American Psycho Movie Review Example | Topics and Well Written Essays - 750 words

American Psycho - Movie Review Example In this essay, Messier analyzes the paradigm related to the term, eloquently propounded by Robert Stam as â€Å"intertextual dialogism†. The relation between the literary texts and its cinematic adaptations in the recent years as claimed by Vartan has suffered from a lack of narration between the literary text and its cinematic adaptation. Vartan claims that it would be a very logo centric approach and straightforward inference if the cinematic adaptations of the literary texts are coined as â€Å"unfaithful to and/or of lesser value† in relation to their mother text from which the cinematic production has evolved without taking into consideration the huge realm of socio-cultural context upon which they evolve and get dispersed. In order to establish this content of the essay, Vartan meticulously chose one of the best cinemas of the recent times, which is an adaptation of a text. American Psycho is a novel by Bret Easton Ellis published in the year 1991. In the year 200 0, director Marry Harron prepared a film out of the Ellis’ text which Vartan and the critics following the same school of thoughts considered for a close reading. They considered both the works from a very close contour resulting into the establishment of the discourse that would give a shape to the interpretations, receptions and dispersions of both the works under a critical and ideological light. Vartan begins the paper by providing a short overview of the text, â€Å"American Psycho† by Ellis and then introduces the amount of controversy inherent within the content of the text before the essay intends to discuss the myriad ways by which the visual poetics of the novel operate as a cut-throat and dissecting critique by figuring the transition of the aesthetics related with the sexual violence which Vartan views as a potent trend in the contemporary consumer culture. Next, the essay takes a turn and launches the comparison of the text by Ellis with that of its cinema tic adaptation of Harron and intends to discuss the shortcomings inherent in the novel which shocks the audiences. Contrarily, according to Vartan, the film by Harron provides a social commentary keeping the plot of the film at a safe distance which actually facilitated it to became more soothing (Messier, â€Å"Visual Poetics, Intertextuality, and the Transfiguration of Ideology: An "Eye" for an "I" in Mary Harron's Cinematic Adaptation of Bret Easton Ellis's "American Psycho"). Vartan in order to establish the propositions suggested by him at the very outset of the essay divides the essay into four sub-sections with intriguing titles like, ‘ Shock and Scandal: American Psycho as Postmodern Pastiche’, ‘ From Pastiche to Parody, or, an â€Å"I† for an â€Å"Eye†, ‘ Transfigurations: Excess, Affect, and the Gaze’ and ‘The Politics of Adaptation: Poetics, Intertextuality, and Ideology’. These sections individually throw elabo rate light on the propositions inherent within the essay required to establish the point of Vartan effectively. The section, ‘ Shock and Scandal: American Psycho as Postmodern Pastiche’ discusses the amount of problem the film, ‘American Psycho’ was about to face long before its date of release owing to the fact that it was a cinematic adaptation of a text which was already condemned as, â€Å" sadistic, pornographic , misogynistic and loathsome†. The negative reception according to Vartan did create an inquisitive pursuit and rage to view the film as it was thought to capture a few of its turbulent sexually violent passages, but Vartan asserts that maintenance of the distance from the stark

What is a strategic control system what role does accounting play in a Essay

What is a strategic control system what role does accounting play in a strategic control system are the strategic management accounting in Caterpillar effecti - Essay Example The process can be considered as a fundamental due to the fact that it involves even the strategic management accounting as one of the essential aspects. Basically SMA is one of the specializing areas in strategic control systems due to the fact that it deals primarily on management accounting issues specifically by using data and information related to finances and accounting (SMA Supplementary Notes 2-3). The strategic control management then can be considered essential in the operation of the whole system and in the specific aspects of the operation as well. In relation to management accounting, the said process can be considered to be involved in the study of the different groups involved in the market along with the market’s behaviour, the determination of most effective solutions and implementation of the said strategies (SMA Supplementary Notes 3). The main role of strategic control system in accounting is evident in the dimensions covered by the SMA. Specifically, it is the planning of possible projects and implementations of different plans related to financial issues. Due to the characteristic of being strategic, there are different alternatives and contingencies that are presented to ensure the success of the operation. It is important to consider though that management accounting is not the sole field wherein strategic control system is involve. Although majority of the companies concentrate on the strategies to improve and achieve profits for the company, the coverage of the system is extensive due to the involvement in different aspects and departments of the company or organization. Based on the study by Ittner & Larcker, the main processes included in the strategic control system are the strategic implementation process, the internal and the external monitoring (3). The said processes are applied

Book Review on Song of the Hummingbird Essay Example | Topics and Well Written Essays - 750 words

Book Review on Song of the Hummingbird - Essay Example We can examine that Aztecs were willing and also trying a lot to make peace by doing work with Spaniards but Spaniards totally against with Aztecs and not willing to unite with them. Author also portrayed that Spaniards createsproblems with Aztecs and also they didn’t even try to understand the Mexican Culture, for this act which pertained to the devil; author saw lot of human sacrifices for this act.Mostly people at that time agree with Limon that people should understand each other because if they are not united then several problems occurred between people just like Aztecs and Spaniards. This story takes place in newly colonized Mexicoafter thirty years of Aztec Empire. Author is continuously against of war, racism and class problems. Limon argue about that people get ways from colonized mind and give the value of beauty and making love and understand each other. Graciela Limon wrote this novel in 1996 about the fall of Aztecs and discuss the end era of Mexicans by Spanish conquers. Author expresses the views of a struggling, indigenous and powerful old woman her name is Huitzitzilin for easiness people pronounce her name â€Å"Hummingbird†. Theme of starting chapters of this book is transcription of Huitzitzilinand this transcription is basically the device of this book. In other chapters it reveals that protagonist of this story is Huitzitzilin or HummingBird was a Nobel Mexica means Aztec at birth. Her age is almost 20 years old when Spaniards arrived in Mexica then after sometime these Spaniards known as Tenochtitlan. In the start of novel author writes that â€Å"Like most of her people she experienced the awe caused by those bearded white men when they first arrived; wonderment that soon gave way to outrage of seeing the devastation of her land, the disruption of her life and the end of civilization as she knew it†. From these points author narrates the powerful images and expresses the passion of a story which is the

Leading Innovation and Change Research Paper Example | Topics and Well Written Essays - 6000 words

Leading Innovation and Change - Research Paper Example We elaborate the role of leader to bring the change and innovation in the organization, its impact on the people working in the organization and other stakeholders, we also discuss the peoples' resistance towards the change and innovation and the leaders strategy to overcome these types of reactions. In other hand we discuss the successful and unsuccessful change and innovation to evaluate the change and innovation, as well as did an assessment of the performance of leader as a leader of innovation and change and also include an action plan to support the further development.This assignment discusses all the aspects related to the innovation and change, like in this study, we critically evaluate the theories of innovation and change and leadership of innovation and change, assess the performance of leader of innovation and change and develop further plans to support the future development related to the innovation and change. The innovation and change are interrelated with each other and the factors, which are affecting both of them are the same. The leadership is necessary for bringing in the innovation and change whether at the individual level or organizational level. The importance of the leadership to bring in the innovation is gradually increasing worldwide because a leader has the ability to give the understanding to others related to something new, ability to convince people towards the improvement and also a leader can replace old ideas and techniques with new ideas and techniques easily (Aitken and Higgs 2010). ... Leader analyzes the situation as where the change is needed and develop an action plan according to the change required in order to bring in and implement the change. Now, the question is how to bring in the change? Who brings in the change? Why to bring in the change? These are some questions, which normally arise while bringing in a change and in its implementation, there are many other questions related to the change and innovation can arise and the person who brings that change has to give the answer of all these questions that can be convincing for the people who have concerns in respect to the coming changes. (Aitken and Higgs 2010) With the passage of time, the world is getting advanced and new technologies are introduced, organizational structure and systems are introduced and new ways of doing diverse works are presented, organizations have to adopt these changes otherwise they will not be able to survive in the digital world. Manager of an organization doing work in any of the situation must face the situation of change, he has to analyze the nature of the change in the initial level of the implementation of change that is the most appropriate way of managing the changing situations. First of all, he should analyze the need of change whether the change is required to take place in the organization or not and then he should find out whether the change it is appropriate according to the organizational environment or not. Since introducing the change, first thing is to recognize the need for this change like if there is a problem in working with the present ways of doing work, the work is not more efficient with the present ways and there are some opportunities existent, which encourage to bring in a

Strategies for Promoting Democracy in Iraq Term Paper

Strategies for Promoting Democracy in Iraq - Term Paper Example This is usually entrenched in the laws of the land. It involves social, cultural and economic conditions that provide free and fair practice of self-determination politically. It hails from a Greek word demokratia whose meaning is â€Å"rule of the people†. This word can be divided into two. Demos meaning â€Å"people† and Kratos meaning â€Å"power†. Democracy contrasts other forms of government like monarchy and aristocracy /oligarchy. Monarchy is where power is held by one individual while aristocracy is where power is held by a group of people. Contemporary governments tend to have a mixture of all these elements. There are several types of democracy (Lijphart 150-250). The two basic forms of democracy are a direct democracy and representative or indirect democracy. In direct democracies, citizens have direct participation in the decision-making process within the government. In indirect  democracies, citizens elect representatives (Lijphart 200-250). He ex plains that in modern democracies, the sovereign power is retained by the people while the political power is exercised through the representatives (200-250). He further explains that an electoral democracy government is determined by popular sovereignty (200-300). People in the country determine who will govern them. They do this by voting. Liberal democracy is one in which both sovereignty and liberties are manifested. The civil society is very active and restraints the government from misusing power. A substantive democracy is a working democracy. Under the representative democracy, we have a parliamentary, presidential and constitutional democracy. Parliamentary democracy is a democracy where the government is chosen by elected representatives (Lijphart 200-250). The government in this case is checked by the legislative parliament elected by people. The legislature can dismiss the prime minister under this system of government. This happens by passing a vote of no confidence. Th e presidential system of democracy is where the public elects the president through general elections (Lijphart 200-250).

Shakespeare and Kafka Essay Example for Free

Shakespeare and Kafka Essay Both Shakespeare and Franz Kafta were the men of their contemporary world, placing the world as they see with irony and forms a juxtaposition of a psychological trauma of man troubled by the societies unwarranted interference and trouble in their lives. Shakespear’s tragedy and Kafta’s trial are the tragedies and both of their protagonists have their tragic end not only due to the circumstances created by the society but also by their own mental traumas and thoughts. But, there are certain differences between the two, Hamlet’s King died more due to his moral dilemma while Kafta have to bear the consequences of the bureaucratic rules and laws of the land. Shakespeare uses the technique of inner struggle of a man who is confounded with the fact that her mother has married with her husband’s murdered and the plans of revenge he intended to take. The Hamlet’s trauma was his thoughts about the various moral issues. The biggest moral issue in front of him came when he got revelation of the truth about his mother’s infidelity. He was fixed in a dilemma was it right to kill his mother as a punishment?. He was caught so much in the dilemma that he delayed taking revenge resulting in the circumstances that proved tragic for him. Though he took revenge in the end but at the cost of his life and also of others to whom he matters the most particularly his beloved. Trial is also same in the sense that it also ended in tragedy yet it is different in the sense that Joseph K was giving unsuccessful fight against the court to denounce his death sentence. It was only in the end he accepted his fate and himself summoned his call of death. If Shakespeare would have written â€Å"The Trial† then Joseph, the main protagonist of the story would have been a man of higher status in the society and would have been more a tale of suffering. His misfortune would have been something exceptional and extraordinary. When Joseph K was informed that he would be arrested, Shakespeare would have turned the character as the most humble without any power to face resistance. Joseph would have been incapable to make quick decisions, though he would have been capable to make indecisive and rash decision but would never have been able to take planned or premeditated action. As in the exact story of Kafka where Joseph is shown making efforts for his release, calling his attorney and even went to the court on the appointed day for the hearings as the demand of the law of the land, Shakespeare would have twisted the story here. He would have made Joseph lament of the tragedy going to befall on him. Joseph would have given himself to mediation and reflection and in this process he would have gone on delaying in his efforts to secure his release. Joseph would have thought too much about the circumstances befallen on him before making efforts of his actions. Shakespeare would have allowed him to commit the actions and commissions and cause him great mental agony and not only physical pain that he was going to endure. And later as â€Å"Hamlet†, he would have tried to find out the main reason behind his alleged arrest and trials and who were responsible for his so tragic fate, which was never revealed to him by Kafka. In tragedy by Shakespeare, the hero normally comes to the realization of truth of which he had been always unaware of and as Aristotle himself said, â€Å"a change from ignorance to knowledge†. (Mcmanus, Online) Therefore in â€Å"The Trial†, Shakespeare would have made Joseph make efforts in gaining the knowledge and reason behind what has made his fate most tragic. Later Joseph would have grown in stature and wisdom, as Shakespeare would have made him realize the fact that reason is not enough. An over reliance on reason and belief and untrammeled free will are hallmarks of the Shakespearian villain, and the heroes learn better. Joseph would have made to realize that he should have resort to much better efforts while dealing with the crises and situations, which are out of his control. On the other hand, Kafka would have also used the elements so typical of him as a writer in Hamlet. He would have created Hamlet as a person who would not be merely pondering and reasoning on the causes of his befallen fate but would have strived to fight for his rights against the unjustified justice. He would have made Hamlet, instead of entering into the moral dilemma of whims and his passion for his mother, strived to take immediate action against the culprits who were responsible for his father’s death. His anguish and emotional trauma would have been very less as compared to the trauma inflicted by Joseph. Where in Hamlet, Shakespeare has created an element of Ghost, Kafka would have made the appearance of divine figure like that of a priest who would have taught Hamlet to accept his fate as what is destined for him has to happen. For Kafka, guilt is a feeling that no man can avoid whatever his or her experience may be and in Hamlet, Kafka would have definitely used the element of guilt. He would have made Hamlet realized his mother and uncle of guilt instead of entering into moral dilemma himself and make them face the trials. Here Hamlet would have been realized the fact that it is not important that an individual is actually guilty of an offence but what is important is that crime has occurred and he knew the culprits and therefore they ought to be put in trial and convicted. Another aspect to it is an issue of revenge. Hamlet wanted to take the revenge of the death of his father that was contradictory to the contemporary society in which Hamlet was written. This revenge implies taking the law in one’s own hands in order to satisfy an inner urge, although in civilized society the function of fighting the wrongs done to individuals belong to the state or the government. If Kafka had written Hamlet, he would have made Hamlet t try to follow the course of law to punish the culprits. But, he would have made Hamlet unsuccessful in his efforts as the law takes its own course and by that time Claudius would have been set free and took an adequate opportunity to bring Hamlet to his tragic death. In other words, death of Hamlet could not have occurred due to the moral flaws in the character of Hamlet but due to the circumstances that were out of his control. In spite of his efforts, Hamlet would never have been able to bring real culprits on the punishment panel. Kafka would have made Hamlet a story of the faults in the justice system of the contemporary society instead of the moral flaw in the character that would have made his end inevitable.   In Kafta;s version of Hamlet too, would have also died in the end but in a different way. Hamlet would have never been able to find truth of the conspiracy behind his father’s death. As with the Mouse Trap’ in Hamlet was the major victory for Hamlet as he was able to contrive his father’s murderer but the parable of Kafka’s would have made Hamlet frustrated as it neither contained any golden rule nor even suggested a mode of behavior under particular conditions. Hamlet would have not learnt anything and died a meaningless life bearing the misery of human existence. In the end Hamlet says, â€Å"the rest is silence†, this dialogue is an exploration of the true nature and life of human beings. Hamlet realized the fact that for all human beings, the ultimate destiny is death. Finally all human beings have to attain the death, which is inevitable and have to accept their ultimate faith. Hamlet confronts, recognizes and accepts the condition of being man and the last death wringed in him final cry of passion. But it is not simply the acceptance of death that Hamlet wants to unveil but within this end is the paradigm of complete mysteries of life and along with it is the mystery of evil. The realization of the reality of the death and the mystery of life he felt in the graveyard where the bodies of scheming politicians, the hollow courtier, the tricky lawyer, emperor or the queen and the beautiful young maidens laid.   Whereas when Joseph K said â€Å"like a dog†, Kafka struck at the contemporary society with a tool of an irony. Joseph K.’s demise is the demise of all human beings who had to face the courts and trials without any question. All the human beings are the dogs in the hands of the society and have to accept the fate what has been destined for them. This is a difference between ‘like a dog’ and ‘the rest is silence’. The similarity between the two lies in the fact that fate is the biggest thing in the life of human beings and every one has to accept their fate. Works Cited McManus, Barbara F. â€Å"Outline of Aristotles Theory of Tragedy in the POETICS†. Internet. (1999). Available: http://www.cnr.edu/home/bmcmanus/poetics.html, October 17, 2008.

High Performance Liquid Chromatography (HPLC) 214

High Performance Liquid Chromatography (HPLC) 214 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. 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   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   b) Quantitative calibration  Ã‚  Ã‚  Ã‚  Ã‚   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. Table-8.1 Important information concerning sample composition and properties 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   Ã‚   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 â€Å"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 (    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 â€Å"chiral† conditions for their separations. Biological Several factors make samples or this kind â€Å"special†; molecular conformation, polar functionality and a wide range of hydrophobicity. Macromolecules â€Å"Big† molecules require column packing with large pores  Ã‚  (>> 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 2.0 Additives (e.g., amine modifiers, ion pair reagents) Do not use initially Flow rate 1.5–2.0 ml/min Temperature 35-45 ºC Sample Size Volumed >25 mL Weightd B : Polar solvent  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   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 pH 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 Quantitation   Ã‚ £ 2% (1 SD) for assays;  £ 5% for less-demanding analysis;  £15% for trace analysis. Pressure 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 (Rs>1.5) 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 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 Beer’s 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  Ã‚  Ã‚   =  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   3 X N / B LOQ  Ã‚  Ã‚   =  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   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 â€Å" 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 2 ºC from room temperature to 80 ºC 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 ½)2 Where ‘tR’ is the retention time of the peak and ‘W ½Ã¢â‚¬â„¢ 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 â€Å"W0.05† is the width of the peak at 5% height and â€Å"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. High Performance Liquid Chromatography (HPLC) 214 High Performance Liquid Chromatography (HPLC) 214 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. 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   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   b) Quantitative calibration  Ã‚  Ã‚  Ã‚  Ã‚   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. Table-8.1 Important information concerning sample composition and properties 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   Ã‚   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 â€Å"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 (    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 â€Å"chiral† conditions for their separations. Biological Several factors make samples or this kind â€Å"special†; molecular conformation, polar functionality and a wide range of hydrophobicity. Macromolecules â€Å"Big† molecules require column packing with large pores  Ã‚  (>> 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 2.0 Additives (e.g., amine modifiers, ion pair reagents) Do not use initially Flow rate 1.5–2.0 ml/min Temperature 35-45 ºC Sample Size Volumed >25 mL Weightd B : Polar solvent  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   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 pH 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 Quantitation   Ã‚ £ 2% (1 SD) for assays;  £ 5% for less-demanding analysis;  £15% for trace analysis. Pressure 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 (Rs>1.5) 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 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 Beer’s 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  Ã‚  Ã‚   =  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   3 X N / B LOQ  Ã‚  Ã‚   =  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   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 â€Å" 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 2 ºC from room temperature to 80 ºC 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 ½)2 Where ‘tR’ is the retention time of the peak and ‘W ½Ã¢â‚¬â„¢ 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 â€Å"W0.05† is the width of the peak at 5% height and â€Å"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.