Calibration of HPLC Shimadzu
Purpose: To lay down a procedure for calibration of HPLC Pump and UV detector.
Responsibility: Chemist (s)
Accountability: Head of Q.C. / Q.A.
Procedure:
Ensure that Equipment is clean.
Pump:
Flow Rate Accuracy:
• Replace the solvent in the pump with clean degassed water. When the solvent in the pump is note miscible with water, it should first be replaced with a third solvent which is miscible with both the solvent and water.
• Prepare a calibrated stop watch, calibrated volumetric flasks (5 and 25 ml), and stainless steel tubes used as flow restrictors.
Water should be thoroughly degassed prior to use.
1 ml/min without load back pressure: (Calibration of HPLC Shimadzu)
Connect a restrictor that gives a back pressure of approximately 10 kg / cm2 at a flow rate of 1 ml / minute. Note that this back pressure is necessary for the pump to properly function.
• Set the flow rate to 1 ml / min.
• Flow water and wait for the back pressure is built and stable.
• Start receiving water from the restrictor in 5 ml volumetric flask, and measure the time required for filling up the flask to mark
• Repeat the measurement described in step 4.3.1.4.4 five times and calculate the average value
5 ml / min without Load Back Pressure:
• Connect a rusticator that gives a back pressure of approximately 10kg/cm2 at a flow rate of 1 ml /minute. Note that this back pressure is necessary for the for the pump to properly function.
• Set the flow rate to 5 ml / min.
• Flow water and wait for the back pressure is built and stable.
• Start receiving water from the restrictor in 25 ml volumetric flask, and measure the time required for filling up the flask to mark.
• Repeat the measurement described in step 4.3.1.5.4 five times and calculate the average value.
1 ml / min with Load Back Pressure:
• Connect a rusticator that gives a back pressure of approximately 100kg/cm2 at a flow rate of 1 ml /minute. Note that this back pressure is necessary for the for the pump to properly function.
• Set the flow rate to 1 ml / min.
• Flow water and wait for the back pressure is built and stable.
• Start receiving water from the restrictor in 25 ml volumetric flask, and measure the time required for filling up the flask to mark.
• Repeat the measurement described in above step five times and calculate the average value.
• The measured deviation in all cases should be less than ±2%.
Calibration: (Calibration of HPLC Shimadzu)
Detector:
Column: C-18, 250x4.6mm, 5µ particle size
Mobile phase: A mixture of 80% methanol and 20 % water.
Flow Rate: 1 ml / minutes
Detector: UV detector,
Sample: 20mcg in methanol
Inject Volume: 20µl,
Procedure:
• Operate the system as per SOP QC-103.
• Set the injection volume 20 µl and detection limit 268 nm.
• After completion of sample save the data.
• Similarly take the observations at 269, 270, 271, 272, 273, 274, 275, & 276 nm and record the observation.
• Print the data and file it.
• Take the five replicate of the sample at 272 nm and record the data.
Acceptance Criteria:
Maxima : 272 ± 1 nm
RSD of RT : NMT 0.5%
RSD of Peak Area : NMT 1.5%
Tailing Factor : NMT 1.5
Validation:
• Mobile Phase: 0.05% Aqueous Acetone
• Flow Rate: 1 ml / minutes
• Operate the HPLC as per SOP / QC-103.
• Press VP button. Touch on auto validation on screen.
• Press VP button. Touch on auto validation on screen.
• Auto validation started.
System Checks all the parameters one by one.
• After completion of auto validation save the data by pressing F3 key and then press F5 to exit the auto validation.
• Print the data and review.
• If all the test are O.K./PASS then validation is complete.
• If any of the tests fails, then inform to service engineer.
