Guidelines for Writing a Lab Report Essay Example for Free

Guidelines for Writing a Lab Report Essay Writing a good lab report is an important goal of your science education, and gives you the opportunity to enhance your writing skills and to communicate your understanding of the scientific process to others. Your lab report for this semester will be a write up of your independent research project. This will follow the standard format for a lab report and should include the following sections: Title Introduction Materials and Methods Results Discussion Conclusion References For this course we are giving extra emphasis to the materials and methods section. This section should include sufficient detail to allow others to reproduce your experiments, without being overly descriptive. A guide to writing each section is as follows: TITLE Name the experiment. The title should be descriptive of what you did or what your data showed. A reader should be able to obtain some understanding of the content of your report from the title. In the research world, scientists scan the table of contents of journals to determine if there are any papers relevant to their research that they should read. Therefore the title is important for getting your work recognized. INTRODUCTION Explain why you choose this project, and what you hoped to learn from it. You will be required to research the background information for your project, and present the current state of knowledge for the topic of your research. In addition, you must explain your rationale for choosing this project, clearly state the objective or hypothesis, and predict the outcome of the experiments if the hypothesis holds true. Example: if an independent research project investigated the effect of hand sanitizers on the growth of E. coli, then the introduction should include background information on hand sanitizers (what they are, how they are used, the ingredients that kill bacteria), and background information on E. coli (what it is, why it is a problem). It would also include the experimental hypothesis, e.g. â€Å"Hand sanitizers will be more effective at killing E. coli than soap.† MATERIALS AND METHODS Describe how you conducted your experiments in sufficient detail that someone else could repeat them, WITHOUT excess detail. First and foremost, this section is NOT simply a list of materials and a step-by-step accounting of what you did. You should write your materials and methods in descriptive form, using past tense (describe what you did). Do not include reasoning in your methods – this belongs in the discussion section. You should describe what you did in enough detail that someone could repeat the experiment if he or she wanted to, but do not use excessive details. As you are doing your experiment, be sure you keep track of what you actually did in your lab notebook, especially any details which vary from the instructions in the lab manual. What you do could have important implications for the results you get, and your interpretation of those results. The Materials and Methods in your lab report should be what you actually did, and not just what the lab manual told you to do. Points to remember: * Organize this section carefully and logically, place the methods in the order in which you ran them. * Use subheadings that break the text into distinct sections (if warranted). Do not use subheadings such as â€Å"Lab 4.† Use a descriptive subheading, such as â€Å"Agarose Gel Electrophoresis† and make use of bold text to distinguish subheadings. * Provide enough information to allow others to repeat the same experiment * Use specific, informative language (quantify whenever possible) * Omit unnecessary information. You do not need to include every possible detail of the time you spent in the lab. Include only those procedures directly pertaining to the results you plan to present in the paper. * Include complete mathematical formulas if appropriate. * Do not make the common error of mixing some of the Results in this section RESULTS Present your data in such a way that someone could go directly to the results section and understand the results of your experiments. The results section will have a text portion and a portion that contains figures, tables, photographs, graphs, etc., depending on what kind of data you have. In the text of your results section, describe the trends and important points of your data. Point out what it is that you want your reader to come away with. Be sure you refer to the relevant figures and/or tables when you are writing your text. Refer to them as if you were citing them. For example: The purity of the enzyme improved with each step of the purification scheme (Table 1). For the data portion (i.e., figures and tables), there are many ways to present your results, and you should think very carefully about which is the best way. Is it a line graph, a bar graph or histogram, a pie chart, a table, a picture or diagram, or is it some combination of these? Whatever you choose, be sure it clearly shows your results. You want your reader to be able to look at your tables, charts, figures, etc. and know exactly what experiment was done for each one. You also want the reader to be able to understand what the results actually are. ALL of your data that relates to the report should be presented (even negative data). Points to remember: * Organize your data carefully and logically. If possible, present results in the same order as the methods. * Use subheadings that break the data into distinct sections (if warranted) * Summarize the data and emphasize important patterns or trends * Do not interpret your data; do not draw conclusions; do not speculate.in the results section (save these issues for the Discussion) * Graphs, drawings, and photos are considered figures. Each figure and table must have a title and be numbered sequentially as they are introduced in the text. (figure 1, figure2, table 1, table2) * Specify units on the axes of graphs and label all columns and rows of tables. * Computer programs, such as Excel, can help you draw graphs and diagrams. If the graphs are hand drawn they must be neat and accurate. * Examples of some of the ways that you can present your data are illustrated at the end of this handout. DISCUSSION Relate your results back to the introduction. Did you add to the current state of knowledge? What did you learn from your experiments? Were there any sources of error? What future experiments might you conduct? Was your hypothesis supported by your data? This section is for an interpretation of your results, e.g. what do your results mean? Why did you think that you obtained these results? What can be learned from this experiment.? Connect your results to the concepts behind the experiments and your hypothesesAre there any questions the experimental design leaves unanswered (related to your hypothesis or not)? How would you improve this experiment in the future? What other experiments would you do now to extend or confirm your results (what is the next step)? You should also indicate if there are any inherent flaws or sources of error in the experimental design. Do not use â€Å"human error† as an explanation. Only discuss experimental errors that you think actually occurred during your experiment. You should also avoid saying that taking more samples or doing more repetitions of the experiment would improve the data. That is almost always true of any experiment, and goes without saying. Points to remember: * Interpret your results; draw attention to your major findings. * Support your conclusions with evidence; convince the reader that your interpretations are sound and that your work represents a valid contribution to the field. * Recognize the importance of negative results. * Address both the advantages and limitations of your methods; What causes may be responsible for your findings? Suggest explanations if you have conflicting or unexpected results. * Go from specific to general; Start with a discussion of your specific results and end with more far-reaching conclusions or predictions; Can you make generalizations? What would the next questions be? * Be aware of phrasing used in scientific discourse, e.g. verbs like suggest, indicate, show, demonstrate; adverbs like possibly, probably, presumably, very likely; auxiliary verbs like may, might, would, could, etc. (qualifiers) CONCLUSIONS Summarize the meaning of your results in two or three sentences. REFERENCES List all of the information sources used for your introduction. Any references that you use should be cited in the text and listed in alphabetical order in a reference section at the end of your report. Use the APA citation style: For citations in the text: (Author, year) goes after the material from a particular source. For references section: Last name, First name. Year. Title of article. Journal. Volume. Page Numbers.

The Jerry Springer Show Essay -- essays research papers

Television has come a long way from the first black and white silent show to its current craze, which happens to be the television talk shows. In the nineteen eighties, the daytime airwaves were monopolised by soap operas and game shows but the trend gradually evolved to talk shows towards the nineties. Being before my time, research appears to imply that the talk show intrusion of our airwaves all seemed to start with the originals such as Geraldo Riveria, Donahue and Sally Jesse Rafael. Surprisingly though, some of the morality and the topics remain somewhat similar throughout the years, such as the improvement of society and the quality of life often show on shows like the Oprah Winfrey Show and the Montel Williams Show. As we approach the darker realms of talk shows, we come to talk shows such as the Jerry Springer show. At first impression, one may get the inherent idea that the show is basically is exploiting the misery and troubles of real life people who do not live the blessed lives that we imagine up in our ideal minds. There is no doubt to me at all that the main aim of such shows is to exploit the lives of these misfits and the more dirt the producers get, the higher their ratings. But unfortunately, it is ironic that when the decrease in society's morals is highlighted in every episode Springer airs the producers are getting richer. Looking deeper at the show, we can safely say that the show is based mainly on relationships between people. Morality is first pu...

International Relations: Philippines and China on the Spratly Islands Issue Essay

For decades, the Spratly Islands in the South China Sea, now called as the Philippine Western Sea in the Philippines, has long been debated by its claimants: Malaysia, Brunei Darussalam, Taiwan, Vietnam, China, and the Philippines (Baker, 2004). Among all these states, the most active and significant actors are the Republic of the Philippines and the People’s Republic of China. There are several reasons for the dispute: (1) the presence of natural gas and oil in some parts of the territories, (2) potential profit for commercial fishing1, (3) potential profit for commercial shipping2, and (4) extension of continental shelf claims – expansion of territory and a boost in the country’s sovereignty3 (Joyner, n.d.). The Spratly’s issue has always been crucial since it directly affects the different state’s national interests on profit and security. For one, the Philippines and China have each asserted their power on claiming the islands by installing their own flags over their claimed areas. The Philippines, following the United Nations Convention on the Laws of the Seas’ – which empowers the International Tribunal for the Law of the Sea – laws on the 200 nautical miles zone, claimed a number of Spratly islands since these islands are within the 200nm distance from Palawan. On the other hand, China claimed most of the Spratly Islands, using historical background as its basis (BBC, 2013). Also, both countries resorted to upgrading their military capabilities in the sense that they both deploy ships to take turns on guarding their claimed areas. These actions caused to increase the tensions and strains in the two countries’ relationship (Encomienda, 2011). Both the International Court of Justice and the International Tribunal for the Law of the Sea are under the United Nation’s efforts of bringing peace and unity among different nations or countries (ITLOS, 2013). Acknowledging the principles introduced by the United Nations Convention on the Laws of the Seas4, both China and the Philippines have participated in ratifying it. However, both also have chosen not to follow the UNCLOS’ guide on settling boundary disputes closely (Yeneza, 2012). Even with the efforts of the Philippines on taking the conflict to the ITLOS and the ICJ and promote diplomacy, it has still been forced to resort to upgrading its military capacity in order to increase security over their claimed areas and to respond to China’s actions on asserting their claim over the Spratly’s. With this, it can be concluded that the international organizations involved as mediators to the issue are insufficient. The guide presented by the UNCLOS is too limited for the varying arguments that both the Philippines and China present in order to fully claim the Spratly Islands. However, these institutions should not be blamed fully for the failure of the conflict resolution. The governments of China and the Philippines have a big role in the conflict; none of the states wants to either compromise or interpret the UNCLOS in the same way (Jinming and Dexia, 2003). These international issues can be better comprehended by the realist theoretical framework. Realism denotes that the state aims to achieve national interest through acquiring more power; it gives more priority to the national interest rather than its ideology. If its national interest could not be gained because of other states, it has the right to go against them in order to assert its power and achieve its national interest (Morgenthau, 2006). Thomas Hobbes (1651), an author on realism, discussed that realism has three core assumptions: (1) the equality of men or states, (2) the interaction of states in anarchy, and (3) the actions taken by the different states are motivated by competition, hesitancy, and progress. In the case of the Philippines and China on the Spratly’s issue, the first assumption of Hobbes can be applied in the sense that neither China nor the Philippines will contend the islands in the basis of which state is more developed or has more alliances. For this territorial issue, using the state’s development or its capacity to enhance its development through the acquisition of the islands does not guarantee the resolution of the issue. China and the Philippines both have equal footing on their claims on the islands (Joyner, n.d.) On the second assumption, Hobbes mentioned of interaction among states. China and the Philippines have their own governments, however, in the Spratly Islands, there are mediators: the International Court of Justice and the International Tribunal for the Law of the Sea. These mediators, as much as they try to pacify the conflicts, were not able to control the conflict; since, states are free of doing what they please to do to assert their power and sovereignty – thus, serving their own national interests (Furtado, 1991). For once, one of the proposed resolutions of China to resolve the conflict – to have the governments of each state deal with the conflict directly instead of having mediators – seem more compatible with the issue. Lastly, it is innate for states to compete with each other to get to the top. Even though every state wants to have good relations with others, one cannot deny even a hint of competition. Being critical and cautious, these states will not go against randomly; they will be vigilant enough to choose who to compete with. Succeeding in the particular competition, the state achieves progress (Hobbes, 1651). In the case of China and the Philippines, one of the main reasons for the fight over the Spratly Islands is profit. Aside from the presence of natural gas and oil, the islands are a good spot for commercial fishing and commercial shipping. Hence, investing in this area can give high returns of investment to the country who owns it. Plus, the prestige of being the first East Asian country to possess oil boosts its international image, let alone lessens its relying on the Middle East for oil and natural gas supply (Yeneza, 2012). Hans J. Morgenthau (2006) and his six principles on political realism can also be related to the case of the Philippines and China on the Spratly’s issue. First of all, the society is ruled by laws that are created based on the nature of man. The UNCLOS (United Nations Convention on the Laws of the Seas) was created to solve and/or prevent conflicts such as the Spratly Islands issue. Therefore, it can be considered that the UNCLOS itself can be the solution to the problem only if that law was considered. However, even though such law – that brought about the existence of the UNCLOS – is implemented and expected to serve for the benefit of men, the men involved in the conflict, or the states rather, chose not to adhere with the UNCLOS (Yeneza, 2012). Secondly, realism is directed towards the use of power5. The Philippines and China demonstrated their power as they both valiantly expressed their claims on the disputed areas and how much they spent for them. As China asserted its claim on the Spratly Islands, the Philippines resorted to securing the areas it claimed (Encomienda, 2011). The third principle, according to Morgenthau (2006), power is not fixed. In the case of the Philippines and China, they controlled each other’s exercise of power on the claimed areas as they both measure and depend on each others’ actions in order to formulate and plan what to do next (Yeneza, 2012). The fourth principle of realism is related to the moral importance of the actions done by the two states – Philippines and China. At times, morality and success do not go together; some states tend to go down the path of impiety in order to achieve its national interests. In realism, states are innate of being mindful of the actions they are about to execute because of other factors that can be affected by its desired course of action. Thus, the Philippines and China can opt to wage war in order to acquire the disputed territories. Conversely, both countries have chosen the path of diplomacy rather than war, knowing full well that morally speaking, waging a war is not right since many civilians will be affected, as well as the fact that the costs brought by the war exceed the benefits that they will acquire from the Spratly Islands. Then, another principle Morgenthau (2006) explained is that states fail to recognize moral aspirations in relation to the moral laws that rule the universe. The state is self-centered; it only sees its own reasons and interests as the valid ones. Being self-centered, it does not give importance to the reasons and interests of other states. If only states can understand and compromise for others’ interests, the Spratly’s issue would have been solved decades ago. As each country present their claims on the areas, it is clear that no one wants to give up (Yeneza, 2012). Lastly, the sixth principle is that political realism is different with other schools of thought that states may adopt (Morgenthau, 2006). The conflict of China and the Philippines on the Spratly’s are grounded for the pursuant of their national interests (Yenez, 2012). Aside from the profit the Spratly’s may offer, another reason it is being fought over is that it affects the matters of security to both countries. Both the Philippines and China believe that in order to pursue security, they must secure their territories; thus, they each lay claim on the Spratly’s (De Castro, 2011). In summary, China and the Philippines have both resorted to upgrading their military capabilities in order to enhance security over their claimed areas. Through the years, China spent so much on military expenditure whereas the Philippines created more military capability programs. In the realist approach, as long as China and the Philippines hold on to their own claims on the Spratly’s, neither the International Court of Justice nor the International Tribunal for the Law of the Sea will be able to calmly resolve the conflict between the two countries. With the Philippines maintaining a good relationship with the other ASEAN members, it garnered the support of other Southeast Asian countries in laying claim to the Spratly’s; thus, giving it more strength on its hold on the islands (Yeneza, 2012). Unless one of them give up on its claim, the conflict will not be resolved easily and without causing further strains among the countries’ relationship (Morgenthau, 2006). There are two possible solutions that can be taken to resolve the conflict: (1) share the claimed areas, whenever it is feasible, and (2) undergo a diversion process. For the first solution to work, the Philippines and China must be allowed to gather the resources and share them equally – e.g. fish and seafood, oil, natural gas, sites for commercial shipping. Bilateral talks should always be done; therefore, the countries must interact directly rather than use a mediator and then promote diplomacy. The second one will be that rather than focusing on the conflict itself, both the Philippines and China should focus on repairing and enhancing their diplomatic ties and cooperation. They may opt to engage in mutually beneficial bilateral agreements and allow their relationship to pacify or stabilize (Yeneza, 2012). In the realist approach, it can be seen that the different international institutions have delayed the resolution of the Spratly’s issue rather than fixing it quickly. The issue requires the states to interact and discuss the matter directly, rather than using a mediator, in which the clauses provided in the guidelines for settling territorial disputes are too limited and can be interpreted in varying ways depending on the ideology adopted by a certain state. REFERENCES: Baker, C. 2004. China-Philippine relations: cautious cooperation. Pacific Center for Security Studies. October 5, 2013. Accessed from: http://apcss.org BBC. 2013. Q&A: South China sea dispute. October 5, 2013. Accessed from: http://www.bbc.co.uk/news/world-asia-pacific-13748349 De Castro, R.C. 2011. Maritime security Asia. October 5, 2013. Accessed from: http://maritimesecurity.asia Encomienda, A.A. 2011. The south China sea: Back to the future through cooperation. October 6, 2013. Accessed from: http://www.southchinaseastudies.org Furtado, X.1991. International Law and the Dispute over the Spratly Islands:Whither UNCLOS? October 5, 2013. Accessed from: http://www.findarticles.com Jinming, L. and Dexia, L. 2003. The dotted line on the Chinese map of the south China sea: A note. October 4, 2013. Accessed from: https://circle.ubc.ca/handle/2429/6494 Joyner, C.C. n.d. The Spratly Islands dispute in the south China sea: Problems, policies and prospects for diplomatic accommodation. South China Sea Virtual Library. October 4, 2013. Accessed from: http://www.southchinasea.org Morgentau, H.J. 2006. Politics among nations: The struggle for power and peace. 7th Ed. New York: McGraw-Hill/Irwin. Yeneza, Christine. 2012. The spratly’s conflict: Foreign policy implications to the people’s republic of china and the republic of the Philippines. Cebu City, Philippines: University of San-Jose Recolectos

High performance liquid chromatography 214 - Free Essay Example

Sample details Pages: 12 Words: 3597 Downloads: 6 Date added: 2017/06/26 Category Statistics Essay Did you like this example? Introduction High performance liquid chromatography 214 is the most widely used of all of the analytical separation techniques. The reasons for the popularity of the method is its sensitivity, ready adaptability to accurate quantitative determinations, suitability for separating non-volatile species or thermally fragile ones, wide spread applicability to substance that are of prime interest to industry, many fields of science and the public. The applications of chromatography have grown explosively in the last fifty years owing not only to the development of several new types of chromatographic techniques but also to the growing need by scientist for better methods for characterizing complex mixtures. Don’t waste time! Our writers will create an original "High performance liquid chromatography 214" essay for you Create order General methodology for the development of new HPLC methods 215-228 HPLC method development follows the series of steps summarized below. Information on sample, objective of separation. Need for special HPLC procedure, sample pretreatment etc. Choice of detector and detector settings. Choosing LC method, preliminary run, estimation of best separation conditions. Optimization of separation conditions. Check for problems or requirement for special procedure. a) Recovery of purified material b) Quantitative calibration c) Qualitative method Validate method for routine laboratory use. A good method development strategy should require only as many experimental runs as are necessary to achieve the desired final result. Finally, method development should be simple as possible, yet it should allow the use of sophisticated tools such as computer modeling if these are available. Before the beginning of method development, it is necessary to review what is known about the sample in order to define the goals of separation. The kinds of sample related information that can be important are summarized in Table-7.1. Number of compounds present in the sample Chemical structures of components Molecular weights of compounds PKa values of compounds UV spectra of compounds Concentration range of various compounds in samples of interest Sample solubility The chemical composition of the sample can provide valuable clues for the best choice of initial conditions for an HPLC separation. Objectives of separation The objectives of HPLC separation need to be specified clearly include. The use of HPLC to isolate purified sample components for spectral identification or quantitative analysis. It may be necessary to separate all degradants or impurities from a product for reliable content assay. In quantitative analysis, the required levels of accuracy and precision should be known (a precision of 1 to 2% is usually achievable). Whether a single HPLC procedure is sufficient for raw material or one or more formulations and / or different procedures are desired for the analysis of formulations? When the number of samples for analysis at one time is greater than 10, a run time of less than 20 min. will be oftenly important. Knowledge on the desired HPLC equipment, experience and academic training the operators have. Sample pretreatment and detection Samples for analysis come in various forms such as: Solutions ready for injections. Solutions that require dilution, buffering, addition of an internal standard or other volumetric manipulation. Solids that must first be dissolved or extracted. Samples that require pretreatment to remove interference and/or protect the column or equipment from damage. Most samples for HPLC analysis require weighing and / or volumetric dilution before injection. Best results are often obtained when the composition of the sample solvent is close to that of the mobile phase since this minimizes baseline upset and other problems. Some samples require a partial separation ( pretreatment) prior to HPLC, because of need to remove interference, concentrate sample analytes or eliminate column killer. In many cases the development of an adequate sample pretreatment can be challenging than achieving a good HPLC separation. The detector selected should sense all sample components of interest. Variable-wavelength ultraviolet (UV) detectors normally are the first choice, because of their convenience and applicability for most samples. For this reason information on the UV spectra can be an important aid for method development. When the UV response of the sample is inadequate, other detectors are available (flourescence, electrochemical, PDA etc.) or the sample can be derivatized for enhanced detection. Developing the method for the separation Selecting an HPLC method and initial conditions If HPLC is chosen for the separation, the next step is to classify the sample as regular or special. Regular samples means typical mixtures of small molecules (2000 Da) that can be separated using more or less standardized starting conditions. Exceptions or special samples are usually better separated with a different column and customized conditions as summarized in Table-8.2. Regular samples can be further classified as neutral or ionic. Table-8.3 summarizes the appropriate experimental conditions for the initial reversed phase separation of regular samples. Samples classified as ionic include acids, bases, amphoteric compounds and organic salts. If the sample is neutral, buffers or additives are generally not required in the mobile phase. Acidic or basic samples, usually require the addition of the buffer to the mobile phase. For basic or cationic samples, less acidic reverse phase columns are recommended (Table-8.4) and amine additives for the mobile phase may be beneficial. Usin g these conditions, the first exploratory run is carried out and then improved systematically as discussed below. Table-8.2 Handling of special sample Sample Requirements Inorganic ions Detection is primary problems; use ion chromatography Isomers Some isomers can be separated by reversed-phase HPLC and are then classified as regular samples; better separations of isomers are obtainable using either (1) normal-phase HPLC or (2) reversed-phase separations with cyclodextrin-silica columns. Enantiomers These compounds require chiral conditions for their separations. Biological Several factors make samples or this kind special; molecular conformation, polar functionality and a wide range of hydrophobicity. Macromolecules Big molecules require column packing with large pores( 10-nm diameters); in addition, biological molecules require special conditions as noted above. Table-8.3 Preferred experimental conditions for the initial HPLC separation Separation variable Preferred initial choice Column Dimensions (length, ID) 15 x 0.46 cm Particle size 5 mma Stationary phase C8 or C18 Mobile phase Solvent A and B Buffer-acetonitrile % B 80-100%b Buffer (compound, pH, concentration) 25mM potassium phosphate 2.0pH3.0e Additives (e.g., amine modifiers, ion pair reagents) Do not use initially Flow rate 1.52.0 ml/min Temperature 35-45C Sample Size Volumed 25 mL Weightd 100 mg B : Polar solvent a 3.5 mm particles are an alternative using a 7.5 cm column b For an initial isocratic run; an initial gradient run is preferred. c No buffer required for neutral samples; for pH2.5, pH-stable columns are recommended. d Smaller values required for smaller-volume columns (e.g., 7.50.46-cm, 3.5-mm column). Table-8.4 Physical properties of silica supports for some C 18 columns Column (mL/mL) Pore diameter (nm) Surface area (m2/g) Percent Porosity Hypersil ODS 12 170 57 LiChrosorb C18 10 355 71 Novapak C18 6 N/Aa N/Aa Nucleosil C18 10 350 69` Symmetry C18 10 335 66 Zorbax ODS 6 300 55 Zorbax Rx, SB, XDB 8 180 50 a N/A : Not available On the basis of the initial exploratory run isocratic or gradient elution can be selected as most suitable. If typical reversed-phase conditions provide insufficient sample retention, suggesting the use of either ion pair on normal phase HPLC. Alternatively, the sample may be strongly retained with 100% acetonitrile as mobile phase, suggesting the use of non-aqueous reversed-phase (NARP) chromatography or normal phase HPLC. Some characteristics of reversed-phase and other HPLC methods are summarized below. Table-8.5 Characteristics of primary HPLC methods Method / description/ columns Preferred method Reversed-phase HPLC Uses water organic mobile phase Columns: C18 (ODS), C8, phenyl, trimethylsilyl (TMS), Cyano First choice for most samples, especially neutral or non-ionisable compounds that dissolve in water-organic mixtures Ion-pair HPLC Uses water-organic mobile phase a buffer to control pH and an ion pair reagent. Column : C18, C8, cyano. Acceptable choice for ionic or ionizable compounds, especially bases or cations. Normal phase HPLC Uses mixtures of organic solvents as mobile phase Columns: Cyano, diol, amino and silica. Good second choice when reversed-phase or ion-pair HPLC is ineffective, first choice for lipophilic samples that do not dissolve well in water-organic mixtures, first choice for mixtures of isomers and for preparative-scale HPLC (silica best) Getting started on method development One approach is to use an isocratic mobile phase of some average solvent strength (e.g., 50%) organic solvent. A better alternative is to use a very strong mobile phase with (80-100% B), then reduce %B as necessary. The initial separation with 100%B results in rapid elution of the entire sample, but few groups will separate. Decreasing solvent strength shows the rapid separation of all components with a much longer run time, with a broadening of later bands and reduced detection sensitivity. Improving the separation and repeatable separation Generally the chromatographers will consider several aspects of the separation, as summarized in Table-8.6. Table-8.6 Objectives of separation in HPLC method development Objectivesa Comment Resolution Precise and rugged quantitative analysis requires that Rs be greater than 1.5. Separation time 5-10 min is desirable for routine procedures. Quantitation 2% (1 SD) for assays; 5% for less-demanding analysis; 15% for trace analysis. Pressure 150 bar is desirable, 200 bar is usually essential (new column assumed) Peak height Narrow peaks are desirable for large signal / noise ratios Solvent consumption Minimum mobile-phase use per run is desirable. a Roughly in order of decreasing importance but may vary with analysis requirements. Separation or resolution is a primary requirement in quantitative HPLC. The resolution (Rs) value should be maximum (Rs1.5) favours maximum precision. Resolution usually degrades during the life of the column and can vary from day to day with minor fluctuations in separation conditions. Therefore, values of Rs = 2 or greater should be the goal during method development for simple mixtures. Such resolution will favour both improved assay precision and greater method ruggedness. Some HPLC assays do not require base line separation of the compounds of interest (qualitative analysis). In such cases only enough separation of individual components is required to provide characteristic retention times for peak identification. The time required for a separation (run time = retention time for base band) should be as short as possible and the total time spent on method development is reasonable (runtimes 5 to 10 minutes are desirable). Conditions for the final HPLC method should be selected so that the operating pressure with a new column does not exceed 170 bar (2500 psi) and upper pressure limit below 2000 psi is desirable. There are two reasons for that pressure limit, despite the fact that most HPLC equipment can be operated at much higher pressures. First, during the life of a column, the back pressure may rise by a factor of as much as 2 due to the gradual plugging of the column by particular matter. Second, at lower pressures 170 bars, pumps, sample values and especially auto samples operate much better, seals last longer, columns tend to plug less and system reliability is significantly improved. For these reasons, a target pressure of less than 50% of the maximum capability of the pump is desirable. When dealing with more challenging samples or if the goals of separation are particularly stringent, a large number of method development runs may be required to achieve acceptable separation. Repeatable separation As the experimental runs described above are being carried out, it is important to confirm that each chromatogram can be repeated. When changing conditions (mobile phase, column, and temperature) between method development experiments, enough time must elapse for the column to come into equilibrium with a new mobile phase and temperature. Usually column equilibration is achieved after passage of 10 to 20 column volumes of the new mobile phase through the column. However, this should be confirmed by carrying out a repeat experiment under the same conditions. When constant retention times are observed in two such back-to-back repeat experiments ( 0.5% or better), it can be assumed that the column is equilibrated and the experiments are repeatable. Completing the HPLC method development The final procedure should meet all the objectives that were defined at the beginning of method development. The method should also be robust in routine operation and usable by all laboratories and personnel for which it is intended. Quantitation and method validation One of the strengths of HPLC is that is an excellent quantitative analytical technique. HPLC can be used for the quantitation of the primary or major component of a sample (including pure samples) for mixture of many compounds at intermediate concentrations and for the assessment of trace impurity concentrations in matrix. Method validation, according to the United States Pharmacopoeia (USP), is performed to ensure that an analytical methodology is accurate, specific, reproducible and rugged over the specified range that an analyte will be analysed. Method validation provides an assurance of reliability during normal use and is sometimes described as the process of providing documented evidence that the method does what it is intended to do. According to USP, the method validation involves eight steps as given below. Precision Accuracy Limit of detection Limit of quantitation Specificity Linearity and range Ruggedness Robustness Precision and accuracy: Already discussed in chapter-1. Linearity The linearity of the method is a measure of how well a calibration plot of response v/s concentration approximates a straight line, or how well the data fit to the linear equation. Y = aX + b Where Y is the response, X is the concentration, a is the slope and b is the intercept of a line fit to the data. Ideally, a linear relationship is preferred (b = 0) because it is more precise, easier for calculations and can be defined with fewer standards. Also, UV detector response for a dilute sample is expected to follow Beers law and be linear. Therefore, a linear calibration gives evidence that the system is performing properly throughout the concentration range of interest. Generally in HPLC, if we are using internal standard, then the linearity plot is to be drawn by taking concentration of the analyte on x-axis and the ratio of area under the curve (AUC) of analyte to AUC of internal standard (IS) on y-axis. The resulting plot slope, intercept and correlation coefficient provide the desired information on linearity. A linearity correlation coefficient above 0.999 is acceptable for most methods. Limit of detection (LOD) The limit of detection (LOD) is the smallest concentration that can be detected reliably. The LOD represents the concentration of analyte that would yield a signal-to-noise (S/N) ratio of 3. Limit of quantitation (LOQ) The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represents the concentration of analyte that would yield a signal-to-noise ratio of 10. LOD and LOQ can be determined by using the following expressions. LOD = 3 X N / B LOQ = 10 X N / B Where N is the noise estimate, is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the drugs. B is the slope of the corresponding calibration curve. The LOD and LOQ values determined during method validation are affected by the separation conditions, columns, reagents and especially instrumentation and data systems. Ruggedness Method ruggedness is defined as the reproducibility of results when the method is performed under actual use conditions. This includes different analysts, laboratories, columns, instruments, sources, chemicals, solvents etc. method ruggedness may not be known when a method is first developed, but insight is obtained during subsequent use of that method. Robustness The concept of robustness of an analytical procedure has been defined by the ICH as a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters. The robustness of a method is the ability to remain unaffected by small changes in parameters such as pH of the mobile phase, temperature, percentage of organic solvent and buffer concentration etc. to determine robustness of the method experimental conditions were purposely altered and chromatographic characteristics were evaluated. To study the pH effect on the retention (K1) of the drug, buffer pH is to be changed by 0.2 units. At certain point, retention will increase at any pH above and below of the pH unit. The effect of temperature on the retention characteristics (K1) of the drug is to be studied by changing the temperature in steps 2C from room temperature to 80C and see the effect of temperature on the resolution and peak shape. Effect of percentage organic strength on retention is to be studied by varying the percentage of organic solvents like acetonitrile, methanol etc. from 0 to 2% while the other mobile phase contents are held constant and observe the K1. At certain point decreases in K1 observed with increase in the level of organic solvent. Effect of buffer concentration should be checked at three concentration levels i.e. 0.025 M, 0.05 M and 0.1 M and observe retention time and resolution. Stability To generate reproducible and reliable results, the samples, standards and reagents used for the HPLC method must be stable for a reasonable time (e.g., One day, one week, one month, depending on the need). For example, the analysis of even a single sample may require 10 or more chromatographic runs to determine system suitability, including standard concentrations to create a working analytical curve and duplicate or triplicate injections of the sample to be assayed. Therefore, a few hours of standard and sample solution stability can be required even for a short (10 min.) separation. When more than one sample is analyzed, automated, over night runs often are performed for better laboratory efficiency. Typically, 24 hours stability is desired for all solutions and reagents that need to be prepared for each analysis. Mobile phases should be chosen to avoid stability problems, especially the use of amine additives or specific solvents. For example, mobile phase containing THF (tetra hydrofuran) are known to be susceptible to oxidation, therefore, the mobile phase should be prepared daily with fresh THF. Some buffered mobile phases cause problems for example, phosphate and acetate provide good media for microbial growth. Sodium oxide (0.1%) is often added to the mobile phase buffer to inhibit such growth, adding more than 5% of organic solvent is also effective. Long term column stability is critical for method ruggedness. Even the best HPLC column will eventually degrade and lose its initial performance, often as a function of the number of samples injected. System suitability System suitability experiments can be defined as tests to ensure that the method can generate results of acceptable accuracy and precision. The requirements for system suitability are usually developed after method development and validation have been completed. The criteria selected will be based on the actual performance of the method as determined during its validation. For example, if sample retention times forms part of the system suitability criteria, their variation (SD) during validation can be determined, system suitability might then require that retention times fall within a 3 SD range during routine performance of the method. The USP (2000) defines parameters that can be used to determine system suitability prior to analysis. These parameters include plate number (N), tailing factor, k and / or a, resolution (Rs) and relative standard deviation (RSD) of peak height or peak area for respective injections. The RSD of peak height or area of five injections of standard solution is normally accepted as one of the standard criteria. For an assay method of a major component, the RSD should typically be less than 1% for these five respective injections. The plate number and / or tailing factor are used if the run contains only one peak. For chromatographic separations with more than one peak, such as an internal standard assay or an impurity method, expected to contain many peaks, some measure of separations such as Rs is recommended. Reproducibility of tR or k value for a specific compound also defines system performance. The column performance can be defined in terms of column plate number N is defined by N = 5.54 (tR / W)2 Where tR is the retention time of the peak and W is the width of the peak at half peak height. The resolution of two adjacent peaks can be calculated by using the formula Rs = 1.18 (t2-t1) / W0.5.1 +W0.5.2 Where t1 and t2 are retention times of the adjacent peaks and W0.5.1 and W0.5.2 are the width of the peaks at half height. Rs = 2.0 or greater is a desirable target for method development. The retention factor k is given by the equation. k = (tR t0) / t0 where tR is the band retention time and t0 is the column dead time. The peak symmetry can be represented in terms of peak asymmetry factor and peak tailing factor, which can be calculated by using the following formula. Peak asymmetry factor = B /A Where B is the distance at 50% peak height between leading edge to the perpendicular drawn from the peak maxima and A is the width of the peak at half height. According to USP (2000) peak tailing factor can be calculated by using the formula T = W0.05 / 2f Where W0.05 is the width of the peak at 5% height and f is the distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 50% of the peak height from the base line.