Cost Of The American Civil War Essay Example | Topics and Well Written Essays - 250 words

Cost Of The American Civil War - Essay Example This research will begin with the statement that soldiers who engage in Civil War are doing so because they believe that their actions will ultimately lead to a better society for them, their family and their friends and countrymen. This essay discusses that many of the Confederate states that seceded from the Union did so because they knew that the abolition of slavery would not only destroy their way of life, but it would drastically hinder the families of the south to be able to properly make a living. The present research has identified that the entire economic system was based upon using African American slaves in the south to create crops that were used to engage in international trade with Europe at the time as well as to provide basic products and money to other families and services in the South. Likewise, the Federal soldiers fought to conserve their country that they believed so much in. From the research, it can be comprehended that the Confederacy was a direct threat to their way of life and the nature of the conflict bridging on war threatened the safety of their land and placed an even greater threat on the lives of friends and family. The researcher states that in both cases, each side fought directly to ensure that their way of life remained intact and that nothing would threaten their financial health or their safety. The real cost of the Civil war came in the form of not only financial cost but also in the cost of lives and resources in America.  

United States v. Jones Essay Example | Topics and Well Written Essays - 1000 words

United States v. Jones - Essay Example In the making of court decisions involving the fourth amendment of the Bill of Rights, three critical aspects are considered. The aspects include the kind of activities considered by the government to constitute searches and seizures, the probable cause constituted by the actions in the government activities, and the appropriate mechanisms of addressing the violations of the fourth amendments (Wetterer 94). In the case between the state and Jones, it is in order to put clear some facts to understand the unfolding of events in the court decision and reasons for such decisions. First, Jones, who is the respondent in the case, is the owner and operator of a nightclub come under suspicion that he could be dealing in drug trafficking. Based on the above occurrence, police after conducting their investigations sort for a warrant from the Federal District Court, which the courts granted for to use of a GPS tracking device on the Jeep registered to Jones’ wife, which, however, driven exclusively by Jones (McDonald and Means 112). However, the officers failed to beat the ten-day deadline issued and instead put the GPS tracking device on the car to track on the car’s movement on the eleventh day while in Maryland, outside of DC, which the permit granted. With the help of satellite, the GPS device was in a position to monitor the car’s movement for twenty-eight day, sending i ts data to a government-monitored computer and producing data comprising of over 2,000 pages over the same period (McDonald and Means 62). From the findings, Jones was charged for conspiracy of cocaine distribution by the government. Concerning the evidence presented to the court by the state, the judges suppressed the data obtained from the GPS while the car was parked in Jones garage while holding the remaining data based on the aspect that Jones had no expectation of privacy whenever the car was in a public place at the pre-trial hearing of the case (Arcila 88).

Corporate social responsibility in the global business community Assignment

Corporate social responsibility in the global business community - Assignment Example Also, it changes the business entrepreneurs perception towards business and society. Today, business is not just about profits, but it is more about profit sharing with society and employees (Kiran & Sharma, 2011). The majority of the world-renowned firms and organizations, for instance, the world bank, as well as European Commissions are mostly actively promoting and supporting the concept of Corporate Social Responsibility. However, Modern corporations attempt to put significant focus on the politics, economy, and the society of a country (Baxi, 2006). Perhaps, the developing nations are supposed to focus more on the Corporate Social Responsibility implementation and from the planning process. In addition, they can form alternatives to lifting the development of the social sector, and a suggested way is to develop viably and strategized public-private partnerships. Currently, the greatest challenge for most governments is on how to create Corporate Social Responsibility practices policies aa well as nurture a framework that is durable and would assist society and companies to translate the public policies to deliverables (Kiran & Sharma, 2011). Therefore, the paper focuses on defining CSR, identifying a global business that implements a CSR strategy, explain why, how, and where the global market they implement their strategy. The overall objective of the paper is to get a better understanding of how and why global companies are becoming more responsible. The paper also include the interpretation and conclusion. Nestle is one of the global renowned and largest, beverage and food companies. Nestle is a very old organization that started its activities almost 130 years ago. Its key success is innovations of its product. Alongside with its innovative capabilities, its brand business acquisitions that have made it be the large food firm all over the world. However, with time the passing, the Nestle company has swiftly grown and penetrated into the production of

Analysis Essay Example | Topics and Well Written Essays - 750 words - 17

Analysis - Essay Example The prices of groceries are determined by the interaction between demand and supply as with any other marketable product. Both the consumers and suppliers rely on quantity and prices as a signal to adjust respective demand and supply of groceries in the market. But as groceries are necessary items and widely available in any market due to competitive market, it has significant buyers purchase decisions. The supply curve plots the relationship between quantity and prices of US grocery industry s that are able to procure and sell products in open market. Assuming that the market structure is perfectly competitive, the basic law of supply states that with the increase in prices of groceries in the market, the suppliers will response positively by increasing supply with the objective to earn more profits. Conversely, as the supply of groceries become abundant the suppliers negatively react by lessening procurement and supply of groceries in the market in order to cut losses that gradually leads to the fall in prices of groceries. Now, in the given case, Albertsons parent Cerberus is buying Safeway for over $9 billion. Many analyst have noted that the move was an attempt to acquire top spot in the US groceries industries. In US, over 19 percent market share is owned by Kroger followed by Safeway which is at present the second largest chain following Kroger. The merger will impact the demand-supply equation because post-merger it is expected that Kroger would operate with 2,640 stores whereas Albertsons would operate with 2,400 stores. The chart depicted above reveals that the volatility in market regarding the supply of groceries modifies the basic supply curve by shifting position. The analysis of supply movements of groceries indicate that when the curve shifts from S1 to S2 (right-side), it is an indication that there is an increase in supply of groceries in market, assuming

Laboratory Testing and Evaluation Report Essay Example | Topics and Well Written Essays - 1500 words

Laboratory Testing and Evaluation Report - Essay Example A theoretical study on their mechanical properties is also made. Samples of both finished materials having 1.2 mm thickness are tested against their mechanical properties which include universal testing and hardness testing. Another study of same grade of material which is kept under different process conditions is also made. Samples of 1.2 mm finished skin rolled SPCC steel versus 1.2 mm annealed SPCC steel were also studied in the same manner. As per discussing the chemical composition of the two materials, JISF (2011) had shown the allowable alloying percentage as given in Table 1. Table 1 Symbol of Grade C (%) Mn (%) P (%) S (%) SPCC steel 0.15 max 0.60 max 0.10 max 0.050 max SPCG steel 0.02 max 0.25 max 0.020 max 0.020 max However, the mill test certificates of the samples reveal following composition as given in Table 2 of the two materials: Table 2 Symbol of Grade C (%) Mn (%) P (%) S (%) SPCC steel 0.10 0.23 0.02 0.009 SPCG steel 0.002 0.17 0.019 0.007 As evident from the ste el grades, commercial SPCC steel is applied in forming applications, and extra deep drawing SPCG steels are meant to be deep drawn. These steels are widely used in various applications. SPCC steel is being used in the making of door hinges, pans, casings, galvanised steels, and profile sheets. Whereas SPCG steel is utilised in deep drawing applications such as motor cycle fuel tank, chain cover, etc. Chemical composition of the alloy steel is incredibly important because each element has its own role in varying the material properties. For instance, rust is a vulnerable defect of steels, and rust is basically the result of iron oxide formation (Blair and Stevens, 1995). Absolute iron is absolutely susceptible to rust. With the introduction of other elements, rust exposure is decreased. Higher percentage of alloying material decreases rust possibility and lowers percentage of alloying material; alloying material increases the risk of rust formation. Therefore, SPCG steel is more susc eptible to rust as compare to SPCC steel. Similarly, elements present in alloying composition also affect mechanical properties of the steel. Mechanical properties are to be assessed in this laboratory testing and material evaluation case study. 1. Structure of Materials Figure 1 (Blair and Stevens, 1995) is the iron-carbon phase diagram. It shows the percentage of Carbon influencing the microstructure of the steel. It is a low-carbon steel which contains carbon between 0 02 % and 0.15 %. Both material grades are the dead mild steel comprises mainly alpha ferrite and the traces of pearlite in its microstructure. These are extremely ductile and are therefore usually utilised as sheets for forming and drawing operations. Its microstructure seems white as of very less insignificant pearlite content steel. Both SPCC and SPCG sample materials lies in pearlite + ferrite region. Figure 1 2. Expected Theoretical Results The chemical composition of these steel is vitally important for assess ing mechanical properties. It is because lower carbon percentage in steel will affect the making of material, lowering its hardness, and a higher percentage of carbon will yield a harder material. Therefore, according to theoretical analysis, SPCC steel should be harder than SPCG steel due to a higher percentage of carbon. JISF (2011) specifies the range for the two material grades, as mentioned in Table 3, of different mechanical prope

Psychology, Theology, and Sprituality in Christian Counseling Essay Example for Free

Psychology, Theology, and Sprituality in Christian Counseling Essay In his conceptual book, Psychology, Theology, and Spirituality in Christian Counseling (1996), McMinn presents a convincing presentation of a multitasking counselor who has developed a niche in the counseling world. New age Christian counselors have developed simultaneous skills that embark on the areas of psychology, theology, and spirituality. This begins our journey down the path of understanding how values and perspectives can be changed as a result of a well rounded, multitasking Christian counselor. With life illustrations and brief counseling scenarios throughout this book, McMinn (1996) provides the reader with an excellent working model of identifying and relating life experiences to Psychology, Theology, and Spirituality. The opposition of modern psychology and theology practices is delivering different contemporary messages concerning mental health. Psychologist Albert Ellis wrote, â€Å"The emotionally healthy individual should primarily be true to himself and not masochistically sacrifice himself for others.† Versus Christian spirituality that identifies and states in scripture that as individuals we are instructed to look out for the interest of others (Phil. 2:4) and to prefer one another in honor (Rom 12:10) (McMinn, 1996). As McMinn works through the concepts of integrating these three distinct disciplines, Christian counseling becomes more complex and multifaceted. Christian counselors set their eyes upon God with an individual’s spiritual growth and mental health in their mind. In addition to placing the pieces of a battered mental health condition focus to the forefront, their client’s eternity of life and knowledge of God is an important piece of the puzzle. A more detailed perspective of psychological and spiritual health, allows every individual to recognize their responsibility to God, to their family and friends and to themselves. As humans, we must understand and have a healthy awareness of brokenness to allow ourselves to experience grace and hope in the midst of our walk on this earth through life’s trials and tribulations with Jesus Christ. Integrating psychology, theology, and spirituality in addition to the niche of multitasking, all three disciplines in unison takes time, energy, loyalty, and dedication of the counseling profession. In the last two thirds of this book, McMinn uses prayer, scripture, sin, confession, forgiveness, and redemption to show us a glimpse of the counseling world. The problem with sin is that it separates us from God; the wonder of redemption is that individuals are brought back into relationship with God. (McMinn, 1996, p. 265). A redemptive Christian counselor has humility and compassion and experiences God’s grace with gratitude. Scripture yanks people out of the grips of sin as a redemptive God shines through. Those who deny sin see no need for spiritual redemption. Confession, allows us to acknowledge our sin and our desperate need for help and through the grace of God we receive forgiveness and experience redemption. Once our affections turn to God, our prayer will never be the same. Our lives will be completed changed and everlasting. â€Å"Bless the Lord, O my Soul, and do not forget all his benefits-who forgives all your iniquity, who heals all your diseases, who redeems your life from the pit, who crowns you with steadfast love and mercy, who satisfies you with good as long as you live so that your youth is renewed like the e agle’s†(Ps. 103:2-5). Response Come to me, all you who are weary and burdened, and I will give you rest. Take my yoke upon you and learn from me, for I am gentle and humble in heart, and you will find rest for your souls. For my yoke is easy and my burden is light.† As I had struggled throughout my chaotic childhood dealing with the presence of alcohol, drugs, fighting, and uncertain living conditions in my circle of life, I developed the ability to discern which people were right for me in my life. My perception of family was skewed. At the age of 7, I lost my grandfather and life’s losses of my protectors continued until 2004 with the death of my first husband Dale. My life was over. My family was a mess. I had just started a new church and I wasn’t sure where God was in my life. My father in law, who preached my husband’s funeral, started providing Christian guidance and support as a lay counselor. We prayed and shared scripture together. At the age of 33, I learned the true meaning of prayer. I knew how to pray and I knew how humbled I had been in my prayers for Dale. In the kindness of his God filled heart, he taught me that humility allows us to see God’s word. As I study and read my Bible, I continue to learn the importance of a Christian family for support and guidance and the necessity to share the story of Jesus Christ. My father in law stepped out in faith to help turn my life around. Through his obedience, I work to help others who are hurting find the power of prayer and scripture in their times of heartache. Reflection As I read through Psychology, Theology, and Spirituality in Christian Counseling (McMinn, 1996), a highlighter was used to mark special passages I needed to keep in the forefront of this class. There are so many key notes to remember in the text, the book was turning a nice shade of yellow. Anyone who is starting out in the counseling field should be required to read this book. McMinn does an excellent job of showing the reader how to integrate our Christian faith and spirituality into the secular world with real life reflections. Christian counselors want to follow the will of God and McMinn provides the examples, guidance, and techniques on how to incorporate God into the counseling session. The major drawback with this work concerns the conclusion. Throughout the reading, it is apparent McMinn is preparing the counselor to learn the skill of multi-tasking. In two pages, McMinn summarizes the whole book. The most important part of integrating the three diverse areas of studies is profound enough for him to provide a greater level of detail to the new counselor concerning the background of multitasking. Success lies in the strategy that is used in multitasking these three distinct practices and beliefs. In dealing with the secular world, this skill is a necessity to bring all three disciplines together. Action I am sending you out like sheep among wolves. Therefore be as shrewd as snakes and as innocent as doves. â€Å"Be on your guard against men; they will hand you over to the local councils and flog you in their synagogues. On my account you will be brought before governors and kings as witnesses to them and to the Gentiles. But when they arrest you, do not worry about what to say or how to say it. At that time you will be given what to say; for it will not be you speaking, but the Spirit of your Father speaking through you. (Matthew 10:16-20). God has given me back tenfold since the loss of my first husband. I have established myself as a praying respiratory therapist at UVA. As Jesus calls me into the counseling field, he is preparing me for the secular world. In my environment, people are at the lowest points of their lives. I use Gods calling on my heart and spirit to reach out in Christian love to these individuals and families. God places pastors, judges, professional basketball players, the rich, the homeless, the curable, the dying, in my path for a reason and a purpose. Life is simple when you are obedient. Go where God calls you. The people I come into contact with are not by accident. I minister to their hearts through their illness and time of need. I pray for God to give me the strength and the right words of his will to deliver to the sick and their families. I pray for each individual that crosses my path to find the love of Jesus Christ and meet him in heaven to spend an everlasting life with our father.

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.