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about HPLC


By: sumithrasivakumar (74 month(s) ago)


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TYPES OF HPLC Mode of chromatography Principle of separation Elution technique Scale of separation Type of Analysis Normal phase Reverse phase Adsorption Ion exchange Size exclusion Affinity Chiral phase Isocratic Gradient Analytical Preparative Qualitative Quantitative


INSTRUMENTATION: Pumps: Solvent delivery system Mixing unit, Gradient controller, and Degassing Injector: Manual and auto injectors Column: Guard and Analytical Detectors Recorder MOBILE PHASE PUMP Damping Device Injection point Column Detector Recorder Gauge



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PUMPS: SOLVENT DELIVERY SYSTEM Solvent is passed through high pressure of 1000 to 3000 psi High purity solvents(HPL grade) and filtered through 0.45µ Particle size of stationary phase is 5 - 10µ Two types of pumps Mechanical pumps: by using piston Pneumatic pumps: High pressure with compressed gas The flow rate is 0.01 -10ml/min For micro bore column : 10-100µl/min For analytical column: 0.5-2.0ml/min DAMPING DEVICE: Used to reduce the noise at high level of sensitivity

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CHECK VALVE: Control the flow rate of solvent and back pressure MIXING UNIT, GRADIENT CONTROLLER: Mixes the solvents at different proportions and pass through column Two types of mixing units Low pressure: needs degassing High pressure: no need degasing GRADIENT CONTROLLER: Isocratic elution: no need Gradient elution: polarity of mobile phase is change, hence it is used to maintain the polarity

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SOVENT DEGASSING: Solvents flows at high pressure to form gas bubbles which interfere the shape of peak The gas bubbles are removed by using following methods: Vacuum filtration: Apply vacuum pressure Helium purging: To pass helium gas but expensive Ultrasonication : to produce vibrations to remove air bubbles INJECTION DEVICE: Manual or Auto injector Septum Injectors: To inject the sample through a rubber septum Stop flow: During injection the mobile phase flow is stopped and sample is injected using valve device Rheodyne injector: mostly used and available in fixed volume loop like 20-50µl.

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GUARD COLUMN: Present before the column and contains small quantity of adsorbent Used to remove any dirt particles or foreign matter present Not involved in separation. ANALYTICAL COLUMN: Very important part decides not only the separation Available in different size and length Separates compounds according to principle Made of stainless steel, glass, polyethylene etc., For standard column: 10-30 cm length, 4-5 mm diameter Shorter column: 3-6 cm length Increase in length of column ,sensitivity of column is increases. Particle size of adsorbent is : 1-10µ

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Functional groups: Normal phase column contain polar stationary phase example- Hydroxyl groups Reverse phase: nonpolar stationary phase eg:Octa decyl silane (C 18 ) Octyl column(C 8 ) Butyl column(C 4 ) DETCTORS: Selection of detectors is based on the property of compounds Classified into two types Solute property Bulk property Eg : UV detector, fluorescence detector , electrochemical detector Eg : Refractive index detector

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DIODE ARRAY DETECTORS: Multiplication of photo electrons by secondary emission of electrons using photo cathode and series of anode Advantage: detects very weak signals UV DETECTOR: Single wavelength and multiple wavelength According to absorbance CHROMATOGRAPHIC PARAMETERS: RETENSION VOLUME: This is the volume of the mobile phase required to elute one half of the compound from column V r = t r X f where f= Flow rate t r = retension time

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RESOLUTION: To measure the extent of separation of two components and baseline is achieved Rs =2(Rt 1 -Rt 2 )/W 1 +W 2 THEORETICAL PLATE: Imaginary valve and the solute is distributed between stationary phase and mobile phase W 1 W 2 Rt 1 Rt 2 Inj point

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HEIGHT EQUIVALENT TO THEOTETICAL PLATE : The thoretical plate and the length of the column is used to calculate the HETP HETP is less___ more efficient HETP is more___ less efficient HETP = length of the column/No. of theoretical plate or by van Deemter equation HETP = A+B/µ + Cµ where A : Eddy diffusion B: Longitudinal diffusion C: Effect of mass transfer which depends oon flow rate µ: Flow rate COLUMN EFFICIENCY (No. of theoretical plates) The efficiency of column is expressed by no. of theoretical plates

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n= 16 R t 2/w2 where n= no. of theoretical plates R t = retension time w = width of peak No. of theoretical plates more efficiency more ASYMMETRY FACTOR: To measure the peak width at 5% height or 10% height Tailing effect is reduced by using more liquid phase Fronting is avoided by using small amount of sample Ideal asymmetry factor 0.95 to 1.05

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APPLICATIONS OF HPLC: Widely used in all industries Qualitative Analysis: Identify the compound by comparing with the retension time To check the purity , if additional peaks are present it contains impurities Calculate the % of impurities by using peak area Quantitative Analysis: Single component analysis Direct comparison method Calibration curve method Internal standard method Multi component analysis To identify multicomponent in one step then quantify by using above any one method Identification and identification of drugs in mixture. Stability studies Purity of compounds

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