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1.0. OBJECTIVE: The objective of this SOP is to ensure that the instrument performs satisfactorily and gives accurate and reproducible data.
2.0. RESPONSIBILITY: 2.1 The Chemist/ Executive - Quality Control shall be: 2.1.1. Responsible for operating the HPLC as per the SOP. 2.1.2. Responsible for timely calibration of HPLC System as per the SOP. 2.1.3 Responsible for ensuring the adherence of SOP.
3.0. ACCOUNTABILITY: Head - Quality Control
4.0. PROCEDURE: 4.1 PROCEDURE FOR GENERAL CLEANING: 4.1.1 Ensure that the power supply to the instrument is switched off and main cord is removed from supply. 4.1.2 Clean the instrument with a clean dry cloth every day. A wet cloth dipped in dilute soap solution may be used occasionally. 4.1.3 Precaution must be taken to clean the instrument immediately with dry cloth.
4.2 OPERATING INSTRUCTIONS: 4.2.1 Ensure that the instrument is properly connected to the power supply. 4.2.2 Fix the column and prepare the mobile phase.
4.3 PREPARATION OF MOBILE PHASE 4.3.1Use HPLC grade solvent / water & AR/Excel/ HPLC grade reagent only.
detector and then system controller.6 Purge the auto injector by pressing purge key on auto sampler display. 4.5 From the ³file menu´ create a new method or load the existing method.2 Double click on the main Menu of SHIMADZU Icon. after then save the schedule.4. The pump will run and indicator will glow. 4.0 %.8 For system suitability test. the standard solution shall be injected 3 times and the RSD shall be calculated and ensure that it is within the limit.5 PROCEDURE TO OPERATE CLOSE UP 4.4. 4.5. 4. 4. 188.8.131.52. 4. 4.1 Switch on pump. 184.108.40.206 Run Sample Schedule.5.45 membrane filter after mixing it in required proportion and de-gas on ultrasonic bath for about 5 minutes. sample volume. and Sample Schedule & Post Run Analysis.9 After every ten injection of sample (5 samples in duplicate).4.4 Set desired flow rate by pressing function key. 4. .clockwise to open the drain valve to run the purge system.3 After the purge is over close the drain valve.1 Switch on the computer monitor and printer.5. batch number.4.4. 4.4 PROCEDURE TO GET STARTED 4. auto injector.4 Open Real Time. 4.4.2 Turn the drain valve knob to 180 in anti. 4. 4. inject six continuous injections of same standard and RSD should not be more than 2.6 Create a new sample schedule and feel sample name.7 Enter the desired wavelength on detector using function key.3. Real Time.5.3 It will show Configuration.4.2 Filter the mobile phase through 0. 4. method name and file name.5 Press pump key.4. 4. otherwise it is specified in standard test procedure.
7.3% acetone with HPLC grade water.2.99 to 1.7.2 4.7 Record the observation in Annexure -1 4.4 Calculate the flow rate by dividing the weight obtained with weight per ml and 10 (run time). 4.6 SHUT DOWN PROCEDURE 4.B 4. 4.3 Switch-off the computer.6.3 Prepare the mobile phase.7. 4.7.1 Disconnect the column and connect the inlet and outlet tubing¶s with a union. 4.1.01 ml / min.6.1. 4.4.1. Prepare 0.2.1.2 From Start button select ³Shut down´ and click yes. 4.7.7 CALIBRATION PROCEDURE.7.7.1 Install union in place of column & flush solvent lines (A&B) at flow rate of 2ml/min with water.7.8 Acceptance criteria: Flow rate should be in between 0. 4. . 4.4 Switch-off CBM. Real Time and then CLASS LC 10 to Software.220.127.116.11.3 Set the flow rate at 1ml / min and collect the mobile phase (water) in a dry preweighed beaker and collect the mobile phase for 10 min. 18.104.22.168 Close Sample Schedule. ³Pump´ key and switch off the pump module.2. auto sampler.7.1.6 Repeat the same procedure for Pump. 4. 4.5 Calculate the corresponding flow rate.1 FOR PUMP: 4. Carry out the experiment in duplicate.2 FOR GRADIENT VALVE: 22.214.171.124.2 Prime all the lines at 5 ml/min flow rate with water and ensure that flow line is free from air bubbles. 4.6 Stop pump by pressing. 4. 4.6.7. weigh the beaker to get the weight of mobile phase.5 Switch-off Detector then.
7.5 STANDARD PREPARATION: 4.01 40. sonicate to dissolve and make up the volume with Methanol to obtain a solution containing about 0.7.3.01 10.3.00 20.3 INSTRUMENT SET UP: 4. Add 100ml of Methanol.00 10.7.7.00 FUNCTION B CONC B CONC B CONC B CONC B CONC B CONC B CONC B CONC B CONC B CONC VALUE 10 10 50 50 90 90 100 100 0 0 4.5.01 30.2. 4.00 40.7.1 Transfer about 50 mg of Uracil to a 250ml volumetric flask.7.4.01 20. 4.2.3 4.00 30.3% acetone in HPLC grade water as mobile phase.7.2 4.02 mg/ml of Uracil. 4.3. The gradient valve test shall be accepted if actual concentration with ±1% of set concentration.01 50.1 Enter the following time program: TIME 0.4 Fill reservoir A with 100% HPLC grade water & reservoir B with 0.4 Use detector at wavelength of 254 nm. Record the printout of gradient valve test as per Annexure . .126.96.36.199 CALIBRATION OF INJECTOR: CHECK FOR PRECISION: Purge the injector system with 100% water to ensure the complete washing of injector.
1 Standard preparation. 5.0 ml/min Injector Volume: 20m litre Detector : 254 nm 4.efficient calculated shall be not less than 0.7.3 Use HPLC grade Methanol as the mobile phase.2 Tabulate the average area against each injection. Plot a graph for area counts vs mlitre the resulting graph shall be linear.5. Mobile phase.7. 188.8.131.52 Inject 10.6.6.5.2 Filter the solution through 0.7 Record the observation in the format as mentioned in Annexure 3.6 mm. Instrument set. 4.5 Affix a calibration status label on the instrument containing ³Calibrated On´.0m : 1. 262 nm 4. 4.5.4 Instrumental Set Up Column Flow rate : Hypersil ODS or equivalent 150 x 184.108.40.206 CALIBRATION OF DETECTOR: 4.3.4 Record the observation as per the Annexure .7.4 Record the results in the detection calibration record as per the Annexure . 30.5. 4.7.3 The largest peak response shall be at 258 nm 2nm. 4. 20.7. 4.7. 4.6 CHECK FOR LINEARITY: 4. 4. .4.7.6 The relative standard deviation for area counts calculated shall not be more than 1.7. 4. 220.127.116.11m membrane filter.7. Calculate the average area counts corresponding to each set of injection.5. The peak areas observed shall be consistent.up same as mentioned in calibration of Injector.7.3 The correlation co.7.7.7. 40 & 50m litre of standard preparation in duplicate. 18.104.22.168.0%.2 Run the chromatograph at different wavelength (252 nm with 2 nm increment) 22.214.171.124.5.7.5 Inject the standard preparation six times in the system. ³Due On´ and ³Signature´.
1 Detector: Once in three months and after each maintenance job.7.7.0 Annexure Annexure ANNEXURES: 1 2 : Format for Calibration Record of Pump (Flow Rate) : Format for Calibration Record of Gradient Proportionate Valve .8. 4. 1 : Head of Department Certified Copy No. 4.3 Injector: Once in six months and after each maintenance job.8. UNDER MAINTENANCE EQUIPMENT SINCE : SIGNATURE: : 4.7.7. 126.96.36.199.8 FREQUENCY OF CALIBRATION: 4.4 Gradient: Once in six months and after each maintenance job in the pump.4. if any discrepancy observed during calibration or operating the instrument and affix µUnder Maintenance¶ label on the instrument.6 Report to Head QC. 5. 4.0 DISTRIBUTION: Quality Control Certified Copy No. 3 : Display copy Near HPLC System Original Copy : Head QUALITY ASSURANCE.8.2 Pump: Once in three months and after each maintenance job.8.0 Trainer Trainees Period TRAINING: ---Head Quality Control Quality Control Chemists and Assistants One day 6. 2 : File copy in HPLC Calibration Data Reference Copy No.
REFERENCES: .Annexure Annexure 3 4 : Format for Calibration Record of Injector : Format for Calibration Record of Detector 8.0 In house.
ANNEXURE 1 FORMAT FOR CALIBRATION RECORD OF HPLC REF.: 00 Effective from: Page No. Checked By: Date: . (W2) Weight of beaker (W1) (W2 W1) / 10 0. / min Mean Vol.01 Weight per ml of water = Calibrated By: Date ________ gm.01 Pump-B Weight of beaker + water after 10 min.: Revision No. SOP NO.0 ml / min.99 to 1. Mean wt. 1 2 Pump-A Weight of beaker + water after 10 min. / min Limit in ml / min. Flow rate adjusted to 1. (W2) Weight of beaker (W1) (W2 W1) / 10 0.99 to 1.: 1 of 1 Calibration Record Instrument No. Make and Model Calibration Date Calibration Due On : : : High Performance Liquid Chromatograph : : Calibration of Pump (Flow Rate): Observed weight in gm.
0.0 (Lt: 9.: 00 Effective from: Page No.9) Value Obtained Actual Value Set In The Program Value: Height of B Concentration Height of a 100 % Concentration Remarks: Satisfactory / Not Satisfactory Checked By: Date : Calibrated By: Date .1) (Lt: 49.1 To 90. Height From Base Line Tine Range To B Concentration Peak.ANNEXURE 2 FORMAT FOR CALIBRATION RECORD OF HPLC REF.3% Acetone HPLC : 254 nm. SOP NO.0 20.: 1 of 1 Calibration Record Instrument No. Make and Model Calibration Date Calibration Due On : : : High Performance Liquid Chromatograph : : Calibration of Gradient Proportionate Valve Parameters: Mobile Phase Reservoir B Wavelength Flow rate : Reservoir A 100% Water with HPLC : 0.5 To 50.9 To 10.5) (Lt: 89.01 To 20.: Revision No.01 To 10.0 10. : 2 ml / min.01 To 30.
Injection no.ANNEXURE 3 FORMAT FOR CALIBRATION RECORD OF HPLC REF.0 % Area % of RSD Limit .: 1 of 1 Calibration Record Instrument No. Make and Model Calibration Date Calibration Due On : : : High Performance Liquid Chromatograph : : Calibration of Injector: 1. Injection volume: 20 ml.: Revision No. SOP NO. à 5ml à 50ml with Methanol. Column: ODS (150 mm x 4. Check for Precision: Standard Preparation: Take ______ mg of Uracil (about 50 mg) à 250 ml. Inject six replicate injection of standard solution and calculate RSD for area of main peak. 5 micron) Wavelength: 254 nm. Flow rate: 1 ml / min.6 mm. Parameters: Mobile Phase: Methanol HPLC grade. 1 2 3 4 5 6 Not More Than 1.: 00 Effective from: Page No.
3 30 ml. 4 40 ml. 2 20 ml. Check for Linearity: Sr.2. Injection volume Area i) ii) i) ii) i) ii) i) ii) i) ii) Not less than 0. Remarks: Satisfactory / Not satisfactory.99 Mean Correlation coefficient Observed results Limit 1 10 ml. Calibrated By: Date Checked By: Date: . No. 5 50 ml.
5 micron) Wavelength: 252. (Limit: 258 nm Remarks: Satisfactory / Not Satisfactory Calibrated By: Checked By: Date Date: 2 nm) .264 nm with 2 nm increment.: Revision No.No.6 mm. No.: 00 Effective from: Page No. Flow Rate: 1 ml / min. Injection Volume: 20ml. Parameters: Mobile Phase: Methanol HPLC grade Column: ODS (150 mm x 4.ANNEXURE 4 FORMAT FOR CALIBRATION RECORD OF HPLC REF.) à250 ml. Sr. à 5ml à 50ml with Methanol.: 1 of 1 Calibration Record : Instrument Sr. : Make and Model : Calibration Date : Calibration Due On : High Performance Liquid Chromatograph Calibration of Detector Standard Preparation: Take _____ mg of Uracil (about 50 mg. 1 2 3 4 5 6 7 Wavelength (nm) 252 254 256 258 260 262 264 Area 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) 1) 2) Mean Largest peak response is observed at __________ nm. SOP NO.
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