HPLC

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/ HIGH PRESSURE LIQUID CHROMATOGRAPHY:

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/ HIGH PRESSURE LIQUID CHROMATOGRAPHY

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HPLC HPLC- It was originally referred to as High Pressure Liquid Chromatography since high pressure is applied using a pumping system to the column. This pressure works by forcing the mobile phase through, at much higher rate increasing the resolution power. Due to its high efficiency and performance High Pressure Liquid Chromatography is referred to as High Performance Liquid Chromatography.

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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

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Advantages of HPLC Simultaneous analysis High resolution High sensitivity Good repeatability Moderate analysis condition Easy to fractionate and purify Not destructive

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Mobile Phase / Stationary Phase A site in which a moving phase ( mobile phase ) and a non-moving phase ( stationary phase ) make contact via an interface that is set up. The affinity with the mobile phase and stationary phase varies with the solute.  Separation occurs due to differences in the speed of motion Strong weak Stationary phase Mobile phase 2 1

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ADSORPTION CHRT: PARTITION CHRT: 6 Principle of HPLC

INSTRUMENTATION::

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 SAMPLE INJECTION Column Detector Recorder Gauge

HPLC-INSTRUMENTATION:

HPLC-INSTRUMENTATION

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9 Outline of LC-2010 System Controller UV detector Auto sampler Column Oven Pump Unit Reservior Tray Degassing Unit Low pressure gradient device

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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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HPLC-PUMP

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Solvent is pumped back and forth by a motor driven piston Two ball check valves which open & close which controls the flow The piston is in direct contact with the solvent Small internal volume 35-400 μ L High output pressure up to 10,000 psi Ready adaptability to gradient elution and constant flow rate

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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 the separation Available in different size and length Separates the compounds according to adsorption 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 the length of column the sensitivity also increases. Particle size of adsorbent is : 1-10µ

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Normal phase Stationary Phase – Polar nature. Eg: SiO2,Al2O3 Mobile Phase – Non-Polar nature. Eg:heptane,hexane,cyclohexane,CHCl3,CH3OH Mechanism: Polar compounds travels slower & eluted slowly due to higher affinity to st.phase Non-polar compounds travels faster & eluted 1 st due to lower affinity to st.phase. This technique is not widely used in pharmaceutical separations. Reverse phase Stationary Phase – Non-Polar nature. Eg: n-octadecyl, n-octyl, ethyl, phenyl diol, hydrophobic polymers. Mobile Phase – Polar nature. Eg: methanol or acetonitrile/water or buffer sometimes with additives of THF or dioxane. Mechanism : Polar compounds travels faster & eluted 1st due to lesser affinity to st.phase Non-Polar compounds travels slower & eluted slowly due to higher affinity to st.phase

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18 Effect of Stationary Phase C18 (ODS) Strong C8 sample sample sample C4 Medium Weak

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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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ABSORBANCE DETECTORS: The UV/Vis source usually comes from a monochromator so the wavelength can be selected, or scanned. If wavelength scanning is desired, the flow is stopped long enough for the scan to take place. Fixed wavelength-measures at single wavelength usually 254 nm. Variable wavelength-measures at single wavelength at a time but can detect over a wide range of wavelengths simultaneously.

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21 Instrumentation of UV-Vis Detector Sample Cell Reference Cell Photodiode Photodiode E in E out E in Grating Light source D2 / W lamp E out M2 M1

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Ultraviolet / Visible Detector Advantage: Sensitivity is high Relative robust to temperature and flow rate change Compatible with gradient elution Disadvantage: Only compounds with UV or visible absorption could be detected. Additional Functions Dual Wavelength mode Wavelength Time Program mode Wavelength Scan mode

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PHOTODIODE ARRAY DETECTORS: Sample Cell 512 Elements Photodiode Array Grating D2 / W lamp One element detects one absorbance at one wavelength.

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PHOTODIODE ARRAY DETECTORS: This is a recent detector which is similar to UV detector which operates from 190-600nm. Radiations of all wavelength fall on the detector simultaneously. The resulting spectra is a three dimensional plot of Response Vs Time Vs Wavelength. ADVANTAGE: The wavelength need not be selected but detector detects the responses of all compounds.

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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 RETENSION TIME ( tr ) The time required to elute one half of compound from column. Rt Sample injection

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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

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COLUMN EFFICIENCY (No. of theoretical plates) The efficiency of column is expressed by no. of theoretical plates 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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