# Calibration of High Performance Liquid Chromatography (isocratic)

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 is applicable 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)

4.1.4    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.

5.1.2      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.    CALIBRATION OF DETECTOR

5.2.1      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

5.3.1      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.

5.3.2      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

Dilution Medium       : Mobile Phase

5.4.1      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 %.