bhargavi - calibration of instruments

Information about bhargavi - calibration of instruments

Published on August 1, 2014

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PowerPoint Presentation: QUALIFICATION AND CALIBRATION OF VARIOUS ANALYTICAL INSTRUMENTS Presented by G.Bhargavi M.Pharmacy 1 st year Pharmaceutical Analysis SPMVV Under guidance of Dr. A. Sridevi M.Pharm PhD., Associate Professor SPMVV PowerPoint Presentation:  CONTENTS Validation Qualification Calibration Calibration of Various Instruments 1) HPLC 2) GAS CHROMATOGRAPHY 3) UV SPECTROPHOTOMER 4) IR SPECTROPHOTOMET 5) PHOTO – FLUOROMETER 6) POLARI,ETER 7) ANALYTICAL BALANCE 8) pH METER 9) MEASURING CYLINDER Conclusion Reference PowerPoint Presentation: VALIDATION Definition: Validation is an analytical procedure that deals with the collection of documented evidence which is eshtablished by the laboratory studies. It contains performance characteristics of the instrument that meet the requirements for the intended analytical applications. PowerPoint Presentation: PROTOCOL It is a written plan which states how validation will be conducted. This protocol identifies the Equipment process parameters Product characteristics Sampling data to be collected No. of validation runs and acceptable test results. PowerPoint Presentation: QUALIFICATION Qualification is a part of validation. It is action of proving and documenting the equipment which are properly installed. Qualification is of 3 types: Installation qualification, Performance qualification, Operation qualification PowerPoint Presentation: INSTALLATION QUALIFICATION PowerPoint Presentation: OPERATION QUALIFICATION PERFORMANCE QUALIFICATION PowerPoint Presentation: CALIBRATION It is set of operations which is eshtablished under specified conditions. It is necessary to ensure the accuracy of the data produced during the process PowerPoint Presentation: CALIBRATION OF HPLC PowerPoint Presentation: Flow rate accuracy Prime all the solvent line with water. Then set the flow rate to 0.5ml/min & wait for 15min to stabilize the system ,ensure that the pressure is stable. Insert the out let tubing into 10ml volumetric flask & start the stop watch. When the lower meniscus reaches the 10ml mark on the flask then stop the stopwatch. Record the elapsed time. Similarly check the flow for 1.0ml/min & 2.0ml/min. Injector accuracy Connect the pump & detector inlet with union. Prepare mobile phase consisting of water and methanol(70:30 v/v). Set the flow rate to 0.5ml/min & column temperature at 25 + 2°c. PowerPoint Presentation: Fill a standard HPLC vial with water & seal the vial properly. Weigh the vial & record the weight as W 1 . Place the vial in chromatographic system & perform 6 injections of 50µl from this vial & again weigh the vial note it as W 2 . Calculate the mean volume injected per injection as follows WAVELENGTH ACCURACY Set the wavelength of the instrument and inject blank, followed by standard preparation and note the absorbance. Mean volume injected = W 1 -W 2 PowerPoint Presentation: SYSTEM PRECISION Standard preparation: Accurately weigh 60mg of caffeine into 100ml of volumetric flask and dissolve in mobile phase and make up to the volume. Transfer 10ml of this solution into a 100ml volumetric flask and dilute to the volume with mobile phase. Procedure: Inject blank followed by standard preparation in 6 replicates and note the peak area and retention time. Now calculate the %RSD of retention time and peak area for 6 replicates. PowerPoint Presentation: PDA DETECTOR ACCURACY Select 3D mode and set the wavelength range as 200-400nm. Inject 20µl of standard preparation once into the chromatographic system. Then record the wavelengths from 202 to 208nm with an interval of 1nm and at 269 to 275nm with an interval of 1nm and note down the absorbance. INJECTOR LINEARITY Inject 5µl of the mobile phase as blank. Then inject 5µl, 10µl, 20µl,50µl,and 80µl of the standard preparation and record the peak area. Then plot a curve for peak area Vs volume injected. PowerPoint Presentation: DETECTOR LINEARITY Standard preparation: Accurately weigh 60mg of caffeine into 100ml volumetric flask and dilute it with mobile phase. Procedure: Inject the blank followed by detector linearity solutions and record responses. Draw a plot between concentration Vs the peak responses. PowerPoint Presentation: COLUMN OVEN TEMPERATURE ACCURACY It is evaluated by using calibrated digital thermometer at 30°c and 60°c. Place the thermometer probe in the column oven and set the temperature at 30°c. Wait till the temperature stabilizes. Record the temperature displayed on the thermometer. Similarly perform the column oven temperature accuracy test at 60°c. PowerPoint Presentation: ACCEPTANCE CRITERIA The acceptance criteria for the calibration parameters are as follows: S.NO PARAMETERS ACCEPTANCE CRITERIA 1) Flow rate accuracy Should be within 2.0ml/min 2) Injector accuracy Mean injected volume should be 50 + 1.0µl 3) Wavelength accuracy Maximum absorbance should be + 2nm 4) System precision %RSD of the retention time & peak area should be <1.0% 5) PDA detector accuracy Maximum absorbance should be at 205 + 2nm and 272 + 2nm 6) Injector linearity Plot should be linear & regression coefficient( R 2 ) should NLT 0.99 7) Detector linearity Plot should be linear 8) Column oven temperature Should be within + 2°c PowerPoint Presentation: CALIBRATION OF GAS CHROMATOGRAPHY PowerPoint Presentation: Flow rate accuracy Connect the digital flow meter to the detector outlet port. Set the carrier gas flow & wait until it reaches the set flow. Note the observed flow in replicate. Repeat the procedure by using other carrier gases such as hydrogen. Then record the result in GC calibration protocol. Column oven temperature accuracy Connect the column to the detector port. Place the thermometer probe in the column oven & set the column oven temperature at 40°c. Wait till the temperature stabilizes. Note the observed temperature & repeat the procedure for 100°c, 150°c, 190°c. PowerPoint Presentation: SYSTEM PRECISION Standard preparation: Transfer 20ml of methanol, ethanol and acetone into 100ml volumetric flask and make up with ethyl acetate. Procedure: Inject blank followed by standard preparation in 6 replicates. Note down the peak areas and retention times. DETECTOR NOISE & DRIFT TEST After GC is ready run the system up to 15min through single run. After completion of run calculate noise and drift through software. PowerPoint Presentation: DETECTOR LINEARITY Procedure: Inject blank, followed by detector linearity solutions and record the peak responses. Draw a plot between concentration Vs peak responses. PowerPoint Presentation: S.NO PARAMETERS ACCEPTANCE CRITERIA 1) Flow rate accuracy Flow rate of gas should be 10% of set flow 2) Column oven temperature Temperature should be within + 2ºc 3) System precision %RSD of retention time should NMT 1.0% & peak area NMT 5.0% 3) Detector linearity Plot should be linear & regression coefficient( R 2 ) should be NLT 0.99 5) Detector noise & Drift test Noise NMT 100µV Drift NMT 2500µV/hr ACCEPTANCE CRITERIA The acceptance criteria for the parameters of gas chromatography are as follows: PowerPoint Presentation: CALIBRATION OF UV SPECTROPHOTOMETER PowerPoint Presentation: SPECTRAL CALIBRATION (visible region) Ensure the socket of the power cord is inserted properly. Then switch on the instrument & allow to stand for 15min to warm up. Keep the dummy cuvette in position of sample holder. Set the wavelength to 485nm & press %T button. Press %T in appropriate direction to adjust 0.00 reading on read out. Now remove dummy cuvette from sample holder & close the lid. By adjusting the coarse & fine control set a reading of around 80.0 on read out. Now set the value of wavelengths in increments of 0.1nm up to 487nm and read the value of % T at each increment. PowerPoint Presentation: Draw a curve %T Vs λ . If the peak value of %T is occuring at λ 486.1 + 0.5 nm, the spectral calibration of the instrument in the visible spectral region is proper. This can be confirmed by repeating the above steps with a maximum value of %T of around 30.0 on the read out & λ setting from 655 to 657nm. If the maximum %T is obtained at a λ 656.2 + 0.5 nm then the spectral calibration of the instrument in the visible region is confirmed to be proper. PowerPoint Presentation: SPECTRAL CALIBRATION (UV region) Keep blank filled cuvette & sample(benzene vapour ) filled cuvette & set the λ to 253nm & press the absorbance button. Adjust blank to 0.000 on the read out using coarse and fine adjustment. Now place sample into the optical path, value of absorbance of sample at the λ set appears on the read out. Again set the values of wavelengths in increments of 0.1nm up to 255nm.Measure the absorbance at each increment. If the maximum absorbance is observed at 253.9 + 0.5nm the spectral calibration of the instrument in UV region is confirmed to be proper. PowerPoint Presentation: PHOTOMETRIC CALIBRATION Place dummy cuvette in sample holder & set %T to “zero”. Now remove cuvette by using coarse and fine control set & press absorbance button. If the maximum absorbance obtained at λ 485nm is 0.398 + 0.002 then the photometric calibration of the instrument is confirmed to be proper. To confirm, repeat above steps, and set 10.0 on read out & again press absorbance button. If the λ at 485nm is 1.000 + 0.002 then it is confirmed the photometric performance in the visible region is proper. UV region: Place 0.1N H 2 SO 4 as blank in cuvette & 60ppm as sample( K 2 Cr 2 O 7 ) . Set exactly to 257nm, if the value of absorbance of sample is 0.864 + 0.005, the instrument is measuring absorbance properly. Visible region: PowerPoint Presentation: TRANSMITTANCE As the value of %T is delivered from absorbance itself, if the instrument is measuring absorbance properly it is deemed that it measures %T properly. CONCENTRATION Place 0.1N H 2 SO 4 as blank & 60ppm K 2 Cr 2 O 7 as standard & 20ppm 0.1N H 2 SO 4 as sample in cuvette . Then press ‘Concentration’ push button and adjust concentration control to 600 for standard on read out. Now place sample holder into optical path, if the value of concentration appearing on the read out for sample is 200 + 5, the instrument is measuring concentration properly. PowerPoint Presentation: WAVELENGTH ACCURACY PowerPoint Presentation: LIMIT OF STRAY LIGHT Weigh accurately 1.2g of dried potassium chloride in 100ml volumetric flask and make up to the mark with distilled water. Measure the absorbance at 200nm. RESOLUTION Prepare 0.02%v/v solution of toluene and make up with hexane. Scan the wavelength from 250 -280 nm. Maximum absorbance is 269nm & minimum absorbance is 266nm. PowerPoint Presentation: CALIBRATION OF IR SPECTROPHOTOMETER PowerPoint Presentation: WAVE NUMBER VERIFICATION The wave number may be verified by recording the spectrum of a polystyrene film, which has transmission maxima at the wave numbers which is shown in the table. PowerPoint Presentation: DETECTOR ENERGY RATIO Record the minimum energy ratio value for at least one of the following measurement points and compare it to the vendor’s specifications. LIMIT: Energy ratio test specification for each spectrometer configuration should be within the manufactures specification. PowerPoint Presentation: NOISE RATIO Record the maximum noise level for each of the following regions. LIMIT: Noise level test specification vary for each spectrometer configuration and it should compare to the manufactures specification. PowerPoint Presentation: CONTAMINATION TEST This test is conducted for ATR instruments. This test checks the presence of peaks that signal contamination problem. To perform this test an automated function of the instrument is necessary. If not available record the background spectrum. LIMITS: PowerPoint Presentation: THROUGHPUT CHECK (ATR) It checks the unexpected reduction of the transmittance. A background spectrum is recorded and the transmittance is measured at 3 wave numbers at 4000, 2600, 1000cm -1. LIMIT: The limit of transmittance for the 3 wave numbers must be 80%. PowerPoint Presentation: CALIBRATION OF PHOTO-FLUROMETER PowerPoint Presentation: Procedure: Ensure the instrument is clean and free from dust. Connect the mains plug of the instrument to the 230AC mains and switch on the instrument and allow to warm up for 30mins. Set the standard coarse control and blank coarse control and insert primary and secondary filters in the appropriate slots marked i.e 366, 513nm. Then insert the blank and set the meter to zero with blank controls and insert the standard and set the meter to 100 with standard control. Insert the sample(1ppm solution of quinine sulphate in 0.1N sulphuric acid)and record the readings. PowerPoint Presentation: CALIBRATION OF POLARIMETER PowerPoint Presentation: Operating procedure: Ensure that the instrument is clean and free from dust and ensure that all parts including optical lens, telescope lens, polarimeter tube are clean. Switch on the instrument and let the sodium lamp illute to its maximum brightness. Then set the alignment of the sodium lamp compartment to the optical bench. Ensure that all settings are done in the zero position, then open the polarimeter tube compartment and place the polarimeter tube, which is filled with sample in such a way as to avoid air bubbles. Specific attention should be paid to temperature control of the sample and note the observations and perform a blank determination in the similar manner. Clean the instrument and keep under cover when not in use. PowerPoint Presentation: Calibration: The instrument is calibrated by using a solution of previously dried sucrose and measuring the optical rotation in a 2dm tube at 25°c by using the concentration given in the table. S.NO CONCENTRATION g/ml ANGLE OF ROTATION OBSERVED READINGS ACCEPTABLE TOLERANCE 1) 10 13.33 + 0.02 2 20 26.61 + 0.02 3) 30 39.86 + 0.02 4) 40 53.06 + 0.02 5) 50 66.23 + 0.02 PowerPoint Presentation: CALIBRATION OF pH METER PowerPoint Presentation: TWO POINT CALIBRATION METHOD This is the method in which pH meter is calibrated with 2 different buffers of known pH and then checking the pH meter against pH 7.00 standard buffer to confirm the calibration. PERSONNEL QUALIFICATION: Personnel are required to be knowledgeable of the procedures in this SOP as well as the operation hints and maintenance of the pH meter. PowerPoint Presentation: PROCEDURE: Check the level of the 3M KCL electrode filling solution(electrical conductor between reference &sample) to ensure that it is within 25mm of the filling hole(small circular hole on the side of the electrode). Then rinse the probe with de-ionized water & blot dry with kimwipes & then place the tip of the electrode into the pH 7.00 buffer solution and press “Cal”. The pH meter will automatically read the endpoint when the reading is stable & appropriate buffer symbol will appear on the display and record the pH value. PowerPoint Presentation: Then rinse the tip of the electrode with de-ionized water and blot with kimwipes . Again place the tip of the electrode in the second calibration buffer i.e either in pH 4.00 or 10.00 and repeat the procedure. Note the endpoint when the reading is stable and appropriate buffer symbol will appear on the display. The pH meter will also display electrode slope value. If the slope is less than 95% or greater than 105% then refer to the troubleshooting section. PowerPoint Presentation: Record the calibration data promptly, legibly and in inedible ink on the record forms. PRECAUTIONS: Do not allow the fill solution to run dry and add fill solution when ever the level falls 25mm below. Always remove the wetting cap and the fill hole plug during calibration. Do not leave the electrode in organic solvents as the tip and body may be damaged. Discard used buffer solutions daily. Do not reuse it. INTERFERENCE: The electrode can be stored for up to one week in pH 7.00 or 4.00 not in distilled water. For longer storage periods remove the wetting cap and fill it with KCL solution and push it onto the tip of the electrode. PowerPoint Presentation: CALIBRATION OF BALANCE PowerPoint Presentation: Many types of balances are used in chemical and biological tests. These balances must be calibrated to ensure the accuracy of the data produced by it. Different types of balances used are as follows: PowerPoint Presentation: Requirements of the balance It should work under optimal conditions like weighing room, weighing bench, temperature, light, air etc. S.NO REQUIREMENTS ACCEPTANCE 1) Weighing bench Should be non-magnetic vibration proof & dust free 2) Temperature Constant temperature should be maintained. Deviations should not exceed 5°c 3) Atmospheric humidity Should be between 40% to 60% 4) Light Should be protected from direct sunlight 5) Weighing vessel Smallest possible weighing vessel used. Weighing vessel and sample it contains should have same temperature. PowerPoint Presentation: PARAMETERS The parameters which are used to check the balance are as follows: VERIFICATION Verification of the balance is performed by placing a suitable weight. Weight should be placed in center of the pan and compare the result with pre defined weight. Weights which used for this always should be same. ACCURACY The accuracy of the balance is checked by weighing at least 3 different weights. It is recommended that weighing is repeated at least 5 times for every weight and compare the results with manufactures specifications PowerPoint Presentation: LINEARITY The results obtained from accuracy can be used to calculate the correlation coefficient and it checks linearity. The correlation coefficient is calculated by comparing the nominal and measured masses of the weights. PRECISION The precision of the balance should be verified by weighing at least 5 times a weight that is equivalent to 50% of the maximum capacity of the balance. It is recommended to repeat the test with a weight that is equivalent to 5% of the maximum capacity of the balance, if the balance is used at the lower range. PowerPoint Presentation: It is an error relating to variations in the positioning of weights on the weighing pan. This test should be carried out using weights equivalent to 30% of the maximum capacity of the balance. The weight should be placed between halfway to 3/4 th of the distance from the center of the pan to its edge and measured at each location in the following sequence: center, front left, back left, back right, front right and again center. ENCENTRICITY PowerPoint Presentation: It is progressive change in the display of the digital readouts of balances. This test is performed only on 5, 6 or 7 decimal scale balances. It is carried out by repeated measurements i.e every 5 mins for 30 mins of a control weight in the morning and afternoon. The mean measurement, standard deviation and relative standard deviation can be calculated from the resulting data. DRIFT TEST PowerPoint Presentation: LINEARITY ERROR Linearity error is the difference between the digital display for a weight that weighs 50% of the full weighing capacity of the instrument. It is tested at least 3 times using four weights of defined masses who aggregate total mass is approximately equal to half of the maximum capacity, depending on the type of balance. First the combined mass of all 4 weights is weighed and recorded. Then 2 sub-sets of weights are made and their masses are recorded. The linearity error of the balance can be calculated by the difference between the combined masses of all 4 weights and sum of the masses from the 2 sub-sets of weights divided by2. PowerPoint Presentation: MEASUREMENT UNCERTAINTY It is a parameter associated with the result of measurement. It is calculated according to the relevant documents. A weight of approximately 50% of the maximum capacity of the balance is weighed at least 10 times and the mean measurement, standard deviations and relative standard deviation are calculated. MINIMUM WEIGHT The minimum weight value depends on the type of the balance used. It determined by technical data and the external calibration certificate. PowerPoint Presentation: S.NO PARAMETERS FREQUENCY 1) Verification Once a week 2) Accuracy Once a year 3) Linearity Once a year 4) Precision Once a year 5) Eccentricity Once a year 6) Linearity error Once in every 6 months 7) Drift test Once in every 6 months 8) Minimum weight Once a year 9) Maximum uncertainty Once a year FREQUENCY Frequency of the parameters to be checked are as follows PowerPoint Presentation: CALIBRATION OF MEASURING CYLINDER PowerPoint Presentation: Procedure: Clean the cylinder to be calibrated to remove grease, dirt by keeping it in chromic acid for 30mins. Rinse the cylinder thoroughly under running tap water then rinse it with distilled water and dry it an oven at 105°c for 30mins. Remove the cylinder from the oven and allow to cool at room temperature for 15mins.Arrange water bath consisting of beaker containing water. Maintain the temperature of water bath at 25°c . Place the cylinder on the single pan balance and press tare switch and note the weight. PowerPoint Presentation: Then transfer the distilled water so that the meniscus of liquid in the cylinder is in level with 1ml mark and weigh the cylinder and note the weight. Similarly transfer water to fill successive 1ml graduations are recorded. Continue this till the nominal capacity of the cylinder is reached. PowerPoint Presentation: S.NO INSTRUMENTS INTERVAL 1 HPLC 3 + 7days 2 Gas chromatography 3 + 7 days 3 UV spectrophotometer Monthly once + 3 days 4 IR spectrophotometer Monthly once + 3 days 5 NMR spectrophotometer 6 + 15 days 6 Fluorimeter 3 + 7 days 7 Polarimeter Monthly once + 3 days 8 pH meter Daily 9 Analytical balance Daily Schedule for calibration various instruments PowerPoint Presentation: CONCLUSION Here by I concluded that the use of qualified analytical instrument provides the confidence that the procedure will generate test data of acceptable quality and it minimizes the risk of contamination. PowerPoint Presentation: REFERENCE Gowrisankar.D ., et al., (2010). Validation and Calibration of Analytical Instruments.vol: 2(2),89-99. Annex-4 Guidelines on Qualification of IR Spectrophotometers.(2007). Standard Operating Procedure for Calibration and Maintenance of pH meters.(2005). Annex-8 Guidelines on Qualification of Balances.(2012). PowerPoint Presentation: Thank you

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