Calibration of High Performance Liquid Chromatography (isocratic)
Calibration of High Performance Liquid Chromatography
1.0 PURPOSE
The purpose of the standard operating procedure is to calibrate the High-Performance Liquid Chromatography (Isocratic HPLC).
2.0 SCOPE
Standard operating operation applies to the staff responsible for the operation and calibration of the High-Performance Liquid Chromatography (Isocratic).
3.0 RESPONSIBILITY
3.1 Section Incharge
3.2 Technical Manager
3.3 Q.A. Manager
4.0 PROCEDURE
4.1 MATERIALS REQUIRED
4.1.1 Instrument under calibration.(Isocratic HPLC)
4.1.2 Acetone (HPLC Grade)
4.1.3 Water (HPLC Grade)
4.1.4 Caffeine (Working Standard)
4.1.5 Calibrated Digital Thermometer
4.1.6 Naphthalene
4.1.7 Calibrated Glassware
4.1.8 Weighing Balance
5.0 PROCEDURE
5.1 CALIBRAION OF PUMP
5.1.1 PRESSURE PULSATIONS TEST
The Pressure Pulsations Test is to verify that the output flow fluctuations are within specification (2% peak to peak of the running pressure).
5.1.1.1 Set the pump flow rate to 1 ml/min.
5.1.1.2 Start the pump and let the pressure stabilize.
5.1.1.3 Observe the minimum and maximum pressure during the 1 min interval
5.1.1.4 Determine the minimum and maximum pressures observed, or difference between them. Determine the mid point pressure (MP) between the max and min, this is the mid point pressure used to compute 2 % peak-to-peak error band.
ACCEPTANCE CRITERIA:
Difference between the minimum and the maximum pressure should be less than 2% of the mid point pressure.
- FLOW ACCURACY TEST
This test is used to prove that the flow rate selected on the pump is delivered in the correct time.
CHECKING FLOW AT 1 ML/MIN (LOW FLOW RATE)
5.1.2.1 Connect the backpressure regulator to the output of the pump.
5.1.2.2 Zero the scale of the weighing balance. Place a 50 ml beaker on a scale and note the reading of the empty beaker (W1).
5.1.2.3 Start the pump and fill the lines with water.
5.1.2.4 Set the pump flow at 1 ml/min.
5.1.2.5 When pressure stabilizes, direct the flow of water into the beaker and simultaneously start the stopwatch.
5.1.2.6 Let the pump run for 5 min.
5.1.2.7 Remove the beaker after 5 min and weigh the beaker containing water (W2).
5.1.2.8 Subtract the final weight with the initial weight of the beaker (W2-W1) & divide the Weight of the water by 5 & with the water density to get the volume delivered per minute.
5.1.2.9 Repeat the above procedure for 10 min.
5.1.3 CHECKING FLOW AT 2 ML/MIN (HIGH FLOW RATE)
5.1.3.1 Connect the backpressure regulator to the output of the pump.
5.1.3.2 Zero the scale of the weighing balance. Place a 50 ml beaker on a scale and note the reading of the empty beaker (W1).
5.1.3.3 Start the pump and fill the lines with water.
5.1.3.4 Set the pump flow at 2 ml/min.
5.1.3.5 When pressure stabilizes, direct the flow of water into the beaker and simultaneously start the stopwatch.
5.1.3.6 Let the pump run for 5 min.
5.1.3.7 Remove the beaker after 5 min and weigh the beaker containing water (W2).
5.1.3.8 Subtract the final weight with the initial weight of the beaker (W2-W1) & divide the weight of the water by 10 & with the water density to get the volume delivered per minute.
5.1.3.9 Repeat the above procedure for 10 min.
ACCEPTANCE CRITERIA:
The volume of water collected should be ±0.5 ml.
5.2.1 CALIBRATION OF DETECTOR
- CAFFEINE WAVELENGTH VERIFICATION AND ACCURACY
Dissolve 10 mg of caffeine WS with 1000 ml of water. Dissolve and mix completely.
5.2.1.1 Place the 1-liter of dilute caffeine mixture on an available reservoir of the pump.
5.2.1.2 In another reservoir place a bottle of HPLC water. Using the HPLC water, purge the pump lines. Connect outlet of pump directly to inlet of the UV/VIS flow cell.
5.2.1.3 Set the UV/VIS Detector wavelength to 300 nm.
5.2.1.4 Start the pump flow rate at 1ml/min with water and pump for 5 minutes.
5.2.1.5 Auto zero the detector with water in the flow cell.
5.2.1.6 Set the wavelength to 205 nm.
5.2.1.7 Start pump at 1 ml/min with dilute caffeine solution. After 5 minutes check the absorbance.
5.2.1.8 Record the observed wavelength maximum.
5.2.2 CHANNEL PARAMETERS
Run Time : 23.00min
Sampling Rate : 2.0000 pts/s
5.2.3 DETECTOR PARAMETERS
Step Time Wavelength Auto zero
1 1.0 300 No
2 4.0 200 No
3 1.0 201 No
4 1.0 202 No
5 1.0 203 No
6 1.0 204 No
7 1.0 205 No
8 1.0 206 No
9 1.0 207 No
10 1.0 208 No
11 1.0 269 No
12 1.0 270 No
13 1.0 271 No
14 1.0 272 No
15 1.0 273 No
16 1.0 274 No
17 1.0 275 No
18 1.0 276 No
19 1.0 277 No
20 1.0 278 No
ACCEPTANCE CRITERIA:
The wavelength maximum at 205 nm and 273 nm should be in the range of ±1 nm.
5.3. CALIBRATION OF COLUMN OVEN
- TEMPERATURE ACCURACY
Measure oven temperature accuracy at three points in the normal operating range of 5°C above ambient to 100°C.
5.3.1.1 Open the oven door. Carefully place the probe of the thermometer in the column holder bracket so the tip of the probe is in the air stream of the oven.
5.3.1.2 Ensure the oven is installed properly and power switched on at the Column Oven. Select a Set Temperature of 40°C for the heat only oven.
5.3.1.3 Monitor the reported oven temperature (on the oven’s display) vs. the temperature record on the thermometer. Allow the system to reach the set point and to stabilize for 15- 30 minutes. Record the temperature displayed on the digital thermometer.
5.3.1.4 Repeat step (c) for a Set Temperature of 60°C, 80°C & 100°C for the oven.
ACCEPTANCE CRITERIA: The value should be within ±1°C.
- TEMPERATURE STABILITY
5.3.2.1 With the set temperature set to 40°C and stabilized, take six further readings of the Digital Thermometer at fifteen-minute intervals. Record the temperature range. The range of the results should be less 1°C.
ACCEPTANCE CRITERIA:
The value should be Max (reading) – Min (reading) <1°C.
5.4. CALIBRATION OF INJECTOR:
The initial system set up conditions:
Wavelength : 260 nm
Flow Rate : 1.0 ml/min
Mobile Phase : Methanol: Water (80:20 v/v)
Column : C18
DilutionMedium : Mobile Phase
- LINEARITY:
Linearity will check that the injector can accurately inject varied concentration of sample. We do this by injecting different concentration of Naphthalene of same injection volumes.
The different concentrations are 10 ppm, 20 ppm, 30 ppm 40 ppm & 50 ppm. There should be linear relationship between the concentration of the sample and area under the peak.
ACCEPTANCE CRITERIA:
The R-squared value should be of 0.999 or greater.
5.4.2 REPEATABILITY (PRECISION)
Repeatability will look at the standard deviation of the area of the Naphthalene peak, the next to last peak in the chromatogram to check Repeatability. The Naphthalene peak from each data file will be averaged and the standard deviation determined. From the standard deviation the RSD is calculated. The purpose of Repeatability is to verify that the injector can repeatedly inject the same volume of sample numerous times.
Inject 6 injections of 20 mL each.
ACCEPTANCE CRITERIA:
The standard deviation should be less than or equal to 0.5 %.
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