new architectural style that emerged in many Western countries in the decade after World War I. It was based on the rational use of modern materials, theRead Morecritical thinking - annotated bibliography1137 Words   |  5 Pagesphilosophy of interior design. New York; Harper Row. The rationalization of how interior design can be affected by the design thinking process is imperative to the comprehension of how interior design itself affects the psychological wellbeing of the end user. Abercrombie considers that conceptualization of the overall desired result is necessary as a starting point, rather than part by part. This human-centered umbrella approach is significant to design thinking as it encourages design to focusRead MoreThe Pros And Cons Of Modern Architecture1171 Words   |  5 PagesWhat comes to people’s mind when they hear the word â€Å"Modern?† How about for Modern †Architecture?† Based on Merriam-Webster’s â€Å"Modern† term, Modern means; â€Å"relating to, or characteristic of a period extending from a relevant remote past to the present time.† On the other hand, â€Å"Modern Architecture† article also defines Modern architecture as; â€Å"A new architectural style that emerged in many Western countries in the decade after World War I. It was based on the rational use of modern materials, theRead MoreInterior Design Is The Process Of Solving Problems Through Creative Development1524 Words   |  7 Pagesenvironment was created by an interior designer in respect to the expectations of the restaurant owner. Interior design is the process of solving problems through creative development w hile considering the business goals of the client and the functionality of the space. As said by Greta Guelich, interior design is to â€Å"plan, coordinate and execute† (Piotrowski, 5). As interior design has grown into an impressively large profession, designers now have the option to work in specific design specialties such asRead MoreReflection About Interior Design1259 Words   |  6 Pagesis that of the Interior Design Field. Interior design has adapted over the past 100 years from basic decoration to now highly skilled and thought out interior spaces that affect the everyday people who experience the space. What I like most about this major is its ability to connect with the human environment. The way it changes your perspective on the building from the inside out. I also enjoy the limitless field of opportunity available, from home to corporate headquarters interior designers areRead MoreMaxey Hayse Design Studios Creates Interior Concepts that Inspire1481 Words    |  6 PagesMaxey Hayse is a hospitality design studio that also works in other areas in interior design such as commercial and residential designs. The firm offers many different types of work for different types of interior design jobs in the tri-state area. In addition, the firm has won a couple of awards for the firms work in the past. One award that the firm has won was from the National Commercial Builders Association for the work the firm did for the Cinder’s Wood Fired Grill located in Mine Hill TownshipRead MoreArchitects Checklist For Rehabilitating Historic Structures1433 Words   |  6 Pagesbuilding is a part of the National Registry. Other questions are what historical documents are available from the building, such as architectural or engineering drawings of the building or local documents or if the structure presents any bad architectural issues such as poor design. In Oklahoma in the early May of 1863, a small clash between Confederate and Union forces broke out during the Civil War. Though the first skirmish was small, it led to the precursor of a larger battle that took place nearlyRead MoreThe Sandringham House970 Words   |  4 PagesBuckingham Palace and Windsor Castle, Sandringham house has its special attractions to royal members for architectural style, living environment, interior or exterior decoration etc. However, this spectacular building was not the same as what it look like at present. More than 3 renowned architects were invited to design and reconstruct this house in history. As time went on and technology developed, almost all elements including style, shape, materials as well as decoration have changed. This part willRead MoreVision Without Action Is A Daydream1307 Words   |  6 Pagesnightmare. My interest in Design, though initially new found, has now been so immense that my duration of study for the entire course of Bachelor of Design has been completed with utmost sincerity and passion. The potential for transforming the mundane into the aesthetically pleasing is what attracts me to design, and after careful thought, I have decided to devote my future career in this field. To accomplish this objective, I have considered to apply for the MS in Interior Design program offered by Oklahoma