SOP Title-Calibration of High Performance Liquid Chromatography (HPLC)
- OBJECTIVE
To lay down the procedure for Calibration of High Performance Liquid Chromatography.
- SCOPE
This SOP is applicable to Calibration of High Performance Liquid Chromatography Quality Control laboratory.
- RESPONSIBILITY
- QC personnel shall be responsible for implementation of this procedure.
- QC Head /designee shall be responsible for compliance of this procedure.
- PROCEDURE :-
- Calibration Frequency: Calibrate the instrument quarterly or after major part replacement.
- HPLC is calibrated for following parameters:
- Flow rate Accuracy of pump
- Injection Volume Accuracy of the pump
- Precision
- Injector Linearity
- Detector Linearity
- Wavelength Accuracy
- Carry over test
- Drift and Noise
- Gradient Proportional Value (GPV Test)
- Oven calibration
- Software Verification
| PARAMETERS | LIMIT |
| Flow rate Accuracy of pump | ± 2.0% from the set value |
| Injection Volume Accuracy of the pump | ± 2.0% from the set value |
| Precision | RSD of Area 2.0% and Retention time
NMT 1.0% |
| Injector Linearity | Correlation Coefficient ≥ 0.999 |
| Detector Linearity | Correlation Coefficient ≥ 0.999 |
| Wavelength Accuracy | Maxima at 205 nm ±2 nm, Minima at 245 nm ± 2 nm and Maxima at 272 nm ± 2 nm. |
| Carry over test | NMT 0.01 % |
| Drift and Noise | Drift ±5000 uV and ± Noise 200 uV |
| Gradient Proportional Value (GPV Test) | 10 % Height (8.0 % to 12.0%) ,50% Height (45% to 55 %) and 90.0% Height (85.0% to 95.0% |
| Oven calibration | ± 2.0°C of set value |
| Software Verification | It should pass |
- PUMP CALIBRATION:
- Remove the column and purge the pump with Distilled water. Set the flow rate 0.5 ml/min. and allow to run for 5 min .Take dry volumetric Flask and note the weight (W1). Collect the eluent carefully into the volumetric flask for exactly 5 minutes by noting the time using a calibrated stop watch. Note the weight (W2) of the bottle with the collected eluent. Calculate the flow rate by the formula given below. Repeat the procedure twice again. Similarly repeat the procedure with flow rates of 1.0 ml/min. and 2.0 ml/min twice from all channels.
(W2 -W1) 1
Flow = —————– x ———— = ————- ml/min.
0.9970 5
Acceptance Criteria: The flow rate shall not vary more than 2.0% from the set value.
- INJECTION VOLUME ACCURACY:
- Fill the HPLC vial with Purified water and allow vial to come at room temperature note the weight of filled vial in gm. Carry out 20 consecutive injection of 5 μl and after the completion of the injection reweight the vial (in gm). Follow the same for 10μl, 20μl, 50 μl using 10 consecutive Injection and 100 μl using 5 consecutive injection.
CHROMATOGRAPHIC CONDITION
Column : Octadecylsilyl silica gel (C18), 5 µ or Equivalent
Dimensions : 250 x 4.60 mm
Wave length : 272 nm
Flow rate : 1.0 ml/minute.
Injection Volume : 5μl, 10μl, 20μl, 50μl & 100μl
Mobile phase : Distilled Water
Run Time : 1.0 min
Calculation :-
(W1 – W2) x 1000
Volume = —————————– = ———- µl
0.9970 x Y
W1 : Initial weight of filled vial
W2 : Initial weight of vial after 10 Injection
Y : No of Consecutive Injection
Density : 0.9970 at 250 C
- PRECISION :-
Weigh accurately 30 mg of caffeine standard and transfer to a 100 ml volumetric flask, dissolve and make up to the volume with mobile phase. Further dilute 10 ml of this above solution to 100 ml, mix and make up to volume with mobile phase. Inject the one Blank Preparation and Six standard preparation. Record the chromatograms.
CHROMATOGRAPHIC CONDITION
Column : Octadecylsilyl silica gel (C18) 5 µ or Equivalent
Dimensions : 250 x 4.60 mm
Wave length : 272 nm
Flow rate : 1.0 ml/minute.
Injection Volume : 20 μl
Mobile phase : (Acetonitrile : Water) (70: 30)
Run Time : 10 .0 min
- INJECTOR LINEARITY :
Weigh accurately 30 mg of caffeine standard and transfer to a 100 ml volumetric flask, dissolve and make up to the volume with mobile phase. Further, dilute 10 ml of this above solution to 100 ml mix and make up to volume with mobile phase. Inject 5µl, 10µl,20 µl, 50 µl and 100 µl 3 times and plot the graph volume against mean area. Find out linearity coefficient.
CHROMATOGRAPHIC CONDITION :
Column : Octadecylsilyl silica gel (C18) 5 µ or Equivalent
Dimensions : 250 x 4.60 mm
Wave length : 272 nm
Flow rate : 1.0 ml/minute.
Injection Volume : 20 μl
Mobile phase :(Acetonitrile : Water) (70: 30)
Run Time :10.0 min
- DETECTOR LINEARITY :
Weigh accurately 30 mg of caffeine standard and transfer to a 100 ml volumetric flask, dissolve and make up to the volume with mobile phase. This solution is (Stock A) further Dilute.
2 ml of the stock A to 50 ml of volumetric flask mix and make up to volume with mobile phase i.e. concentration of 12 ppm.
4 ml of the stock A to 50 ml of volumetric flask mix and make up to volume with mobile phase i.e. concentration of 24 ppm.
6 ml of the stock A to 50 ml of volumetric flask mix and make up to volume with mobile phase i.e. concentration of 36 ppm.
8 ml of the stock A to 50 ml of volumetric flask mix and make up to volume with mobile phase i.e. concentration of 48 ppm.
10 ml of the stock A to 50 ml of volumetric flask mix and make up to volume with mobile phase i.e. concentration of 60 ppm.
CHROMATOGRAPHIC CONDITION :
Column :Octadecylsilyl silica gel (C18) 5 µ or Equivalent
Dimensions : 250 x 4.60 mm
Wave length : 272 nm
Flow rate :1.0 ml/minute.
Injection Volume :20 μl
Mobile phase : (Acetonitrile : Water) (70: 30)
Run Time : 10.0 min
- WAVELENGTH ACCURACY :-
Weigh accurately 30 mg of caffeine standard and transfer to a 100 ml volumetric flask, dissolve and make up to the volume with mobile phase. Further, dilute 10 ml of this above solution to 100 ml mix and make up to volume with mobile phase. Inject the one Blank Preparation and Six standard preparation. Record the chromatograms.
CHROMATOGRAPHIC CONDITION :
Column : Octadecylsilyl silica gel (C18) 5 µ or Equivalent
Dimensions : 250 x 4.60 mm
Wave length : 201 nm to 209 nm, 241 nm to 249 nm and 268 nm to 276 nm.
Flow rate : 1.0 ml/minute.
Injection Volume : 20 μl
Mobile phase : (Acetonitrile : Water) (70: 30)
Run Time : 10.0 min
- CARRY OVER TEST :-
- Weigh accurately 30 mg of caffeine standard and transfer to a 100 ml volumetric flask, dissolve and make up to the volume with mobile phase.
CHROMATOGRAPHIC CONDITION :
Column : Octadecylsilyl silica gel (C18) 5 µ or Equivalent
Dimensions : 250 x 4.60 mm
Wave length : 272 nm
Flow rate : 1.0 ml/minute.
Injection Volume : 20 μl
Mobile phase : (Acetonitrile : Water) (70: 30)
Run Time : 60.0 min
- GRADIENT PROPIOTINAL VALVE :-
CHROMATOGRAPHIC CONDITION :
Pump A : 0.3 % acetone in distilled water.
Pump B : Distilled water.
Flow rate : 1 ml/min.
Wavelength : 254 nm.
Column : No column, but union guard column directly connected to the detector.
B.CONC : 100 %
Time Programmer :-
| Time in min. | Function | Conc. in % |
| 0.01 | B.Conc | 90.0 |
| 10.00 | B.Conc | 90.0 |
| 10.01 | B.Conc | 50.0 |
| 20.00 | B.Conc | 50.0 |
| 20.01 | B.Conc | 10.0 |
| 30.00 | B.Conc | 10.0 |
| 30.01 | B.Conc | 0.0 |
| 40.00 | B.Conc | 0.0 |
| 40.01 | B.Conc | 100.0 |
| 50.01 | Stop | – |
Where, A10, A50, A90, A100 in the graph, are the distance from the base line to the I, II, III, IV step in centimeters respectively.
Specification: Pump A concentration ratio of 10%, 50% and 90 %.
Precautions for Measurement:
Ensure that the mobile phase is degassed.
Open the drain valve and wash the flow line for 5 minutes, with the flow rate set 5 ml / min and B.CONC parameter to 100 %.
Then set the flow rate to 3 ml / min, close the drain valve and wash the flow line for 20 minutes.
Last, set the flow rate to 2 ml/ min and the B.CONC parameter to 100 and check that the baseline has become stable, then start measurement.
- Oven Calibration:
- Carry out Oven Cabinet calibration at 30°C, 40°C, 50°C and 60°C with the help of suitable calibrated thermometer and record the observation.
- Software Verification
- Go to the tools bar and select “Tools”, than select “Check program files”, a window “Check the program files” will display.
- Click on execute and wait for few seconds.
- Now click on Browse & than take the report print. All the parameter of software verification should be pass.
- Calibration report shall be prepared as per Annexure.
- After the successful completion of calibration. Calibration label shall be affixed on the instrument.
- ABBREVIATION(s)
| Abbreviation | Full Description |
| QC | Quality Control |
| QA | Quality Assurance |
| SOP | Standard Operating Procedure. |
| µl | Micro liter |
| µm | Micrometer |
| % | Percentage |
| °C | Degree Centigrade |
| RSD | Relative standard Deviation |
| mv | milli volt |
| GPV | Gradient Proportional Valve |
*Note – Ready to use SOP available in “DOWNLOAD” Section.