Calibration of High Performance Liquid Chromatography (HPLC) SOP

HPLC Calibration SOP | Calibration of HPLC by Caffeine | HPLC Calibration Parameters | HPLC Calibration Procedure |

HPLC Calibration SOP covers below points:
- Precautions
- HPLC Calibration parameters
  - Performance of pump for constant flow rate (pump calibration)
  - Wavelength accuracy test for UV detector
  - Linearity of detector response
  - Calibration of injector & loop
    - Precision and Linearity of Injector and Loop
    - Auto Sampler Injector Calibration
  - Calibration for gradient program test
  - Column oven temperature
  - Carry over test
- HPLC Calibration format

1.0 OBJECTIVE :

To define the Calibration procedure for High Performance Liquid Chromatography.

2.0 SCOPE :

This procedure is applicable to HPLC.

3.0 RESPONSIBILITY :

Quality Control Officer and above

4.0 ACCOUNTABILITY:

Quality Control Head

5.0 PROCEDURE :

5.1 PRECAUTIONS

Ensure that the plug is connected to the mains.
Check and ensure that the instrument is clean and calibration is valid.
Ensure that the pump, column, detector & processors are properly connected.
Set mobile phase and waste solution bottle in place.
Ensure that insert the end of solvent tube into solvent bottle.
Ensure that HPLC is connected with UPS and have battery backup.
Before you start the analysis of a sample. Ensure that ,
- Suitable column is installed,
- System is purged and equilibrated,
- Solvent bottles are filled by the mobile phase or by solvent,
- Confirm the wavelength, flow rate , column temperature etc.

5.2 CALIBRATION

Perform the calibration of instrument as per procedure given below.
Frequency for calibration of HPLC system: Half Yearly.

PERFORMANCE OF PUMP FOR CONSTANT FLOW RATE
- Start the pump with ON/OFF key.
- Purge all the port with degassed water for 10 minutes.
- Connect the Union Instead of Column.
- Select Pump – A from instrument for calibration.
- Set the flow 0.25 ml /min. and start the pump to stabilize the system.
- When the system gets stabilized, Weigh the empty 20 ml volumetric flask and put at the outlet of Detector end. Start the stopwatch immediately after starting the run.
- After 5 minutes, take out the volumetric flask and weigh the volumetric flask containing water, measure the time by using calibrated stop watch.
- Calculate the flow deliver by pump per minute using the density of water 0.99602 gm/ml at 25°C by using following formula.
  - Actual flow = Weight of water in gm / Time X 0.99602
  - Where 0.99602 is density of water at 25°C.
  - Repeat twice the same steps
- Repeat above steps by changing flow rate at 0.5 ml/min, 1.0 ml/min, 1.5 ml/min., 2.0 ml/min. and actual flow delivered by pump A is determined.
- Repeat above steps for Calibration of Pump-B, Pump-C and Pump-D.
- Confirm and record the results.
- Acceptance criteria: Mean of three replicate should be as follows;

Flow rate	Acceptance criteria
0.25 ml / minute	0.25 ml ± 0.1 ml
0.5 ml / minute	0.5 ml ± 0.1 ml
1.0 ml / minute	1.0 ml ± 0.1 ml
1.5 ml / minute	1.5 ml ± 0.1 ml
2.0 ml / minute	2.0 ml ± 0.1 ml

WAVELENGTH ACCURACY TEST FOR UV DETECTOR:
- CHROMATOGRAPHIC CONDITIONS:
- The chromatographic conditions are as follows;
  - Column : C₁₈(250 mm × 4.6 mm)5 µm
  - Column temperature : 25⁰C
  - Flow rate : 1.0 ml/ min
  - Mobile phase : Methanol (70) : HPLC water (30)
  - Injection Volume : 20 µl
- Prepare the 20 ppm of caffeine in HPLC water as follow;
  - Caffeine Stock Solution: Weigh accurately about 50 mg of Caffeine In 100 ml volumetric flask, add 60 ml of HPLC water. Sonicate for 15 min. and diluted to volume with HPLC water.
  - Dilute 2 ml of Caffeine stock solution to 50 ml with HPLC water.
- Inject the preparation at 269, 270, 271, 272, 273,274, 275, 276 and 277 nm.
- Same time check that the maximum area should be detected at the wavelength 272 nm ± 2.0 nm.
- Confirm and record the results.

LINEARITY OF DETECTOR RESPONSE
Chromatographic conditions: The chromatographic conditions are as follows;
- Column : C₁₈(250 mm × 4.6 mm)5 µm
- Column temperature : 25⁰C
- Flow rate : 1.0 ml/min.
- Mobile phase : Methanol (70) : HPLC water (30)
- Wavelength : 272 nm
- Injection volume : 20 µl
Then prepare the level solution of 20, 30, 40, 50 and 60 ppm with the help of the dilution of Caffeine stock solution.
- 20 ppm solution: Dilute 2.0 ml of the Caffeine stock solution to 50 ml with HPLC water.
- 30 ppm solution: Dilute 3.0 ml of the Caffeine stock solution to 50 ml with HPLC water.
- 40 ppm solution: Dilute 4.0 ml of the Caffeine stock solution to 50 ml with HPLC water.
- 50 ppm solution: Dilute 5.0 ml of the Caffeine stock solution to 50 ml with HPLC water.
- 60 ppm solution: Dilute 6.0 ml of the Caffeine stock solution to 50 ml with HPLC water.
Inject the each solution three times; calculate the mean area RSD%. (NMT 2.0%)
Plot the graph of concentration against area for the linearity of detector response.
Calculate the correlation coefficient and correlation coefficient should not be less than 0.998.
Remark the performance of detector in terms of linearity is satisfactory or not & note the results.

CALIBRATION OF INJECTOR & LOOP
Precision and Linearity of Injector and Loop
- Chromatographic conditions: The chromatographic conditions are as follows;
  - Column : C₁₈(250 mm × 4.6 mm)5 µm
  - Column temperature : 25⁰C
  - Flow rate : 1.0 ml/min.
  - Mobile phase : Methanol (70) : HPLC water (30)
  - Wavelength : 272 nm
  - Injection volume : 5 µl ; 10 µl; 20 µl; 50 µl and 100 µl
- Prepare the 20 ppm of caffeine in HPLC water by diluting 2 ml of stock solution to 50 ml with HPLC water and inject the triplicates of 5 µl, 10 µl, 20 µl, 50 µl and 100 µl of sample preparation.
- Plot the graph of Injection volume against area.
- Calculate correlation coefficient should not be less than 0.998.
- Remark :The performance of Injector & Loop in terms of Linearity and Precision is satisfactory or not & note the results.
Auto Sampler Injector Calibration
- Chromatographic conditions: The chromatographic conditions are as follows;
  - Column : Union
  - Flow : 1.0 ml / min
  - Wavelength : 254 nm
  - Mobile phase : Water
  - Injection volume : 5 ml/10 ml/20 ml/50 ml/100 ml
  - Run time : 2 minutes
- Fill HPLC vial with HPLC water .
- Weigh accurately filled vial & note down the weight of filled vial (A).
- Inject 10 injections of equal volume about 5 ml of HPLC water from the same vial.
- Reweigh the same vial & note down the weight (B).
- The difference in the weighing gives the weight of water injected. Maintain temperature at 25°C during whole procedure.
- Determine the volume of water injected by assuming that the weight of 1 ml of purified water at 25°C when weighed in air of density 0.0012 gm per ml is 0.99602.
  - Volume of water per Inj. = A – B x 0.99602 x 1000 / 10
- Repeat the same procedure by taking injection volume 10 ml, 20 ml, 50 ml & 100 ml instead of 5 ml.
- Acceptance criteria: Volume in ml of individual injection should be ± 2% of defined volume.

CALIBRATION FOR GRADIENT PROGRAM TEST
- High pressure gradient elute delivery of 2 mobile phases (HPLC water and 10 ppm caffeine solution) is performed, the absorbance produced by various set concentrations is measured and the measurements are used to calculate the actual concentrations.
- Purge all the port with HPLC water.
- Replace the column with Union.
- Equilibrate system up to minimum 1 hour.
- Before gradient program test is carried out, Parameters set are as below,
  - Pump A – C Receiver : 500 ml HPLC water.
  - Pump B – D Receiver : 500 ml 10 ppm caffeine
  - Pumping mode : Gradient system ( A, B, C, D )
  - Flow Rate : 1 ml/min
  - UV Detector : 272 nm
  - Temperature : 25°C
  - Stop Time : 35 minutes.

GRADIENT PROGRAME TEST TIME

Calculate the observed mobile phase composition C_x (in % B and in % D ) from H_x values by the following formula
- C_x (%) = H_x x 100 / H₁₀₀
  - Where,
  - X : For programmed step height (10%,50%,90%,100%)
  - H_x : Height of respective plateaus in mm
  - C_x : The observed mobile phase composition
  - H₁₀₀: Height for 100% peak.
Acceptance criteria : The observed mobile phase composition (Cx) shall be within ± 2.0 of the set composition.
Remark the performance of gradient composition as satisfactory or not & note the results.

COLUMN OVEN TEMPERATURE
- Place a Calibrated digital temperature thermometer probe inside the column heater.
- Ensure that the thermometer probe does not contact any inside surface of oven.
- Close the column compartment properly.
- Set column compartment temperature 30°C .
- Take sufficient time to the oven temperature to reach and stabilize at set temperature.
- Record the result.
- Repeat the procedure for 40°C & 60°C and record the results.

CARRY OVER TEST
- Chromatographic conditions: The chromatographic conditions are as follows;
  - Column : C₁₈(250 mm × 4.6 mm) 5 µm
  - Column temperature : 25°C
  - Flow rate : 1.0 ml/min.
  - Mobile phase : Methanol (70) : water (30)
  - Wavelength : 272 nm
  - Injection volume : 20 µl
- Sample preparation: 100 ppm solution : Dilute 10 ml of Caffeine Stock solution to 50 ml with HPLC water.
- Calculate carryover by the formula;
  - Carryover (%) = Carryover Area in blank x 100 / Test Area
- Acceptance criteria: Not more than 0.01%.

6.0 ABBREVIATIONS:

Abbreviation	Expanded form
ml	Milliliter
min	Minute
⁰C	Degree Celsius
nm	Nanometer
%	Percentage
Abs	Absorbance
UPS	Uninterruptible Power Supply
ppm	Parts Per Million
gm	Gram
mm	Millimeter
µl	Microliter
µm	Micrometer
µ	Micron