High Performance Liquid Chromatography Experiment

tall Performance Liquid Chromatography ExperimentINTRODUCTIONpharmaceutic Analysis may be destined as the application of analytic occasions consumptiond to determine the purity, safety and quality of doses and chemicals. The term Pharmaceutical synopsis is otherwise called quantitative pharmaceutical chemistry. Pharmaceutical psycho synopsis includes twain qualitative and quantitative analysis of drugs and pharmaceutical substances starts from masses drugs to the finished dosage forms. In the juvenile practice of medicine, the analytic systems ar apply in the analysis of chemical constituents found in human body whose altered tightnesss during illness states serve as diagnostic aids and in like manner used to go the medical agents and their metabolites found in biological system.Qualitative inorganic analysis seeks to establish the front man of given element or inorganic escalate in a sample.Qualitative organic analysis seeks to establish the presence of a given f unctional group or organic involved in a sample.QuantitativeQuantitative analysis seeks to establish the nitty-gritty of a given element or compound in a sample.The term quality as applied to a drug mathematical product has been defined as the sum of all federal agents, which contribute directly or indirectly to the safety, goodness and reliability of the product. These properties ar built into drug products by dint of research and during exhibit by procedures collectively referred to as choice 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 flairrn analytical chemistry is categorized by two variant approaches much(prenominal) as analytical targets or analytical rules.INTRODUCTION FOR CHROMATOGRAPHYHigh consummation fluent chrom atography is the process, which sepe pass judgment mixture containing two or much comp integritynts under luxuriously pressure. In this the stationary pattern is packed in towboat one end of which is accustomed to a source of pressurized liquid erratic strain.High exercise liquid chromatography is the profuseed growing analytical technique for the analysis of drug. Its simplicity, spunky specificity and big jog 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 column chromatography in which the stationary anatomy is consists of a gauzy particles (3-5om) packing contained in a column with a small bore (2-5mm), one end of which is attached to source of pressurized liquid eluent( planetary mannikin).Different Types of Principles According to the courses involved, HPLC can be categorise into some(prenominal) types, which ar as fol pet ty(a)s nonemal stagecoach Chromatography (NPC)Reverse Phase Chromatography (RPC)Liquid Solid Chromatography or adsorption HPLCLiquid Liquid Chromatography or Partition HPLCIon exchange Chromatography or Ion exchange HPLCSize exclusion or gel permeation or steric exclusion HPLC1. Normal Phase Chromatography (NPC) In normal conformation chromatography, the stationary level is more polar and so the fluid phase, and the mobile phase is a mixture of organic solvents with erupt added weewee (e.g. isopropane with hexane) and the column packing is either an inorganic adsorbent (silica) argon a polar bonded phase (cyanno, diol, amino) on a silica support. sample distribution 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 mixtur e of organic and aqueous phase. Reverse-phase chromatography is typically more convenient and rugged than the other forms of liquid chromatography and is more likely to vector sum in a satisfactory final insularism. 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. orchestrationRECORDERSCHEMATIC DIAGRAM OF HPLCa. Pumps Pumps are needed to deliver a constant decrease of mobile phase at pressures ranging from 1 550 gin mill pumps capable of pressure up to 6000 psi provide a wide range of persist rates of mobile phase, typically from 0.01-10ml min-1. Low flow rates (10-100l min-1) are used with micro bore columns, mediocre flow rates (0.5-2ml min-1) are used with conventional analytical HPLC columns, and fast flow rates are used for preparative or rig preparative columns and for s lurry packing techniques.Mechanical pumps of the reciprocating piston type view a pulsating supply of mobile phase. A damping device is there fore necessitate to smooth out the pulses so that excessive noise at juicy levels of sensitivity or low pressure does non detract from signal detection of small quantities of sample. This type of pump is mostly used.Dual piston reciprocating pumps get out 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 pull ahead when use a flow sensitive sensor such as ultraviolet or refractive business leader detector.b. Injection SystemsInjection ports are of two basic types, (A) those in which the sample with injected directly into the column and (B) those in which the sample is deposited beforehand the column inlet and hence swept by a valving action into the column by the mobile phase.c. ColumnsHPLC c olumns are make of high-pitched quality stainless steel, polish internally to a reverberate finish. Standard analytical columns are 4-5 mm internal diam and 10-30 cm in distance. Shorted columns (3-6 cm) containing a smaller particles size packing material (3 or 5 m) recruit similar or better efficiencies, in damage of the number of theoretical plates ( about 7000), that those of 20 cm columns containing 10 m sec particles and are used an short analysis time and highest throughput of samples are required. small 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 rove solvents are used. HPLC are also being carried out on the semi preparative scales by utilise columns of 7-10 mm or 20-40 mm internal diameter respectively.d. DetectorsThe most astray used detectors for liquid chromatography areDetectorAnalytesSolvent Requirem entsCommentsUV-VisibleAny with chromophoresUV-grade non UV sorb solventsHas degree of selectivity and useful 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 gradients moderate sensitivityConductivityCharged or polar compoundsMobile phase mustiness be conductingExcellent for ion exchange compoundsElectrochemicalReadily change or reduced compounds, specially biological samplesMobile phase must be conductingVery selective and sensitiveMass-SpectrometerBroad range compounds mustiness use volatile solvents or volatile buffersHighly sensitive. Many modes available. postulate trained personTheoretical principles of HPLCa. Retention time The time is required between the injection point and the placard 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 of 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 determine are too low it is likely that the solutes may be adequately resolved and for high k care fors the analysis time is too long. It can be cipher bytr t0k = -t0tr = Retention time, t0 = Dead time.c. shadowing factor imminent study of a chromatographic 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 re response. Tailing is mainly due to deteriorated column, overloading column, extra column-volumes, and incompatibility of sample with exemplification and/or mobile phase. Practically it can be calculated or obstinate at 10% of the total b loom height. It must not be greater than 2.0d. Resolution The degree of separation of one component from other is described by the resolution. It is generally denoted by Rs. It is measured as the dissimilarity in retention time and the arithmetic mean of the two extremum widths.tr2 tr1Rs = 0.5(w1 + w2)tr2 = Retention time of first inflorescence 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 goodly 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 walloping number of plates in the column.f. Height equivalent to theoretical plate (HETP) subject of plates directly proportional to t he 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. land 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 system snap offmentThe wide variety of equipment, columns, eluent and running(a) parameters involved makes high performance liquid chromatography (HPLC) mode development come out 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 best separation conditionsOptimize separation conditionsCheck for problems or requirement for special procedure ecesis for release to routine laboratoryThe future(a) must be considered when developing an HPLC methodKeep it uncomplicatedTry the most frequent columns and stationary phases firstThoroughly investigate binary mobile phases before going on to ternaryThink of the factors that are likely to be significant in achieving the desired resolution.Mobile phase piece of music, for example, is the most mighty way of optimizing selectivity whereas temperature has a minor effect and would only achieve small selectivity changes. pH will only significantly affect the retention of light-headed acids and bases.VALIDATION OF ANALYTICAL METHOD IN PHARMACEUTICAL ANALYSIS verification is documented evidence, which is completed to ensure that an analytical method is accurate, reproducible and heavy-armed over the specific range. The quality of the analytical info is a name factor in the success of a drug development program. The process of method development and validatio n has a direct impact on the quality of these data. mode validationMethod validation is the process to put up that analytical procedure employed for a specific test is adequate for its intended use. Method needs to be validated or revalidated in advance 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 information on the validation of analytical proceduresMETHOD VALIDATION PARAMETERSSPECIFICITY.ACCURACY.PRECISION.LINEARITY.ROBUSTNESS.SOLUTION STABILITY.The goal of the validation process is to gainsay the method and determine the limit of allowed variability for the conditions needed to run the method. The followi ng statistical parameters are to be determined to validate the create method. correlativity coefficient(r)When the changes in one variable are associated 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 = moment of deviationsFor practical interpretation 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 variation (C.V) or percent relative blood line deviation (%RSD).C.V OR %RSD = 100* S/ XCriteria for Validation of the MethodCHARACTERISTICSACCEPTABLE RANGESpecificityNo InterferenceAc curacyRecovery (98-102%)precisenessRSD LinearityCorrelation Coefficient(r)0.99Range80-120%Stability24h or 12hDRUG inditeRIZATRIPTAN BENZOATEStructureChemical name N,N diethyl -5-(1H-1,2,4-triazol-1-1-ylmetyl)-1HIndole-3 Ethanamine monobenzoateMolecular Formula C15H19N5.C6H5COOHMolecular exercising weight 391.47Description White crystalline powderMelting point 178-1800Csolvability Sparingly soluble in water and methanolStorage transmit 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.Pharmacokinetics tightnessCompletely absorbed from GI tract, absolute bioavailability is 45% plasma peak concentration attained with in 1-1.5 hours (conventional diggings )or 1.6-2.5 hours (orally disintegrating tablet)after oral administration.DistributionCrosses placenta and is dist ributed in to milk in animal, no studies in pregnant or breast feeding women.MetabolismMetabolized principally via oxidative deamination by Mao-A to an inactive indole acetic acid metaboliteEliminationExcreted principally in urine(14% of dose as unchanged drug and 51 % a indole acetic acid metaboliteAdverse doDry mouthDizzinessPain tightness/pressure in neck/throat/jaw.NauseaChest painParasthesiaFatigue venereal infection and administrationThe dose range of Rizatriptan benzoate is 10-30mg orally once daily.Rizatriptan benzoate can be administer orally disintegrating tablet with out meals.LITERATURE REVIEWSasmitha Kumar et al has been certain UV spectroscopic method for estimation of Rizatriptan benzoate.The drug shows maximum 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 determination of Rizatriptan benzoate. The proposed method utilized column L1 inertsil ODS-3v, 250 nmx4.6 mm having particle size, 5m. The mobile 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 determination of Rizatriptan benzoate in bulk powder and in pharmaceutical formulations. The method utilizes c18 column having prop 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 ambient 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 parentage with metyl tertiary butyl ether, the analytes separated usig amobile phase consisting of 0.05%v/v triehylamine in water slumping ph 2.75 with 85% phosphoric acid and acetonitrile.fluroscence detection was performed at an annoyance 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 absor bance 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 approximate the Rizatriptan benzoateThe plan of the proposed work includes the following stepsTo undertake solvability studies for analytical studies of Rosuvastatin calciumDevelop sign 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.data-basedNEW RP-HPLC METHOD FOR THE ESTIMATION OF RIZATRIPTAN BENZOATE IN TABLET DOSAGE modelA 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 modify with tri ethylamine. T he 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 HPLC clean pump 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 standardizeds 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 literature su rvey 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 amend 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 excerpt 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. mixing and a djust PH 5.0 with orthophosporic acid. Filter with 0.45u nylon filter.Preparation of mobile phaseThe mobile phase was prepared by mixing Buffer Methanol (8020). the solution was then filtered through 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. Add about 50ml of diluent and sonicate to dissolve. Make up the volume with diluent. Mix well. pass on dilute 5.0ml of the above solution to 250ml with diluent, mix well.Preparation of sample solutionWeight and depute 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. Further dilute 5ml of the solution to 250 ml with diluent and mix well.CONCLUSIONThe rating of obtained values sug gests 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 espouse for routine estimation of Rizatriptan benzoate in tablet dosage form.