Hplc by ikrm khan and groupe
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samia ,urooj ,ahsan ,hasnain,momo baqai institue of pharmaceutical sceiences
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Hplc by ikrm khan and groupe
- 3. INTRODUCTION OF HPLC HPLC offers a combination of speed, reproducibility and sensitivity. HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution.
- 4. TERMINOLOGIES FOR HPLC Resolving Power: The extent of separation of the compounds present in the mixture across the column. Theoretical plates: An imaginary division of the column , equal to the length of column. Stationary phase: The phase which remains fixed in the column e.g. C18,silica Mobile phase: carries the sample through the stationary phase as it moves through the column.
- 6. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM Solvent Solvent Delivery System (Pump) Injector Column Detectors Waste Collector Recorder (Data Collection)
- 7. SOLVENT On basis of solvent two types of systems are made: i. Isocratic system: same mobile phase runs throughout the elution of sample. e.g. in QC. ii. Gradient system: strength of mobile phase is increased with time during sample elution. e.g. for complex mixtures. In reverse phase mostly water and acetonitrile or methanol is used as polar solvents. The mobile phase must be degassed to eliminate the formation of air bubbles.
- 8. PUMP The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL /min).
- 9. COLUMN Considered as “heart of HPLC”. Its inner is lined with stationary phase, which separates the sample components of interest using various physical and chemical parameters. The stationary phase can be polar or non-polar.
- 10. INJECTOR The injector serves to introduce the liquid sample into the flow stream of the mobile phase. Manual Injector Auto sampler
- 11. DETECTOR: The detector can see (detect) the individual molecules that comes out (elute) from the column. UV-Visible detectors, photo diode array detector, fluorescence detector are mostly used.
- 12. COMPUTER: Frequently called the data system.
- 13. PRINCIPLE The process involves the interaction of the compounds in the analyte (which travels along with a mobile phase) across an immobile surface (stationary phase). The different component in the mixture pass through the column and differentiates due to differences in their partition behavior between the mobile phase and the stationary phase. The differential wash out or elution of compounds is basis of HPLC.
- 14. WORKING 14 Separation in based upon differential migration between the stationary and mobile phases. Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column. Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Waste
- 30. THE CHROMATOGRAM 30 Injection to tR mAU time tR to - elution time of unretained peak tR- retention time – the time the eluent reaches the detector Area or height is proportional to the quantity of analyte.
- 32. ORDER OF CALIBRATION Detector pump injector column Detector i. Wavelength accuracy ii. Detector linearity Pump i. Flow rate accuracy ii. Pressure test Injector i. Injector accuracy ii. Carryover iii. Injector linearity Column i. Temperature accuracy
- 34. Wavelength Accuracy: •Select 3D mode and set the wavelength range as 200-400nm. •Inject 20 μl caffine of standard preparation once into the chromatographic system. •Extract and record the chromatograms at wavelengths of 202 to 208nm with an interval of 1nm and at 269 to 275 nm with an interval of 1nm. •Note down the height or absorbance. • Acceptance criteria: The maximum absorbance should be at 205±2nm and 272±2nm.
- 35. Detector Linearity: Standard Preparation: Accurately weigh and transfer about 60mg of Caffeine into a 100ml volumetric flask. Dissolve and dilute to the volume with mobile phase. 6 linearity solutions are prepared in different concentrations of the drug. Procedure: Inject blank, followed by Detector linearity solutions and record the peak responses of Caffeine standard plot between the concentration Vs the peak responses. Acceptance criteria: The plot should be linear and regression coefficient (R2) should not be less than 0.99.
- 37. Flow Rate Accuracy: 1. Prime all the solvent lines with water. 2. Set the flow rate to 0.500 ml/m. 3.Wait for about 15 m to stabilize the system and ensure that the pressure is stable. 4.Insert the outlet tubing into a 10 ml volumetric flask and start the stop watch simultaneously. 5. Stop the stopwatch when the lower meniscus reaches the 10 ml mark on the flask. 6. Record the elapsed time. 7. Similarly check the flow for 1.0 ml/m and 2.0 ml/m. Acceptance criteria: The time taken to collect the water should be with in ± 2.0% of the actual value. 37
- 38. Pressure Test : •The first step of the pressure test is to plug the outlet of the pump using a dead-nut and by setting the automatic pump shutdown pressure to 6,000 psi. •The pump-head pressure signal output is connected to a recorder. Pressurize the pump by pumping methanol at 1 mL/min. The pressure inside the pump head increases quickly as the outlet of the pump is blocked. •The pressure will gradually rise to the shutdown pressure if the check valves are able to hold the mobile phase in the pump chamber as would be normally expected . If the check valve is not functioning properly, the pressure will fluctuate at about 3,000 psi instead of reaching the shutdown pressure. 38
- 40. Injector Accuracy: 1. Connect the pump and detector inlet with union. 2. Prepare mobile phase consisting of a mixture of water and Methanol (70:30) 3. Set a flow rate of 0.5 ml/m and a run time of 1 m. 4. Set the column temperature at 25± 2°C. 5.Fill a standard HPLC vial to 2/3rd with water. Seal the vial properly with a cap. 6. Weigh the vial and record the weight as W1 grams. 7. Place the vial in the chromatographic system and perform 6 injections of 50μl volume from this vial. 8. Weigh the vial again and note the weigh after the injections as W2 grams. 9. Calculate the mean volume injected per injection as follows: •Mean injected volume (μl) = (W1 – W2) × 100/6 •Acceptance criteria: The mean injected volume should be 50.0±1.0 μl. 40
- 41. Carryover : • Small amounts of analyte may get carried over from the previous injection and contaminate the next sample to be injected. • The carryover will affect the accurate quantitation of the subsequent sample. • The effectiveness of the cleaning can be evaluated by injecting a blank after a sample that contains a high concentration of analyte. • The response of the analyte found in the blank sample expressed as a percentage of the response of the concentrated sample can be used to determine the level of carryover. 41
- 42. Injector Linearity: • Standard Preparation: Accurately weigh and transfer about 60mg of Caffeine into a 100ml volumetric flask. Dissolve and dilute to the volume with mobile phase. Transfer 10ml of Standard Preparation into a 100ml volumetric flask and dilute to the volume with mobile phase. • Procedure: Inject 5 μl of the mobile phase as blank injection. Inject 5 μl, 10 μl, 20 μl, 50 μl and 80 μl of the Standard Preparation and record the peak areas. • Plot a curve for the volume injected Vs peak area • Acceptance criteria: The plot should be linear and regression coefficient (R2) should not be less than 0.99.
- 44. i. It is evaluated with a calibrated digital thermometer at 30°Cand 60°C .Place the thermometer probe in the column oven and set the column oven temperature at 30°C.Wait till the temperature stabilizes. Record the temperature displayed on the thermometer. Similarly performs the column oven temperature accuracy test at 60°C. Acceptance criteria: The resulting oven temperature from the thermometer display should be within ±2°C of the set temperature. COLUMN OVEN TEMPERATURE ACCURACY
- 45. SUMMARY