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 rateAcceptance 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 Cx (in % B and in % D ) from Hx values by the following formula
    • Cx (%) = Hx x 100 / H100
      • Where,
      • X   : For programmed step height (10%,50%,90%,100%)
      • Hx : Height of respective plateaus in mm
      • Cx : 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:
AbbreviationExpanded form
mlMilliliter
minMinute
0CDegree Celsius
nmNanometer
%Percentage
AbsAbsorbance
UPSUninterruptible Power Supply
ppmParts Per Million
gmGram
mmMillimeter
µlMicroliter
µmMicrometer
µMicron

7.0 ANNEXURES:
Annex. No.                           Title
01Calibration Of High Performance Liquid Chromatography

8.0 SOP REFERENCES
  •  Manual of High Performance Liquid Chromatography

END OF THE SOP

For HPLC Basics- Principle and Scope click link: HPLC Basics- Principle and Scope


ANNEXURES :

Annex. No. 01 Calibration Of High Performance Liquid Chromatography