Calibration of High Performance Liquid
The purpose of the standard operating procedure is
to calibrate the High Performance
Liquid Chromatography (Isocratic HPLC).
operating operation is applicable to the staff responsible for the operation
of the High Performance Liquid
3.1 Section Incharge
3.2 Technical Manager
3.3 Q.A. Manager
under calibration.(Isocratic HPLC)
5.1 CALIBRAION OF PUMP
The Pressure Pulsations Test is to verify
that the output flow fluctuations are within
specification (2% peak to peak of the running
188.8.131.52 Set the pump flow rate to 1 ml/min.
184.108.40.206 Start the pump and let the pressure
220.127.116.11 Observe the minimum and maximum pressure
during the 1 min interval
18.104.22.168 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
Difference between the minimum and the maximum
pressure should be less than 2% of the
mid point pressure.
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)
Connect the backpressure regulator to
the output of the pump.
22.214.171.124 Zero the
scale of the weighing balance. Place a 50 ml beaker on a scale and note the
of the empty beaker (W1).
126.96.36.199 Start the pump and fill the lines with water.
188.8.131.52 Set the pump flow at 1 ml/min.
pressure stabilizes, direct the flow of water into the beaker and
Let the pump run for 5 min.
Remove the beaker after 5 min and weigh
the beaker containing water (W2).
the final weight with the initial weight of the beaker (W2-W1) & divide the
of the water by 5 & with the water density to get the volume delivered per minute.
Repeat the above procedure for 10 min.
5.1.3 CHECKING FLOW AT 2 ML/MIN (HIGH FLOW RATE)
the backpressure regulator to the output of the pump.
the scale of the weighing balance. Place a 50 ml beaker on a scale and note the
reading of the empty beaker (W1).
the pump and fill the lines with water.
the pump flow at 2 ml/min.
184.108.40.206 When pressure stabilizes, direct the flow of
water into the beaker and simultaneously
the pump run for 5 min.
the beaker after 5 min and weigh the beaker containing water (W2).
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.
the above procedure for 10 min.
volume of water collected should be ±0.5 ml.
WAVELENGTH VERIFICATION AND ACCURACY
10 mg of caffeine WS with 1000 ml of water. Dissolve and mix completely.
the 1-liter of dilute caffeine mixture on an available reservoir of the pump.
220.127.116.11 In another reservoir place a bottle of HPLC
water. Using the HPLC water, purge the
lines. Connect outlet of pump directly to inlet of the UV/VIS flow cell.
the UV/VIS Detector wavelength to 300 nm.
the pump flow rate at 1ml/min with water and pump for 5 minutes.
zero the detector with water in the flow cell.
the wavelength to 205 nm.
Start pump at 1 ml/min with dilute
caffeine solution. After 5 minutes check the
Record the observed wavelength maximum.
5.2.2 CHANNEL PARAMETERS
Run Time : 23.00min
Sampling Rate :
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
1.0 204 No
7 1.0 205 No
8 1.0 206 No
9 1.0 207 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
1.0 276 No
1.0 277 No
1.0 278 No
The wavelength maximum at 205 nm and 273 nm should be in the range of ±1 nm.
5.3. CALIBRATION OF COLUMN OVEN
Measure oven temperature accuracy at three
points in the normal operating range of
5°C above ambient to 100°C.
Open the oven door. Carefully place the probe of the thermometer in the
so the tip of the probe is in the air stream of the oven.
18.104.22.168 Ensure the oven is installed properly and
power switched on at the Column Oven.
a Set Temperature of 40°C
for the heat only oven.
the reported oven temperature (on the oven’s display) vs. the temperature
on the thermometer. Allow the system to reach the set point and to stabilize
minutes. Record the temperature displayed on the digital thermometer.
22.214.171.124 Repeat step (c) for a Set Temperature of 60°C, 80°C & 100°C for the oven.
The value should be within ±1°C.
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.
The value should be Max (reading) – Min (reading) <1°C.
5.4. CALIBRATION OF INJECTOR:
initial system set up conditions:
Wavelength : 260 nm
Rate : 1.0 ml/min
Phase : Methanol:
Water (80:20 v/v)
Column : C18
will check that the injector can accurately inject varied concentration of
sample. We do this by injecting different concentration of Naphthalene of same
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.
The R-squared value should be of 0.999 or greater.
5.4.2 REPEATABILITY (PRECISION)
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.
6 injections of 20 mL
The standard deviation should be less than or equal to 0.5 %.