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.

Low self esteem

I was flicking through some featured articles on my IPad last night when I happened to come across Rod Liddle's piece: ‘our children urgently need less self-esteem'. I asked myself, do our children need less self-esteem? Preposterous. Of course not, in fact I believe that they need more self-esteem. Low self-esteem is one of the major underlying problems of crime, bullying, unemployment†¦ honestly, the list could go on. Children with low self-esteem suffer from depression and a sense of insignificance and generally have a pessimistic view on all aspects of life. Whereas children with high self-esteem are positive and seem to do better in life than those who lack confidence. Why? Why should children need less self-esteem when it is already clear that high self-esteem seems to be a better trait in children? Honestly, I really do think this article is just a '50 year old twat' ranting about the younger generation, however I have an urge to criticize his opinions because his article is just too fallacious. Rod Liddles article was a response to the riots earlier last year in the summer, and yes I do agree that they were outrageous and caused a huge calamity nationally, but, like the teachers I would say the kids need more self-esteem rather than less. This is due to the fact that a majority of the rioters probably had low self-esteem to start with and therefore gave into peer pressure to follow some idiots smashing up the shop windows of Poundland just because it seemed like the ‘cool' thing to do. Even if some kids have self-esteem which sky rockets to the sky, acting as role models for the rioters, it is probably the parents fault, and the upbringing they had which means it is Liddles generation to blame. I believe that the riots were not started by some random, over-confident teens wanting to take over the nation but were sparked by an outrage when the judicial system failed to explain the accidental shooting and killing of Mark Duggan. So obviously it is his generation who started the riots In the first place by unjustly ignoring people's anger, consequently allowing the riots to start. Also, Liddle states that the judicial system is made too liberal saying â€Å"if anything goes wrong with a modern child's life, it is someone else's fault: teachers, or the Police, or society†. In this he is totally contradicting himself, because the police force is made up of his generation or maybe slightly younger, but all still being adults, so really he is criticizing the wrong people. In addition, according to Wikipedia, police arrested 3,100 people which is a fairly reasonable amount of people. Meaning the judicial system is not actually that soft, they do lock up a fair amount of wrong doers. As for the teachers, well I'm sure almost everyone on the planet has had a detention or some form of punishment for doing something wrong in school. And if you do something horrifically bad then you are expelled with no exceptions. So tell me again, Liddle, where children are not disciplined? The riots were not caused only by low self-esteem by also by desperation due to the current economic situation our country is in now. Many people are now broke, poor and depressed, and obviously some people want to do something about it. So when the opportunity arises where you can take whatever you can get hold of, whether you actually want it or get some money out of it then I'm guessing the mind-set is ‘why not? , other people are doing it'. The state of the economy had to be caused somehow, some time ago and after some research it turns out that it was in fact Liddles generation who caused this total slump. Apparently, ‘back then' houses were cheaper and loaning from the bank was easier, causing people to have too much self-esteem and consumer confidence. So when people decided they were rich they started buying and buying and buying until they had no money to give back to the banks. Now in the present day all of us have to pay for their expenses and suffer in this drudgingly, slow sink into an economic depression. Lastly, I am convinced that Liddle is totally unreliable and his use of hyperbole is excessive, he is clearly not a modern day teen and cannot say anything about self-esteem in kids because he simply is not one. Liddle is pretty much 30 years past his prime and cannot accurately state the minds of our modern youths. Many teens are low in self-esteem but merely hide behind a mask to make it seem as though they are ‘significant' and know it, but in fact they want to curl up in a ball and cry about life.

Famous Creative Thinkers Essay

In this assignment there are several great creative thinkers to choose from for completion. After reading through the list James Hal Cone and Grace Hopper became the choices. The reasoning was these individuals were so different in their paths of success, or for better word accomplishments. Both are extraordinary people; however their journeys are profoundly different, however both are instrumental in worthy contributions in society and the world. Throughout this paper we will uncover and discover Cone and Hopper purpose, passion, and for one even pain. James Hal Cone was born August 5, 1938 in Fordyce Arkansas, to Charles and Lucy Cone. Early in Cone childhood he was introduced to religion and had a strong spiritual guidance. James was an intelligent child who went on to graduate from high school at age 16, at which time he became a minister. Cone beginnings started with being brought up in a segregated part of Arkansas, where people of color were discriminated against and treated u nfairly. James early in life reflected upon the social injustices of the poor, blacks, and women. Cone took the treatment to heart early along with his religious background and became a minister at 16, starting to address the differences of treatment. During his early life at college he was a minister at several churches. However during his early period of college and ministry the civil rights movement started evolving and he noticed Martin Luther King. Through that introduction of Dr. King he realized his true direction was ministry and attended Theological Seminary getting a M.A. and Ph.D. James acquired four degrees in a seven year span, a man on a mission with an extraordinary mind. Two things directed Hal’s path pain of discrimination and coming to know Jesus. After graduation with his Ph.D. in theology, Hal went on to be a professor of religious studies, still believing in nonviolence. During this period changes came with Malcolm X, northern riots, and Stokely Carmichael’s   call to â€Å"Black Power† (This Far by Faith, 2015). The direction of society from two important men King and X, initiated Hal’s influence of empowering African Americans Christianity from slavery, segregation and justice in society. Hal knew through theology he could address the issue and started writing introducing black liberation theology. What Hal orchestrated was self-worth with assimilation of the black power movement, addressing social and racial justice for black people, freeing them from oppression economically and spiritually. Ideally Hal wanted to empower people through Christianity beliefs; however he addressed the separate treatment of the poor, oppressed and blacks in the Christian community. Hal eventually wrote many books that were supported and criticized because of his criticism of white theologians not addressing the struggle and differential treatment of the African American people as far back as slavery. Hal also went to speak in China and Latin America concerning the lack of address of people of color being oppressed, poor and oppression as Christians. He took a stand against segregation and mistreatment of all people on a theologian podium. James Hal took theology using it as a tool towards the individuality that contributed to the history, existence, and civilization of black people. Hal thinking really was devised from his thought of how can people be Christians, but be ok with the injustices of segregation. With Hal coming of age during the civil rights movement had a major effect on his social consciousness. This was a period (civil rights movement) where it was obvious that black people were being mistreated, even among white Christians. Hal took the stand against the treatment even though it was not a popular decision. For James it became unacceptable for Christians to treat people differently than what the bible taught, â€Å"Love your neighbor as you love yourself†. Even though it appeared Hal writings and views were racially motivated, by earlier content, Hal knew his sentiments was to empower Christian people to realize that in spite of their skin color they were of value and deserve to be treated accordingly. Through it all the process that Hal used to devise his plan of action came from evaluating and analyzing the information obtained from his personal experience, observation and communication. Armed with those factors of critical thinking he creatively started changing how people of color, women, oppressed, and poor people thought about themselves spiritually. Hal later realized that terms used to address white Christians was impropriate  and that he could have addressed the issue differently, still standing on his beliefs, that there should be no racism or segregation in the church. James Hal Cone the pioneer for making people aware of the segregation that should never be acceptable in the theologian Christian community, God created all people equal and God is in respect of no man. Grace Hopper computer scientist and that is speaking lightly of her accomplishments and creativity. Grace Hopper was born December 6, 1906, as Grace Brewster Murray in New York. Born during a period that girl were not normally educated, Grace parents believed just the opposite. Grace was not a traditional girl, on one account she was fascinated with the working of a clock, at seven she dismantled the clock. Her actions were based on how does the alarm clock work? As the story goes she dismantles all the clocks in her home, first sign of her tenacity, innovation and perseverance (Hopper Biography- Mac Tutor of Mathematics, 2015). Grace attended private school and went to earn a college degree in mathematics from Vassar College. From 1928 to 1931 she achieved marriage, along with a M.A. degree from Yale University. Three years later Grace was an associate professor with a Ph. D., however with all her accomplishments her real passion was to join the military. Opportunity presented itself when the United States entered the Second World War, unbeknownst to Grace she was too old and to slight in weight. However not one to take no for an answer she persuaded the Navy to enlist her at the old age of 37, unheard of especially for a woman. Mission accomplished she’s a Navy woman with her first orders to start working on the Harvard Mark I computer, she was elated. The computer took her back to childhood, now she really could dismantle the computer and make adjustments, becoming the first woman to program the Mark I. She is in her element and making strives, moving forward. Grace went on to play a significant role in the creation of the Mark II and III computer program. After retiring from the Navy she developed with a team the UNIVAC computer, along with an upgrade compiler. While still being employed at a computer corporation, Grace and team developed the first English – language data processing computer (Hopper Biography –Mac Tutor Mathematics, 2015). Grace was the pioneer behind the first English language computer being in existence. Before the team and Grace developed the language computer, computers were  only mathematically designed. There was only word numbers, no words had come into computer existence. Now computers with business language existed, which people in the computer world thought was not possible. The COBOL (common business – oriented language computer came into existence in 1959 changing the world. Grace had creative ideas before the time frame of computers starting with an alarm clock. With her ability for mathematics, brilliant mind, and education she was able to accomplish the beginning of the computer language, which have gone on to universal success. Grace had no restraints, what she set out to do she did. In spite of the times she had courage, direction, daring, and most important she did not have an established pattern for her creativity thinking. At all levels she challenged herself with a range of ideas; from discovery, defining, designing, and developing the computer with the English language. She was the lady behind the computer bug (Berni Dwan, Irish Times, 2001). Her biggest obstacle was being a female in an area of men and times when women were not so easily accepted in the corporate world, especially the unknown like computers. References Blake, John. â€Å"America’s ‘Angriest’ Theologian Faces Lynching Tree.† CNN Belief Blog. http://religion.blogs.cnn.com/2012/04/21/americas-angriest-theologian-faces-lynching-tree/?hpt=hp_c1 (accessed April 23, 2012). â€Å"James H. Cone.† Union Theological Seminary in the City of New York. http://www.utsnyc.edu//Page.aspx?pid=353 (accessed June 15, 2011). BLACK THEOLOGY AND IDEOLOGY: DEIDEOLOGICAL DIMENSIONS IN THE THEOLOGY OF JAMES H. CONE (Book). By: Hayes, Diana L., Theological Studies, 00405639, Dec2003, Vol. 64, Issue 4 http://www.pbs.org/thisfarbyfaith/people/james_cone.html Grace Hopper. (2015). The Biography.com website. Retrieved 01:15, Mar 23, 2015, from http://www.biography.com/people/grace-hopper-21406809 http://www-history.mcs.st-andrews.ac.uk/Biographies/Hopper.html Programming’s amazing grace she developed the first program to translate computer instructions from english into machine language and gave the world the computer `bug’. berni dwan looks back at the life and work of grace hopper. (2001, May 21). Irish Times Retrieved from http://search.proquest.com/docview/309366344?accountid=358 Famous Creative Thinkers

The Influence of the American Automobile - 1321 Words

The Influence of the American Automobile There’s hardly a person alive in America today that hasn’t ridden in an automobile of some sort at some point in their life. We’re all connected to each other by roads crisscrossing roads and highways all across country, and yet few people understand how we got to this point. They simply accept their magic metal box will work when they put the key in to start their car. Long ago, this country once had a great love affair with the American Automobile, and it was a turbulent, passion filled, amazing ride. If someone asks, â€Å"What was the first American car?† Many would answer, â€Å"Ford’s Model-T.† However, Henry Ford had been manufacturing cars for over a decade before the introduction of the Model-T. The first American car was most likely the Baushke Autymobile built in 1894. During that time period there was a boom in start-up manufactures of the new â€Å"Horseless Carriage† machines, ever ybody in the coach and bicycle industry was jumping at the chance to build one. Dozens of manufactures came and went, but a few of those early start-ups still exist today. These early automobiles were loud, uncomfortable, and often dangerous, but that didn’t stop the dreamers and innovators from pushing forward. Henry Ford was one such visionary. He knew early on the automobile industry was going to change America. He strategically positioned himself to be at the forefront of this new invention sweeping the nation.Show MoreRelatedThe Automobile Industry Influenced The American Economy1012 Words   |  5 PagesIn 1769, the first automobile, a steam-powered carriage that would carry up to four people at two miles per hour, was created. 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