To lay down a procedure for calibrating the HPLC Shimadzu, (Prominence– i LC – 2030).
SCOPE
This procedure applies to the HPLC Shimadzu (Prominence– i LC – 2030) in the Quality Control Laboratory.
RESPONSIBILITY
Execution: Chemist and above – QC department.
Checking: Asst. Manager and above – QC department
FREQUENCY
Calibration shall be done every six months after spare parts' maintenance or replacement.
CALIBRATION PROCEDURE
1. Calibration of the pump:
A. Flow rate Accuracy
- Connect the inlet and outlet tubing with a union.
- Purge the line with water and ensure that the flow line is free from air bubbles.
- Set the flow rate at 0.5ml / min, collect the mobile phase (water) in a dry 10mL volumetric flask and fill the volumetric flask up to the mark, note down the time.
- Calculate the flow rate by using the formula (Volume of water/ Time in a sec) x 60
- Repeat the above procedure for 1.0ml/min, 2.0ml/min, and 3.0ml/min. Record the results in Attachment-I.
- Acceptance Criteria: ± 2% of the flow rate
B. Flow rate Consistency.
- Record the RSD of the retention time of the uracil or caffeine peak under the test injector repeatability and record the results in Attachment-I.
- Acceptance Criteria: % RSD should not be more than 1%
C. For gradient valve
- Install a union in place of the column & flush solvent lines (A) at a flow rate of 2ml/min with water.
- Fill reservoir A with 100% HPLC grade water and reservoirs B, C & D with 0.3% acetone in water as mobile phase.
- Set the following time program.
- Use the detector at a wavelength of 254 nm.
- Record the value of the gradient valve test in Attachment-I and repeat the procedure for C and D lines.
2. Calibration of the Injector:
A. Volume Accuracy
- Take HPLC-grade water in one of the vials and stopper with septa.
- Weigh the vial with water (W1).
- Place the vial in the sample compartment. Set the system to inject 10 injections with a 10µl- injection volume.
- Take the vial and reweigh it (W2).
- The difference in weight gives the weight of water drawn by the auto-injector. (W1-W2).
- Calculate the quantity withdrawn by the auto-injector per injection.
- Calculate the volume withdrawn by the auto-injector per injection by the formulae (W1-W2)/10/ 0.99602, Where 0.99602 is the density of water at 25°C.
- The volume of water drawn by the auto-injector per injection should be within ± 0.5 % of the set value.
- Repeat the procedure with 20µl and 50µl.
B. Repeatability of the Results
Chromatographic condition
Column : BDS Hypersil or equivalent 250 x 4.6 mm, 5.0µm
Flow rate: 1.0 ml/min
Injector Volume: 20µl
Wavelength: 254 nm
Mobile phase: Methanol
Standard preparation (20ppm):
- Transfer about 25 mg of Uracil or caffeine to a 100ml volumetric flask, add 70ml of Methanol, sonicate to dissolve, and make up the volume with Methanol. Dilute 4 ml of the resulting solution to 50 ml with the mobile phase.
- Inject the standard preparation five times and record the observed area in Attachment-I.
Acceptance Criteria: % RSD of the area of Uracil or caffeine should not be more than 1%.
C. Injector Linearity
- For chromatographic conditions and standard preparation refer to section B.
- Inject 10, 20, 30, 40 & 50µl of the standard preparation in duplicate. Calculate the average area counts corresponding to each set of injections.
- Plot a graph of the mean area against injection volume and calculate the correlation coefficient.
Acceptance Criteria: Correlation factor - NLT 0.999.
3. Calibration of the Detector:
A. Detector Linearity
For chromatographic conditions refer to section B (Repeatability of the Results).
Standard stock solution (250ppm):
- Transfer about 25 mg of Uracil or caffeine to a 100ml volumetric flask, add 70ml of Methanol, sonicate to dissolve, and make up the volume with Methanol.
- Take 2ml, 4ml, 6ml, 8ml, and 10ml of stock solution respectively in 50 ml of the volumetric flask, and dilute up to the mark with methanol.
- Inject all the solutions in duplicate and record the peak response in Attachment-I.
- Plot the graph of concentration against the mean area of Uracil or caffeine and calculate the correlation factor.
Acceptance Criteria: Correlation factor - NLT 0.999
4. Calibration of the Column Oven:
- Set the column oven temperature to 30°C.
- Allow the system to attain set temperatures.
- Read the temperatures using the data logger.
- Repeat the same for oven temperature 50°C.
The acceptable temperature range for the column oven is + 2°C.
ATTACHMENTS
Attachments 1: Calibration Record of HPLC.
ABBREVIATIONS
UV – Ultra Violet
HPLC – High-Performance Liquid Chromatography
QC – Quality Control
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