High Performance Liquid Chromatography Experiment

High Performance suave Chromatography ExperimentINTRODUCTIONPharmaceutical Analysis may be defined as the application of uninflected procedures use to determine the purity, safety and gauge of do medicatess and chemicals. The term Pharmaceutical compendium is otherwise called quantifiable pharmaceutical chemistry. Pharmaceutical outline includes both qualitative and quantitative synopsis of drugs and pharmaceutical substances starts from bulk drugs to the finished dosage forms. In the modern practice of medicine, the analytical rules atomic number 18 used in the analysis of chemical constituents fix in valet de chambre body whose altered densitys during disease states serve as diagnostic aids and also used to analyze the medical agents and their metabolites found in biological system.Qualitative in original analysis seeks to establish the presence of given element or inorganic compound in a sample.Qualitative organic analysis seeks to establish the presence of a given f unctional group or organic compound in a sample.QuantitativeQuantitative analysis seeks to establish the amount of a given element or compound in a sample.The term quality as applied to a drug product has been defined as the sum of all factor outs, which contribute directly or indirectly to the safety, effectiveness and reliability of the product. These properties be built into drug products with research and during process by procedures collectively referred to as Quality control. Quality control guarantees with in reasonable limits that a drug productsIs free of impurities.Is physically and chemically stableContains the amount of active ingredients as stated on the label andProvides optimal release of active ingredients when the product is administered.Most modern analytical chemistry is categorized by deuce different approaches much(prenominal) as analytical targets or analytical methods.INTRODUCTION FOR CHROMATOGRAPHYHigh process liquid chromatography is the process, which seperates mixture containing two or more atoms under high up obligate. In this the stationary class is packed in tugboat one end of which is attach to a descent of pressurized liquid meandering(a) variant.High carrying out liquid chromatography is the fasted growing analytical technique for the analysis of drug. Its simplicity, high specificity and wide range of sensitivity makes its ideal for the analysis of many drugs in both dosage form and biologic fluids.HPLC is also known as High performance liquid chromatography. It is essential form towboat chromatography in which the stationary phase is consists of a small particles (3-5om) packing contained in a towboat with a small bore (2-5mm), one end of which is attached to source of pressurized liquid eluent(mobile phase).Different Types of Principles According to the phases involved, HPLC can be classified into several types, which atomic number 18 as followsNormal Phase Chromatography (NPC) annul Phase Chromatography (RPC)Liquid Solid Chromatography or adsorption HPLCLiquid Liquid Chromatography or Partition HPLCIon substitution Chromatography or Ion exchange HPLCSize exclusion or gel interpenetration or steric exclusion HPLC1. Normal Phase Chromatography (NPC) In normal phase chromatography, the stationary phase is more polar then the mobile phase, and the mobile phase is a mixture of organic solvents with out added water (e.g. isopropane with hexane) and the column packing is either an inorganic adsorbent (silica) are a polar bonded phase (cyanno, diol, amino) on a silica support. Sample retention in normal phase chromatography increases with the polarity of mobile phase decreases. They are eluted in the order of increasing polarities.2. Reverse Phase Chromatography (RPC) In reverse-phase chromatography, the stationary phase is less polar than the mobile phase and the mobile phase is a mixture of organic and aqueous phase. Reverse-phase chromatography is typically more convenient and rug ged than the other forms of liquid chromatography and is more likely to result in a satisfactory final detachment. High performance RPC columns are efficient, stable and reproducible. In this, the solutes are eluted in the order of their decreasing polarities. These are prepared by treating the surface silanol group of site with an organic chloro silane reagent.INSTRUMENTATION recording machineSCHEMATIC DIAGRAM OF HPLCa. Pumps Pumps are required to deliver a constant time period of mobile phase at pressures ranging from 1 550 bar affectionatenesss capable of pressure up to 6000 psi provide a wide range of flow rates of mobile phase, typically from 0.01-10ml min-1. Low flow rates (10-100l min-1) are used with micro bore columns, intermediate flow rates (0.5-2ml min-1) are used with conventional analytical HPLC columns, and fast flow rates are used for preparative or semi preparative columns and for slurry packing techniques.Mechanical pumps of the reciprocating piston type view a pulsating supply of mobile phase. A damping device is thither fore required to smooth out the pulses so that excessive noise at high levels of sensitivity or low pressure does not cut back from detection of small quantities of sample. This type of pump is mostly used.Dual piston reciprocating pumps produce an almost pulse free flow because the two pistons are conservatively faced so that as one is filling the other is pumping. These pumps are more expensive than single piston pumps but are of benefit when development a flow sensitive detector such as ultraviolet or refractive index detector.b. Injection SystemsInjection ports are of two canonic types, (A) those in which the sample with injected directly into the column and (B) those in which the sample is deposited before the column inlet and then swept by a valving action into the column by the mobile phase.c. ColumnsHPLC columns are made of high quality stainless steel, polish internally to a mirror finish. Standard analytic al columns are 4-5 mm internal diameter and 10-30 cm in space. Shorted columns (3-6 cm) containing a smaller particles sizing packing material (3 or 5 m) produce similar or conk out efficiencies, in terms of the number of theoretical habitations (about 7000), that those of 20 cm columns containing 10 m irregular particles and are used an short analysis snip and highest throughput of samples are required. Micro bore columns of 1-2 mm internal diameter and 10-25 cm in length have certain advantages of lower detection limits and lower consumption of solvent, the latter(prenominal) being important if expensive HPLC grade solvents are used. HPLC are also being carried out on the semi preparative scales by using columns of 7-10 mm or 20-40 mm internal diameter respectively.d. DetectorsThe most widely used detectors for liquid chromatography areDetectorAnalytesSolvent RequirementsCommentsUV-VisibleAny with chromophoresUV-grade non UV absorbing solventsHas storey of selectivity and us eful for many HPLC applicationsFluorescenceFluorescent compoundsUV-grade non UV absorbing solventsHighly selective and sensitive, often used to analyze derivitized compoundsRefractive indexCompounds with different RI than mobile phaseCannot run mobile phase gradientsLimited sensitivityConductivityCharged or polar compoundsMobile phase must be conductingExcellent for ion exchange compoundsElectrochemicalReadily oxidized or reduced compounds, specially biological samplesMobile phase must be conductingVery selective and sensitiveMass-Spectrometer capacious range compoundsMust use volatile solvents or volatile buffersHighly sensitive. Many modes available. Needs trained person suppositional principles of HPLCa. Retention time The time is required between the injection point and the gratuity supreme is called the retention time. It is denoted as the Rt. It is mainly useful for the qualitative analysis for the identification of compound.b. Capacity factor It represents the molar ratio o f the compound in the stationary phase and the mobile phase. It is independent of column length and mobile phase flow rate. It is denoted as the k. It should be kept 1-10. If k values are too low it is likely that the solutes may be adequately resolved and for high k values the analysis time is too long. It can be calculate bytr t0k = -t0tr = Retention time, t0 = Dead time.c. Tailing factor Closer study of a chromatographicalal show that the Gaussian forms is usually not completely symmetrical. The graph spread out to a greater or lesser extent, forming a tail. It reduces the column plate number which intern influences the resolution. Tailing is mainly due to deteriorated column, overloading column, extra column-volumes, and incompatibility of sample with standard and/or mobile phase. Practically it can be calculated or determined at 10% of the total peak height. It must not be greater than 2.0d. Resolution The degree of judicial separation of one component from another is desc ribed by the resolution. It is generally denoted by Rs. It is measured as the difference in retention time and the arithmetic mean of the two peak widths.tr2 tr1Rs = 0.5(w1 + w2)tr2 = Retention time of first peak w1 = width of first peaktr1 = Retention time of second peak w2 = width of second peake. Theoretical plates It is important property of the column. It reflects its quality of separation and its ability to produce sharp, narrow peak and achieving skinny resolution of peak. N denotes it.3500 X L (cm)Theoretical plates = -dp(m)L = length of the column in cm, dp = diameter of the particle (m)It follows that if the exchange is fast and efficient, the theoretical plate will be small in size and there will be large number of plates in the column.f. Height equivalent to theoretical plate (HETP) Number of plates directly proportional to the column length (L) and inversely proportional to the diameter of the particles (dp). The value of H is a criterion for the quality of a column. Lower the HETP, higher is the efficiency of the column. Its value depends upon particle size, flow rate, viscosity of mobile phase.H = L/NL = Length of column, N = No. of theoretical plateHPLC method developmentThe wide variety of equipment, columns, eluent and operational parameters involved makes high performance liquid chromatography (HPLC) method development seem complex. The main objective of method development is to obtain a good separation with minimum time and effort. Based on the goal of separation, the method development is preceded. The steps involved areInformation on sample, define separation goalsNeed for special HPLC procedure, sample pretreatment, etc.Choose detector and detector settingsChoose LC method, preliminary runEstimate better(p) separation conditionsOptimize separation conditionsCheck for problems or requirement for special procedureValidation for release to routine laboratoryThe following must be considered when developing an HPLC method prevail it chil dlikeTry the most common columns and stationary phases firstThoroughly investigate binary mobile phases before going on to ternary bring forward of the factors that are likely to be significant in achieving the desired resolution.Mobile phase composition, for example, is the most powerful way of optimizing selectivity whereas temperature has a minor effect and would single achieve small selectivity changes. pH will only significantly affect the retention of weak acids and bases.VALIDATION OF ANALYTICAL METHOD IN PHARMACEUTICAL psychoanalysisValidation is documented evidence, which is completed to ensure that an analytical method is accurate, reproducible and robust over the specific range. The quality of the analytical data is a mark factor in the success of a drug development program. The process of method development and validation has a direct impact on the quality of these data.method acting validationMethod validation is the process to confirm that analytical procedure emplo yed for a specific test is suitable for its intended use. Method needs to be validated or revalidatedBefore their introduction into routine useWhenever the conditions changes for which the method has been validated , e.g., instrument with different characteristicsWhenever the method is changed, and the change is outside the original scope of the method.Depending on the use of the assay, different parameters will have to be measured during the assay validation. ICH and several regulatory bodies and Pharmacopoeia have published teaching on the validation of analytical proceduresMETHOD VALIDATION PARAMETERSSPECIFICITY.ACCURACY.PRECISION.LINEARITY.ROBUSTNESS.SOLUTION STABILITY.The goal of the validation process is to challenge the method and determine the limit of allowed variability for the conditions required to run the method. The following statistical parameters are to be determined to validate the authentic method.Correlation coefficient(r)When the changes in one variable are as sociated or followed by changes in the other, it is called correlation. The numerical measure of correlation is called the coefficient of correlation and is defined by the relation. (x x) (y -y)r = (x -x) 2 (y -yRegression equationRegression equation= I + aCY2 Y1a = slope = X2 X1I = Intercept = regression a CAs a percentage of mean absorbance.3. Standard DeviationS = (X- X) 2/N 1Where, X = observed valuesX = Arithmetic mean = X/NN = Number of deviationsFor practical reading material it is more convenient to express S in terms of percent of the approximate average of the range of analysis is used in the calculation of S. This is called co-efficient of rendering (C.V) or percent relative standard deviation (%RSD).C.V OR %RSD = 100* S/ XCriteria for Validation of the MethodCHARACTERISTICSACCEPTABLE RANGESpecificityNo enlistmentAccuracyRecovery (98-102%)PrecisionRSD LinearityCorrelation Coefficient(r)0.99Range80-120%Stability24h or 12hDRUG PROFILERIZATRIPTAN BENZOATEStructure Chemical name N,N diethyl -5-(1H-1,2,4-triazol-1-1-ylmetyl)-1HIndole-3 Ethanamine monobenzoateMolecular formulation C15H19N5.C6H5COOHMolecular weight 391.47Description White crystalline powderMelting point 178-1800CSolubility Sparingly soluble in water and methanol computer storage Air tight container protect from light.Drug Category Anti migraine drugTHERAPEUTIC RATIONALRIZATRIPTAN BENZOATECLINICAL PHARMACOLOGYMechanism of actionRizatriptan binds with high affinity to human 5-HTIB and 5-HTID receptors leading to cranial blood vessel constriction.PharmacokineticsAbsorptionCompletely absorbed from GI tract, absolute bioavailability is 45% plasma peak concentration attained with in 1-1.5 hours (conventional tablet )or 1.6-2.5 hours (orally disintegrating tablet)after oral administration.DistributionCrosses placenta and is distributed in to milk in animal, no studies in pregnant or nursing women.MetabolismMetabolized generally via oxidative deamination by Mao-A to an inactive indo le acetic acid metaboliteEliminationExcreted principally in urine(14% of dose as unchanged drug and 51 % a indole acetic acid metaboliteAdverse effectsDry mouthDizzinessPain tightness/pressure in neck/throat/jaw.Nausea boob painParasthesiaFatigueDosage and administrationThe dose range of Rizatriptan benzoate is 10-30mg orally once daily.Rizatriptan benzoate can be administer orally disintegrating tablet with out meals. literary works REVIEWSasmitha Kumar et al has been developed UV spectroscopic method for estimation of Rizatriptan benzoate.The drug shows utmost absorption at 277 nm and 281 nm and obeys beer-lamberts law in the concentration of 0.5-20 g/ml at 277 nm and 0.5-80 g/ml at 281 nm respectively. The percentage recovery was found to be 97-100%.Madhukar et al has been developed reverse phase high performance liquid chromatographic method for mark of Rizatriptan benzoate. The proposed method utilized column L1 inertsil ODS-3v, 250 nmx4.6 mm having particle size, 5m. The mob ile phases were comprised of A, B of Acetonitrile and buffer pH 6.5 at UV detection 225 nm.The method shows recovery 96.64-97.71Sachin jagthap et al has been developed stability indicating reversed phase high performance liquid chromatographic method for the finale of Rizatriptan benzoate in bulk powder and in pharmaceutical formulations. The method utilizes c18 column having dimension 250mmx4.6 mm having particle size,5.0 m using a mobile phase 0.01M sodium dihydrogen phosphate buffer Methanol , at a flow rate 1ml/min at close temperature and detected at 225 nm.and the method was validated according to ICH guidelinesQuizi zhang et al has been developed, a high performance liquid chromatographic method for the determination of Rizatriptan benzoate in human plasma.using asingle step liqid liqid extraction with metyl tertiary butyl ether, the analytes separated usig amobile phase consisting of 0.05%v/v triehylamine in water adjusting ph 2.75 with 85% phosphoric acid and acetonitrile .fluroscence detection was performed at an excitation wavelength of 225 nm and an emission wavelength of 360 nm.The linearity for rizatriptan was within the concentration range of 0.5-50ng/ml.Rajendra Kumar et al has been developed and validated stability a stability indicating high performance liquid chromatographic method for Rizatriptan benzoate.The force degradation studies were performed on bulk sample of Rizatriptan benzoate. The method utilizes a zorbax SB-CN column with dimension of 250 mmx4.6 mm, 5um column. The mobile phase consists of a mixture of aqueous potassium dihydrogen ortho phosphate (ph3.4), acetonitrile and methanol.Rauza bagh et al has been developed a spectroscopic method for analysis of Rizatriptan benzoate in bulk and tablet dosage form. The Rizatriptan benzoate shows maximum absorbance at 225 nm. Beers law was obeyed in the concentration range of 1-10g/ml.AIM AND PLAN OF WORKThe present aim is to develop a new simple and rapid analytical method to estimate the Rizatriptan benzoateThe plan of the proposed work includes the following stepsTo undertake solubility studies for analytical studies of Rosuvastatin calciumDevelop initial chromatographic conditions.Setting up of initial chromatographic conditions for the assay of Rosuvastatin calcium Optimization of initial chromatographic conditions.Validation of the developed HPLC Analytical method according to ICH method validation parameters.EXPERIMENTALNEW RP-HPLC METHOD FOR THE ESTIMATION OF RIZATRIPTAN BENZOATE IN TABLET DOSAGE FORMA simple reverse phase HPLC methods was developed for the determination of Rizatriptan benzoate in tablet dosage form. Zorbax Eclipse XBD C18 (250 cm - 4.6 mm) column in isocratic mode with mobile phase Buffer ph 5.0 Methanol (8020) was used and pH-3 correct with tri ethylamine. The flow rate was 1.0 ml/min and UV detection at 225nm. The retention time 3.0 min. The proposed method was also validated.EXPERIMENTAL1. InstrumentationShimadzu LC-10A HPLCVacuum pum p Gelmon scienceElico SL-164 double beam UV-Visible spectrophotometerUltra sonicator 3.5L 100(pci)2. ChemicalsWater HPLC gradeMethanol HPLC grade (Merck)Potassium dihydrogen orthophosphate(AR Grade)Triethylamine (AR Grade)5.1 OPTIMIZATION1. Selection of wavelengthAfter solubility study for the drug solvent was selected and appropriate concentration of Rizatriptan benzoate standards with solvent were prepared. The solution were then scanned by using doubl beam UV-Visible spectrophotometer the range between 200-400nm.The overlain spectra for the both drug were observed and maximum wavelength was finally selected.2. Selection of mobile phaseTo develop a prcised and robust HPLC method for determination of Rizatriptan benzoate , its standard solution were injected in the HPLC system. After books survey and solubility data different composition of mobile phase of different flow rates were employed in order to determine the best condition for effective separation of drugs.3. Selection of columnInitially different C8 and C18 columns were tried for selected composition of mobile phase and quality of peaks were observed for the drugs. Finally the column was fixed upon the satisfactory results of various system suitability parameters such as column efficiency, retention time, tailing factor / peak asymmetry of the peaks.Other parameters such as flow rate, column temperature etc. were selected by varying its value up to certain levels and results were observed. The value at satisfactory results were obtained has been selected for the method. The final selection of chromatographic conditions as followsOptimized chromatographic conditionsPreparation of Buffer ph 5.0Dissove 2.76 gm of potassium dihydrogen orthophosphate in 1000ml of HPLC water plus 5.0 mlof Triethylamine. Mix and adjust PH 5.0 with orthophosporic acid. slobber with 0.45u nylon filter.Preparation of mobile phaseThe mobile phase was prepared by mixing Buffer Methanol (8020). the solution was then filtered t hrough 0.45m membrane filter and sonicated.Preparation of standard stock solutionStandard solution of the pure drug was prepared by dissolving 73.0 mg of Rizatriptan benzoate in 100ml volumetric flask. The drugs were dissolved by using mobile phase as a diluent. summarise about 50ml of diluent and sonicate to dissolve. Make up the volume with diluent. Mix well. encourage dilute 5.0ml of the above solution to 250ml with diluent, mix well.Preparation of sample solutionWeight and transfer 10 intact tablet in into a100ml volumetric flask. Add about 50ml of diluent and sonicate for 15 min and make up the volume with diluent. Mix well, filter through 25 mm 0.45 u nylon , discard 4ml filtrate. pass on dilute 5ml of the solution to 250 ml with diluent and mix well.CONCLUSIONThe evaluation of obtained values suggests that the proposed HPLC methods provide simple, precise, rapid and robust quantitative analytical method for determination of Rizatriptan benzoate in tablet dosage form. The mobile phase is simple to prepare and economical. After validating proposed method as per ICH guidelines and correlating obtained values with the standard values, satisfactory results were obtained.Hence, the method can be easily and conveniently adopted for routine estimation of Rizatriptan benzoate in tablet dosage form.