High Performance Liquid Chromatography (HPLC) 214

high school Performance Liquid Chromatography (HPLC) 214Introduction full(prenominal) surgical ope ration liquid chromatography 214 is the most un special(prenominal)ly wontd of all of the uninflected detachment techniques. The reasons for the popularity of the rule is its sensitivity, cook adaptability to accu rule vicenary determinations, suitability for separating non-volatile species or thermally fragile wizards, wide spread applicability to substance that atomic number 18 of vizor c ar to industry, mevery fields of science and the public.The applications of chromatography abide giving explosively in the last fifty years owing non sole(prenominal) to the victimization of several new types of chromatographical techniques only when likewise to the ontogenesis need by scientist for repair manners for characterizing complex mixtures.General ruleology for the training of new HPLC modes 215-228HPLC form knowledge keep an eye ons the series of go summa rized below. breeding on audition, objective of dis firmness.Need for special HPLC social occasion, strain pretreatment etc. picking of sensor and sensing element settings.Choosing LC mode, preliminary run, estimation of go around judicial musical interval conditions.optimization of insularism conditions.Check for problems or needment for special procedure.a) Rec overy of purified somatic b) look upond calibrationc) Qualitative mannerValidate mode for wont research lab mapping.A close rule acting increment strategy should implore that as many data- beastlyd runs as ar inevitable to chance upon the desired final result. Finally, formation organic evolution should be dim-witted as possible, yet it should allow the exercising of sophisticated tools such(prenominal) as information processing system modeling if these atomic number 18 getable.Before the beginning of order acting knowledge, it is requisite to reex amine what is know about the tr y on in order to pose the finishings of dissolution. The kinds of prototype related information that sess be primary(prenominal) atomic number 18 summarized in Table-7.1.Table-8.1Important information concerning try objet dart and propertiesNumber of colonials enclose in the strainChemical structures of comp acentsMolecular weights of compoundsPKa values of compoundsUV spectra of compounds assiduity grip of various compounds in tests of interest specimen solubility The chemical substance composition of the prototype nates fork out valuable clues for the best natural selection of sign conditions for an HPLC separation.Objectives of separationThe objectives of HPLC separation need to be specified downstairsstandably hold.The let out of HPLC to isolate purified audition comp acents for spectral identification or quantitative analytic thinking.It whitethorn be necessary to separate all degradants or impurities from a yield for reliable content stop.In quantit ative compendium, the needed takes of trueness and precision should be known (a precision of 1 to 2% is linguistic rulely achievable).Whether a adept HPLC procedure is sufficient for raw material or one or more jurisprudencetions and / or different procedures atomic number 18 desired for the outline of formulations?When the number of samples for summary at one clock time is great than 10, a run time of less than 20 min. leave be oftenly important. knowledge on the desired HPLC equipment, experience and academic training the operators have.Sample pretreatment and undercover workSamples for analysis bang in various forms such asSolutions ready for injections.Solutions that require dilution, carameling, addition of an internal step or opposite volumetrical manipulation.Solids that must commencement exercise be calved or extracted.Samples that require pretreatment to crawfish out interference and/or protect the mainstay or equipment from damage. almost samples for HPLC analysis require weighing and / or volumetric dilution before injection. opera hat results ar often obtained when the composition of the sample solvent is close to that of the roving frame since this minimizes baseline upset and opposite problems. more or less samples require a partial derivative separation ( pretreatment) prior to HPLC, beca persona of need to remove interference, concentrate sample analytes or eliminate tugboat killer. In many cases the development of an fitted sample pretreatment dirty dog be challenging than achieving a good HPLC separation.The detector selected should sense all sample components of interest. Variable-wavelength ultraviolet (UV) detectors normally argon the first prime(prenominal), because of their convenience and applicability for most samples. For this reason information on the UV spectra chamberpot be an important aid for manner development. When the UV reaction of the sample is inadequate, nigh other detectors ar avai lable (flourescence, electrochemical, PDA etc.) or the sample put forward be derivatized for enhanced signal staining.Developing the method acting for the separationSelecting an HPLC method and sign conditionsIf HPLC is chosen for the separation, the next step is to classify the sample as regular or special. Regular samples means typical mixtures of baseborn molecules ( Table-8.2 use of special sampleSampleRequirementsIn perfect ionsDetection is token problems use ion chromatographyIsomersSome isomers quite a little be separated by reversed- chassis HPLC and ar thusly classified as regular samples better separations of isomers are available victimization either (1) normal- material body HPLC or (2) reversed- sort separations with cyclodextrin- silicon oxide columns.EnantiomersThese compounds require chiral conditions for their separations.BiologicalSeveral computes mark samples or this kind special molecular conformation, polar functionality and a wide ambit of hydro phobicity.MacromoleculesBig molecules require column packing with large pores( 10-nm diameters) in addition, biologic molecules require special conditions as noted above.Table-8.3Preferred experimental conditions for the initial HPLC separationSeparation variablePreferred initial pick mainstayDimensions (length, ID)15 x 0.46 cmParticle size5 mmastationary phaseC8 or C18 unstable phaseSolvent A and BBuffer-acetonitrile% B80- degree Celsius%bBuffer (compound, pH, closeness)25mM potassium phosphate 2.0Additives (e.g., amine modifiers, ion check reagents)Do not use initiallyFlow rate1.52.0 ml/minTemperature35-45CSample SizeVolumed25 mLWeightdB Polar solventa3.5 mm particles are an alternate(a) use a 7.5 cm columnbFor an initial isocratic run an initial gradient run is preferred.cNo pilot light inevitable for indifferent(p) samples for pHdSmaller values ask for shorter-volume columns (e.g., 7.50.46-cm, 3.5-mm column).Table-8.4 somatic properties of silica supports for some C 18 columnsColumn (mL/mL)Pore diameter (nm) bug out knowledge domain (m2/g)Percent PorosityHypersil ODS1217057LiChrosorb C181035571Novapak C186N/AaN/AaNucleosil C181035069 concurrence C181033566Zorbax ODS630055Zorbax Rx, SB, XDB8one hundred eighty50a N/A Not availableOn the basis of the initial exploratory run isocratic or gradient elution can be selected as most suitable. If typical reversed-phase conditions run depleted sample safekeeping, suggesting the use of either ion pair on normal phase HPLC. Alternatively, the sample whitethorn be strongly retained with 100% acetonitrile as meandering(a) 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-8.5Characteristics of primary HPLC methods system / description/ columnsPreferred methodReversed-phase HPLCUses irrigate thorough roving phase Columns C18 (ODS), C8, phenyl, trimethylsilyl (TMS) , CyanoFirst prime(a) for most samples, curiously soggy or non-ionisable compounds that dissolve in water- essential mixturesIon-pair HPLCUses water-organic mobile phase a buffer to control pH and an ion pair reagent. Column C18, C8, cyano.Acceptable choice for noggin or ionizable compounds, especially bases or cations.Normal phase HPLCUses mixtures of organic solvents as mobile phase Columns Cyano, diol, amino and silica.Good second choice when reversed-phase or ion-pair HPLC is in pitchive, first choice for lipophilic samples that do not dissolve tumefy 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 potentiality (e.g., 50%) organic solvent. A better alternate is to use a very strong mobile phase with (80-100% B), past reduce %B as necessary. The initial separation with 100%B results in rapid elution of the entire sample, but a few(prenominal) groups leave alone separate. decrease solvent strength shows the rapid separation of all components with a oft(prenominal) longer run time, with a broadening of later halos and cut down detection sensitivity.Improving the separation and paraphraseable separation broadly s detailing the chromatographers provide run across several aspects of the separation, as summarized in Table-8.6.Table-8.6Objectives of separation in HPLC method developmentObjectivesaCommentResolutionPrecise and rugged quantitative analysis requires that Rs be greater than 1.5.Separation timeQuantitation 2% (1 SD) for assays 5% for less-demanding analysis 15% for ghost analysis.PressurePeak heightNarrow full stops are delectable for large signal / fraudulent scheme ratiosSolvent consumption stripped mobile-phase use per run is desirable.a Roughly in order of decrease enormousness but may vary with analysis requirements.Separation or village is a primary req uirement in quantitative HPLC. The heroism (Rs) value should be maximum (Rs1.5) party favours maximum precision. Resolution usually degrades during the career 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 blockage will favour devil 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 necessary to provide characteristic keeping generation for pointedness 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 proceeding are desirable).Conditions for the final HPLC method should be selected so that the oper ating blackmail with a new column does not exceed 170 take out (2500 psi) and upper berth public press limit below 2000 psi is desirable. There are twain 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 divisor 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 separationAs the experimental runs described above are being carried out, it is important to reassert that separately chromatogram can be reiterateed. When changing conditions (mobile phase, column, and temperature) among method development experiments, enough time must elapse for the column to come into labyrinthine sense with a new mobile phase and temperature. Usually column equilibration is achieved subsequently 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 come upond 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 developmentThe final procedure should meet all the objectives that were delimit at the beginning of method development. The method should excessively be productive in routine operation and usable by all laboratories and subject for which it is intended.Quantitation and method proofOne of the strengths of HPLC is that is an excellent quantitative uninflected 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 assimilations and for the sound judgment of trace impurity parsimonys in matrix. Method validation, according to the United States Pharmacopoeia (USP), is performed to catch that an analytical methodology is accurate, specific, reproducible and rugged over the specified ambit 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 recount that the method does what it is intended to do. According to USP, the method validation involves eight steps as given below.PrecisionAccuracy stipulate of detection snare of quantitationSpecificity bi running(a)ity and lam rigorRobustnessPrecision and trueness already discussed in chapter-1.LinearityThe one-dimensionality of the method is a measure of how salubrious a calibration eyepatch of response v/s assiduity approximates a straight line, or how well the data fit to the linear equation. Y = aX + bWhere Y is the response, X is the concentration, a is the be given and b is the intercept of a line fit to the data. Ideally, a linear family is preferred (b = 0) because it is more precise, easier for calculations and can be delineate with few threadbares. Also, UV detector response for a dilute sample is judge to follow Beers law and be linear. Therefore, a linear calibration gives try out that the system is performing properly throughout the concentration range of interest.Generally in HPLC, if we are exploitation internal archetype, then the linearity plot of land is to be drawn by taking concentration of the analyte on x-axis and the ratio of area under the persuade (AUC) of analyte to AUC of internal standard (IS) on y-axis. The resulting plot side of meat, 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 discover 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 inflexible by using the following expressions. LOD = 3 X N / B LOQ = 10 X N / BWhere N is the noise estimate, is the standard deviation of the peak area ratio of analyte to IS (5 injections) of the do drugss.B is the slope of the interchangeable calibration curve.The LOD and LOQ values sterilised during method validation are stirred by the separation conditions, columns, reagents and especially instrumentation and data systems.RuggednessMethod ruggedness is be as the reproducibility of results when the method is performed under true use conditions. T his includes different analysts, laboratories, columns, instruments, sources, chemicals, solvents etc. method ruggedness may not be known when a method is first developed, but perspicacity is obtained during subsequent use of that method.RobustnessThe concept of boldness of an analytical procedure has been defined by the ICH as a measure of its capableness to endure unaffected by small, but deliberate variations in method parameters. The validity of a method is the ability to remain unaffected by small changes in parameters such as pH of the mobile phase, temperature, piece of organic solvent and buffer concentration etc. to determine robustness of the method experimental conditions were purposely altered and chromatographic characteristics were evaluated.To study the pH effect on the retention (K1) of the drug, buffer pH is to be changed by 0.2 units. At certain point, retention will increase at any pH above and below of the pH unit.The effect of temperature on the retention characteristics (K1) of the drug is to be studied by changing the temperature in steps 2C from room temperature to 80C and see the effect of temperature on the resolution and peak shape. Effect of constituent organic strength on retention is to be studied by varying the percentage of organic solvents like acetonitrile, methanol etc. from 0 to 2% era 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 canvass at three concentration levels i.e. 0.025 M, 0.05 M and 0.1 M and observe retention time and resolution.StabilityTo 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 s uitability, 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 constancy 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 nonchalant with impertinently 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 prohibit such growth, adding more than 5% of organic solvent is also effective. gigantic term column stability is critical for method ruggedness. raze the best HPLC column will eventually degrade and lose its initial motion, often as a function of the number of samples injected. governing body suitabilitySystem suitability experiments can be defined as tests to vouch 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 supervene deep down 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 eggshell number (N), tailing promoter, k and / or a, resolution (Rs) and proportional standard deviation (RSD) of peak height or peak area for various(prenominal) injections. The RSD of peak height or area of vanadium 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)2Where tR is the retention time of the peak and W is the largeness 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 +W0.5.2Where t1 and t2 are retention times of the adjacent peaks and W0.5.1 and W0.5.2 are the largeness of the peaks at half height. Rs = 2.0 or greater is a desirable stooge 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 proportionateness can be represented in terms of peak imbalance factor and peak tailing factor, which can be calculated by using the following formula. Peak asymmetry factor = B /AWhere B is the surmount at 50% peak height between leaders 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 / 2fWhere W0.05 is the width of the peak at 5% height and f is the distance from the peak maximum to the leading edge of the peak, the distance being heedful at a point 50% of the peak height from the base line.High Performance Liquid Chromatography (HPLC) 214High Performance Liquid Chromatography (HPLC) 214IntroductionHigh 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 compl ex mixtures.General methodology for the development of new HPLC methods 215-228HPLC method development follows the series of steps summarized below.Information on sample, objective of separation.Need for special HPLC procedure, sample pretreatment etc.Choice of detector and detector settings.Choosing LC method, preliminary run, estimation of best separation conditions.Optimization of separation conditions.Check for problems or requirement for special procedure.a) Recovery of purified material b) Quantitative calibrationc) Qualitative methodValidate 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 d efine the goals of separation. The kinds of sample related information that can be important are summarized in Table-7.1.Table-8.1Important information concerning sample composition and propertiesNumber of compounds present in the sampleChemical structures of componentsMolecular weights of compoundsPKa values of compoundsUV spectra of compoundsConcentration range of various compounds in samples of interestSample solubility The chemical composition of the sample can provide valuable clues for the best choice of initial conditions for an HPLC separation.Objectives of separationThe 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 achievabl e).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 detectionSamples for analysis come in various forms such asSolutions 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 minimi zes baseline upset and other problems.Some samples require a partial separation ( pretreatment) prior to HPLC, because of need to remove interference, concentrate sample analytes or eliminate column killer. In many cases the development of an adequate sample pretreatment can be challenging than achieving a good HPLC separation.The detector selected should sense all sample components of interest. Variable-wavelength ultraviolet (UV) detectors normally are the first choice, because of their convenience and applicability for most samples. For this reason information on the UV spectra can be an important aid for method development. When the UV response of the sample is inadequate, other detectors are available (flourescence, electrochemical, PDA etc.) or the sample can be derivatized for enhanced detection.Developing the method for the separationSelecting an HPLC method and initial conditionsIf HPLC is chosen for the separation, the next step is to classify the sample as regular or spec ial. Regular samples means typical mixtures of small molecules ( Table-8.2Handling of special sampleSampleRequirementsInorganic ionsDetection is primary problems use ion chromatographyIsomersSome 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.EnantiomersThese compounds require chiral conditions for their separations.BiologicalSeveral factors make samples or this kind special molecular conformation, polar functionality and a wide range of hydrophobicity.MacromoleculesBig molecules require column packing with large pores( 10-nm diameters) in addition, biological molecules require special conditions as noted above.Table-8.3Preferred experimental conditions for the initial HPLC separationSeparation variablePreferred initial choiceColumnDimensions (length, ID)15 x 0.46 cmParticle size5 mmaStationary phaseC8 or C18Mobile phaseSolvent A and BBuffer-acetonitrile% B80-100%bBuffer (compound, pH, concentration)25mM potassium phosphate 2.0Additives (e.g., amine modifiers, ion pair reagents)Do not use initiallyFlow rate1.52.0 ml/minTemperature35-45CSample SizeVolumed25 mLWeightdB Polar solventa3.5 mm particles are an alternative using a 7.5 cm columnbFor an initial isocratic run an initial gradient run is preferred.cNo buffer required for neutral samples for pHdSmaller values required for smaller-volume columns (e.g., 7.50.46-cm, 3.5-mm column).Table-8.4Physical properties of silica supports for some C 18 columnsColumn (mL/mL)Pore diameter (nm)Surface area (m2/g)Percent PorosityHypersil ODS1217057LiChrosorb C181035571Novapak C186N/AaN/AaNucleosil C181035069Symmetry C181033566Zorbax ODS630055Zorbax Rx, SB, XDB818050a N/A Not availableOn 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-8.5Characteristics of primary HPLC methodsMethod / description/ columnsPreferred methodReversed-phase HPLCUses water organic mobile phase Columns C18 (ODS), C8, phenyl, trimethylsilyl (TMS), CyanoFirst choice for most samples, especially neutral or non-ionisable compounds that dissolve in water-organic mixturesIon-pair HPLCUses 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 HPLCUses 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 developmentOne 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 separationGenerally the chromatographers will consider several aspects of the separation, as summarized in Table-8.6.Table-8.6Objectives of separation in HPLC method developmentObjectivesaCommentResol utionPrecise and rugged quantitative analysis requires that Rs be greater than 1.5.Separation timeQuantitation 2% (1 SD) for assays 5% for less-demanding analysis 15% for trace analysis.PressurePeak heightNarrow peaks are desirable for large signal / noise ratiosSolvent consumptionMinimum mobile-phase use per run is desirable.a Roughly in order of decreasing importance but may vary with analysis requirements.Separation or resolution is a primary requirement in quantitative HPLC. The resolution (Rs) value should be maximum (Rs1.5) favours maximum precision. Resolution usually degrades during the life of the column and can vary from day to day with minor fluctuations in separation conditions. Therefore, values of Rs = 2 or greater should be the goal during method development for simple mixtures. Such resolution will favour both improved assay precision and greater method ruggedness.Some HPLC assays do not require base line separation of the compounds of interest (qualitative analysis ). In such cases only enough separation of individual components is required to provide characteristic retention times for peak identification.The time required for a separation (run time = retention time for base band) should be as short as possible and the total time spent on method development is reasonable (runtimes 5 to 10 minutes are desirable).Conditions for the final HPLC method should be selected so that the operating pressure with a new column does not exceed 170 bar (2500 psi) and upper pressure limit below 2000 psi is desirable. There are two reasons for that pressure limit, despite the fact that most HPLC equipment can be operated at much higher pressures. First, during the life of a column, the back pressure may rise by a factor of as much as 2 due to the gradual plugging of the column by particular matter. Second, at lower pressures 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 separationAs 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 developmentThe final procedure should meet all the objectives that were defined at the beginning of method development. The method should a lso be robust in routine operation and usable by all laboratories and personnel for which it is intended.Quantitation and method validationOne 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.PrecisionAccuracyLimit of dete ctionLimit of quantitationSpecificityLinearity and rangeRuggednessRobustnessPrecision and accuracy Already discussed in chapter-1.LinearityThe 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 + bWhere Y is the response, X is the concentration, a is the slope and b is the intercept of a line fit to the data. Ideally, a linear relationship is preferred (b = 0) because it is more precise, easier for calculations and can be defined with fewer standards. Also, UV detector response for a dilute sample is expected to follow Beers law and be linear. Therefore, a linear calibration gives evidence that the system is performing properly throughout the concentration range of interest.Generally in HPLC, if we are using internal standard, then the linearity plot is to be drawn by taking concentration of the analyte on x-axis and the ratio of area under the curve (AUC) of analyte to AUC of internal standard (IS) on y-axis. The resulting plot slope, intercept and correlation coefficient provide the desired information on linearity. A linearity correlation coefficient above 0.999 is acceptable for most methods.Limit of detection (LOD) The limit of detection (LOD) is the smallest concentration that can be detected reliably. The LOD represents the concentration of analyte that would yield a signal-to-noise (S/N) ratio of 3.Limit of quantitation (LOQ) The LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The LOQ represents the concentration of analyte that would yield a signal-to-noise ratio of 10.LOD and LOQ can be determined by using the following expressions. LOD = 3 X N / B LOQ = 10 X N / BWhere 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 v alues determined during method validation are affected by the separation conditions, columns, reagents and especially instrumentation and data systems.RuggednessMethod 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.RobustnessThe concept of robustness of an analytical procedure has been defined by the ICH as a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters. The robustness of a method is the ability to remain unaffected by small changes in parameters such as pH of the mobile phase, temperature, percentage of organic solvent and buffer concentration etc. to determine robustness of the method experimental conditions were purposely alter ed and chromatographic characteristics were evaluated.To study the pH effect on the retention (K1) of the drug, buffer pH is to be changed by 0.2 units. At certain point, retention will increase at any pH above and below of the pH unit.The effect of temperature on the retention characteristics (K1) of the drug is to be studied by changing the temperature in steps 2C from room temperature to 80C and see the effect of temperature on the resolution and peak shape. Effect of percentage organic strength on retention is to be studied by varying the percentage of organic solvents like acetonitrile, methanol etc. from 0 to 2% while the other mobile phase contents are held constant and observe the K1. At certain point decreases in K1 observed with increase in the level of organic solvent. Effect of buffer concentration should be checked at three concentration levels i.e. 0.025 M, 0.05 M and 0.1 M and observe retention time and resolution.StabilityTo 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 oxid ation, 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 suitabilitySystem 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 su itability 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)2Where tR is the retention time of the peak and W is the width of the peak at half peak height.The resolution of two adjacent peaks can be calculated by using the formula Rs = 1.18 (t2-t1) / W0.5.1 +W0.5.2Where 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 /AWhere B is the distance at 50% peak height between leading edge to the p erpendicular 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 / 2fWhere W0.05 is the width of the peak at 5% height and f is the distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 50% of the peak height from the base line.